CN113880959B - Polygonatum sibiricum homogeneous polysaccharide and preparation method and application thereof - Google Patents
Polygonatum sibiricum homogeneous polysaccharide and preparation method and application thereof Download PDFInfo
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Abstract
A polygonatum sibiricum homogeneous polysaccharide and a preparation method and application thereof belong to the technical field of separation and purification of Chinese herbal medicine macromolecular polysaccharide. Based on the problems of the related separation and purification of polygonatum polysaccharide, the composition of polygonatum polysaccharide, the initial structural representation and less research on pharmacological action, in order to further research the polygonatum polysaccharide, a water extraction and alcohol precipitation method is adopted to extract rhizome of polygonatum to obtain crude polygonatum polysaccharide, and then a cation and anion series exchange resin column and a Sephacyrl S400 gel column are used to further purify the crude polygonatum polysaccharide, so that the efficiency of separating and purifying the polygonatum homogeneous polysaccharide is improved, and the polygonatum homogeneous polysaccharide is obtained. The polygonatum sibiricum homogeneous polysaccharide has no impurities such as protein, nucleic acid, polypeptide and the like, has uniform components and higher purity, has good effect on treating nephritis, can be used for preparing a medicament for treating nephritis, and provides another feasible method for treating nephritis.
Description
Technical Field
The invention belongs to the technical field of separation and purification of Chinese herbal medicine macromolecular polysaccharide, and particularly relates to polygonatum uniform polysaccharide and a preparation method and application thereof.
Background
Rhizoma Polygonati is dried rhizome of Liliaceae plants including rhizoma Polygonati, rhizoma Polygonati Yunnanensis, and rhizoma Polygonati Odorati. As one of the traditional Chinese medicinal materials in China, the Chinese medicinal composition is also a famous medicinal and edible medicinal material, has the effects of tonifying qi and blood and strengthening kidney, and has high medicinal value and commercial value. The polysaccharide is the component with the highest content in Polygonatum plants, and has effects of resisting aging, resisting tumor, enhancing immunity, reducing blood lipid and blood sugar, etc. However, many reports on polygonatum polysaccharides in the prior art relate to the functions of polygonatum polysaccharides, and few studies on polygonatum homopolysaccharides have been made. Therefore, crude polysaccharide is extracted from the rhizoma polygonati medicinal material, and the rhizoma polygonati polysaccharide is separated and purified, which is beneficial to further research on the rhizoma polygonati polysaccharide.
Disclosure of Invention
The invention provides a polygonatum uniform polysaccharide based on the problems of separation and purification of polygonatum polysaccharide, composition of polygonatum polysaccharide, primary structure representation and less research on pharmacological action, and aims to further research the polygonatum polysaccharide.
Further defined, the ratio of mannose, fucose and galactose is 1.
The invention also provides a preparation method of the polygonatum uniform polysaccharide, which comprises the following steps:
s1, extracting rhizome of polygonatum sibiricum by using distilled water, and concentrating the obtained filtrate to obtain an extracting solution;
s2, mixing the extracting solution obtained in the step S1 with an ethanol solution, fully stirring, standing, performing suction filtration to obtain a precipitate, and drying the precipitate to obtain crude polygonatum polysaccharide;
and S3, purifying the crude polygonatum polysaccharide by sequentially utilizing an anion-cation series exchange resin column and a Sephacyrl S400 gel column to obtain the polygonatum homogeneous polysaccharide.
Further limiting, the extraction in S1 is that rhizome of Polygonatum sibiricum and distilled water are mixed according to a ratio of 1g:3 mL-1 g: mixing materials and liquid in a ratio of 4mL, decocting for 1-2 h in boiling water, filtering, and collecting filtrate and filter residue; extracting the filter residue for 1-2 times repeatedly according to the operation, and then combining the filtrate obtained by each extraction.
Further defined, the volume ratio of the extracting solution S2 to the 95% ethanol solution is 1:3 to 1:4; the standing time is 10 to 14 hours.
Further limiting, S2 further comprises the step of carrying out alcohol precipitation for 1-2 times by using an ethanol solution with the volume fraction of 95% after standing to obtain a precipitate.
And 3, further limiting, the method for purifying the anion-cation series exchange resin column comprises the steps of taking crude polygonatum sibiricum polysaccharide obtained in the step 2, preparing a polysaccharide solution with distilled water to be 3%, centrifuging, taking the supernatant, adding the supernatant into the FPA90Cl and FPC3500 anion-cation series exchange resin column, eluting by sequentially utilizing distilled water, 0.5mol/L NaCl and 1mol/L NaCl, and sequentially concentrating, dialyzing, concentrating and freeze-drying the components obtained by elution to obtain a water-washing component.
Further defined, the dialysis is; dialyzing with flowing water for 15-50 h, and dialyzing with distilled water for 20-28 h.
Further, the purification method of the Sephacyrl S400 gel column S3 is that the water washing component obtained by purifying the anion and cation series exchange resin column S3 is prepared into a polysaccharide solution with the concentration of 3 percent, after centrifugation, the supernatant is taken and added into the Sephacyrl S400 gel column, and distilled water and 0.1 mol.L are sequentially used -1 、0.2mol·L -1 、0.3mol·L -1 、0.4mol·L -1 、0.5mol·L -1 、1mol·L -1 Gradient elution is carried out on NaCl at a flow rate of 2mL/min -1 Collecting the washing component and 0.1 mol.L -1 、0.2mol·L -1 And NaCl salt washing the components, dialyzing with distilled water, concentrating under reduced pressure, and freeze-drying to obtain the polygonatum sibiricum homogeneous polysaccharide.
The invention also provides application of the polygonatum uniform polysaccharide in preparing a medicine for treating nephritis.
The invention has the beneficial effects that:
the invention extracts the rhizome of the polygonatum by a water extraction and alcohol precipitation method to obtain crude polygonatum polysaccharide, the method has high yield, and the polysaccharide structure is not easy to change; and (3) passing the crude polysaccharide through an anion-cation series exchange resin column to remove protein, pigment and other impurities, and then passing through an ion exchange agar gel column to obtain the polygonatum uniform polysaccharide. Ultraviolet full-wavelength scanning spectrum shows that the polygonatum uniform polysaccharide has no impurities such as protein, nucleic acid, polypeptide and the like; the polysaccharide characteristic peak is displayed in the infrared spectrum, and the structure of the polygonatum uniform polysaccharide is confirmed; when the molecular weight of the polysaccharide is measured by an HPLC-ELSD method, a map shows a single peak and has symmetrical peak shapes, which indicates that the purified polysaccharide component is uniform and has higher purity, and the measured molecular weight is about 4000Da, which indicates that the polysaccharide component is a macromolecular component. In addition, the polygonatum uniform polysaccharide obtained by the invention has good effect on treating nephritis, can be used for preparing a medicament for treating nephritis, and provides another feasible method for treating nephritis.
Drawings
FIG. 1 is a standard graph of total polysaccharide;
FIG. 2 is a graph of uronic acid standard curves;
FIG. 3 is a graph of a protein standard curve;
FIG. 4 is a graph showing the elution profile of PSP-1S of the sample;
FIG. 5 is a PSP-1S ultraviolet full wavelength scan;
FIG. 6 is a PSP-1S infrared scan;
FIG. 7 is a graph of dextran standard curves;
FIG. 8 is a PSP-1S chromatogram;
FIG. 9 is a graph showing the results of PMP pre-column derivatization HPLC analysis;
FIG. 10 is a graph showing the results of HPLC analysis of PSP-1S completely hydrolyzed PMP derivatives;
FIG. 11 is a graph showing the results of histopathological morphological observation of kidney tissues in each group.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, and the procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for those specifically mentioned below, and the present invention is not particularly limited thereto.
Example 1: preparation method of polygonatum sibiricum homogeneous polysaccharide
Extraction of polygonatum polysaccharide
The method for extracting the polygonatum polysaccharide by adopting distilled water extraction and ethanol precipitation comprises the following specific steps: taking 2.6kg of dried rhizome of the processed polygonatum sibiricum medicinal material, adding 500g of the medicinal material each time, and mixing according to the material-liquid ratio of 1g of polygonatum sibiricum to distilled water: adding 3mL of distilled water, decocting for 90min under slightly boiling state, filtering to obtain medicinal liquid, extracting the residue for 2 times, mixing medicinal liquids, and concentrating to obtain extractive solution; according to the volume ratio of the extracting solution to the 95% ethanol solution by volume fraction of 1:4, slowly adding a 95% ethanol solution into the extracting solution while stirring, standing overnight after the completion, repeating the precipitation twice, then performing suction filtration on the precipitate, taking the precipitate, putting the precipitate into an oven for drying, weighing and storing to obtain 510g of crude Polygonatum Sibiricum Polysaccharide (PSP), wherein the yield is 19.62%.
Content determination of crude polysaccharide of rhizoma polygonati
(1) Content determination of rhizoma polygonati total polysaccharide
The content determination of the rhizoma polygonati total polysaccharide adopts a sulfuric acid-phenol method, and comprises the following specific steps:
(1) drawing of standard curve
The reagents were added as shown in Table 1, shaken, left to stand for 10min, cooled in boiling water bath for 15min, and measured at 490nm, the measurement results are shown in Table 2, and the standard curve is shown in FIG. 1.
TABLE 1 preparation of the corresponding reagents (Unit: mL)
TABLE 2 polysaccharide Standard Curve measurements
(2) Calculation of conversion factor
Taking 0.2mL of crude polysaccharide sample solution, filling the solution into a 10mL test tube with distilled water to 2mL, preparing three parts, treating according to the same method, measuring the absorbance at 490nm, and calculating the conversion coefficient according to the following formula:
in the above formula, w is the mass of polysaccharide, C is the glucose concentration, and D is the dilution factor.
The absorbance of the sample was 0.427; calculating according to the above formula to obtain a conversion coefficient of 1.44, substituting into the formula (wherein w is polysaccharide mass, C is glucose concentration, D is dilution factor, and f is conversion coefficient), and calculating to obtain rhizoma Polygonati crude polysaccharide with content of 62.58%.
(2) Uronic acid content determination
This experiment required 0.4mg/mL uronic acid control solution. The reagents were added as shown in Table 3, and the mixture was subjected to water bath at 85 ℃ for 20min, allowed to cool, shaken up, and measured after 2 hours at a wavelength of 530nm, the measurement results are shown in Table 4, and the standard curve is shown in FIG. 2.
TABLE 3 preparation of the corresponding reagents
TABLE 4 uronic acid Standard Curve measurements
Taking 1mL of 0.4mg/mL sample solution in a test tube with a stopper, measuring the absorbance by the same method, and substituting the sample solution into a standard curve to calculate the content of uronic acid. The absorbance of the sample was 0.233; the uronic acid content in the polygonatum polysaccharide is calculated to be 18.27%.
(3) Protein content determination
The experiment adopts a Coomassie brilliant blue G-250 method to determine the protein content in crude polysaccharide of rhizoma polygonati, and comprises the following steps:
0.5mg/mL bovine serum albumin standard solution was prepared in 0.9% NaCl solution as solvent, and after adding the corresponding reagents according to Table 5, shaking was immediately followed by standing at room temperature for 15min, and absorbance at 590nm was measured, and the results of protein measurement are shown in Table 6 and the standard curve of measurement is shown in FIG. 3.
TABLE 5 preparation of corresponding reagents
TABLE 6 protein Standard Curve measurements
100mg of PSP was weighed out precisely, and dissolved in a 100mL volumetric flask using 0.9% NaCl solution. Taking 1mL of sample, operating the same method to obtain an absorbance, and inputting the absorbance into a standard curve. The absorbance of the sample is 0.058, and the protein content in the polygonatum polysaccharide is 1.55%.
(II) separation and purification of crude polysaccharide of rhizoma Polygonati
Preparing crude rhizoma Polygonati polysaccharide 80g into 3% polysaccharide solution with distilled water, centrifuging, collecting supernatant, and passing through FPA90Cl and FPC3500 anion-cation series exchange resin column. First, distilled water was used as an eluent, and the flow rate was 10 mL/min -1 Tracking and detecting by phenol-sulfuric acid method, and using 0.5 mol.L when sugar-free component flows out -1 NaCl elution is carried out until no sugar component flows out, and then 1.0 mol.L is changed -1 Eluting with NaCl until sugar-free component flows out, and eluting with 2.0 mol.L -1 And (5) washing the column by NaCl. Concentrating each component, dialyzing with 3500Da dialysis bag, dialyzing with running water for 48 hr, dialyzing with distilled water for 24 hr, concentrating the solution in bag under reduced pressure, and lyophilizing to obtain water-washed component and 0.5 mol.L -1 NaCl salt wash fractions, recorded as: PSP-1 and PSP-2.
Dissolving the PSP-1 obtained in the step into double distilled water to prepare polysaccharide solution with the concentration of 3%, centrifuging, and purifying the supernatant by using a Sephacyrl S-400 gel column. Distilled water and 0.1 mol.L are used in sequence -1 、0.2mol·L -1 、0.3mol·L -1 、0.4mol·L -1 、0.5mol·L -1 、1mol·L -1 Gradient elution is carried out on NaCl at a flow rate of 2mL/min -1 10 mL/tube automatic part of effluentCollecting with a collector, separating with a phenol-sulfuric acid method to obtain color, drawing elution curve with number-optical density of elution tubes, mixing samples with the same peak position to obtain water, 0.1 mol.L -1 、0.2mol·L -1 And a NaCl component. Eluting the components with distilled water, mixing the components with the same peak position (figure 4), dialyzing with distilled water, concentrating the solution in the dialysis bag under reduced pressure, freeze-drying, and concentrating the eluate under reduced pressure to obtain PSP-1S, enriching the sample for multiple times, and freeze-drying to obtain fluffy white floccule and light yellow fluffy floccule which are rhizoma Polygonati homogeneous polysaccharide.
Detecting the obtained polygonatum uniform polysaccharide PSP-1S:
(1) Purified fraction ultraviolet full wavelength scanning
In the ultraviolet full-wavelength scanning chart, whether an ultraviolet absorption peak exists at 200-300 nm can judge the existence of protein and nucleic acid in the polysaccharide. Polysaccharide component PSP-1S is prepared into 0.1mg/mL solution, full-wavelength scanning is carried out within the range of 200-400 nm, the result is shown in figure 5, and as can be seen from figure 5, an ultraviolet scanning graph has no obvious absorption peaks at 260nm and 280nm, and the purified polysaccharide is preliminarily shown to have no impurities such as nucleic acid, protein, polypeptide and the like.
(2) Purified fraction infrared spectroscopy
Mixing the raw materials in a ratio of 1:200, mixing polysaccharide powder PSP-1S and KBr powder, fully and uniformly mixing the mixture in an agate mortar, and grinding the mixture into fine powder. After tabletting, the mixture is heated in the infrared of 4000 to 400cm -1 The wavelength range was scanned and the results analyzed (see fig. 6). The obtained map shows that PSP-1S has polysaccharide characteristics. The infrared spectrum shows that the three polysaccharides are 1100-1000 cm -1 ，1600～1400cm -1 ，3000～2800cm -1 ，3500～3300cm -1 All have characteristic absorption peaks of polysaccharide, 3500-3300 cm -1 A broad peak appears at the position, which is an O-H stretching vibration absorption peak and indicates that intermolecular and intramolecular hydrogen bonds exist; at 3000-2800 cm -1 The weak absorption peak between them is C-H stretching vibration absorption peak, 1630-1609 cm -1 The weak absorption peak between the two is C = O asymmetric stretching vibration, 1425-1416 cm -1 (C = O symmetric stretching vibration) is the characteristic peak of protonated carboxyl groups in uronic acids. 1145cm -1 ，1091～1096cm -1 ，1045～1014cm -1 Is also a characteristic peak of uronic acids.
(3) Molecular weight determination of purified fractions
Measuring molecular weight by HPLC-ELSD method, dissolving dextran standard substances with different molecular weights and PSP-1S in mobile phase to obtain 2mg/ml solution, and filtering with 0.45 μm microporous membrane. A standard curve is plotted with retention time tR on the abscissa and lgM on the ordinate (FIG. 7). The obtained chromatogram is shown in FIG. 8, and the chromatogram has only one peak and symmetrical peak shape, which indicates that the components are uniform and have high purity. The PSP-1S retention time was 12.283min. Substituting into standard curve, and preliminarily calculating polysaccharide molecular weight at about 4000 Da.
(4) Monosaccharide composition analysis
(1) Preparation of control solutions
11 monosaccharide reference substances such as Man, rha, galUA, glc, gal, ara and the like are accurately weighed and respectively prepared into standard monosaccharide aqueous solutions of 1 mmol/L. In addition, accurately weighing 11 monosaccharide reference substances to prepare a mixed standard monosaccharide aqueous solution with the concentration of 1 mmol/L.
(2) Preparation of polysaccharide acid hydrolysis sample solution
Weighing 10mg of refined polysaccharide, adding 2mL of 2mol/L trifluoroacetic acid solution, filling the solution into an N2 sealed tube, hydrolyzing for 180min under the condition of 120 ℃ water bath to obtain a hydrolysis sample solution, adding methanol, evaporating to dryness under reduced pressure, and repeating for 3 times until the trifluoroacetic acid is completely removed. Adding 2mL of distilled water for dissolving for later use.
(3) PMP derivatization of standard monosaccharide, mixed monosaccharide, and polysaccharide hydrolyzed samples
Precisely transferring 1.4mL of each of a standard monosaccharide reference substance, a mixed monosaccharide reference substance and a hydrolysis sample solution of polysaccharide, sequentially adding 700 mu L of PMP methanol solution and 0.3mol/L NaOH solution with the same volume, heating in a water bath at 70 ℃ for 40min for reaction, taking out, cooling the sample to room temperature, adding 700 mu L of 0.3mol/L hydrochloric acid for neutralization, uniformly mixing with an isovolumetric isoamyl acetate solvent, extracting for 3 times by using a separating funnel, discarding the supernatant, extracting for 1 time by using isovolumetric chloroform, discarding a chloroform extraction layer, preserving a methanol-water phase, filtering by using a filter membrane, and reserving the sample for later use.
(4) Liquid phase conditions
DIKMA C18 column; mobile phase A:0.1mol/L phosphate buffer (pH 6.7), mobile phase B: acetonitrile; flow rate: 1.0mL/min; detection wavelength: 245nm; sample introduction: 10 mu L of the solution; the elution conditions are shown in Table 7.
TABLE 7 HPLC elution conditions
(5) The high performance liquid analysis results of PMP pre-column derivatization are shown in FIG. 9, and the high performance liquid analysis results of PSP-1S complete acid hydrolysis of PMP derivatives are shown in FIG. 10.
The method for calculating the molar ratio of monosaccharide composition in polysaccharide comprises the following steps: and calculating by an internal standard plus correction factor method.
Mannose: fucose: galactose = 1.
The method comprises the steps of adopting anion-cation tandem exchange resin, sephacryl S-400 gel and the like as carriers, respectively carrying out gradient elution by using water and NaCl with different concentrations, obtaining a polysaccharide component PSP-1S after dialysis and freeze drying, and identifying the purity of the polysaccharide component PSP-1S through infrared spectroscopy, infrared spectroscopy and High Performance Liquid Chromatography (HPLC) to verify that the polysaccharide component is homogeneous polysaccharide. PMP pre-column derivatization HPLC analysis results: mannose: fucose: galactose = 1.
Experimental research on treatment of nephritis by using polygonatum sibiricum polysaccharide
The main drugs and experimental animals involved in this experiment were as follows:
medicine preparation: doxycycline hydrochloride injection (Zhejiang Haizhen pharmacy Co., ltd.); prednisone acetate tablets (pharmaceutical industry group, ltd, south, guangdong).
Experimental animals: normal healthy SD rats, male, 200 + -20 g, with a certification number of SCXK (Liao) 2015-0001, purchased from Liaoning Biotechnology Limited. The temperature of the environment for animal experiments is 20 ℃, the humidity is 60%, and the animals can freely drink and eat water.
The animal experiment in the experiment is approved by the animal experiment ethics committee of the university of traditional Chinese medicine of Heilongjiang.
(1) Preparation of polygonatum polysaccharide extract
Selecting radix rehmanniae extract, and making into decoction pieces according to pharmacopoeia and Chinese medicine processing teaching material method. The preparation method of polysaccharide is the same as above.
(2) Animal grouping, modeling and administration
After 3 days of ambient acclimatization at room temperature, the animals were randomly divided into 4 groups (10 rats per group) according to the weight average: a normal group, a model group, a positive drug group (prednisone acetate tablets) and a treatment group (rhizoma polygonati polysaccharide dosage water extract). Except for the normal control group, 6.5mg/kg of adriamycin is injected into the tail vein at one time, and 3.0mg/kg of adriamycin is injected into the tail vein again after two weeks. And after 1 week, measuring the quantitative rate of the urinary protein for 24h to be more than 80mg, and determining that the molding is successful. In the experimental process, the rats in the treatment group are respectively administrated with 216mg/kg of extract every day, the prednisone acetate positive control group is administrated by intragastric administration of 10mg/kg every day, and the normal group and the model group are intragastric administered with distilled water every day. The experimental period is 5 weeks, and 24h urine of rats is collected at 1w, 3w and 5w after molding respectively for quantitative analysis of urine protein.
(3) Sample collection and detection
(1) 24 hour urine protein quantification
After 1w, 3w, and 5w of the drug administration, urine was collected for 24 hours, and 24 hours of urine protein was measured using a full-automatic biochemical analyzer, and the 24 hours of urine protein quantitation = urine protein concentration × 24 hours of urine volume.
(2) Examination of biochemical indices related to renal function
Each group of rats was anesthetized with the abdominal cavity five weeks after the administration. Blood was collected from the abdominal aorta, and the blood sample was centrifuged at 3500r/min at 4 ℃ for 10min to separate the serum. The full-automatic biochemical analyzer is used for measuring 3 biochemical indexes of total serum protein (TP), serum Albumin (ALB) and creatinine (ALB).
(3) Observation of renal histopathology and morphology
i taking kidney tissue
After the experiment was completed, the left kidney was fixed with 10% neutral formalin solution and left for HE detection.
ii HE staining
Picking kidney, fixing part of kidney tissue with 10% neutral formalin solution, embedding paraffin into section, adding xylene, hydrating, staining with hematoxylin-eosin (HE), dehydrating with gradient alcohol, clearing xylene, sealing with resin adhesive, and examining pathological morphology under optical microscope.
(4) As a result, the
(1) 24 hour urine protein quantification
The results of the 24h urine protein quantitative analysis of each group are shown in table 1, and it can be seen from table 8 that the 24h urine protein amount of the rats in the model group is obviously increased from 3 weeks, and has a significant difference (p < 0.01) compared with the blank group; after 5 weeks, the polygonatum polysaccharide, the positive medicine group and the model group have significant differences (p is less than 0.05, p is less than 0.01), and the positive medicine group and the polygonatum polysaccharide have no significant differences.
TABLE 8 quantitative analysis results of 24-h urine protein in each group
(2) Examination of biochemical indices related to renal function
The results of the biochemical indexes related to kidney function of each group are shown in table 9, and compared with the model group, the normal group has significantly increased Cr, significantly decreased ALB and TP and has statistical significance (p is less than 0.01); compared with the model group, the rhizoma polygonati polysaccharide group and the positive drug Cr are reduced to a certain extent, ALB and TP are obviously improved (p is less than 0.01), which shows that the rhizoma polygonati polysaccharide has the function of obviously improving renal function, and the positive drug group and the rhizoma polygonati polysaccharide have no obvious difference.
TABLE 9 examination results of various groups of biochemical indicators related to kidney function
#### △△ p<0.01, △ p<0.05,compared with Control group；**p<0.01,*p<0.05,compared with Model group
(3) Observation of renal histopathology and morphology
The morphologic observation of renal tissue in each group is shown in fig. 11, and from the morphologic observation of the kidney, no obvious lesion was observed in glomerulus and renal tubule of the rats in the normal control group. The glomerulus of the model group is slightly atrophic, a little inflammatory cells infiltrate, the wall of the sacculus is slightly thickened, the sacculus cavity is slightly expanded, even a cavity appears in a local area, a protein tube type appears in a medullary area, and epithelial cells fall into the lumen; obvious fibrosis of the interstitium is visible, the focal inflammatory cells infiltrate the surrounding of the fibroplasia and the atrophic renal tubules are surrounded by the visible fibrosis tissue; mesangial cell proliferation, capillary congestion.
The administration group has the hyperplasia degree of mesenteric cells and mesenteric matrixes, the capillary hemangiosphere is adhered to the balloon wall, and the focal thickening degree of the balloon wall is lighter than that of the model group; blood stasis and bleeding of blood vessels among tissues, slight deformation of renal tubule lumens, and obvious improvement on the vacuole and fibrosis degree of kidney tissues compared with a model group.
Claims (2)
1. The application of polygonatum uniform polysaccharide in preparing a medicine for treating nephritis is characterized in that the preparation method of polygonatum uniform polysaccharide comprises the following steps:
s1, mixing rhizome of rhizoma polygonati with distilled water according to a ratio of 1g: mixing materials and liquid in a ratio of 3mL, decocting for 1-2 h in boiling water, filtering and collecting filtrate and filter residue; extracting the filter residue repeatedly for 1-2 times according to the operation, combining the filtrate obtained by each extraction, and concentrating the obtained filtrate to obtain an extracting solution;
s2, mixing the extracting solution obtained in the S1 with an ethanol solution with the volume fraction of 95% according to the proportion of 1:4, fully stirring, standing for 10-14 h, performing suction filtration to obtain a precipitate, and drying the precipitate to obtain crude polygonatum polysaccharide;
s3, purifying the crude polygonatum sibiricum polysaccharide by sequentially utilizing an anion-cation series exchange resin column and a Sephacyrl S400 gel column to obtain polygonatum sibiricum homogeneous polysaccharide; taking crude polygonatum sibiricum polysaccharide obtained in S2, preparing a 3% polysaccharide solution by using distilled water, centrifuging, taking supernatant, adding the supernatant into an FPA90Cl and FPC3500 anion-cation series exchange resin column, eluting by using distilled water, 0.5mol/L NaCl and 1mol/L NaCl in sequence, and concentrating, dialyzing, concentrating and freeze-drying each component obtained by eluting in sequence to obtain a water-washing component; preparing a water washing component obtained by purifying an S3 cation and anion series exchange resin column into a polysaccharide solution with the concentration of 3%, centrifuging, taking the supernatant, adding the supernatant into a Sephacyrl S400 gel column, sequentially carrying out gradient elution by using distilled water, 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L, 0.5mol/L and 1mol/L NaCl, carrying out the flow rate of 2mL/min, collecting the water washing component, dialyzing by using the distilled water, carrying out reduced pressure concentration, and carrying out freeze drying to obtain the polygonatum uniform polysaccharide;
s3, the active ingredients of the polygonatum uniform polysaccharide prepared by the method consist of mannose, fucose and galactose, wherein the ratio of the mannose, the fucose and the galactose is 1.
2. The use of claim 1, wherein S2 further comprises precipitating with 95% by volume ethanol for 1-2 times after the precipitate is obtained by standing.
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