Renal tea acidic polysaccharide, and preparation method and application thereof

Abstract

The application discloses a renal tea acidic polysaccharide and a preparation method and application thereof. The renal tea acidic polysaccharide is composed of arabinose, rhamnose, xylose, glucose, mannose and galactose, and the mass percentages of the components are 1.48%, 0.54%, 0.38%, 2.79%, 42.93% and 51.87% respectively. Main derivatives of the renal tea acidic polysaccharide are 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol and 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol, and main connection modes are t-Glc(p), t-Gal(p), 4-GalA(p) and 4-Glc(p). The renal tea acidic polysaccharide provided by the application has anti-hyperuricemia activity in a dose range of 50-200 mg / kg, and exceeds a positive drug allopurinol in a dose range of 100-200 mg / kg. The method for preparing the renal tea acidic polysaccharide is simple in operation, high in extraction process efficiency, environmental protection and low in cost, and is worth popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide extraction technology, specifically relating to a kidney tea acidic polysaccharide, its preparation method, and its application. Background Technology

[0002] Hyperuricemia (HUA) is a common metabolic disease caused by abnormally high blood uric acid levels due to disordered purine metabolism. Increased uric acid synthesis and / or decreased excretion from any cause can lead to HUA. Currently, there are two main types of chemical drugs for treating HUA: one type reduces excessive uric acid production, such as allopurinol and febuxostat; the other type promotes uric acid excretion, such as benzbromarone and probenecid. While these drugs are relatively effective, they have limited target areas, significant adverse reactions and side effects, marked gastrointestinal reactions, and poor patient tolerance and compliance. Patients often experience liver and kidney dysfunction. Traditional Chinese medicine (TCM) has fewer adverse reactions and is inexpensive. Therefore, as a current research hotspot, developing highly effective and safe TCM for treating HUA based on traditional anti-hyperuricemia drugs has significant value and far-reaching implications.

[0003] With the development of modern separation and analysis techniques, a large number of polysaccharides have been isolated and identified, and their pharmacological activities have been extensively studied. The biological activities of polysaccharides mainly focus on immunomodulation, antitumor, antiviral, anticoagulant, hypoglycemic, and antioxidant effects. However, research on their anti-hyperuricemia activity is relatively limited. This invention aims to provide a renal tea acidic polysaccharide with anti-hyperuricemia activity. Summary of the Invention

[0004] The first objective of this invention is to provide a kidney tea acidic polysaccharide, and the second objective of this invention is to provide a method for preparing the kidney tea acidic polysaccharide and its application.

[0005] The first objective of this invention is achieved as follows: a renal tea acidic polysaccharide, composed of arabinose, rhamnose, xylose, glucose, mannose, and galactose, with the following mass percentages: 1.48%, 0.54%, 0.38%, 2.79%, 42.93%, and 51.87%, respectively; the main derivatives of the renal tea acidic polysaccharide are 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol, and 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol, with the main linkage modes being t-Glc(p), t-Gal(p), 4-GalA(p), and 4-Glc(p).

[0006] The second objective of this invention is achieved by the method for preparing the kidney tea acidic polysaccharide, which is carried out according to the following steps: 1) After pulverizing the dried kidney tea medicinal material, pass it through a No. 2 sieve. After defatting with acetone by reflux at 65-70℃ for 3-4 times, filter it. After the residue is naturally dried in a ventilated place, add distilled water to the residue and heat it to reflux at 80-90℃ for 3-4 times, each time for 1-2 hours. Filter it, and after the residue is naturally dried in a ventilated place, add 0.2 mol / L NaOH solution and heat it to reflux at 50-60℃ for 3-5 times. Combine the filtrates. 2) Adjust the pH of the filtrate obtained in step 1) to 7-8 with 0.02 mol / L HCl solution, concentrate it to a certain volume under reduced pressure, add anhydrous ethanol to adjust the alcohol concentration of the solution to 80-90% while stirring, let it stand for 24 h, centrifuge at 3000-3500 r / min for 10-20 min, collect the precipitate, and obtain the crude acidic polysaccharide of kidney tea. 3) Dissolve the crude acidic polysaccharide of kidney tea in distilled water, add chloroform and n-butanol, sonicate at 500 W for 30-40 min, centrifuge at 3000-3500 r / min for 10-20 min, repeat the above process 4-6 times, collect and combine the supernatant, concentrate under reduced pressure, dissolve in distilled water, and obtain the target kidney tea acidic polysaccharide after dialysis and concentration.

[0007] The application of the renal tea acidic polysaccharide is as an active ingredient in the preparation of drugs for treating hyperuricemia.

[0008] The beneficial effects of this invention are as follows: 1. This invention provides a novel drug with good efficacy against hyperuricemia—rencha acidic polysaccharide. It exhibits anti-hyperuricemia activity within a dosage range of 50-200 mg / kg, and surpasses the positive control drug allopurinol within a dosage range of 100 mg / kg-200 mg / kg. Furthermore, rencha acidic polysaccharide has low toxicity and a certain protective effect on the kidneys, making it a promising new traditional Chinese medicine ingredient for anti-HUA (hyperuricemia).

[0009] 2. The method for preparing acidic polysaccharides from kidney tea according to this invention—the dilute alkali extraction method—utilizes the alkali's destructive effect on cell walls, which facilitates the leaching of acidic polysaccharides and thus shortens the extraction time. The acidic polysaccharides are stable in the alkali solution, reducing polysaccharide loss during extraction. The extraction conditions are easy to control, the operation is simple, and the extraction process is highly efficient, environmentally friendly, and low-cost, making it worthy of widespread application. Attached Figure Description

[0010] Figure 1 The high-performance gel permeation chromatography (GPC) chromatogram of the acidic polysaccharide from the kidney tea prepared in Example 1 is shown. Figure 2 The standard curve for determining the total sugar content of the kidney tea acidic polysaccharide prepared in Example 1; Figure 3 The standard curve for determining the protein content of the acidic polysaccharide in kidney tea prepared in Example 1; Figure 4 The monosaccharide composition analysis spectrum of the acidic polysaccharide prepared in Example 1 is shown below; Figure 5 The total ion current spectrum was used to analyze the linkage mode of the acidic polysaccharide prepared in Example 1. Figure 6 Infrared spectrum of the kidney tea acidic polysaccharide prepared in Example 1; Figure 7 The figures in the middle and lower right corners show the effects of different concentrations of CSP-A (50 mg / kg, 100 mg / kg and 200 mg / kg) prepared in Example 1 on serum uric acid (UA), xanthine oxidase (XOD), creatinine (CRE) and blood urea nitrogen (BUN) in hyperuricemic mice. Detailed Implementation

[0011] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention in any way. Any modifications or substitutions made based on the technical teachings of the present invention shall fall within the protection scope of the present invention.

[0012] This invention discloses a kidney-derived acidic polysaccharide composed of arabinose, rhamnose, xylose, glucose, mannose, and galactose, with the following mass percentages: 1.48%, 0.54%, 0.38%, 2.79%, 42.93%, and 51.87%, respectively. The main derivatives of this kidney-derived acidic polysaccharide are 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol, and 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol, with the main linkage modes being t-Glc(p), t-Gal(p), 4-GalA(p), and 4-Glc(p).

[0013] This invention also provides a method for preparing the aforementioned kidney tea acidic polysaccharide, which is implemented according to the following steps: 1) After pulverizing the dried kidney tea medicinal material, pass it through a No. 2 sieve. After defatting with acetone by reflux at 65-70℃ for 3-4 times, filter it. After the residue is naturally dried in a ventilated place, add distilled water to the residue and heat it to reflux at 80-90℃ for 3-4 times, each time for 1-2 hours. Filter it, and after the residue is naturally dried in a ventilated place, add 0.2 mol / L NaOH solution and heat it to reflux at 50-60℃ for 3-5 times. Combine the filtrates. 2) Adjust the pH of the filtrate obtained in step 1) to 7-8 with 0.02 mol / L HCl solution, concentrate it to a certain volume under reduced pressure, add anhydrous ethanol to adjust the alcohol concentration of the solution to 80-90% while stirring, let it stand for 24 h, centrifuge at 3000-3500 r / min for 10-20 min, collect the precipitate, and obtain the crude acidic polysaccharide of kidney tea. 3) Dissolve the crude acidic polysaccharide of kidney tea in distilled water, add chloroform and n-butanol, sonicate at 500 W for 30-40 min, centrifuge at 3000-3500 r / min for 10-20 min, repeat the above process 4-6 times, collect and combine the supernatant, concentrate under reduced pressure, dissolve in distilled water, and obtain the target kidney tea acidic polysaccharide after dialysis and concentration.

[0014] In step 1), the mass-to-volume ratio of kidney tea herbs to acetone is 1:8-10.

[0015] In step 1), the ratio of medicinal residue to distilled water is 1:8-10 g / ml, and the mass-volume ratio of medicinal residue to 0.2 mol / L NaOH solution is 1:6-8 g / ml.

[0016] In step 3), the volume of chloroform is 1 / 3 to 1 / 5 of the volume of the polysaccharide solution, and the volume of n-butanol is 1 / 3 to 1 / 5 of the volume of chloroform.

[0017] In step 3), the ultrasonic power is 500 W.

[0018] The present invention further provides the application of the aforementioned kidney tea acidic polysaccharide as an active ingredient in the preparation of drugs for treating hyperuricemia.

[0019] The anti-hyperuricemia drug comprises the renal tea acid polysaccharide as an active agent and its pharmaceutically effective carrier.

[0020] Example 1 1) Pulverize 500 g of dried kidney tea medicinal material and pass it through a No. 2 sieve. Add 4000 mL of acetone and heat at 65℃ for reflux three times to defatted the material. After filtration, air-dry the residue at room temperature in a ventilated place. Add 4000 mL of distilled water to the dried residue and heat at 80℃ for reflux three times, each time for 1 hour. After filtration, air-dry the residue at room temperature in a ventilated place. Add 3000 mL of 0.2 mol / L NaOH solution and heat at 50℃ for reflux three times to extract the residue. Combine the filtrates.

[0021] 2) Add 0.02 mol / L HCl to the filtrate to adjust the pH to 7, concentrate the extract under reduced pressure to a certain volume, add anhydrous ethanol to adjust the alcohol concentration of the solution to 80%, stir while adding, let stand for 24 h, centrifuge at 3500 r / min for 10 min, collect the precipitate, and obtain the crude acidic polysaccharide of kidney tea.

[0022] 3) Dissolve the crude acidic polysaccharide of *Tea stenoptera* in distilled water to a certain volume. Accurately measure the volume of the crude acidic polysaccharide solution, add 1 / 5 volume of chloroform, then add 1 / 5 volume of n-butanol, and sonicate at 500 W for 30 min. Centrifuge at 3500 r / min for 10 min. Repeat the above process 4 times. Collect and combine the supernatants, concentrate under reduced pressure, freeze-dry for 24 h, dissolve again in distilled water, and dialyze with distilled water in a 3400 Da dialysis bag for 48 h. After concentration and freeze-drying for 24 h, 51.26 g of acidic polysaccharide of *Tea stenoptera* (CSP-A) was obtained, with an extraction rate of 10.25%. High-performance gel permeation chromatography (HPGPC) purity detection showed a single symmetrical peak (…). Figure 1 This indicates that the CSP-A prepared in this embodiment is a homogeneous polysaccharide component.

[0023] Example 2 1) Pulverize 500 g of dried kidney tea medicinal material and pass it through a No. 2 sieve. Add 4500 mL of acetone and heat at 67℃ for reflux to defatted 3 times. After filtration, place the residue in a ventilated place at room temperature to dry. Add 4500 mL of distilled water to the dried residue and heat at 85℃ for reflux extraction 3 times, 1.5 h each time. After filtration, place the residue in a ventilated place at room temperature to dry. Add 3500 mL of 0.2 mol / L NaOH solution and heat at 55℃ for reflux extraction 4 times. Combine the filtrates.

[0024] 2) Add 0.02 mol / L HCl to the filtrate to adjust the pH to 7.5, concentrate the extract under reduced pressure to a certain volume, add anhydrous ethanol to adjust the alcohol concentration of the solution to 85%, stir while adding, let stand for 24 h, centrifuge at 3250 r / min for 15 min, collect the precipitate, and obtain the crude acidic polysaccharide of kidney tea.

[0025] 3) Dissolve the crude acidic polysaccharide of *Tea stenoptera* in distilled water to a certain volume. Accurately measure the volume of the crude acidic polysaccharide solution, add 1 / 3 volume of chloroform, then add 1 / 3 volume of n-butanol, and sonicate at 500 W for 35 min. Centrifuge at 3250 r / min for 15 min. Repeat the above process 5 times, collect and combine the supernatants, concentrate under reduced pressure, freeze-dry for 24 h, dissolve again in distilled water, and dialyze with distilled water in a 3400 Da dialysis bag for 48 h. After concentration and freeze-drying for 24 h, 58.37 g of acidic polysaccharide of *Tea stenoptera* (CSP-A) was obtained, with an extraction rate of 11.67%.

[0026] Example 3 1) Crush 500 g of dried kidney tea medicinal material and pass it through a No. 2 sieve. Add 5000 mL of acetone and heat at 70℃ for reflux four times to defatted the material. After filtration, place the residue in a ventilated place at room temperature to dry. Add 5000 mL of distilled water to the dried residue and heat at 90℃ for reflux four times, each time for 2 hours. After filtration, place the residue in a ventilated place at room temperature to dry. Add 4000 mL of 0.2 mol / L NaOH solution and heat at 60℃ for reflux five times to extract. Combine the filtrates.

[0027] 2) Add 0.02 mol / L HCl to the filtrate to adjust the pH to 8, concentrate the extract under reduced pressure to a certain volume, add anhydrous ethanol to adjust the alcohol concentration of the solution to 90%, stir while adding, let stand for 24 h, centrifuge at 3000 r / min for 20 min, collect the precipitate, and obtain the crude acidic polysaccharide of kidney tea.

[0028] 3) Dissolve the crude acidic polysaccharide of *Tea styracifolium* in distilled water to a certain volume. Accurately measure the volume of the crude acidic polysaccharide solution, add 1 / 4 volume of chloroform, then add another 1 / 4 volume of chloroform. Sonicate at 500 W for 40 min, centrifuge at 3000 r / min for 20 min, repeat the above process 6 times, collect and combine the supernatants, concentrate under reduced pressure, freeze-dry for 24 h, dissolve again in distilled water, and dialyze with distilled water in a 3400 Da dialysis bag for 48 h. After concentration and freeze-drying for 24 h, 53.58 g of acidic polysaccharide of *Tea styracifolium* (CSP-A) was obtained, with an extraction rate of 10.72%.

[0029] Experimental Example 1: Determination of Total Sugar Content in Kidney Tea Acidic Polysaccharide (CSP-A) Determination method: Phenol-sulfuric acid method 1) Preparation of 6% phenol solution: Accurately weigh 15 g of solid phenol, add 250 mL of distilled water and dissolve it completely in a 60℃ water bath. Store in a brown ground glass bottle away from light for later use.

[0030] 2) Preparation of 0.1 mg / mL CSP solution: Accurately weigh 10 mg of the renal tea acidic polysaccharide prepared in Example 1, add distilled water to a volumetric flask to make up to 100 ml, and prepare a 0.1 mg / mL renal tea acidic polysaccharide solution for testing.

[0031] 3) Preparation of 0.1 mg / mL glucose standard solution: Accurately weigh 10 mg of glucose standard, add distilled water to a volumetric flask to make up to 100 mL, and prepare a 0.1 mg / mL glucose standard solution for later use.

[0032] 4) Construction of the glucose standard curve: Accurately measure 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of glucose standard solution into 12 mL glass test tubes, and add distilled water to bring the volume to 1 mL. Perform three replicates for each concentration. Add 0.5 mL of 6% phenol solution to each test tube, then slowly add 2.5 mL of concentrated sulfuric acid. Shake the test tubes rapidly and allow them to cool naturally until the color change stabilizes. Measure the absorbance at a wavelength of 490 nm. Plot the standard curve with the mass of the glucose standard solution on the x-axis and absorbance A on the y-axis.

[0033] 5) Determination of total sugar content in CSP-A samples: Take 1 mL of 0.1 mg / mL CSP-A solution, add 0.5 mL of 6% phenol solution and 2.5 mL of concentrated sulfuric acid solution, and measure its absorbance at a wavelength of 490 nm. Calculate the total sugar content in CSP-A based on the standard curve.

[0034] Results: The total sugar content of the prepared kidney tea acidic polysaccharide was determined using the phenol-sulfuric acid method. The CSP-A standard curve for the polysaccharide was obtained as follows: y = 2.2255x + 0.0967, R 2 =0.9968. The standard curve is as follows: Figure 2 As shown, the total sugar content of CSP-A was 88.34% according to the standard curve.

[0035] Experimental Example 2: Determination of Protein Content in Kidney Tea Acidic Polysaccharide (CSP-A) Determination method: Coomassie brilliant blue method 1) Preparation of Coomassie Brilliant Blue G-250 solution: Accurately weigh 25 mg of Coomassie Brilliant Blue G-250 and place it in a 250 mL brown volumetric flask. Add 25 mL of 95% ethanol to dissolve it completely, then add 25 mL of 85% phosphoric acid. Add distilled water to the mark and shake well.

[0036] 2) Preparation of 0.1 mg / mL CSP solution: Accurately weigh 10 mg of the renal tea acidic polysaccharide prepared in Example 1, add distilled water to a volumetric flask to make up to 100 ml, and prepare a 0.1 mg / mL renal tea acidic polysaccharide solution for testing.

[0037] 3) Preparation of 0.1 mg / mL protein standard solution: Accurately weigh 10 mg of protein standard, add distilled water to a volumetric flask to make up to 100 mL, and prepare a 0.1 mg / mL protein standard solution for later use.

[0038] 4) Construction of the protein standard curve: Accurately pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of protein standard solution into glass test tubes, respectively, and top up with distilled water to 1 mL. Perform three replicates for each concentration. Add 5 mL of Coomassie Brilliant Blue G-250 solution to each test tube, mix well, and measure the absorbance at 595 nm using a UV spectrophotometer. Plot the protein standard curve with the mass of the protein standard solution on the x-axis and the absorbance A on the y-axis.

[0039] 5) Protein content determination of CSP-A samples: Accurately measure 1 mL of 0.1 mg / mL CSP solution and add 5 mL of Coomassie Brilliant Blue G-250 solution. Set up 3 replicates, and measure the absorbance at 595 nm using a UV spectrophotometer. Calculate the average value and calculate the protein content in CSP according to the standard curve.

[0040] Results: The protein content in CSP was determined using the Coomassie Brilliant Blue method. A standard curve was plotted with the concentration of bovine serum albumin standard solution on the x-axis and absorbance (A) on the y-axis. The standard curve is shown below. Figure 3As shown, the standard equation y = 0.0088x - 0.0129 (R²) is obtained from the regression curve. 2 =0.9971). Based on the standard curve, the protein content in CSP-A was calculated to be 2.17%.

[0041] Experimental Example 3: Monosaccharide Composition Analysis of Kidney Tea Acidic Polysaccharide (CSP-A) Determination method: GC-MS analysis of polysaccharide acetylation derivatives 1) Hydrolysis of polysaccharides: Accurately weigh 5 mg of the kidney tea acidic polysaccharide prepared in Example 1 and place it in an ampoule. Add 4 mL of 2 mol / L TFA, seal the ampoule, and place it in a 105℃ ultra-high temperature electric heating oven for hydrolysis for 4 h. After cooling to room temperature, concentrate to dryness by rotary evaporation, then concentrate to dryness again by adding methanol. Repeat the operation 4-5 times to remove all TFA.

[0042] 2) Preparation of acetylated derivatives: 5 mg each of the TFA-free sample and monosaccharide standards (rhamnose, mannose, galactose, fucose, arabinose, xylose, fructose, and glucose) were placed in an ampoule. 5 mg of hydroxylamine hydrochloride was added, followed by 1 mL of pyridine solution. The mixture was reacted at 90 °C for 30 min. After cooling the reaction solution to room temperature, 1 mL of acetic anhydride was added, and the reaction was continued at 90 °C for 3 h to prepare the acetylated derivatives. The reaction product was cooled to room temperature, dried under nitrogen in a 70 °C water bath, and then redissolved in 1 mL of chloroform. 1 μL of the product was analyzed by GS-MS.

[0043] 3) GC-MS Analysis: An Agilent 8890 GC-MS system equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) was used. GC conditions: High-purity helium as carrier gas; injection port temperature 250℃; programmed temperature ramp: initial temperature 100℃, increased to 200℃ at 5℃ / min, held for 1 min, then increased to 250℃ at 10℃ / min, held for 5 min; carrier gas flow rate 1.0 mL / min, 1 μL injection at a splitless flow rate. Mass spectrometry conditions: interface temperature 280℃; quadrupole temperature: 150℃; ionization mode: EI; electron impact energy: 70 eV; solvent extension time: 5 min; full scan range: 50-800 m / z; mode: selected ion scan (SIM).

[0044] 4) Establishment of standard curves: Stock solutions containing mixed monosaccharide standards of rhamnose (Rha), arabinose (Ara), fructose (Fru), xylose (Xyl), mannose (Man), glucose (Glc), fucose (Fuc), and galactose (Gal) were prepared at concentrations of 18, 36, 72, 144, 288, and 576 μg / mL, respectively. Acetylated derivatives were prepared and analyzed by GC-MS. Linear regression was performed on each monosaccharide standard with mass concentration ρ (μg / mL) as the abscissa and peak area y of the derivative as the ordinate. The linear range was 18–576 μg / mL (Table 1).

[0045] Results: CSP-A was acid-hydrolyzed into different monosaccharides, which were then acetylated to obtain polysaccharide derivatives for GC-MS analysis. Monosaccharide standards were used to determine the composition of CSP-A (arabinose, rhamnose, xylose, glucose, mannose, and galactose) by retention time and ion fragmentation. Figure 4 The percentages of each component were: 1.48%, 0.54%, 0.38%, 2.79%, 42.93%, and 51.87%. Fucose was not detected (Table 2).

[0046] Table 1 Regression equations for monosaccharide standards Table 2. Percentage of each monosaccharide component in CSP-A Experiment Example 4: Analysis of Monosaccharide Linkage Modes of Kidney Tea Acidic Polysaccharide (CSP-A) Determination method: GC-MS analysis of polysaccharide derivatives 1) CSP-A Derivatization: Weigh 5 mg of the renal tea acidic polysaccharide prepared in Example 1, dissolve in 1 mL of pure water, add 200 µL of 0.2 M MES, then add 500 µL of 200 mg / mL carbodiimide, and react at room temperature for 2 h. Add 1 mL of 2 M imidazole and 1 mL of 70 mg / mL NaBD4, and react for 3 h. Add 300 μL of glacial acetic acid to terminate the reaction. Dialyze the sample for 48 h, and after dialysis, freeze-dry the sample for methylation treatment. Dissolve in 1 mL of DMSO in the freeze-dried sample. Add 30 mg of NaOH and incubate for 30 min. Add 250 μL of iodomethane solution, purge with nitrogen, and react in the dark for 1 h. Add another 250 μL of iodomethane solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the washing with water 3 times. Take the lower dichloromethane phase and dry it under nitrogen. Add 1 mL of 2M TFA and react at 121℃ for 120 min. Dry under nitrogen at 30℃. Add 1 mL of freshly prepared 1 M NaBD4 (ammonia solution). Incubate with magnetic stirring at room temperature for 2.5 h. Terminate the reaction by adding 300 μL of acetic acid and dry under nitrogen. Dry twice in a 40℃ water bath using 2 mL of 5% (vol / vol) acetic acid-methanol solution with nitrogen, then twice again in a 40℃ water bath using 2 mL of methanol. Add 1.5 mL of acetic anhydride, vortex to mix, and react at 100℃ for 2.5 h. Add 2 mL of water and let stand for 10 min. Add 1 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat washing with water 3 times. Take the lower dichloromethane phase for analysis.

[0047] 2) GC-MS Analysis: The gas chromatography system used was an Agilent 7890A gas chromatography system (Agilent Technologies, USA), with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA). The carrier gas was high-purity helium (purity not less than 99.999%), the flow rate was 1.0 mL / min, and the injection port temperature was 260℃. The injection volume was 1 μL, split injection, split ratio 10:1, and solvent delay 2.2 min. The temperature program was: 50℃ held for 1.0 min, increased to 130℃ at 50℃ / min, increased to 230℃ at 3℃ / min, and held for 2 min. The mass spectrometry system used was an Agilent 5977B quadrupole mass spectrometer (Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source had an inlet temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The scanning mode was full scan (SCAN), with a mass scan range (m / z) of 30-600.

[0048] Results Analysis: CSP-A was acid-hydrolyzed into different monosaccharides, and then methylated to obtain polysaccharide derivatives for GC-MS analysis. Based on the relative retention time and mass spectrum of each chromatographic peak, and by comparing with literature data and the database of the Complex Carbohydrates Research Center at the University of Georgia, the sugar residue type corresponding to each chromatographic peak was analyzed. The content of each component was calculated using the peak area corresponding to the sugar residue type (relative molar amount = peak area / molecular weight; *relative molar ratio (%) = relative molar amount / sum of relative molar amounts of all components). The results are shown in Table 3 and [Table data would be inserted here]. Figure 5 The results showed that CSP-A and CSP-N were mainly composed of six monosaccharide derivatives, which was consistent with the results of the monosaccharide composition analysis. Among them, the main derivatives of CSP-A were: 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol, and 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol, with the main linkage modes being t-Glc(p), t-Gal(p), 4-GalA(p), and 4-Glc(p).

[0049] Table 3. Connection methods, derivative names, and relative molar ratios in CSP-A Experimental Example 5: Infrared Spectroscopic Analysis of Kidney Tea Acidic Polysaccharide (CSP-A) Determination method: Weigh 2 mg of CSP-A, mix it thoroughly with KBr, press it into a thin film, and detect it in an FTIR instrument at a wavelength of 4000 nm–400 nm.

[0050] Results analysis: such as Figure 6 As shown: 3411.76 cm -1 The signal peak at 2925.34 cm⁻¹ is generated by the -OH stretching vibration. -1 The signal peak at 1716.74 cm⁻¹ is generated by the stretching vibration of CH. -1 and 1632.49 cm -1 The signal peak at 1410.25 cm⁻¹ is generated by the asymmetry of C=O. -1 The absorption peak at 1246.24 cm⁻¹ is caused by the CO stretching vibration. -1 The signal peak at 1149.19 cm⁻¹ represents the bending vibration of CH. -1 1072.83 cm -1 The signal peak at 1000 cm⁻¹ -1 -1200 cm -1 This region is generated by two CO stretching vibrations: COH and COC of the sugar ring; 940.02 cm -1 The absorption peak at 832.79 cm⁻¹ is a characteristic peak of β-glycosidic bonds. -1 The signal absorption peak at 770.78 cm⁻¹ is due to the CH-angle vibration of the epimerism of the α-terminal group of pyranose. -1 It is a symmetrical ring stretching vibration of the pyran ring.

[0051] Example 1: Detection of the anti-hyperuricemia activity of acidic polysaccharides from *Tea lycopene*. Methods for evaluating drug efficacy: 1) Eighty-four 18-22 g SPF-grade KM mice were housed in an SPF-grade animal room with suitable temperature and humidity. After one week of acclimatization, the mice were randomly divided into a normal control group (n=12) and a model group (n=72). The normal control group was given a 0.5% sodium carboxymethyl cellulose (0.5% CMC-Na) solution, while the model group was given a suspension of 100 mg / kg adenine and 400 mg / kg potassium oxonate dissolved in 0.5% sodium carboxymethyl cellulose. The modeling agent was administered daily at 9:00 AM for 14 days to establish a mouse model of hyperuricemia. On day 14, after administration of the modeling agent, mice were allowed normal water intake but fasted for 12 hours. Ten mice from the model group were randomly selected, and blood was collected from the orbital sinus. Serum was centrifuged to obtain serum uric acid. A uric acid assay kit was used to evaluate the success of the modeling (serum uric acid concentration >110 mg / kg). (μmol / L indicates successful modeling). Fifty mice with successful modeling were randomly divided into a model group, an allopurinol group (All group), and low, medium, and high dose CSP-A drug groups, with 10 mice in each group. At 9:00 AM, the normal group continued to receive 0.5% sodium carboxymethyl cellulose, while the other groups received the modeling agent. At 1:00 PM, the normal group and the model group received 0.5% sodium carboxymethyl cellulose, the All group received 30 mg / kg of allopurinol, and the CSP-AL, CSP-AM, and CSP-AH groups received 50 mg / kg, 100 mg / kg, and 200 mg / kg of CSP-A dissolved in 0.5% sodium carboxymethyl cellulose, respectively. After 7 consecutive days of administration, blood was collected from the orbital sinus after the last administration, with normal water intake and fasting for 12 hours. After standing for 30 minutes, the cells were centrifuged at 3500 r / min for 10 minutes, and the supernatant was used to detect hyperuricemia-related indicators.

[0052] Results Analysis: From Figure 7 It can be seen that, compared with the normal control group, the levels of UA, XOD, CRE, and BUN in the model group were significantly increased. p <0.001 indicates that the HUA mouse model was successfully established, and the mice had severely impaired renal function. Compared with the model group, the levels of UA, XOD, CRE, and BUN in the drug-treated group were significantly reduced ( p <0.05), especially in the CSP-A group ( p <0.001). Furthermore, the levels of BUN and CRE in the CSP-AM and CSP-AH groups were lower than those in the positive control group (All group). These results indicate that CSP-A possesses anti-HUA activity and a certain degree of renal function protection. In particular, the CSP-AM and CSP-AH groups showed better effects in reducing serum UA and XOD than the positive control group. However, the results of renal function impairment indicators (CRE and BUN) showed that CSP-A had a weaker adverse effect on mouse renal function than the positive control group.

[0053] In summary, the renal tea acidic polysaccharide (CSP-A) provided by this invention exhibits good anti-hyperuricemia activity, and its activity is superior to that of the positive control drug allopurinol within a dose range of 100 mg / kg-200 mg / kg. Furthermore, it has low toxicity, a broad safety margin, and a certain degree of kidney protection, making it a promising alternative to allopurinol.

Claims

1. A kidney-tonifying acidic polysaccharide, characterized in that, The renal tea acidic polysaccharide is composed of arabinose, rhamnose, xylose, glucose, mannose, and galactose, with the following mass percentages: 1.48%, 0.54%, 0.38%, 2.79%, 42.93%, and 51.87%, respectively. The main derivatives of the renal tea acidic polysaccharide are 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol, and 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl The preparation method of the renal tea acidic polysaccharide includes galactitol and 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol, with the main linkage modes being t-Glc(p), t-Gal(p), 4-GalA(p), and 4-Glc(p); the method of preparing the renal tea acidic polysaccharide is carried out according to the following steps: 1) After pulverizing the dried kidney tea medicinal material, pass it through a No. 2 sieve. After defatting with acetone by reflux at 65-70℃ for 3-4 times, filter it. After the residue is naturally dried in a ventilated place, add distilled water to the residue and heat it to reflux at 80-90℃ for 3-4 times, each time for 1-2 hours. Filter it, and after the residue is naturally dried in a ventilated place, add 0.2mol / L NaOH solution and heat it to reflux at 50-60℃ for 3-5 times. Combine the filtrates. 2) Adjust the pH of the filtrate obtained in step 1) to 7-8 with 0.02 mol / L HCl solution, concentrate it under reduced pressure to a certain volume, add anhydrous ethanol to adjust the alcohol concentration of the solution to 80-90% while stirring, let it stand for 24 hours, centrifuge at 3000-3500 r / min for 10-20 min, collect the precipitate, and obtain the crude acidic polysaccharide of kidney tea. 3) Dissolve the crude acidic polysaccharide of kidney tea in distilled water, add chloroform and n-butanol, sonicate for 30-40 min, centrifuge at 3000-3500 r / min for 10-20 min, repeat the above process 4-6 times, collect and combine the supernatant, concentrate under reduced pressure, dissolve in distilled water, place in a 3400 Da dialysis bag and dialyze with distilled water for 48 h, and concentrate to obtain the target acidic polysaccharide of kidney tea.

2. The method for preparing the kidney tea acidic polysaccharide according to claim 1, characterized in that, Follow these steps to achieve the following: 1) After pulverizing the dried kidney tea medicinal material, pass it through a No. 2 sieve. After defatting with acetone by reflux at 65-70℃ for 3-4 times, filter it. After the residue is naturally dried in a ventilated place, add distilled water to the residue and heat it to reflux at 80-90℃ for 3-4 times, each time for 1-2 hours. Filter it, and after the residue is naturally dried in a ventilated place, add 0.2mol / L NaOH solution and heat it to reflux at 50-60℃ for 3-5 times. Combine the filtrates. 2) Adjust the pH of the filtrate obtained in step 1) to 7-8 with 0.02 mol / L HCl solution, concentrate it under reduced pressure to a certain volume, add anhydrous ethanol to adjust the alcohol concentration of the solution to 80-90% while stirring, let it stand for 24 hours, centrifuge at 3000-3500 r / min for 10-20 min, collect the precipitate, and obtain the crude acidic polysaccharide of kidney tea. 3) Dissolve the crude acidic polysaccharide of kidney tea in distilled water, add chloroform and n-butanol, sonicate for 30-40 min, centrifuge at 3000-3500 r / min for 10-20 min, repeat the above process 4-6 times, collect and combine the supernatant, concentrate under reduced pressure, dissolve in distilled water, place in a 3400 Da dialysis bag and dialyze with distilled water for 48 h, and concentrate to obtain the target acidic polysaccharide of kidney tea.

3. The kidney tea acidic polysaccharide according to claim 1, characterized in that, In step 1), the mass-to-volume ratio of kidney tea herbs to acetone is 1:8~10.

4. The kidney-tea acidic polysaccharide according to claim 1, characterized in that, In step 1), the ratio of medicinal residue to distilled water is 1:8~10 g / mL, and the mass-volume ratio of medicinal residue to 0.2 mol / L NaOH solution is 1:6~8 g / mL.

5. The kidney tea acidic polysaccharide according to claim 1, characterized in that, In step 3), the volume of chloroform is 1 / 3 to 1 / 5 of the volume of the polysaccharide solution, and the volume of n-butanol is 1 / 3 to 1 / 5 of the volume of chloroform.

6. The kidney tea acidic polysaccharide according to claim 1, characterized in that, In step 3), the ultrasonic power is 500W.

7. The use of the renal tea acidic polysaccharide as an active ingredient in the preparation of drugs for treating hyperuricemia, as described in claim 1.

8. The application according to claim 7, wherein the antihyperuricemia drug comprises the renal tea acid polysaccharide as an active agent and its pharmaceutically effective carrier.

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