A neutral polysaccharide and its preparation method and application

Abstract

The invention relates to the field of polysaccharide preparation, and discloses a neutral polysaccharide and a preparation method and application thereof. The preparation method comprises the following steps: S1: crushing wild-cultivated Huoshan dendrobium, and extracting with ethanol; S2: evaporating the medicinal residues to dryness, extracting with water, concentrating, and precipitating with alcohol to obtain crude Huoshan dendrobium polysaccharide; S3: dissolving the crude polysaccharide in water, and repeatedly deproteinizing to obtain refined polysaccharide; S4: dissolving the refined polysaccharide in water, separating, eluting, collecting the eluate, concentrating, and freeze-drying to obtain total neutral polysaccharide; S5: further separating the total neutral polysaccharide to obtain two purified polysaccharide components NDHP-1 and NDHP-2; S6: performing ultrafiltration centrifugal separation on the polysaccharide component NDHP-2 to purify and obtain the neutral polysaccharide NDHP-2-1; the invention purifies a neutral polysaccharide NDHP-2-1 from Huoshan dendrobium, and the molecular weight is 20.42. kDa, mainly composed of glucose (95.46%) and a small amount of mannose (4.54%), can promote the release of NO and anti-inflammatory factors, and has good anti-inflammatory activity.

Description

Technical Field

[0001] The invention relates to the field of polysaccharide preparation, and more specifically, to a neutral polysaccharide and a preparation method and application thereof. Background Art

[0002] Inflammation is a common physiological reaction of the human body when it is stimulated. In most cases, it is beneficial and can activate the immune system to help the body return to normal. It is the body's automatic defense reaction. Redness, swelling, heat, pain and dysfunction are the five main manifestations of its clinical symptoms. However, when inflammation attacks the body's own tissues, it is harmful to the human body, such as allergic asthma, colitis, arthritis, diabetes, etc., and can cause death in severe cases. At present, chemical drugs are mainly used for the treatment of inflammation, which have adverse reactions such as hypersensitivity, arrhythmia, hematopoietic changes and male breast development. Studies have found that plant polysaccharides have unique advantages in preventing and treating inflammation, such as multiple pathways, multiple targets, low toxicity and few side effects. Finding natural anti-inflammatory active plant polysaccharides is of great significance to improving and preventing inflammatory diseases.

[0003] Huoshan Dendrobium (CZ Tang et SJ Cheng) is a perennial herbaceous plant of the genus Dendrobium in the orchid family. It is a rare and authentic Anhui medicinal material and is listed as the "top ten Anhui medicines". It is slightly cold in nature, sweet in taste, and enters the stomach and kidney meridians. It has many functions such as benefiting the stomach and producing fluid, harmonizing yin and yang, nourishing yin and tonifying the kidney, nourishing the skin, clearing the throat, clearing the voice and improving eyesight, relieving heat and invigorating qi, nourishing the stomach and clearing heat. It is recorded in the "Shennong Bencao Jing" that it can "eliminate numbness and relieve qi", "take it for a long time to thicken the stomach and intestines, lighten the body and prolong life".

[0004] Under natural conditions, the germination rate of Huoshan Dendrobium is low and its growth is slow. In addition, due to excessive artificial excavation, the wild resources have been drastically reduced and are on the verge of extinction. Due to the long-term endangered resources of Huoshan Dendrobium, the separation and identification of its chemical components have not been systematically studied due to the lack of experimental materials. Most of the existing raw materials for studying the chemical components of Huoshan Dendrobium come from protocorms cultured in laboratory tissues or plants artificially cultured under controlled laboratory conditions. In the absence of external environmental pressure, the secondary metabolites produced by Huoshan Dendrobium tissue culture may be different from those in the natural environment. The polysaccharides of Huoshan Dendrobium cultivated in simulated wild have attracted great attention from biologists due to their diverse biological properties, including anti-inflammatory and antioxidant properties. There is no report on how to prepare the neutral polysaccharides of Huoshan Dendrobium cultivated in simulated wild. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a neutral polysaccharide and a preparation method and application thereof.

[0006] The present invention provides a method for preparing a neutral polysaccharide, comprising the following steps:

[0007] S1: crushing the dried wild-cultivated stems of Dendrobium huoshanense and then extracting them with ethanol;

[0008] S2: The drug residue is evaporated to dryness, extracted with water, concentrated, and anhydrous ethanol is added for alcohol precipitation to obtain crude polysaccharides of Dendrobium huoshanense;

[0009] S3: Dissolve the crude polysaccharide in water, repeatedly deproteinize using the Sevag method, and concentrate and freeze-dry the resulting solution to obtain refined polysaccharide;

[0010] S4: dissolving the refined polysaccharide in water, then separating and eluting, collecting the eluate, concentrating and freeze-drying to obtain total neutral polysaccharide;

[0011] S5: Subsequently, the total neutral polysaccharide was further separated using a gel column Sephadex G-200 with distilled water as the mobile phase to obtain two purified polysaccharide fractions, which were named NDHP-1 and NDHP-2;

[0012] S6: Finally, the polysaccharide component NDHP-2 is ultrafiltered and centrifuged using an ultrafiltration centrifuge tube, and the filtrate is further purified to obtain the neutral polysaccharide NDHP-2-1.

[0013] Preferably, in step S1, the dried simulated wild-cultivated stems of Dendrobium huoshanense are crushed and then extracted with 95% ethanol for 3 hours.

[0014] Preferably: in step S2, 800 L of water is added, extraction is performed at 80° C. for 4 h, and the extraction is repeated 3 times. The filtrates are combined and concentrated, and 4 times the volume of anhydrous ethanol is added, and the extraction is performed at 4° C. for 24 h to obtain crude polysaccharides of wild-cultivated Dendrobium huoshanense.

[0015] Preferably: in step S4, the refined polysaccharide is dissolved in water, separated with DEAE-52 cellulose, eluted with distilled water, 0.1 mol / LNaCl solution, 0.2 mol / LNaCl solution and 0.3 mol / LNaCl solution, respectively, at a flow rate of 5 mL / min, the eluate is collected, the water elution portion is collected, concentrated and freeze-dried to obtain total neutral polysaccharides.

[0016] Preferably: in step S5, the flow rate of distilled water is 0.5 mL / min;

[0017] Preferably: in step S6, the molecular weight cut-off of the ultrafiltration centrifuge tube is 30 kDa.

[0018] The present invention provides a neutral polysaccharide, which is prepared by the above-mentioned preparation method. The molecular weight of the neutral polysaccharide is 20.42 kDa, and the chemical structure of the neutral polysaccharide is as follows:

[0019] .

[0020] The invention provides an application of a neutral polysaccharide, which is applied in preparing a therapeutic agent for treating inflammatory diseases.

[0021] The beneficial effects of the present invention are as follows: the present invention purifies a neutral polysaccharide from the simulated wild cultivated Dendrobium huoshanense, the molecular weight of which is 20.42 kDa, and which is mainly composed of glucose (95.46%) and a small amount of mannose (4.54%). The polysaccharide can promote the release of NO and anti-inflammatory factors, and has good anti-inflammatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Flow chart of separation and purification of Dendrobium huoshanense in Example 4 of the present invention;

[0023] Figure 2 is the DEAE elution curve diagram in Example 4 of the present invention;

[0024] Figure 3 is the elution curve of Sephadex G-200 in Example 4 of the present invention;

[0025] Figure 4 It is the ultraviolet spectrum diagram in Example 5 of the present invention;

[0026] Figure 5 is the NDHP-2-1HPGPC chromatogram in Example 5 of the present invention;

[0027] Figure 6 is a monosaccharide composition diagram of NDHP-2-1 in Example 5 of the present invention;

[0028] Figure 7 is the infrared spectrum in Example 5 of the present invention;

[0029] Figure 8 is a screenshot of the methylation analysis results of NDHP-2-1 in Example 5 of the present invention;

[0030] Fig. 9 is a screenshot of the chemical shift values ​​of the sugar residues of NDHP-2-1 in Example 5 of the present invention;

[0031] Fig.10 It is a partial screenshot of the nuclear magnetic resonance spectrum of NDHP-2-1 in Example 5 of the present invention;

[0032] Fig.11 This is another partial screenshot of the nuclear magnetic resonance spectrum of NDHP-2-1 in Example 5 of the present invention;

[0033] Fig.12 is the HSQC spectrum of NDHP-2-1 in Example 5 of the present invention;

[0034] Fig.13is the COSY spectrum of NDHP-2-1 in Example 5 of the present invention;

[0035] Fig.14 is the HMBC spectrum of NDHP-2-1 in Example 5 of the present invention;

[0036] Fig.15 is a structural diagram of NDHP-2-1 in Example 5 of the present invention;

[0037] Fig.16 This is a graph showing the test results of NDHP-2-1 on lipopolysaccharide-induced RAW264.7 cell viability in Example 6 of the present invention;

[0038] Fig.17 This is a graph showing the test results of NDHP-2-1 in Example 6 of the present invention on NO production in RAW264.7 cells induced by lipopolysaccharide;

[0039] Fig.18 This is a test result diagram of the effect of NDHP-2-1 on lipopolysaccharide-induced TNF-α expression in RAW264.7 cells in Example 6 of the present invention;

[0040] Fig.19 This is a test result diagram of the effect of NDHP-2-1 in Example 6 of the present invention on lipopolysaccharide-induced IL-6 (D) expression in RAW264.7 cells. DETAILED DESCRIPTION

[0041] The subject matter described herein will now be discussed with reference to example embodiments. It should be understood that the discussion of these embodiments is only to enable those skilled in the art to better understand and implement the subject matter described herein, and the functions and arrangements of the elements discussed may be changed without departing from the scope of protection of the contents of this specification. Each example may omit, replace or add various processes or components as needed. In addition, the features described relative to some examples may also be combined in other examples. Example 1

[0042] In this embodiment, a method for preparing a neutral polysaccharide is proposed, comprising the following steps:

[0043] S1: The dried wild-cultivated Dendrobium huoshanense was crushed and then extracted with 95% ethanol for 3 hours;

[0044] S2: The residue was evaporated to dryness, 800 L of water was added, and extraction was performed at 80 °C for 4 h. This process was repeated three times. The filtrates were combined and concentrated. Four times the volume of anhydrous ethanol was added and the mixture was precipitated at 4 °C for 24 h to obtain crude polysaccharides from wild-cultivated Dendrobium huoshanense.

[0045] S3: Dissolve the crude polysaccharide in water and repeatedly remove protein using the Sevag method to obtain refined polysaccharide.

[0046] S4: Dissolve the refined polysaccharide in water, separate it with DEAE-52 cellulose, elute with distilled water, 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution and 0.3 mol / L NaCl solution, respectively, at a flow rate of 5 mL / min, and collect the eluate. Collect the water elution part, concentrate it and freeze-dry it to obtain the total neutral polysaccharide.

[0047] S5: Subsequently, the neutral polysaccharide was further separated using a gel column Sephadex G-200 with distilled water as the mobile phase to obtain two purified polysaccharide fractions, which were named NDHP-1 and NDHP-2;

[0048] S6: Finally, the polysaccharide component NDHP-2 was separated by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa. The filtrate was further purified to obtain the neutral polysaccharide NDHP-2-1. Example 2

[0049] In this example, a neutral polysaccharide is proposed, which is prepared by the preparation method of Example 1. The molecular weight of the neutral polysaccharide is 20.42 kDa, and the chemical structure of the neutral polysaccharide is as follows:

[0050] . Example 3

[0051] In this example, a neutral polysaccharide is proposed for use in the preparation of a therapeutic agent for treating inflammatory diseases.

[0052] Materials and reagents in the following examples:

[0053] The dried stems of wild-cultivated Dendrobium huoshanense were provided by Anhui Jiuxianzun Huoshan Dendrobium Co., Ltd. Dextran standards (molecular weight: 5 kDa-2000 kDa) were purchased from Beijing Wokai Biotechnology Co., Ltd. Glucose (Glc), arabinose (Ara) and mannose (Man), trifluoroacetic acid (TFA), DEAE-52 cellulose, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and iodomethane were purchased from Shanghai Aladdin Co., Ltd., China. Lipopolysaccharide and dexamethasone (DXM) were purchased from Sigma-Aldrich Trading Co., Ltd. Sephadex G-200 and Sephadex G-100 were purchased from Beijing Solebao Biotechnology Co., Ltd. Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. Penicillin and streptomycin were from Wuhan Punosai Life Science Co., Ltd., China. The NO detection kit was from Shanghai Biyuntian Biotechnology Co., Ltd., China. All reagents used in the experiment were analytical grade reagents. Example 4

[0054] In this example, a neutral polysaccharide was prepared according to the preparation method in Example 1, and it was named NDHP-2-1. The specific process is as follows:

[0055] 80 kg of dried wild-cultivated stems of Dendrobium huoshanense were crushed and then extracted with 95% ethanol (1200 L, 3 h). The residue was evaporated to dryness, 800 L of water was added, and the extraction was carried out at 80 °C for 4 h, which was repeated 3 times. The filtrate was combined and concentrated, and 4 times the volume of anhydrous ethanol was added, and the precipitation was carried out at 4 °C for 24 h to obtain crude polysaccharides (CDHP) of wild-cultivated Dendrobium huoshanense. The protein in CDHP was removed by Sevag method. The obtained solution was concentrated and freeze-dried to obtain refined polysaccharides (RDHP). RDHP was dissolved in water and separated by DEAE-52, and eluted with distilled water, 0.1 mol / L NaCl, 0.2 mol / L NaCl and 0.3 mol / L NaCl solutions (2.6 cm × 50 cm) at a flow rate of 5 mL / min. The eluate was collected in 5 mL / tube, and the water elution part was collected, concentrated and freeze-dried to obtain total neutral polysaccharides (NDHP). Subsequently, NDHP was further separated using a Sephadex G-200 gel column with distilled water as the mobile phase (flow rate 0.5 mL / min) to obtain the purified polysaccharide components NDHP-1 and NDHP-2. Finally, NDHP-2 was ultrafiltrated and centrifuged using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, and the filtrate was further purified by Sephadex G-100 to obtain the neutral polysaccharide NDHP-2-1.

[0056] Wherein: The extraction yield (R) is calculated by the following formula:

[0057]

[0058] Wherein, W1 is the dry weight of RDHP, NDHP and NDHP-2-1, and W is the dry weight of the stem of wild-cultivated Dendrobium huoshanense.

[0059] The extraction of crude polysaccharides was carried out by defatting with ethanol, water extraction and alcohol precipitation, e.g. Figure 1 As shown. The obtained precipitate was further freeze-dried with a freezer and named CDHP. CDHP was further purified by Sevag deproteinization, DEAE ion exchange chromatography, Sephadex G-200, ultrafiltration centrifuge tube, and Sephadex G-100. Relative to the dried Huoshan Dendrobium, the yield of RDHP was 12.75%. The eluent of the DEAE column was distilled water, the flow rate was 5 ml / min, 5 ml / tube, and the absorbance was detected by the anthrone-sulfuric acid assay method to draw the elution curve. The elution curve is shown in Figure 2As shown. One component was obtained, named NDHP, with a yield of 4.49%. The eluent of Sephadex G-200 was distilled water, the flow rate was 0.5 ml / min, 5 ml / tube, and the absorbance was detected by the anthrone-sulfuric acid determination method to draw the elution curve. The elution curve is shown in Figure 3 As shown. Two components were obtained, named NDHP-1 and NDHP-2. The yields of NDHP-1 and NDHP-2 were 0.72% and 0.23%, respectively. Finally, NDHP-2 was further purified using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa and Sephadex G-100 to obtain the NDHP-2-1 fraction. The yield of NDHP-2-1 was 0.18%. Example 5

[0060] In this example, the NDHP-2-1 prepared in Example 4 was tested and analyzed.

[0061] 1. The total sugar content was determined by the anthrone-sulfuric acid method, and the protein content was determined by the Coomassie brilliant blue method.

[0062] The total sugar and protein of NDHP-2-1 were detected by anthrone-sulfuric acid method and Coomassie brilliant blue method, and their contents were 97.62% and 0.60%, respectively. The UV spectrum scanning results showed that there were no absorption peaks at 260 and 280nm, indicating that there were no impurities such as protein and nucleic acid in NDHP-2-1 ( Figure 4 ). This result is consistent with the results of protein content analysis.

[0063] 2. Molecular weight determination

[0064] The relative molecular weight of NDHP-2-1 was detected by high performance gel permeation chromatography (HPGPC). Standard dextran (5000Da, 10000Da, 20000Da, 40000Da, 70000Da, 150000Da, 280000Da, 1000000Da, 2000000Da) and polysaccharide samples were prepared into a solution with a concentration of 2mg / mL with double distilled water, filtered with a 0.45μm filter membrane, and analyzed on the machine. The liquid phase conditions were chromatographic column: TSK-GELGMPWXL (7.8*300mm), differential refractometer, column temperature 25℃; mobile phase: double distilled water, flow rate 0.5mL / min, injection volume 20μL. The standard curve was drawn with the retention time of the corresponding standard sample on the chromatographic column as the horizontal axis. The relative molecular weight of NDHP-2-1 was calculated.

[0065] The homogeneity of NDHP-2-1 was determined by HPGPC-RID ( Figure 5The HPGPC-RID spectrum of polysaccharide NDHP-2-1 showed a single peak symmetry, indicating that NDHP-2-1 is a high-purity polysaccharide. The standard curve was drawn with the peak time of each molecular weight standard dextran as the horizontal axis (X) and the logarithm of the molecular weight of each standard dextran as the vertical axis (Y). The linear regression equation of the standard dextran was Y=-0.3368X+9.4331 (R 2 =0.9902), the molecular weight of NDHP-2-1 was calculated to be 20.42 kDa. Figure 6 As shown, monosaccharide composition analysis showed that NDHP-2-1 was mainly composed of glucose (95.46%) and contained a small amount of mannose (4.54%).

[0066] 3. Monosaccharide composition analysis

[0067] The monosaccharide composition of NDHP-2-1 was determined by PMP pre-column derivatization. 5 mg of NDHP-2-1 was hydrolyzed with 3 ml of 2 mol / L trifluoroacetic acid (TFA) at 120°C for 2 h. After removal of TFA, the hydrolyzate was derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP). The derivatized products were analyzed using an Agilent Extend-C18 column (5 μm, 4.6 mm × 250 mm) and a 250 nm detector. The mobile phase was 0.02 mol / L ammonium acetate (A) and 100% acetonitrile (B) at a flow rate of 1.0 mL / min and a column temperature of 30°C. The mobile phase conditions were: 0-5 min, 84% A; 5-17 min, 84-81% A; 17-30 min, 81-78% A; 30-35 min, 78-84% A; 35-40 min, 84% A.

[0068] like Figure 7 As shown in the figure, the NDHP-2-1 component exhibits typical infrared characteristics of sugars. In the FT-IR spectrum of NDHP-2-1, at 3390 cm -1 and 2928 cm -1 The absorption peaks observed at 1152 cm -1 and 1077 cm -1 The absorption peak observed at 892 cm -1 and 853 cm -1 The absorption at corresponds to the stretching vibration of CH in the glycosidic bond, indicating that NDHP-2-1 contains polysaccharides with β- and α-glycosidic bonds, respectively.

[0069] 4. Methylation analysis

[0070] Reagent pretreatment: Add 3A molecular sieves to CH3I and DMSO, dry for 24 hours, and remove as much water as possible from the reagents.

[0071] NDHP-2-1 (5 mg) was dissolved in NaOH / DMSO (50.0 mg / 3.0 mL) solution for 1 h, 0.5 mL of CH3I was added, stored in the dark for 1 h, and the reaction was quenched with distilled water. The reaction solution was dialyzed for 24 h to remove residual reagents. This process was repeated three times until the absorption peak corresponding to the hydroxyl group in the infrared detection sample disappeared, indicating that NDHP-2-1 was completely methylated. The methylated sample was hydrolyzed with 2 mol / L TFA (2 mL) at 120 °C for 2 h. After removing TFA, the hydrolyzate was reduced with NaBD4 and finally acetylated with acetic anhydride. The acetylated polysaccharide was dissolved in dichloromethane and analyzed using a Thermo ISQ1300 GC-MS (Thermo FisherScientific, USA) with an electron ionization source (EI) and a DB-5 MS fused silica capillary column (30 m × 0.25 mmI.D, × 0.25 μm). The injection volume was 1 µL, the split ratio was 10:1, and high-purity helium was used as the carrier gas. The temperature program started at 140 °C, increased to 230 °C at a rate of 3 °C / min, and maintained for 2 min. Detection was performed in SCAN mode, and the mass scanning range (m / z) was 30-550.

[0072] After NDHP-2-1 was methylated, it was analyzed by GC-MS. By comparing with existing literature and standard databases, the glycosidic bond types of these monosaccharides were analyzed. Figure 8 As shown, the major sugar residues identified in NDHP-2-1 were 1,4-Glcp, T-Glcp, 1,3,4-Glcp, and 1,6-Glcp.

[0073] 5. UV-Vis and IR spectroscopy and NMR analysis

[0074] Weigh 1 mg of dry NDHP-2-1 sample and 100 mg of dry potassium bromide, grind them evenly and press them into tablets at 400-4000 cm -1 Infrared scanning was performed using a UV-visible spectrometer (TU-1950, Beijing Puxi General Instrument Co., Ltd.).

[0075] The ultraviolet and visible (UV / Vis) absorbance of NDHP-2-1 (0.5 mg / mL) was measured in the range of 200–400 nm by a TU-1950 spectrophotometer (Shimadzu, Japan).

[0076] 10.0 mg of polysaccharide sample was accurately weighed and dissolved in D2O. The sample was detected by Bruker Avance AV-600 NMR spectrometer. 13 C, 1 H, HMBC, HSQC and COSY NMR spectra.

[0077] The application of one-dimensional and two-dimensional NMR spectroscopy helps to more deeply analyze the types of glycosidic bonds within sugar chains, as well as the peak characteristics and relationships between different isosaccharide residues. Fig.10 and Fig.11 As shown in Figure 2, the sugar residues of NDHP-2-1 showed hydrogen anomaly signals in the range of δ 4.54 to δ 5.20 ppm, while δ C The carbon anomaly signals appeared in the range of 98 to δ 100 ppm, indicating that there were two configurations, β- and α-, in the sugar residues of NDHP-2-1. 13 C NMR spectrum showed that δ C There are six isomeric carbon signals at chemical shifts of 98.04, 99.53, 92.14, 99.53, 92.10 and 95.87 ppm, corresponding to residues A, B, C, D, E and F, respectively. By integrating methylation analysis, NMR data and literature data, all the residues of polysaccharide NDHP-2-1 were identified. 13 C and 1 H chemical shift data are systematically classified, as shown in the attached Fig. 9 shown.

[0078] Residue A: In HSQC ( Fig.12 ) spectrum, δ H 5.40 / δ C The anomeric signal at 98.04 ppm (H1 / C1) indicates that residue A is in the α-configuration. Fig.13 ), based on the observed HH coupling signals, the chemical shift signals of H-2 to H-6 in residue A were determined to be δ H 3.61, 3.54, 3.87, 3.89 and 4.01 ppm. By coupling signal analysis of HSQC spectra, the chemical shift signals of C-2 to C-6 in residue A were determined to be δ C 76.68, 71.43, 81.20, 69.55 and 63.34 ppm. It is noteworthy that the downfield chemical shifts of C1 and C4 indicate substitutions at the O-1 and O-4 positions of the sugar ring, indicating that residue A is →4)-α-D-Glcp-(1→.

[0079] Residue B: In HSQC ( Fig.12 ) spectrum, δ C The anomeric carbon signal at 99.53 ppm is δ H The coupling signal of the anomeric hydrogen at 5.40 ppm indicates that residue B is in the α-configuration. Based on the coupling signal observed in the COSY spectrum ( Fig.13 ), the chemical shift signals from H-2 to H-6a in residue B are δ H 3.56, 3.73, 3.44, 3.84 and 3.71 ppm. Fig.12 ) spectrum, these signals are respectively δ C The carbon signals corresponding to 71.00, 72.50, 69.14, 72.34 and 63.72 ppm were connected. Therefore, residue B was identified as →6)-α-D-Glcp-(1→.

[0080] Residue C: Residue C has δ H 5.41 / δ C The terminal group signal is 92.14 ppm (H1 / C1). The chemical shifts of residue C from H2 / C2 to H6a / C6 are δ H 3.81 / δ C 72.71, δ H 3.46 / δ C 69.16, δ H 3.82 / δ C 72.34, δ H 3.56 / δ C 72.50ppm ( Fig.13 ) and HSQC ( Fig.12 ). Residue C was identified as T-α-D-Glcp-(1→.

[0081] Residue D: In HSQC( Fig.12 ) spectrum, δ C The anomeric carbon signal at 99.53 ppm is δ H The coupling signal of the anomeric hydrogen at 5.41 ppm indicates that the residue D is in the α-configuration. Based on the coupling signal observed in the COSY spectrum ( Fig.13 ), the chemical shift signals from H-2 to H-6a in residue D are δ H 3.55, 3.82, 3.63, 3.82 and 3.66 ppm. Fig.12 ) spectrum, these signals are respectively δ C The carbon signals corresponding to 72.50, 81.20, 76.73, 72.50 and 63.84 ppm were connected. Therefore, residue D was identified as →3,4)-α-D-Glcp-(1→.

[0082] Except for residues A, B, C, and D, HSQC( Fig.12 ) Spectra also observed δ H 5.22 / δ C 92.10 ppm (H1 / C1) and δ H 4.63 / δ C There are two anomeric coupling signals at 95.87 ppm (H1 / C1), corresponding to residues E and F. The chemical shifts of residues E and F correspond to the chemical shifts of the terminal carbons of the α and β configurations of Glcp. Fig.12 ) and COSY( Fig.13 ) The chemical shift signals from H2 / C2 to H6a / C6 in the spectrum are δ H 3.55 / δ C 71.00, δ H 3.72 / δ C 72.01, δ H 3.65 / δ C 76.73, δ H 3.82 / δ C 72.71 and δ H 3.65 / δ C 62.32 ppm. Similarly, the HSQC of residue F ( Fig.12 ) and COSY( Fig.13 ) spectra show that the signals from H2 / C2 to H6a / C6 are δ H 3.23 / δC 73.92, δ H 3.46 / δ C 75.70, δ H 3.75 / δ C 76.96, δ H 3.46 / δ C 72.34 and δ H 3.81 / δ C 60.05 ppm. Residue E was identified as →4)-α-D-Glcp and residue F was identified as →4)-β-D-Glcp.

[0083] In HMBC( Fig.14 ) spectrum, residue A H1 and residue B C6 are δ H 5.40 / δ C There is a coupling signal at 63.72 ppm, and residue A C4 is coupled with residue A H1 and residue B H1. δ C 81.20 / δ H There is a coupling signal at 5.40 ppm. There is a coupling signal between C6 of residue B and H1 of residue D. δ C 63.72 / δ H The coupling signal at 5.41 ppm is between C3 of residue D and H1 of residue B. δ C 81.20 / δ H The coupling signal at 5.40 ppm exists between C1 of residue C and H1 of residue D. δ C 92.14 / δ H Coupled signal at 3.66 ppm.

[0084] Therefore, according to the above results, it can be inferred that the polysaccharide is mainly composed of →6)-β-D-Glcp-(1→, →4)-α-D-Glcp-(1→, →3,4)- α-D-Glcp-(1→ as the main chain and α-D-Glcp-(1→ as the branch chain. The structure is as follows Figure 6 shown. Example 6

[0085] In this embodiment, the anti-inflammatory activity of NDHP-2-1 prepared in Example 4 was analyzed.

[0086] 1. Cell culture and cell viability analysis

[0087] RAW264.7 macrophages were cultured in high-glucose DMEM complete medium (containing 10% FBS and 1% double antibody solution) at 37°C in a 5% CO2 incubator. The CCK-8 method was used to evaluate the cytotoxic effect of polysaccharides on RAW264.7 macrophages. The cells were seeded in 96-well plates at a density of 5 × 10 4 Cells / mL, 100 μL per well, incubated for 12 h, then NDHP-2-1 solution with concentrations of 6.25, 12.5, 25, 50, 100, 400, 800 μg / mL was added for 24 h, and the control group was treated with an equal volume of DMEM medium. CCK8 solution (10 μL) was added for 2 h, and cell viability was measured by absorbance at 450 nm. Lipopolysaccharide was used as a positive control at a concentration of 1 μg / mL. The cell viability calculation formula is as follows:

[0088] Cell survival rate (%) = A1 / A2 × 100%

[0089] Where A1 and A2 are the absorbance of the sample and the control group, respectively.

[0090] like Fig.16 As shown, the results showed that compared with the control and positive drug ( Fig.16 Compared with DEX (dexamethasone in 5% paraformaldehyde, DEX is dexamethasone), NDHP-2-1 had no effect on cell viability in the concentration range of 12.5 to 200 μg / mL. When the polysaccharide concentration was increased to 400 μg / mL, a significant decrease in cell viability was observed, which may be attributed to the osmotic pressure changes caused by the increased drug concentration. Therefore, concentrations of 12.5, 25, 50, 100, and 200 μg / mL were used in subsequent experiments.

[0091] 2. Effect of NDHP-2-1 on NO secretion in RAW264.7 cells

[0092] RAW264.7 cells (8 × 10 5 The cells were cultured in 96-well plates and pre-incubated with NDHP-2-1 at concentrations of 6.25, 12.5, 25, 50, and 100 μg / mL for 2 h, and then treated with lipopolysaccharide at a concentration of 1 μg / mL for 24 h. The nitric oxide (NO) content in the supernatant was determined using a Griess kit (Biyuntian Biotechnology Co., Ltd.). The control group was treated with DMEM, and the positive control group was treated with dexamethasone (100 μg / mL).

[0093] 3. Effect of NDHP-2-1 on cytokine secretion in RAW264.7 cells

[0094] RAW264.7 cells (1 × 10 5 Cells were cultured in 96-well plates with 1 μg / mL NDHP-2-1 solution at different concentrations for 12 h, and then stimulated with 1 μg / mL lipopolysaccharide for 24 h. The supernatant was collected and centrifuged at 1000 × g for 20 min to remove cell debris. ELISA kits (Elerite Biotechnology Co., Ltd., Wuhan, China) were used to detect the concentrations of IL-6 and TNF-α.

[0095] Inflammation is associated with abnormal expression of inflammatory cytokines, including NO, IL-6, and TNF-α. We investigated the effect of NDHP-2-1 on the levels of inflammatory cytokines in cells stimulated by lipopolysaccharide. Figure 17-Figure 19 As shown, the results showed that cellular inflammation increased significantly after LPS stimulation, indicating that it plays a key role in inducing excessive inflammatory responses. As the concentration of NDHP-2-1 increased, the effect became more obvious. NDHP-2-1 has a significant anti-inflammatory effect. Figure 16-Figure 19 LPS in the above is the abbreviation of lipopolysaccharide. When lipopolysaccharide enters the body, it can stimulate immune cells (such as macrophages) to release a variety of pro-inflammatory cytokines (such as IL-6, TNF-α, IL-10, etc.) by activating pattern recognition receptors, thereby inducing an inflammatory response. Due to this characteristic, LPS is often used in scientific research as an inducer of an inflammatory model to simulate the inflammatory response in the body or to study the effects of anti-inflammatory drugs.

[0096] A neutral polysaccharide NDHP-2-1 was purified from Dendrobium huoshanense and its structure was characterized and identified. The results showed that NDHP-2-1 was a homogeneous polysaccharide with a molecular weight of 20.42 kDa, mainly composed of glucose (95.46%) and a small amount of mannose (4.54%); the main chain was α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3,4)- α-D-Glcp-(1→) and the branch chain was α-D-Glcp-(1→). In addition, the anti-inflammatory activity of NDHP-2-1 was studied by RAW264.7 cells. The results showed that NDHP-2-1 had no toxic effect on macrophages at a concentration below 200 μg / mL, and promoted the release of NO and anti-inflammatory factors, indicating that NDHP-2-1 had good anti-inflammatory activity.

[0097] The above describes an embodiment of the present invention, but this embodiment is not limited to the above specific implementation methods. The above specific implementation methods are merely illustrative and not restrictive. Under the guidance of this embodiment, ordinary technicians in this field can also make many forms, all of which are protected by this embodiment.

Claims

1. A method for preparing a neutral polysaccharide, characterized in that: The steps include: S1: crushing the dried wild-cultivated stems of Dendrobium huoshanense and then extracting them with ethanol; S2: The drug residue is evaporated to dryness, extracted with water, concentrated, and anhydrous ethanol is added for alcohol precipitation to obtain crude polysaccharides of Dendrobium officinale; S3: Dissolve the crude polysaccharide in water and repeatedly deproteinize it using the Saveg method to obtain refined polysaccharide; S4: dissolving the refined polysaccharide in water, then separating and eluting, collecting the eluate, concentrating and freeze-drying to obtain total neutral polysaccharide; S5: Subsequently, the total neutral polysaccharide was further separated using a gel column Sephadex G-200 with distilled water as the mobile phase to obtain two purified polysaccharide components, which were respectively named NDHP-1 and NDHP-2; S6: Finally, the polysaccharide component NDHP-2 is ultrafiltered and centrifuged using an ultrafiltration centrifuge tube, and the filtrate is further purified to obtain the neutral polysaccharide NDHP-2-1; In step S1, the dried wild-simulated cultivated stems of Dendrobium huoshanense were crushed and then extracted with 95% ethanol for 3 hours; In step S2, 800 L of water was added, and the mixture was extracted at 80°C for 4 h, which was repeated 3 times. The filtrates were combined and concentrated, and 4 times the volume of anhydrous ethanol was added, and the mixture was precipitated at 4°C for 24 h to obtain crude polysaccharides from Dendrobium huoshanense. In step S4, the refined polysaccharide is dissolved in water, separated with DEAE-52 cellulose, eluted with distilled water, 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution and 0.3 mol / L NaCl solution, respectively, at a flow rate of 5 mL / min, and the eluate is collected, and the water elution portion is collected, concentrated and freeze-dried to obtain total neutral polysaccharides; In step S5, the flow rate of distilled water is 0.5 mL / min; In step S6, the molecular weight of the ultrafiltration centrifuge tube is 30 kDa; The molecular weight of the neutral polysaccharide prepared by the preparation method is 20.42 kDa, and the chemical structural formula of the neutral polysaccharide is as follows: 。 2. An application of a neutral polysaccharide prepared by the preparation method as claimed in claim 1, characterized in that: The neutral polysaccharide is used in preparing a therapeutic agent for treating inflammatory diseases.

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