Divaricate saposhnikovia root polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses saposhnicovia divaricata polysaccharide as well as a preparation method and application thereof, and belongs to the field of natural polymers. The method has the advantages that the average relative molecular weight is measured through GPC, monosaccharide composition is analyzed through GC-MS, it is found that the average relative molecular weight of the saposhnicovia divaricata polysaccharide RSP-1 is 111125 Da, the protein content of the saposhnicovia divaricata polysaccharide RSP-1 is 1.29%, the saposhnicovia divaricata polysaccharide RSP-1 does not contain uronic acid and is neutral polysaccharide, and it is found that trace beta-pyranose exists. Meanwhile, the saposhnicovia divaricata polysaccharide RSP-1 disclosed by the invention can be used for remarkably relieving liver fat infiltration, reducing the number of ballooning and necrotic cells and relieving alcoholic liver injury.

Description

Technical Field

[0001] The invention relates to the technical field of natural polymers, in particular to a preparation method and application of fangfeng polysaccharide capable of protecting alcoholic liver damage. Background Art

[0002] Saposhnikovia divaricata (Turcz.) Schischkin is a perennial herbaceous plant in the genus Saposhnikovia, family Apiaceae. It prefers cool climates and is cold- and drought-tolerant. It is primarily grown in Hebei, Heilongjiang, Sichuan, and Inner Mongolia Autonomous Region. The root, used raw, is pungent, sweet, and slightly warm. It has the effects of dispelling wind and relieving exterior symptoms, relieving dampness and relieving pain, and relieving spasms. It is widely used in Traditional Chinese Medicine and was first listed as a top-grade herb in the Shennong's Herbal Classic. Saposhnikovia root also has a long history of folk use. Currently, it is primarily used as a traditional Chinese medicine for treating colds, rheumatic pain, and tetanus. It contains a rich variety of chemical compounds and is a good source of various medicinal natural products. However, its current utilization is relatively low. Aside from its use as a traditional Chinese medicine, most of the root is used as fertilizer or simply discarded after harvesting. Therefore, it is necessary for us to conduct in-depth research on the windproof root resources and make rational use of them, which will help to better utilize the windproof root and increase its added value.

[0003] Numerous compounds with broad activity have been discovered in the root of Saposhnikovia divaricata, primarily polysaccharides, coumarins, chromones, and volatile oils. These compounds have been shown to possess pharmacological properties in areas such as dispelling wind and relieving exterior symptoms, providing anti-inflammatory and analgesic effects, regulating immunity, combating tumors, reducing fever, and developing anti-aging and natural antimicrobial agents. Saposhnikovia divaricata polysaccharides are a key active component of the root, including saponikovan A, B, C, XC-1, and XC-2, with demonstrated pharmacological activities including immunomodulatory, anti-tumor, and antioxidant properties. Currently, research on saposhnikovia divaricata polysaccharides is primarily focused on the root, while research on polysaccharides from other parts of the root is still in its infancy.

[0004] Chinese patent document CN110117333B discloses a method for preparing a fangfeng polysaccharide and its use in preparing a drug for treating diabetes or hyperlipidemia. Chinese patent document CN112745398A discloses a method for preparing a fangfeng polysaccharide and its use in improving the yield of crude fangfeng polysaccharide. Chinese patent document CN103249231A discloses a method for preparing a fangfeng polysaccharide and its use in preparing a liposome immunopotentiator. Chinese patent document CN110144016A discloses a method for preparing a fangfeng polysaccharide and optimizing the extraction technology to expand its scope of application. Chinese patent document CN101601705A discloses a method for preparing a fangfeng polysaccharide. Chinese patent document CN1065751C discloses a method for preparing a fangfeng polysaccharide. Currently, there are no relevant patent reports on the use of fangfeng polysaccharide in reducing the toxic and side effects of chemotherapy drugs, lowering blood sugar, anti-fatigue, anti-coagulation, anti-thrombosis, and protecting kidney damage. Summary of the Invention

[0005] The purpose of the present invention is to provide a fangfeng polysaccharide capable of protecting alcoholic liver damage in view of the deficiencies in the prior art.

[0006] Another object of the present invention is to provide a method for preparing fangfeng polysaccharide capable of protecting alcoholic liver damage.

[0007] Another object of the present invention is to provide a use of fangfeng polysaccharide for protecting alcoholic liver damage.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A method for separating and extracting fangfeng polysaccharide comprises the following steps:

[0010] (1) Grinding, crushing, and sieving the dried root of Radix Saposhnikoviae var. elegans, and adding deionized water to obtain a Radix Saposhnikoviae var. elegans extract;

[0011] (2) Sevage reagent was added to the extract of Saposhnikovia divaricata to remove protein and the solution was concentrated, and then the concentrate was decolorized with macroporous resin D101;

[0012] (3) adding 85% ethanol to the decolorized solution to precipitate the polysaccharide, and collecting the precipitate by centrifugation; dissolving the precipitate in distilled water, and freeze-drying to obtain crude polysaccharide;

[0013] (4) The crude polysaccharide was purified and separated by macroporous resin HP-20 chromatography column, and the eluate containing the highest polysaccharide content was selected, combined, concentrated, dialyzed, and freeze-dried to obtain the fangfeng polysaccharide;

[0014] (5) The fangfeng polysaccharide was prepared into a solution, separated by Sephadex G-200 column, eluted with 0.1 mol / L NaCl solution, and the eluate was concentrated, dialyzed, and freeze-dried to obtain fangfeng polysaccharide RSP-1.

[0015] As a preferred embodiment of the present invention, in step (1), the dried radix fangfeng (Saposhnikovia divaricata) root is crushed and passed through a 60-mesh sieve, and then a certain proportion of deionized water is added to the radix fangfeng (Saposhnikovia divaricata) root powder, the mixture is placed in an extraction container, and the mixture is heated and extracted at 100° C. for 2-3 hours. After the extraction is completed, the mixture is filtered while hot and the extract is collected. The extraction can be repeated 2-3 times, and the extracts are combined;

[0016] As a further preferred embodiment of the present invention, in step (1), the dried radix fangfeng (Saposhnikovia divaricata) root is crushed and passed through a 60-mesh sieve, the sieved radix fangfeng (Saposhnikovia divaricata) root powder is soaked in 95% ethanol at 4°C for 10-12h, filtered and dried on a tray at 70°C for 1.5-3h, and then extracted with deionized water at a material-liquid ratio of 1:10 at 100°C for 2h. After the extraction is completed, the extract is filtered while hot and collected, and the extraction is repeated 2-3 times. The extracts are combined and concentrated to 1 / 4 of the volume using a rotary evaporator to obtain a concentrated solution.

[0017] As a preferred embodiment of the present invention, in step (2), 3 volumes of Sevage reagent are added to the extract obtained in step (1), wherein the Sevage reagent is chloroform: n-butanol = 3:1, the mixture is vigorously shaken for 30 minutes, centrifuged at 4000 rpm for 20 minutes, the supernatant is taken, and the operation is repeated 5 times; then, the organic reagent is removed by concentration under reduced pressure at 60°C and the solution is concentrated, and then the concentrate is decolorized with macroporous resin D101.

[0018] As a further preferred embodiment of the present invention, in step (2), 3 volumes of Sevage reagent are added to the concentrated solution obtained in step (1) for multiple extractions, wherein the Sevage reagent is chloroform: n-butanol = 3:1, and the organic reagent is removed by concentration under reduced pressure at 55-65°C and the solution is concentrated, followed by precipitation treatment; decolorization is performed using macroporous resin D101 at 35°C with a resin:solution ratio of 1:2 (v / v) for 2 hours.

[0019] As a preferred embodiment of the present invention, in step (3), 3 volumes of 85% ethanol are added to the decolorized concentrate, the solution is allowed to stand at 4°C for 12-24 hours to precipitate polysaccharides, and the precipitate is collected by centrifugation at 10,000 rpm, 15 minutes, and 4°C; the precipitate is dissolved in distilled water, and freeze-dried to obtain crude polysaccharides.

[0020] As a further preferred embodiment of the present invention, 3 volumes of 85% ethanol are added to the decolorized solution in step (3), precipitated overnight at 4°C, and the precipitate is collected by centrifugation at 10,000 rpm for 15 min at 4°C. The precipitate is redissolved in distilled water, 85% ethanol is added for precipitation at 4°C, and the precipitate is collected by centrifugation again. This is repeated several times, and the precipitate is freeze-dried to obtain freeze-dried crude polysaccharide.

[0021] As a preferred embodiment of the present invention, in step (4), 0.2 g of crude polysaccharide is prepared into a solution with a concentration of 5 mg / mL, the sample volume is 2 mL, and the solution is purified and separated by a macroporous resin HP-20 chromatography column, and eluted with 0, 0.05, 0.1, 0.3, 0.4, 0.5, and 0.6 mol / L NaCl solutions in sequence, and collected by an automatic fraction collector at a flow rate of 1 mL / min, and collected every 5 mL until there is no obvious polysaccharide content in the eluate; the polysaccharide content in the eluate is detected by an ultraviolet spectrophotometer at a wavelength of 280 nm, and the component containing a higher polysaccharide content is selected for further purification; the eluates of the collected components are combined and concentrated, dialyzed, and freeze-dried to obtain polysaccharides.

[0022] As a preferred embodiment of the present invention, in step (5), the polysaccharide is configured into a solution with a concentration of 100 mg / mL, the sample volume is 2 mL, separated by a Sephadex G-200 column, eluted with a 0.1 mol / L Nacl solution, and collected by an automatic fraction collector at a flow rate of 1 mL / min, 4 min per tube; the phenol-sulfuric acid method is used for detection, and tubes 9-14 are collected. The collected eluates are combined and concentrated, dialyzed, and freeze-dried to obtain the fangfeng polysaccharide RSP-1.

[0023] As a preferred embodiment of the present invention, the separation and extraction method comprises the following steps:

[0024] (1) The root of Radix Saposhnikoviae was crushed by a crushing machine and then passed through a 60-mesh sieve to obtain the root powder;

[0025] (2) adding water to the radix fangfengensis powder, extracting the radix fangfengensis powder at 100°C for 2 h in a solid-liquid ratio of 1:10 to obtain a water extract, and concentrating under reduced pressure at 60°C to obtain a concentrate;

[0026] (3) The aqueous extract of the root of Chinese prickly ash powder was deproteinized by the Sevage method, decolorized with macroporous resin D101 at 35°C for 2 h at a resin:solution ratio of 1:2 (v / v), precipitated with 85% ethanol, and finally freeze-dried to obtain crude polysaccharide;

[0027] (4) 0.2 g of crude polysaccharide was prepared into a solution with a concentration of 5 mg / mL, and the sample volume was 2 mL. It was purified and separated by a macroporous resin HP-20 column chromatography column, and eluted with 0, 0.05, 0.1, 0.3, 0.4, 0.5, and 0.6 mol / L NaCl solutions in sequence. The fractions were collected by an automatic fraction collector at a flow rate of 1 mL / min, and collected every 5 mL until there was no obvious polysaccharide content in the eluate. The polysaccharide content in the eluate was detected by an ultraviolet spectrophotometer at a wavelength of 280 nm, and the fractions containing a higher polysaccharide content were selected for further purification.

[0028] (5) The eluates from step (4) are combined and concentrated, dialyzed, and freeze-dried to obtain polysaccharides;

[0029] (6) The polysaccharide was prepared into a solution with a concentration of 100 mg / mL, and the sample volume was 2 mL. The solution was separated by Sephadex G-200 column, eluted with 0.1 mol / L NaCl solution, and collected by an automatic fraction collector at a flow rate of 1 mL / min for 4 minutes per tube. The phenol-sulfuric acid method was used for detection, and tubes 9-14 were collected.

[0030] (7) The eluates from step (6) were combined, concentrated, dialyzed, and freeze-dried to obtain the Fangfeng polysaccharide RSP-1.

[0031] The fangfeng polysaccharide is prepared according to the separation and extraction method.

[0032] As a preferred embodiment of the present invention, the fangfeng polysaccharide has an average relative molecular weight of 111125Da, a protein content of 1.29%, is free of uronic acid, and is a neutral polysaccharide containing β-pyranose. Its structure is primarily composed of 1,4-α-D-glucose (Glc) as the main chain skeleton, with side chains containing 1,6-β-D-glucose, 1,3-β-D-glucose, and 1,3-α-D-galactose.

[0033] The invention relates to the use of the fangfeng polysaccharide in the preparation of a liver protection preparation.

[0034] As a preferred embodiment of the present invention, the liver protection preparation inhibits liver tissue damage.

[0035] As a preferred embodiment of the present invention, the liver protection preparation reduces the production of liver inflammatory factors.

[0036] The invention relates to the use of the fangfeng polysaccharide in the preparation of auxiliary medical drugs.

[0037] The advantages of the present invention are:

[0038] 1. The present invention separates a new polysaccharide from the root of Radix Parsnips, measures its average relative molecular weight, analyzes its monosaccharide composition and determines its specific active uses.

[0039] 2. GPC analysis of the average relative molecular weight of fangfeng polysaccharide revealed it to be 111,125 Da. GC-MS, methylation, and NMR analysis revealed that the main monosaccharides of fangfeng polysaccharide are 0.653% mannose and 99.347% glucose, with trace amounts of β-pyranose. Its structure is primarily composed of 1,4-α-D-glucose (Glc) as the backbone, with side chains consisting of 1,6-β-D-glucose, 1,3-β-D-glucose, and 1,3-α-D-galactose.

[0040] 3. Fangfeng polysaccharide significantly inhibited liver tissue damage and reduced liver cell fat accumulation and the production of inflammatory factors in the liver. HE staining results showed that the liver cell wall structure of mice in the normal group was intact and the adipocytes had regular morphology. The model group showed steatosis, hepatocyte spheroidization, disorder, inflammatory cell infiltration, numerous fat droplets, and extensive collagen expression around the portal vein. The liver cells of mice treated with medium and high doses of Fangfeng polysaccharide and the positive control group were neatly arranged, and steatosis and liver fat accumulation were significantly reduced. This suggests that Fangfeng polysaccharide can alleviate the pathological changes of alcoholic liver injury and reduce the changes in steatosis and accumulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : Flow chart of extraction of fangfeng polysaccharide.

[0042] Figure 2 : Desorption curves of Radix Saposhnikoviae Polysaccharide on Sephadex G-200 fast flow column.

[0043] Figure 3 : Elution curve of fangfeng polysaccharide on gel filtration chromatography column.

[0044] Figure 4 : Infrared spectroscopic spectrum of fangfeng polysaccharide.

[0045] Figure 5 : Ion chromatogram of standard monosaccharide components.

[0046] Figure 6 : Ion chromatogram of monosaccharide components in Radix Saposhnikoviae.

[0047] Figure 7 : Total ion current scan of RSP-1 under electron impact ionization.

[0048] Figure 8 : 1H spectrum of RSP-1 in D2O solution.

[0049] Figure 9: 13C spectrum of RSP-1 in D2O solution.

[0050] Figure 10 : HSQC spectrum of RSP-1 in D2O solution.

[0051] Figure 11 : HMBC spectrum of RSP-1 in D2O solution.

[0052] Figure 12 : COSY spectrum of RSP-1 in D2O solution.

[0053] Figure 13 : NOESY spectrum of RSP-1 in D2O solution.

[0054] Figure 14 :Structural composition of RSP-1.

[0055] Figure 15 :Pathological sections of mouse liver and intestinal tissue (H&E staining, oil red staining) DETAILED DESCRIPTION

[0056] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0057] Example 1 Preparation of Radix Saposhnikoviae Polysaccharide

[0058] (1) The roots of Saposhnikovia divaricata (Turcz.) Schischkin were collected, the dried roots were ground, pulverized, and passed through a 60-mesh sieve. A certain proportion of deionized water (1:10, w / v) was then added to the powdered Saposhnikovia root. The mixture was placed in an extraction container and heated at 100°C for 2 h. After the extraction was completed, the extract was filtered while hot and collected. The extraction was repeated three times and the extracts were combined.

[0059] (2) Add 3 volumes of Sevage reagent to the extract, wherein the Sevage reagent is chloroform: n-butanol = 3:1 (v:v), shake vigorously for 30 minutes, centrifuge at 4000 rpm for 20 minutes, take the supernatant, and repeat the operation 5 times; then concentrate under reduced pressure at 60°C to remove the organic reagent and concentrate the solution; use macroporous resin D101 for decolorization at 35°C with a resin: solution ratio of 1:2 (v / v) for 2 hours; then add 3 volumes of 85% ethanol, let it stand at 4°C for 12 hours to precipitate polysaccharides, and collect the precipitate by centrifugation at 10000 rpm, 15 minutes, and 4°C; dissolve the precipitate in distilled water and freeze-dry to obtain crude polysaccharide.

[0060] 0.2 g of crude polysaccharide was prepared into a solution with a concentration of 5 mg / mL, the sample volume was 2 mL, and it was purified and separated by a macroporous resin HP-20 chromatography column, and eluted with 0, 0.05, 0.1, 0.3, 0.4, 0.5, and 0.6 mol / L NaCl solutions in sequence, and collected by an automatic fraction collector at a flow rate of 1 mL / min, collecting every 5 mL until there was no obvious polysaccharide content in the eluate; the polysaccharide content in the eluate was detected by an ultraviolet spectrophotometer at a wavelength of 280 nm, and the fraction containing a higher polysaccharide content was selected for further purification; the eluates of the collected fractions were combined, concentrated, dialyzed, and freeze-dried to obtain the fangfeng polysaccharide;

[0061] The fangfeng polysaccharide was prepared into a solution with a concentration of 100 mg / mL, the sample volume was 2 mL, separated by Sephadex G-200 column, eluted with 0.1 mol / L Nacl solution, and collected by automatic fraction collector at a flow rate of 1 mL / min, 4 minutes per tube; the phenol-sulfuric acid method was used for detection, and tubes 9-14 were collected, combined, concentrated, dialyzed, and freeze-dried to obtain fangfeng polysaccharide RSP-1.

[0062] Example 2 Characterization of Radix Saposhnikoviae Polysaccharides (I) Determination of the Average Relative Molecular Weight of Polysaccharides

[0063] Molecular weights were determined using a Waters 1515 Infinity size exclusion chromatography and gel permeation chromatography system equipped with a Waters 2410 refractometer (Waters, CA, USA) and a gel filtration column (OHpak SB-803HQ, Shodex, Tokyo, Japan). Dextran standards (1, 5, 12, 25, 50, 80, 150, 270, 410, and 670 kDa, Sigma-Aldrich) were used for calibration. Elution was performed with 0.05 M ammonium acetate at a rate of 0.65 mL / min and maintained at 40 ± 1°C.

[0064] Table 1 Results of GPC determination of Radix Saposhnikoviae Polysaccharide

[0065]

[0066] (2) Analysis of monosaccharide composition of polysaccharides

[0067] 1. Hydrolyze polysaccharides to monosaccharides: Accurately weigh 5.0 mg of purified polysaccharide RSP-1 from Radix Saposhnikoviae Polysaccharides and place them into two separate ampoules. Add 2.0 mL of trifluoroacetic acid (TFA) solution, seal the bottles, and hydrolyze at 120°C for 8 h. Rapidly dry the hydrolyzed product in a vacuum oven. Add 2 mL of methanol and evaporate to dryness under reduced pressure. Repeat this process three times until no TFA residue remains. The dried product is then ready for use.

[0068] 2. Derivatization of Hydrolyzed Monosaccharides: Add 0.1 mL of acetic anhydride to each of the hydrolyzed products and continue dissolving in water for 30 minutes. After acetylation and derivatization, concentrate to dryness under reduced pressure. Add 1 mL of chromatography-grade chloroform to each tube and dissolve by shaking. Filter through a 0.45 μL microporous filter before injecting the sample for analysis. Derivatization of standard monosaccharides (glucose and mannose) proceeds in the same manner as above.

[0069] 3. Analytical conditions: GC-MS system: Agilent, 77890A-5975C, USA; DB-5MS (30 mm × 0.25 mm × 0.25 μm, Agilent); N2 flow rate: 1 mL / min; temperature program: inlet temperature: 270 °C, ion source temperature: 230 °C, column temperature: 100 °C for 2 min, then increased to 190 °C at 20 °C / min, then increased to 260 °C at 20 °C / min, and finally increased to 300 °C at 10 °C / min and maintained for 4 min.

[0070] (3) Infrared spectroscopy analysis

[0071] The dry RSP-1 powder was pressed into thin sheets for analysis. The spectrum of RSP-1 was determined by FT-IR. Figure 4 As shown, the FTIR spectrum used for the characterization of RSP-1 shows the peak of polysaccharide at 3399.54 cm -1 It is a strong absorption peak of OH stretching vibration of intermolecular or intramolecular hydrogen bonds; 2928.54cm -1 The medium-intensity peak near 1642.23cm is the CH stretching vibration absorption peak of methyl (-CH3) and methyl (-CH2); -1 It is the characteristic absorption peak of amide carbonyl. 1450~1200cm -1 Middle 1424.64cm -1 and 1372.30cm -1 The absorption peak at 1154.71cm is the angle-varying vibration absorption peak of CH, which together with the stretching vibration of CH constitutes the characteristic absorption of the sugar ring; -1 is the stretching vibration of pyranose COC; 1150~1010cm -1 、1069.32cm -1 and 1019.74cm -1 The absorption at the two positions is the bending vibration of the CO bond in the COH or COC structure; 834.35 cm -1 The peak is the vibration peak of carbohydrate molecules, 895.79 cm -1 The characteristic region of β-pyranose bond, 840.70 cm -1 The CH angle vibration peak of the α-terminal group of pyranose is 760.83 cm -1The symmetric ring stretching vibration peaks of the pyran ring are shown.

[0072] (IV) Methylation analysis

[0073] RLP-1 was methylated according to the Ciucanu & Kerek method (Ciucanu & Kerek, 1984). An HP-5MS fused silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA) was used, coupled with gas chromatography-mass spectrometry (Agilent 5977B; Agilent Technologies, USA). The detailed temperature program was as follows: the initial oven temperature was 50°C, maintained for 1 min, then raised to 230°C at a rate of 3°C / min and maintained for 2 min. The temperature of the split injector was 260°C, helium was used as the carrier gas, and the flow rate was 1.0 mL / min. Figure 5 、 Figure 6 HPLC chromatograms of monosaccharide standards and RSP-1. Figure 7 The total ion chromatogram revealed peak profiles of methylated products. By comparing the retention times and spectra of these products with the spectral database of the Center for Complex Carbohydrate Research, we determined the glycosyl residues present in RSP-1, which primarily contained 1,4-linked Glc, with minor amounts of 1,3,4- and 1,3,6-linked Glc.

[0074] Table 2 GC-MS analysis of methylation of Radix Saposhnikoviae Polysaccharides

[0075]

[0076] Nuclear magnetic resonance (NMR) analysis

[0077] RSP-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 Spectromete, Germany). The spectra obtained are shown in FIG. Figure 8-13 As shown, Figure 8 、 9Complete hydrogen and carbon chemical shift data for all major sugar residues are provided, helping to elucidate the linkages between different sugar units. Monosaccharide composition and methylation analysis revealed that the main monosaccharides identified were Man and Glc. Methylation analysis revealed a predominant presence of 1,4-linked Glc, with minor amounts of 1,3,4- and 1,3,6-linked Glc. Based on the results of monosaccharide composition and methylation analysis of RSP-1, as well as analysis using NMR techniques such as 1H-NMR, 13C-NMR HSQC, and 1H-1H COSY, multiple isotopic signals were observed. Through comprehensive analysis including monosaccharide composition evaluation, methylation study and NMR evaluation, we found that this polysaccharide sample showed many different isotope signal peaks, which are valuable for structural elucidation; by correlating the heteronuclear multiple bond protons observed in the remote HMBC spectrum with the carbon atoms or hydrogen atoms of each sugar residue, considering that the two protons adjacent to the connection site between adjacent sugar residues are likely to produce stronger NOE signals due to spatial proximity, we can use the HMBC remote correlation spectrum and NOESY spectrum to further infer the connection order between sugar residues. The HMBC correlation spectrum and NOESY spectrum of the RSP-1 sample are illustrated in Figure 2. Figure 11 and Figure 13Several coupling signals can be identified from these figures: 1HNMR spectrum detected a strong anomeric proton signal at δH 5.41, which is due to the type of →3)-glcp-(1→ residue, and is presumed to be α-configuration. In the 13C NMR spectrum, δC 95-110ppm is the chemical shift region of the heteromeric carbon, and the signals below δC 85ppm belong to the non-terminal carbon. The telomeric carbon signal of RSP-1 is less than δC 103ppm, which is the α-telomeric carbon signal. In addition, there is no signal in the δC value range of 170-175ppm, indicating the absence of uronic acid. 13C nuclear magnetic resonance spectrum analysis shows that the chemical shift is less than δC 85ppm, which is caused by the C2-C6 proton signal region on the glycoside ring. The δC values ​​are 76.82, 73.31, 71.50, 71.15 and 60.43ppm, corresponding to C3, C2, C4, C5 and C6, respectively. Among them, the chemical shift of C3 is approximately δC The HMBC spectrum of δH 5.41ppm and δC 99.58ppm were closely related. The cross peaks in the 1H-1H COSY spectrum also confirmed the chemical shift of H. The strong cross peak signal near δH 5.41 / δC 76.82ppm in the HMBC spectrum corresponds to the correlation between H1 and C3 in the residue, indicating the presence of a (→3)-α-D-Glcp(1→3)-α-D-Glcp(1→ chain mode) in the repeating backbone unit. Finally, the 1H-1H NOESY spectrum showed that there was almost no cross peak signal between H and H in the C space, indicating a straight chain structure.

[0078] Table 3 1H and 13C chemical shift assignments of various sugar residues in polysaccharide samples ("--" indicates undetermined or not detected)

[0079]

[0080] Combining the analysis of monosaccharide composition, methylation results, and one-dimensional and two-dimensional NMR information, it can be inferred that the polysaccharide sample is a simple polysaccharide, e.g. Figure 14 shown.

[0081] Example 3 Use of Fangfeng Polysaccharide

[0082] Materials and reagents: Radix Saposhnikoviae Polysaccharide RSP-1 (prepared in Example 1), hematoxylin & eosin staining kit, 4% paraformaldehyde, gradient ethanol, xylene.

[0083] Instruments: paraffin embedding machine (Leica Histo Core Arcadia), paraffin sectioner (Leica 2255), light microscope.

[0084] 1. Fangfeng polysaccharide protects the liver and reduces lipid metabolism disorders

[0085] All mice were housed under controlled environmental conditions (25°C and a 12 / 12-hour light / dark cycle) with free access to food and water. After a one-week acclimation period, the mice were randomly divided into five groups (n=6 per group): a normal control (ND) group, an ALD model group (administered orally with 56% alcohol liquor, 7 ml / kg twice daily, morning and evening, for one week), a medium- and high-dose saposhnikovia polysaccharide group, and a positive control group. The entire animal experiment lasted 16 weeks. All mice received high-pressure water and a normal diet for one week. Based on the results of the pilot study, the intervention doses for the medium- and high-dose saposhnikovia polysaccharide groups were set at 50 mg / kg (body weight) / day and 150 mg / kg (body weight) / day, respectively; the positive control group received compound glycyrrhizin tablets at 100 mg / kg (body weight) / day. Drug intervention was administered once daily from week 2 to week 16. During the experimental period from week 2 to week 16, the experimental animals received normal drinking water and a normal diet. After the intervention, the mice were dissected, the liver tissue was separated, the blood on the liver tissue surface was rinsed with saline, the capsule was peeled off, and the liver tissue was fixed in 4% paraformaldehyde. The liver tissue fixed in paraformaldehyde was removed, paraffin sections were prepared, and the pathological morphology of the liver tissue was observed by HE staining. Figure 15 As shown in the results, fangfeng polysaccharide at high concentrations can significantly improve liver pathological changes, including fatty degeneration, hepatocyte spheroidization and disorder, inflammatory cell infiltration, a large number of fat droplets, and extensive expression of collagen around the portal vein. This indicates that fangfeng polysaccharide can, to a certain extent, alleviate liver pathological changes and reduce the occurrence of fat tissue disorder in the liver.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A method for separating and extracting fangfeng polysaccharide RSP-1, characterized in that: The fangfeng polysaccharide is prepared by the following steps: (1) Grinding, crushing, and sieving the dried root of Radix Saposhnikoviae var. elegans, and adding deionized water to obtain a Radix Saposhnikoviae var. elegans extract; (2) The extract of Saposhnikovia divaricata was concentrated and Sevage reagent was added to the solution to remove protein, and then the concentrate was decolorized with macroporous resin D101; (3) adding 85% ethanol to the decolorized solution to precipitate the polysaccharide, and collecting the precipitate by centrifugation; dissolving the precipitate in distilled water, and freeze-drying to obtain crude polysaccharide; (4) The crude polysaccharide was purified and separated by macroporous resin HP-20 chromatography column, and the eluate containing the highest polysaccharide content was selected, combined, concentrated, dialyzed, and freeze-dried to obtain the fangfeng polysaccharide; (5) The fangfeng polysaccharide was prepared into a solution, separated by Sephadex G-200 column, eluted with 0.1 mol / L NaCl solution, and the eluate was concentrated, dialyzed, and freeze-dried to obtain pure fangfeng polysaccharide RSP-1.

2. The separation and extraction method according to claim 1, wherein Step (1) crushing the dried fangfeng root and passing it through a 60-mesh sieve, then adding a certain proportion of deionized water (10:1, w / w) to the fangfeng root powder, mixing, placing in an extraction container, heating and extracting at 100° C. for 2-3 hours, after the extraction is completed, filtering while hot, collecting the extract, repeating the extraction 2-3 times, and combining the supernatant extracts; using a rotary evaporator to concentrate the total extract to 1 / 4 of the volume at 55° C. to obtain a concentrated solution.

3. The separation and extraction method according to claim 1, wherein Step (2) adding 3 volumes of Sevage reagent to the extract obtained in step (1), wherein the Sevage reagent is chloroform: n-butanol = 3:1, vigorously shaking for 30 minutes, centrifuging at 4000 rpm for 20 minutes, taking the supernatant, and repeating the operation 5 times; then concentrating under reduced pressure at 60°C to remove the organic reagent and concentrate the solution, and then decolorizing the concentrate with macroporous resin D101; preferably, adding 3 volumes of Sevage reagent to the concentrated solution obtained in step (1) for multiple extractions, wherein the Sevage reagent is chloroform: n-butanol = 3:1, concentrating under reduced pressure at 55-65°C to remove the organic reagent and concentrate the solution, and then performing precipitation treatment; decolorizing with macroporous resin D101 at 35°C with a resin: solution ratio of 1:2 (v / v) for 2 hours.

4. The separation and extraction method according to claim 1, wherein Step (3) adding 3 times the volume of 85% ethanol to the decolorized concentrated solution, standing at 4°C for 12-24 hours to precipitate polysaccharides, and centrifuging at 10,000 rpm for 15 minutes at 4°C to collect the precipitate; dissolving the precipitate in distilled water and freeze-drying to obtain crude polysaccharide; preferably, adding 3 times the volume of 85% ethanol to the decolorized solution, precipitating at 4°C overnight, centrifuging at 10,000 rpm for 15 minutes at 4°C to collect the precipitate, redissolving the precipitate in distilled water, adding 85% ethanol at 4°C to precipitate, and centrifuging again to collect the precipitate, repeating the process multiple times, and freeze-drying the precipitate to obtain freeze-dried crude polysaccharide.

5. The separation and extraction method according to claim 1, wherein Step (4) taking 0.2 g of crude polysaccharide, preparing a solution with a concentration of 5 mg / mL, loading 2 mL, purifying and separating by macroporous resin HP-20 chromatography column, eluting with 0, 0.05, 0.1, 0.3, 0.4, 0.5, and 0.6 mol / L NaCl solutions in sequence, collecting by an automatic fraction collector, at a flow rate of 1 mL / min, collecting every 5 mL until there is no obvious polysaccharide content in the eluate; detecting the polysaccharide content in the eluate at a wavelength of 280 nm using an ultraviolet spectrophotometer, and selecting the component containing a higher polysaccharide content for further purification; and combining the eluates of the collected components, concentrating, dialyzing, and freeze-drying to obtain polysaccharides.

6. The separation and extraction method according to claim 1, wherein Step (5) The polysaccharide was prepared into a solution with a concentration of 100 mg / mL, the sample volume was 2 mL, separated by Sephadex G-200 column, eluted with 0.1 mol / L NaCl solution, and collected by an automatic fraction collector at a flow rate of 1 mL / min, 4 min per tube; the phenol-sulfuric acid method was used for detection, and tubes 9-14 were collected. The collected eluates were combined and concentrated, dialyzed, and freeze-dried to obtain the windproof polysaccharide RSP-1.

7. The method for preparing fangfeng polysaccharide according to claim 1, characterized in that: The preparation method comprises the following steps: (1) the root of the Chinese catalpa is crushed by a crushing machine, and then passed through a 60-mesh sieve to obtain the root of the Chinese catalpa powder; (2) water is added to the root of the Chinese catalpa powder, and the root of the Chinese catalpa powder is extracted at 100°C for 2 hours according to a material-liquid ratio of 1:10 to obtain a water extract, and the water extract is concentrated under reduced pressure at 60°C to obtain a concentrate; (3) the water extract of the root of the Chinese catalpa powder is subjected to the Sevage method to remove protein, precipitated with 85% ethanol, and finally freeze-dried to obtain a crude polysaccharide; (4) 0.2 g of the crude polysaccharide is taken, prepared into a solution with a concentration of 5 mg / mL, the sample volume is 2 mL, and the solution is purified and separated by a macroporous resin HP-20 chromatography column, and eluted with 0, 0.05, 0.1, 0.3, 0.4, 0.5, and 0.6 mol / L NaCl solutions in sequence, and collected by an automatic fraction collector at a flow rate of 1 mL / min. min, collect every 5 mL until there is no obvious polysaccharide content in the eluate; detect the polysaccharide content in the eluate at a wavelength of 280 nm using an ultraviolet spectrophotometer, and select the fraction containing a higher polysaccharide content for further purification; (5) the eluates from step (4) are combined and concentrated, dialyzed, and freeze-dried to obtain polysaccharides; (6) the polysaccharide is prepared into a solution with a concentration of 100 mg / mL, the sample volume is 2 mL, separated by a Sephadex G-200 column, eluted with a 0.1 mol / L NaCl solution, and collected by an automatic fraction collector at a flow rate of 1 mL / min, 4 min per tube; the phenol-sulfuric acid method is used for detection, and tubes 9-14 are collected; (7) the eluates from step (6) are combined and concentrated, dialyzed, and freeze-dried to obtain the Fangfeng polysaccharide RSP-1.

8. The fangfeng polysaccharide prepared by the separation and extraction method according to any one of claims 1 to 4.

9. The fangfeng polysaccharide according to claim 1, characterized in that The fangfeng polysaccharide has an average relative molecular weight of 111125Da, a protein content of 1.29%, does not contain uronic acid, and is a neutral polysaccharide.

10. Use of the fangfeng polysaccharide according to claim 8 in a protective preparation for alcoholic liver disease.

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