Purified poria cocos polysaccharide with kidney protection activity as well as preparation method and application of purified poria cocos polysaccharide

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a preparation method of purified poria cocos polysaccharide with kidney protection activity. The preparation method comprises the following steps: adding water into poria cocos, performing ultrasonic treatment, and performing suction filtration to obtain supernate 1 and precipitate; adding water, beta-glucanase and cellulase into the precipitate, performing enzymolysis, and performing suction filtration to obtain supernate 2; and finally, mixing the supernate 1 and the supernate 2 to obtain the poria cocos extracting solution. Compared with a water extraction process, the process disclosed by the invention has the advantages that the polysaccharide molecular weight can be effectively reduced, the antioxidant activity is improved, the purified poria cocos polysaccharide FL-1 is obtained through deproteinization and sephadex column chromatography separation and purification, and the purified poria cocos polysaccharide FL-1 is evaluated through a renal tubular epithelial cell aging model and a zebra fish kidney injury model. The cell activity can be obviously improved, the content of beta-galactosidase is reduced, and the content of NAD < + > is improved; the occurrence rate of the renal edema of the zebra fish can be obviously reduced, and the glomerular filtration function of the zebra fish is improved.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a method for preparing purified polysaccharides from Poria cocos with renal protective activity. Background Technology

[0002] Poria cocos polysaccharides, as the core active ingredient in Poria cocos sclerotia, have been proven to possess various physiological functions, including immunomodulation, anti-tumor activity, antioxidant activity, and anti-aging. They account for over 90% of the dry weight of Poria cocos and have broad application prospects in medicine, functional foods, and other fields. The bioactivity of polysaccharides is closely related to their structural characteristics, such as molecular weight distribution, glycosidic bond type, and monosaccharide composition. However, natural Poria cocos polysaccharides mainly exist in the form of alkali-soluble β-glucan, with a low proportion of water-soluble components. Furthermore, the dense cell wall structure of Poria cocos severely restricts its efficient extraction and bioavailability improvement, becoming a core bottleneck for industrial application.

[0003] Currently, domestic and international technologies for preparing Poria cocos polysaccharides mainly fall into two categories: one is direct extraction technology, including traditional water extraction and alcohol precipitation, alkali extraction, and recently developed methods such as ultrasound-assisted extraction, enzymatic hydrolysis, green solvent extraction, and bio-fermentation; the other is structural modification technology, including carboxymethylation modification, sulfation modification, and hydrogen peroxide degradation, aiming to improve the water solubility and bioactivity of polysaccharides through chemical or physical means. However, all of the above technologies have significant drawbacks and are difficult to meet the needs of high-value development.

[0004] Traditional water extraction and alcohol precipitation methods are only efficient at extracting water-soluble polysaccharides, with poor dissolution effects on alkali-soluble polysaccharides. While alkali extraction can improve yield, the alkaline environment easily leads to polysaccharide backbone breakage and destruction of active conformations, and residual alkali poses safety risks. Green solvent extraction methods face challenges in solvent recovery and low recycling rates. Bio-fermentation methods involve complex strain selection, sensitive process parameters, uneven product molecular weight distribution, and safety risks such as potential pathogenic strains producing toxins and the accumulation of metabolic byproducts. Chemical modification technologies such as carboxymethylation and sulfation require the use of toxic reagents such as chloroacetic acid and chlorosulfonic acid, which can easily leave residues. Hydrogen peroxide degradation methods may produce harmful degradation products such as formaldehyde, all of which fail to meet the safety requirements for food and cosmetic raw materials.

[0005] Chinese invention patent application CN 105132491A discloses a method for extracting Poria cocos polysaccharides, including the following steps: (1) After crushing Poria cocos, add 4-6 times the amount of water to expand it, add yeast and lactic acid bacteria at 1%-5% (w / w) in a ratio of 1:2, and carry out fermentation at a temperature of 28℃-31℃, pH 3.1-4.0, for 12-16 hours; (2) Mix the fermentation liquid evenly, add a compound enzyme preparation, which includes cellulase, hemicellulase, amylase and pectinase, at an addition amount of 1.2%-2.5% (w / w), at a temperature of 36℃-42℃, pH 3.5-4.2, for 10-12 hours; (3) Precipitate the reaction product with ethanol, add ethanol to make the alcohol content of the reaction product reach 80% (w / w), spray dry the precipitate, and obtain about 70.2% water-soluble Poria cocos polysaccharides.

[0006] Another Chinese invention patent application, CN106399420A, discloses a Poria cocos health-promoting yogurt and its preparation method. It first provides a Poria cocos polysaccharide enzymatic hydrolysate, prepared using an enzymatic hydrolysis method. Through optimization of various process parameters, the yield of Poria cocos polysaccharides can reach over 13.20%. This invention uses this Poria cocos polysaccharide enzymatic hydrolysate, or the supernatant and precipitate obtained after centrifugation, as raw materials to prepare a set-type Poria cocos yogurt. By adjusting the amount added and the content of sugar and fermentation yogurt, after process optimization, a set-type Poria cocos yogurt with a delicate texture, small, uniform, smooth curds, no bubbles, and excellent taste can be prepared, and the fermentation cycle can be greatly shortened. In addition, this yogurt also has the aroma of Poria cocos and the health benefits of Poria cocos polysaccharides, meeting the diverse needs of consumers.

[0007] Ultrasonic-assisted extraction and enzymatic hydrolysis currently offer advantages such as mild conditions, high safety, mature equipment, and controllable enzyme preparation costs. However, existing technologies are mostly simple combinations without parameter matching tailored to the structural characteristics of Poria cocos polysaccharides (e.g., molecular weight regulation and preservation of active conformations), resulting in limited improvement in polysaccharide yield and activity. Due to the complex structure of polysaccharides, containing multiple monosaccharide units and different branched structures, the yield of purified Poria cocos polysaccharides obtained using existing extraction and purification processes is low and the composition is heterogeneous, leading to unreliable drug activity. Therefore, a stable and efficient extraction process is urgently needed to obtain purified Poria cocos polysaccharides with high activity and uniform composition.

[0008] Chronic kidney disease and its related complications such as renal fibrosis and glomerulosclerosis have become a major global public health problem. Studies have confirmed that some plant polysaccharides can exert renal protective effects through pathways such as anti-oxidative stress, inhibition of inflammatory responses, and regulation of autophagy. However, there is a lack of experimental evidence to show whether enzymatic modification of Poria cocos polysaccharides can enhance this targeted efficacy; the correlation mechanism between the structural characteristics of polysaccharides (such as molecular weight range, monosaccharide composition, and sugar residue linkage) and renal protective activity is unclear, which cannot provide theoretical support for the targeted preparation of highly active polysaccharides.

[0009] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: Precise control of the process is difficult: the enzymatic hydrolysis parameters (enzyme source and type, temperature, time, etc.) need to be precisely matched with the dense cell wall and polysaccharide structure characteristics of Poria cocos. While improving water solubility and yield, it is also necessary to ensure activity and avoid destruction of active conformation or product heterogeneity. Structure-activity relationship verification is difficult: There is a lack of clear evidence of the correlation between the structural characteristics (molecular weight, glycan linkage, etc.) of Poria cocos polysaccharides (especially after enzymatic hydrolysis and modification) and their renal protective activity. A reliable model needs to be established and the mechanism of action needs to be elucidated. Industrialization is challenging: while pursuing high activity, it is essential to ensure that the process does not use toxic reagents, maintains batch stability, and keeps costs under control, in order to meet the food industry's requirements for safety, uniformity, and economy. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention provides a purified polysaccharide from Poria cocos with renal protective activity, its preparation method, and its applications. Addressing the core deficiencies of existing extraction processes that fail to consider both structure and targeted activity, and whose renal protective efficacy is unclear, this invention proposes the following research approach: By optimizing the complex enzymatic hydrolysis process of Poria cocos polysaccharides, high-yield, highly water-soluble polysaccharide components are prepared; its renal protective activity is systematically evaluated using a zebrafish kidney injury model and a renal tubular epithelial cell senescence model; the structural characteristics of the active polysaccharide are analyzed using modern chromatographic techniques; and finally, the structure-renal protective activity relationship of Poria cocos polysaccharides is clarified, providing a safe and practical technical solution for its in-depth development and industrial application in functional foods, special diets, and other fields.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] The definition of standard chemical terms can be found in the reference "Pharmacopoeia of the People's Republic of China (2020 Edition): China Medical Science and Technology Press: May 2020: 1st Edition".

[0014] Unless otherwise stated, conventional methods within the scope of the art, such as centrifugation, reflux, concentration, resuspension, etc., shall be used.

[0015] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0016] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0017] The term "Poria" used in this article refers to the dried sclerotium of the fungus *Poria cocos* (Schw.) Wolf, belonging to the Polyporaceae family. It is mostly harvested from July to September. After being dug up, the soil and sand are removed, and the fungus is piled up to "sweat," then spread out to dry until the surface is dry. This process of "sweating" is repeated several times until wrinkles appear and most of the internal moisture is lost. It is then air-dried, and this is called "Poria cocos pieces." Alternatively, fresh Poria cocos can be cut into different parts and air-dried, resulting in "Poria cocos peel" and "Poria cocos chunks," respectively. It has the effects of promoting diuresis and eliminating dampness, strengthening the spleen and calming the mind. It is used for edema with scanty urine, phlegm retention with dizziness and palpitations, spleen deficiency with poor appetite, loose stools and diarrhea, restlessness, palpitations, and insomnia.

[0018] The objective of this invention is achieved through the following technical solution: On the one hand, the present invention provides a method for preparing purified polysaccharides from Poria cocos with renal protective activity, comprising the following steps: (1) Add water to Poria cocos, sonicate, filter, and obtain supernatant 1 and precipitate; (2) Add water, β-glucanase and cellulase to the precipitate, enzymatically hydrolyze it, filter it, and obtain supernatant 2; (3) Mix supernatant 1 and supernatant 2 to obtain Poria cocos extract, and then purify it.

[0019] Among them, the technical characteristic is that the mass ratio of Poria cocos to water is 1:10-20; Among them, the preferred mass ratio of Poria cocos to water is 1:12-18; Among them, the preferred mass ratio of Poria cocos to water is 1:14-16; Among them, the preferred mass ratio of Poria cocos to water is 1:14, 1:15, or 1:16. Among them, the temperature of the ultrasound is 90-100℃, preferably 92-98℃, further preferably 94-96℃, most preferably 94℃, 95℃, 96℃, and most preferably 95℃.

[0020] The duration of the ultrasound is 0.5-2 hours, preferably 0.6-1.8 hours, preferably 0.8-1.5 hours, preferably 0.9-1.3 hours, and preferably 1 hour.

[0021] In some embodiments of the present invention, the mass ratio of the precipitate to water in step (2) is 1:5-15, preferably 1:6-13, even more preferably 1:8-12, most preferably 1:8, 1:10, 1:12, and most preferably 1:10.

[0022] In some embodiments of the present invention, after adding water, the pH is adjusted to 5-6 with citric acid, preferably 5.2-5.8, preferably 5.3-5.7, even more preferably 5.4-5.6, even more preferably 5.4, 5.5, 5.6, and most preferably 5.5.

[0023] In some embodiments of the present invention, the enzymatic hydrolysis time is 3-5 hours, preferably 3.5-4.5 hours, and more preferably 4 hours.

[0024] In some embodiments of the present invention, the enzymatic hydrolysis temperature is 40-60°C, preferably 42-58°C, preferably 44-56°C, preferably 48-50°C, preferably 48°C, 50°C, and most preferably 50°C.

[0025] In some embodiments of the present invention, the total amount of β-glucanase and cellulase added in step (2) is 3-5% of the mass of Poria cocos, preferably 3.2-4.8%, preferably 3.5-4.5%, preferably 3.8-4.2%, and preferably 4%; In some embodiments of the present invention, the mass ratio of β-glucanase to cellulase is 3-5:1, preferably 3:1, 4:1, or 5:1, and most preferably 4:1.

[0026] In some embodiments of the present invention, the β-glucanase is derived from Trichoderma listeri and Trichoderma viride in a mass ratio of 1:0.5-2; preferably 1:0.6-1.8, more preferably 1:0.8-1.6, and preferably and / or the cellulase is derived from Trichoderma viride.

[0027] In some embodiments of the present invention, the purification step in step (3) includes alcohol precipitation, removal of proteins, elution by G100 dextran gel column chromatography, and freeze-drying to obtain purified polysaccharides. The Poria cocos extract is concentrated and precipitated with alcohol to obtain a polysaccharide precipitate; then, a Poria cocos polysaccharide solution is obtained.

[0028] In some embodiments of the present invention, the alcohol precipitation includes concentrating the Poria cocos extract to 1 / 2 to 1 / 4 of its original volume, preferably 1 / 2, 1 / 3, or 1 / 4, and most preferably 1 / 3.

[0029] In some specific embodiments of the present invention, anhydrous ethanol is added until the final volume fraction of ethanol is 75-85%, preferably 78-82%, preferably 79-81%, and preferably 80%.

[0030] In some specific embodiments of the present invention, precipitation is carried out at 0-6℃ for 10-15 hours, and the polysaccharide precipitate is collected by centrifugation. Preferably, precipitation is carried out at 2-5℃ for 11-14 hours, and preferably, precipitation is carried out at 3-4℃ for 12-13 hours.

[0031] In some embodiments of the present invention, the step of removing proteins includes dissolving the polysaccharide precipitate in water, adding an equal volume of 3-6 wt% trichloroacetic acid solution, letting it stand for 3-5 hours, centrifuging, and concentrating to obtain a Poria cocos polysaccharide solution.

[0032] In some embodiments of the present invention, the Poria cocos polysaccharide solution is prepared into a solution with a mass concentration of 10 mg / mL, and subjected to 3 cm × 30 cm G100 dextran gel chromatography with water elution at a flow rate of 0.4-0.6 mL / min. The eluent is collected, and the absorbance is measured according to the phenol-sulfuric acid method for determining polysaccharides. An elution curve is plotted, and the polysaccharide solution is collected according to the peaks. The solution is then freeze-dried to obtain purified polysaccharides.

[0033] Secondly, the present invention provides a purified polysaccharide of Poria cocos prepared by the preparation method described above.

[0034] In some embodiments of the present invention, the purified polysaccharide of Poria cocos includes one or more of purified polysaccharide-1, purified polysaccharide-2, and purified polysaccharide-3 of Poria cocos, and / or, the weight-average molecular weight of purified polysaccharide-1 is 25,000-30,000, preferably 29,483, and the distribution coefficient Mw / Mn is 2-3, preferably 2.17; Preferably, the weight-average molecular weight of the purified polysaccharide-2 from Poria cocos is 10,000-15,000, more preferably 14,683, and the distribution coefficient Mw / Mn is 1.5-2.0, more preferably 1.74. Preferably, the weight-average molecular weight of the purified Poria cocos polysaccharide-3 is 15349 and 2471, and the distribution coefficients Mw / Mn are 1.09 and 1.40, respectively; and / or, the monosaccharides in the structural composition of the purified Poria cocos polysaccharide-1 include mannose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose, with molar percentages of 30-32%, 0.2-0.4%, 9-10%, 44-46%, 0.06-0.08%, 0.5-0.7%, and 13-15%, respectively.

[0035] Thirdly, the present invention provides a purified polysaccharide of Poria cocos prepared by the preparation method described above, or the use of the purified polysaccharide of Poria cocos in the preparation of drugs with antioxidant activity and / or renal protective activity and / or prevention and treatment of kidney diseases.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Through careful selection of enzyme types, the present invention unexpectedly discovered that using cellulase and β-glucanase from different strains to perform compound enzymatic hydrolysis of Poria cocos can effectively reduce the molecular weight of polysaccharides, improve antioxidant activity, and increase the polysaccharide yield by 3.8 times compared with water extraction.

[0037] (2) The present invention obtains the purified polysaccharide FL-1 from Poria cocos by separating and purifying it through gel column chromatography to remove proteins and dextran. It has the strongest antioxidant activity in vitro. Evaluation by renal tubular epithelial cell senescence model and zebrafish kidney injury model showed that the purified polysaccharide FL-1 from Poria cocos can significantly improve cell viability, reduce β-galactosidase content, and increase NAD+ content; it can significantly reduce the incidence of renal edema in zebrafish and improve the glomerular filtration function of zebrafish.

[0038] (3) The purified polysaccharide FL-1 obtained in this invention is a heteropolysaccharide composed of seven monosaccharides, with galactose being the most abundant, followed by mannose, fucose, glucose, arabinose, galacturonic acid, and xylose. Its structure is a complex polysaccharide with 1,6-galactose as the main chain, containing 1,2-branched galactose and terminal mannose, exhibiting high branching degree and structural diversity. The 1,2-branched galactose and abundant terminal mannose give FL-1 a multivalent, multi-epitope surface structure. This complex "dendritic" conformation is similar to many natural biological signaling molecules. This structure provides the material basis for more effectively binding to or interacting with multiple receptors on the cell surface, thereby exerting multiple pharmacological activities such as cell protection, anti-aging, and improvement of kidney function. Attached Figure Description

[0039] Figure 1 The elution curve of Poria cocos polysaccharide by column chromatography is shown.

[0040] Figure 2 This is a chromatogram of monosaccharide standards.

[0041] Figure 3 The chromatogram of purified polysaccharides from Poria cocos.

[0042] Figure 4 This is the total ion chromatogram.

[0043] Figure 5 The cell viability graph shows that, compared with the normal group, ##P<0.01; compared with the model control group, P < 0.05 P < 0.01.

[0044] Figure 6 The relative fluorescence intensity of β-galactosidase is shown. Note: Compared with the normal group, ##P<0.01; compared with the model control group, P < 0.05 P < 0.01.

[0045] Figure 7 The value represents NAD+ content. Note: Compared with the normal group, ##P<0.01; compared with the model control group, P < 0.05 P < 0.01.

[0046] Figure 8 This is a graph of glomerular filtration function. Note: Compared with the normal group, ###P<0.001; compared with the model group, P < 0.001. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to specific embodiments. All materials used in the following experiments are commercially available conventional raw materials.

[0048] Experimental materials: The suppliers of β-glucanase are Xiasheng and Dongheng Huadao; the suppliers of cellulase and hemicellulase are Xiasheng; the supplier of HK-2 cells is the Cell Bank of the Chinese Academy of Sciences; the suppliers of CCK-8 kit, SPiDER-β Gal kit, and NAD / NADH Assay Kit-WST are Beiren Chemical Technology (Beijing) Co., Ltd.; the supplier of aristolochic acid is Shanghai Aladdin Biochemical Technology Co., Ltd.; the supplier of the fluorescent marker (dextran, tetramethylrhodamine, 10000MW neutral) is Invitrogen, USA. The suppliers of zebrafish have the experimental animal use license number: SYXK (Zhe) 2022-0004, and the feeding management meets the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethical review number: IACUC-2025-202508150003-01.

[0049] Experimental methods: (1)The method for determining the content of pachyman polysaccharide and calculating the yield refers to "SN / T 4260-2015 Determination of crude polysaccharides in exported plant-derived foods - Phenol-sulfuric acid method" to detect the content of pachyman polysaccharide. Ethanol was used for the pretreatment to remove substances such as monosaccharides, disaccharides, and oligosaccharides. A correction factor was used in the calculation process to avoid affecting the detection results of polysaccharides. The yield of pachyman polysaccharide was calculated by the following formula: Yield of pachyman polysaccharide / % = Mass of pachyman polysaccharide / Mass of pachyman sample × 100.

[0050] (2)In vitro antioxidant experiment: Calculation of DPPH inhibition rate.

[0051] Prepare a 0.2 mM working solution of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) with absolute ethanol, and prepare sample solutions of different concentrations with pure water. Take 2 mL of the sample to be measured at different concentrations and 2 mL of DPPH solution, mix well, and react in the dark at room temperature for 30 min. Measure the absorbance at a wavelength of 517 nm.

[0052] DPPH radical scavenging rate (%) = [1 - ( A i - A j ) A c × 100.

[0053] Among them, A c is the absorbance value of 2 mL of absolute ethanol plus 2 mL of DPPH working solution; A i is the absorbance value of adding 2 mL of DPPH to 2 mL of the sample to be measured at different concentrations; A j Add 2 mL of anhydrous ethanol to 2 mL of the solution of the sample corresponding to the specified concentration and measure the absorbance.

[0054] (3) Molecular weight determination: High-performance gel permeation chromatography was used for detection. Chromatographic conditions: Waters high-performance liquid chromatograph with differential detector, two polymer matrix water-soluble SEC (GFC) columns (8×300 mm) in series, mobile phase was 0.05M NaCl solution, flow rate was 0.5mL / min, column temperature was 40℃, and injection volume was 30μL.

[0055] Example 1 A Poria cocos polysaccharide, prepared by the following steps: (1) Weigh 100g of Poria cocos, add 15 times the amount of water, extract by ultrasonication at 95℃ for 1h, filter and take the supernatant and precipitate; (2) Add 10 times the amount of water to the precipitate, adjust the pH to 5-6 with citric acid, add 4% of the weight of Poria cocos compound enzyme [β-glucanase (the mass ratio of Trichoderma lichrysogenum supplied by Xia Sheng and the mass ratio of Trichoderma viride supplied by Dong Heng Hua Dao is 1:1) and the mass ratio of cellulase is 4:1], enzymatically hydrolyze for 4 hours at 50℃, then at 95℃ for 20 minutes to inactivate the enzyme, filter and take 2 supernatants; (3) Combine the two supernatants, concentrate and freeze dry to obtain Poria cocos polysaccharide.

[0056] The yield of Poria cocos polysaccharide was 1.31%, the DPPH inhibition rate was 58.34%, and the solid content yield was 7.13%.

[0057] Example 2 A Poria cocos polysaccharide, prepared by the following steps: (1) Weigh 100g of Poria cocos, add 10 times the amount of water, extract by ultrasonication at 90℃ for 1.5h, filter and take the supernatant 1 and precipitate; (2) Add 5 times the amount of water to the precipitate, adjust the pH to 5 with citric acid, add 5% of the mass of Poria cocos compound enzyme [β-glucanase (from the source of Trichoderma lichrysogenum supplied by Xia Sheng and the source of Trichoderma viride supplied by Dong Heng Hua Dao with a mass ratio of 1:0.5) and cellulase with a mass ratio of 3:1], enzymatically hydrolyze for 5 h at 40℃, then at 95℃ for 20 min to inactivate the enzyme, filter and take 2 supernatants; (3) Combine the two supernatants, concentrate and freeze dry to obtain Poria cocos polysaccharide.

[0058] The yield of Poria cocos polysaccharide was 1.23%, the DPPH inhibition rate was 56.47%, and the solid content yield was 7.09%.

[0059] Example 3 A Poria cocos polysaccharide, prepared by the following steps: (1) Weigh 100g of Poria cocos, add 20 times the amount of water, extract by ultrasonication at 98℃ for 0.5h, filter and take the supernatant and precipitate; (2) Add 15 times the amount of water to the precipitate, adjust the pH to 6 with citric acid, add 3% of the mass of Poria cocos compound enzyme [β-glucanase (the mass ratio of Trichoderma lichrysogenum supplied by Xia Sheng and the mass ratio of Trichoderma viride supplied by Dong Heng Hua Dao is 1:2) and the mass ratio of cellulase is 5:1], enzymatically hydrolyze for 3 hours at 60℃, then at 95℃ for 20 minutes to inactivate the enzyme, filter and take 2 supernatants; (3) Combine the two supernatants, concentrate and freeze dry to obtain Poria cocos polysaccharide.

[0060] The yield of Poria cocos polysaccharide was 1.27%, the DPPH inhibition rate was 57.82%, and the solid content yield was 7.22%.

[0061] Comparative Examples 1-8 The only difference between the comparative examples and Example 1 is the type or source of the enzyme, as detailed in Table 1 below. Furthermore, the total amount of enzyme added in Comparative Examples 1-8 is the same as that in Example 1. The final yield of Poria cocos polysaccharides and the DPPH scavenging rate are shown in Table 1.

[0062] Table 1

[0063] Results Analysis: β-glucanases from different brands and strains significantly affected the yield of Poria cocos polysaccharides. Cellulase also significantly improved the polysaccharide yield. The optimal β-glucanase was selected from *Trichoderma listeri* and *Trichoderma viride* sources (mass ratio 1:1), and the cellulase was selected from *Trichoderma viride*. Studies showed that β-glucanases from *Trichoderma listeri* and *Trichoderma viride* sources had a significant synergistic effect in the extraction of Poria cocos polysaccharides, and a strong synergistic interaction also occurred between cellulase and β-glucanase.

[0064] Comparative Examples 9-17 The difference between this comparative example and Example 1 is that the extraction method is different, as shown in Table 2 below. The final yield of Poria cocos polysaccharide and DPPH scavenging rate are shown in Table 2.

[0065] Table 2

[0066] Comparative Examples 18-23 The difference between the comparative example and Example 1 is that the proportion of enzymes is different, as shown in Table 3.

[0067] Table 3

[0068] Compared with the water extraction process of Comparative Example 13, the process of Example 1 of this invention can effectively reduce the molecular weight of polysaccharides, improve antioxidant activity, and increase the polysaccharide yield by 3.8 times, as shown in Table 4 below.

[0069] Table 4

[0070] Example 4 Take the Poria cocos polysaccharide solution prepared in Example 1, concentrate it to 1 / 3 of the original volume, add anhydrous ethanol, control the final volume fraction of ethanol to 80%, precipitate at 4℃ for 12h, and collect the polysaccharide precipitate by centrifugation. The polysaccharide precipitate was added to pure water and stirred until completely dissolved. Then, an equal volume of 5 wt% trichloroacetic acid was added, and the mixture was allowed to stand at room temperature for 4 hours. The protein was removed by centrifugation, and the supernatant was dialyzed, concentrated, and freeze-dried to obtain Poria cocos polysaccharide powder.

[0071] Poria cocos polysaccharide powder was prepared into a solution with a mass concentration of 10 mg / mL. The solution was then subjected to G100 dextran gel chromatography (3 cm × 30 cm) with pure water elution at a flow rate of 0.5 mL / min. Eighty tubes were collected, each containing 5 mL. The absorbance was measured using the phenol-sulfuric acid method to determine the polysaccharide content. An absorbance elution curve was plotted, and the polysaccharide solution was collected based on the peak. The solution was then freeze-dried to obtain purified Poria cocos polysaccharide.

[0072] Example 5 Take the Poria cocos polysaccharide solution prepared in Example 1, concentrate it to 1 / 4 of the original volume, add anhydrous ethanol, control the final volume fraction of ethanol to 85%, precipitate at 0℃ for 10h, and collect the polysaccharide precipitate by centrifugation. The polysaccharide precipitate was added to pure water and stirred until completely dissolved. Then, an equal volume of 6wt% trichloroacetic acid was added, and the mixture was allowed to stand at room temperature for 3 hours. The protein was removed by centrifugation, and the supernatant was dialyzed, concentrated, and freeze-dried to obtain Poria cocos polysaccharide powder.

[0073] Poria cocos polysaccharide powder was prepared into a solution with a mass concentration of 10 mg / mL. The solution was then subjected to G100 dextran gel chromatography (3 cm × 30 cm) with pure water elution at a flow rate of 0.6 mL / min. 5 mL of the eluent was collected per tube. The absorbance was measured using the phenol-sulfuric acid method to determine the polysaccharide content. An absorbance elution curve was plotted, and the polysaccharide solution was collected based on the peak. The solution was then freeze-dried to obtain purified Poria cocos polysaccharide.

[0074] I. Characterization of purified polysaccharides from Poria cocos (1) Elution curve of purified polysaccharides from Poria cocos as shown in Figure 1 Figure 1As shown. Three purified polysaccharides from Poria cocos were collected, designated FL-1, FL-2, and FL-3. The polysaccharide content was determined according to the above-mentioned standard, "SN / T 4260-2015 Determination of Crude Polysaccharides in Exported Plant-Derived Foods by Phenol-Sulfuric Acid Method". Ethanol was used for pretreatment to remove monosaccharides, disaccharides, and oligosaccharides. Correction coefficients were used in the calculations to avoid affecting the polysaccharide detection results. The yield of Poria cocos polysaccharides was calculated using the following formula: The yield of Poria cocos polysaccharides (%) = the mass of Poria cocos polysaccharides / the mass of Poria cocos sample × 100.

[0075] The purity of polysaccharides in FL-1, FL-2, and FL-3 was determined to be 96.1%, 93.4%, and 95.1%, respectively.

[0076] (2) The relative molecular weights of the Poria cocos polysaccharides, purified Poria cocos polysaccharides FL-1, FL-2, and FL-3 obtained before column chromatography in Example 4 were determined. The relative molecular weights of FL-1 and FL-2 were evenly distributed. FL-1 showed the strongest in vitro antioxidant activity. The specific results are shown in Tables 5-6.

[0077] Table 5

[0078] Table 6

[0079] (3) Characterization of the structure of purified polysaccharides from Poria cocos - determination of monosaccharide composition The purified polysaccharides from Poria cocos were derivatized using PMP, and their monosaccharide composition was analyzed. Chromatographic conditions: Thermo U3000 high-performance liquid chromatography system, Agilent ZORBAX Eclipse XDB-C18 (4.6) column. The column was 250 nm long and 5 μm wide. The mobile phase was acetonitrile: phosphate buffer (12 g / L potassium dihydrogen phosphate, pH adjusted to 6.8 with 2 M NaOH) with isocratic elution. The volume ratio of acetonitrile to phosphate buffer was 17:83. The flow rate was 0.8 ml / min. The column temperature was 30 ºC. The detection wavelength was 250 nm. The injection volume was 10 μL.

[0080] The chromatogram of the monosaccharide standard is shown below. Figure 2 Example 1: Chromatogram of purified polysaccharide from Poria cocos (see Figure 1). Figure 3 The molar percentage of monosaccharides is shown in Table 7.

[0081] Table 7. Molar percentage of monosaccharide composition

[0082] Results analysis: FL-1 is a heteropolysaccharide composed of 7 monosaccharides, with galactose having the highest content, followed by mannose, fucose, glucose, arabinose, galacturonic acid, and xylose.

[0083] (4) Methylation analysis Chromatographic conditions: Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA), HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas was high-purity helium (purity not less than 99.999%), flow rate was 1.0 mL / min, and the injection port temperature was 260℃. Injection volume was 1 μL, split injection, split ratio 10:1, solvent delay 2.2 min. Temperature program: 50℃ held for 1.0 min, increased to 130℃ at 50℃ / min, increased to 230℃ at 3℃ / min, held for 2 min, increased to 290℃ at 15℃ / min, held for 5 min.

[0084] Mass spectrometry conditions: 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.

[0085] The total ion chromatogram is shown below. Figure 4 The GC-MS analysis results of FL-1 after methylation are shown in Table 8.

[0086] Table 8 GC-MS analysis results after FL-1 methylation

[0087] Results analysis: 1,6-Galp and 1,2,6-Galp together account for nearly 50%, indicating that FL-1 has a 1,6-linked galactose main chain with 1,2-branching. Terminal mannose (t-Manp) and 1,2-Manp together account for about 28%, suggesting that mannose functions as a terminal or branched glycosyl group in the structure. 1,3-Fucp and t-Fucp together account for about 10%, and glucose (Glcp) linkages are diverse but low in content: including 1,6-, 1,4-, and 1,3,6- linkages, but the total amount is not high, possibly serving as an auxiliary structural unit. Structural speculation suggests that FL-1 may be a complex polysaccharide with a 1,6-galactose main chain, containing 1,2-branched galactose and terminal mannose, exhibiting high branching degree and structural diversity.

[0088] The 1,2-branched galactose and abundant terminal mannose give FL-1 a multivalent, multi-epitope surface structure. This complex "dendritic" conformation is similar to many natural biological signaling molecules. This structure enables it to bind or interact more effectively with multiple receptors on the cell surface, thus providing the material basis for its multiple pharmacological activities, including cell protection, anti-aging, and improvement of kidney function.

[0089] II. Pharmacodynamic Study: Evaluation of the Nephroprotective Effect of Purified Polysaccharides from Poria cocos 2.1 HK-2 cell senescence model HK-2 cells in logarithmic growth phase were seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. The cells were then divided into control, model, and sample groups. The control group received complete culture medium, the model group received complete culture medium containing D-galactose, and the sample groups received complete culture medium containing D-galactose and different concentrations of Poria cocos samples. Incubation continued for another 24 hours. Cell viability, β-galactosidase fluorescence intensity, and NAD+ content were measured according to the kit instructions.

[0090] 2.2 Zebrafish Kidney Injury Model (1) Determination of maximum detection dose Wild-type AB strain zebrafish, 2 days post-fertilization, were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Different concentrations of the sample were administered in water, and both normal and model groups were established. Except for the normal group, all other experimental groups were given aristolochic acid in water to establish a zebrafish kidney injury model. After treatment at 28℃ for 2 days, the maximum detectable dose of FL-1 in the model zebrafish was determined.

[0091] (2) Incidence of renal edema Wild-type AB strain zebrafish, 2 days post-fertilization, were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Different safe concentrations of the sample were administered in water, along with a positive control (33.3 µg / mL) of renal failure-relieving capsules. A normal control group and a model group were also established. Except for the normal control group, all other experimental groups were given aristolochic acid in water to establish a zebrafish kidney injury model. After treatment at 28℃ for 2 days, each experimental group was observed under a dissecting microscope. The number of zebrafish with renal edema was counted, and the incidence of renal edema in each experimental group was calculated. The statistical analysis results of this index were used to evaluate the renal protective efficacy of FL-1 (incidence of renal edema).

[0092] (3) Glomerular filtration capacity Except for the normal group, all other experimental groups were given aristolochic acid in water to establish a zebrafish kidney injury model. After treatment at 28°C for 1 day, each experimental group was intravenously injected with fluorescent markers (dextran, tetramethylrhodamine, 10000MW neutral). After treatment at 28°C for another day, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D3.20 advanced image processing software. The fluorescence intensity of the zebrafish was analyzed, and the statistical analysis results of this index were used to evaluate the nephroprotective efficacy (glomerular filtration) of FL-1.

[0093] 2.3 Data Processing Statistical analysis was performed using Excel 2021. Data are expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons between groups, and p < 0.05 was considered statistically significant. Charts were generated using Origin 2021 software.

[0094] 2.4 Results and Discussion (1) HK-2 cell viability The highest safe concentrations of Poria cocos polysaccharide, FL-1, FL-2, and FL-3 were 6 mg / mL, 3 mg / mL, 3 mg / mL, and 8 mg / mL, respectively. The effects of Poria cocos polysaccharide on HK-2 cell viability are shown in [the table below]. Figures 5-7 The purified polysaccharide FL-1 from Poria cocos significantly improved the viability of damaged HK-2 cells, exhibiting the strongest effect. FL-1 reduced β-galactosidase levels and increased NAD+ levels, indicating that FL-1 can improve the senescent state of HK-2 cells.

[0095] (2) Evaluation of the protective effect of zebrafish kidney Maximum detection dose determination: At a concentration of FL-1 of 250 μg / mL, the survival rate of zebrafish was 100%, similar to that of the model control group.

[0096] The incidence of renal edema is shown in Table 9. Purified polysaccharide FL-1 significantly reduced the incidence of renal edema in zebrafish.

[0097] Table 9. Incidence of renal edema

[0098] Note: Compared with the normal group, ### P <0.001; compared with the model group, P <0.001.

[0099] The glomerular filtration rate of purified polysaccharide FL-1 from Poria cocos is shown in the figure. Figure 8Purified polysaccharide FL-1 can significantly improve the glomerular filtration function of zebrafish.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing purified polysaccharides from Poria cocos with renal protective activity, characterized in that, Includes the following steps: (1) Add water to Poria cocos, sonicate, filter, and obtain supernatant 1 and precipitate; (2) Add water, β-glucanase and cellulase to the precipitate, enzymatically hydrolyze it, filter it, and obtain supernatant 2; (3) Mix supernatant 1 and supernatant 2 to obtain Poria cocos extract.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of Poria cocos to water is 1:10-20; the temperature of the ultrasound is 90-100℃; and the duration of the ultrasound is 0.5-2h.

3. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of the precipitate to water is 1:5-15. After adding water, the pH is adjusted to 5-6 with citric acid. The enzymatic hydrolysis time is 3-5 hours and the enzymatic hydrolysis temperature is 40-60℃.

4. The preparation method according to claim 1, characterized in that, In step (2), the total amount of β-glucanase and cellulase added is 3-5% of the mass of Poria cocos; the mass ratio of β-glucanase to cellulase is 3-5:1, the source of β-glucanase is Trichoderma listeri and Trichoderma viride, and the mass ratio of the two is 1:0.5-2; and / or the source of cellulase is Trichoderma viride.

5. The preparation method according to claim 1, characterized in that, The preparation method also includes step (4) purification operation, which specifically includes alcohol precipitation of Poria cocos extract, removal of protein, G100 dextran gel column chromatography, elution, and freeze drying to obtain purified polysaccharide.

6. The preparation method according to claim 5, characterized in that, The alcohol precipitation process involves concentrating the Poria cocos extract to 1 / 2 to 1 / 4 of its original volume, adding anhydrous ethanol to a final ethanol volume fraction of 75-85%, precipitating at 0-6°C for 10-15 hours, and then centrifuging to collect the polysaccharide precipitate.

7. The preparation method according to claim 5, characterized in that, The step of removing proteins includes dissolving the polysaccharide precipitate in water, adding an equal volume of 3-6 wt% trichloroacetic acid solution, letting it stand for 3-5 hours, centrifuging, and concentrating to obtain a Poria cocos polysaccharide solution.

8. The preparation method according to claim 5, characterized in that, The Poria cocos polysaccharide solution was prepared to a mass concentration of 10 mg / mL and subjected to chromatography on a 3 cm × 30 cm G100 dextran gel column with water elution at a flow rate of 0.4-0.6 mL / min. The eluent was collected, and the absorbance was determined according to the phenol-sulfuric acid method for polysaccharide determination. An elution curve was plotted, and the polysaccharide solution was collected according to the peaks. The solution was then freeze-dried to obtain purified polysaccharide.

9. A purified polysaccharide of Poria cocos prepared by the preparation method according to any one of claims 1-8, characterized in that, The purified polysaccharides from Poria cocos include one or more of purified polysaccharides from Poria cocos-1, purified polysaccharides from Poria cocos-2, and purified polysaccharides from Poria cocos-3, and / or, the weight-average molecular weight of purified polysaccharide from Poria cocos-1 is 29483, and the distribution coefficient Mw / Mn is 2.17; and / or, the weight-average molecular weight of purified polysaccharide from Poria cocos-2 is 14683, and the distribution coefficient Mw / Mn is 1.74; and / or, the weight-average molecular weight of purified polysaccharide from Poria cocos-3 is 15349 and 2471, and the distribution coefficients Mw / Mn are 1.09 and 1.40, respectively; and / or, the monosaccharides in the structural composition of purified polysaccharide from Poria cocos-1 include mannose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose, with molar percentages of 30-32%, 0.2-0.4%, 9-10%, 44-46%, 0.06-0.08%, 0.5-0.7%, and 13-15%, respectively.

10. The use of a purified polysaccharide of Poria cocos prepared by any one of claims 1-8 or the purified polysaccharide of Poria cocos prepared by claim 9 in the preparation of a drug having antioxidant activity and / or renal protective activity and / or preventing and treating kidney diseases.

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