Structural identification method of rhizoma bletillae neutral polysaccharide

Abstract

The invention relates to the technical field of biological pharmacy, in particular to the field of IPC C08B37, and more particularly relates to a structure identification method of bletilla striata neutral polysaccharide. Comprising the following steps: step 1, sequentially extracting, separating and purifying rhizoma bletillae crude polysaccharide to obtain rhizoma bletillae polysaccharide; step 2, pretreating the rhizoma bletillae polysaccharide, and performing homogeneity and molecular weight determination; 3, determining the content of total sugar in the rhizoma bletillae polysaccharide by adopting a phenol-sulfuric acid method, determining the chemical composition in the rhizoma bletillae polysaccharide by adopting a trifluoroacetic acid method in combination with gas chromatography-mass spectrometry, determining the content of uronic acid in the rhizoma bletillae polysaccharide component by adopting an m-hydroxybiphenyl method, and determining the content of protein in the rhizoma bletillae polysaccharide by adopting a Bio-rad protein quantitative method; step 4, carrying out methylation analysis on the bletilla striata polysaccharide; and step 5, analyzing monosaccharide components and bonding modes of the bletilla striata polysaccharide by using a nuclear magnetic resonance technology.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, in particular to the field of IPC C08B37, and more specifically to a method for structural identification of neutral polysaccharides from Bletilla striata. Background Art

[0002] Polysaccharides are a natural high-molecular compound and a basic substance that constitutes organisms. They exist in the cell membranes of most living organisms and participate in various life activities of cells, playing an indispensable role in organisms. Bletilla striata (Thunb.) Reichb.f. is the dried tuber of Bletilla striata, a traditional Chinese medicine of the orchid family. Its main active ingredient with a high content is Bletilla striata polysaccharide. On the one hand, studies have shown that the optimal relative molecular mass range for different polysaccharides to exert immune effects is different. A relative molecular mass that is too large may not be conducive to the polysaccharide molecules passing through the cell membrane of the organism and entering its interior to exert the corresponding biological activity. A relative molecular mass that is too small may destroy the polymeric structure of the polysaccharide to produce the best activity, thereby affecting or weakening the activity of the polysaccharide components to varying degrees. Therefore, it is very necessary to carry out structural identification of polysaccharides after separation and purification, which can lay a solid foundation for subsequent activity research of polysaccharides at this relative molecular mass; on the other hand, most natural polysaccharide components in Chinese medicinal materials coexist with proteins in the same or similar parts. Therefore, in order to explore the activity of polysaccharides, it is necessary to further separate and purify the extracted polysaccharide components to ensure that the polysaccharide components used in the experiment are relatively uniform, free of or with less impurities, which is conducive to further determining the biological activity of such polysaccharide components through research; in addition, the separation and purification of polysaccharides is also beneficial to quality control and structure-activity relationship research in the subsequent related development of polysaccharides.

[0003] CN108051432A discloses a method for identifying anti-hypoxia polysaccharides of Pleurotus eryngii, including molecular weight determination, polysaccharide content determination, structural identification and anti-hypoxia functional characteristics determination of three source polysaccharides, so as to obtain multiple parameters such as molecular weight, polysaccharide content, polysaccharide structural characteristics and anti-hypoxia functional characteristics of Pleurotus eryngii anti-hypoxia polysaccharides, thereby providing data support for the practical application of anti-hypoxia polysaccharides of Pleurotus eryngii, exerting its efficacy and providing help for the healthy development of mankind. However, the structure of the anti-hypoxia polysaccharides of Pleurotus eryngii in the invention is relatively simple, and no determination method is provided for complex polysaccharides. Summary of the invention

[0004] The present invention provides a method for structural identification of neutral polysaccharides from Bletilla striata, comprising the following steps:

[0005] Step 1: sequentially extracting, separating and purifying crude polysaccharides from Bletilla striata to obtain Bletilla striata polysaccharides;

[0006] Step 2: pre-treat the polysaccharide from Bletilla striata and measure its homogeneity and molecular weight;

[0007] Step 3: using the phenol-sulfuric acid method to determine the total sugar content in the Bletilla striata polysaccharide, using the trifluoroacetic acid method combined with gas chromatography-mass spectrometry to determine the chemical composition of the Bletilla striata polysaccharide, using the m-hydroxybiphenyl method to determine the uronic acid content in the Bletilla striata polysaccharide component, and using the Bio-rad protein quantitative method to determine the protein content in the Bletilla striata polysaccharide;

[0008] Step 4: Methylation analysis of Bletilla striata polysaccharides;

[0009] Step 5: Use nuclear magnetic resonance technology to analyze the monosaccharide components and bonding patterns of Bletilla striata polysaccharides.

[0010] The step 1 is specifically as follows: taking Bletilla striata medicinal material, extracting it with water, concentrating the filtrate and adding anhydrous ethanol to obtain a mixture, filtering the mixture, drying the sediment, and then adding water to dissolve it again to obtain a primary product; dialyzing the primary product with distilled water, concentrating and drying it to obtain Bletilla striata crude sugar; dissolving the Bletilla striata crude sugar in distilled water and separating and purifying it using an anion exchange column, concentrating and freeze-drying it to obtain a first neutral polysaccharide; separating and purifying the first neutral polysaccharide using a gel column, concentrating and freeze-drying it to obtain.

[0011] The water extraction temperature is 50-90° C., the water extraction time is 50-150 min, the material-liquid ratio of the water extraction is 1:(5-30), and the number of water extractions is 2-5 times.

[0012] Preferably, the water extraction temperature is 60-80°C, the water extraction time is 60-120 min, the material-liquid ratio of the water extraction is 1:(10-20), and the number of water extractions is 2-4 times.

[0013] The pretreatment is specifically as follows: dissolving the polysaccharide of Bletilla striata in a solution containing 0.01-0.05 wt % NaN 3 0.05-0.5MNaNO 3 In the aqueous solution, the concentration of the Bletilla striata polysaccharide is 0.2-2 mg / mL, and then filtered using a 0.1-1 μm filter membrane.

[0014] Preferably, the pretreatment is specifically: dissolving the polysaccharide of Bletilla striata in a solution containing 0.01-0.03wt% NaN 3 0.05-0.2M NaNO 3 In the aqueous solution, the concentration of the Bletilla striata polysaccharide is 0.5-1.5 mg / mL, and then filtered using a 0.3-0.8 μm filter membrane.

[0015] The step 2 uses a differential refractive index detector, a laser scattering detector and a high performance liquid chromatograph to perform homogeneity and molecular weight determination.

[0016] The present application has found that the use of a differential refractive index detector, a laser scattering detector and a high performance liquid chromatograph for homogeneity and molecular weight determination in step 2 can effectively improve the accuracy of the detection. Most of the natural polysaccharide components in Bletilla striata coexist with proteins in the same or similar parts. Therefore, in order to explore the activity of polysaccharides, it is necessary to further separate and purify the extracted polysaccharide components to ensure that the polysaccharide components used in the experiment are relatively uniform and contain no or less impurities, which is conducive to further determining the biological activity of such polysaccharide components through research.

[0017] The high performance liquid chromatograph used a Shodex OHpakSB HQ series gel chromatography column.

[0018] Preferably, the model of the gel chromatography column includes at least one of SB-802HQ, SB-802.5HQ, SB-803HQ, SB-804HQ, SB-805HQ, SB-806HQ, SB-806MHQ, and SB-809-HQ.

[0019] Further preferably, the gel chromatography column is a series of the following types of chromatography columns: SB-805HQ, SB-804HQ, SB-803HQ and SB-809-HQ.

[0020] The chromatographic parameters of the high performance liquid chromatograph are as follows: the mobile phase is a 0.1-0.8 wt % dimethyl sulfoxide aqueous solution, the flow rate is 0.1-0.5 mL / min, and the elution gradient is isothermal for 100-150 min.

[0021] Preferably, the chromatographic parameters of the high performance liquid chromatograph are as follows: the mobile phase is a 0.3-0.8 wt % dimethyl sulfoxide aqueous solution, the flow rate is 0.1-0.4 mL / min, and the elution gradient is isothermal for 100-130 min.

[0022] The gas chromatography-mass spectrometry chromatography system is a combination of ion chromatography and electrochemical detector.

[0023] The Bio-rad protein quantification method uses bovine serum protein as a control for external standard method testing.

[0024] This experiment intends to use the phenol-sulfuric acid method to determine the total sugar content in the polysaccharide, the trifluoroacetic acid (TFA) method to cut the sugar chain in the polysaccharide, and use GC (gas chromatography) to determine its monosaccharide components, and use GC to determine the differences between different monosaccharide components, so as to clarify the single chain components and their contents of the target polysaccharide. This can maximize the integrity of the sugar chain while maintaining its internal structure.

[0025] The step 4 specifically includes: methylating the Bletilla striata polysaccharide, then oxidizing it with sodium borohydride, and then acetylating it to obtain a structurally stable sugar alcohol-acetate compound, and then using gas chromatography-mass spectrometry to determine the position and content ratio of the glycosidic bonds in the polysaccharide.

[0026] The temperature program of the gas chromatography in step 4 is: the initial column temperature is 120-160°C, maintained for 1-4 minutes, and the column is heated to 220-240°C at a heating rate of 2-5°C / min, and maintained for 2-5 minutes; the ion source of the mass spectrometer is an electron bombardment ion source.

[0027] Preferably, the temperature raising program of the gas chromatography in step 4 is: the initial column temperature is 130-150°C, maintained for 1-3 min, the column is heated to 220-230°C at a heating rate of 2-4°C / min, and maintained for 3-5 min; the ion source of the mass spectrometer is an electron bombardment ion source.

[0028] The step 5 is specifically as follows: dissolving the monosaccharide components of Bletilla striata polysaccharide in heavy water, and using nuclear magnetic resonance technology to conduct H-NMR, C-NMR, heteronuclear single quantum region correlation spectroscopy and heteronuclear multi-carbon correlation spectroscopy for research.

[0029] Beneficial effects:

[0030] 1. In step 2, a differential refractive index detector, a laser scattering detector and a high performance liquid chromatograph are used to measure the homogeneity and molecular weight, which can effectively improve the accuracy of the detection.

[0031] 2. The phenol-sulfuric acid method was used to determine the total sugar content in Bletilla striata polysaccharide, the trifluoroacetic acid method combined with gas chromatography-mass spectrometry was used to determine the chemical composition of Bletilla striata polysaccharide, the m-hydroxybiphenyl method was used to determine the uronic acid content in Bletilla striata polysaccharide, and the Bio-rad protein quantification method was used to determine the protein content in Bletilla striata polysaccharide, which can maximize the integrity of the sugar chain while maintaining its internal structure.

[0032] 3. Methylation analysis of Bletilla striata polysaccharides can accurately detect the position and content ratio of glycosidic bonds in the target polysaccharides.

[0033] 4. The monosaccharide components and bonding patterns of Bletilla striata polysaccharides were analyzed using nuclear magnetic resonance technology, which can improve the detection accuracy and sensitivity in the process of studying the overall structure of the polysaccharide components.

[0034] 5. This study used the external standard method to quantitatively determine the total sugar content, and drew a fitting curve based on different concentrations of glucose standards to improve the convenience of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1This is the elution curve of BSP on the DEAE-650 column in Example 1.

[0036] Figure 2 This is the elution curve of BSP-1 on Sepharose 6B column in Example 1.

[0037] Figure 3 This is the absolute molecular weight dot diagram of BSP-2 in Example 1.

[0038] Figure 4 This is the absolute molecular weight analysis chart of BSP-2 in Example 1.

[0039] Figure 5 This is a molecular configuration analysis diagram of BSP-2 in Example 1.

[0040] Figure 6 It is the chromatogram of the standard product in Example 1.

[0041] Figure 7 This is the chromatogram of the BSP-2 sample in Example 1.

[0042] Figure 8 This is the chromatographic integration result of the BSP-2 sample in Example 1.

[0043] Fig. 9 This is the total ion current diagram of the BSP-2 sample in Example 1.

[0044] Fig.10 For Example 1 1 H-NMR (one-dimensional hydrogen spectrum) spectrum.

[0045] Fig.11 For Example 1 13 C-NMR (one-dimensional carbon spectrum) spectrum.

[0046] Fig.12 This is the COSY (hydrogen-hydrogen correlation spectrum) spectrum in Example 1.

[0047] Fig.13 It is the NOESY (NOE-type two-dimensional nuclear magnetic resonance spectrum) spectrum in Example 1.

[0048] Fig.14 This is the HSQC (carbon-hydrogen correlation spectroscopy) spectrum in Example 1.

[0049] Fig.15 This is the HMBC (carbon-hydrogen correlation) spectrum in Example 1. DETAILED DESCRIPTION

[0050] Example 1

[0051] A method for structural identification of neutral polysaccharides from Bletilla striata comprises the following steps:

[0052] Step 1: sequentially extracting, separating and purifying crude polysaccharides from Bletilla striata to obtain Bletilla striata polysaccharides;

[0053] Step 2: pre-treat the polysaccharide from Bletilla striata and measure its homogeneity and molecular weight;

[0054] Step 3: using the phenol-sulfuric acid method to determine the total sugar content in the Bletilla striata polysaccharide, using the trifluoroacetic acid method combined with gas chromatography-mass spectrometry to determine the chemical composition of the Bletilla striata polysaccharide, using the m-hydroxybiphenyl method to determine the uronic acid content in the Bletilla striata polysaccharide component, and using the Bio-rad protein quantitative method to determine the protein content in the Bletilla striata polysaccharide;

[0055] Step 4: Methylation analysis of Bletilla striata polysaccharides;

[0056] Step 5: Use nuclear magnetic resonance technology to analyze the monosaccharide components and bonding patterns of Bletilla striata polysaccharides.

[0057] Step 1 is as follows:

[0058] Extraction of crude polysaccharide of Bletilla striata: Take 1.5 kg of dried medicinal materials of Bletilla striata, add distilled water at a mass volume ratio of 1:14 (g:mL), extract at a constant temperature of 68°C water bath for 3 times, each time for 94 minutes, filter through four layers of gauze and then filter by suction, then combine the filtrates of 3 times, and concentrate the filtrates by rotary evaporation to make its volume reach about 1 / 5 of the original. Add anhydrous alcohol until the final mass fraction reaches 85%, and place it in a refrigerator at 4°C overnight. After filtration, freeze-dry the sediment, then add distilled water to dissolve it again, and stir it fully at room temperature until it is completely dissolved. Put the sample solution into a dialysis bag with a molecular weight of 7000Da, dialyze with distilled water, then collect the solution in the bag, centrifuge at 4000r / min for 20 minutes, concentrate and dry the supernatant, so as to obtain BSP, weigh it and store it at -20°C for standby use.

[0059] Isolation and purification of neutral polysaccharides: 2 g of BSP was added to distilled water and mixed to dissolve it in water, then centrifuged at 4000 r / min for 15 minutes to remove the insoluble matter, and then the precipitate was collected and about 5 mL of supernatant was taken out; the sample was loaded onto a filled anion exchange column (DEAE-650M, 55 mm 30 cm), rinsed with water, measured with phenol, and graded and recovered to obtain component BSP-1. The elution curve is shown in the following figure. Figure 1As shown, the components from 196 to 294 nm were collected with a yield of 21.69%. BSP-1 was concentrated and lyophilized. 500 mg BSP-1 was added with an appropriate amount of purified water to completely dissolve at room temperature, and then placed in a dialysis bag with a molecular weight of 7000 Da; after dialysis with distilled water, the solvent in the bag was collected, 4000 r / min, 20 min, the supernatant was concentrated, and then the supernatant was concentrated to a certain volume and added to a uniformly filled gel chromatography (Sepharose6 B, 45 mm x 90 cm), and then lyophilized with H 2 O for elution, 14 mL in each tube, and then use the sulfuric acid phenol method to determine the sugar components. Collect until there is no sugar. The elution curve is as follows Figure 2 As shown, the components from 900 to 1086 nm were collected to obtain component BSP-2, which was numbered and weighed, with a yield of 68.19%. The components were stored in a drying cylinder for later use.

[0060] Step 2 specifically includes: using the laser scattering detector (DAWN HELEOS II) of Optilab T-rEX (Wyatt Technology, USA) and Dyatt Technology II (Wyatt Technology, USA).

[0061] Sample pretreatment: The sample was dissolved in a solution containing 0.02% NaN 3 (W / W) 0.1M NaNO 3 The (W / W) solution was filtered with a 0.45 μm filter membrane and then sent to the machine for measurement.

[0062] Determination of polysaccharide homogeneity: Determine whether the polysaccharide component is uniform based on the peak shape on the chromatographic peak.

[0063] Calculation of molecular weight of polysaccharide components: Calculate its relative molecular mass based on the standard curve.

[0064] Chromatographic conditions: Ohpak SB-805HQ (300x8mm), Ohpak SB-804HQ (300x8mm), Ohpak SB-803HQ and 300x8mm Ohpak SB-809-HQ in series. The temperature is preferably 60°C. The sample volume is about 100 μL. Use mobile phase B (0.5wt% dimethyl sulfoxide), flow rate 0.3mL / min, isothermal 120min elution gradient.

[0065] Step 3 specifically includes: calculating the total sugar content of Bletilla striata polysaccharide according to the phenol-sulfuric acid method.

[0066] Standard: Glucose. Standard preparation: Prepare 100 μg / mL glucose standard solution, use it as the mother solution, dilute with distilled water to obtain a series of concentration gradients, the specific preparation is as shown in the following table. Pipette 0.4mL of the mixed sample into a new test tube and measure it simultaneously with the sample according to the determination method.

[0067] Table 1

[0068]

[0069] Accurately weigh the standard sample, add 1mL of sterile water, ultrasonically extract for 10 minutes, and centrifuge at 12000r / min for 10 minutes. Add 0.1mL of supernatant to a new test tube and dilute with sterile water. Mix the standard solution phenol and concentrated sulfuric acid in a glass tube, stir thoroughly, and measure the absorbance by UV spectroscopy at 490nm; use the obtained absorption intensity and content to draw the corresponding standard curve. Take about 1mg of the polysaccharide component BSP-2 of Bletilla striata, add 1mL of deionized water to make it a 1mg / mL sample solution, dilute it to a concentration between 0.2-0.8mg / mL, and then measure the absorbance according to the same detection method as the aforementioned standard, and import it into the corresponding standard curve to obtain the total sugar in the separated polysaccharide component.

[0070] Analysis of monosaccharide composition of BSP-2

[0071] The standard varieties are shown in Table 2:

[0072] Table 2

[0073] name English name Abbreviation CAS Number Fucose Fucose Fuc 2438-80-4 Rhamnose Rhamnose Rha 10030-85-0 Arabinose Arabinose Ara 5328-37-0 Galactose Galactose Gal 26566-61-0 glucose Glucose Glc 50-99-7 Xylose Xylose Xyl 58-86-6 Mannose Mannose Man 3458-28-4 fructose Fructose Fru 57-48-7 Ribose Ribose Rib 50-69-1 Galacturonic acid Galacturonic Acid Gal-UA 14982-50-4 Glucuronic acid Glucuronic Acid Glc-UA 6556-12-3 Mannosyl acid Mannuronic Acid Man-UA 6814-36-4 Gulunic acid GμLuronic Acid GμL-UA 15769-56-9

[0074] Preparation of standard substances: Accurately weigh 10 mg each of L-rhamnose (Rha), L-fucose (Fuc), L-arabinose (Ara), mannose (Man), D-glucose (Glc), D-galactose (Gal), and D-xylose (Xyl) standards, add appropriate amount of distilled water to prepare 10 mg / mL standard solution mother solution single standard, then take appropriate amount of standard stock solution single standard and mix them to prepare standard mixed standards with the highest index concentration of 60 μg / mL, 50 μg / mL or 40 μg / mL, and prepare the series of standards required for the instrument according to the following concentration gradient, as shown in Table 3.

[0075] Table 3

[0076]

[0077]

[0078] Sample processing

[0079] Prepare by adding 100 μL and 50 μL of a mixture of 5 mg / mL inositol to 50 μL of the white and polysaccharide monomer solutions, respectively. Weigh the BSP-2 polysaccharide using a chromatography column, add 1 mL of 2MTFA to acidify, 121°C, 2h. Dry with nitrogen. Then add 99.99% methanol to the sample for washing, then blow dry, and wash repeatedly with methanol 2-3 times. Finally, add sterile water to completely dissolve it in the solution, and transfer it to a column chromatography bottle for determination. Extract the sample solution to a gas-mass chromatography column, and determine its monosaccharide component using the reference substance as a control.

[0080] The chromatography system used was the Thermo ICS5000+ ion chromatography system (ICS5000+, Thermo Fisher Scientific, USA), and the corresponding electrochemical detector was used to achieve rapid and accurate determination of the target polysaccharide monomer components. The Dionex PA20 (150*3.0 mm, 10 μm) liquid chromatography column was selected. The sample volume was about 5 μL. 2 O), mobile phase (0.1M sodium hydroxide), mobile phase (0.1M sodium hydroxide), mobile phase (0.1M sodium hydroxide, 0.2MNaAc), mobile phase with a flow rate of 0.5μL / min. The temperature was controlled below 30°C. From the order of elution, A / B / C (95:5:0, V / V), 26minA / B / C (85:5:10, V / V), 42minA / B / C (60:0:40, V / V), 52.1minA / B / C (95:5:0, V / V), 52.1min(95:5:0, V / V).

[0081] Standard curve: the horizontal axis is the content of the reference substance, and the vertical axis is the peak area.

[0082] Analyze the measured monosaccharide components and contents: compare each monosaccharide component with the standard, substitute the retention time into the standard curve, and obtain the proportion of each monosaccharide component.

[0083] Calculation of uronic acid content in polysaccharide components of Bletilla striata: This experiment intends to use the m-hydroxybiphenyl method to calculate the content of uronic acid in polysaccharide components of Bletilla striata. The standard substance used is α-D-galacturonic acid. The standard substance is made into a 1 mg / mL solution. After gradient dilution, the concentrations of 200μg / mL, 40μg / mL, 20μg / mL, 10μg / mL, 5μg / mL, and 3μg / mL are prepared according to the required concentration. Take more than 0.2mL of solution, then add 1mL of 0.01M sulfuric acid-sodium tetraborate aqueous solution in sequence, cool to room temperature, heat the solution for 5 minutes, and then cool to room temperature. Add 30μL of 0.2wt% sodium hydroxide solution to the control group, add 30μL of 0.1wt% m-hydroxybiphenyl solution to the sample group, shake well and place at room temperature, and use UV spectroscopy to determine the absorbance of each target component at 520nm. After the determination is completed, draw a standard curve with concentration and absorbance as the horizontal axis. The polysaccharide component was prepared into a 1 mg / mL solution, which was diluted to a certain concentration until the measured absorbance was 0.2 to 0.8, and then the absorbance A was measured in the same way as the standard, and the uronic acid content was calculated from the standard curve.

[0084] Determination of protein content in polysaccharide fraction of Bletilla striata

[0085] This experiment used bovine serum protein as a control and used the Bio-rad (ultraviolet spectrophotometry) protein quantification method to analyze the protein in the polysaccharide component of Bletilla striata. In the preparation of the standard solution, deionized water was used to make bovine serum protein as a standard, and its concentration was 1 mg / mL, and then it was diluted in sequence to a system of 0, 5, 10, 20, and 40 μg / mL as a standard. 20 μL of the above-mentioned liquid was added to a 0.2 mL kit for detecting protein, and it was allowed to occur at 200°C for 0.5 hours, and its absorbance at 560 nm was measured by UV method. The protein content and absorbance in the sample were determined by the standard sample determination method, and a standard line was drawn with this as the horizontal axis. 1 mg of BSP-2 component was added to an appropriate amount of deionized water, and it was prepared into a 1 mg / mL solution. After uniform mixing, it was diluted to a certain concentration to ensure that the absorption value measured by the ultraviolet spectrometer was between 0.2 and 0.8. The absorption coefficient A of the polysaccharide component solution was determined using the same steps as the reference substance, and the protein content was calculated based on the standard curve.

[0086] Step 4 is as follows:

[0087] Methylation analysis of polysaccharide components of Bletilla striata: First, the free hydroxyl groups in the polysaccharide are methylated, then oxidized by sodium borohydride, and then acetylated to obtain structurally stable sugar alcohol-acetate compounds. The fragment ions are determined by gas chromatography to infer the position and content ratio of the glycosidic bonds in the target polysaccharide.

[0088] Weigh about 2-3 mg of Bletilla polysaccharide sample BSP-2, after complete drying, add 500 μL of dimethyl sulfoxide again, after uniformity, add about 1 mg of sodium hydroxide solid powder thereto, stir it evenly for 30 minutes at room temperature, then add about 50 μL of iodide solution, and let it react completely for 1 hour. Then, add 1 mL of deionized water and 2 mL of dichloromethane again, use a vortex instrument to fully mix, then centrifuge, and discard the corresponding aqueous phase, wash with water for many times, at least 3 times, absorb the lower dichloromethane phase and blow dry with nitrogen, after the above operation, chloroform is mixed, dry it with nitrogen in a fume hood, and then add 100 μL of 2M trifluoroacetic acid. Reaction was carried out for 1.5 hours in a metal solution at 121 ° C. The solution was dried with nitrogen in a fume hood, then washed with methanol, and repeated 4 times. After washing with methanol (water-free), the solvent was evaporated in a reduced pressure rotary device, and repeated 3 times. 50 μL of ammonia water (2M) was added to the sediment gradually, and 50 μL of 1M sodium borohydride was slowly added, mixed thoroughly, and allowed to react at room temperature for at least 2.5 hours. 20 μL of acetic acid was used to terminate the reaction, and nitrogen was also used to blow dry. The mixture was cleaned with more than 250 μL of methanol, and the number of washes was not less than two times. After washing, nitrogen was blown dry. 250 μL of acetic anhydride was added, mixed, and allowed to react at a temperature of 100°C for 2.5 hours. Then 1 mL of deionized water was added, and the mixture was allowed to stand at room temperature for at least 10 minutes. Then 500 μL of dichloromethane was added, and the mixture was stirred and centrifuged to remove the aqueous phase. The mixture was washed several times, at least three times. On this basis, it was analyzed by gas chromatography-mass spectrometry.

[0089] The sample solution was sampled and sent to the gas-mass spectrometer to measure the monosaccharide components with the reference substance. An Agilent-gas chromatograph (Agilent7890A) was used. Agilent, USA), and the chromatography column used was BPX70 (30μmx0.25μm, Australia). 1μl was injected, and high-purity helium was used as the carrier gas, and the separation ratio was 10:1. At the starting temperature of 140°C, the starting temperature of the column temperature box was set to 2.0min, and then the column temperature box was heated to 230°C at a heating rate of 3°C / min, and then the heating time was continued for 3min.

[0090] The mass spectrometry system uses a Tetrast-bar mass spectrometer (Agilent 5977B) produced by Aiglent Inc. (Agilent 5977B, Agilent Technologies, USA), and the actual spectrum detection system is also equipped with an electron bombardment ion source (EI) and a MassHunter workstation for analysis. The electron bombardment ion source (EI) is used to chromatograph the components to be tested, and the components to be tested are fully scanned (SCAN), with a mass spectrum (m / z) range of 50-350. Therefore, the concentration and attribution of the fragment ions in the product can be determined based on the retention time, peak area ratio and other characteristics of the product.

[0091] Step 5 is as follows: NMR spectrum of Bletilla striata polysaccharide: prepare the sample BSP-2 into a 40 mg / mL solution to scan the Bletilla striata polysaccharide components, add 1 mL of analytical pure heavy water to make it completely dissolved in water, and then use NMR (600 MHz) to study HNMR, C-NMR, heteronuclear single quantum region correlation spectrum, heteronuclear multi-carbon correlation spectrum, etc. in sequence.

[0092] Performance Testing Methods and Data

[0093] 1. The homogeneity and molecular weight of Bletilla striata polysaccharide components were determined. Figure 3 As shown, the sample peak presents a symmetrical sharp peak, indicating that the uniformity of BSP-2 is good.

[0094] 2. The chromatography results of step 2 were analyzed using ASTRA6.1 software. The following is the absolute molecular weight analysis of the sample ( Figure 3 ) diagram and molecular configuration analysis curve ( Figure 4 ), the molecular weight of BSP-2 is shown in Table 4.

[0095] The molecular weight analysis diagram: The red line represents multi-angle laser scattering (LS, in V), and the intensity of the scattered light is directly proportional to the molecular size and relative molecular mass of the object. The response size of the differential signal represented by RIU (in RIU) depends on the type, concentration and relative molecular mass of the sample. The black line represents the relative molecular mass of the two signals. The inorganic salt phase has a solvent peak near 65min of the mobile phase, and the DMSO phase has a solvent peak near 102min of the mobile phase. Figure 3 As shown in the figure, through the absolute molecular weight analysis chart of the sample, it can be preliminarily inferred that the multi-angle laser light scattering signal of the sample is strongest between the sample retention time of about 55-57min, indicating that the molecular size and molecular weight of the sample here reach the maximum value, and this is basically consistent with the peak value of the post-column elution refractive index of the sample, which once again verifies that this is the peak value of the sample molecular weight and concentration and the substance type is basically unified.

[0096] Molecular configuration diagram: With logarithm (molecular mass) as the horizontal axis and logarithm (RMS Radius) as the vertical axis, the slope is used as the benchmark and the slope is used as the reference. A slope of 1 represents that the substance is rod-shaped, and a slope of 0.5-0.6 represents that the substance is a disordered line cluster. When the slope is 1 / 3, the substance is spherical. Figure 4 ,Analysis shows that the slope of the molecular configuration diagram of the sample is between 0.5-0.6, indicating that the sample configuration is an irregular coil.

[0097] 3. The external standard method was used to quantitatively determine step 3, and a fitting curve was drawn based on different concentrations of glucose standards, as shown in Table 5 and Figure 5 shown.

[0098] The total sugar content in the sample can be calculated based on the standard curve:

[0099] Total sugar content (mg / g) = C*V / M, where C is the result calculated according to the standard curve; M is the actual weighed mass; and V is the total volume of the extract. The chemical composition analysis results of the Bletilla striata polysaccharide samples are shown in Table 6.

[0100] The total sugar content of the purified Bletilla striata polysaccharide component is close to 100%, the uronic acid content is low, and it contains almost no protein, which means that the Bletilla striata polysaccharide component does not contain carboxyl groups in its structure and is a neutral sugar.

[0101] Analysis of monosaccharide composition of BSP-2

[0102] Qualitative results: Chromatographic data were processed using the Chromeleon software. Figure 6 The ion chromatogram of the standard sample is shown. Figure 7 The ion chromatogram of the sample.

[0103] According to the monosaccharide composition analysis results of the purified Bletilla striata polysaccharide fraction ( Figure 8 ), compared with the monosaccharide standard spectrum used, the polysaccharide component BSP-2 is mainly composed of mannose (Man) and glucose (Glc), and the composition ratio is 2:1. In addition, there are a small amount of galactose (Gal) and arabinose (Ara) and a trace amount of rhamnose (Rha). The specific results are shown in Table 7.

[0104] 4. Methylation analysis results of polysaccharide components of Bletilla striata:

[0105] Qualitative results: According to Fig. 9The analysis results showed that the main chain of BSP-2 was mainly composed of 4-Man(p) and 4-Glc(p) residues. The ratio was about 3:1, indicating that the BSP-2 components were mainly composed of mannose residues and highly branched xyloglucan. In addition, some terminally connected t-Man(p) and t-Glc(p) residues and a small amount of 3,4-Glc(p), 2,4-Glc(p), and 4,6-Glc(p) residues were detected, suggesting that BSP-2 may be a typical galactosyl mannose and contain a small amount of glucose galactan.

[0106] Quantitative results: as shown in Table 8.

[0107] Note:

[0108] Relative molar amount = peak area / molecular weight;

[0109] Relative molar ratio (%) = relative molar amount / total relative molar amounts of each component.

[0110] 5. NMR analysis results of homogeneous components of Bletilla striata polysaccharide: 1 H-NMR (one-dimensional hydrogen spectrum), 13 C-NMR (one-dimensional carbon spectrum), COSY (hydrogen-hydrogen correlation spectrum), NOESY (NOE type two-dimensional nuclear magnetic resonance spectrum), HSQC (carbon-hydrogen correlation spectrum) and HMBC (carbon-hydrogen correlation spectrum) analysis.

[0111] 1 H-NMR is shown in the figure: The one-dimensional hydrogen spectrum detection results show the glycosidic bond structure of the target polysaccharide. In the nuclear magnetic resonance hydrogen spectrum, the signals of polysaccharides are mainly concentrated in the range of 3-6ppm. Most of the isomeric hydrogen signals of glycosidic bonds are located at δ4.3-4.8ppm, while most of the isomeric hydrogen signals of glycosidic bonds are located at δ4.8-5.8ppm. Our previous research found that there are multiple different types of sugar residues in this sample, among which there are multiple different types of sugar residues in the anomeric signal area of ​​δ4.3-5.4ppm, and their chemical shifts are δ5.12, 4.85, 4.47, and 4.45ppm respectively. The H2-H6 chemical shifts of each sugar residue on the sugar ring are determined by COSY and HSQC spectra, but there is a strong overlap between them. Therefore, we also need to use COSY and HSQC spectra to determine their exact structure. In the sample, there is a strong signal peak around δ4.71ppm, which is the solvent peak.

[0112] 13 CNMR: 13 C NMR spectra showed that the ectopic carbon chains of the polysaccharide were in the range of 95-110 ppm. 13By analyzing the intersection peaks of C-NMR and HSQC spectra, we found a new HSQC signal: δ4.69 / 100.14, 4.47 / 102.51, 4.68 / 99.97, 5.12 / 93.83, 4.45 / 102.69 and 4.85 / 93.64ppm. Based on the previous work, this project intends to propose A(A)-β-D-Manp-(1, residue B is 4)-β-D-Glcp-(1, residue C is β-D-Manp-(1, residue C is)-α-D-Glcp-(1, residue F is 3,4)-α-D-Glcp-(1) through bioinformatics analysis and bioinformatics analysis. And determine their 1 H, 13 The chemical shift of C.

[0113] COSY: Fig.12 As shown, the adjacent or interlaced proton hydrogen and diagonal peak related peaks are marked accordingly in the figure; NOESY: Fig.12 As shown; HSQC: Fig.12 As shown; HMBC: Fig.12 shown.

[0114] As shown in Table 9, the NMR signal attribution process of the main sugar residues is as follows:

[0115] Sugar residue A: The anomeric signal 4.69 / 100.14ppm (H1 / C1) suggests that the A residue may be a β-structured mannose residue. The cross peaks of the H1 and HSY spectra of the A residue determined by 1HNMR are 4.69 / 4.06ppm respectively. On this basis, the H3-H6 sequence was analyzed using the COSY spectrum, and the chemical shifts of its H3-H6 corresponded to δ3.74ppm, δ3.75ppm, 3.5ppm, 3.51(3.59)ppm. On this basis, the positions of the C2-C6 atoms on the sugar ring were determined according to the HSQC signal, and the positions of the C2-C6 atoms on the sugar ring were determined (δ69.97ppm, 71.25ppm, 76.5ppm, 74.5ppm, 66.65ppm). Combined with relevant literature and the above methylation experiment, we speculate that the structure of the A-type sugar residue A is likely to be 4)-β-D-Manp-(1), that is, the O-1 and O-4 positions of the sugar ring are replaced.

[0116] Sugar residue B: The anomeric signal 4.47 / 102.51ppm (H1 / C1) suggests that residue B is likely to be a glucose residue in beta conformation. Based on the chemical shift of H1 of sugar residue B determined by 1HNMR at δ3.29ppm, COSY spectrum was used to cross-analyze H2-H6, and H2-H6 (same as sugar residue A, the same below) was obtained, and the chemical shifts of H2-H6 were δ3.29ppm, 3.61ppm, 3.6ppm, 3.8ppm. On this basis, according to the HSQC signal, the chemical shifts of C2-C6 on the sugar ring were determined, and the positions of C2-C6 in the sugar ring were determined, namely δ72.84ppm, 73.94ppm, 78.45ppm, 71.84ppm, 60.19ppm. Combined with relevant literature and the above-mentioned methylation study [8], it is speculated that the sugar site of B may be 4)-β-D-Glcp-(1), and the chemical shifts of the C1 and C4 sugar rings are biased towards the low-frequency magnetic field, suggesting that the sugar site of B may be 4)-β-D-Glcp-(1).

[0117] Sugar residue C: The anomeric signal 4.68 / 99.97ppm (H1 / C1) suggests that residue C is likely to be a β-configuration mannose residue. Based on the H1 chemical shift δ4.68ppm of sugar residue C determined by 1HNMR, the H2~H6 signals can be determined in sequence by the COSY spectrum cross peaks. Sugar residue C: The anomeric signal δ4.68 / 99.97ppm (H1 / C1) indicates that residue C may be a β-configuration mannose residue. Based on the H1 chemical shift δ4.68ppm of sugar residue C determined by 1HNMR, the H2~H6 signals can be determined in sequence by the COSY spectrum cross peaks, and the H2-H5 chemical shifts of sugar residue C are assigned to δ3.96ppm, δ4.06ppm, δ4.1ppm, δ3.97ppm and δ3.69(3.62)ppm, respectively. After assigning the chemical shifts of hydrogen on the sugar ring, the chemical shifts of C2 to C6 on the sugar ring can be assigned by HSQC signals, which are δ69.84ppm, δ71.25ppm, δ69.8ppm, δ70.43ppm and δ60.41ppm, respectively. The chemical shift of C1 shifts to the downfield, indicating that the residue is substituted at the O-1 position of the sugar ring. According to the corresponding literature reports and the above methylation analysis results [7], it is inferred that the sugar residue C may be β-D-Manp-1.

[0118] Sugar residue D: The anomeric signal δ5.12 / 93.83ppm (H1 / C1) indicates that residue D may be an α-configuration glucose residue. Based on the chemical shift of sugar residue DH1 determined by 1HNMR at δ5.12ppm, COSY spectrum was used to analyze H2-H6, and it was found that the chemical shifts of H2-H6 corresponded to δ3.92ppm, δ3.44ppm, 3.6ppm, 3.23ppm, and 3.98ppm. On this basis, HSQC was used to analyze the H on the sugar ring and the position of C2-C6 on the sugar ring C2-C6 was determined to be δ69.44ppm, 73.94ppm, 72.5ppm, 73.25ppm, and 60.27ppm. Combined with relevant literature and the above-mentioned methylation study [9], it is speculated that residue D is α-D-GlCP-(1), and the C-terminal C position has a large change, indicating that the O-1 position of the sugar ring has been replaced.

[0119] Sugar residue E: The anomeric signal δ4.45 / 102.69ppm (H1 / C1) indicates that residue E may be a glucose residue of β-configuration. Based on the H1 chemical shift of sugar residue E determined by 1HNMR at δ4.45ppm, the H2-H6 signals were identified sequentially by the cross peaks in the COSY spectrum, and the chemical shifts of H2-H6 were δ3.38ppm, 3.73ppm, 3.84ppm, 3.59ppm, and 3.44ppm. On this basis, the chemical shifts of C2-C6 on the sugar ring were determined according to the HSQC signal, and the positions of C2-C6 in the sugar ring were determined, namely δ74.93ppm, 72.2ppm, 76.72ppm, 71ppm, and 69.47ppm. Among them, there are chemical shifts at the O-1, O-4, and O-6 positions on the C1, C4, and C6 sugar rings, and it is speculated that the E site is 4,6)-β-D-Glcp-(1).

[0120] Sugar residue F: The anomeric signal δ4.85 / 93.64ppm (H1 / C1) indicates that residue F may be an α-configuration glucose residue. The chemical shifts of H2-H6 of sugar residue F determined by 1HNMR are δ4.85ppm, H2-H6 chemical shifts are δ3.94ppm, H2-H6, 3.58ppm, 3.61ppm, 3.5ppm. On this basis, HSQC was used to analyze the H on the sugar ring and determine the positions of C2-C6 on the sugar ring at different positions (δ70.27ppm, 76.37ppm, 78.47ppm, 72.01ppm, 60.44ppm). Our previous research found that: 1, 3, 4, 4 amino acid residues are located at the O-1, O-3, and O-4 positions of the sugar ring, respectively. Combined with relevant literature [9,10], it is speculated that the F site is: (3,4)-α-D-Glcp-(1). Combining the one-dimensional NMR and two-dimensional NMR spectra, the sugar residue signals of the samples were assigned, as shown in Table 2-6 below.

[0121] By determining the 13C and 1H positions of different sugar sites and analyzing them through HMBC spectra: A-H1 and residue A-C4 have two cross peaks, 4.69 / 76.5ppm and E-C6. There is an overlapping peak position of cross peak 4.47 / 76.5ppm between sugar residue B-H1 and A-C4 amino acid residue. There is an overlapping peak position of cross peak 4.68 / 76.72ppm between sugar residue C-H1 and E-C4 residue. Based on the NOESY spectrum, we inferred the interaction between different amino acid residues, among which there are cross peaks 4.47 / 3.75ppm and 4.47 / 3.63ppm between B-H1 and A-H4. The results show that there is an intersection peak of cross peak 4.45 / 3.63ppm between sugar residue E-H1 and B-H4. Because the content of residue D and residue F is very small, they are not reflected in the structural formula.

[0122] Combining 1D NMR and 2D NMR data with methylation studies, we speculate that its main skeleton is: (1)-β-D-Manp-(1,4)-α-D-Glcp-(1,6)-β-D-GlCP-(1>,)-α(1,4)-beta-D-GlCP-(1)-β–D-Glcp-(1,7)-β(4,6)—β-D-GlCP-"(1O-4 position), thus speculating that its skeleton is as shown in the following formula:

[0123] Table 4

[0124]

[0125] Table 5

[0126]

[0127] Table 6

[0128] Sample name Mass(mg) Uronic acid content (%) Protein content Total sugar content (mg / g) Bletilla striata polysaccharide 5.04 3.1 Tr. 968.6619

[0129] Table 7

[0130]

[0131]

[0132] Table 8

[0133]

[0134] Table 9

[0135]

Claims

1. A method for structural identification of neutral polysaccharides from Bletilla striata, characterized in that: The following steps are involved: Step 1: sequentially extracting, separating and purifying crude polysaccharides from Bletilla striata to obtain Bletilla striata polysaccharides; Step 2: pre-treat the polysaccharide from Bletilla striata and measure its homogeneity and molecular weight; Step 3: using the phenol-sulfuric acid method to determine the total sugar content in the Bletilla striata polysaccharide, using the trifluoroacetic acid method combined with gas chromatography-mass spectrometry to determine the chemical composition of the Bletilla striata polysaccharide, using the m-hydroxybiphenyl method to determine the uronic acid content in the Bletilla striata polysaccharide component, and using the Bio-rad protein quantitative method to determine the protein content in the Bletilla striata polysaccharide; Step 4: Methylation analysis of Bletilla striata polysaccharides; Step 5: Use nuclear magnetic resonance technology to analyze the monosaccharide components and bonding patterns of Bletilla striata polysaccharides.

2. The method for structural identification of neutral polysaccharides from Bletilla striata according to claim 1, characterized in that: The pretreatment is specifically as follows: dissolving the polysaccharide from Bletilla striata in a 0.05-0.5M NaNO3 aqueous solution containing 0.01-0.05wt% NaN3, wherein the concentration of the polysaccharide from Bletilla striata is 0.2-2mg / mL, and then filtering using a 0.1-1μm filter membrane.

3. The method for structural identification of neutral polysaccharides from Bletilla striata according to claim 2, characterized in that: The step 2 uses a differential refractive index detector, a laser scattering detector and a high performance liquid chromatograph to perform homogeneity and molecular weight determination.

4. The method for structural identification of neutral polysaccharides from Bletilla striata according to claim 3, characterized in that: The high performance liquid chromatograph used a Shodex OHpakSB HQ series gel chromatography column.

5. The method for structural identification of neutral polysaccharides from Bletilla striata according to claim 4, characterized in that: The chromatographic parameters of the high performance liquid chromatograph are as follows: the mobile phase is a 0.1-0.8 wt % dimethyl sulfoxide aqueous solution, the flow rate is 0.1-0.5 mL / min, and the elution gradient is isothermal for 100-150 min.

6. The method for structural identification of neutral polysaccharides from Bletilla striata according to claim 1 or 5, characterized in that: The gas chromatography-mass spectrometry chromatography system is a combination of ion chromatography and electrochemical detector.

7. The method for structural identification of neutral polysaccharides from Bletilla striata according to claim 6, characterized in that: The Bio-rad protein quantification method uses bovine serum protein as a control for external standard method testing.

8. The method for structural identification of neutral polysaccharides from Bletilla striata according to claim 1 or 5, characterized in that: The step 4 specifically includes: methylating the Bletilla striata polysaccharide, then oxidizing it with sodium borohydride, and then acetylating it to obtain a structurally stable sugar alcohol-acetate compound, and then using gas chromatography-mass spectrometry to determine the position and content ratio of the glycosidic bonds in the polysaccharide.

9. The method for structural identification of neutral polysaccharides from Bletilla striata according to claim 8, characterized in that: The temperature program of the gas chromatography in step 4 is: the initial column temperature is 120-160°C, maintained for 1-4 minutes, and the column is heated to 220-240°C at a heating rate of 2-5°C / min, and maintained for 2-5 minutes; the ion source of the mass spectrometer is an electron bombardment ion source.

10. The method for structural identification of neutral polysaccharides from Bletilla striata according to claim 1, characterized in that: The step 5 is specifically as follows: dissolving the monosaccharide components of Bletilla striata polysaccharide in heavy water, and using nuclear magnetic resonance technology to conduct H-NMR, C-NMR, heteronuclear single quantum region correlation spectroscopy and heteronuclear multi-carbon correlation spectroscopy for research.

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