Neutral heteropolysaccharide of hosta plantaginea as well as extraction method and application of neutral heteropolysaccharide

Abstract

The invention relates to the technical field of medicine, and discloses hosta plantaginea flower neutral heteropolysaccharide, an extraction method and application thereof, the hosta plantaginea flower neutral heteropolysaccharide is HPFP-1 and HPFP-2, monosaccharide of the HPFP-1 is composed of glucose and fructose, the HPFP-1 is mainly structurally characterized in that 1)-beta-D-Fruf-(2-> and a small amount of 1, 6)-beta-D-Fruf-(2->, 6)-alpha-D-Glcp are connected with each other to form a main chain, and the main chain is connected with the beta-D-Fruf-(2->, and the beta-D-Fruf-(2->, 6)-alpha-D-Glcp. The branched chain is mainly formed by connecting beta-D-Fruf-(2-) to the O-6 site of sugar residue-> 1, 6)-beta-D-Fruf-(2-) as a side chain. According to the application disclosed by the invention, on an LPS (Lipopolysaccharide)-induced Raw264.7 cell inflammation model, HPFP-1 and HPFP-2 can be used for reducing the level of NO, TNF-alpha (Tumor Necrosis Factor-alpha) and IL-6 (Interleukin-6) by inhibiting NF-kappa B signal channels (p-65, p65, p-I kappa B and p-I kappa B).

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a hosta flower neutral heteropolysaccharide, an extraction method and application thereof. Background Art

[0002] Acute pharyngitis is an acute inflammation of the pharyngeal mucosa, submucosal tissue and lymphoid tissue. Its symptoms are dryness, burning, roughness, pain in the pharynx, and pain when swallowing. The pain in the pharynx can radiate to the ears and neck on both sides. If the inflammation involves the larynx, symptoms such as cough and hoarseness may occur. It is often part of an upper respiratory tract infection and can occur alone or secondary to acute rhinitis. The causes of acute pharyngitis include viruses (such as influenza viruses, adenoviruses, etc.), bacterial infections (such as streptococci, staphylococci, etc.), and physical and chemical factors, such as high temperature, dust, smoke, irritating gases, tobacco, alcohol, spicy foods, etc. Acute pharyngitis is a common disease. For acute pharyngitis caused by bacteria, antibiotics (such as amoxicillin) can be used to kill bacteria and indirectly relieve the inflammatory state. It is worth noting that although acute pharyngitis caused by viruses and physical and chemical factors is self-limiting, the attack period will still cause great pain to the patient. At present, there is still a lack of specific drugs to treat or relieve acute pharyngitis. More importantly, repeated acute inflammation can turn into chronic inflammation and then cause canceration of the nasopharyngeal mucosa. Therefore, it is of great value to develop drugs to treat acute pharyngitis.

[0003] Hosta is a perennial root plant of the genus Hosta in the Asparagaceae family. It is native to China, Japan, the United States and other countries, and is also cultivated in Europe. As a commonly used medicine in traditional Chinese medicine, Hosta flowers are used to treat sore throat, hoarseness, and relieve cough. Hosta flowers contain polysaccharides, flavonoids, steroids, monoterpenoid saponins, and phenylpropanoids. Modern pharmacological studies have shown that Hosta flower extracts have anti-inflammatory, analgesic, antioxidant, anti-tumor, antibacterial, and anti-chronic prostatitis effects. Hosta flower extracts have anti-AP effects by inhibiting the levels of inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 in rat serum. Hosta flower extracts can reduce rat pharyngeal damage by inhibiting NF-κB, MAPKs, JAK-STAT, and PI3K-Akt pathways. Polysaccharides are high-molecular carbohydrates composed of more than 10 monosaccharides connected by glycosidic bonds. Polysaccharides composed of the same monosaccharides are called homopolysaccharides, such as starch, cellulose and glycogen; polysaccharides composed of different monosaccharides are called heteropolysaccharides, such as gum arabic which is composed of pentose and galactose.

[0004] Currently, there is little research on hosta polysaccharides. In order to clarify the structure and activity of hosta polysaccharides, the present invention provides a hosta neutral heteropolysaccharide, an extraction method and application thereof. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention can be achieved through the following technical solutions:

[0006] On the one hand, the embodiment of the present application provides a neutral heteropolysaccharide of Hosta, wherein the neutral heteropolysaccharide of Hosta is HPFP-1, and the monosaccharide of HPFP-1 is composed of glucose and fructose, and the molar ratio of monosaccharides is: 17.08:82.92;

[0007] The HPFP-1 is mainly composed of a structure of: →1)-β-D-Fruf-(2→ and a small amount of →1,6)-β-D-Fruf-(2→, →6)-α-D-Glcp connected to each other to form a main chain, and the main branch chain is composed of β-D-Fruf-(2→ connected to the O-6 position of the sugar residue →1,6)-β-D-Fruf-(2→ as a side chain.

[0008] On the other hand, the present application provides a neutral heteropolysaccharide of Hosta, wherein the neutral heteropolysaccharide of Hosta is HPFP-2, wherein the monosaccharides of HPFP-2 are composed of arabinose, rhamnose, galactose, glucose, xylose, mannose and glucuronic acid, and the molar ratio of monosaccharides is 24.90:6.87:59.56:3.03:2.29:1.38:1.95;

[0009] The HPFP-2 is mainly composed of a main chain of structures such as →3,6)-β-D-Galp-(1→, →5)-α-L-Araf-(1→, →3)-β-D-Galp-6-ome(1→, →6)-β-D-Galp-(1→, →4)-α-D-Galp-(1→), and the main branch chain is formed by α-L-Araf-(1→ connected to the O-3 position of the sugar residue →3,6)-β-D-Galp-(1→, and β-D-Galp-(1→ connected to the O-3 position of the sugar residue →3,6)-β-D-Galp-(1→).

[0010] A method for extracting neutral heteropolysaccharides from hosta flowers, the method comprising the following steps:

[0011] S1. Grind and sieve the dried Hosta flower product after drying, add anhydrous ethanol, stir to extract fat-soluble components, and centrifuge to obtain a precipitate;

[0012] S2, adding pure water to the precipitate, extracting in a water bath, collecting the supernatant after centrifugation, precipitating and extracting twice, combining the two supernatants to obtain an extract;

[0013] S3, concentrating the extract by vacuum rotary evaporation, adding anhydrous ethanol for overnight alcohol precipitation, and collecting the precipitated solid by centrifugation to obtain crude polysaccharide;

[0014] S4, adding pure water to the crude polysaccharide obtained in S3 to fully dissolve the crude polysaccharide, adding papain, enzymolysis overnight, adding 1 / 4 volume of chloroform and n-butanol to the aqueous phase, mixing well, collecting the upper aqueous phase; adding petroleum ether to the liquid, mixing well, collecting the lower aqueous phase, adding macroporous resin AB-8 to the aqueous phase, mixing well, adsorbing overnight, collecting the liquid, dialyzing with a 3000Da dialysis bag for 24 hours to remove small molecular components, and freeze-drying the polysaccharide solution to obtain crude polysaccharide;

[0015] S5, dissolving the crude polysaccharide obtained in S4 in pure water, centrifuging and taking the supernatant to purify through an ion exchange column at a flow rate of 4 ml / min; sequentially using pure water, 0.1 mol / L, 0.2 mol / L and 0.3 mol / L NaCl solution for isocratic elution, collecting one tube for every 15 ml, monitoring by sulfuric acid-phenol method, and drawing an ion purification elution curve; combining the eluates of each collection tube corresponding to the same elution peak, and concentrating; dialyzing for 48 hours with a 3000Da dialysis bag for desalination to obtain the ion-purified polysaccharide;

[0016] S6. Take the polysaccharide after ion purification, add pure water to dissolve; centrifuge and take the supernatant to pass through a gel chromatography column for separation and purification at a flow rate of 1 ml / min; elute with pure water, collect one tube for every 12 ml, and collect all the eluate; monitor with sulfuric acid-phenol method, and draw a gel purification elution curve; combine the eluates from each collection tube corresponding to the same elution peak, concentrate, and freeze-dry; use sulfuric acid-phenol method to detect the purity of the polysaccharide after gel purification, and obtain neutral heteropolysaccharides HPFP-1 and HPFP-2 from Hosta.

[0017] Furthermore, in S2, extraction was performed in a water bath at 60° C. for 4 h, and centrifugation was performed at 6000 g for 10 min.

[0018] Furthermore, in S3, the vacuum rotary evaporation is concentrated to 1 / 10 of the original volume, four times the volume of anhydrous ethanol is added for overnight alcohol precipitation, the precipitated solid is collected by centrifugation at 8000g for 10 minutes, and the soluble total sugar content and purity of the crude polysaccharide extract are determined by the sulfuric acid-phenol method. The specific operation is: accurately weigh a small amount of polysaccharide freeze-dried powder, add water to dissolve it, add sulfuric acid-phenol reagent, react in the dark for 10 minutes, and determine the absorbance at 490nm.

[0019] Furthermore, in S4, the crude polysaccharide obtained is measured for total soluble sugar content and purity using the sulfuric acid-phenol method.

[0020] Furthermore, in S5, the sulfuric acid-phenol method is used to detect the content and purity of the polysaccharide after ion purification.

[0021] On the other hand, the embodiments of the present application provide the use of the above-mentioned neutral heteropolysaccharide of Hosta odorata in the preparation of anti-inflammatory drugs for improving inflammatory damage.

[0022] Compared with the prior art, the technical solution of this application has the following beneficial effects:

[0023] In the LPS-induced Raw264.7 cell inflammation model of the present invention, HPFP-1 and HPFP-2 may reduce the levels of NO, TNF-α and IL-6 by inhibiting the NF-κB signaling pathway (p-65, p65, p-IκB, p-IκB). Therefore, the hosta flower polysaccharide is one of the components of the hosta flower that exerts an anti-inflammatory effect, and HPFP can be used as a dietary supplement or a potential anti-inflammatory drug for treating inflammatory damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0025] Figure 1 : Figure A is the ion purification elution curve of neutral heteropolysaccharide from Hosta odorata; Figure B is the gel purification elution curve of neutral heteropolysaccharide HPFP-1 from Hosta odorata; Figure C is the gel purification elution curve of neutral heteropolysaccharide HPFP-2 from Hosta odorata.

[0026] Figure 2 : Figure A is the ultraviolet spectrum of the neutral heteropolysaccharide HPFP-1 from Hosta flower; Figure B is the ultraviolet spectrum of the neutral heteropolysaccharide HPFP-2 from Hosta flower; Figure C is the infrared spectrum of the neutral heteropolysaccharide HPFP-1 from Hosta flower; Figure D is the infrared spectrum of the neutral heteropolysaccharide HPFP-2 from Hosta flower.

[0027] Figure 3 : A and B are the absolute molecular weight analysis diagrams of HPFP-1; C and D are the absolute molecular weight analysis diagrams of HPFP-2.

[0028] Figure 4 : Figure A is the ion chromatogram of the monosaccharide composition of HPFP-1; Figure B is the ion chromatogram of the monosaccharide composition of HPFP-2.

[0029] Figure 5 : Figure A is the 1H-NMR spectrum of HPFP-1; Figure B is the 13C NMR spectrum of HPFP-1; Figure C is the COSY spectrum of HPFP-1; Figure D is the HSQC spectrum of HPFP-1; Figure E is the NOESY spectrum of HPFP-1; and Figure F is the HMBC spectrum of HPFP-1.

[0030] Figure 6 : Figure A is the 1H-NMR spectrum of HPFP-2; Figure B is the 13C NMR spectrum of HPFP-2; Figure C is the COSY spectrum of HPFP-2; Figure D is the HSQC spectrum of HPFP-2; Figure E is the NOESY spectrum of HPFP-2; and Figure F is the HMBC spectrum of HPFP-2.

[0031] Figure 7 : Figure A is the X-ray diffraction pattern of HPFP-1; Figure B is the X-ray diffraction pattern of HPFP-2.

[0032] Figure 8 : A and B show the appearance of HPFP-1; C and D show the appearance of HPFP-2.

[0033] Fig. 9 : Figure A shows the effect of HPFP-1 on the proliferation of RAW 264.7 macrophages; Figure B shows the effect of HPFP-2 on the proliferation of RAW 264.7 macrophages; Figures C, D, and E show the effect of HPFP-1 on the production of NO, TNF-α, and IL-6 by RAW 264.7 macrophages stimulated by LPS; Figures F, G, and H show the effect of HPFP-1 on the production of NO, TNF-α, and IL-6 by RAW 264.7 macrophages stimulated by LPS.

[0034] Fig.10 : Figure A shows the effect of different concentrations of HPFP-1 on the expression of proteins in the NF-κB signaling pathway; Figure B shows the effect of different concentrations of HPFP-2 on the expression of proteins in the NF-κB signaling pathway. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0036] 1. Materials and Methods

[0037] 1.1 Materials and reagents

[0038] Dried lily of the valley was obtained from Inner Mongolia University for Nationalities. Petroleum ether, chloroform, anhydrous ethanol, n-butanol, phenol, sulfuric acid, sodium chloride, sodium hydroxide, and sodium nitrate were purchased from Sinopharm Chemical Reagent Co., Ltd. Papain, glucose, potassium bromide, monosaccharide standards (Fucose, Rhamnose, Arabinose, Galacto- ose, Glucose, Xylose, Mannose, Fructose, Ribose, Galacturonic Acid, Glucuronic Acid, Mannuronic Acid, and Guluronic Acid), dimethyl sulfoxide, sodium borodeuteride, and acetic anhydride were purchased from Sigma, and dichloromethane / DCM, trifluoroacetic acid / TFA, ammonia water, methyl iodide, and acetic acid were purchased from Shanghai Anpu Company. Deuterium oxide (heavy water) was purchased from Aladdin. Penicillin-streptomycin solution and fetal bovine serum were purchased from hyclone, DMEM medium supplemented with 10% fetal bovine serum and 2% penicillin-streptomycin solution, and LPS was purchased from invivogen. Elisa detection kits (NO, TNF-α, IL-1β, IL-6) were purchased from Jiangsu Jingmei Biotechnology Co., Ltd. NO detection kit was purchased from Biyuntian Biotechnology Co., Ltd. Primary antibodies (GAPDH, p65, p-p65, IκB, p-IκB) were purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0039] 1.2 Extraction and purification of polysaccharides

[0040] The dried sample / raw material was crushed with a grinder and passed through a 60-mesh sieve. Anhydrous ethanol was added, the fat-soluble components were extracted by stirring, centrifugation, and the precipitate was collected. Pure water was added to the precipitate, extracted in a 60°C water bath for 4 hours, centrifuged (6000g, 10min), and the supernatant was collected. The precipitate was extracted twice. The two extracts were combined, vacuum rotary evaporation concentrated to 1 / 10 of the original volume, and four times the volume of anhydrous ethanol was added for alcohol precipitation overnight. Centrifugation (8000g, 10min) was used to collect the precipitated solid, i.e., crude polysaccharide. The soluble total sugar content and purity of the crude polysaccharide extract were determined by the sulfuric acid-phenol method. The specific operation was as follows: accurately weigh a small amount of polysaccharide freeze-dried powder, dissolve it in water, add sulfuric acid-phenol reagent, react for 10 minutes in the dark, and measure the absorbance at 490nm.

[0041] Add pure water to the crude polysaccharide to fully dissolve it, add papain, and enzymolyze overnight. Add 1 / 4 volume of chloroform and n-butanol to the aqueous phase, mix thoroughly, and collect the upper aqueous phase. Add petroleum ether to the liquid, mix thoroughly, and collect the lower aqueous phase. Add macroporous resin AB-8 to the aqueous phase, mix thoroughly, and adsorb overnight. Collect the liquid, dialyze it with a 3000Da dialysis bag for 24 hours to remove small molecular components, and freeze-dry the polysaccharide solution. The soluble total sugar content and purity of the crude polysaccharide were determined by the sulfuric acid-phenol method.

[0042] 1.3 Ion exchange chromatography and dextran gel chromatography

[0043] 1.3.1 Ion exchange chromatography separation

[0044] The AKTA purification system of General Electric (China) Healthcare Group (GE, China) was used, and the weak anion exchange chromatography column DEAE seplife FF was purchased from Xi'an Lanxiao Technology New Materials Co., Ltd. The crude polysaccharide obtained in 2.2 was dissolved in pure water. Centrifugation (10000g, 10min), the supernatant was purified by ion exchange column, the flow rate was 4ml / min; pure water, 0.1mol / L, 0.2mol / L and 0.3mol / L NaCl solution were used in turn for gradient elution, and one tube was collected for every 15ml. The sulfuric acid-phenol method was used for monitoring, and the ion purification elution curve was drawn. The eluates of each collection tube corresponding to the same elution peak were combined and concentrated. Desalination was performed by dialyzing with a 3000Da dialysis bag for 48h. The content and purity of the polysaccharide after ion purification were detected by the sulfuric acid-phenol method.

[0045] 1.3.2 Sephadex gel chromatography separation

[0046] The AKTA purification system of the Life Sciences Department of General Electric (China) Healthcare Group was used. The Sephacryl S-400HR gel chromatography column of GE Company was selected for gel purification. The polysaccharide sample after ion purification was added with pure water and dissolved. Centrifugation (10000g, 10min), the supernatant was separated and purified by gel chromatography column, the flow rate was 1ml / min; pure water was used for elution, and one tube was collected for every 12ml, and all the eluents were collected. The sulfuric acid-phenol method was used for monitoring, and the gel purification elution curve was drawn. The eluents of each collection tube corresponding to the same elution peak were combined, concentrated, and freeze-dried. The purity of the polysaccharide after gel purification was detected by the sulfuric acid-phenol method.

[0047] 1.4 Homogeneity and molecular weight determination

[0048] The chromatographic system used was a 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). The sample was dissolved in a 0.1 mol / L NaNO3 aqueous solution (containing 0.02% NaN3) with a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm before being tested on the machine. Gel exclusion chromatography columns Ohpak SB-805HQ (300×8 mm) and Ohpak SB-803HQ (300×8 mm) were used in series. The column temperature was 45°C, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6 mL / min, and the isocratic elution was 75 min.

[0049] 1.5 Fourier-transforminfrared (FT-IR) spectroscopic analysis and UV–visible (UV–vis) spectroscopic analysis

[0050] The spectra were analyzed by using a Nicolet iZ-10 Fourier transform infrared spectrometer with a scanning range of 4000-450 cm-1. 1 mg of the sample was weighed and mixed with 200 mg of potassium bromide, and then pressed into a sheet with a thickness of 1 mm and tested on the spectrometer.

[0051] The polysaccharide solution was quantitatively analyzed using a Thermo Fisher (USA) multifunctional microplate reader. The microplate reader was Corning (USA), and the absorbance difference between the wells of the parallel experiment was required to be less than 0.02 at the target wavelength. The scanning start wavelength was 200nm, and the end wavelength was 1000nm. An appropriate amount of polysaccharide sample was weighed and dissolved in pure water to prepare a 5mg / mL polysaccharide solution, which was then tested on the machine.

[0052] 1.6 Monosaccharide composition analysis

[0053] The chromatographic system used is the Thermo ICS5000 ion chromatography system (ICS5000, Thermo Fisher Scientific, USA), and the monosaccharide components are analyzed and detected using an electrochemical detector. Take a clean chromatographic bottle, weigh an appropriate amount of polysaccharide sample, add 1 ml of 2M TFA acid solution, and heat at 60°C for 1 hour. Pass nitrogen and blow dry. Add 99.99% methanol to wash, blow dry again, and repeat the methanol washing 2-3 times. Add an appropriate amount of sterile water to dissolve, and transfer to a chromatographic bottle for testing. Use Dionex TMCarboPac TM PA20 (150*3.0mm, 10μm) liquid chromatography column; injection volume is 5μl. Mobile phase A (H2O), mobile phase B (0.1mol / L NaOH), mobile phase C (0.1mol / L NaOH, 0.2mol / L NaAc), flow rate 0.5ml / min; column temperature was 30℃; elution gradient: 0min phase A / phase B / phase C (95:5:0, V / V), 26min phase A / phase B / phase C (85:5:10, V / V), 42min phase A / phase B / phase C (85:5:10, V / V), 42.1min phase A / phase B / phase C (60:0:40, V / V), 52min phase A / phase B / phase C (60:40:0, V / V), 52.1min phase A / phase B / phase C (95:5:0, V / V), 60min phase A / phase B / phase C (95:5:0, V / V).

[0054] 1.7 Methylation analysis

[0055] Agilent Technologies Inc. CA, USA 6890A-5977B gas spectrometer was used, and the autosampler model was G4567A. The mass spectrometry system used was the quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA) of Aiglent, USA, equipped with an electron impact ion source (EI) and a MassHunter workstation. Take 2 mg of sample and add 500 μl DMSO to dissolve. Add 1 mg NaOH and incubate for 30 min. Add 50 μL iodomethane solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, mix, centrifuge, and discard the aqueous phase. Repeat the water wash 3 times. Take the lower dichloromethane phase and blow it dry with nitrogen. Add 100 μL 2 mol / L trifluoroacetic acid and react at 60 ° C for 1 h. Evaporate to dryness at 30 ° C. Add 50μL 2mol / L ammonia water, 50μL 1mol / L NaBD4, mix well, and react at room temperature for 2.5h. Add 20μl acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250μl methanol, blow dry with nitrogen. Add 250μL acetic anhydride, vortex mix well, and react at 100℃ for 2.5h. Add 1mL water and let stand for 10min. Add 500μL dichloromethane, vortex mix well, centrifuge, discard the aqueous phase, and repeat the water washing 3 times. Take the lower dichloromethane phase and detect it on GC-MS. 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 programmed to 230℃ at 3℃ / min and maintained for 3min. An electron impact ion source (EI) was used to detect the analytes in full scan (SCAN) mode with a mass scan range (m / z): 50-350.

[0056] 1.8 Nuclear magnetic resonance spectroscopy analysis

[0057] The target was quantitatively analyzed using a Bruker (Germany) 500MHz NMR spectrometer with a scanning temperature of 25°C. Liquid probe QXI 1H / 31P / 13C / 15N 5mm quadruple resonance reverse detection probe (Z-gradient, ATM Acc). Take an appropriate amount of purified polysaccharide and fully dissolve it in HPFP-2O to prepare a polysaccharide solution with a concentration of 40 mg / mL. Transfer the dissolved solution to an NMR tube and add 0.5 mL. Place the NMR tube in the NMR spectrometer to scan the one-dimensional 1H spectrum, 13C spectrum, and the two-dimensional COSY, HSQC, HMBC, and NOESY spectra.

[0058] 1.9 Field Emission Scanning Electron Microscope Scanning

[0059] A Hitachi Regulus 8100 high-resolution field emission scanning electron microscope was used. The polysaccharide sample was passed through a 100-mesh sieve, and a small amount was taken on a conductive carbon tape. After gold spraying, the sample was scanned and photographed using an electron microscope at a magnification of 5000 times.

[0060] 1.10 X-ray diffraction

[0061] The analysis was performed using an X'Pert Pro X-ray diffractometer (PANalytical, Netherlands). The purified polysaccharide sample was dried, crushed, passed through a 100-mesh sieve, and 20 mg of the sample was weighed on the stage, pressed and spread evenly, and then tested on the machine.

[0062] 1.11 Evaluation of the inhibitory effects of HPFP-1 and HPFP-2 on macrophage activation

[0063] 1.11.1 RAW264.7 cell culture

[0064] RAW264.7 macrophages were purchased from the Cell Bank of the Chinese Academy of Sciences and maintained in DMEM medium supplemented with 10% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin. The cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0065] Cell viability assay

[0066] The MTT method was used to determine the effect of polysaccharides on cell viability. RAW264.7 cells (90 μL) were plated in 96-well plates, with 5×10 5 , placed in a humidified incubator (37°C, 5% CO2), after the cells adhered, different concentrations of HPFP-1 and HPFP-2 (final concentrations: 12.5, 25, 50, 100, 200 and 500 μg / mL) were added to the wells, with a final volume of 100 μL / well, and an equal volume of DMEM medium was added to the control group, and three replicate wells were paralleled. After 24 hours of drug incubation, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated for 4 hours. The culture medium was discarded, 100 μL of DMSO was added to each well, and shaken in the dark for 10 minutes to dissolve the formazan. The absorbance at 570 nm was detected by an enzyme marker. Cell viability calculation method: Cell viability = A1 / A0×100%, where A0 is the absorbance value of the control group and A1 is the absorbance value of the sample group.

[0067] 1.11.3 Detection of nitric oxide (NO) content

[0068] The effects of HPFP-1 and HPFP-2 on LPS-induced NO production in RAW264.7 cells were evaluated using a nitric oxide assay kit (NO Assay Kit). RAW264.7 cells (1 mL) were seeded in 6-well plates (5 × 10 5 Different concentrations of polysaccharides (0, 12.5, 25, 50 μg / mL) were added to the wells and incubated for 2 h, and then the cells were stimulated with LPS at a final concentration of 5 μg / mL (except for the control group). After 12 h of stimulation, the NO content in the supernatant was detected using Griess reagent. The absorbance of the reaction product was measured at a wavelength of 540 nm using an enzyme marker.

[0069] 1.11.4 Detection of IL-2 and IL-6 expression

[0070] ELISIA detection kits were used to detect the expression levels of inflammatory factors in RAW264.7 cells induced by LPS by HPFP-1 and HPFP-2. The cell supernatants of each group in 2.11.3 were collected and the expression levels of IL-2 and IL-6 were detected according to the instructions of the ELISIA detection kits.

[0071] 1.11.5 Western Blot

[0072] To explore the anti-inflammatory mechanism of HPFP-1 and HPFP-2, western blot was used to detect the activation of NF-κB pathway. RAW264.7 cells (1 mL) were seeded in 6-well plates (5 × 10 5 / well). The cells were divided into a blank group, a positive drug group, and a drug-treated group. One hour before LPS stimulation, the positive drug group was treated with dexamethasone (5 μg / mL), and the drug-treated group was treated with HPFP-1 and HPFP-2 (12.5, 25, 50 μg / mL). The cells were stimulated with LPS (5 μg / mL) for 0.5 h, and total protein was extracted after incubation. The expression of 65, p-p65, IκB, P-IκB, and GADPH proteins was analyzed by western blot according to the literature method.

[0073] 1.11.6 Statistical analysis

[0074] All experiments were repeated at least 3 times. Data are expressed as mean ± standard deviation (n ≥ 3). The differences were statistically significant:

[0075] *P<0.05 vs. blank group, ^P<0.05 vs. LPS group.

[0076] 2. Results

[0077] 2.1 Extraction, purification and separation of Hosta polysaccharides

[0078] Using water-soluble alcohol precipitation method, 48g of crude polysaccharide was extracted from 0.446kg of Hosta flower dried product, with a content of 55.6% and a yield of 10.8%. After impurities were removed, the content of the obtained crude polysaccharide product was 77.0%. Take 5g of crude polysaccharide product and pass it through ion exchange chromatography (such as Figure 1 A) and gel chromatography (as shown in Figure 1 B, C), the obtained HPFP-1 was 0.625 g, the yield was 12.5%, the content was 90.7%, and the HPFP-2 was 0.234 g, the yield was 4.68%, and the content was 92.2%.

[0079] 2.2 UV and IR spectra analysis of HPFP-1 and HPFP-2

[0080] By UV scanning analysis, HPFP-1 and HPFP-2 had no absorption at 260nm and 280nm, indicating that the two polysaccharides did not contain nucleic acids and proteins (such as Figure 2 A, B). By infrared spectroscopy analysis (as Figure 2 C, D), HPFP-1, HPFP-2 at 3410.67cm -1 ,3419.04cm -1 There is absorption, this absorption peak is the stretching vibration absorption peak of OH, which is the characteristic peak of sugars. The two polysaccharides are at 2932.52cm -1 , 2931.60cm -1 There is absorption at 1027.49 cm-1, which is attributed to the stretching vibration of CH. -1 , 1075.96cm -1 There is absorption, indicating the presence of CO bonds. The carbonyl absorption peak most commonly appears in the region of 1755-1670 cm -1 , and the absorption is very strong, often the first strong peak in the IR spectrum. HPFP-1 and HPFP-2 have no absorption in this range. -1 , 1635.55cm -1 There is absorption but it is not strong, so it can be inferred that the two polysaccharides have no carbonyl or a small amount of carbonyl. It is further shown that the two polysaccharides do not contain uronic acid or contain a small amount of uronic acid, and both are neutral polysaccharides. This result is consistent with the results of monosaccharide composition analysis and glycosidic bond analysis.

[0081] 2.3 Molecular weight of HPFP-1 and HPFP-2

[0082] The absolute molecular weights of the two polysaccharides were determined using a gel chromatography-differential multi-angle laser light scattering system (e.g. Figure 3A, B, C, D). Mn is the number average molecular weight, Mw is the weight average molecular weight, and the polydispersity is (Mw / Mn). HPFP-1 has Mn: 3.115 kDa, Mw: 3.503 kDa, and polydispersity: 1.124. HPFP-2 has Mn: 76.656 kDa, Mw (kDa): 112.709, and polydispersity: 1.470.

[0083] 2.4 Analysis of monosaccharide composition of HPFP-1 and HPFP-2

[0084] The results of monosaccharide composition analysis are shown in the figure ( Figure 4 A, B). HPFP-1 is mainly composed of fructose (Fru) and glucose (Glc), with a molar ratio of 17.08:82.92. HPFP-2 is composed of arabinose (Ara), rhamnose (Rha), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), and glucuronic acid (Glc-UA), with a molar ratio of 24.90:6.87:59.56:3.03:2.29:1.38:1.95.

[0085] Table 1 GS-MS data of HPFP-1 methylation analysis

[0086]

[0087] 2.5 HPFP-1 and HPFP-2 methylation analysis

[0088] Methylation analysis showed the types, connection modes and molar ratios of sugar residues in HPFP-1 and HPFP-2. HPFP-1 has five types of glycosidic bonds: Fruf-(2→, Glcp-(1→, →1)Fruf-(2→, →6)-Glcp-(1→, →1, 6)-Fruf-(1→, with a molar ratio of 25.32:2.22:49.95:8.22:14.28 (Table 1).

[0089] Table 2 GS-MS data of HPFP-2 methylation analysis

[0090]

[0091]

[0092] HPFP-2 has 16 types of glycosidic bonds: Rhap-(1→, Araf-(1→, Arap-(1→, Xylp-(1→, →2)-Rhap-(1→, Glcp-(1→, →3)-Araf-(1→, Galp-(1→, →5)-Araf-(1→, →4)-Xylp-(1→, →2,4)-Rhap-(1→, →3)-Galp-(1→ ,→4)-Galp-(1→,→4)-Glcp-(1→,→6)-Galp-(1→,→3,6)-Galp--(1→, with a molar ratio of 1.19:15.41:1.15:1.16:1.23:1.90:1.53:12.56:3.54:1.23:2.08:12.28:6.00:1.45:6.79:30.49 (Table 2).

[0093] 3.6 Analysis of HPFP-1 and HPFP-2 NMR spectra

[0094] NMR spectroscopy plays an important role in determining sugar residues, substitution sites, and sugar attachment order. 1D NMR spectroscopy includes 1 H NMR and 13 C NMR, 2D NMR spectra include COSY, NOESY, HSQC, HMBC. The spectra and analysis process of HPFP-1 and HPFP-2 obtained by NMR scanning are as follows:

[0095] 2.6.1 NMR spectrum analysis of HPFP-1

[0096] like Figure 5 HPFP-1 did not detect a significant absorption peak in the range of δ4.4-5.3ppm (δ4.71ppm is the HOD solvent peak), which is consistent with the fructose signal, indicating that the sample contains fructose structure. There is a weak absorption peak at δ5.31ppm, which belongs to α-configuration glucose and is recorded as sugar residue D. The non-anomeric hydrogen signal is mainly concentrated in δ

[0097] In the 3.1-4.2 ppm region, due to the serious overlap of individual signals, it is necessary to combine COSY and HSQC spectra to assign the H2-H6 chemical shifts of each sugar residue separately.

[0098] HPFP-1 mainly identified four coupling signal peaks in the anomeric carbon region, with chemical shifts of δ103.64, 103.87, 103.15 and δ92.10 ppm, which were recorded as sugar residues A, B, C and D respectively. 1 H NMR, 13The cross peaks in the anomeric region of the C NMR spectrum and the HSQC spectrum determined that the anomeric signal of the sugar residue D was δ5.31 / 92.10ppm; sugar residues A, B, and C had no cross peaks in the anomeric region of the HSQC spectrum, which was consistent with the structural characteristics of fructose. Combining the sample bonding structure (methylation) information, anomeric signals, and comprehensive literature reports, it was inferred that sugar residue A was →1)-β-D-Fruf-(2→, residue B was β-D-Fruf-(2→, residue C was →1,6)-β-D-Fruf-(2→, and residue D was →6)-α-D-Glcp. 1 H and 13 The chemical shifts were assigned and the results are shown in Table 3.

[0099] The process of assigning the NMR signals of the main sugar residues is as follows: Sugar residue A: 13 C NMR showed two groups of anomeric carbon signals at around δ92ppm and δ103ppm, while 1 In H NMR, only a group of obvious anomeric proton signals were observed at δ5.31ppm. Combined with the fact that sugar residue A has no cross peak in the anomeric region of the HSQC spectrum and ketose does not contain anomeric protons, it is shown that residue A may be a fructose residue, and its anomeric carbon signal is δ103.64ppm. For pyranose residues with glycosidic bonds, the anomeric carbon signals of the α configuration are mainly distributed in the range of 90–100ppm, and the anomeric carbon signals of the β configuration are mainly distributed in the range of 100-106ppm. Combining the HSQC spectrum and literature reports, the chemical shifts of sugar residue A were assigned. The results are shown in Table 3. It is inferred that sugar residue A is →1)-β-D-Fruf-(2→.

[0100] Similar to the above method, it can be inferred that sugar residues B and C may also be fructose residues. Combining the methylation analysis results and literature reports, it is inferred that sugar residue B may be β-D-Fruf-(2→, and C may be →1,6)-β-D-Fruf-(2→.

[0101] Sugar residue D: The anomeric signal δ5.31 / 92.10ppm (H1 / C1) indicates that residue D may be an α-configuration glucose residue. In the COSY spectrum, H2 (3.47ppm) of residue C was determined according to the cross peak δ5.31 / 3.47ppm, H3 (3.65ppm) of residue C was determined according to the cross peak δ3.47 / 3.65ppm, H4 (3.84ppm) of residue C was determined according to the cross peak δ3.65 / 3.84ppm, H5 (3.75ppm) of residue C was determined according to the cross peak δ3.84 / 3.75ppm, H6 (3.38ppm) of residue C was determined according to the cross peak δ3.75 / 3.38ppm, and H6 (3.83ppm) of residue C was determined according to the cross peak δ3.75 / 3.83ppm, so that the chemical shifts of hydrogen on the sugar ring can be assigned. Then, the chemical shift of C on the sugar ring was assigned by HSQC signal. The C1 chemical shift of residue C was δ92.10ppm, the C2 chemical shift of residue C was δ71.08ppm, the C3 chemical shift of residue C was δ72.38ppm, the C4 chemical shift of residue C was δ71.50ppm, the C5 chemical shift of residue C was δ74.37ppm, and the C6 chemical shift of residue C was δ69.13ppm. The chemical shift of HPFP-1 shifted to the low field, indicating that the residue was substituted at the O-6 position of the sugar ring. Combined with the methylation analysis results and literature reports, it was inferred that sugar residue D might be →6)-α-D-Glcp.

[0102] Combining the one-dimensional NMR and two-dimensional NMR spectra, the sugar residue signals of the samples were assigned, as shown in Table 3:

[0103] Table 3 Sugar residues 1 H and 13 Chemical shift of C

[0104]

[0105] 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 δ103.64 / 3.71ppm between C2 of sugar residue A and H1 of sugar residue A, there is a cross peak of δ103.64 / 3.63ppm between C2 of sugar residue A and H1 of sugar residue C, there is a cross peak of δ103.87 / 3.64ppm between C2 of sugar residue B and H6 of sugar residue C, there is a cross peak of δ103.15 / 3.83ppm between C2 of sugar residue C and H6 of sugar residue D. According to the NOESY spectrum, there is a cross peak of δ5.31 / 3.59ppm between H1 of sugar residue D and H1 of sugar residue A.

[0106] Therefore, based on the analysis of 1D and 2D NMR information and methylation results, it is inferred that the polysaccharide is mainly composed of →1)-β-D-Fruf-(2→ and a small amount of →1,6)-β-D-Fruf-(2→, →6)-α-D-Glcp connected to each other to form the main chain, and the branch chain is mainly composed of β-D-Fruf-(2→ connected to the O-6 position of the sugar residue →1,6)-β-D-Fruf-(2→ as a side chain. Therefore, the possible structure of HPFP-1 is speculated as follows:

[0107]

[0108] 2.6.2 NMR spectrum analysis of HPFP-2

[0109] like Figure 6 The HPFP-2 hydrogen spectrum signals are mainly concentrated between δ3.0 and 5.5ppm. Multiple coupling signal peaks are identified in the δ4.3-5.4ppm anomeric signal region, indicating that this sample contains a variety of sugar residues, and the chemical shifts of the corresponding anomeric hydrogens are δ4.42, 4.47, 4.56, 4.63, 4.94, 5.02, 5.15, 5.17, etc. The non-anomeric hydrogen signals are mainly concentrated in the δ3.1-4.2ppm region. Due to the serious overlap of individual signals, the H2-H6 chemical shifts of each sugar residue need to be attributed in combination with the COSY spectrum. Among them, the strong signal peak near δ4.71ppm is the solvent peak. The signal peak near δ3.42ppm is the signal of hydrogen of O-CH3.

[0110] HPFP-2 identified multiple signal peaks in the anomeric carbon region. 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: δ4.42 / 103.39, 5.17 / 109.22, 4.56 / 104.34, 4.63 / 103.62, 4.47 / 103.12, 4.94 / 99.36, 5.02 / 107.38, 5.15 / 98.86 ppm, and were recorded as sugar residues A, B, C, D, E, F, G, and H, respectively. Combining the sample bonding structure (methylation) information, anomeric signals and literature reports, it is speculated that sugar residue A is →3,6)-β-D-Galp-(1→, sugar residue B is α-L-Araf-(1→, sugar residue C is β-D-Galp-(1→, sugar residue D is →3)-β-D-Galp-6-ome-(1→, sugar residue E is →6)-β-D-Galp-(1→, sugar residue F is →4)-α-D-Galp-(1→, sugar residue G is →5)-α-L-Araf-(1→, sugar residue H is →2,4)-α-L-Rhap-(1→. The 1H and 13C chemical shifts were assigned, and the results are shown in Table 6.7. Among them, the signal peak near δ59.31ppm is the signal of the carbon of O-CH3.

[0111] Table 4 Sugar residues 1 H and 13 Chemical shift of C nd: Abbreviation for "not detected", meaning not identified.

[0112] 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. Since the HMBC spectrum is weak, it is mainly determined based on the NOESY spectrum. There is a cross peak of δ4.42 / 3.85ppm between H1 of sugar residue A and H6 of sugar residue A, a cross peak of δ4.42 / 3.87ppm between H1 of sugar residue A and H5 of sugar residue G, a cross peak of δ5.17 / 3.67ppm between H1 of sugar residue B and H3 of sugar residue A, a cross peak of δ4.56 / 3.67ppm between H1 of sugar residue C and H3 of sugar residue A, and a cross peak of δ4.70 / 3.70ppm between H1 of sugar residue D and H6 of sugar residue A. There is a cross peak between H1 and H6 of sugar residue E at δ4.63 / 3.84 ppm, there is a cross peak between H1 of sugar residue E and H4 of sugar residue F at δ4.47 / 3.86 ppm, there is a cross peak between H1 of sugar residue F and H6 of sugar residue A at δ4.94 / 3.85 ppm, there is a cross peak between H1 of sugar residue G and H3 of sugar residue D at δ5.01 / 3.22 ppm, there is a cross peak between H1 of sugar residue G and H5 of sugar residue G at δ5.01 / 3.87 ppm.

[0113] Therefore, based on the one-dimensional and two-dimensional NMR information and the methylation results, it was inferred that the polysaccharide was mainly composed of →3,6)-β-D-Galp-(1→, →5)-α-L-Araf-(1→, →3)-β-D-Galp-6-ome(1→, →6)-β-D-Galp-(1→, →4)-α-D-Galp-(1→, etc., which were connected to each other to form the main chain. The side chains were mainly composed of α-L-Araf-(1→ connected to the O-3 position of the sugar residue →3,6)-β-D-Galp-(1→, β-D-Galp-(1→ connected to the O-3 position of the sugar residue →3,6)-β-D-Galp-(1→, etc. The possible structure of HPFP-2 is inferred to be as follows:

[0114]

[0115] 3.7 Morphology and X-ray diffraction analysis of HPFP-1 and HPFP-2

[0116] HPFP-1 was observed to have a rough surface and irregular fragments under magnifications of 5000 and 10000 times. Figure 8 A, B). Under magnification of 5000 and 10000, it was observed that the surface of HPFP-2 was relatively flat with circular hole-like depressions ( Figure 8 C,D).

[0117] X-ray diffraction (XRD) analysis is often used to determine the crystalline or amorphous structure of polysaccharides. The crystal structure of polysaccharides determines physical properties such as solubility and flexibility. The diffraction peaks of HPFP-1 and HPFP-2 are both broad peaks with a diffraction angle of about 19°, but there are no visible crystal peaks, indicating that the structures of HPFP-1 and HPFP-2 are semi-crystalline structures ( Figure 7 A, B).

[0118] 2.8 In vitro anti-inflammatory studies of HPFP-1 and HPFP-2

[0119] 2.8.1 Effects of HPFP-1 and HPFP-2 on cell viability

[0120] HPFP-1 and HPFP-2 had no significant toxicity to RAW264.7 cells in the range of 12.5-500 μg / mL. The proliferation rate of macrophages was slightly reduced at the two concentrations of 200 and 500 μg / mL. The reason may be that the high concentration of polysaccharides affects the osmotic pressure of the cell culture medium and thus has a certain effect on cell proliferation.

[0121] 2.9.2 Detection of expression levels of inflammatory factors such as NO, TNF-α, and IL-6

[0122] The levels of NO, TNF-α, and IL-6 reflect the inflammatory level of LPS-induced RAW264.7 cells. Compared with the model group, 12.5, 25, 50, and 100 μg / mL of HPFP-1 and HPFP-2 can inhibit the production of inflammatory factors NO, TNF-α, and IL-6 in RAW264.7 cells induced by LPS, and the inhibition level and concentration gradient are not linear ( Fig. 9 ).

[0123] 2.9.3 Western-blot analysis of NF-κB signaling pathway

[0124] The NF-κB signaling pathway is related to innate immunity, adaptive immunity, inflammation, and stress response. p65, p-p65, p-IκB, and IκB are key proteins in the NF-κB pathway. The activation of the pathway is determined by the high or low expression of the four proteins. The LPS-induced macrophage RAW.264.7 inflammation model was used to evaluate the anti-inflammatory level of HPFP-1 and HPFP-2 in vitro. As shown in the figure. Compared with the control group, there was no significant difference in the expression of p65 and IκB bands in the model group (under the stimulation of 5ng / mL LPS), while the p-p65 and p-IκB bands became significantly thicker and darker, proving that the NF-κB signaling pathway was successfully activated. Compared with the model group, at 12.5, 25, 50, and 100 mg / mL HPFP-1 and HPFP-2 concentrations, there was no significant difference in the expression of p65 and IκB bands. The p-p65 and p-IκB bands became significantly lighter to varying degrees, and in a concentration-dependent manner, indicating that HPFP-1 and HPFP-2 can reduce the level of inflammation in vitro by inhibiting the NF-κB pathway ( Fig.10 ).

[0125] In summary, this application confirms the anti-inflammatory activity of Hosta polysaccharides and analyzes the structure of Hosta polysaccharides. Through water-soluble alcohol precipitation and column chromatography separation, we obtained two polysaccharides with significant structural differences, namely HPFP-1 and HPFP-2. HPFP-1 is composed of glucose and fructose with a molar ratio of 17.08:82.92, a weight-average molecular weight of 3.503 kDa, and a structure of: →1)-β-D-Fruf-(2→ and a small amount of →1,6)-β-D-Fruf-(2→, →6)-α-D-Glcp are interconnected to form a main chain, and the main branch chain is composed of β-D-Fruf-(2→ connected to the O-6 position of the sugar residue →1,6)-β-D-Fruf-(2→ as a side chain. HPFP-2 is composed of arabinose (Ara), rhamnose ( Rha), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), glucuronic acid (Glc-UA), the molar ratio is: 24.90: 6.87: 59.56: 3.03: 2.29: 1.38: 1.95, the weight average molecular weight is 112.709 kDa. HPFP-2 structure is mainly composed of →3,6)-β-D-Galp-(1→, →5)-α-L-Araf-(1→, →3)-β-D-Galp-6-ome (1→, →6)-β-D-Galp-(1→, →4)-α-D-Galp-(1→ etc. are connected to each other to form the main chain, and the main branch chain is composed of α-L-Araf-(1→ connected to the O-3 position of the sugar residue →3,6)-β-D-Galp-(1→, β-D-Galp-(1→ connected to the O-3 position of the sugar residue →3,6)-β-D-Galp-(1→ etc. HPFP increases the level of NO in rats with acute pharyngitis, decreases the levels of TNF-α, IL-1β, IL-6, and reduces inflammation HPFP can improve the inflammatory state of acute pharyngitis in rats by infiltrating neutrophils and macrophages. At the cellular level, the two polysaccharides can inhibit the expression of inflammatory factors such as NO, TNF-α, and IL-6 in Raw264.7 macrophages induced by LPS by inhibiting NF-κB activation. Therefore, the results of the study show that Hosta polysaccharide is one of the components of Hosta that exerts anti-inflammatory effects, and HPFP can be used as a dietary supplement or a potential anti-inflammatory drug for the treatment of inflammatory damage.

[0126] The invention concept is described in detail using specific examples herein, and the description of the above embodiments is only used to help understand the core idea of ​​the invention. It should be pointed out that any obvious modification, equivalent substitution or other improvement made by a person of ordinary skill in the art without departing from the invention concept should be included in the protection scope of the present invention.

Claims

1. A neutral heteropolysaccharide from Hosta odorata, characterized in that: The neutral heteropolysaccharide of Hosta is HPFP-1, and the monosaccharide of HPFP-1 is composed of glucose and fructose, and the molar ratio of monosaccharides is 17.08:82.92; The HPFP-1 is mainly composed of a structure of: →1)-β-D-Fruf-(2→ and a small amount of →1,6)-β-D-Fruf-(2→, →6)-α-D-Glcp connected to each other to form a main chain, and the main branch chain is composed of β-D-Fruf-(2→ connected to the O-6 position of the sugar residue →1,6)-β-D-Fruf-(2→ as a side chain.

2. A neutral heteropolysaccharide from Hosta odorata, characterized in that: The hosta neutral heteropolysaccharide is HPFP-2, the monosaccharides of the HPFP-2 are composed of arabinose, rhamnose, galactose, glucose, xylose, mannose and glucuronic acid, and the molar ratio of the monosaccharides is 24.90:6.87:59.56:3.03:2.29:1.38:1.95; The HPFP-2 is mainly composed of a main chain of structures such as →3,6)-β-D-Galp-(1→, →5)-α-L-Araf-(1→, →3)-β-D-Galp-6-ome(1→, →6)-β-D-Galp-(1→, →4)-α-D-Galp-(1→), and the main branch chain is formed by α-L-Araf-(1→ connected to the O-3 position of the sugar residue →3,6)-β-D-Galp-(1→, and β-D-Galp-(1→ connected to the O-3 position of the sugar residue →3,6)-β-D-Galp-(1→).

3. A method for producing neutral heteropolysaccharides from Hosta odorifera according to claim 1 or 2, characterized in that: The extraction method comprises the following steps: S1. Grind and sieve the dried Hosta flower product after drying, add anhydrous ethanol, stir to extract fat-soluble components, and centrifuge to obtain a precipitate; S2, adding pure water to the precipitate, extracting in a water bath, collecting the supernatant after centrifugation, precipitating and extracting twice, combining the two supernatants to obtain an extract; S3, concentrating the extract by vacuum rotary evaporation, adding anhydrous ethanol for overnight alcohol precipitation, and collecting the precipitated solid by centrifugation to obtain crude polysaccharide; S4, adding pure water to the crude polysaccharide obtained in S3 to fully dissolve the crude polysaccharide, adding papain, enzymolysis overnight, adding 1 / 4 volume of chloroform and n-butanol to the aqueous phase, mixing well, collecting the upper aqueous phase; adding petroleum ether to the liquid, mixing well, collecting the lower aqueous phase, adding macroporous resin AB-8 to the aqueous phase, mixing well, adsorbing overnight, collecting the liquid, dialyzing with a 3000Da dialysis bag for 24 hours to remove small molecular components, and freeze-drying the polysaccharide solution to obtain crude polysaccharide; S5, dissolving the crude polysaccharide obtained in S4 in pure water, centrifuging and taking the supernatant to purify through an ion exchange column at a flow rate of 4 ml / min; sequentially using pure water, 0.1 mol / L, 0.2 mol / L and 0.3 mol / L NaCl solution for isocratic elution, collecting one tube for every 15 ml, monitoring by sulfuric acid-phenol method, and drawing an ion purification elution curve; combining the eluates of each collection tube corresponding to the same elution peak, and concentrating; dialyzing for 48 hours with a 3000Da dialysis bag for desalination to obtain the ion-purified polysaccharide; S6. Take the polysaccharide after ion purification, add pure water to dissolve; centrifuge and take the supernatant to pass through a gel chromatography column for separation and purification at a flow rate of 1 ml / min; elute with pure water, collect one tube for every 12 ml, and collect all the eluate; monitor with sulfuric acid-phenol method, and draw a gel purification elution curve; combine the eluates from each collection tube corresponding to the same elution peak, concentrate, and freeze-dry; use sulfuric acid-phenol method to detect the purity of the polysaccharide after gel purification, and obtain neutral heteropolysaccharides HPFP-1 and HPFP-2 from Hosta.

4. The method for producing neutral heteropolysaccharides from Hosta odorifera according to claim 3, characterized in that: In the S2, extraction was performed in a water bath at 60° C. for 4 h and centrifuged at 6000 g for 10 min.

5. The method for producing neutral heteropolysaccharides from Hosta odorifera according to claim 3, characterized in that: In the S3, vacuum rotary evaporation is performed to concentrate the extract to 1 / 10 of the original volume, four times the volume of anhydrous ethanol is added for overnight alcohol precipitation, and the precipitated solid is collected by centrifugation at 8000g for 10 minutes. The soluble total sugar content and purity of the crude polysaccharide extract are determined by the sulfuric acid-phenol method. The specific operation is as follows: accurately weigh a small amount of polysaccharide freeze-dried powder, add water to dissolve it, add sulfuric acid-phenol reagent, react in the dark for 10 minutes, and determine the absorbance at 490nm.

6. The method for producing neutral heteropolysaccharides from Hosta odorifera according to claim 3, characterized in that: In S4, the crude polysaccharide obtained is measured for total soluble sugar content and purity using the sulfuric acid-phenol method.

7. The method for producing neutral heteropolysaccharides from Hosta odorifera according to claim 3, characterized in that: In S5, the sulfuric acid-phenol method is used to detect the content and purity of the polysaccharide after ion purification.

8. Use of the neutral heteropolysaccharide of Hosta odorata as claimed in any one of claims 1 to 2 in the preparation of anti-inflammatory drugs for improving inflammatory damage.

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