Extraction method and application of lanceleaf lilyturf tuber polysaccharide SDLN1

Abstract

The invention relates to an extraction method and application of Lepidodendron simsii polysaccharide SDLN1, can effectively solve the medicinal problem of Lepidodendron simsii polysaccharide, and adopts the technical scheme that the structure of the Lepidodendron simsii polysaccharide SDLN1 is as shown in the specification: # imgabs0. The Lepidodendron simsii polysaccharide with a brand new structure is extracted, and further research shows that the Lepidodendron simsii polysaccharide SDLN1 has a wide application prospect. The lanceleaf lilyturf herb polysaccharide extracted by the invention has a certain protection effect on lung tissues of LPS-induced ALI model mice, can be used as an active ingredient of an anti-inflammatory drug to be applied to preparation of the anti-inflammatory drug, and is an innovation in the extraction method of the lanceleaf lilyturf herb polysaccharide.

Description

1. Technical Field

[0001] The invention relates to the field of medicine extraction, in particular to a method for extracting Bulbophyllum polysaccharide SDLN1 and application thereof. 2. Background Technology

[0002] Bulbophyllum is a common Chinese herbal medicine in my country, which has antibacterial, anti-inflammatory, antioxidant and anti-cholinesterase activities. Polysaccharide is one of the main active ingredients of Bulbophyllum. At present, the research on Bulbophyllum is mostly focused on resources, identification or small molecule compounds, and there are few studies on polysaccharides. At present, there is no research on the extraction and preparation methods of Bulbophyllum polysaccharides. Therefore, the separation, purification and structural analysis of Bulbophyllum polysaccharides need further research.

[0003] Acute lung injury (ALI) is a common response to various infectious and non-infectious injuries. There are many causes of the disease. It can be caused by inflammatory stress. In severe cases, it can cause acute respiratory distress syndrome and multiple organ failure, endangering the patient's life. Polysaccharides are natural high molecular weight compounds that are generally found in animals and plants, and are mainly distributed in animal cell membranes and plant cell walls. Pharmacological studies have shown that polysaccharides have antiviral, anti-inflammatory, antioxidant, and anti-tumor biological activities. Therefore, they have attracted more and more attention from researchers, especially in terms of anti-inflammatory effects. Bulbophyllum can nourish yin and reduce fire, clear away heat and reduce swelling. It is commonly used in folk medicine to treat sore throat, pneumonia, tuberculosis, etc. At present, the main active ingredient of Bulbophyllum is still unclear, and there has been no research report on whether Bulbophyllum polysaccharide is its main active ingredient. Therefore, it is very necessary to conduct structural analysis and application research on its polysaccharides in order to obtain Bulbophyllum polysaccharide components with anti-inflammatory effects. III. Summary of the invention

[0004] In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide a method for extracting Bulbophyllum polysaccharide SDLN1 and its application, which can effectively solve the medicinal problem of Bulbophyllum polysaccharide.

[0005] The technical solution provided by the present invention is that the structure of the Bulbophyllum polysaccharide SDLN1 is as follows:

[0006]

[0007] The method for extracting Bulbophyllum polysaccharide comprises the following steps:

[0008] 1) Extraction of defatted Bulbophyllum

[0009] Take a dried Bulbophyllum sample and crush it, and then perform a petroleum ether degreasing treatment to obtain a defatted Bulbophyllum;

[0010] 2) Extraction of crude Bulbophyllum polysaccharide

[0011] The defatted Bulbophyllum orchid extracted in step 1) is added to water, and then subjected to heating and reflux extraction; after the extraction is completed, the extract is concentrated to obtain a concentrated solution, and ethanol is added to the concentrated solution for alcohol precipitation to obtain a crude Bulbophyllum orchid polysaccharide;

[0012] 3) Extraction of Bulbophyllum polysaccharide

[0013] The crude Bulbophyllum polysaccharide extracted in step 2) is redissolved in water, and then subjected to Sevag method deproteinization, decolorization and dialysis treatment to obtain Bulbophyllum refined crude polysaccharide;

[0014] 4) Purification of crude polysaccharide from Bulbophyllum

[0015] The refined crude polysaccharide of Bulbophyllum ovata extracted in step 3) is subjected to separation and purification treatments on a DEAE-52 cellulose column and a Sephadex G-100 column in turn, and freeze-dried to obtain the Bulbophyllum ovata polysaccharide SDLN1.

[0016] The application of the Bulbophyllum polysaccharide SDLN1 in the preparation of anti-inflammatory drugs.

[0017] The present invention extracts a Bulbophyllum polysaccharide with a completely new structure, and further studies have shown that the Bulbophyllum polysaccharide extracted by the present invention has a certain protective effect on the lung tissue of LPS-induced ALI model mice, can be used as an active ingredient of anti-inflammatory drugs in the preparation of anti-inflammatory drugs, and is an innovation in the Bulbophyllum polysaccharide extraction method. IV. Description of the drawings

[0018] Figure 1 This is the elution curve of the 0.1Nacl part of the Bulbophyllum polysaccharide of the present invention.

[0019] Figure 2 This is the infrared characteristic map of SDLN1 extracted by the present invention.

[0020] Figure 3 This is the ion chromatogram of the standard product of the present invention.

[0021] Figure 4 This is the ion chromatogram of the sample of the present invention.

[0022] Figure 5 The 1H NMR spectrum (A), 13C nuclear magnetic resonance spectrum (B), HSQC spectrum (C), COSY spectrum (D), HMBC spectrum (E), and NOESY spectrum (F) of SDLN1 of the present invention are shown.

[0023] Figure 6 The effect of SDLN1 on the total protein, lung tissue and TNF-α and IL-6 levels in bronchoalveolar lavage fluid of mice in each group; Note: Compared with the blank group, *P<0.05, **P<0.01; Compared with the model group, #P<0.05, ## P<0.01.

[0024] Figure 7 The following are the pathological changes in lung tissue of mice in each group of the present invention (HE staining, A: blank group B: model group C: DEX group D: SDLN1-L group E: SDLN1-H group). V. Specific implementation methods

[0025] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples.

[0026] Example 1

[0027] The present invention, when implemented, specifically comprises the following steps:

[0028] 1) Extraction of defatted Bulbophyllum

[0029] Take 2 kg of dried Bulbophyllum and grind it into powder, add petroleum ether at a solid-liquid ratio of 1:20, reflux and extract at 90°C for 1 hour, filter and obtain defatted Bulbophyllum;

[0030] 2) Extraction of crude Bulbophyllum polysaccharide

[0031] The defatted Bulbophyllum residue extracted in step 1) is evaporated to dryness, distilled water is added according to a solid-liquid ratio of 1:20, hot water is refluxed and extracted 3 times, each time for 1 hour, and filtered; the filtrate is concentrated to 2000 mL under reduced pressure, ethanol is added to make the alcohol content reach 80%, and the mixture is allowed to stand overnight, the precipitate is collected by centrifugation, and vacuum freeze-dried to obtain a crude Bulbophyllum polysaccharide;

[0032] 3) Extraction of Bulbophyllum polysaccharide

[0033] The crude polysaccharide of Bulbophyllum orchid extracted in step 2) is dissolved in distilled water, and deproteinized by Sevag method (chloroform: n-butanol=4:1), the volume ratio of polysaccharide solution to Sevag reagent is 3:1, and then depigmented by H2O2 method, dialyzed to remove small molecules, and the liquid is collected and vacuum freeze-dried to obtain the refined crude polysaccharide of Bulbophyllum orchid;

[0034] 4) Purification of crude polysaccharide from Bulbophyllum

[0035] 100 mg of the refined crude polysaccharide of Bulbophyllum orchid extracted in step 3) was dissolved in 10 ml of water, added to the equilibrated DEAE-52 cellulose column, and eluted in sequence with a NaCl gradient solution (0-0.5 Mol / L), and the eluates of Bulbophyllum orchid polysaccharide with different gradients were collected, concentrated, dialyzed, and freeze-dried to obtain different elution fractions of polysaccharide; the polysaccharide fractions were tracked by the phenol-sulfuric acid method, and the peaks containing polysaccharides were collected and freeze-dried. According to the results of each elution fraction, the 0.1 NaCl elution site was selected and further purified using a gel purification system, and the polysaccharide fractions were collected, concentrated, dialyzed, and freeze-dried to obtain Bulbophyllum orchid polysaccharide SDLN1.

[0036] The extraction method of the present invention extracts a Bulbophyllum polysaccharide with a new structure, and studies have shown that the Bulbophyllum polysaccharide SDLN1 can reduce the inflammatory response of LPS-induced ALI model mice, has a certain protective effect on the lung tissue of LPS-induced ALI model mice, and can be used in anti-inflammatory drugs. The relevant test data are as follows:

[0037] 1. Structural characterization of the polysaccharide SDLN1 from Bulbophyllum japonicum

[0038] 1.1 Molecular weight determination

[0039] The molecular weight of the polysaccharide was determined by gel chromatography-differential-multi-angle laser light scattering system, the liquid phase system was U3000 (Thermo, USA), the differential detector was Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was DAWN HELEOSⅡ (Wyatt technology, CA, USA). Gel exclusion chromatography columns Ohpak SB-805HQ (300×8mm) and Ohpak SB-803HQ (300×8mm) were connected in series. The column temperature was 45℃, the injection volume was 100μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6mL / min, and the elution gradient was isocratic for 75min. The chromatographic data were processed using the software ASTRA6.1. The results showed that the absolute molecular weight of the polysaccharide was 39.937KDa.

[0040] 1.2 Fourier transform infrared spectroscopy (FT-IR) analysis

[0041] The dried SDLN1 was combined with KBr in a ratio of 1:100 to obtain a transparent pressed sheet, which was then scanned using FT-IR (Nicolet 6700, Thermo) in the range of 4000-400 cm-1. The infrared spectrum was as follows: Figure 2As shown in the figure, it can be seen that there is a strong and broad absorption peak at 3412.89cm-1, which is a strong absorption peak of OH stretching vibration of hydrogen bonds between or within polysaccharide molecules, and is a characteristic peak of sugars. The peak at 2934.85cm-1 is the CH stretching vibration absorption peak, and there is an absorption peak at 1637cm-1, which may be attributed to C=O asymmetric stretching vibration. There are absorption peaks at 1402m-1 and 1050cm-1, which may be attributed to CO stretching vibration.

[0042] 1.3 Monosaccharide composition analysis

[0043] The SDLN1 sample was added with 1 ml of 2M TFA acid solution and hydrolyzed at 121°C for 2 h, dried with nitrogen, rinsed with methanol, dried with nitrogen, repeated 2-3 times, dissolved with distilled water, and transferred to a chromatographic bottle for testing. The monosaccharide components were analyzed and detected using the Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA). The specific chromatographic conditions are as follows: Dionex TM CarboPac TM PA20 (150*3.0mm, 10μm) liquid chromatography column; injection volume is 5μl. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 ml / min; column temperature was 30°C; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V). The results showed that SDLN1 was mainly composed of fructose, rhamnose, arabinose, galactose, glucose, xylose and mannose with a molar ratio of 4.66:10.18:9.96:27.86:15.32:10.56:19.49:1.97. The chromatograms of the standard and sample are shown in Figure 2. Figure 3-Figure 4 shown.

[0044] 1.4 Methylation analysis

[0045] Take a small amount of SDLN1 sample (2-3 mg), add 500 μl DMSO to dissolve. Add 1 mg NaOH, incubate for 30 min, then add 50 μl iodomethane solution to react for 1 h. After the reaction is completed, add 1 ml water and 2 ml dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat 3 times. Then aspirate the lower dichloromethane phase and blow it dry with nitrogen, then add 100 μl 2M TFA, react at 121°C for 90 min, and evaporate to dryness at 30°C. Then add 50 μl 2M ammonia water, 50 μl 1M NaBD4, mix, react at room temperature for 2.5 h, terminate the reaction with 20 μl acetic acid, blow dry with nitrogen, wash twice with 250 μl methanol, and blow dry with nitrogen. Then add 250 μl acetic anhydride, vortex to mix, react at 100°C for 2.5 h, add 1 ml water and let stand for 10 min. Then add 500μl of dichloromethane, vortex mix, centrifuge, discard the aqueous phase, and repeat the water washing 3 times. Take the lower dichloromethane phase and detect it on the GC-MS machine. Agilent gas chromatograph was used for analysis. The chromatographic conditions are as follows: chromatographic column: BPX70 (30m×0.25mm×0.25μm, SGE, Australia). The injection volume is 1μl, the split ratio is 10:1, the carrier gas is high-purity helium, and the flow rate is 1.5ml / min; the initial temperature of the column oven is 140℃ for 2.0min, and the temperature is raised to 230℃ at 3℃ / min and maintained for 3min. Electron bombardment ion source (EI), mass scanning range (m / z): 50-350. The results of mass spectrometry identification of ion fragments (as shown in Table 1) showed that SDLN1 contained 18 different types of bonds, among which galactose residues occupied the largest proportion, followed by t-Gal(p), 3-Gal(p), 4-Gal(p), 2,4-Gal(p) and 3,6-Gal(p) sugar bonds, followed by mannose residues and glucose residues.

[0046] Table 1 Results of SDLN1 methylation analysis

[0047]

[0048]

[0049] 1.5 NMR analysis

[0050] A 50 mg sample of SDLN1 was dissolved in 0.5 mL of D2O and freeze-dried, and the process was repeated twice. The sample was then dissolved in 0.5 mL of D2O and 1H-NMR, 13C-NMR, chemical shift correlation (COSY), heteronuclear single quantum correlation (HSQC), two-dimensional NOE spectrum (NOESY) and heteronuclear multicarbon correlation (HMBC) (e.g. Figure 5 shown).

[0051] As can be seen from the figure, the sample hydrogen spectrum signal is mainly concentrated between δ3.0 and 5.5ppm, and multiple coupling signal peaks are identified in the δ4.3-5.4ppm anomeric signal area, indicating that this sample contains a variety of sugar residues, and the chemical shifts of the corresponding anomeric hydrogen are δ4.41, 4.5, 4.61, 4.62, 4.95, 5.06, 5.21, 5.39ppm, etc. The non-anomeric hydrogen signals are mainly concentrated in the δ3.1-4.2ppm area. Due to the serious overlap of individual signals, it is necessary to combine COSY and HSQC spectra to attribute the H2-H6 chemical shifts of each sugar residue. Among them, the strong signal peak near δ4.71ppm is the solvent peak. The signal peak near δ3.16ppm is the signal of hydrogen of O-CH3.

[0052] Multiple signal peaks were identified in the anomeric carbon region of the sample. Combined with the cross peaks in the anomeric region of the 13C NMR spectrum and the HSQC spectrum, the anomeric signals present in the sample were determined to be: δ5.06 / 108.87, 4.41 / 103.29, 4.5 / 101.85, 4.61 / 103.55, 5.39 / 97.21, 4.62 / 101.02, 5.21 / 97.64, 4.95 / 99.03 ppm, and were recorded as sugar residues A, B, C, D, E, F, G, and I, respectively. Combining the sample bonding structure (methylation) information, anomeric signals and literature reports, it was inferred that sugar residue A was α-L-Araf-(1→), sugar residue B was →4)-β-D-Galp-(1→, sugar residue C was →3,4)-β-D-Glcp-6-OMe-(1→, sugar residue D was →3)-β-D-Glcp-(1→, sugar residue E was →3)-α-D-Galp-(1→[8], sugar residue F was →4)-β-D-Manp-(1→, sugar residue G was →2)-α-D-Manp-(1→, sugar residue I was →3,6)-α-D-Galp-(1→. The 1H and 13C chemical shifts were assigned, and the results are shown in Table 2.

[0053] Table 2 Sugar residues 1 H and 13 Chemical shift of C

[0054]

[0055] nd: Abbreviation for "not detected", meaning not identified.

[0056] According to the chemical shifts of 13C and 1H of each sugar residue in the sample, the structure and connection mode of the polysaccharide were analyzed in combination with HMBC and NOESY spectra. According to the HMBC spectrum, there is a cross peak of δ5.06 / 82.9ppm between H1 of sugar residue A and C4 of sugar residue C. According to the NOESY spectrum, there is a cross peak of δ5.06 / 4.23ppm between H1 of sugar residue A and H4 of sugar residue C, there is a cross peak of δ5.06 / 3.5ppm between H1 of sugar residue A and H2 of sugar residue G, there is a cross peak of δ4.41 / 3.38ppm between H1 of sugar residue B and H4 of sugar residue B, there is a cross peak of δ4.41 / 4.06ppm between H1 of sugar residue B and H4 of sugar residue F, there is a cross peak of δ4.5 / 4.09ppm between H1 of sugar residue C and H3 of sugar residue I, there is a cross peak of δ4.5 / 4.09ppm between H1 of sugar residue D and H4 of sugar residue E. There is a cross peak at H3 at δ4.61 / 3.64 ppm, a cross peak at H1 of sugar residue E and H4 of sugar residue B at δ5.39 / 3.38 ppm, a cross peak at H1 of sugar residue F and H3 of sugar residue C at δ4.62 / 3.89 ppm, a cross peak at H1 of sugar residue G and H6 of sugar residue I at δ5.21 / 3.95 ppm, a cross peak at H1 of sugar residue G and H6 of sugar residue I at δ5.21 / 4.01 ppm, and a cross peak at H1 of sugar residue I and H3 of sugar residue D at δ4.95 / 3.73 ppm. Therefore, based on the one-dimensional and two-dimensional NMR information and the analysis of methylation results, it is inferred that the polysaccharide is mainly composed of →4)-β-D-Galp-(1→, →3,4)-β-D-Glcp-6-OMe-(1→ and →3)-β-D-Glcp-(1→, etc., which are connected to each other to form the main chain, and the side chain is mainly composed of α-L-Araf-(1→ connected to the sugar residue →3,4)-β-D-Glcp-6-OMe-(1→ O-4, etc. Therefore, the possible structure of the polysaccharide chain is as follows:

[0057]

[0058] 2. Pharmacological effects of SDLN1 polysaccharide from Bulbophyllum japonicum on acute lung injury (ALI)

[0059] 2.1 Methods

[0060] 2.1.1 Establishment of ALI mouse model and drug administration

[0061] Forty 6-week-old SPF male C57BL / 6J mice were randomly divided into a blank group, a model group, a dexamethasone group (DEX group), a low-dose SDLN1 administration group (SDLN1-L group, 20 mg / kg / d) and a high-dose SDLN1 administration group (SDLN1-H group, 40 mg / kg / d) after 7 days of adaptive feeding, with 8 mice in each group. The DEX group was intraperitoneally injected with 10 mg / kg DEX before modeling, and the SDLN1-H group and the SDLN1-L group were treated with SDLN1 for 3 days before modeling. Subsequently, intratracheal instillation of LPS was used to establish the ALI model, that is, ALI was induced by intratracheal administration of Escherichia coli 055:B5 lipopolysaccharide. After 24 hours of LPS infusion, the mice were killed, and the left lung was lavaged twice with PBS to obtain the alveolar lavage fluid (BALF). At the same time, the right lung tissue was divided into two parts, one part was immersed in 10% formalin solution for histopathological examination, and the other part was used for the determination of inflammatory factors.

[0062] 2.1.2 Detection of total protein in bronchoalveolar lavage fluid by BCA method

[0063] The alveolar lavage fluid was obtained and the total protein concentration was determined using a BCA protein concentration detection kit according to the instructions of the kit.

[0064] 2.1.3 Detection of TNF-α and IL-6 levels by enzyme-linked immunosorbent assay (ELISA)

[0065] The concentrations of TNF-α and IL-6 in the supernatant of BALF and lung tissue extracts were measured using a kit. The BALF was centrifuged and the supernatant was taken for later use. The lung tissue was weighed, and then the tissue was homogenized with PBS, centrifuged at 12000g for 15 minutes at 4°C, and the supernatant was taken for later use.

[0066] 2.1.4 Pathological changes in lung tissue

[0067] The right lung tissue of mice was fixed with 10% formalin, embedded in paraffin, sliced ​​and stained with hematoxylin-eosin (HE), and histopathologically observed under a microscope.

[0068] 2.1.5 Statistical methods

[0069] The experimental data were analyzed using SPSS22.0 software and Graphpad Prism 8.0 software. The measurement data were expressed as mean ± standard deviation (xˉ±s). The t test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between multiple groups. P<0.05 was considered statistically significant.

[0070] 2.2 Results

[0071] 2.2.1 Total protein in bronchoalveolar lavage fluid, TNF-α, and IL-6 levels in lung tissue and bronchoalveolar lavage fluid

[0072] Compared with the control group, the levels of BCA, TNF-α and IL-6 in BALF of the LPS group were significantly increased (p<0.05), and the levels of TNF-α and IL-6 in lung homogenate tissue were significantly increased (p<0.05), indicating that a certain inflammatory response occurred in the lungs of the model group mice. After treatment with the Bulbophyllum polysaccharide SDLN1, the levels of inflammatory factors decreased significantly, among which the levels of BCA, IL-6 in lung homogenate tissue, and TNF and IL-6 in BALF of the SDLN1-H group were significantly decreased (p<0.05). The level of TNF in BALF of the SDLN1-L group was significantly decreased (p<0.05), and the levels of BCA, TNF, IL-6 in lung homogenate tissue, and IL-6 in BALF showed a decreasing trend, indicating that the Bulbophyllum polysaccharide SDLN1 can alleviate the inflammatory response of LPS-induced ALI model mice, such as Figure 6 shown.

[0073] 2.2.2 Pathological observation of mouse lung tissue

[0074] The lung tissue structure of mice in the blank group was intact, with no obvious pathological changes. Compared with the blank group, the alveolar epithelial cells of mice in the model group proliferated, the alveolar septum was significantly thickened, and a large number of inflammatory cells infiltrated the interstitium. A certain degree of improvement was observed in the low-dose group of Bulbophyllum polysaccharide, with some alveolar epithelial cells proliferating, the alveolar septum was thickened, and more inflammatory cells infiltrated the interstitium. The improvement effect of the low- and high-dose groups of Bulbophyllum polysaccharide was more obvious, and the inflammatory cells in the lung tissue were significantly reduced, indicating that Bulbophyllum polysaccharide has a certain protective effect on the lung tissue of LPS-induced ALI model mice. Figure 7 shown.

[0075] Conclusion

[0076] In summary, the present invention extracts a Bulbophyllum polysaccharide SDLN1 with a completely new structure, and studies have shown that the Bulbophyllum polysaccharide SDLN1 can reduce the inflammatory response of LPS-induced ALI model mice, has a certain protective effect on the lung tissue of LPS-induced ALI model mice, can be applied to the production and development of anti-inflammatory drugs, develops a new structure and new use of Bulbophyllum polysaccharide, has practical clinical application and promotion value, and has huge economic and social benefits.

[0077] It should be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A Bulbophyllum polysaccharide SDLN1, characterized in that: The structure of the Bulbophyllum polysaccharide SDLN1 is shown below:

2. The method for extracting the Bulbophyllum polysaccharide SDLN1 according to claim 1, characterized in that: The following steps are involved: 1) Extraction of defatted Bulbophyllum Take a dried Bulbophyllum sample and crush it, and then perform a petroleum ether degreasing treatment to obtain a defatted Bulbophyllum; 2) Extraction of crude Bulbophyllum polysaccharide The defatted Bulbophyllum orchid extracted in step 1) is added to water, and then subjected to heating and reflux extraction; after the extraction is completed, the extract is concentrated to obtain a concentrated solution, and ethanol is added to the concentrated solution for alcohol precipitation to obtain a crude Bulbophyllum orchid polysaccharide; 3) Extraction of Bulbophyllum polysaccharide The crude Bulbophyllum polysaccharide extracted in step 2) is redissolved in water, and then subjected to Sevag method deproteinization, decolorization and dialysis treatment to obtain Bulbophyllum refined crude polysaccharide; 4) Purification of crude polysaccharide from Bulbophyllum The refined crude polysaccharide of Bulbophyllum ovata extracted in step 3) is subjected to separation and purification treatments on a DEAE-52 cellulose column and a Sephadex G-100 column in turn, and freeze-dried to obtain the Bulbophyllum ovata polysaccharide SDLN1.

3. The method for extracting Bulbophyllum polysaccharide SDLN1 according to claim 2, characterized in that: The following steps are involved: 1) Extraction of defatted Bulbophyllum Take 2 kg of dried Bulbophyllum and grind it into powder, add petroleum ether at a solid-liquid ratio of 1:20, reflux and extract at 90°C for 1 hour, filter and obtain defatted Bulbophyllum; 2) Extraction of crude Bulbophyllum polysaccharide The defatted Bulbophyllum residue extracted in step 1) is evaporated to dryness, distilled water is added according to a solid-liquid ratio of 1:20, hot water is refluxed and extracted 3 times, each time for 1 hour, and filtered; the filtrate is concentrated to 2000 mL under reduced pressure, ethanol is added to make the alcohol content reach 80%, and the mixture is allowed to stand overnight, the precipitate is collected by centrifugation, and vacuum freeze-dried to obtain a crude Bulbophyllum polysaccharide; 3) Extraction of Bulbophyllum polysaccharide The crude polysaccharide of Bulbophyllum orchid extracted in step 2) is dissolved in distilled water, and deproteinized by Sevag method with chloroform: n-butanol = 4:1, the volume ratio of polysaccharide solution to Sevag reagent is 3:1, and then depigmented by H2O2 method, dialyzed to remove small molecules, and the liquid is collected and vacuum freeze-dried to obtain the refined crude polysaccharide of Bulbophyllum orchid; 4) Purification of crude polysaccharide from Bulbophyllum 100 mg of the refined crude polysaccharide of Bulbophyllum orchid extracted in step 3) was dissolved in 10 ml of water, added to the equilibrated DEAE-52 cellulose column, and eluted in sequence with a NaCl gradient solution of 0-0.5 Mol / L, and the eluates of Bulbophyllum orchid polysaccharide with different gradients were collected, concentrated, dialyzed, and freeze-dried to obtain different elution fractions of polysaccharide; the polysaccharide fractions were tracked by the phenol-sulfuric acid method, and the peaks containing polysaccharides were collected and freeze-dried. According to the results of each elution fraction, the 0.1 NaCl elution site was selected and further purified using a gel purification system, and the polysaccharide fractions were collected, concentrated, dialyzed, and freeze-dried to obtain Bulbophyllum orchid polysaccharide SDLN1.

4. Use of the Bulbophyllum polysaccharide SDLN1 according to claim 1 in the preparation of anti-inflammatory drugs.

5. Use of the Bulbophyllum polysaccharide SDLN1 extracted by the extraction method according to any one of claims 2 to 3 in the preparation of anti-inflammatory drugs.

Images