Preparation of high-purity sugarcane leaf polysaccharide and application of high-purity sugarcane leaf polysaccharide in improvement of diabetic cardiomyopathy

Abstract

The invention discloses a preparation method of high-purity sugarcane leaf polysaccharide, which comprises the following steps: separating sugarcane leaf crude polysaccharide by using an alkaline H2O2 method, and then separating and purifying by using a DEAE-Sepharose Fast Flow anion exchange column to obtain the high-purity sugarcane leaf polysaccharide SLP-2. The monosaccharide composition of the high-purity sugarcane leaf polysaccharide SLP-2 is mainly Xyl, Ara and Gal, and the main chain structure of the high-purity sugarcane leaf polysaccharide SLP-2 is composed of glycosidic bonds such as Araf-(1-), Xylp-(1-),-> 3, 4)-Galp-(1-),-> 5)-Araf-(1-),-> 4)-Xylp-(1-),-> 4)-Glcp-(1-),-> 3, 4)-Xylp-(1-) and the like. The research on the efficacy of a diabetic cardiomyopathy rat model induced by feeding with streptozotocin combined with high-sugar and high-fat feed finds that the high-purity sugarcane leaf polysaccharide SLP-2 has the effects of reducing the blood sugar of the diabetic cardiomyopathy rat and improving the heart function of the diabetic cardiomyopathy rat. In conclusion, the preparation process is simple, the polysaccharide composition and structure are clear, and the SLP-2 has good effects of reducing blood sugar and improving diabetic cardiomyopathy and heart injury, has a certain application prospect in the field of medicines, and is worthy of further research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of sugarcane leaf polysaccharide, and particularly relates to preparation of high-purity sugarcane leaf polysaccharide and application thereof in improving diabetic cardiomyopathy. BACKGROUND

[0002] Sugarcane leaf is the leaf of Saccharum sinensis Roxb. belonging to the Gramineae family, is a traditional Yao medicine and folk medicine in Guangxi, has a long history of use as medicine, and is recorded in the second volume of Guangxi Yao Medicine Quality Standard. The Yao medicine uses sugarcane leaf decoction to treat diabetes (diabetes mellitus), but the active substance of the medicine is still unknown. The sugarcane leaf contains rich polysaccharides, and researches show that the crude polysaccharide of the sugarcane leaf has good antioxidant, anti-inflammatory and improvement effects on cardiovascular-related necrotic apoptosis. In addition, the uniform polysaccharides ASLW and ASLM in the sugarcane leaf have good antibacterial activity, and SPL70-1 has good antitumor activity. At present, it has not been reported whether the sugarcane leaf polysaccharide can be used for treating diabetic cardiomyopathy. SUMMARY

[0003] The technical problem to be solved by the present application is to provide preparation of high-purity sugarcane leaf polysaccharide and application thereof in improving diabetic cardiomyopathy.

[0004] To solve the above technical problem, the present application adopts the following technical scheme:

[0005] The preparation method of the high-purity sugarcane leaf polysaccharide separates the crude polysaccharide of the sugarcane leaf by the alkaline H2O2 method, and then separates and purifies the high-purity sugarcane leaf polysaccharide SLP-2 by using a DEAE-Sepharose Fast Flow anion exchange column. The monosaccharide composition of the high-purity sugarcane leaf polysaccharide SLP-2 mainly includes Xyl, Ara and Gal, and the main chain structure thereof is composed of glycosidic bonds such as Araf-(1→, Xylp-(1→, →3,4)-Galp-(1→, →5)-Araf-(1→, →4)-Xylp-(1→, →4)-Glcp-(1→, →3,4)-Xylp-(1→.

[0006] The above preparation method comprises the following steps:

[0007] (1) Pretreatment: the sugarcane leaf is soaked in a NaOH solution;

[0008] (2) Crude polysaccharide extraction: H2O2 solution is added, and then the pH is adjusted to alkaline by using a NaOH solution, extraction is performed, and the filter residue is discarded;

[0009] (3) Crude polysaccharide purification: the pH of the extraction liquid is adjusted by using an HCl solution, insoluble impurities are removed by suction filtration, the pH of the filtrate is continuously adjusted by using a solution, anhydrous ethanol is added for alcohol precipitation, and the precipitate is washed with anhydrous ethanol;

[0010] (4) dissolving the precipitate in pure water, dialysis, and freeze-drying to obtain crude polysaccharide SCLP from sugarcane leaves;

[0011] (5) SLP-2 purification: SCLP is purified using an anion exchange column, eluted with deionized water, 0.2M NaCl, and 1.0M NaCl in sequence, and the deionized water and 1.0M NaCl elution components are discarded, the 0.2M elution component is enriched, and after concentration, desalination by dialysis, and freeze-drying, high-purity sugarcane leaf polysaccharide SLP-2 is obtained.

[0012] In step (1), the sugarcane leaves are added with 1% NaOH solution at a material-to-liquid ratio of 1:40 (w / v) and soaked at 55°C for 2h.

[0013] In step (2), 3% H2O2 solution is added at a material-to-liquid ratio of 1:40 (w / v), and then 1% NaOH solution is used to adjust the pH to 11, and extraction is carried out at 45°C for 12h, and the filter residue is discarded.

[0014] In step (3), the extraction liquid is adjusted to pH 8.5 with 6mol / L HCl solution, and insoluble impurities are removed by suction filtration, and the filtrate is continuously adjusted to pH 5.5 with 6mol / L HCl solution, and anhydrous ethanol is added to an alcohol content of 80% for alcohol precipitation, and the precipitate is washed with anhydrous ethanol.

[0015] In step (4), the precipitate is dissolved in pure water, dialyzed for 48h with a pore size of 8-14kDa, and freeze-dried to obtain crude polysaccharide SCLP from sugarcane leaves.

[0016] In step (5), SCLP is purified using a DEAE-Sepharose Fast Flow anion exchange column, eluted with deionized water, 0.2M NaCl, and 1.0M NaCl in sequence, and the deionized water and 1.0M NaCl elution components are discarded, the 0.2M elution component is enriched, and after concentration, desalination by dialysis for 48h with a 3.5kDa membrane, and freeze-drying, high-purity sugarcane leaf polysaccharide SLP-2 is obtained.

[0017] High-purity sugarcane leaf polysaccharide SLP-2 obtained by the above preparation method.

[0018] Use of the above high-purity sugarcane leaf polysaccharide SLP-2 in the preparation of a drug or health food for treating diabetic cardiomyopathy.

[0019] In order to fully develop and utilize the sugarcane leaf resources, the inventors have conducted in-depth research and established a preparation method of high-purity sugarcane leaf polysaccharide, the crude polysaccharide is separated from sugarcane leaves by an alkaline H2O2 method, and then the high-purity sugarcane leaf polysaccharide SLP-2 is obtained by separation and purification with a DEAE-Sepharose Fast Flow anion exchange column; the monosaccharide composition of the high-purity sugarcane leaf polysaccharide SLP-2 mainly comprises Xyl, Ara and Gal, and the main chain structure thereof is composed of glycosidic bonds such as Araf-(1→, Xylp-(1→, →3,4)-Galp-(1→, →5)-Araf-(1→, →4)-Xylp-(1→, →4)-Glcp-(1→, →3,4)-Xylp-(1→, etc. The pharmacodynamic study on a diabetic cardiomyopathy rat model induced by feeding with a high-sugar and high-fat feed combined with streptozotocin shows that the high-purity sugarcane leaf polysaccharide SLP-2 has the effects of reducing the blood glucose level of the diabetic cardiomyopathy rat and improving the cardiac function thereof. In conclusion, the preparation process of the present application is simple, the polysaccharide composition and structure are clear, the SLP-2 has good effects of reducing blood glucose and improving cardiac injury of diabetic cardiomyopathy, and has certain application prospect in the medical field, and is worthy of further research. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is one of structural characterization combination charts, in the chart: A, DEAE Fast Flow elution curve of SCLP, B, HPGPC chromatogram of SLP-2, SLP-2-1, SLP-2 and SLP-2-3, C, UV graph of SLP-2-1, SLP-2-2 and SLP-2-3, D, IR graph of SLP-2-1, SLP-2-2 and SLP-2-3, E, chromatogram of SLP-2-1, SLP-2-2, SLP-2-3 and standard monosaccharide, F, GC-MS chromatogram of SLP-2-1, SLP-2-2 and SLP-2-3.

[0021] Figure 2 is a result chart of the repair effect of SLP-2 on high-sugar injury of myocardial cells and the regulation effect on NF-κB / NLRP3-mediated inflammation, in the chart: A, glucose consumption of HepG2 cells in each elution component, B, influence of SLP-2 and SCLP on the activity of H9c2 cells injured by high-sugar+TNF alpha, C, influence of SLP-2 on the expression of related genes in cells induced by high-sugar+TNF alpha.

[0022] Figure 3 is a blood glucose level change chart after administration.

[0023] Figure 4Figure 6 is a columnar chart of echocardiography data related to SLP-2 administration, in which: *P<0.05 compared with the blank group; ***P<0.001; #P<0.05 compared with the model group; ##P<0.01.

[0024] Figure 5 Figure 7 is a M-mode echocardiography chart, in which: K-the blank group; M-the model group; MET-the metformin group; L-the low-dose group; Z-the middle-dose group; H-the high-dose group.

[0025] Figure 6 Figure 8 is a graph of heart / body ratio results.

[0026] Figure 7 Figure 9 is a graph of inflammatory factor expression levels in rat serum.

[0027] Figure 8 Figure 10 is a graph of NLRP3, ASC, and IL-1β mRNA relative expression amounts in rat heart tissue.

[0028] Figure 9 Figure 11 is a graph of p50 immunohistochemical results in each group.

[0029] Figure 10 Figure 12 is a graph of p65 immunohistochemical results in each group.

[0030] Figure 11 Figure 13 is a graph of the effects of SLP-2 on NF-κB p65, NF-κB p50, NLRP3, and Caspase-1 protein expression in the heart tissue of diabetic rats. DETAILED DESCRIPTION

[0031] I. Isolation, purification, and structural identification of SLP-2

[0032] 1. Materials and methods

[0033] 1.1 Materials

[0034] Sugarcane leaves were collected from Wuming, Nanning, Guangxi, and identified by Professor We Songji, Director of the Medicinal Plant Teaching and Research Office of Guangxi University of Chinese Medicine, as the leaves of Saccharum sinensis Roxb. of the family Poaceae; DEAE-Fast-Flow and DEAE-Sepharose Cl-6B were purchased from Cytiva, USA; 1-phenyl-3-methyl-5-pyridinone (PMP), monosaccharide standards: mannose (Man), arabinose (Ara), glucose (Glc), glucuronic acid (GlcA), galacturonic acid (GalA), ribose (Rib), xylose (Xyl) were purchased from Sigma Chemical; rhamnose (Rha) and fucose (Fuc) were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China); and the remaining reagents were all of analytical purity.

[0035] 1.2 Extraction of crude polysaccharides from sugarcane leaves

[0036] Air-dried sugarcane leaves were added with a 1% NaOH solution at a solid-liquid ratio of 1:40 (w / v) at 55°C for 2 hours. After cooling to room temperature, the extract was filtered and the filtrate removed. Pre-treated sugarcane leaves were added with a 3% H2O2 solution at a solid-liquid ratio of 1:40 (w / v), the pH adjusted to 11 with 1% NaOH, and the extract was removed at 45°C for 12 hours. The extract was filtered, and the pH of the filtrate was adjusted to 8.5 with a 6 mol / L HCl solution. Insoluble (silicate) impurities were removed by suction filtration. The filtrate was further adjusted to pH 5.5 with a 6 mol / L HCl solution, concentrated, and anhydrous ethanol was added to 80% alcohol content. The extract was allowed to settle and filtered. The precipitate was washed with anhydrous ethanol and evaporated to remove alcohol. The precipitate was dissolved in pure water and dialyzed for 48 hours (8-14 kDa). The extract was freeze-dried to obtain crude sugarcane leaf polysaccharide (SCLP).

[0037] 1.3 Isolation and purification of SCLP

[0038] A DEAE-Sepharose Fast Flow anion exchange column ( SCLP was isolated and purified using a gradient elution method (40 cm x 40 cm) using deionized water, followed by 0.2, 0.4, 0.6, 0.8, and 1.0 M NaCl solutions. Elution fractions were collected using an automatic fraction collector, and the elution peaks were tracked using the phenol-sulfuric acid method. The absorbance was measured at 490 nm, and an elution curve was plotted. Repeated loading was performed, and three major components (3.5 kDa) were enriched according to the elution curve. Each component was dialyzed against deionized water for 48 hours and freeze-dried to yield three polysaccharide fractions: SLP-1, SLP-2, and SLP-3.

[0039] Among them, SLP-2 is the main component of SCLP, which was further purified using Sepharose CL-6B gel column ( SLP-2 was further purified using a 0.2M NaCl solution (170 cm × 170 cm). The eluate was collected using an automated fraction collector at a controlled flow rate of approximately 24 mL / h and 15 min / tube. High-performance gel permeation chromatography (HPGPC) was used to monitor and enrich and purify SLP-2-1, SLP-2-2, and SLP-2-3 based on molecular weight.

[0040] 1.4 Determination of physical and chemical properties

[0041] The total sugar content of the sugarcane leaf polysaccharide was determined by using the phenol-sulfuric acid method with anhydrous glucose as the standard; the uronic acid content of the sugarcane leaf polysaccharide was determined by using the m-hydroxydiphenyl colorimetric method with galacturonic acid as the standard; and the protein content of the sugarcane leaf polysaccharide was determined by using the Coomassie brilliant blue method with bovine serum albumin (BSA) as the standard.

[0042] 1.5 Determination of homogeneity and molecular weight of sugarcane leaf polysaccharide

[0043] According to the method and conditions of Yuan QX et al. (Qingxia Y, Kunling L, Jinwen H, et al. Simulated digestion, dynamic changes during fecal fermentation and effects on gut microbiota of Avicennia marina (Forssk.) Vierh. fruit non-starch polysaccharides [J]. Food Chemistry: X, 2022, 16: 100475-100475.), the HPGPC method was used to determine SLP-2-1, SLP-2-2, and SLP-2-3, and the purity was determined according to the number of peaks and the shape of the peaks, and the retention time of the sample was recorded. Then, the standard curve (retention time as X, and the logarithmic value of the molecular weight as Y) was drawn using the dextran series standard (D1, D2, D3, D4, D5, D6), and the molecular weight of the sample was calculated based on the retention time.

[0044] 1.6 UV spectrum scanning and infrared spectrum analysis of sugarcane leaf polysaccharide

[0045] (1) An appropriate amount of sugarcane leaf polysaccharide sample was dissolved in deionized water, and the UV spectrophotometer (UV-VIS SPECTROPHOTOMETER, UV-2600, SHIMADZU) was used for scanning with a wavelength range of 200-800 nm.

[0046] (2) An appropriate amount of dried sugarcane leaf polysaccharide sample was taken, and the KBr tabletting method was used for infrared (FT-IR, L1600400, Perkin Elmer) scanning in the range of 4000-400 cm -1

[0047] 1.7 Analysis of monosaccharide composition of sugarcane leaf polysaccharide

[0048] ​Reference Tang H et al. (Hao T, Jinwen H, Qingxia Y, et al. A regular Chlorella mannogalactan and its sulfated derivative as a promising anticoagulant: Structural characterization and anticoagulant activity [J]. Carbohydrate Polymers, 2023, 314: 120956-120956.) method and conditions, using trifluoroacetic acid (TFA) to completely acid hydrolyze sugarcane leaf polysaccharide, and then using 1-phenyl-3-methyl-5-pyridine ketone (PMP) to complete the pre-column derivatization experiment, and using HPLC method to detect the monosaccharide composition. Then use 10 kinds of monosaccharide standard mixture for PMP derivatization, draw standard curve, calculate the molar ratio of monosaccharide composition of sample polysaccharide.

[0049] 2.9 Methylation of sugarcane leaf polysaccharide and GC-MS analysis

[0050] Reference Yuan QX et al. (Yuan Q, Zhao L, Cha Q, et al. Structural Characterization and Immunostimulatory Activity of a Homogeneous Polysaccharide from Sinonovacula constricta [J]. Journal of Agricultural and Food Chemistry, 2015, 63(36): 7986-7994.) methylation method, dissolve the sugarcane leaf polysaccharide sample in DMSO, then add NaOH and CH3I, and carry out methylation reaction. Repeat 4 times to completely methylate the polysaccharide sample, then carry out complete acid hydrolysis, reduction and acetylation to obtain polysaccharide methylation derivative. GC-MS analysis was used.

[0051] GC-MS test conditions: Agilent Technologies 7890B GC System, capillary column 30 m*0.25 mm*0.25 μm, flow rate 1.0 mL / min, injection port temperature 260℃, injection amount 1 μL, split injection, split ratio 10:1, solvent delay 2.2 min; the program temperature conditions are: 50℃ for 1.0 min, 50℃ / min to 130℃, 3℃ / min to 230℃, hold for 2 min.

[0052] 1.8 NMR analysis of sugarcane leaf polysaccharide

[0053] The sugarcane leaf polysaccharide sample was dissolved in D2O and then freeze-dried, repeated three times, and then fully dissolved in 600 μL D2O (containing internal standard), transferred to a nuclear magnetic resonance tube, and analyzed by nuclear magnetic resonance (Bruker AvanⅢHD 500M spectromnyer) to obtain 1Dspectra ( 1 H NMR and 13 C NMR, 13 C spectrum with time-lapse scanning) and 2D spectra (HSQC, HMBC, DEPT-135, TOCSY, NOESY, COSY, HSQC-TOCSY) spectra.

[0054] 2. Experimental Results

[0055] 2.1 Results of extraction, separation and purification of sugarcane leaf polysaccharides

[0056] SCLP was extracted using alkaline hydrogen peroxide method, and DEAE Sepharose Fast Flow column chromatography was used to separate SCLP using different concentrations of NaCl. The elution curve was drawn using the phenol-sulfuric acid method ( Figure 1 A), the main component was enriched as the 0.2 M NaCl elution fraction (named SLP-2), which was further purified by Sepharose Cl-6B column chromatography and monitored by HPGPC. Three polysaccharide components (named: SLP-2-1, SLP-2-2, and SLP-2-3) were enriched according to molecular weight distribution. The yields, based on dry SCLP, were 10.69%, 34.01%, and 10.50%, respectively.

[0057] SLP-2-1, SLP-2-2, and SLP-2-3 showed symmetrical peaks in HPGPC, indicating that SLP-2-1, SLP-2-2, and SLP-2-3 were all polysaccharides with good homogeneity. The molecular weights calculated using the dextran standard curve were approximately 102.15 kDa, 17.55 kDa, and 4.32 kDa, respectively. Figure 1 B) The calibration curve formula for calculating the molecular weight (Da) is: y = -0.306x + 9.5258, (y is the logarithm of the molecular weight, x is the retention time).

[0058] 2.2 Physicochemical properties of sugarcane leaf polysaccharides

[0059] The phenol-sulfuric acid method, m-hydroxybiphenyl colorimetric method, and Coomassie brilliant blue method were used to determine the total sugar, uronic acid, and protein contents of crude polysaccharides and purified fraction polysaccharides. The test results are shown in Table 1.

[0060] Table 1 Physicochemical properties of SCLP, SLP-2-1, SLP-2-2 and SLP-2-3

[0061]

[0062] 2.3 UV and IR spectra of SLP

[0063] The UV and IR spectra of SLP were scanned by UV-Vis spectrophotometer and Fourier infrared spectrometer. The UV spectra of SLP-2-1, SLP-2-2 and SLP-2-3 had no obvious absorption peaks at 260 nm and 280 nm (Fig. 2A), which indicated that the purified SLP-2-1, SLP-2-2 and SLP-2-3 almost contained no nucleic acid and protein, which was consistent with the results of protein content determination. Figure 1 The IR spectra of SLP-2-1, SLP-2-2 and SLP-2-3 were basically the same (Fig. 2B). The characteristic absorption peak at 3334 cm -1 was produced by the stretching vibration of O-H in hydroxyl; the signal at 2930 cm -1 was produced by the stretching vibration of C-H; the signal at 2102 cm -1 was produced by the aliphatic C-H bond; the absorption peaks at 1610 cm -1 and 1418 cm -1 belonged to the asymmetric and symmetric COO- stretching vibration; the signal at 1041 cm -1 was produced by the deformation vibration of C-O-H, which was the characteristic absorption of pyranose; the absorption at 895 cm -1 indicated the existence of β-type glycoside. Figure 1

[0064] 2.4 Monosaccharide composition analysis of SLP

[0065] According to the HLPC analysis, SLP-2-1, SLP-2-2 and SLP-2-3 were mainly composed of Xyl, Ara and Gal, and contained a small amount of Man, Rha, GlcA, GalA and Glc (Fig. 3), which indicated that SLP-2-1, SLP-2-2 and SLP-2-3 were the same type of heteropolysaccharide, and the molar ratio of the specific monosaccharide composition was shown in Table 1. Figure 1

[0066] 2.5 Methylation and GC-MS analysis of SLP

[0067] After the SLP samples were subjected to complete methylation, complete acid hydrolysis, reduction and acetylation, the sample derivatives were obtained, which were analyzed by GC-MS. The total ion chromatograms of SLP-2-1, SLP-2-2 and SLP-2-3 were shown in Fig. 4. Figure 1 ​​F) and each fragment ion peak. According to the partially methylated sugar alcohol acetate (PMAA) data in the Composite Carbohydrate Research Center (CRCC) Spectral Database at the University of Georgia, the main linkage mode and molar ratio of the glycosidic bond of SLP-2-1, SLP-2-2, SLP-2-3 are shown in Table 2, SLP-2-1, SLP-2-2, SLP-2-3 are mainly composed of Araf-(1→, Xylp - (1→,→3,4)-Galp - (1→,→5)-Araf-(1→,→4)-Xylp - (1→,→4)-Glcp - (1→,→3,4)-Xylp - (1→,→3,4)-Xylp

[0068] Table 2 GC-MS results of methylation of SLP-2-1, SLP-2-2 and SLP-2-3

[0069]

[0070] 2.6 Nuclear magnetic resonance analysis

[0071] Based on the structural information obtained from the above physicochemical analysis, the sugar structure connection mode of SLP-2-3 can be further clarified by 1D and 2D NMR. In 1In the H spectrum, the 10 signals at 5.40, 5.30, 5.30, 5.40, 4.64, 4.59, 4.56, 4.53, 4.50, 4.49 ppm are easily identified by HSQC spectrum and marked as x and a-i. According to DEPT-135 spectrum and the anomeric hydrogen signal greater than 4.8 ppm, it indicates that the residues x, a, b, c are in α configuration, and the anomeric hydrogen signal less than 4.8 ppm indicates that the residues d, e, f, g, h, i are in β configuration. According to the cross signals in the COSY spectrum at 5.40 / 4.17, 5.30 / 4.18, 5.30 / 3.59, 5.40 / 4.17, 4.64 / 3.45, 4.59 / 3.26, 4.56 / 3.29, 4.53 / 3.46, 4.50 / 3.30, 4.49 / 3.28 ppm, the chemical shifts of each sugar residue H-2 are easily determined, and then the C-1 and C-2 chemical shifts of each sugar residue are determined according to the HSQC spectrum. Through the comprehensive analysis of COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY and HMBC spectra of each sugar residue, the chemical shifts of H3-5 / 6 and their corresponding C3-5 / 6 are obtained. According to the relatively low field chemical shift of C-1 (110.5-111.6 ppm), the sugar residues a, b are identified as α-Araf, and the C-5 chemical shift of sugar residue b is obviously higher than that of sugar residue a (unsubstituted Araf residue), so the sugar residue b is determined as →5)-α-Araf-(1→. Similarly, according to the relatively low field chemical shift of C-1 (99.3-104.4 ppm), the sugar residues d, e, g, h, i are identified as β-Xylp, and the C-4 chemical shift of sugar residues d, g is obviously higher than that of sugar residues e, h, so the sugar residues d, g are identified as →4)-β-Xylp - (1→, and the C-3 / 4 chemical shift of sugar residue i is obviously higher than that of sugar residues e, h, so it is determined as →3,4)-β-Xylp - (1→. Residue c is determined as →3,4)-β-Galp - (1→, which is due to its lower field chemical shift value of C-3 / 4 than the unsubstituted Galp sugar residue, and residue f is identified as →3)-β-Glcp(1→ for the same reason. The weak signal of sugar residue x has a larger anomeric carbon chemical shift at 111.6 ppm, and the H / C3-5 signal is too weak to be observed, which is speculated to be a terminal Araf sugar residue connected to →3)-β-Glcp(1→.

[0072] The detailed C, H signals of x, a-i were assigned according to the correlation signals in COSY, HSQC-TOCSY, NOESY and HMBC spectra, and the results are shown in Table 3. According to the correlation signals at 5.40 / 3.44, 5.40 / 79.2 ppm, →3,4)-β-Xylp - (1→ (sugar residue i) is connected to terminal α-Araf-(1→ (sugar residue a). The signals at 5.30 / 3.44, 4.56 / 3.44, 5.30 / 79.2, 4.15 / 79.2 ppm confirm that →3,4)-β-Xylp - (1→ (sugar residue i) is connected to terminal α-Araf-(1→ (sugar residue a). The signals at 5.30 / 3.44, 4.56 / 3.44, 5.30 / 79.2, 4.15 / 79.2 ppm confirm that →3,4)-β-Xylp - (1→ (sugar residue i) is connected to terminal α-Araf-(1→ (sugar residue a). The signals at 5.30 / 3.44, 4.56 / 3.44, 5.30 / 79.2, 4.15 / 79.2 ppm confirm that →3,4)-β-Xylp - (1→ (sugar residue i) is connected to terminal α-Araf-(1→ (sugar residue a). The signals at 5.30 / 3.44, 4.56 / 3.44, 5.30 / 79.2, 4.15 / 79.2 ppm confirm that →3,4)-β-Xylp - (1→ (sugar residue i) is connected to terminal α-Araf-(1→ (sugar residue a). The signals at 5.30 / 3.44, 4.56 / 3.44, 5.30 / 79.2, 4.15 / 79.2 ppm confirm that →3,4)-β-Xylp - (1→ (sugar residue i) is connected to terminal α-Araf-(1→ (sugar residue a). The signals at 5.30 / 3.44, 4.56 / 3.44, 5.30 / 79.2, 4.15 / 79.2 ppm confirm that →3,4)-β-Xylp - (1→ (sugar residue i) is connected to terminal α-Araf-(1→ (sugar residue a). The signals at 5.30 / 3.44, 4.56 / 3.44, 5.30 / 79.2, 4.15 / 79.2 ppm confirm that →3,4)-β-Xylp - (1→ (sugar residue i) is connected to terminal α-Araf-(1→ (sugar residue a). The signals at 5.30 / 3.44, 4.56 / 3.44, 5.30 / 79.2, 4.15 / 79.2 ppm confirm that →3,4)-β-Xylp

[0073] The above spectrum signal assignment illustrates the glycosidic bond connection between various residues of SLP-2-3. In addition, according to the previous physical and chemical analysis results and 1 H et al., it can be known that SLP-2-1, SLP-2-2 and SLP-2-3 are identical in the characterization information except for the molecular weight, and it can be judged that SLP-2-1, SLP-2-2 and SLP-2-3 have the same primary structure, and therefore SLP-2 composed of them also has a similar primary structure, but has a wider molecular weight distribution.

[0074] Table 3 Carbon and hydrogen spectrum results of SLP-2-3

[0075]

[0076]

[0077] Note: Bold indicates glycosylation position, t represents terminal, ND represents not detected.

[0078] II. Improvement of SLP-2 on diabetic cardiomyopathy and its mechanism

[0079] 1. Materials and methods

[0080] 1.1 Experimental reagents

[0081] Streptozotocin (STZ), Sigma, batch number S0130; Metformin (Met), Merck Pharmaceuticals (Jiangsu) Co., Ltd., batch number ACE0016; Rat TNF-alpha Elisa kit, batch number E-EL-R2856, Rat IL-1β Elisa kit, batch number E-EL-R0012, Wuhan Elabscience Biotechnology Co., Ltd.; Rat Caspase-1 Elisa kit, batch number CSB-EL004543RA, CUSABIO; MTS kit, batch number 0000571012, Promega; total RNA extraction kit, batch number 0000632414, GOTaq Master Mix, batch number 0000602761, Prolmage Biotechnology Co., Ltd.; reverse transcription kit, batch number 0000630156, Roche; primers, batch number 20231104, Guangzhou Ruibo Biotechnology Co., Ltd.

[0082] 1.2 Effect of SLP-2 on high glucose-induced H9c2 myocardial cell injury

[0083] 1.2.1 Effect of SLP-2 on H9c2 cell viability injured by high glucose + TNFα

[0084] Take the logarithmic growth period H9c2 cells according to 5×10 3cells / 100 μL were inoculated in 96-well plates, and after adhering for 24 h, the medium in the wells was aspirated, each well was treated with 100 mM high glucose solution + TNF alpha 20 ng / mL for 48 h, the medium in the wells was then aspirated, SLP-2 and SCLP were added for intervention for 48 h, the medium in the wells was then aspirated, 100 μL of DMEM medium containing 10% MTS was added to each well, and after 2 h of light shielding, the OD value of each well was determined at 490 nm wavelength, and the cell viability was calculated. Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) x 100%.

[0085] 1.2.2 Effect of SLP-2 on inflammatory factors TNF alpha and IL-1 beta

[0086] The cells were inoculated in 24-well plates at 2.5 x 10 4 cells / 500 uL, and after adhering for 24 h, the medium in the wells was aspirated, each well was treated with 100 mM high glucose solution + TNF alpha 20 ng / mL for 48 h, the medium in the wells was then aspirated, SLP-2 and SCLP were added for intervention for 48 h, and the cell supernatant was collected in a new EP tube and centrifuged, and the supernatant was placed in a new EP tube. The content of TNF-a and IL-1 beta in the supernatant was detected according to the steps in the instruction manual.

[0087] 1.2.3 qPCR detection of the expression of key genes in the NF-kappa B and NLRP3 inflammasome pathways in the SLP-2 intervention high glucose + TNF alpha model

[0088] The cells were inoculated in 6-well plates at 1 x 10 5 cells / mL, and after 48 h of treatment with high glucose combined with TNF alpha, SLP-2 (0.12, 0.24, 0.48 ug / mL) and SCLP (60 ug / mL) were added for treatment for 48 h, the cell precipitate was collected, total RNA was extracted according to the steps in the kit, and after denaturation and reverse transcription, the expression of the genes was detected by qPCR.

[0089] 1.3 Intervention effect of SLP-2 on diabetic cardiomyopathy rat models and influence on the NF-kappa B / NLRP3 pathway

[0090] Male SD rat model was established to evaluate diabetic cardiomyopathy induced by high-fat high-sugar feeding combined with streptozotocin. Healthy 4-5-week-old male SD rats were selected and bred in the SPF level experimental animal center of Guangxi University of Chinese Medicine, with temperature controlled at 25±2℃ and relative humidity of 55-60%. The rats were purchased from Hunan Slaik Jingda Experimental Animal Co., Ltd. (Quality Certificate No. SCXK(Xiang)2021-0002). All experiments were conducted in accordance with the standard ethical guidelines and approved by the Research Ethics Committee of Guangxi University of Chinese Medicine (Approval No. DW20220706-182).

[0091] After an adaptation period of 1 week, the rats were fed with high-fat high-sugar diet (feed provided by Yizhenganli Maoguo Biological Technology Co., Ltd., product number 2024GZTL0728) for 4 weeks, followed by a single intraperitoneal injection of streptozotocin (STZ, 50 mg / kg). One week later, rats with fasting blood glucose ≥11.1 mmol / L and obvious symptoms of "polyuria, polydipsia, polyphagia and weight loss" were considered to have successfully established the diabetic model. After the diabetic model rats continued to be fed with high-fat high-sugar diet for 4 weeks, color Doppler ultrasound examination of the heart showed that the myocardial function began to weaken, and the treatment of sugarcane leaf crude polysaccharide and metformin was started. The STZ solution was prepared using 0.1M sodium citrate buffer as the solvent.

[0092] The successfully modeled rats were randomly divided into model group (Model group), metformin administration positive control group (Met group) and different concentration sample group (SLP-2 group), with 6 rats in each group. In addition, the rats in the positive control group were given metformin (90 mg / kg body weight / day) by gavage; the rats in the sample group were given SLP-2 (35.75, 71.50 and 143 mg / kg body weight / day) by gavage at the same time. A normal control group (Normal group) was also established, which was not induced by STZ and was given normal diet and water throughout the process. Each group of animals was given gavage according to the above scheme for 5 days, and then serum was obtained by collecting rat blood. In addition, rat heart tissue was collected for subsequent experiments.

[0093] 1.3.2 Biochemical index determination

[0094] After grouping, the body weight of the rats was measured, and the initial body weight and blood glucose evaluation were obtained by tail blood collection. The body weight, food and water intake of the rats were recorded every week, and the blood glucose level was measured by tail vein. After STZ injection, the rats were fed with high-fat high-sugar diet for 4 weeks, and echocardiography was performed to determine whether the DCM model was successful. At the 5th week of administration, echocardiography was performed again to determine the corresponding cardiac function indicators (LVIDd, LVIDs, EDV, ESV, EF). After the rats were sacrificed, the heart was weighed and the heart-to-body weight ratio was calculated.

[0095] 1.3.3 Study on the regulatory effect of SLP-2 on NF-κB / NLRP3 pathway-mediated inflammation

[0096] The levels of inflammatory factors in rat serum were detected by ELISA to evaluate the inhibitory effect of SLP-2 on systemic inflammatory response. The distribution of NF-κB p50 and p65 in myocardial tissue was detected by immunohistochemistry to analyze the regulatory effect of SLP-2 on the NF-κB signaling pathway. At the same time, qPCR and WB techniques were used to detect the expression of related genes and proteins in the NF-κB and NLRP3 inflammasome pathways, respectively, to study the regulatory mechanism of SLP-2 at the molecular level.

[0097] 1.4 Statistical methods

[0098] SPSS 19.0 statistical software was used to process the experimental data, and the means of multiple groups were compared using the One-Way ANOVA test, with P < 0.05 considered statistically significant.

[0099] 2 Results

[0100] Experiments have shown that SLP-2 not only has a protective effect on the high-glucose-induced H9c2 cell inflammatory injury model, but also significantly improves blood sugar and cardiac function in a rat model of diabetic cardiomyopathy. Its mechanism of action is related to inflammation mediated by the NF-κB / NLRP3 inflammasome. The specific results are as follows:

[0101] 2.1 Protective effect of SLP-2 on the high glucose-induced inflammatory injury model of H9c2 cells

[0102] Sugarcane leaf polysaccharide (SCLP) and its main enriched fraction SLP-2 have a significant protective effect on H9c2 cardiomyocyte inflammatory damage induced by high glucose and TNFα. Among the three main components of SCLP, SLP-1, SLP-2 and SLP-3, separated by DEAE-Sepharose Fast Flow anion exchange column, SLP-2 has the highest content. The experimental results showed that both SCLP and SLP-2 can significantly increase the glucose consumption of HepG2 cells in a dose-dependent manner ( Figure 2 A). In addition, SCLP and SLP-2 can significantly improve the viability of H9c2 cardiomyocytes ( Figure 2 B). Further molecular biological analysis showed that SCLP and SLP-2 could significantly downregulate the mRNA levels of inflammation-related genes (ASC, caspase-1, p50, NLRP3) ( Figure 2 C), indicating that it has anti-inflammatory effects. These results suggest that SLP-2 has a significant pharmacological effect in improving DCM, and its mechanism may be related to the inhibition of NF-κB / NLRP3-mediated inflammatory response.

[0103] 2.2 Interventional effect of SLP-2 on diabetic cardiomyopathy rat model and its impact on NF-κB / NLRP3 pathway

[0104] (1) SLP-2 administration can effectively reduce blood glucose in DCM rats

[0105] like Figure 3 As shown, after 5 weeks of SLP-2 administration, blood glucose levels in the metformin and medium-dose groups decreased significantly after the second week compared with the model group (P < 0.05). After three weeks of administration, blood glucose levels in the metformin, low-dose, and medium-dose groups decreased significantly compared with the model group (P < 0.001). After four weeks of administration, blood glucose levels in the metformin and medium-dose groups decreased significantly compared with the model group (P < 0.05). After five weeks of administration, blood glucose levels in the metformin, medium-dose, and high-dose groups decreased significantly compared with the model group (P < 0.05). This suggests that SLP-2 has a certain hypoglycemic effect on rats with diabetic cardiomyopathy.

[0106] Table 4 Changes in blood glucose in rats 5 weeks after administration (X±SD, n=6)

[0107]

[0108] Compared with the blank group: ***P<0.001; compared with the model group: # P<0.05, ### P<0.001

[0109] (2) SLP-2 administration significantly improved cardiac function in DCM rats

[0110] After establishing the diabetic model, the rats were fed a high-fat, high-sugar diet to establish a diabetic cardiomyopathy model. After four weeks of this diet, cardiac ultrasound examinations were performed. The model group developed symptoms such as polydipsia, polyphagia, polyuria, and weight loss, indicating a successful diabetic model. The cardiac ultrasound results are shown in Table 5. Compared with the control group, the LVIDd and LVIDs in the model group were significantly increased (P < 0.01), indicating impaired cardiac function and confirming the successful establishment of a diabetic cardiomyopathy model.

[0111] When the rat model of diabetic cardiomyopathy was established, SLP-2 and positive drug (metformin) were given for treatment. Daily gavage administration, and after 5 weeks, cardiac color Doppler detection was performed again. Compared with the blank group, the model group LVIDd, LVIDs, EDV, FS, ESV, EF indicators showed significant difference (P<0.05), indicating that the cardiac function was damaged; compared with the model group, the cardiac indicators of each group decreased, among which, the FS value of L-SLP-2 group increased significantly (P<0.01), and the EF value of L-SLP-2 and H-SLP-2 groups had significant difference (P<0.05). There was no statistical difference in LVIDd, LVIDs, EDV, ESV indicators, but from the statistical column chart, it can be seen that SLP-2 treatment still has the trend of improving the above indicators.

[0112] Table 5 results of cardiac color Doppler indicators after administration (X ± SD, n = 6)

[0113]

[0114] As Figure 6 shown, the heart / body ratio of the model group had an increasing trend compared with the blank group (P<0.001), and the heart / body ratio of the Met group had a decreasing trend compared with the model group (P<0.05), and there was no statistical difference in other groups, but from the statistical column chart ( Figure 6 ), it can be seen that SLP-2 treatment still has the trend of reducing the heart / body ratio.

[0115] (3) Study on the regulation of SLP-2 on NF-κB / NLRP3 pathway

[0116] Sugarcane leaf polysaccharide (SCLP) and its main component SLP-2 have significant improvement effect on diabetic cardiomyopathy (DCM). The results show that SLP-2 can inhibit the expression of TNFα, Caspase-1, IL-1β, inflammatory factors in the serum of diabetic cardiomyopathy rats ( Figure 7 ). Reduce the mRNA expression level of IL-1β, NLRP3, ASC in the heart tissue of diabetic cardiomyopathy rats ( Figure 8 ); Can significantly down-regulate the expression amount of NF-κB p50 ( Figure 9 ) and p65 protein ( Figure 10 ); Also can significantly down-regulate the expression level of NLRP3 protein (P<0.001), and can down-regulate the expression amount of NF-κB p50, NF-κB p65, Caspase-1 protein ( Figure 11 ). It is shown that after SLP-2 treatment, the expression of NF-κB, NLRP3 inflammasome pathway related genes and proteins can be inhibited, and the production of inflammatory factors can be reduced, which suggests that SLP-2 may play a role in treating DCM in rats by inhibiting the inflammation mediated by NF-κB / NLRP3 pathway.

Claims

1. A method for preparing high-purity sugarcane leaf polysaccharide, characterized by: Crude sugarcane leaf polysaccharides were separated by an alkaline H2O2 method, and then separated and purified using a DEAE-Sepharose Fast Flow anion exchange column to obtain high-purity sugarcane leaf polysaccharide SLP-2; the monosaccharide composition of the high-purity sugarcane leaf polysaccharide SLP-2 was mainly Xyl, Ara, and Gal, and its main chain structure was composed of glycosidic bonds such as Araf-(1→,Xylp-(1→,→3,4)-Galp-(1→,→5)-Araf-(1→,→4)-Xylp-(1→,→4)-Glcp-(1→,→3,4)-Xylp-(1→ 2. The preparation method according to claim 1, wherein The following steps are involved: (1) Pretreatment: Sugarcane leaves were soaked in NaOH solution; (2) Extraction of crude polysaccharides: Add H2O2 solution, adjust the pH to alkaline with NaOH solution, extract, and discard the residue; (3) Purification of crude polysaccharides: The pH of the extract was adjusted with HCl solution, and insoluble impurities were removed by filtration. The pH of the filtrate was further adjusted with solution, and anhydrous ethanol was added to allow the precipitate to stand and precipitate was washed with anhydrous ethanol. (4) Dissolve the precipitate in pure water, dialyze, and freeze-dry to obtain crude sugarcane leaf polysaccharide SCLP; (5) SLP-2 purification: SCLP was purified using an anion exchange column, eluted with deionized water, 0.2 M NaCl, and 1.0 M NaCl in sequence. The deionized water and 1.0 M NaCl elution fractions were discarded, and the 0.2 M elution fraction was enriched. After concentration, the salt was removed by dialysis, and the high-purity sugarcane leaf polysaccharide SLP-2 was obtained by freeze-drying.

3. The preparation method according to claim 1, wherein In step (1): sugarcane leaves were added with 1% NaOH solution at a solid-liquid ratio of 1:40 (w / v) and soaked at 55° C. for 2 h.

4. The preparation method according to claim 1, characterized in that In step (2): add 3% H2O2 solution at a material-liquid ratio of 1:40 (w / v), then adjust the pH to 11 with 1% NaOH solution, extract at 45°C for 12 hours, and discard the residue.

5. The preparation method according to claim 1, characterized in that In step (3), the pH of the extract is adjusted to 8.5 with a 6 mol / L HCl solution, and insoluble impurities are removed by filtration. The pH of the filtrate is further adjusted to 5.5 with a 6 mol / L HCl solution, and anhydrous ethanol is added until the alcohol content reaches 80%, and the mixture is allowed to stand for alcohol precipitation, and the precipitate is washed with anhydrous ethanol.

6. The preparation method according to claim 1, characterized in that In step (4): the precipitate is dissolved in pure water, dialyzed for 48 hours with a pore size of 8-14 kDa, and freeze-dried to obtain the crude sugarcane leaf polysaccharide SCLP.

7. The preparation method according to claim 1, characterized in that In step (5): SCLP was purified using a DEAE-Sepharose Fast Flow anion exchange column, eluted with deionized water, 0.2 M NaCl, and 1.0 M NaCl in sequence, the deionized water and 1.0 M NaCl elution fractions were discarded, the 0.2 M elution fraction was enriched, concentrated, dialyzed at 3.5 kDa for 48 h for desalination, and freeze-dried to obtain high-purity sugarcane leaf polysaccharide SLP-2.

8. High-purity sugarcane leaf polysaccharide SLP-2 obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the high-purity sugarcane leaf polysaccharide SLP-2 according to claim 8 in the preparation of medicines or health foods for treating diabetic cardiomyopathy.

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