Composition for reflecting polygonatum sibiricum polysaccharide structure evolution relation before and after polygonatum sibiricum processing and preparation method and application thereof

Abstract

The invention belongs to the field of traditional Chinese medicine processing and polysaccharide development, and particularly relates to a polygonatum sibiricum polysaccharide composition which is produced by processing and converting raw polygonatum sibiricum and has a specific structure evolution relation, a preparation method of the composition and application of the composition in preparation of products for prevention, improvement, adjuvant therapy or treatment of kidney-related diseases. According to the invention, a group of polygonatum sibiricum polysaccharide composition group with a specific structure evolution relationship is successfully constructed and defined, and experimental data clearly proves that compared with crude polysaccharide, the composition group disclosed by the invention integrally shows remarkable kidney protection activity. The achievement not only provides a key scientific material basis and direct evidence for the traditional Chinese medicine theory of rhizoma polygonati processing synergy, but also converts the component group into a kidney protection function factor which can be accurately developed and is controllable in quality, and lays a solid material basis for developing a new generation of rhizoma polygonati products.

Description

A composition reflecting the structural evolution of Polygonatum polysaccharides before and after processing, its preparation method and application Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine processing and polysaccharide development, specifically involving a group of Polygonatum polysaccharide compositions with specific structural evolution relationships produced by processing and transforming raw Polygonatum, as well as the preparation method and application of the compositions. Background Technology

[0002] Polygonatum rhizome, a perennial herb belonging to the genus Polygonatum in the Liliaceae family, is a traditional Chinese medicine. As one of the original sources of Polygonatum, it is included in the 2025 edition of the Chinese Pharmacopoeia (Part I). It possesses the functions of "tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys," and is commonly used for symptoms such as spleen and stomach qi deficiency, fatigue, insufficient essence and blood, soreness and weakness of the lower back and knees, and thirst. Modern chemical analysis shows that polysaccharides are one of the most important water-soluble active components of Polygonatum, considered an important material basis for its tonifying effects.

[0003] Ancient texts record that raw Polygonatum rhizome "stabbed at the throat," requiring processing before medicinal use to reduce toxicity and enhance efficacy. Among these methods, "nine steaming and nine drying" is the most representative traditional processing technique. The *Complete Collection of Ancient and Modern Medicine* states, "Fresh Polygonatum rhizome should be boiled in water without stirring until thoroughly cooked. Then, it should be filtered, dried in the sun, steamed again, and dried for a long time. If it is indeed steamed and dried nine times, consuming it can prolong life, and long-term consumption can lead to ascension to heaven," demonstrating the ancients' high regard for the life-prolonging effects of processed Polygonatum rhizome. Benefiting from its definite clinical efficacy and its "medicine and food homology" attribute, Polygonatum rhizome is widely used in the medical and health industries. However, the booming development of the industry contrasts sharply with the ambiguity of its scientific connotation.

[0004] Although existing studies have shown that processing not only alters the physical properties of Polygonatum slices but also profoundly affects the chemical composition and structure of polysaccharides, current research on polysaccharides from raw and processed Polygonatum focuses primarily on extraction process optimization and activity screening. However, systematic separation, fine structural characterization, and parallel comparative studies of the purified polysaccharide components of both are still limited. This has resulted in an unclear material basis and mechanism of action for the "enhanced efficacy" of processed Polygonatum, which further restricts the in-depth development of modern, high-value-added products based on highly active polysaccharides from processed Polygonatum. Summary of the Invention

[0005] To overcome the defects and shortcomings of existing technologies, this invention provides a systematic technical solution aimed at fundamentally revealing the material basis and transformation law of the enhanced efficacy of processed Polygonatum odoratum. Its core lies in establishing a complete technical chain from "systematic comparative study before and after processing" to "multi-component characterization" and "in vitro activity verification," successfully obtaining a group of Polygonatum odoratum polysaccharide compositions with specific evolutionary relationships.

[0006] Specifically, the present invention is achieved through the following technical solutions: In the first aspect, the present invention provides a Polygonatum polysaccharide composition having a specific structural evolution relationship and renal protective effect before and after processing, the composition comprising one or more of the following: homogeneous polysaccharides PRP-1 and / or PRP-2 derived from raw Polygonatum, and homogeneous polysaccharides PPRP-1, PPRP-2 and / or PPRP-3 derived from processed Polygonatum.

[0007] Alternatively, in the above composition, PRP-1 and PRP-2 are composed of fructose and glucose, with →1)-β-D-Fruf-(2→) as the main chain; PPRP-1 is composed of fructose, glucose, and arabinose, with →5)-α-L-Araf-(1→) added to the RPP-1 / PRP-2 composition; PPRP-2 and PPRP-3 are composed of arabinose, galactose, galacturonic acid, and rhamnose, containing α-L-Araf-(1→, →5)-α-L-Araf-(1→, →4)-β-D-Galp-(1→, →4)-α-D-GalAp-6-O-Me-(1→, →4)-α-D-GalAp-(1→, →2,4)-α-L-Rhap-(1→).

[0008] Alternatively, in the above composition, in the raw polysaccharide, PRP-1 and PRP-2 are both fructans, and their weight-average molecular weight ranges from 2.0 to 3.0 kDa; in its monosaccharide composition, the molar ratio of fructose to glucose ranges from 90%:10% to 99%:1%.

[0009] Alternatively, in the above composition, the fructans in PRP-1 and PRP-2 in the polysaccharide product are degraded after processing to form PPRP-1 with a smaller molecular weight and containing trace amounts of arabinose. The weight-average molecular weight of PPRP-1 ranges from 1.5 to 2.5 kDa. During the processing, pectin polysaccharides PPRP-2 and PPRP-3 from the plant cell wall are also released. The weight-average molecular weight of PPRP-2 and PPRP-3 ranges from 8 to 25 kDa, and the sum of their molar contents of galactose and galacturonic acid reaches 70% to 75%.

[0010] As an optional approach, in the above composition, the homogeneous polysaccharide PPRP-2 derived from Polygonatum sibiricum is the core highly active component, with a weight-average molecular weight of 8-14 kDa. In the monosaccharide composition, the molar proportions of galactose and galacturonic acid are not less than 40% and 15%, respectively. Its fine chemical structure includes the following structural units: with →4)-α-D-GalAp-(1→、→2,4)-α-L-Rhap-(1→、→4)-α-D-GalAp-6-O-Me-(1→、→3, 5)-α-L-Araf-(1→ etc. as the main chain and α-L-Araf-(1→ etc. as the side chain.

[0011] Alternatively, in the above composition, the weight-average molecular weights of PRP-1, PRP-2, PPRP-1, PPRP-2, and PPRP-3 are 2.619 kDa, 2.608 kDa, 1.894 kDa, 11.084 kDa, and 21.499 kDa, respectively.

[0012] Alternatively, in the above compositions, the molar ratio of glucose to fructose in the monosaccharide composition of PRP-1 is 5.52:94.48; the molar ratio of glucose to fructose in the monosaccharide composition of PRP-2 is 5.47:94.53; the molar ratio of arabinose, glucose, and fructose in the monosaccharide composition of PPRP-1 is 0.41:6.64:92.95; the molar ratio of rhamnose, arabinose, galactose, glucose, and galacturonic acid in the monosaccharide composition of PPRP-2 is 8.68:15.31:57.61:0.60:17.80; and the molar ratio of rhamnose, arabinose, galactose, and galacturonic acid in the monosaccharide composition of PPRP-3 is 15.03:12.08:48.65:24.25.

[0013] Those skilled in the art should know that any polysaccharide composition that embodies the raw product component dominated by fructan, the degraded and enriched product component, and their synergistic evolution should be considered as an equivalent of the present invention.

[0014] In a second aspect, the present invention provides a method for preparing the composition described in the first aspect, comprising the following steps: Step 1, raw material selection: using processed Polygonatum sibiricum as raw material; Step 2, crude polysaccharide extraction: taking the processed Polygonatum sibiricum selected in Step 1, defatting with ethanol, hot water extraction, alcohol precipitation, and protein removal to obtain crude Polygonatum sibiricum polysaccharide; Step 3, separation of polysaccharide components: separating the crude polysaccharide obtained in Step 2 by ion exchange column chromatography to obtain polysaccharide components; Step 4, screening of active components: performing primary structural identification, including ultraviolet, infrared, molecular weight, and monosaccharide composition, and parallel comparative analysis of in vitro renal protective activity on each polysaccharide component obtained in Step 3 and the homogeneous polysaccharide extracted from raw Polygonatum sibiricum, and screening out the component with the best activity; Step 5, fine identification of target components: performing methylation and NMR analysis on the component with the best activity screened in Step 4 to obtain the homogeneous polysaccharide as described in any one of claims 1 to 5.

[0015] As an optional method, in the above preparation method, in step 1, the prepared Polygonatum is Polygonatum that has undergone multiple steaming treatments; in step 2, the volume concentration of ethanol during defatting is 70-90%, and the volume concentration of ethanol during fractional alcohol precipitation is 50%-80%; in step 3, the packing material for the ion exchange column chromatography is DEAE-52 cellulose packing material.

[0016] As an optional approach, in the above preparation method, the raw material processing can employ alternative methods such as steaming or alcoholic distillation; the extraction step can utilize auxiliary methods such as ultrasound or microwave; the ion exchange packing material used for separation and purification can be similar products such as DEAE-Sephadex A-25, and the gel packing material can be Sephacryl S-300 HR; drying can be achieved using vacuum drying or spray drying. The process parameters for each step, such as temperature, time, and concentration, can be routinely adjusted according to equipment and production requirements.

[0017] In a third aspect, the present invention provides the use of the composition described in the first aspect above, or the composition prepared by the preparation method described in the second aspect above, in the preparation of products for the prevention, improvement, adjunctive treatment or treatment of kidney-related diseases.

[0018] Alternatively, in the above applications, the kidney-related diseases include diabetic nephropathy or renal fibrosis.

[0019] Alternatively, in the above applications, the product may be a medicine, health supplement, or functional food.

[0020] As an alternative, in the above applications, when the polysaccharide obtained by the present invention is prepared into a composition, its effective content can be adjusted within a wide range of 0.1%-99%, and it can be combined with a variety of conventional excipients for use in sub-healthy individuals or patients with chronic kidney disease who have kidney maintenance needs.

[0021] As an alternative approach, in the above applications, the product may, for example, combine polysaccharides with prebiotics or specific probiotics to form a compound powder, which indirectly supports renal tubular health and helps delay the progression of fibrosis by regulating the gut microbiota and intervening in the gut-kidney axis; secondly, it may be compounded with exogenous antioxidants such as polyphenols into soft capsules to build a synergistic antioxidant network, thereby comprehensively regulating blood sugar and blood lipid levels; thirdly, it may use polysaccharides as the main active ingredient to help patients with diabetic nephropathy improve various indicators.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. The present invention uses 70-90% ethanol to defatt the raw and steamed Polygonatum odoratum, water extraction-fractional alcohol precipitation to obtain crude polysaccharides, and then separates and purifies homogeneous polysaccharides (PRP-1, PRP-2, PPRP-1, PPRP-2, PPRP-3) by DEAE-52 cellulose column separation. The obtained polysaccharides have good homogeneity, simple production process, high purity (above 90%), and good product color.

[0023] 2. The structures of the polysaccharides obtained in this invention have been clearly characterized. The molecular weights of the homogeneous polysaccharides are 2.619 kDa, 2.608 kDa, 1.894 kDa, 11.084 kDa, and 21.499 kDa, respectively, indicating that the Polygonatum polysaccharides underwent degradation and polymerization after steaming. Structural analysis shows that PRP-1 and PRP-2 are composed of fructose and glucose, with →1)-β-D-Fruf-(2→) as the main chain; PPRP-1 is composed of fructose, glucose, and arabinose, with the addition of →5)-α-L-Araf-(1→) on the basis of RPP-1 / PRP-2; PPRP-2 and PPRP-3 are composed of arabinose, galactose, galacturonic acid, and rhamnose, including α-L-Araf-(1→, →5)-α-L-Araf-(1→, →4)-β-D-Galp-(1→, →4)-α-D-GalAp-6-O-Me-(1→, →4)-α-D-GalAp-(1→, →2,4)-α-L-Rhap-(1→), etc.

[0024] 3. The five homogeneous polysaccharides from Polygonatum provided in this invention all exhibit certain antioxidant and anti-fibrotic activities against high glucose-induced NRK-52E cells, with PPRP-2 showing superior activity. Each homogeneous polysaccharide has the potential for application in renal protection and the treatment of diabetic nephropathy.

[0025] 4. This invention delves into the differences in polysaccharide components and activities before and after processing of Polygonatum odoratum, constructing a complete chain of evidence connecting "traditional processing experience" with "modern scientific mechanisms," providing a theoretical basis for scientifically explaining the connotation of Polygonatum odoratum processing, and also facilitating the development of specific products for raw and steamed Polygonatum odoratum. Attached Figure Description

[0026] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1: Separation diagram of raw and processed Polygonatum polysaccharides.

[0027] Figure 2: UV scan images of raw and processed Polygonatum polysaccharides.

[0028] Figure 3: Infrared spectra of raw and processed Polygonatum polysaccharides.

[0029] Figure 4: HPLC chromatograms of raw and processed Polygonatum polysaccharides.

[0030] Figure 5: Monosaccharide composition chromatograms of raw and processed Polygonatum polysaccharides.

[0031] Figure 6: Effects of raw and processed Polygonatum polysaccharides on the viability of high glucose-induced NRK-52E cells.

[0032] Figure 7: Effects of raw and processed Polygonatum polysaccharides on the antioxidant (SOD, MDA, GSH) and antifibrotic (FN, COL1, α-SMA) levels of high glucose-induced NRK-52E cells.

[0033] Figure 8: NMR spectrum of PPRP-2.

[0034] Figure 9: Effects of raw and processed Polygonatum on body weight gain and fasting blood glucose (FBG) in DKD mice.

[0035] Figure 10: Effects of raw and processed Polygonatum on renal function in DKD mice.

[0036] Figure 11: Effects of raw and processed Polygonatum sibiricum on HE sections of kidney tissue from DKD mice (×400).

[0037] Figure 12: Effects of raw and processed Polygonatum on Masson sections of kidney tissue from DKD mice (×400). Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0039] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0040] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0041] Example 1: A parallel comparison-based method for separating Polysaccharides from Polygonatum sibiricum and its preliminary characterization 1.1 Preparation of raw materials and extraction of crude polysaccharides 1.1.1 Processing and preparation of raw and processed Polygonatum sibiricum Select fresh Polygonatum sibiricum without mold, remove the fibrous roots and wash it clean. Take a suitable size and place it in a steamer and steam for 20 minutes until thoroughly cooked. Take it out immediately and let it air dry naturally until the skin is slightly wrinkled. Then slice and air dry.

[0042] To prepare Polygonatum, select fresh Polygonatum without mold, remove the fibrous roots, wash it, take a suitable size, place it in a steamer and steam for 3 hours, simmer for 40 minutes, and then dry it in a cool and ventilated place until the outer skin is slightly dry. This is a product that has been steamed and dried. Then, depending on the specific situation, add distillate or an appropriate amount of boiling water to soak it, and repeat the steaming, simmering, and drying process. Repeat this process multiple times until the product is naturally dried until the surface is slightly wrinkled, then slice it and air dry it.

[0043] 1.1.2 Preparation of crude polysaccharides from raw and processed Polygonatum sibiricum: Accurately weigh appropriate amounts of raw and processed Polygonatum sibiricum slices, add 70-90% ethanol solution at a material-to-liquid ratio of 1:10 (g / mL), reflux for 1 h, filter and collect the residue, add distilled water at a material-to-liquid ratio of 1:8 (g / mL), reflux for 1 h, repeat the extraction twice, combine the extracts, filter and concentrate the filtrate to 1-1.4 g / mL by rotary evaporation, then use the Sevag method (dichloromethane: n-butanol = 4:1, v / v) to remove free proteins from the polysaccharides. Add the Sevag reagent and extract to a sealed container at a ratio of 1:3, shake vigorously for 15 min, centrifuge at 4000 r / min for 10 min, remove the lower milky protein liquid, collect the upper aqueous phase, repeat at least 4 times until the lower milky white layer disappears. Add 95% ethanol to the treated extract, fractionate the ethanol precipitation to a final ethanol concentration of 30-80% (v / v), precipitate overnight at 4°C, centrifuge, combine the precipitates, and freeze-dry to obtain crude polysaccharides PRP and PPRP.

[0044] 1.2 Separation of PRP and PPRP 1.2.1 Acquisition of PRP-purified polysaccharides The supernatant from dissolved and centrifuged PRP was loaded onto a DEAE-52 cellulose-packed chromatography column (3.0 cm × 30 cm). Elution was performed sequentially with pure water and 0.05 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, and 0.5 M NaCl solutions, collecting 10 mL per tube, for each elution gradient using 3 column volumes. Using the sulfuric acid-anthrone method, the absorbance of different eluents was measured at 582 nm every 5 tubes. Fractions with the same absorbance peak were combined, concentrated under reduced pressure at 50 °C, dialyzed through a dialysis bag, and then concentrated and lyophilized. PRP was enriched into two fractions: one in distilled water (PRP-1) and the other in 0.05 mol / L NaCl solution (PRP-2).

[0045] 1.2.2 PPRP Purification: PPRP was purified using the same chromatographic conditions as in 1.2.1. PPRP was enriched into three fractions: pure water, 0.2 mol / L NaCl solution, and 0.3 mol / L NaCl solution, labeled PPRP-1, PPRP-2, and PPRP-3, respectively.

[0046] The separation of PRP and PPRP is shown in Figure 1, and the yield is shown in Table 1.

[0047] 1.3 Preliminary Characterization of Five Polysaccharides 1.3.1 UV and FT-IR Analysis Five dried polysaccharides were prepared into sample solutions of 1.0 mg / mL. Using water as a blank solvent, UV scanning was performed in the range of 200-400 nm. The results are shown in Figure 2. No obvious absorption was observed at 260 nm and 280 nm for each polysaccharide. Therefore, it can be determined that the polysaccharides obtained after preliminary separation and purification contain almost no protein and nucleic acid impurities.

[0048] Weigh 1 mg of each dried homogeneous polysaccharide, grind it with spectroscopically pure KBr, and prepare transparent tablets. Spectroscopy was performed using a Fourier transform infrared spectrometer at 400-4000 cm⁻¹. -1 Infrared spectral scanning analysis was performed within the specified range. The results are shown in Figure 3: 3400 cm⁻¹ -1 A broad and strong absorption peak near 1400 cm⁻¹ -1 The stretching and deformation vibrations of OH are located at 2930 cm⁻¹. -1 The stretching vibration of CH is located at 1640 cm. -1 The position represents the CO stretching vibration (carboxyl group) or the presence of bound water, 1020 cm. -1 The peak at this point is generated by the stretching vibration of the COC glycosidic bond in the sugar ring. Furthermore, PPR-2 and PPR-3 exhibit distinct carboxyl vibration peaks at 1740 cm⁻¹. -1 933 cm -1 809 cm -1 The peak at this location is a characteristic peak of β-D-furanose.

[0049] 1.3.2. Determination of total sugar, uronic acid, and protein content: An anthrone-sulfuric acid method was used with anhydrous glucose as the standard to establish a standard curve for determining the total sugar content of each polysaccharide; a phloroglucinol method was used with galacturonic acid as the standard to establish a standard curve for determining the uronic acid content of each polysaccharide; and a BCA kit was used to determine the protein content of each polysaccharide according to the manufacturer's instructions. The results are shown in Table 1.

[0050] Table 1. Yield of polysaccharides and determination of polysaccharide, uronic acid, and protein content. 1.3.3 Purity and Molecular Weight Determination: High-performance gel permeation chromatography (HPGPC) was used to determine the average molecular weight and homogeneity of the polysaccharides. The sample concentration was 5 mg / mL, dissolved, and filtered through a 0.22 μm filter membrane for later use. The detection system was a Thermo U3000 system equipped with a differential detection (RID) detector. The chromatographic column was a BRT105-103-101 tandem gel column (8 × 300 mm). The mobile phase was 0.5 M NaCl solution, the flow rate was 0.7 mL / min, the column temperature was 40℃, and the injection volume was 100 μL. A molecular weight standard curve was plotted using GBW(E) series glucose standards. The results are shown in Figure 4. The peaks of each polysaccharide were single and symmetrical, indicating homogeneous polysaccharides. The molecular weight results are shown in Table 2.

[0051] Table 2. Molecular weight of polysaccharides 1.3.4 Monosaccharide Composition Determination: Ion chromatography (IC) was used to analyze the monosaccharide composition of five polysaccharides. 2 mg of each polysaccharide sample was accurately weighed and placed in an ampoule. 1 ml of 2M TFA solution was added. PRP-1, PRP-2, and PPRP-1 underwent partial acid hydrolysis (heating at 60°C for 1 hour); PPRP-2 and PPRP-3 underwent complete acid hydrolysis (heating at 121°C for 2 hours). Nitrogen gas was purged, and the mixture was dried. Methanol was added for washing, and the mixture was dried again. This methanol washing process was repeated 2-3 times. The mixture was dissolved in sterile water and transferred to a chromatographic bottle for analysis.

[0052] Accurately pipette 1 mg of each monosaccharide into a 10 mL volumetric flask and add 0.3 mol / L NaOH solution to prepare the standard solution for each monosaccharide. Accurately measure the same volume of each monosaccharide stock solution and mix well to prepare the mixed standard solution for monosaccharides. Accurately measure 200 μL of each of the above solutions to prepare the mixed standard solution. Take 200 μL of the mixed standard solution and add 200 μL of 0.5 mol / L PMP methanol solution and 100 μL of 0.3 mol / L NaOH solution, and repeat the process as above.

[0053] Chromatographic conditions: Dionex™ CarboPac™ PA20 (150×3.0 mm, 10 μm) liquid chromatography column was used; the injection volume was 5 μL. Mobile phases A (H₂O), B (0.1 M NaOH), and C (0.1 M NaOH, 0.2 M NaAc) were used at a flow rate of 0.5 mL / min. The column temperature was 30 °C. The elution gradients were as follows: 0 min A / B / C (95:5:0, v / v), 26 min A / B / C (85:5:10, v / v), 42 min A / B / C (85:5:10, v / v), 42.1 min A / B / C (60:0:40, v / v), 52 min A / B / C (60:40:0, v / v), 52.1 min A / B / C (95:5:0, v / v), and 60 min A / B / C (95:5:0, v / v). The elution results are shown in Figure 5 and Table 3.

[0054] Table 3 Monosaccharide composition of polysaccharides Example 2: Screening of the activity of five homogeneous polysaccharides from Polygonatum sibiricum against high glucose-induced NRK-52E cells 2.1 Cell Culture Frozen NRK-52E cells (purchased from Wuhan Saiweier Biotechnology Co., Ltd., catalog number STCC30003P) were rapidly thawed in a 37℃ water bath, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in high glucose DMEM medium (4.5 g / L) containing 10% serum, mixed well, and transferred to a 75 cm medium. 2 In culture flasks, cells were cultured at 37°C in an incubator with 5% CO2. Once the cells reached the logarithmic growth phase, they were cultured for two more generations in a high-glucose medium containing 5% serum, and then cultured in a low-glucose medium (1.0 g / L).

[0055] 2.2 Effect of polysaccharides on cell viability as determined by the CCK-8 assay: When NRK-52E cells reached the logarithmic growth phase, they were digested with trypsin, the supernatant was discarded, and a cell suspension was prepared using a low-glucose medium (containing 5% fetal bovine serum). The cells were then subjected to a 1×10⁻⁶ ppm assay. 5 Cells were seeded at a density of [number] cells / well in 96-well plates. When the cells reached 70-80% confluence, they were cultured in serum-free, low-glucose medium for 2 hours. The old medium was then discarded and replaced with serum-free medium containing the drug. After 48 hours of drug administration, the medium was replaced with complete medium containing 10% CCK8 (containing 5% fetal bovine serum) and incubated for 2 hours. The absorbance was measured at 450 nm. The calculation formula is shown below: Experimental wells (containing high-glucose culture medium, 5.4 g / L) Control wells (containing low-glucose medium, 1.0 g / L) Blank wells (without culture medium) were grouped as follows: NG group (normal glucose, low sugar), HG group (high glucose, high sugar), polysaccharide groups with different concentrations (12.5, 25, 50, 100 μg / mL), and Met group (Metformin, 5 μg / mL).

[0056] Data are expressed as mean ± standard deviation. Because the data violated normality (confirmed by the Shapiro–Wilk test) and homogeneity of variance (confirmed by Levene's test), intergroup comparisons were performed using the Kruskal–Wallis H test; for significant results, post-hoc analysis was performed using Dunn's test (corrected by the Holm-Bonferroni method). Statistical analysis and graph creation were performed using Origin (version 2024) and GraphPad Prism (version 8.0) software, while nuclear magnetic resonance spectroscopy analysis was performed using MestReNova (version 14.0) software.

[0057] The results are shown in Figure 6: Compared with the NG group, cell viability was significantly reduced in the HG group (76.82%). Compared with the HG group, the Met group, PPR-1, and PPR-2 (2.5-100 μg / mL) consistently and significantly increased cell proliferation (p<0.05); PRP-1, PRP-2, and PPRP-3 showed significantly enhanced viability at concentrations of 25, 50, and 100 μg / mL, respectively (p<0.05). There were no significant differences among the polysaccharide groups compared with the Met group (p>0.05). At 100 μg / mL, the effects of all treatment groups (each polysaccharide group and the Met group) were similar (p>0.05).

[0058] 2.3 Detection of oxidative damage indicators (SOD, MDA, GSH) NRK-52E cells 2×10 5 Cells were seeded in 6-well plates and cultured in low-glucose medium containing 5% serum for 12 h. The original medium was then discarded. The NG group was treated with low-glucose serum-free medium, while other groups were treated with high-glucose serum-free medium. The drug-treated groups were treated with polysaccharides containing 12.5, 25, 50, and 100 μg / mL of each, and 5 mM of Met, respectively. Cells were cultured for another 48 h. Cell lysates were then collected, and SOD, MDA, and GSH levels were measured according to the kit instructions.

[0059] The results are shown in Figures 7A-7C. Compared with the NG group, the HG group showed significantly lower SOD and GSH levels (p < 0.05) and significantly higher MDA levels (p < 0.05). Compared with the HG group, all treatment groups improved this condition to some extent, indicating that each homogeneous polysaccharide can reduce high glucose-induced oxidative damage to NRK-52E cells. Among them, PPRP-2 showed better effects at 100 μg / mL.

[0060] 2.4 Detection of Fibrin Indicators (FN / COL1 / α-SMA): The cell culture method was the same as that used for oxidative damage detection. Cell culture medium was collected. ELISA kits were used to detect fibronectin (FN), collagen type I (COL1), and α-smooth muscle actin (α-SMA) according to the manufacturer's instructions.

[0061] The results are shown in Figures 7D-7F. Compared with the NG group, the expression of FN, COL1, and α-SMA in the HG group was significantly increased (p<0.05). Compared with the HG group, all treatment groups reduced the content of various fibrotic proteins to some extent, indicating that each homogeneous polysaccharide can alleviate high glucose-induced fibrotic damage to NRK-52E cells. For FN and COL1, compared with the HG group, all doses of polysaccharide groups and the Met group significantly reduced their expression (p<0.05). Compared with PRP, PPRP-2 significantly reduced FN expression in the range of 12.5-50 μg / mL (p<0.05). At 12.5-50 μg / mL and 100 μg / mL, PPRP-2 also showed better effects on COL1 expression than PRP-1 and PRP-2 (p<0.05). For α-SMA protein, the therapeutic effect of polysaccharides is dose-dependent. For example, the expression of PRP-1 and PPRP-3 began to decrease significantly at 50 μg / mL (p<0.05).

[0062] 2.5 Structure-activity relationship analysis: Based on the combined results of cell viability and the expression of fibrotic proteins FN and COL1, PPRP-2 showed particularly outstanding effects. This invention determined 100 μg / mL as the optimal concentration for polysaccharide activity. At this concentration, all polysaccharide components exhibited significant activity, with PPRP-2 showing the most significant effect, its activity slightly superior to other groups, possibly related to its unique structural characteristics. Compared to PRP-1, PRP-2, and PPRP-1, PPRP-2 is composed of galactose, galacturonic acid, arabinose, and rhamnose, with a uronic acid content of 17.80%, which can improve oxidative stress damage to some extent. As for PPRP-3, although it has a similar monosaccharide composition to PPRP-2, its high molecular weight (21.5 kDa) leads to high steric hindrance, hindering effective cellular uptake and thus reducing activity. In addition, the degree of esterification is another key factor affecting polysaccharide activity. Compared to PPRP-3 (degree of esterification of 0.92), PPRP-2 has a lower degree of esterification (0.76), which means that it has more free carboxyl groups exposed on its molecular chain and carries a higher negative charge density. The higher charge density helps to enhance the antioxidant capacity of polysaccharides.

[0063] Example 3: Fine Structure Identification of the Dominant Component PPRP-2 3.1 Methylation Analysis Methylation was used to analyze the glycoside linkages of the polysaccharide, and partially methylated aldosterone acetate derivatives (PMAA-MS) were analyzed. Detection conditions: GC-MS analysis was performed using a Shimadzu gas chromatography system equipped with an HP-5 MS column (25 m x 0.22 mm x 0.25 μm), high-purity helium as the carrier gas, with an injection volume of 1 μL and a split ratio of 10:1. The GC temperature program was as follows: 0–2 min, 80℃; 2–5 min, 80–140℃; 5–6 min, 140℃; 6–13.5 min, 140–155℃; 13.5–32.5 min, 155–250℃; 32.5–37.5 min, 250℃. A high-precision all-metal molybdenum quadrupole detection system was adopted, with an electron impact ion source (EI) temperature of 170℃, an electron energy of 70 eV, and a quadrupole temperature of 240℃, recording the spectrum in full scan mode.

[0064] The methylation results are shown in Table 4. Combined with the results of the monosaccharide composition experiment, the sugar residues of PPRP-2 include Araf-(1→、→1)-Araf-(5→、Galp-(1→、→3,5)-Araf-(1→、→2,4)-Rhap-(1→、→4)-Galp-(1→、→4)-Glcp-(1→、→4,6)-Galp-(1→、→4,6)-Galp-(1→、→4)

[0065] Table 4. Results of methylation analysis of various polysaccharides 3.2 NMR Analysis: Weigh 50 mg of thoroughly dried PPRP-2 sample, dissolve it in D2O, transfer it to an NMR tube, and then perform NMR analysis on the NMR spectrometer. 1 H, 13 C. Scanning of DEPT-135, HH COSY, HSQC, HMBC and NOESY spectra.

[0066] The results of monosaccharide composition and methylation experiments showed that PPRP-2 no longer contained fructose (Figure 8C), but instead contained an increase in galactose, galacturonic acid, rhamnose, arabinose, etc. 1 ¹H NMR (Figure 8A) shows that PPRP-2 contains terminal group signals at δ 5.26, 5.25, 5.13, 5.10, 4.97, 4.65, and 4.44, and also contains both α- and β-glycosidic bonds. δ 1.27 is the characteristic signal of rhamnose H6; δ 3.82 is the proton signal of -OMe; and the presence of δ 2.09 and 2.19 indicates that PPRP-2 may have acetyl substitution. 13 The δ 53.05 and 20.41 in the C NMR (Figure 8B) also indicate the possible presence of methoxy and acetyl groups. δ 176.72, 173.30, and 171.13 represent the carboxyl carbon signals of α-D-GalAp, acetyl, and methylated α-D-GalAp, respectively. 13CNMR and HSQC (Figure 8D) spectra, with end base signals including δ 5.24 / 109.31, 5.10 / 107.36, 5.14 / 107.12, 4.64 / 104.63, 4.44 / 103.69, 4.96 / 100.27, 5.10 / 99.29, and 5.26 / 98.37. Combining the literature and the COSY spectrum (Figure 8E), the terminal group signals are inferred to be glycosidic bonds α-L-Araf-(1→(I),→5)-α-L-Araf-(1→(G),→3,5)-α-L-Araf-(1→(K),→4)-β-D-Galp-(1→(L),β-D-Galp-(1→(M),→4)-α-D-GalAp-6-O-Me-(1→(N),→4)-α-D-GalAp-(1→(P),→2,4)-α-L-Rhap-(1→(Q)) and→4)-α-D-Glcp-(1→(R). The HMBC (Figure 8F) and NOESY (Figure 8G) spectra can be further analyzed to determine the connection sites and sequences between each sugar residue. Specifically, the crossover signal δ in the HMBC spectrum... δ values ​​of 5.24 / 83.06 and 3.62 / 104.43 suggest that sugar residues IH1 may be linked to KC3, and RH4 to LC1; δ values ​​of 4.18 / 100.27, 4.39 / 77.79, 4.13 / 107.61, and 3.73 / 107.61 suggest that LH6 may be linked to NC1, PH4 to QC2, QH4 to JC1, and KH5 to JC1. In the NOESY spectrum, δ... Values ​​5.10 / 4.46, 5.14 / 3.73, and 4.44 / 3.73 indicate the possible presence of →4)-α-D-GalAp-(1→4)-α-D-GalAp-6-O-Me-(1→、→1)-α-L-Araf-(5→1)-α-L-Araf-(3,5→、1)-α-L-Araf-(5→1)-β-D-Galp. The structure of PPRP-2 may be as shown in Figure 8H.

[0067] Table 5. Polysaccharides 1 H and 13 C's signal attribution nd: Not detected Example 4: Comparative study on the efficacy of raw and processed Polygonatum sibiricum aqueous extract and its crude polysaccharide on diabetic kidney disease (DKD) mice 4.1 Experimental methods (1) Animal source C57BL / 6 male mice (20 ± 2 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd., license number: No.2107262231101483157, and were acclimatized for one week.

[0068] (2) Preparation of the drug: Aqueous extract: Weigh appropriate amounts of raw Polygonatum and processed Polygonatum slices, add water and reflux for 1 hour at a material-to-liquid ratio of 1:10 (g / mL), repeat twice, and filter. Combine the two extracts and concentrate under reduced pressure using a rotary evaporator at 50℃. Store frozen and dissolve before use.

[0069] Crude polysaccharide: Accurately weigh appropriate amounts of raw and processed Polygonatum sibiricum slices, add 70% ethanol solution at a material-to-liquid ratio of 1:10 (g / mL), reflux for 1 h, filter and collect the residue, add distilled water at a material-to-liquid ratio of 1:8 (g / mL), reflux for 1 h, repeat the extraction twice, combine the aqueous extracts, filter and concentrate the filtrate by rotary evaporation, then use the Sevag method (dichloromethane: n-butanol = 4:1, v / v) to remove free proteins in the polysaccharide, add Sevag reagent and extract at a ratio of 1:3 to a sealed container, shake vigorously for 15 min, centrifuge at 4000 r / min for 10 min, remove the lower milky protein liquid, collect the upper aqueous phase, repeat more than 4 times until the lower milky white layer disappears. Add 95% ethanol to the treated extract, fractionate the ethanol precipitation to a final ethanol concentration of 80% (v / v), precipitate overnight at 4°C, centrifuge, combine the precipitates, freeze dry to obtain crude polysaccharides PRP and PPRP, which are reconstituted before use.

[0070] (3) Grouping, Modeling, and Drug Administration of Experimental Animals: Male mice were randomly divided into a control (Con) group and a model group. The model group was fed a high-sugar, high-fat diet supplemented with 2.5% glucose water; the normal group was fed a regular diet. After 4 weeks, all mice were fasted for 12 hours. On the second day, their weight was measured and blood glucose was measured by blood collection from the tail vein. When the fasting period reached 16 hours, the model group was injected intraperitoneally with STZ solution. After the injection, the mice were fasted for 2 hours before being fed again. This was repeated 5 times. After the blood glucose stabilized for 3 days, a fasting blood glucose level ≥ 11.1 mmol / L and positive urine protein were considered as successful modeling.

[0071] The experimental animals were divided into 7 groups, with 6 animals in each group. The positive control group (Met) was administered 0.35 g / kg of Met aqueous solution by gavage daily; the dosage of raw Polygonatum sibiricum aqueous extract (PR) and processed Polygonatum sibiricum aqueous extract (PPR) was 4.0 g / kg; the dosage of raw Polygonatum sibiricum crude polysaccharide (PRP) and processed Polygonatum sibiricum crude polysaccharide (PPRP) was 0.61 and 0.16 g / kg, respectively (equivalent to a crude drug dosage of 4 g / kg).

[0072] The control group (Con) and the model group (DKD) were given the same volume of water. The Con group was fed regular feed and drank normal water, while the other groups were fed high-fat feed and drank glucose water, with no restriction on water intake.

[0073] (4) Detection indicators: During the experimental period, the growth status of mice was observed, and the weight and fasting blood glucose (FBG) of mice were measured. Urine of mice was collected from metabolic cages in the last week. After 6 weeks of continuous administration, 1% sodium pentobarbital solution was injected into the peritoneum, and blood was collected from the orbital cavity. The following indicators of renal function were detected in plasma: creatinine (CRE), blood urea nitrogen (BUN), urine protein to urine creatinine ratio (ACR), and kidney pathological sections (HE staining and Masson staining).

[0074] (5) Statistical test methods: Data are presented as mean ± standard deviation (Mean ± SD). The statistical significance method is one-way ANOVA, and p < 0.05 is considered statistically significant. GraphPad Prism (v8.0) is used to draw graphs.

[0075] 4.2 Experimental Results (1) Basic Animal Conditions There were no significant differences in body weight, mental state, and fur luster among the mice in each group before modeling. During the experiment, the mice in the blank group showed a significant increase in body weight, good mental state, and quick response. Their fur was shiny and dense, and their bedding was dry. The mice in the other groups showed obvious polyphagia, polydipsia, and polyuria. They were lethargic, their fur was dull and became sparser in the later stages, and their bedding was relatively damp. The mice in each drug-treated group showed varying degrees of relief.

[0076] (2) Effects on mouse body weight: After the experiment, the weight gain of DKD mice in each group was significantly lower than that in the control group (p < 0.05). Compared with the DKD group, the weight of mice in the PPR, PRP, and PPRP groups increased significantly, and there was no significant difference in the growth trend among them (p > 0.05). Although the weight of mice in the PR and Met groups did not increase, the trend of continued weight loss was significantly slowed down (p < 0.05). This indicates that processed Polygonatum sibiricum has a significantly stronger ability to maintain the survival of mice than raw Polygonatum sibiricum, and crude polysaccharide is the main active ingredient of Polygonatum sibiricum. The weight gain of mice in each group before and after administration is shown in Figure 9A.

[0077] (3) Effects on blood glucose in mice After 6 weeks of treatment with raw and processed Polygonatum sibiricum and Met, the FBG of mice in each group was significantly lower than that in the DKD group (Figure 9B). Among them, the PPR group was significantly lower than the PR group (p<0.05), and there was no significant difference with the polysaccharide part and the Met group (p>0.05).

[0078] (4) Effects on renal function in mice. Figures 10A-10C show that, compared with the Con group, the BUN, CRE levels, and ACR ratio of mice in the DKD group were significantly increased (p<0.05), indicating that the mice in this group had obvious renal dysfunction. Compared with the DKD group, all drug-treated groups could effectively improve renal function. Among them, in terms of BUN, PRP and PPRP were significantly stronger than those in the water extract group (PR and PPR).

[0079] (5) Effects on mouse kidney pathological sections 1) HE staining: The kidney tissue structure of mice in the Con group was normal, with clear and normal morphology of glomeruli and renal tubules, and no pathological changes in the renal interstitium. In mice in the DKD group, glomeruli were hypertrophic, the capsule was narrowed (blue arrow), mild dilation of renal tubules was common (black arrow), and inflammatory cell infiltration was observed in the renal interstitium (green arrow). Compared with the DKD group, all treatment groups showed improvement (Figure 11).

[0080] 2) Masson staining: Compared with the Con group, the DKD group mice showed obvious fibrotic lesions in the glomeruli and tubulointerstitium (blue area). Compared with the DKD group, the degree of renal fibrosis in each treatment group was significantly alleviated, but the polysaccharide group was significantly stronger than the aqueous extract group (p<0.05). In addition, the PPR group and PPRP group were significantly better than the PR group and PRP group, respectively (p<0.05), as shown in Figure 12.

[0081] 4.3 Conclusion The aqueous extracts of raw and processed Polygonatum sibiricum, along with their crude polysaccharides, all showed certain therapeutic effects in regulating abnormal glucose and lipid metabolism, improving renal function, and alleviating renal damage in DKD mice. The overall effect of the processed Polygonatum sibiricum was superior to that of the raw product. Furthermore, the therapeutic effect of the crude polysaccharide was almost identical to that of the aqueous extract, sufficiently demonstrating that polysaccharide is the main active component of Polygonatum sibiricum in treating DKD mice.

[0082] In summary, existing research on the effects of processing on Polygonatum polysaccharides is rather one-sided, generally lacking a systematic comparison of the chemical structure and biological activity of polysaccharides before and after processing. This leads to key issues such as unclear material basis for the enhanced efficacy of Polygonatum polysaccharides through processing and lack of quality control standards. This invention provides a systematic technical solution that aims to fundamentally reveal the material basis and transformation law of the enhanced efficacy of Polygonatum polysaccharides through processing.

[0083] First, this invention constructs a complete chain of evidence from raw material processing to active products. This invention does not simply apply processed Polygonatum sibiricum, but rather, through rigorously designed comparative experiments, systematically reveals the chemical profile of the remodeled Polygonatum sibiricum polysaccharides through steaming, ultimately leading to a significant improvement in its bioactivity.

[0084] Secondly, this invention establishes a systematic separation and purification strategy guided by "processing enhancement". Based on a clear understanding of the differences in activity before and after processing, this invention achieves a clear and targeted approach. The core of this invention's strategy lies in the systematic parallel comparison of polysaccharide components before and after processing. Through the optimized combination of "ion exchange separation - parallel activity screening - multi-level structural characterization", the goal is no longer to locate a single active ingredient, but to successfully construct and define a group of compositions with specific structural evolution relationships, consisting of raw polysaccharides and processed polysaccharides. This transforms the complex processing process into a clearly visible and scientifically definable material transformation system.

[0085] Finally, the superiority of this invention is strongly supported by experimental data. This invention successfully constructed and defined a group of Polygonatum polysaccharide compositions with specific structural evolution relationships. Experimental data clearly confirm that, compared with polysaccharides derived from raw materials, the compositions of this invention exhibit significant overall renal protective activity. Specifically, the core component PPRP-2 demonstrates better renal protective activity against high glucose-induced NRK-52E cells; in vivo pharmacodynamic experiments verified that processed Polygonatum significantly improves the debilitating state of DKD mice, and that crude polysaccharide is the main active component of Polygonatum. This achievement not only provides a crucial scientific material basis and direct evidence for the traditional Chinese medicine theory of "enhanced efficacy through processing of Polygonatum," but also transforms this group of components into precisely developable and quality-controllable renal protective functional factors, laying a solid material foundation for the development of a new generation of Polygonatum products.

[0086] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A Polygonatum polysaccharide composition exhibiting a specific structural evolution relationship and renal protective effect before and after processing, characterized in that, The composition comprises one or more of the following: homogeneous polysaccharides PRP-1 and / or PRP-2 derived from raw Polygonatum sibiricum, and homogeneous polysaccharides PPRP-1, PPRP-2 and / or PPRP-3 derived from processed Polygonatum sibiricum.

2. The composition according to claim 1, characterized in that, PRP-1 and PRP-2 are composed of fructose and glucose, with →1)-β-D-Fruf-(2→) as the main chain; PPRP-1 is composed of fructose, glucose, and arabinose, and adds →5)-α-L-Araf-(1→) to the RPP-1 / PRP-2 base; PPRP-2 and PPRP-3 are composed of arabinose, galactose, galacturonic acid, and rhamnose, containing α-L-Araf-(1→, →5)-α-L-Araf-(1→, →4)-β-D-Galp-(1→, →4)-α-D-GalAp-6-O-Me-(1→, →4)-α-D-GalAp-(1→, →2,4)-α-L-Rhap-(1→).

3. The composition according to claim 1 or claim 2, characterized in that, In the raw polysaccharides, PRP-1 and PRP-2 are both fructans, and their weight-average molecular weight ranges from 2.0 to 3.0 kDa; in their monosaccharide composition, the molar ratio of fructose to glucose ranges from 90%:10% to 99%:1%.

4. The composition according to claim 1 or claim 2, characterized in that, In the polysaccharide product, the fructans in PRP-1 and PRP-2 are degraded after processing to form PPRP-1, which has a smaller molecular weight and contains trace amounts of arabinose. The weight-average molecular weight of PPRP-1 ranges from 1.5 to 2.5 kDa. During the processing, pectin polysaccharides PPRP-2 and PPRP-3 from plant cell walls are also released. The weight-average molecular weight of PPRP-2 and PPRP-3 ranges from 8 to 25 kDa, and the sum of their molar contents of galactose and galacturonic acid reaches 70% to 75%.

5. The composition according to claim 4, characterized in that, The homogeneous polysaccharide PPRP-2 derived from Polygonatum sibiricum has a weight-average molecular weight of 8-14 kDa. In its monosaccharide composition, the molar proportions of galactose and galacturonic acid are not less than 40% and 15%, respectively. Its fine chemical structure includes the following structural units: with →4)-α-D-GalAp-(1→、→2,4)-α-L-Rhap-(1→、→4)-α-D-GalAp-6-O-Me-(1→、→3, 5)-α-L-Araf-(1→) as the main chain and α-L-Araf-(1→) as the side chain.

6. A method for preparing the composition according to any one of claims 1 to 5, characterized in that, Includes the following steps: Step 1, Raw material selection: Using processed Polygonatum sibiricum as raw material; Step 2, Extraction of crude polysaccharide: The processed Polygonatum sibiricum selected in Step 1 is defatted with ethanol, extracted with hot water, precipitated with alcohol, and protein removed to obtain crude Polygonatum sibiricum polysaccharide; Step 3, Separation of polysaccharide components: The crude polysaccharide obtained in Step 2 is separated by ion exchange column chromatography to obtain polysaccharide components; Step 4, Screening of active components: The polysaccharide components obtained in Step 3 are compared with the homogeneous polysaccharide extracted from raw Polygonatum sibiricum using primary structure identification including ultraviolet light, infrared light, molecular weight, and monosaccharide composition, and in vitro renal protective activity, to screen out the components with the best activity; Step 5, Fine identification of target components: The components with the best activity screened in Step 4 are subjected to methylation and NMR analysis to obtain the homogeneous polysaccharide as described in any one of claims 1 to 5.

7. The preparation method according to claim 6, characterized in that, In step 1, the prepared Polygonatum is Polygonatum that has undergone steaming treatment; in step 2, the volume concentration of ethanol during defatting is 70-90%, and the volume concentration of ethanol during fractional alcohol precipitation is 50%-80%; in step 3, the packing material for the ion exchange column chromatography is DEAE-52 cellulose packing material.

8. The use of the composition of any one of claims 1 to 5 or the composition prepared by the preparation method of claim 6 in the preparation of products for the prevention, improvement, adjunctive treatment or treatment of kidney-related diseases.

9. The application according to claim 8, characterized in that, The kidney-related diseases include diabetic nephropathy or kidney fibrosis.

10. The application according to claim 8, characterized in that, The product is a medicine, health product, or functional food.

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