Raspberry leaf polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses raspberry leaf polysaccharide as well as a preparation method and application thereof. The average relative molecular weight of the raspberry leaf polysaccharide is 44755 Da, and the raspberry leaf polysaccharide is mainly prepared from mannose, glucose, rhamnose, glucosamine, galactosamine, glucuronic acid, galactose, xylose and arabinose according to the molar ratio of (3.138): (0.894): (12.349): (4.590): (26.498): (18.681): (22.842): (2.690): (8.317). Besides, methylation and nuclear magnetic resonance analysis show that the raspberry leaf polysaccharide is complex polysaccharide, and it can be reasonably deduced that the raspberry leaf polysaccharide is mainly composed of a large number of HG structural domain-like structures and a part of RG-I structures with side chains. Meanwhile, the raspberry leaf polysaccharide disclosed by the invention can be used for inhibiting the injury of kidney tissues and reducing the generation of kidney inflammatory factors.

Description

Technical Field

[0001] The present invention relates to the technical field of natural polymers, and specifically, to a preparation method and application of raspberry leaf polysaccharide with kidney injury protection. Background Art

[0002] Raspberry (Rubus ideaus L.) is a perennial small shrub deciduous fruit tree of the genus Rubus in the Rosaceae family. The roots, stems, and leaves of raspberry can all be used as medicine. There are detailed records about the medicinal value of raspberry in the earliest Chinese pharmacopoeias such as "Compendium of Materia Medica" and "Shennong's Herbal Classic". The tea made from raspberry leaves has a long history both at home and abroad. Currently, raspberry is used as the main raw material to make fruit juice, jam, fruit wine, etc. The content of active metabolites in raspberry leaves is rich, and it is a good source of various medicinal natural products. However, the current utilization rate of raspberry leaves is very low. Except for a few raspberry leaves being made into raspberry leaf tea, most raspberry leaves are treated as fertilizers or directly discarded after fruit picking. Therefore, it is necessary for us to conduct in-depth research and rational utilization of raspberry leaf resources, which helps to better utilize raspberry leaves and increase the added value of raspberry.

[0003] A large number of compounds with extensive activities have been found in raspberry, mainly polyphenols, flavonoids, and terpenoids, including ellagic acid, raspberry ketone, epicatechin, rutin, ferulic acid, chlorogenic acid, asiatic acid, and diterpenoids and triterpenoids. They have been proven to have pharmacological activities in improving muscle activity, anti-thrombosis, hypoglycemic, anti-inflammatory and antibacterial, antioxidant, anti-tumor, anti-obesity, liver protection, heart protection, and treating gastric ulcer and other diseases. Currently, the research on raspberry polysaccharide focuses on raspberry fruits, and there are few reports on raspberry leaves.

[0004] Chinese patent document CN104825387A discloses a raspberry fruit polysaccharide oral liquid, Chinese patent document CN104800174A discloses a raspberry fruit polysaccharide lozenge, CN104758272A discloses a raspberry fruit polysaccharide capsule and the applications of the above three in anti-cancer, reducing the toxic and side effects of chemotherapy drugs, hypoglycemic, and anti-fatigue effects. Chinese patent document CN104873532A discloses the application of raspberry polysaccharide in anti-tumor. Chinese patent document number CN101822731A discloses the application of a raspberry leaf extract in the preparation of anticoagulant and anti-thrombotic drugs. Currently, there is no report on the preparation method and application of a raspberry leaf polysaccharide with kidney injury protection. Summary of the Invention

[0005] The purpose of the present invention is to provide a raspberry leaf polysaccharide with kidney injury protection for the deficiencies in the prior art.

[0006] Another object of the present invention is to provide a method for preparing raspberry leaf polysaccharide with kidney injury protection.

[0007] Another object of the present invention is to provide the use of raspberry leaf polysaccharide with kidney injury protection.

[0008] The object of the present invention can be achieved by the following technical solutions:

[0009] A method for separating and extracting raspberry leaf polysaccharide, comprising the following steps:

[0010] (1) Crush the dried raspberry leaves, soak them in 95% ethanol, filter and dry, extract with hot dd H2O at 75°C for multiple times, and concentrate the combined extract to obtain a concentrated solution;

[0011] (2) Remove proteins from the concentrated solution by the Sevage method and decolorize with macroporous resin D101;

[0012] (3) Precipitate the decolorized solution with 75% and 95% ethanol in sequence, and freeze-dry to obtain crude polysaccharide;

[0013] (4) Dissolve the crude polysaccharide in distilled water, separate it with a DEAE-Sepharose fast flow column, elute with 200 ml of distilled water and 0.1 - 0.5 M sodium chloride solution in sequence, with a flow rate of 1 mL / min, collect the eluate with an automatic collector, combine the eluate containing polysaccharide components, concentrate under reduced pressure at 50 - 60°C, dialyze with a molecular weight cut-off value of 3500 Da for 48 h, and freeze-dry to obtain raspberry leaf polysaccharide.

[0014] As a preference of the present invention, in step (1), crush the dried raspberry leaves and pass through a 100-mesh sieve, soak the sieved raspberry leaf powder in 95% ethanol at 4°C for 10 - 12 hours, filter and dry on a tray at 70°C for 1.5 - 3 h, then extract with 45 - 55 times the volume of hot dd H2O at 75°C for 1 - 2 h. After two rounds of extraction, combine the supernatant and concentrate it to 1 / 4 of the volume using a rotary evaporator to obtain a concentrated solution.

[0015] As a preference of the present invention, in step (2), add Sevage reagent to the concentrated solution for multiple extractions. The Sevage reagent is chloroform: n-butanol = (2 - 4):1 (v / v), concentrate and remove the organic reagent under reduced pressure at 55 - 65°C and concentrate the solution, and then decolorize with macroporous resin D101.

[0016] As a preference of the present invention, in step (3), 75% ethanol is added to the decolorized solution, and the mixture is precipitated overnight at 4°C. The precipitate is collected by centrifugation, redissolved in distilled water, 95% ethanol is added, and the mixture is precipitated at 4°C. Centrifugation is carried out again to collect the precipitate. The operation is repeated multiple times, and the precipitate is freeze-dried to obtain freeze-dried crude polysaccharide.

[0017] As a further preference of the present invention, in step (4), the crude polysaccharide is dissolved in distilled water, and the mass-volume ratio of the crude polysaccharide to distilled water is 1 g:(9 - 12) ml. The sample loading volume is 5 ml. DEAE-Sepharose fast flow column separation is adopted, and the column is eluted successively with 200 ml of distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 M sodium chloride solutions at a flow rate of 1 mL / min. The eluate is collected by an automatic collector, 1 tube every 5 min, and tubes No. 1 to No. 60 are collected. After combination, the mixture is concentrated under reduced pressure at 55°C, dialyzed for 48 - 50 h, and freeze-dried to obtain raspberry leaf polysaccharide.

[0018] As a further preference of the present invention, the separation and extraction method comprises the following steps:

[0019] (1) Raspberry leaves are ground, crushed, and passed through a 100-mesh sieve to obtain raspberry leaf powder; 1 L of 95% (v / v) ethanol is added to every 300 g of raspberry leaf powder, and the mixture is stirred to make full contact. The mixture is refluxed at 70°C for 4 h, filtered through filter paper, and the residue is collected and dried at 50 ± 0.1°C for 24 h to obtain residue powder; the residue is extracted with 50 times the volume of hot dd H2O at 75°C for 2 h. After two rounds of extraction for 4 h each, the supernatant is combined and concentrated to 1 / 4 of the volume using a rotary evaporator to obtain a concentrated solution;

[0020] (2) 3 - 4 times the volume of Sevage reagent is added to the concentrated solution. The Sevage reagent is chloroform:n-butanol = 4:1 (v / v). The mixture is vigorously shaken for 30 - 45 min, centrifuged at 4000 rpm for 20 min, and the supernatant is taken. The operation is repeated 5 times. The organic reagent is removed by concentration under reduced pressure at 60°C and the solution is concentrated. Subsequently, the solution is decolorized with macroporous resin D101 at a resin:solution ratio of 1:2 (v / v) for 2 h;

[0021] (3) 3 times the volume of 75% ethanol is added to the decolorized solution, and the mixture is precipitated at 4°C for 12 h. Centrifugation is carried out at 4000 rpm at room temperature for 20 min, and the precipitate is collected. The precipitate is redissolved in distilled water, 3 volumes of 95% ethanol are added, and the mixture is precipitated at 4°C for 48 h. Centrifugation is carried out under the same conditions again to collect the precipitate. The operation is repeated three times, and the precipitate is freeze-dried to obtain freeze-dried crude polysaccharide;

[0022] Step (4): Dissolve the crude polysaccharide in distilled water at a mass-volume ratio of 1 g:10 ml. The sample loading volume is 5 ml. Separate it using a DEAE-Sepharose fast flow column, and elute it successively with 200 ml of distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 M sodium chloride solutions at a flow rate of 1 mL / min. Collect the eluate with an automatic collector, 1 tube every 5 min, collect tubes No. 1 to No. 60, concentrate it under reduced pressure at 55 °C after merging, dialyze for 48 h, with the molecular weight cut-off value for dialysis = 3500 Da, and freeze-dry to obtain raspberry leaf polysaccharide.

[0023] The raspberry polysaccharide prepared according to the described separation and extraction method.

[0024] As a preference of the present invention, the average relative molecular weight of the raspberry polysaccharide is 44755 Da, and it is mainly composed of mannose, glucose, rhamnose, glucosamine, galactosamine, glucuronic acid, galactose, xylose, and arabinose with a molar ratio of 3.138:0.894:12.349:4.590:26.498:18.681:22.842:2.690:8.317. Its structure is mainly composed of a large number of HG domain-like structures and some RG-I structures with side chains.

[0025] The application of the raspberry leaf polysaccharide described in the present invention in the preparation of a kidney protection preparation.

[0026] As a preference of the present invention, the kidney protection preparation inhibits the damage of kidney tissues.

[0027] As a preference of the present invention, the kidney protection preparation reduces the production of kidney inflammatory factors.

[0028] The application of the raspberry leaf polysaccharide described in the present invention in the preparation of a chemotherapy adjuvant drug.

[0029] The advantages of the present invention are as follows:

[0030] 1. The present invention first isolates a polysaccharide from raspberry leaves, measures its average relative molecular weight respectively, analyzes its monosaccharide composition, and determines its specific active uses.

[0031] 2. Raspberry leaf polysaccharide, as determined by GPC, has an average relative molecular weight of 44755 Da. Through comprehensive analysis of the results of GC-MS, methylation, and NMR, raspberry polysaccharide is a complex polysaccharide mainly composed of mannose, glucose, rhamnose, glucosamine, galactosamine, glucuronic acid, galactose, xylose, and arabinose, with a molar ratio of 3.138:0.894:12.349:4.590:26.498:18.681:22.842:2.690:8.317. Its structure is mainly composed of a large number of HG domain-like structures and some RG-I structures with side chains.

[0032] 3. Raspberry leaf polysaccharide can significantly inhibit kidney tissue damage and reduce the production of kidney inflammatory factors. The results of HE staining showed that the cyst wall structure of mice in the normal group was intact, and the renal tubules and glomeruli had regular morphology; in the model group, there were obvious inflammatory cell infiltrations, the cyst wall structure was blurred, some renal tubules were edematous and necrotic, and the glomeruli had irregular morphology; in the low, medium, and high-dose groups of raspberry leaf polysaccharide and the positive control group, the inflammatory cell infiltrations were reduced, the cyst wall structure was relatively intact, some renal tubules had mild edema and necrosis, and the glomeruli had relatively regular morphology. This indicates that raspberry leaf polysaccharide can, to a certain extent, alleviate the pathological changes of kidney injury and reduce the production of kidney inflammatory factors. Description of the Drawings

[0033] Figure 1 : Flow chart for the extraction of raspberry leaf polysaccharide.

[0034] Figure 2 : Elution curve of raspberry leaf polysaccharide on a DEAE-Sepharose fast flow column.

[0035] Figure 3 : Elution curve of raspberry leaf polysaccharide on a gel filtration chromatography column.

[0036] Figure 4 : Infrared spectrogram of raspberry leaf polysaccharide.

[0037] Figure 5 : Ion chromatograms of standard monosaccharides and raspberry leaf monosaccharide components.

[0038] Figure 6 : Scanning diagram of the total ion current chromatogram of RLP-1 under electron impact ionization.

[0039] Figure 7 : 1H NMR spectrum of RLP-1 in D2O solution.

[0040] Figure 8 : 13C NMR spectrum of RLP-1 in D2O solution.

[0041] Figure 9 : DEPT diagram of RLP-1 in D2O solution.

[0042] Figure 10 : HSQC spectrum of RLP-1 in D2O solution.

[0043] Figure 11 : COSY spectrum of RLP-1 in D2O solution.

[0044] Figure 12 : HMBC spectrum of RLP-1 in D2O solution.

[0045] Figure 13 : Structural composition of RLP-1.

[0046] Figure 14 : Pathological section of mouse kidney tissue (H&E staining). Detailed implementation mode

[0047] The following combines examples to make a detailed description of the specific implementation mode provided by the present invention.

[0048] Example 1 Preparation of raspberry leaf polysaccharide

[0049] Collect dry raspberry (Rubus idaeus L.) leaves, grind and crush the dried raspberry leaves, and pass through a 100-mesh sieve after grinding. Soak the powder in absolute ethanol at 4°C for 12 hours. After filtration, dry the tray at 70°C for 2h, and then extract with 50 times the volume of hot dd H2O at 70°C for 2h. After two rounds of extraction for 4h, combine the supernatant and concentrate it to 1 / 4 of the volume using a rotary evaporator.

[0050] Add 3 times the volume of Sevage reagent to the concentrated solution. The Sevage reagent is chloroform: n-butanol = 4:1 (v / v). Vigorously shake for 30 min, centrifuge at 4000 rpm for 20 min, take the supernatant, repeat the operation 5 times, concentrate and remove the organic reagent under reduced pressure at 60°C and concentrate the solution, and then decolorize with macroporous resin D101 at a ratio of resin: solution of 1:2 (v / v) for 2 hours.

[0051] Add 3 times the volume of 75% ethanol to the decolorized solution, precipitate overnight (12h) at 4°C, centrifuge (4000 rpm, 20 min, room temperature) to collect the precipitate, redissolve the precipitate in distilled water, add 3 times the volume of 95% ethanol and precipitate at 4°C for 48h, centrifuge again (4000 rpm, 20 min, 4°C) to collect the precipitate, repeat the operation three times, and lyophilize the precipitate to obtain freeze-dried crude polysaccharide. Using glucose as the standard, the polysaccharide content was determined by the phenol-sulfuric acid method. Using BSA as the standard, the protein content was determined by the Lowry method.

[0052] 1 g of crude polysaccharide was dissolved in 10 ml of distilled water, with a sample loading volume of 5 ml. It was separated using a DEAE-Sepharose fast flow column and eluted successively with 200 ml of distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 M sodium chloride solutions at a flow rate of 1 mL / min. 5 mL of the eluate was collected using an automatic collector (1 tube every 5 min), detected by the phenol-sulfuric acid method, and tubes 1 to 60 were collected. After merging, it was concentrated under reduced pressure at 55 °C, dialyzed (molecular weight cut-off = 3500 Da) for 48 h, and freeze-dried to obtain raspberry leaf polysaccharide.

[0053] Example 2 Characterization of Raspberry Leaf Polysaccharide

[0054] I) Determination of the average relative molecular weight of polysaccharide

[0055] The relative molecular weight was determined using a Waters 1515 Infinity size exclusion chromatography and gel permeation chromatography system equipped with a Waters 2410 refractive index detector (Waters, CA, USA) and a gel filtration chromatography column (OHpak SB-803HQ, Shodex, Tokyo, Japan). Calibration was performed using dextran standards (1, 5, 12, 25, 50, 80, 150, 270, 410, and 670 kDa, Sigma-Aldrich). Elution was carried out with 0.05 M ammonium acetate solution at a rate of 0.65 mL / min and maintained at 40 ± 1 °C.

[0056] Table 1 Results of GPC determination of raspberry leaf polysaccharide

[0057] Name RT (min) Mp (Da) Mw (Da) Mn (Da) Distribution coefficient (Mw / Mn) Raspberry leaf polysaccharide 36.650 35621 44755 29674 66.30

[0058] II) Monosaccharide composition analysis of polysaccharide

[0059] 1. Hydrolysis of polysaccharide into monosaccharides: RLP-1 (5.3 mg) was hydrolyzed with 1 mL of 2 M trifluoroacetic acid (TFA) at 121 °C for 2 h to obtain the complete hydrolysis product of RLP-1. Then, the hydrolysis product of RLP-1 was co-concentrated with absolute methanol three times to completely remove the residual TFA.

[0060] 2. Derivatization of Hydrolyzed Monosaccharides: The dried RLP-1 hydrolysis product was dissolved in 5 mL of distilled water and reacted with pmp-methanol solution (0.5 M) and sodium hydroxide solution (NaOH, 0.2 M) at 70 °C for 1 h. The reaction product was neutralized with hydrochloric acid solution (HCl, 0.5 M), and then extracted with chloroform three times to remove the extra PMP, obtaining the PMP derivative of RLP-1 (aqueous phase). Monosaccharide standards including mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, glucosamine hydrochloride, galactosamine hydrochloride, and caramel were prepared into a 0.5 mg / mL solution and derivatized with PMP by the method described above.

[0061] 3. Subsequently, the PMP derivative of FOP80-1 was further analyzed using a high-performance liquid chromatography system (U3000, Thermo Fisher, USA) equipped with a ZORBAX Eclipse XDB-C18 chromatographic column (2.1 x 50 mm, 1.8 μm, Agilent Technologies, USA). The mobile phase was acetonitrile-phosphate buffer (17:83, v / v, pH = 6.8), and the flow rate was 0.8 mL / min. The detection wavelength was set at 250 nm.

[0062] (III) Infrared Spectroscopy Analysis

[0063] The dry RLP-1 powder was pressed into a thin film for analysis by a tablet press (Zou et al., 2017). The spectrum of RLP-1 was measured using FT-IR (Nicolet 6700, USA). As Figure 4 shown, the FTIR spectrum was used for the characterization of RLP-1, showing peaks of polysaccharides. There was a strong and broad absorption peak at 3400.58 cm -1 , which was a strong absorption peak of the stretching vibration of O-H in intermolecular or intramolecular hydrogen bonds of polysaccharide molecules. The medium-intensity peak near 2918.51 cm -1 was the absorption peak of the C-H stretching vibration of methyl (-CH3) and propionyl (-CH2); the peak at 1735.88 cm -1 was the stretching vibration of C=O in the sugar acid structure; the peak at 1417.58 cm -1 was the deformation vibration of -OH; the peak at 1237.83 cm -1 was the out-of-plane bending vibration of C-H, which together with the stretching vibration of C-H constituted the characteristic absorption of the sugar ring; in the range of 1150 - 1010 cm -1 , 1099.62 cm -1 , 1076.93 cm -1 , 1024.75 cm -1The strong absorption peaks that appear further prove the existence of pyranosides. These three absorptions are the bending vibrations of C-O-h or C-O-c in the C-O bond structure; the characteristic region at 890.28 cm -1 is considered to be the β-pyranoside bond in polysaccharides; at 760.34 cm -1 a symmetric ring stretching vibration peak is shown.

[0064] (IV) Methylation analysis

[0065] RLP-1 was methylated according to the Ciucanu & Kerek method (Ciucanu & Kerek, 1984). An HP-5MS fused silica capillary chromatographic column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA) was used, and gas chromatography-mass spectrometry (Agilent 5977B; Agilent Technologies, USA) was performed. The detailed temperature program was as follows: the initial oven temperature was 50 °C, held for 1 min, and then heated at a rate of 3 °C / min to 230 °C and held for 2 min. The split injector temperature was 260 °C, helium was used as the carrier gas, and the flow rate was 1.0 mL / min. As Figure 5 are the HPLC chromatograms of monosaccharide standards and RLP-1. Peak 1 = Man, 2 = Rib, 3 = Rha, 4 = GlcA, 5 = GalA, 6 = Glc, 7 = Gal, 8 = Xyl, and 9 = Ara. Figure 6 The total ion chromatogram shows the peak profiles of the methylation products. By comparing the retention times and spectra of these products with the spectral database of the Complex Carbohydrate Research Center, we determined the sugar residues present in RLP-1. Gas chromatography-mass spectrometry analysis showed that there were 12 sugar residues in RLP-1, namely t-Ara (6.067%), t-Xyl (12.053%), 2-rha (3.633%), 4-xyl (3.323%), T-Glcp (1.887%), t-Gal (16.795%), 2,4-rha (3.137%), 4-gal (40.269%), 4-glc (3.795%), 6-Gal (2.598%), 2,4-man (2.631%), 4,6-gal (3.811%).

[0066] Table 2 GC-MS analysis of methylation of raspberry leaf polysaccharide

[0067]

[0068]

[0069] Nuclear magnetic resonance (NMR) analysis

[0070] RLP-1 was dissolved in D2O for NMR analysis. 1D-NMR (1H NMR and 13C NMR) and 2D-NMR (HSQC, HMBC, 1H-1H COSY and 1H-1H NOESY) spectra were recorded using an NMR instrument (Bruker AVANCE HD III 600 MHz Spectrometer, Germany). The resulting spectra are as Figures 7 - 11 shown, Figure 7 , 8 providing complete hydrogen and carbon chemical shift data for all major sugar residues, which helps to clarify the linkages between different sugar units. In the results of monosaccharide composition detection and methylation analysis, the major monosaccharides identified were GalA, Gal, Ara, and Rha. Methylation analysis showed the presence of 1,4-galpa, 1,3,6-galp, 1,3-galp, 1,6-galp, T-Araf, 1,3-araf, 1,2,4-rhap; 1,2-Rhap; and 1-4-Glcp; as well as T-GalpA. We speculated that RLP-1 might contain a large number of hg-type domains and some rg-i type pectin domains. Based on the results of the monosaccharide composition and methylation analysis of RLP-1, and the analysis using NMR techniques such as 1H-NMR, 13C-NMR HSQC, and 1H-1H COSY, we observed the presence of multiple isotopic signals. Correlating these observations with the existing literature [5-7]; the prominent signals around δ 98-104 ppm mainly originated from the carbon signals of α-GalpA sugar residues. In addition, a strong signal around δ 104-108 ppm might be related to the β-Galp carbon signal, while another signal around δ 106-113 ppm might mainly be related to the α-Araf carbon signal. Through comprehensive analysis including monosaccharide composition assessment, methylation study, and nuclear magnetic resonance assessment, we found that this polysaccharide sample showed many different isotopic signal peaks, which were valuable for structure elucidation: δ 4.81 / 103.29 ppm, δ 4.89 / 102.88 ppm, δ 4.97 / 101.88 ppm, δ 4.49 / 106.41 ppm, δ 4.45 / 107.11 ppm, δ 4.35 / 105.26 ppm, δ 4.30 / 106.00 ppm, δ 5.09 / 112.40 ppm, δ 5.02 / 109.89 ppm, δ 5.09 / 101.89 ppm, δ 5.09 / 101.89 ppm, δ 4.35 / 105.26 ppm, δ 4.35 / 105.26 ppm.

[0071] By correlating the heteronuclear multiple-bond protons observed in the distal HMBC spectrum with the carbon atoms of each sugar residue or with the hydrogen atoms of each sugar residue, and considering that two adjacent protons at the linkage site between adjacent sugar residues are likely to generate strong NOE signals due to spatial proximity, we can further deduce the linkage order between sugar residues using the HMBC long-range correlation spectrum and the NOESY spectrum. The HMBC correlation spectrum and the NOESY spectrum of the RLP-1 sample are illustrated respectively in Figure 12and in the figures. Several coupling signals can be identified from these figures: (1) The cross peaks (ge1,4h-1 / ge1,4h-4) in the NOESY spectrum represent the a→4)-α-d-galpa-6-ome-(1→4)-α-d-galpa-6-ome-(1→ linkage; (2) The cross peaks (ga1,4h-1 / ge1,4h-4) in the NOESY spectrum are for the a→4)-α-d-galpa-6-(1→4)-α-d-galpa-6-ome-(1→ linkage; (3) In the NOESY spectrum, the H-1 (δ4.81 ppm) peak of the sugar residue GE1,4 and the H-3 (δ3.86 ppm) peak of the sugar residue GE1,3,4 show a cross peak (GE1,4h-1 / GE1,3,4H-3), indicating the presence of the a→4)-α-d-galpa-6-ome-(1→3,4)-α-d-galpa-6-ome-(1→ bond, and the bonding site is at the O-3 position; (4) The NOESY spectrum shows that the H-1 of At (δ5.09 ppm) overlaps with the H-3 of GE1,3,4 (δ3.86 ppm), indicating the presence of the α–L–Araf-(1→3,4)-α-d-galpa-6-ome-(1→ linkage, and the bonding point is at the O-3 position; (5) The HMBC spectrum shows that the C-1 (δ101.89 ppm) of the sugar residue Rha1,2,4 overlaps with the H-4 (δ4.30 ppm) of the sugar residue GE1,4, and the NOESY spectrum shows that the H-1 (δ4.81 ppm) of the sugar residue GE1,4 overlaps with the H-2 (δ4.05 ppm) of the sugar residue Rha1,2,4, indicating the presence of the →4)-α-d-galpa-6-ome-(1→2,4)-α-l-rhap-(1→ linkage, and the bonding point is at the O-2 position; (6) The NOESY spectrum shows that the H-1 (δ4.45 ppm) of the sugar residue G1,3 overlaps with the H-3 (δ3.57 ppm) of the sugar residue G1,3,6, indicating the presence of the →3-β-d-galp-(1→ linkage, where the bonding point is at the O-3 position; (7) The NOESY spectrum shows that the H-1 (δ4.30 ppm) of the sugar residue G1,6 overlaps with the H-6 (δ3.92 ppm) of the sugar residue G1,3,6, indicating the presence of the →6)-β-d-galp-(1→3,6)-β-d-galp-(1→ linkage, where the bonding point is at the O-6 position; (8) In the NOESY spectrum, there are cross peaks (G1,3,6H-1,2,4H-4 at δ4.35 ppm and δ3. between G1,3,6H-1 / Rha1,2,4H-4 and H-1 (δ4.35 ppm) and Rha1,2,4 and H-4 (δ3.58 ppm).At 58 ppm, it indicates the presence of an a→3,6)-β-d-galp-(1→2,4)-α-l-rhap-(1→ bond, and the bonding site is at the O-4 position; (9) The HMBC spectrum shows a cross-peak (Glc1,4 C-1 / Glc1,4 H-4) between C-1 (δ 105.26 ppm) and H-4 (δ 3.51 ppm) of Glc1,4, indicating the presence of a (1→4)-β-d-glcp-(1→4)-β-d-glcp-(1→) bond.

[0072] Table 3 Chemical shift assignments of 1H and 13C of each sugar residue in the polysaccharide sample ("--" indicates undetermined or undetected)

[0073]

[0074]

[0075] Combined with the analysis of monosaccharide composition, methylation results, one-dimensional and two-dimensional nuclear magnetic resonance information, it can be inferred that the polysaccharide sample is a complex polysaccharide, and it can be reasonably inferred that it is mainly composed of a large number of HG domain-like structures and some RG-I structures with side chains, as Figure 13 shown.

[0076] Example 3 Uses of Raspberry Leaf Polysaccharide

[0077] Materials and reagents: Raspberry polysaccharide (prepared in Example 1), hematoxylin & eosin staining kit, 4% paraformaldehyde, gradient ethanol, xylene.

[0078] Instruments: Paraffin embedding machine (Leica HistoCore Arcadia), paraffin slicer (Leica 2255), optical microscope.

[0079] I. Protective effect of raspberry leaf polysaccharide on the kidney and reduction of the production of kidney inflammatory factors

[0080] Sixty C57BL / 6 mice were adaptively fed for 1 week and then grouped. Among them, CK mice were the normal group, and DDP mice (cisplatin-induced renal injury model group: single injection of 20 mg / kg (body weight)) were divided into the model group, low, medium, and high-dose groups of raspberry leaf polysaccharide, and the positive control group, with 10 mice in each group. According to the results of the preliminary experiment, the intervention doses of the low, medium, and high-dose groups of raspberry leaf polysaccharide were set as: 50 mg / kg (body weight) / day, 100 mg / kg (body weight) / day, and 200 mg / kg (body weight) / day respectively; positive control group: (Jinkui Shenqi Pills 100 mg / kg (body weight) / day). The drug intervention lasted for 12 days, once a day. Mice in the normal group and the model group were gavaged with an equal amount of distilled water. After the intervention, the mice were dissected, the kidney tissues were separated, the blood on the surface of the kidney tissues was rinsed clean with normal saline, the capsule was removed, and the kidney tissues were fixed in 4% paraformaldehyde. The kidney tissues fixed in paraformaldehyde were taken out, paraffin sections were prepared, and the pathological morphology of the kidney tissues was observed by HE staining. The results are as Figure 14 shown. Raspberry leaf polysaccharide can significantly improve the pathological changes of the kidney under high-concentration conditions, including a more complete cyst wall structure, mild necrosis and edema of the renal tubules, a relatively regular glomerular morphology, and a reduction in the infiltration of inflammatory cells. It shows that raspberry leaf polysaccharide can, to a certain extent, alleviate the pathological changes of the kidney and reduce the production of renal inflammatory factors.

[0081] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for separating and extracting raspberry leaf polysaccharide, characterized in that, It includes the following steps: (1) Crush the dry raspberry leaves, soak them in 95% ethanol, filter and dry, extract with hot dd H2O at 75°C for multiple times, and concentrate the combined extract to obtain a concentrated solution; (2) Remove proteins from the concentrated solution by the Sevage method and decolorize with macroporous resin D101; (3) Precipitate the decolorized solution successively with 75% and 95% ethanol, and freeze-dry to obtain crude polysaccharide; (4) Dissolve the crude polysaccharide in distilled water, separate it using a DEAE-Sepharose fast flow column, elute successively with 200 ml of distilled water and 0.1 - 0.5 M sodium chloride solution at a flow rate of 1 mL / min, collect the eluate with an automatic collector, combine the eluate containing polysaccharide components, concentrate under reduced pressure at 50 - 60°C, dialyze with a molecular weight cut-off value of 3500 Da for 48 h, and freeze-dry to obtain raspberry leaf polysaccharide.

2. The separation and extraction method according to claim 1, wherein (1) Crush and sieve the dry raspberry leaves through a 100-mesh sieve, soak the sieved raspberry leaf powder in 95% ethanol at 4°C for 10 - 12 hours, filter and dry on a tray at 70°C for 1.5 - 3 h, then extract with 45 - 55 times the volume of hot dd H2O at 75°C for 1 - 2 h. After two rounds of extraction, combine the supernatant and concentrate it to 1 / 4 of the volume using a rotary evaporator to obtain a concentrated solution.

3. The separation and extraction method according to claim 1, wherein (2) Add Sevage reagent to the concentrated solution for multiple extractions. The Sevage reagent is chloroform:n-butanol = (2 - 4):1 (v / v). Concentrate under reduced pressure at 55 - 65°C to remove the organic reagent and concentrate the solution, and then decolorize with macroporous resin D101.

4. The separation and extraction method according to claim 1, wherein (3) Add 75% ethanol to the decolorized solution, precipitate overnight at 4°C, centrifuge to collect the precipitate, redissolve the precipitate in distilled water, add 95% ethanol and precipitate at 4°C, centrifuge again to collect the precipitate, repeat the operation multiple times, and freeze-dry the precipitate to obtain freeze-dried crude polysaccharide.

5. The separation and extraction method according to claim 1, characterized in that, (4) Dissolve the crude polysaccharide in distilled water, with a mass-volume ratio of crude polysaccharide to distilled water of 1 g:(9 - 12) ml, a sample loading volume of 5 ml, separate it using a DEAE-Sepharose fast flow column, elute successively with 200 ml of distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 M sodium chloride solution at a flow rate of 1 mL / min, collect the eluate with an automatic collector, collect every 5 min per tube, collect tubes 1 - 60, combine and concentrate under reduced pressure at 55°C, dialyze for 48 - 50 h, and freeze-dry to obtain raspberry leaf polysaccharide.

6. The separation and extraction method according to any one of claims 1-5, characterized in that, (1) Grind and crush the raspberry leaves to obtain raspberry leaf powder by passing through a 100-mesh sieve; add 1 L of 95% (v / v) ethanol to every 300 g of raspberry leaf powder, stir to make them fully contact, reflux at 70°C for 4 h, filter through filter paper, collect the residue, dry the residue at 50 ± 0.1°C for 24 h to obtain residue powder; extract with 50 times the volume of hot dd H2O of the residue powder at 75°C for 2 h. After two rounds of 4-h extractions, combine the supernatant and concentrate it to 1 / 4 of the volume using a rotary evaporator to obtain a concentrated solution. ​ (2) Add 3 - 4 volumes of Sevage reagent to the concentrated solution. The Sevage reagent is chloroform:n - butanol = 4:1 (v / v). Vigorously shake for 30 - 45 min, centrifuge at 4000 rpm for 20 min, take the supernatant, repeat the operation 5 times, concentrate under reduced pressure at 60 °C to remove the organic reagent and concentrate the solution. Subsequently, decolorize with macroporous resin D101 at a resin:solution ratio of 1:2 (v / v) for 2 hours; (3) Add 3 volumes of 75% ethanol to the decolorized solution, precipitate at 4 °C for 12 h, centrifuge at 4000 rpm at room temperature for 20 min, collect the precipitate, redissolve the precipitate in distilled water, add 3 volumes of 95% ethanol and precipitate at 4 °C for 48 h, centrifuge under the same conditions again, collect the precipitate, repeat the operation three times, and lyophilize the precipitate to obtain freeze - dried crude polysaccharide; (4) Take the crude polysaccharide and dissolve it in distilled water. The mass - volume ratio of the crude polysaccharide to distilled water is 1 g:10 ml, the sample loading volume is 5 ml. Separate it using a DEAE - Sepharose fast flow column, elute successively with 200 ml of distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 M sodium chloride solutions at a flow rate of 1 mL / min. Collect the eluate with an automatic collector, 1 tube every 5 min, collect tubes 1 - 60, concentrate under reduced pressure at 55 °C after merging, dialyze for 48 h, the molecular weight cut - off value of dialysis = 3500 Da, and freeze - dry to obtain raspberry leaf polysaccharide.

7. Raspberry polysaccharide prepared by the separation and extraction method according to any one of claims 1 - 5.

8. The raspberry leaf polysaccharide according to claim 7, wherein The average relative molecular weight of the raspberry polysaccharide is 44755 Da, and it is mainly composed of mannose, glucose, rhamnose, glucosamine, galactosamine, glucuronic acid, galactose, xylose, and arabinose with a molar ratio of 3.138:0.894:12.349:4.590:26.498:18.681:22.842:2.690:8.

317. Its structure is mainly composed of a large number of HG - domain - like structures and some RG - I structures with side chains.

9. Use of the raspberry leaf polysaccharide according to claim 7 in the preparation of a kidney protection preparation.

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