Nine-processed rhizoma polygonati polysaccharide, preparation method thereof and application of nine-processed rhizoma polygonati polysaccharide in preparation of medicine for treating renal injury

Abstract

The invention provides processed rhizoma polygonati polysaccharide, a preparation method thereof and application of the processed rhizoma polygonati polysaccharide in preparation of a medicine for treating renal injury. The nine-processed polygonatum sibiricum polysaccharide is named as NPCP and is composed of fructose and glucose, the NPCP is straight-chain inulin type polysaccharide with the polymerization degree of 12, and the structure of the NPCP is Glcp-(1-) and Fruf-(2-terminal,-1)-Fruf-(2-main chain. According to the obtained uniform nine-process polygonatum polysaccharide NPCP, the protective effect on adenine-induced HK-2 cell injury is achieved by improving the content of renal function markers, improving oxidative stress injury, reducing expression of proinflammatory cytokines, improving intestinal flora disorder and up-regulating short-chain fatty acid in the intestinal tract; more treatment directions are provided for effectively improving adenine-induced renal toxicity by the nine-process polygonatum polysaccharide, and theoretical and experimental bases are provided for clinically finding effective preparation of drugs for treating renal injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polysaccharide prepared from polygonatum sibiricum and a preparation method thereof, and an application thereof in the preparation of a drug for treating kidney damage. Background Art

[0002] The kidney is an organ of vertebrates and part of the urinary system. It is responsible for filtering impurities in the blood, maintaining the balance of body fluids and electrolytes, and finally producing urine to be discharged from the body through the urethra. It also has endocrine functions to regulate blood pressure. The kidney is an important organ for maintaining life activities. Kidney damage refers to renal ischemia, hypoxia, renal parenchymal fibrosis, proteinuria, and progressive decline in renal function due to spasm, sclerosis, degeneration, and degeneration of renal arterioles. The main causes of kidney damage include trauma, kidney stones, nephritis, irregular work and rest, bad living habits, etc. Kidney damage may cause body edema, abnormal urine, back pain, decreased renal function, renal failure and other hazards, which may endanger illness in severe cases. For humans, kidney damage accounts for about 65% of urogenital tract damage. The edema, pain, and even uremia caused by it seriously affect people's quality of life. At present, there is no effective treatment for kidney damage in clinical practice, and it is difficult for targeted drugs to play a synergistic role. Mainstream clinical drugs such as hormones, antibiotics and antihypertensive drugs all need to be used for a long time and require high concentrations. However, high doses and high concentrations often bring serious adverse reactions. For example, non-steroidal anti-inflammatory drugs, hormone drugs, penicillin antibiotics, ACEI, immunosuppressive drugs, etc. often lead to more serious side effects. Therefore, it is very important to improve the safety and effectiveness of drugs for the treatment of kidney diseases.

[0003] Polygonatum sibiricum contains abundant polysaccharide components, and its chemical structure is complex. The content, structure and activity of polygonatum sibiricum polysaccharide from different sources and varieties vary greatly. Currently, there is no research on the use of processed Polygonatum sibiricum polysaccharide alone for the treatment of renal damage. Summary of the invention

[0004] In view of this, the present invention proposes a nine-processed polygonatum polysaccharide and a preparation method thereof and an application thereof in the preparation of a drug for treating kidney injury.

[0005] The technical solution of the present invention is achieved in this way:

[0006] A processed polygonatum polysaccharide, named NPCP, is composed of fructose and glucose. NPCP is a straight-chain inulin-type polysaccharide with a degree of polymerization of 12, and has a structure of Glcp-(1→ and Fruf-(2→ ends, →1)-Fruf-(2→ main chain, as shown in Formula I;

[0007]

[0008] Furthermore, the NPCP is a neutral polysaccharide with a weight-average molecular weight of 2053 Da.

[0009] A preparation method of polygonatum sibiricum polysaccharide processed nine times. An extract is obtained by extracting polygonatum sibiricum processed nine times through water extraction and alcohol precipitation method, followed by decolorization treatment with D101 macroporous adsorption resin, enzymatic hydrolysis, and removal of proteins using Sevage reagent to obtain a crude polygonatum sibiricum polysaccharide processed nine times. Then, it is eluted and purified through a DEAE-52 cellulose anion exchange chromatography column and a Sephadex G-25 gel chromatography column to obtain the target polygonatum sibiricum polysaccharide NPCP.

[0010] Furthermore, the specific preparation method includes:

[0011] S1. Dry and crush polygonatum sibiricum processed nine times, add the polygonatum sibiricum processed nine times powder to deionized water for extraction, repeat 2 - 4 times, centrifuge to recover the supernatant, concentrate under reduced pressure, add absolute ethanol to the concentrated solution for alcohol precipitation, and centrifuge to collect the alcohol precipitate.

[0012] S2. Dissolve the alcohol precipitate completely, centrifuge to remove impurities, concentrate under reduced pressure, filter, perform decolorization treatment on the concentrated solution using D101 macroporous adsorption resin, add papain for enzymatic hydrolysis, inactivate by centrifugation, use Sevage reagent to remove proteins, filter, dialyze, and freeze-dry to obtain a crude polygonatum sibiricum polysaccharide processed nine times, labeled as NPCPs.

[0013] S3. Take the NPCPs solution, load it onto a DEAE-52 cellulose anion exchange chromatography column, and elute with deionized water, 0.05 M NaCl, and 0.1 M NaCl solutions at a flow rate of 1 mL / min to initially obtain a crude neutral polysaccharide NPCPs eluted with deionized water.

[0014] S4. Purify using a Sephadex G-25 gel chromatography column, elute with deionized water at a flow rate of 0.5 mL / min, and freeze-dry to obtain a uniform polygonatum sibiricum polysaccharide NPCP.

[0015] Furthermore, in step S1, the material-liquid ratio of the polygonatum sibiricum processed nine times powder to deionized water is 1:10 - 20 g / mL; the extraction temperature is 90 - 100 °C, and the time is 2 - 4 h; the volume ratio of absolute ethanol to the concentrated solution is 3 - 5:1; the alcohol precipitation is carried out at 0 - 4 °C for 12 - 24 h.

[0016] Furthermore, in step S2, the addition amount of papain is 2 wt% - 4 wt% of the concentrated solution, and the enzyme activity is 8×10 5 u / g. The temperature of the enzymatic hydrolysis is 50 - 55 °C, and the time is 1 - 3 h; the dialysis is carried out at 0 - 4 °C using a dialysis bag with a cut-off molecular weight of 1 kDa for 48 - 72 h.

[0017] Application of nona-processed polygonatum polysaccharide in preparing medicament for treating kidney injury

[0018] Furthermore, the medicament for treating kidney injury comprises the nona-processed polygonatum polysaccharide and pharmaceutically acceptable excipients

[0019] Furthermore, the medicament for treating kidney injury contains 200 - 400 mg / kg of uniform nona-processed polygonatum polysaccharide NPCP

[0020] Furthermore, the nona-processed polygonatum polysaccharide improves kidney injury through the following ways

[0021] (1) Improving the content of renal function markers, and reducing the contents of serum creatinine, blood urea nitrogen and uric acid

[0022] (2) Improving oxidative stress injury, reducing the contents of reactive oxygen species and malondialdehyde in vivo, and increasing the contents of SOD superoxide dismutase and catalase

[0023] (3) Reducing the expressions of pro-inflammatory cytokines TNF-α, IL-1β and IL-6

[0024] (4) Improving intestinal flora disorder, increasing the abundances of Rikenella and Parasutterella genera, and reducing the abundances of ASF356 and Blautia genera

[0025] (5) Up-regulating short-chain fatty acids in the intestine, and increasing the contents of acetic acid, propionic acid, isobutyric acid and butyric acid

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows

[0027] The uniform nona-processed polygonatum polysaccharide NPCP obtained in the present invention realizes the protective effect on adenine-induced HK-2 cell injury by improving the content of renal function markers, improving oxidative stress injury, reducing the expression of pro-inflammatory cytokines, improving intestinal flora disorder and up-regulating short-chain fatty acids in the intestine, providing more treatment directions for the effective improvement of adenine-induced nephrotoxicity by nona-processed polygonatum polysaccharide. The present invention provides a theoretical and experimental basis for clinically searching for effective preparation of medicaments for treating kidney injury Description of the Drawings

[0028] Figure 1 It is the isolation and purification curve diagram of nona-processed polygonatum polysaccharide NPCP, wherein A is the glucose standard curve, B is the DEAE-52 elution curve, and C is the Sephadex G-25 elution curve

[0029] Figure 2 It is the HPGPC chromatogram of nona-processed polygonatum polysaccharide NPCP

[0030] Figure 3UV and IR spectra of nona-processed polygonatum polysaccharide NPCP, where A is the UV spectrum and B is the IR spectrum.

[0031] Figure 4 Monosaccharide composition analysis chart of nona-processed polygonatum polysaccharide NPCP, where A is the monosaccharide standard and B is the monosaccharide composition chart of the NPCP sample.

[0032] Figure 5 Standard curve chart drawn by HPGPC of nona-processed polygonatum polysaccharide NPCP, where A is the LogMp-T standard curve chart, B is the LogMw-T standard curve chart, C is the LogMn-T standard curve chart, and D is the HPGPC chromatogram.

[0033] Figure 6 Total ion mass spectrometry signal chart of PMAAs of nona-processed polygonatum polysaccharide NPCP.

[0034] Figure 7 Second-order mass spectrometry chart of 2,5-di-O-acetyl-1,3,4,6-tetra-O-methyl(mannitol,glucitol) of NPCP.

[0035] Figure 8 Second-order mass spectrometry chart of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol of NPCP.

[0036] Figure 9 Second-order mass spectrometry chart of 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl(mannitol,glucitol) of NPCP.

[0037] Figure 10 Nuclear magnetic resonance chart of nona-processed polygonatum polysaccharide NPCP, where A is 1 1H-NMR spectrum, B is 13 13C-NMR spectrum, C is the HSQC two-dimensional spectrum, D is the COSY two-dimensional spectrum, E is the HMBC two-dimensional spectrum, and F is the NOESY two-dimensional spectrum.

[0038] Figure 11 Saccharide chain structure chart of nona-processed polygonatum polysaccharide NPCP.

[0039] Figure 12 Influence chart of nona-processed polygonatum polysaccharide NPCP on the viability of HK-2 cells.

[0040] Figure 13 Influence chart of nona-processed polygonatum polysaccharide NPCP on oxidative stress in an in vitro CKD model, where A is the SOD content, B is the catalase CAT, and C is the malondialdehyde MDA.

[0041] Figure 14 It is a diagram showing the effect of nona-processed polygonatum polysaccharide NPCP on the levels of inflammatory factors in an in vitro CKD model. Among them, A is the TNF-α level, B is the IL-1β level, and C is the IL-6 level.

[0042] Figure 15 It is a diagram showing the effect of nona-processed polygonatum polysaccharide NPCP on the renal function of CKD mice. Among them, A is serum creatinine CREA, B is blood urea nitrogen BUN, and C is blood uric acid BUA.

[0043] Figure 16 It is a diagram of kidney staining of CKD mice by different staining methods.

[0044] Figure 17 It is a diagram showing the effect of nona-processed polygonatum polysaccharide NPCP on oxidative stress in CKD mice. Among them, A is the green fluorescence of reactive oxygen species ROS, B is the content of SOD, C is catalase CAT, and D is malondialdehyde MDA.

[0045] Figure 18 It is a diagram showing the effect of nona-processed polygonatum polysaccharide NPCP on the levels of inflammatory factors. Among them, A is the TNF-α level, B is the IL-1β level, and C is the IL-6 level.

[0046] Figure 19 It is a diagram showing the effect of nona-processed polygonatum polysaccharide NPCP on the intestinal flora. Among them, A is the Rank-Abundance curve, B is the Sobs index, C is the Shannon index, D is the Venn diagram of OTUs of mouse intestinal microbiota, E is the Shannon index, F is the Chao index, G is the Sobs index, H is the principal coordinate analysis (PCoA), I is the barplot analysis, J is the phylum-level analysis, K is the genus-level analysis, L is the LEfSe analysis, and M is the Cladogram analysis.

[0047] Figure 20 It is a diagram showing the effect of nona-processed polygonatum polysaccharide NPCP on short-chain fatty acids. Among them, A is the acetic acid content, B is the propionic acid content, C is the butyric acid content, and D is the isobutyric acid content. Detailed implementation manners

[0048] To better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0049] The experimental methods used in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0050] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels unless otherwise specified.

[0051] Example 1

[0052] S1. Dry and pulverize the nine-processed polygonatum sibiricum, add the nine-processed polygonatum sibiricum powder to deionized water according to a solid-liquid ratio of 1:15 g / mL, treat it at 95 °C for 3 h, repeat 3 times, centrifuge at 8000 rpm for 10 min to recover the supernatant, concentrate under reduced pressure, add the concentrated solution to absolute ethanol, and the volume ratio of absolute ethanol to the concentrated solution is 4:1. Perform alcohol precipitation at 4 °C for 12 h, and centrifuge at 5000 rpm for 10 min to collect the alcohol-precipitated product;

[0053] S2. Add the alcohol-precipitated product to deionized water to fully dissolve the precipitate, centrifuge at 20000 rpm for 15 min to remove impurities, concentrate under reduced pressure, filter through a 0.22 μm microporous membrane, perform decolorization treatment with D101 macroporous adsorption resin, then add 3 wt% papain (enzyme activity of 8×10 5 u / g), carry out enzymatic hydrolysis at 50 °C for 3 h, heat at 95 °C for 10 min to inactivate the enzyme, centrifuge at 20000 rpm for 15 min to remove the inactivated enzyme and denatured protein, use Sevage reagent to further remove proteins, concentrate after no obvious protein absorption peak is detected by ultraviolet detection, filter through a 0.22 μm microporous membrane, dialyze at 4 °C using a dialysis bag with a molecular weight cut-off of 1 kDa for 72 h, change the water outside the dialysis bag every 8 h, and freeze-dry to obtain the crude product of nine-processed polygonatum sibiricum polysaccharide, labeled as NPCPs;

[0054] S3. Take the NPCPs solution, load it onto a DEAE-52 cellulose anion exchange chromatography column (2.5 cm × 40 cm), and elute with deionized water, 0.05 M NaCl, and 0.1 M NaCl solutions at a flow rate of 1 mL / min respectively. Collect one tube every 5 min. After the eluate is detected by anthrone-sulfuric acid reagent, combine and concentrate, and freeze-dry to preliminarily obtain the crude product of neutral polysaccharide NPCPs eluted with deionized water;

[0055] S4. Purify using a Sephadex G-25 gel chromatography column (1.6 cm × 60 cm), elute with deionized water at a flow rate of 0.5 mL / min, collect one tube every 6 min. After the eluate is detected by anthrone-sulfuric acid reagent, combine and concentrate, and freeze-dry to obtain the homogeneous nine-processed polygonatum sibiricum polysaccharide NPCP.

[0056] Example 2

[0057] Identify the nine-processed polygonatum sibiricum polysaccharide NPCP prepared in Example 1.

[0058] 1. Determination of the content of nine-processed polygonatum sibiricum polysaccharide NPCP

[0059] Dissolve 0.2 g of anthrone in 100 mL of concentrated sulfuric acid to prepare the color-developing solution, and make up to 100 mL with 0.1 g of anhydrous glucose to prepare a 1 mg / mL stock solution. During the construction of the standard curve, after diluting the standard solution in the range of 0 - 1.0 mL stepwise and making up to 2 mL with distilled water, mix it thoroughly with 6 mL of the color-developing solution respectively. After heat treatment in a boiling water bath for 10 min and then cooling, the absorbance values of each group are detected by a spectrophotometer at a wavelength of 620 nm. When measuring the sample, make up the NPCP to be detected to 2 mL and react with the color-developing solution according to the same steps. Finally, calculate the sugar content by the standard curve method. The results are as Figure 1 shown.

[0060] Results: The extraction rate of NPCPs was 8.52%. The anthrone-sulfuric acid method was used to determine the polysaccharide content, and the relationship between its concentration and absorbance was determined by plotting a glucose standard curve. See Figure 1 A. The standard curve was obtained by fitting the experimental data, and linear regression analysis was performed based on the data. Finally, the linear regression equation y = 2.16x + 0.045 was obtained, and the correlation coefficient R 2 was 0.9969. According to this curve, the polysaccharide content of NPCPs was 93.62%. See Figure 1 B. The water elution fraction was obtained after NPCPs were separated by a DEAE-52 cellulose column. See Figure 1 C. The eluates were further purified by a Sephadex G-25 gel chromatography column, combined, and freeze-dried to obtain NPCP.

[0061] 2. Purity determination of the polysaccharide NPCP from Polygonatum sibiricum Red. var. multinodum Makino

[0062] Dissolve NPCP in deionized water to prepare a 1 mg / mL solution, with an injection volume of 50 μL. Use an Lc-10Avp high-performance gel permeation chromatography (HPGPC) system (Shimadzu, Japan), equipped with an RID-10A differential refractive index detector and assembled with a TSKgel G3000PWXL chromatographic column (7.8×300 mm). Elution program: The mobile phase is deionized water, the column oven temperature is 30 °C, and the flow rate is 0.7 mL / min. The results are as Figure 2 shown.

[0063] Results: See Figure 2 the HPGPC chromatogram. It can be seen that NPCP is a single symmetric narrow peak, with a retention time of 12.45 min, and the purity shows >99.5%, indicating that the NPCP of the present invention is a highly pure and homogeneous polysaccharide.

[0064] 3. UV and IR spectrum determination

[0065] Take a small amount of NPCP and dissolve it in deionized water to prepare a 1 mg / mL solution. Using deionized water as a control, perform a full ultraviolet wavelength scan at 200 - 600 nm. If there are no obvious absorption peaks at 260 nm and 280 nm, it indicates that nucleic acids and proteins have been removed. Weigh a small amount of the polysaccharide sample, mix it evenly with 200 mg of potassium bromide, and press it into a sample with a thickness of 1 mm. Then perform an infrared spectrum scan in the range of 4000 - 450 cm -1 range. The results are as Figure 3 shown.

[0066] Results: Refer to Figure 3 the ultraviolet spectrum of A. It can be seen that there are no obvious ultraviolet absorption peaks at 260 nm and 280 nm, indicating that the NPCP of the present invention does not contain nucleic acids and proteins.

[0067] Refer to Figure 3 the infrared spectrum of B. It can be seen that the absorption peak in the range of 3600 - 3200 cm -1 is the stretching vibration of -OH, which is a characteristic absorption peak of carbohydrate substances. Specific analysis: The stretching vibration peak at 3278.73 cm -1 is the characteristic absorption peak of O-H of sugar. The absorption peak at 2932.69 cm -1 is the stretching vibration of C-H. The peak at 1632.45 cm -1 is the stretching vibration of C=O. The weak infrared absorption peak at 815.11 cm -1 is the C-H stretching vibration of α-D-Glc. The peak value at 918.29 cm -1 indicates the symmetric stretching vibration of the furan ring. The characteristic peak value at 864.08 cm -1 indicates the asymmetric vibration of C-H of the furan ring, indicating that there is a furanose structure in the NPCP of the present invention. The peak at 1046.33 cm -1 is the C-O stretching vibration, which is the pyranose structure.

[0068] 4. Determination of monosaccharide composition

[0069] Hydrolyze 5 mg of NPCP with 2 M trifluoroacetic acid (TFA) at 121 °C for 2 h. After the sample is dried with nitrogen, wash it repeatedly with methanol 2 - 3 times, filter and dry it. Dissolve the obtained residue in deionized water, filter it through a 0.22 μm microporous membrane, and further analyze the sample extract by high performance anion exchange chromatography (HPAEC) combined with a CarboPac PA - 20 anion exchange column (3 × 150 mm, Dionex), equipped with a pulsed amperometric detector (PAD, Dionex ICS 5000+ system). Detection conditions: flow rate is 0.5 mL / min, injection volume is 5 μL, solvent system A is pure water, solvent system B is 0.1 M NaOH, and solvent system C is a mixture of 0.1 M NaOH and 0.2 M NaAc.

[0070] Gradient elution: 0 min: solvent system A, B, C = 95:5:0;

[0071] 26 min: solvent system A, B, C = 85:5:10;

[0072] 42 min: solvent system A, B, C = 85:5:10;

[0073] 42.1 min: solvent system A, B, C = 60:0:40;

[0074] 52 min: solvent system A, B, C = 60:40:0;

[0075] 52.1 min: solvent system A, B, C = 95:5:0;

[0076] 60 min: solvent system A, B, C = 95:5:0.

[0077] Use chromeleon 7.2 CDS (Thermo Scientific) to analyze the data. The results are as Figure 4 shown.

[0078] Results: See Figure 4 , by comparing the retention times of the acid hydrolysis products of NPCP of the present invention with those of monosaccharide standards, it can be seen that the NPCP of the present invention is composed of fructose and glucose.

[0079] 5. Molecular weight determination

[0080] Dextrans with different relative molecular weights were selected as standards, dissolved in 0.05 M NaCl solution to prepare a standard solution with a concentration of 5 mg / ml, and filtered through a 0.22 μm microporous membrane for later use. The standards were separated and detected by a high-performance gel permeation chromatography tandem column (3 polymer matrix water-soluble SEC (GFC) chromatographic columns (8 × 300 mm) in series), and a calibration curve was established by plotting the logarithm of the molecular weight against the retention time.

[0081] 5 mg of the NPCP sample was dissolved in 1 mL of 0.05 M NaCl solution, filtered, and analyzed under the same chromatographic conditions. The retention time (T) measured was substituted into the calibration equation to calculate its molecular weight. Chromatography: mobile phase 0.05 M NaCl solution; flow rate 0.65 mL / min; column temperature 40 °C; injection volume: 30 μL. The results are as Figure 5 shown.

[0082] Results: Refer to Figure 5 The standard curve drawn by HPGPC in 2 can give the peak molecular weight calibration curve equation of LogMp - T: Log Mp = -0.1725T + 10.6517, R

[0083] The weight-average molecular weight calibration curve equation of LogMw - T: Log Mw = -0.1811T + 11.0551, R 2 = 0.9936;

[0084] The number-average molecular weight calibration curve equation of LogMn - T: Log Mn = -0.1722T + 10.5589, R 2 = 0.9943;

[0085] Substituting gives Mp = 1891 Da, Mw = 2053 Da, Mn = 1573 Da.

[0086] 6. Methylation determination

[0087] Mix 1 mL of deionized water with 2 mL of dichloromethane and vortex. After centrifugation, discard the aqueous phase. Repeat the water washing step three times. Collect the lower dichloromethane phase and dry it under a nitrogen stream. Subsequently, add 1 mL of 2 M TFA and react at 120 °C for 60 min. Then add 1 mL of freshly prepared 1 M NaBD4, incubate with magnetic stirring at room temperature for 2.5 h, and add 300 μL of acetic acid to terminate the reaction. Add 2 mL of 5% v / v acetic acid in methanol to the sample, dry it twice under a nitrogen stream at 40 °C, then use 2 mL of methanol and perform two nitrogen drying treatments at the same temperature. Then add 1.5 mL of acetic anhydride and mix well by vortexing. Next, heat the reaction system to 100 °C and react for 2.5 h. After the reaction is completed, add 2 mL of water and let it stand for 10 min. Then add 1 mL of dichloromethane and vortex thoroughly. After centrifugation, discard the aqueous phase and repeat the water washing three times to ensure complete removal of impurities.

[0088] Use an Agilent gas chromatography-mass spectrometry (GC-MS) system in combination with a public database of partially methylated alditol acetates (PMAAs) to analyze and compare the glycosidic bond types of polysaccharides. Chromatographic conditions: HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm); carrier gas is high-purity helium (purity ≥ 99.999%); flow rate is 1.0 mL / min; injection port temperature is 260 °C; injection volume is 1 μL; split ratio is 10:1; solvent delay is 2.2 min. Temperature programming: Hold at 50 °C for 1.0 min, then increase the temperature to 130 °C at a rate of 50 °C / min, and then increase the temperature to 230 °C at a rate of 3 °C / min and hold for 2 min. Mass spectrometry conditions: Equipped with an electron impact ionization source (EI) and a MassHunter workstation; injection port temperature is 230 °C; quadrupole temperature is 150 °C; electron energy is 70 eV; use the full scan mode (SCAN), and the mass scan range (m / z) is 30 - 600. Compare the obtained polysaccharide mass spectrometry fragments with the public database of partially methylated alditol acetates (PMAAs) to analyze the bonding mode of sugar residues.

[0089] Through GC-MS analysis and comparison with the standard database, the total ion chromatogram is obtained, as Figure 6 shown.

[0090] Results: Based on Figure 6 the total ion mass spectrometry signals of PMAAs of NPCP, combined with Figures 7 - 9 the secondary mass spectrometry diagram, the sugar residue composition of NPCP can be obtained, as shown in Table 1.

[0091] Table 1

[0092]

[0093]

[0094] Result: As can be seen from Table 1, NPCP is composed of three sugar residues.

[0095] 7. Nuclear magnetic resonance analysis

[0096] Take an appropriate amount of NPCP sample and dissolve it in D2O to a final concentration of 40 mg / mL. Scan 1D and 2D NMR ( 1 H, 13 C, COSY, NOESY, HMBC and HSQC) at 25 °C using a Bruker AVANCE NEO 500M spectrometer system (Bruker, Germany) and record the relevant data. The results are as Figure 10 shown.

[0097] Result: Refer to Figure 10 the 1 1H-NMR spectrum of A. There is a signal peak at δ 5.22 in the range of 3.00 ppm - 5.00 ppm, indicating the presence of an α-configured glycoside. Combining the monosaccharide composition and methylation results, it shows that this is the anomeric hydrogen proton signal of the α-D-Glcp fragment, which is consistent with the result of the presence of a pyranose conformation in NPCP corresponding to the peak at 815.11 cm Figure 3 in B. Fructose belongs to furanose, and the connection mode of fructose is usually β-D-Fruf-(2→, →1)-β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, so there is no anomeric hydrogen proton signal. -1 Refer to

[0098] the Figure 10 13C-NMR spectrum of B. There are a total of three sets of anomeric carbon signals, which is consistent with the methylation result of having three residue fragments, indicating that δ 100.47 is the C-1 signal of α-D-Glcp, while δ 101.6 and δ 105.1 are the isomeric carbon atoms of β-D-Fruf residues in different chemical environments. 13 According to

[0099] the Figure 10The HSQC two-dimensional spectrum of C and the COSY two-dimensional spectrum of 10D were used to assign the chemical shifts of the hydrogen protons of their residue fragments, as shown in Table 2. The chemical shifts of the carbon signals, δ100.47, 72.25, 75.2, 73.5, 71.2, 62.3, correspond to C1, C2, C3, C4, C5, C6 of fragment A; the chemical shifts corresponding to C1, C3, C4, C5, C6 of fragment B are δ62.3, 101.6, 78.1, 75.3, 83.0, 64.0; finally, the chemical shifts of δ62.3, 105.1, 78.8, 78.6, 79.0, 62.7 were assigned to fragment C.

[0100] Table 2

[0101]

[0102]

[0103] According to Figure 10 The HMBC two-dimensional spectrum of D and the NOESY two-dimensional spectrum of 10F were used to connect the residue fragments of each group. There is an obvious correlation signal between C2 (δ101.6) of fragment B and H-1 (δ5.22) of fragment A; there is a cross peak between C2 (δ105.1) of fragment C and H-1 (δ3.64) of fragment B.

[0104] Therefore, the NPCP of the present invention has Glcp-(1→ and Fruf-(2→ at the ends, a main chain of →1)-Fruf-(2→, and is a linear inulin-type polysaccharide with a degree of polymerization of 12, and its structure is as Figure 11 shown.

[0105] Example 3

[0106] 1. In vitro experiment

[0107] Cell model establishment: The medium used for HK-2 cells is DMEM / F12 medium containing 10% fetal bovine serum, and the culture conditions are 37°C and 5% CO2 incubator.

[0108] Drug treatment: HK-2 cells were treated with 10 ng / mL of TGF-β1 to establish an in vitro CKD model.

[0109] A. Cell viability assay: Logarithmically growing HK-2 cells were divided into a blank control group, a model group, and polysaccharide NPCP treatment groups at different concentrations (100, 200, 300, 400, 500 μg / mL), and cultured for another 24 h. The model group and the polysaccharide NPCP treatment groups at different concentrations were used to treat HK-2 cells with 500 μg / mL thiazolyl blue (MTT) for 4 h. The formed blue azo groups were dissolved in 10% SDS - 5% isobutanol - 0.01 M HCl, and the absorbance was measured at 570 nm with a reference of 630 nm to calculate cell viability. The results are as Figure 9 shown.

[0110] Among them, cell viability = (A 实验孔 - A 空白孔 ) / (A 对照孔 - A 空白孔 ) × 100%

[0111] Conclusion: NPCP did not show obvious cytotoxicity. As Figure 12 can be seen, compared with the blank control group, the viability of HK-2 cells in the model group decreased significantly (P < 0.01). Compared with the model group, the NPCP administration groups at 200, 300, 400, and 500 μg / mL could significantly increase the viability of HK-2 cells (P < 0.01), and the cell viability was the highest at 400 μg / mL.

[0112] B. Using a kit, TNF-α, IL-6, IL-1β, SOD, CAT, and MDA were measured according to the instructions of the manual. The results are as Figures 13 - 14 shown.

[0113] Conclusion: As Figure 13 can be seen from the in vitro experimental results, compared with the blank control group, the contents of SOD and catalase (CAT) in the model group decreased significantly (P < 0.01), and the content of malondialdehyde (MDA) increased significantly (P < 0.01). Compared with the model group, the contents of SOD and catalase (CAT) in the low-dose NPCP group and the high-dose NPCP group increased significantly (P < 0.01), and the level of malondialdehyde (MDA) decreased significantly (P < 0.01), indicating that NPCP of the present invention has an effect of inhibiting oxidative stress.

[0114] As Figure 14The results of in vitro experiments showed that when HK-2 cells were treated with TGF-β1 in vitro, the model group showed higher inflammation levels (TNF-α, IL-1β, IL-6) than the control group (P<0.01), while NPCP treatment could significantly reduce the expression of proinflammatory cytokines TNF-α, IL-1β and IL-6 (P<0.01), and showed dose dependence. This showed that NPCP could inhibit the body's inflammatory response in the CKD in vitro model.

[0115] 2. In vivo experiments

[0116] 2.1 CKD mouse modeling: 40 SPF mice were purchased, weighing 50±5g, and adaptively fed for 7 days. The mice were randomly divided into 4 groups, including a blank control group, a model group, a low-dose NPCP group (200mg / kg), and a high-dose NPCP group (400mg / kg), with 10 mice in each group. The blank control group was always gavaged with normal saline, and the model group, the low-dose NPCP group (200mg / kg), and the high-dose NPCP group (400mg / kg) were gavaged with adenine 250mg / kg every day for 14 days, followed by gavage every other day for 14 days. Then the model group was gavaged with normal saline, and the low-dose NPCP group (200mg / kg) and the high-dose NPCP group were gavaged with the corresponding NPCP dose every day for 28 days, and the mice were anesthetized and killed for sampling.

[0117] 2.2 Sample collection: Fresh feces of mice were collected one day before the mice were killed and placed in an ultra-low temperature refrigerator for intestinal flora and short-chain fatty acid experiments.

[0118] The mice were fasted but not watered one day before being killed and anesthetized with 10% tribromoethanol. The eyeballs were removed and blood was collected. The blood was left at room temperature for 2 hours to coagulate and then centrifuged at 4°C, 4000 rpm for 15 minutes to collect the upper serum and store it for later use.

[0119] Subsequently, the mice were killed by cervical dislocation and dissected, and the kidneys were collected for later use. Part of the kidneys were fixed in paraformaldehyde solution for histopathological staining; part of the kidney tissue was added to pre-cooled PBS solution at a ratio of 1:9 g / mL, mixed and ground, and centrifuged at 4000 rpm for 10 min to collect the upper tissue homogenate.

[0120] 2.3 Detect serum biochemical indicators (BUN, BUA, CREA, TNF-α, IL-6, IL-1β, SOD, CAT, MDA) according to the kit instructions. The results are as follows: Figure 15 shown.

[0121] Conclusion: From Figure 15It can be seen that, compared with the blank control group, the contents of serum creatinine (CREA), blood urea nitrogen (BUN) and blood uric acid (BUA) in the model group were significantly increased (P<0.01), indicating a decrease in renal function in the model group mice. Compared with the model group, after NPCP intervention treatment, the levels of CREA content, blood urea nitrogen (BUN) content and blood uric acid (BUA) content were significantly decreased (P<0.01). It indicates that the NPCP of the present invention can improve the decline in renal function of adenine-induced CKD mice.

[0122] 2.4 Histopathological observation: Tissue samples were taken and fixed in 10% paraformaldehyde, dehydrated, paraffin-embedded and sectioned (with a thickness of 4-5 μm).

[0123] For HE staining, after dewaxing and rehydrating the sections, they were placed in HE staining solution, dehydrated and mounted after staining;

[0124] For PAS staining, after HE staining, the sections were oxidized with periodic acid, then stained with Schiff reagent, and finally counterstained with hematoxylin;

[0125] The steps of Masson staining included dewaxing and hydrating the paraffin sections first, then staining the muscle and collagen areas with Weigert iron hematoxylin and Alcian blue staining, and finally dehydrating and mounting.

[0126] Finally, observation was made under the microscope, and the results were as Figure 16 shown.

[0127] Conclusion: As Figure 16 can be seen, after adenine gavage, the kidneys of CKD mice showed severe dilation of renal tubules, atrophy of glomeruli and a large amount of inflammatory infiltration. It can be observed from the Masson staining that the mice in the model group had relatively severe fibrosis. After treatment with low-dose NPCP and high-dose NPCP, the pathological changes of the kidneys could be improved to varying degrees, the cell structure was relatively complete, the arrangement was relatively regular and the cell gaps were clearly distinguishable. It indicates that the NPCP of the present invention can effectively improve kidney damage in CKD mice.

[0128] 2.5 ROS fluorescence staining: The tissue to be treated was washed with pre-cooled PBS buffer, and the tissue was cut into pieces about 1 mm with ophthalmic scissors 3The small pieces were continuously rinsed in pre-chilled PBS to remove cell debris. Filter through a 300-mesh nylon mesh, and gently operate with an ophthalmic forceps or a scraper, rubbing the tissue while rinsing until it is completely dissociated, and collect the cell suspension. Centrifuge the cell suspension at 500 rpm for 10 min, discard the supernatant and wash the precipitate with PBS. Resuspend the cell precipitate with an appropriate concentration of DCFH-DA solution, adjust the cell concentration, incubate at 37 °C for 40 min, centrifuge at 1000 rpm for 10 min, wash with PBS to remove unbound DCFH-DA, and finally resuspend the cells and observe under a fluorescence microscope. The results are as Figure 17 shown.

[0129] Conclusion: It can be seen from Figure 17 that, compared with the blank control group, the model group showed strong green fluorescence of reactive oxygen species (ROS). Under the intervention treatment of low-dose NPCP and high-dose NPCP, the green fluorescence gradually weakened, indicating that the NPCP of the present invention can reduce the ROS content in CKD mice and has good antioxidant ability.

[0130] Compared with the blank control group, the SOD content and catalase (CAT) content in the model group were significantly decreased (P < 0.01), and the content of malondialdehyde (MDA) was significantly increased (P < 0.01). Compared with the model group, the SOD content and catalase (CAT) content in the low-dose NPCP group and high-dose NPCP group were significantly increased (P < 0.01), and the level of malondialdehyde (MDA) was significantly decreased (P < 0.01). It shows that the NPCP of the present invention has the effect of inhibiting oxidative stress, can restore the body's redox homeostasis, and effectively improve the oxidative stress injury of CKD mice.

[0131] 2.6 Effect on the inflammatory level of CKD mice: See Figure 18 that, compared with the blank control group, the model group showed a higher inflammatory level (TNF-α, IL-1β, IL-6) (P < 0.01). After NPCP intervention treatment, the expression of pro-inflammatory cytokines was significantly decreased (P < 0.01), and it showed a dose dependence. It shows that the NPCP of the present invention can inhibit the body's inflammatory response and effectively improve the inflammatory injury of CKD mice.

[0132] 2.7 Effect on the intestinal flora of CKD mice

[0133] 16S rRNA gene sequencing: On the last three days of the experiment, feces were collected and quickly frozen in liquid nitrogen, and total fecal DNA was extracted for PCR amplification of the 16S rRNA gene. The PCR product was purified using the Qiagen gel extraction kit (Germantown, USA), and the library quality was checked and sequenced. After quality control, 50,000 high-quality sequences were obtained for each sample, and sequences with a similarity of more than 97% were classified into the same operational taxonomic units (OTUs). The Venn diagram results showed that there were significant differences in the number of OTUs among the normal group, model group, and NPCP group, indicating that there were differences in the similarity of the intestinal flora of each group of mice.

[0134] from Figure 19 The α-diversity analysis of EG showed that the diversity of intestinal flora was significantly reduced in the model group, while the richness and diversity of the flora were restored after NPCP intervention treatment. Figure 19 In the β diversity of G, PCoA analysis showed that due to the influence of disease and NPCP, the intestinal flora of the three groups of mice were separated from each other, and the overall structure of the intestinal flora of mice changed. Firmicutes and Bacteroidetes are the two main bacterial genera in mouse feces. The increase in F / B may lead to the aggravation of intestinal inflammation, thereby affecting the systemic immune status. Inflammatory response is one of the main causes of chronic kidney disease progression. Figure 19 IK further showed that the NPCP group could reverse the imbalance of the two genera caused by adenine in mice, reduce the F / B ratio, and improve the intestinal disorder of mice. The heat map analysis at the genus level showed that compared with the blank control group, the abundance of ASF356, Blautia and other genera in the model group increased significantly. These genera will increase the metabolic burden and intestinal environment in chronic kidney disease, reverse their protective effect and accelerate renal fibrosis, which was significantly reduced after NPCP treatment; the abundance of Rikenella, Parasutterella and other genera in the intestine of the model group decreased. These genera are involved in bile acid metabolism, and their absence aggravates the accumulation of hydrophobic bile acids, directly damages renal tubular epithelial cells, and also increases the production of indoxyl sulfate (IS) and p-cresol sulfate (PCS), leading to tubular mitochondrial dysfunction and oxidative stress, which aggravates kidney damage. After NPCP treatment, the abundance of Rikenella, Parasutterella and other genera in the intestine of the drug-treated group increased. Figure 19 LEfSe analysis of LM showed that NPCP was enriched with bacteria that could produce SCFAs, while the model group was enriched with some pathogenic bacterial genera.

[0135] 2.8 Effects on short-chain fatty acids: The short-chain fatty acids in mouse feces were quantified by GC-MS using an Agilent 7890A / 5975C (Agilent Technologies, USA). 100 mg of fecal samples were mixed with 1 mL of 0.005 mol / L sodium hydroxide solution and 50 μL of 2-methylbutyric acid, and reacted at 4 °C for 2 h. The reaction mixture was centrifuged and derivatized after mixing with distilled water and isopropanol / pyridine solution (3:2, V / V). Then the samples were extracted with n-hexane and analyzed by GC-MS. The injection volume was 1 μL, and the results are as Figure 20 shown.

[0136] It can be seen from Figure 20 that compared with the blank control group, the levels of acetic acid, propionic acid, isobutyric acid and butyric acid in the intestines of mice in the model group decreased significantly (P < 0.01). After the NPCP intervention treatment of the present invention, the levels of short-chain fatty acids such as acetic acid, propionic acid, isobutyric acid and butyric acid increased significantly (P < 0.05), indicating that the NPCP of the present invention can effectively up-regulate the short-chain fatty acids in the intestine.

[0137] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A processed polygonatum sibiricum polysaccharide, characterized in that: Named NPCP, it is composed of fructose and glucose in a molar ratio of 1:

11. NPCP is a straight-chain inulin-type polysaccharide with a degree of polymerization of 12, and the structure is Glcp-(1→ and Fruf-(2→ ends, →1)-Fruf-(2→ main chain, as shown in Formula Ⅰ; 2. The processed polygonatum sibiricum polysaccharide according to claim 1, characterized in that: The NPCP is a neutral polysaccharide with a weight average molecular weight of 2053 Da.

3. The method for preparing the polysaccharide of nine processed polygonatum according to claim 1, characterized in that: The extract was extracted from the processed polygonatum by water extraction and alcohol precipitation, and the extract was depigmented by D101 macroporous adsorption resin, enzymatically hydrolyzed, and the protein was removed by Sevage reagent to obtain the crude polygonatum polysaccharide. It was then eluted and purified by DEAE-52 cellulose anion exchange chromatography column and Sephadex G-25 gel chromatography column to obtain the target polygonatum polysaccharide NPCP.

4. The method for preparing the processed polygonatum sibiricum polysaccharide according to claim 3, characterized in that: The specific preparation method includes: S1. Dry and crush the processed polygonatum sibiricum, add the processed polygonatum sibiricum powder into deionized water for extraction, repeat the process 2-4 times, recover the supernatant by centrifugation, concentrate under reduced pressure, add anhydrous ethanol to the concentrated solution for alcohol precipitation, and collect the alcohol precipitate by centrifugation; S2, fully dissolving the alcohol precipitate, centrifuging to remove impurities, concentrating under reduced pressure, filtering, depigmenting the concentrate with D101 macroporous adsorption resin, adding papain for enzymolysis, inactivating centrifugation, removing protein with Sevage reagent, filtering, dialyzing, and freeze-drying to obtain a crude product of Polygonatum cyrtonema polysaccharide, which is labeled as NPCPs; S3, taking the NPCPs solution, loading it onto a DEAE-52 cellulose anion exchange chromatography column, eluting it with deionized water, 0.05M NaCl and 0.1M NaCl solution at 1 mL / min, and preliminarily obtaining a crude neutral polysaccharide NPCPs product eluted with deionized water; S4. Purification was performed using a Sephadex G-25 gel chromatography column, eluted with deionized water at 0.5 mL / min, and freeze-dried to obtain uniform processed polygonatum sibiricum polysaccharide NPCP.

5. The method for preparing the processed polygonatum sibiricum polysaccharide according to claim 4, characterized in that: In step S1, the solid-liquid ratio of the nine-processed polygonatum powder to deionized water is 1:10-20 g / mL; the extraction temperature is 90-100° C., and the time is 2-4 hours; the volume ratio of the anhydrous ethanol to the concentrated solution is 3-5:1; and the alcohol precipitation is carried out at 0-4° C. for 12-24 hours.

6. The method for preparing the processed polygonatum sibiricum polysaccharide according to claim 4, characterized in that: In step S2, the amount of papain added is 2wt%-4wt% of the concentrate, and the enzyme activity is 8×10 5 The enzymatic hydrolysis temperature is 50-55°C and the time is 1-3h; the dialysis is performed at 0-4°C using a dialysis bag with a molecular weight cutoff of 1 kDa for 48-72h.

7. Use of the processed polygonatum sibiricum polysaccharide according to claim 1 in the preparation of a drug for treating kidney damage.

8. The use according to claim 7, characterized in that The kidney injury medicine comprises the polygonatum sibiricum polysaccharide and pharmaceutically acceptable excipients.

9. The use according to claim 8, characterized in that The kidney injury medicine contains 200-400 mg / kg of uniform processed polygonatum sibiricum polysaccharide NPCP.

10. The use according to claim 7, characterized in that The nine-processed polygonatum polysaccharide improves kidney damage through the following pathways: (1) Improve the levels of renal function markers and reduce blood creatinine, blood urea nitrogen and uric acid levels; (2) Improve oxidative stress damage, reduce the content of reactive oxygen and malondialdehyde in the body, and increase the content of SOD superoxide dismutase and catalase; (3) reduce the expression of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6; (4) Improve intestinal flora disorder, increase the abundance of Rikenella and Parasutterella, and reduce the abundance of ASF356 and Blautia; (5) Upregulate short-chain fatty acids in the intestine and increase the levels of acetate, propionate, isobutyrate, and butyrate.

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