Boletus polysaccharide as well as preparation method and application thereof
By extracting and purifying boletes, the boletes polysaccharide BEP-2-1 with immunomodulatory function was prepared, which solved the problem of insufficient research on boletes polysaccharides in the prior art, and achieved effective relief of immune depression in CTX-induced mice.
Patent Information
- Application Number
- CN202510308733.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-17
- Publication Date
- 2025-06-24
AI Technical Summary
In the prior art, there are few studies on porcini polysaccharides in regulating immune function, especially in relieving the low immunity of mice induced by CTX.
By extracting and purifying delicious boletus, a pyran polysaccharide with an α and β configuration was prepared. The specific steps include wall crushing, ethanol extraction, ion exchange chromatography and gel column separation, and finally the purified boletus polysaccharide BEP-2-1 was obtained.
The boletus polysaccharide showed significant immunomodulatory function in animal experiments, which can alleviate the immune depression induced by CTX, improve immune indicators in the spleen, thymus and blood, and demonstrate its potential in developing as a novel immunomodulatory agent.
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Figure CN120192436A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of the application of fungal polysaccharides, and particularly relates to a bolete polysaccharide, a preparation method thereof, and an application thereof. Background Art
[0002] Boletus edulis Bull:Fr. belongs to the family Boletaceae and the genus Boletus, and is an important medicinal and edible fungus. It is rich in high-quality protein, various mineral elements (such as copper, iron, manganese, zinc, etc.), vitamins (vitamin B, vitamin C, vitamin E), polyphenols, polysaccharides, terpenoids and other bioactive components, and has been widely used in the fields of leisure foods, beverages, seasonings, health products, etc. Modern pharmacological studies have found that Boletus edulis has various effects such as antioxidant, anti-tumor, anti-inflammatory, and immune-enhancing, and has good medicinal value.
[0003] In the existing references, the research on bolete polysaccharides mainly focuses on: liver protection, blood sugar lowering, anti-tumor, anti-inflammatory, etc., but there is little research on immune regulation. Summary of the Invention
[0004] Aiming at the deficiencies of the existing technology, in this paper, Boletus edulis is extracted and purified, and the purified bolete polysaccharide (BEP-2-1) is selected through analysis. Its structure is analyzed, and animal experiments are used to study its immunomodulatory activity.
[0005] The object of the present invention can be achieved by the following technical solutions:
[0006] A preparation method of a bolete polysaccharide, the preparation method comprising the following steps:
[0007] S1. The bolete powder is broken and pulverized, and then 95% ethanol is added and mixed evenly. After reflux and centrifugal precipitation, a centrifugal supernatant is obtained;
[0008] S2. The centrifugal supernatant is rotary evaporated to 1 / 5 of the original volume, and then 4 times the volume of 95% ethanol is added for alcohol precipitation; the precipitate is collected, Savage reagent is added, and then it is shaken and centrifuged to remove the lower-layer protein precipitate; the supernatant is rotary evaporated to remove the organic reagent; then the rotary-evaporated solution is filled into a dialysis bag and placed in distilled water for freeze-drying to obtain the crude bolete polysaccharide;
[0009] S3. The crude polysaccharide from Boletus is redissolved with deionized water and loaded onto an anion exchange chromatography column. Then, isocratic elution is performed, and all eluents are collected. The sugar content in each eluent is detected using the phenol-sulfuric acid method, and an elution curve is plotted. The collected elution peak fractions are subjected to dialysis and freeze-drying, and then redissolved in deionized water. Subsequently, they are loaded onto a gel column, eluted with deionized water, and the polysaccharide content is also detected using the phenol-sulfuric acid method. The elution peak fractions are collected, dialyzed, concentrated, and freeze-dried to obtain purified Boletus polysaccharide.
[0010] Further, in step S1, 25 ml of ethanol needs to be added to every 1 g of Boletus powder. The reflux temperature is 60 °C. The conditions for centrifugal precipitation are a rotation speed of 4,000 r / min and a centrifugation time of 10 min.
[0011] Further, in step S2, the Savage reagent contains chloroform:n-butanol, and the volume ratio of chloroform:n-butanol is 4:1.
[0012] Further, in step S3, the anion exchange chromatography column is a DEAE seplife FF anion exchange chromatography column, and the flow rate in the chromatography column is 4 ml / min. The eluents for isocratic elution are pure water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions in sequence. The gel column is a Sephacryl S-400HR gel column, and the flow rate in the gel column is 1 ml / min.
[0013] A kind of Boletus polysaccharide, which is prepared by the preparation method of the above-mentioned Boletus polysaccharide.
[0014] Further, the Boletus polysaccharide belongs to pyranose polysaccharide with α and β configurations in terms of structure. The weight-average molecular weight of the Boletus polysaccharide is 2.728×10 3 Da, and the number-average molecular weight of the Boletus polysaccharide is 2.551×10 3 Da.
[0015] Further, the Boletus polysaccharide contains glucose, mannose, and galactose, and the molar percentages of glucose, mannose, and galactose are 93.99%, 4.91%, and 1.10% respectively.
[0016] The application of the above-mentioned Boletus polysaccharide in the preparation of a drug for relieving thymic atrophy.
[0017] The application of the above-mentioned Boletus polysaccharide in the preparation of a drug for relieving spleen enlargement caused by CTX.
[0018] The application of the above-mentioned Boletus polysaccharide in the preparation of a drug for improving the reduced level of thymic immunoglobulin.
[0019] Advantages of the present invention: By studying the immunomodulatory effect of boletus polysaccharide on CTX-induced immunosuppression in mice, the present invention proves the immunomodulatory function of boletus polysaccharide by detecting the indexes of mouse spleen, thymus and blood, and solves the technical problem of developing boletus polysaccharide into a new potential immunomodulator. Brief Description of the Drawings
[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.
[0021] Figure 1 It is the elution curve graph of DEAE fiber column in the present invention;
[0022] Figure 2 It is the elution curve graph of Sephacryl S-400HR gel column in the present invention;
[0023] Figure 3 It is the Fourier infrared spectrum graph of BEP-2-1 polysaccharide component in the present invention;
[0024] Figure 4 It is the ultraviolet spectrum scanning graph of BEP-2-1 and water in the present invention;
[0025] Figure 5 It is the molecular weight distribution chromatogram of BEP-2-1 in the present invention;
[0026] Figure 6 It is the ion chromatogram of monosaccharide standard product and BEP-2-1 in the present invention;
[0027] Figure 7 It is the nuclear magnetic resonance spectrum of BEP-2-1 in the present invention; where (A) is the 1H-NMR spectrum; (B) is the COSY spectrum; (C) is the HSQC spectrum; (D) is the 13C-NMR spectrum; (E) is the HMBC spectrum; (F) is the NOESY spectrum;
[0028] Figure 8 It is the body weight growth rate of mice in the present invention;
[0029] Figure 9 It is the influence of BEP-2-1 on the thymus and spleen indexes of CTX-induced immunosuppressed mice in the present invention
[0030] (Note: Compared with NC, p < 0.001, ##p < 0.01; compared with MC, **p < 0.01, *p < 0.05);
[0031] Figure 10 Effect of BEP-2-1 on hematological indexes of CTX-induced immunosuppressed mice in the present invention (Note: compared with NC, p < 0.001, ##p < 0.01; compared with MC, ***p < 0.001, **p < 0.01, *p < 0.05).
[0032] Figure 11 Effect of BEP-2-1 on immunoglobulins in the thymus of CTX-induced immunosuppressed mice in the present invention; Figure A shows IgA in the thymus; Figure B shows IgM in the thymus; (Note: compared with NC, p < 0.001, ##p < 0.01; compared with MC, **p < 0.01, *p < 0.05). Detailed implementation manners
[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0034] 1. Preparation of polysaccharide from Boletus edulis
[0035] 1.1 Extraction of polysaccharide from Boletus edulis
[0036] Take an appropriate amount of pulverized Boletus edulis powder after breaking the cell wall and add 95% ethanol, and mix at a ratio of 1:25. Reflux at 60°C for 2 hours to remove pigments, fats and small molecules in the raw materials. Centrifuge to extract the precipitate (4,000 r / min, 10 min), use water as the extraction solution, reflux at 80°C for 2 hours according to the solid-liquid ratio of 1:25, centrifuge to extract the precipitate (4,000 r / min, 10 min), and repeat this step twice. Rotate and evaporate the centrifuged supernatant to 1 / 5 of the original volume, then add 4 times the volume of 95% ethanol and perform alcohol precipitation for 12 hours. Collect the precipitate, add Savage reagent (4:1 chloroform: n-butanol), shake for 20 minutes, centrifuge and remove the lower protein precipitate. The supernatant is rotated and evaporated at 40°C to remove the organic reagent. The solution after rotary evaporation is filled into a dialysis bag (2500 Da), placed in distilled water for 72 hours, and then freeze-dried.
[0037] 1.2 Separation and purification
[0038] The crude polysaccharide of Boletus was redissolved with deionized water and loaded onto a DEAE seplife FF anion exchange chromatography column at a flow rate of 4 ml / min. Gradient elution was carried out successively with pure water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions, and one tube was collected every 15 ml. All the eluates were collected. The sugar content in the eluates of each test tube was detected by the phenol-sulfuric acid method and an elution curve was plotted. After dialysis and freeze-drying of the collected elution peak fractions.
[0039] After preliminary extraction and purification of Boletus, BEP was obtained. The elution curve of BEP separated by a DEAE-cellulose column is as Figure 1 shown. Two obvious elution peaks (BEP-1, BEP-2) were obtained. Since the solubility of BEP-1 was poor, BEP-2 was selected for further purification. After concentrating, dialyzing, and freeze-drying BEP-2, the yield was calculated: 7.33%.
[0040] It was redissolved in deionized water and then loaded onto a Sephacryl S-400HR gel column at a flow rate of 1 ml / min. Elution was carried out with deionized water as the eluent. The polysaccharide content was also detected by the phenol-sulfuric acid method and the elution peak fractions were collected. After dialysis, concentration, and freeze-drying, purified Boletus polysaccharide was obtained. The elution curve of BEP-2 through a Sephacryl S-400HR gel column is as Figure 2 shown. A main elution peak was obtained, and the peak appeared relatively late, indicating a relatively small molecular weight. After concentrating and freeze-drying it, a white flocculent substance was finally obtained. It was named BEP-2-1, and the yield of BEP-2-1 was: 37.8%.
[0041] 2. Structural analysis of Boletus polysaccharide
[0042] 2.1 Spectral analysis
[0043] The Fourier transform infrared spectrum of the polysaccharide was measured (Nicolet iZ-10, Thermo, USA). The polysaccharide sample was mixed with KBr powder and pressed into a 1-mm pellet, and FT-IR measurement was carried out in the range of 4000 - 400 cm-1.
[0044] The polysaccharide sample was dissolved in pure water to prepare a 5 mg / mL polysaccharide solution, and a multifunctional microplate reader (Multiskan GO, Thermo, USA) was used to quantitatively analyze the polysaccharide solution. Scanning wavelength: 200 nm - 1000 nm, with pure water as the blank control for UV–vis measurement.
[0045] BEP-2-1 was identified by infrared spectroscopy, as Figure 3As shown, an obvious characteristic peak of polysaccharide appears at 3400 cm-1. The absorption peaks at 2931.08 cm-1 and 1402.17 cm-1 are attributed to C-H stretching vibration. The absorption peak at 1625.76 cm-1 is the asymmetric stretching vibration of C=O. There are three absorption peaks at 1000 - 1200 cm-1, indicating the presence of pyranose ring. Some studies have shown that the absorption peaks in the range of 820 cm-1 - 890 cm-1 contain the characteristic absorption peaks of α and β glycosidic bonds. It can be preliminarily judged that this polysaccharide should be a pyranose polysaccharide with α and β configurations.
[0046] BEP-2-1 was identified by ultraviolet spectroscopy. As Figure 4 can be seen, compared with the blank control, the sample has no obvious absorption in the wavelength range of 200 - 400 nm, indicating that the sample hardly contains impurities such as pigments, nucleic acids and proteins.
[0047] 2.2 Molecular weight
[0048] The sample was dissolved in 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) with a final concentration of 1 mg / mL, filtered through a filter with a pore size of 0.45 μm, and then detected by machine.
[0049] The molecular weight of the polysaccharide from Boletus edulis was detected by gel chromatography - differential - multi - angle laser light scattering system. Gel permeation chromatography columns Ohpak SB - 805HQ (300×8 mm) and Ohpak SB - 803HQ (300×8 mm) were connected in series. The column temperature was 45 °C, the injection volume was 100 μL, the mobile phase A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient: isocratic for 75 min.
[0050] As Figure 5 Calculated by the formula, the weight - average molecular weight (Mw) and number - average molecular weight (Mn) of Bep - 2 - 1 were 2.728×103 Da and 2.551×103 Da respectively. The polydispersity (Mw / Mn) of Bep - 2 - 1 was 1.069. Compared with the polysaccharides from Boletus edulis extracted by other researchers, the molecular weight of this polysaccharide was lower. It is speculated that the reason for this difference is caused by the different growth environments of Boletus edulis.
[0051] 2.3 Monosaccharide composition
[0052] The sample was pretreated. An appropriate amount of polysaccharide sample was weighed, 1 ml of 2M TFA acid solution was added, and heated at 121 °C for 2 hours. Nitrogen was passed through and dried. 99.99% methanol was added for washing, and then dried again. The methanol washing was repeated 2 - 3 times. Sterile water was added for dissolution and transferred to a chromatographic vial for detection by machine.
[0053] An ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA) was used to analyze and detect monosaccharide components using an electrochemical detector. Dionex TM CarboPac TM PA20 (150 * 3.0 mm, 10 μm) liquid chromatography column was used; the injection volume was 5 μl. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), the flow rate was 0.5 ml / min; the column temperature was 30 °C; elution gradient: 0 min, phase A / phase B / phase C (95:5:0, V / V), 26 min, phase A / phase B / phase C (85:5:10, V / V), 42 min, phase A / phase B / phase C (85:5:10, V / V), 42.1 min, phase A / phase B / phase C (60:0:40, V / V), 52 min, phase A / phase B / phase C (60:40:0, V / V), 52.1 min, phase A / phase B / phase C (95:5:0, V / V), 60 min, phase A / phase B / phase C (95:5:0, V / V).
[0054] Ion chromatography was used to analyze the monosaccharide composition of BEP-2-1. As Figure 6 shown. Bep-2-1 is composed of glucose, mannose, and galactose. The molar percentages of the main monosaccharides of BEP-2-1 calculated based on the molar concentrations and peak areas of the monosaccharide standards and internal standards were 93.99%, 4.91%, and 1.10% respectively. Among them, glucose had the highest proportion.
[0055] 2.4 Methylation
[0056] The polysaccharide sample was dissolved in DMSO. The solution was methylated with CH3I in DMSO / NaOH. The completely methylated over-methylated product was hydrolyzed with 2 mol / L TFA at 121 °C for 1.5 h, reduced with NaBD4, and acetylated with acetic anhydride for 2.5 h (100 °C).
[0057] The analytical instrument for this experiment was an Agilent 7890A-5977B gas chromatography-mass spectrometry instrument from Agilent Technologies Inc., CA, USA, and the model of the automatic sampler was G4567A.
[0058] Chromatographic system (Agilent 7890A; Agilent Technologies, USA), chromatographic column: BPX70 (30 m × 0.25 mm × 0.25 μm, SGE, Australia). The injection volume was 1 μl, the split ratio was 10:1, the carrier gas was high-purity helium, and the flow rate was 1.5 ml / min; the initial temperature of the column oven was maintained at 140 °C for 2.0 min, and then programmed to rise to 230 °C at a rate of 3 °C / min and held for 3 min.
[0059] Mass spectrometry system (Agilent 5977B; Agilent Technologies, USA), equipped with an electron impact ionization source (EI) and a MassHunter workstation. The electron impact ionization source (EI) was used, and the analytes were detected in the full scan (SCAN) mode, and the mass scan range (m / z): 50 - 350.
[0060] Methylation analysis was used to determine the linkage mode of glycosidic bonds. Combining the monosaccharide composition and proportion of BEP-2-1 and the GC-MS detection results, the methylation and main glycosidic bonds of BEP-2-1 are shown in Table 1, and the content proportion of substances in some methylated sugars is basically consistent with the monosaccharide composition results.
[0061] Table 1 Methylation analysis results of BEP-2-1
[0062]
[0063] 2.5 Nuclear magnetic resonance
[0064] The sample was dissolved in 0.5 mL of D2O to a final concentration of 40 mg / mL, and the dissolved solution was transferred to a nuclear magnetic resonance tube with an addition amount of 0.5 mL. A nuclear magnetic resonance spectrometer (AVANCE NEO 500M, Bruker, Germany) was used to scan the sample at 500 MHz.
[0065] BEP-2-1 was analyzed by NMR. 1H-NMR mainly solves the configuration problem of glycosidic bonds in the polysaccharide structure. The signals of polysaccharides in 1H-NMR are concentrated at 3 - 6 ppm. From the 1H-NMR spectrum ( Figure 7 A), it can be seen that multiple coupling signal peaks were identified in the anomeric signal region at δ 4.3 - 5.4 ppm, indicating that this sample contains multiple sugar residues. The chemical shifts corresponding to the anomeric hydrogens are δ 5.34, 5.3, 4.9, 4.76, and 4.47 ppm respectively. Due to severe overlap of individual signals, COSY ( Figure 7 B) and HSQC ( Figure 7C) The 1H-NMR spectra were used to assign the chemical shifts of H2-H6 of each sugar residue. Among them, the strong signal peak near δ4.71 ppm was the solvent peak. The 13C-NMR spectra can provide information on sugar residues to determine the linkage positions of sugar chains and certain specific groups. According to the figure ( Figure 7 D) Multiple signal peaks were identified in the anomeric carbon region of the sample. Combining the cross-peaks in the anomeric region of the 13C-NMR spectra and HSQC spectra, the anomeric signals present in the sample were determined to be: δ5.34 / 99.6, 4.9 / 98.53, 5.3 / 99.73, 4.76 / 102.52, 4.47 / 102.79 ppm, and were denoted as sugar residues A, B, C, D, and E, respectively. Combining the sample bonding structure information, anomeric signals, and comprehensive literature reports, the sugar residues were speculated and their 1H-NMR and 13C-NMR chemical shifts were assigned. The results are shown in Table 2.
[0066] Combined with the HMBC (Figure s7E) spectra, the structural and linkage modes in this polysaccharide were analyzed: there were cross-peaks δ5.34 / 76.68 ppm between H1 of sugar residue A and C4 of sugar residue A, δ99.6 / 3.58 ppm between C1 of sugar residue A and H4 of sugar residue A, δ4.76 / 84.03 ppm between H1 of sugar residue D and C4 of sugar residue C, δ102.52 / 3.73 ppm between C1 of sugar residue D and H4 of sugar residue C, δ4.47 / 68.08 ppm between H1 of sugar residue E and C6 of sugar residue C, and δ102.79 / 3.46 ppm between C1 of sugar residue E and H6 of sugar residue C. Further combined with the NOESY spectra ( Figure 7 F) The linkage order of each residue in this polysaccharide was judged and speculated. There were cross-peaks δ5.34 / 3.58 ppm between H1 of sugar residue A and H4 of sugar residue A, δ5.34 / 3.73 ppm between H1 of sugar residue A and H4 of sugar residue C, δ4.9 / 3.46 ppm between H1 of sugar residue B and H6 of sugar residue C, δ4.9 / 3.54 ppm between H1 of sugar residue B and H6 of sugar residue C, δ4.9 / 3.85 ppm between H1 of sugar residue B and H6 of sugar residue E, δ5.3 / 3.58 ppm between H1 of sugar residue C and H4 of sugar residue A, δ5.3 / 3.78 ppm between H1 of sugar residue C and H3 of sugar residue D, δ4.76 / 3.73 ppm between H1 of sugar residue D and H4 of sugar residue C, δ4.47 / 3.46 ppm between H1 of sugar residue E and H6 of sugar residue C, and δ4.47 / 3.54 ppm between H1 of sugar residue E and H6 of sugar residue C.
[0067] Table 2 Chemical shifts of 1H and 13C of each sugar residue
[0068]
[0069]
[0070] 2.6 Scanning Electron Microscope
[0071] The molecular morphology of the polysaccharide was observed using a scanning electron microscope (Zeiss Merlin Compact, Germany). Under high vacuum conditions, images were observed at a voltage of 1.0 kV, with magnification factors of 100 times and 4000 times respectively.
[0072] 3 Animal Experiments
[0073] 3.1 Modeling
[0074] After 7 days of adaptive feeding, the mice were randomly divided into 6 groups, with 12 mice in each group. The normal group (NC) received only 0.9% saline treatment throughout the experiment. After the other 5 groups of mice were intraperitoneally injected with cyclophosphamide (CTX 80 mg / kg BW) for 3 consecutive days, the model group (MC) received intragastric administration of 0.9% saline, the positive control group (PC) received intragastric administration of 10 mg / kg BW levamisole hydrochloride (LH), and the polysaccharide from Boletus edulis was divided into low (L-BEP-2-1, 50 mg / kg BW), medium (M-BEP-2-1, 100 mg / kg BW), and high (H-BEP-2-1, 200 mg / kg BW) dose groups, which received intragastric administration of the polysaccharide from Boletus edulis. After 24 consecutive days of intragastric treatment, on the third day after injecting CTX, CTX 60 mg / kg BW was injected every 10 days to prevent the recovery of immune function. On the 28th day after the intervention, the experiment ended and the mice were sacrificed.
[0075] 3.2 Spleen and Thymus Indices
[0076] The body weight of the mice was recorded before sacrifice. Blood was collected from the orbital cavity of the mice and centrifuged at 3000 rpm / min at 4°C for 15 min using a refrigerated centrifuge (5424R, eppendorf, Germany) to collect the serum, which was stored at -80°C for later use. After the mice were sacrificed, their intact thymus and spleen were taken, rinsed with pre-cooled saline, placed on filter paper to absorb the excess moisture, and immediately weighed. Organ index = organ weight / body weight.
[0077] As Figure 8 shown, compared with the NC group, the body weights of the other groups decreased significantly after CTX intervention. After starting the intragastric experiment 3 days after continuous intraperitoneal injection of CTX, it was found from the seventh day of the experiment that the body weights of the mice in each group began to show a gradual recovery trend, but the recovery trend of the body weight in the MC group was relatively slow compared with other groups. The group with the most obvious effect was the H-BEP-2-1 group.
[0078] As Figure 9 shown, compared with the NC group, the thymus index of the MC group decreased significantly, indicating that CTX caused thymus atrophy in mice. Compared with the MC group, the thymus index of the PC and BEP-2-1 groups showed a significant increase, indicating that BEP-2-1 could effectively alleviate the symptoms of thymus atrophy in immune-suppressed mice, and the M-BEP-2-1 group had the most obvious effect (p < 0.01). Compared with the NC group, the spleen index of the MC group increased significantly, which was consistent with the symptom of spleen enlargement in mice caused by CTX in the literature. Compared with the MC group, the spleen index of the PC and BEP-2-1 groups decreased significantly, indicating that BEP-2-1 could alleviate the phenomenon of spleen enlargement caused by CTX, and the M-BEP-2-1 group had the best effect (p < 0.01).
[0079] 3.3 Blood indexes
[0080] A hematology analyzer was used to detect blood counts. The analysis included white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), lymphocytes (Lym), neutrophils (Neu), and platelet count (PLT).
[0081] The ability to detect hematopoiesis is usually evaluated by measuring routine blood indexes such as white blood cells, red blood cells, and hemoglobin. The results of the hematological indexes are as Figure 10 shown. Compared with the NC group, the values of neutrophils (Neu), red blood cells (RBC), hemoglobin (HGB), white blood cells (WBC), and lymphocytes (Lym) in the MC group decreased significantly. Under the influence of BEP-2-1, the levels of Neu, RBC, HGB, WBC, and Lym increased significantly. Among them, Neu (p < 0.001), WBC (p < 0.01), and Lym (p < 0.05) had the best effect at high doses, and RBC (p < 0.05) and HGB (p < 0.01) had the best effect at medium doses.
[0082] 3.4 Thymus immunoglobulins
[0083] Take 20 mg of thymus tissue and homogenize it with pre-cooled PBS solution at a ratio of 1:9 (w / v). After centrifugation, collect the supernatant of the tissue homogenate and detect the contents of immunoglobulin A (IgA) and immunoglobulin M (IgM) in the spleen according to the ELISA kit.
[0084] The levels of thymus immunoglobulins (IgA and IgM) were detected using an ELISA kit, and the effect of BEP-2-1 on CTX-induced immunosuppression was also detected. Compared with the NC group, the levels of thymus immunoglobulins in the MC group decreased significantly, while BEP-2-1 could significantly improve the decreased levels of immunoglobulins. After intragastric administration of BEP-2-1, by Figure 10It can be seen that the levels of thymic immunoglobulin A (IgA) and immunoglobulin M (IgM) in the BEP-2-1 group were significantly higher than those in the MC group.
[0085] 3.5 Statistical analysis
[0086] The experimental data were analyzed using SPSS 27 and expressed as mean ± standard deviation (SD). The statistical differences between groups were evaluated by LSD test. A P value < 0.05 was considered statistically significant.
[0087] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
[0088] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.
Claims
1. A method for preparing boletus polysaccharide, characterized in that: The preparation method comprises the following steps: S1. Crush the porcini powder, add 95% ethanol and mix evenly, reflux and centrifuge to obtain a centrifugal supernatant; S2, rotary evaporating the centrifugal supernatant to 1 / 5 of the original volume, then adding 4 times the volume of 95% ethanol to precipitate; collecting the precipitate, adding Savage reagent, and then shaking and centrifuging to remove the lower layer of protein precipitate; rotary evaporating the supernatant to remove the organic reagent; then putting the rotary evaporated solution into a dialysis bag, adding distilled water, and freeze-drying to obtain crude porcini polysaccharide; S3. The crude porcini polysaccharide was re-dissolved in deionized water and loaded onto an anion exchange chromatography column; isocratic elution was then performed and all eluates were collected; the sugar content in each eluate was detected by the phenol-sulfuric acid method and an elution curve was drawn; the collected elution peak components were dialyzed and freeze-dried; they were redissolved in deionized water and then loaded onto a gel column; deionized water was used as the eluent for elution, and the polysaccharide content was detected by the phenol-sulfuric acid method and the elution peak components were collected. Purified porcini polysaccharide was obtained after dialysis, concentration and freeze-drying.
2. The method for preparing boletus polysaccharide according to claim 1, characterized in that: In step S1, 25 ml of ethanol is added to every 1 g of boletus powder; the temperature during reflux is 60° C.; and the conditions for centrifugal precipitation are a rotation speed of 4,000 r / min and a centrifugal time of 10 min.
3. The method for preparing boletus polysaccharide according to claim 1, characterized in that: In step S2, the Savage reagent contains chloroform:n-butanol, and the volume ratio of chloroform:n-butanol is 4:
1.
4. The method for preparing boletus polysaccharide according to claim 1, characterized in that: In step S3, the anion exchange chromatography column is a DEAE seplife FF anion exchange chromatography column, and the flow rate in the chromatography column is 4 ml / min; the eluents during isocratic elution are pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions, respectively; the gel column is a Sephacryl S-400HR gel column, and the flow rate in the gel column is 1 ml / min.
5. A boletus polysaccharide, characterized in that: The boletus polysaccharide is prepared by the preparation method of the boletus polysaccharide according to any one of claims 1 to 4.
6. The boletus polysaccharide according to claim 5, characterized in that: The boletus polysaccharide is a pyranose polysaccharide with α and β configurations in structure; the weight average molecular weight of the boletus polysaccharide is 2.728×10 3 Da, the number average molecular weight of the boletus polysaccharide is 2.551×10 3 Da.
7. The boletus polysaccharide according to claim 5, characterized in that: The boletus polysaccharide contains glucose, mannose and galactose, and the molar percentages of glucose, mannose and galactose are 93.99%, 4.91% and 1.10% respectively.
8. Use of a boletus polysaccharide according to any one of claims 5 to 7 in the preparation of a medicament for alleviating thymic atrophy.
9. Use of a boletus polysaccharide according to any one of claims 5 to 7 in the preparation of a medicament for alleviating splenomegaly caused by CTX.
10. Use of a boletus polysaccharide according to any one of claims 5 to 7 in the preparation of a medicament for improving a decreased level of thymic immunoglobulin.