Separation and purification and structural analysis method of physalis alkekengi calyx polysaccharide with anti-inflammatory activity and application of physalis alkekengi calyx polysaccharide

Abstract

The invention discloses a separation and purification and structural analysis method of physalis alkekengi calyx polysaccharide with anti-inflammatory activity and application of the physalis alkekengi calyx polysaccharide, and relates to the technical field of polysaccharide separation and purification. Comprising the following steps: extracting physalis alkekengi calyx crude polysaccharide from physalis alkekengi calyx, performing separation and purification treatment on the physalis alkekengi calyx crude polysaccharide, and performing structural analysis on the physalis alkekengi calyx refined polysaccharide; the physalis alkekengi calyx polysaccharide is extracted through water extraction and alcohol precipitation, deproteinization, dialysis and freeze-drying technologies, separation and purification operation is carried out by means of DEAE-52 cellulose and a sephadex column, the purity of the obtained extract can reach 90% or above, the method is simple, convenient, feasible, stable and reliable, high operability is achieved, the technological process is simple and clear, the separation effect is excellent, and the method is suitable for industrial production. The method can be recycled for multiple times, has excellent chemical stability, meets the strict requirements of industrial large-scale production, and can accurately analyze the structural characteristics of the physalis alkekengi calyx polysaccharide by combining a plurality of detection technologies.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide separation and purification, and in particular to a separation, purification and structural analysis method of a calyx polysaccharide of Pleurotus eryngii with anti-inflammatory activity and application thereof. Background Art

[0002] As a perennial herbaceous plant of the Solanaceae family, Physalis alkekengi L.var.franchetii (Mast.)Makino has extremely rich resource reserves in my country and has a wide range of pharmacological effects. Its important position is highlighted in the first volume of the 2020 edition of the Chinese Pharmacopoeia. Tracing back to the ancient Shennong's Herbal Classic, there are records showing that the fruit and calyx of Physalis alkekengi L.var.franchetii (Mast.)Makino have significant effects such as clearing heat, calming the nerves, and invigorating qi. In the field of clinical practice in my country, a variety of formulas, preparations or decoctions containing Physalis alkekengi L.var.franchetii (Mast.)Makino are widely used in the treatment of sore throat, pharyngitis, acute and chronic laryngitis, and suppurative tonsillitis, with remarkable results.

[0003] In-depth research on the chemical composition of Lantern Festival shows that it contains a variety of nutrients such as polysaccharides, flavonoids, steroids, mineral elements, vitamins and amino acids. These components work synergistically to give Lantern Festival multiple functions such as anti-inflammatory, thirst-quenching and hemostasis. They can play an active and effective role in relieving sore throat, dryness and treating diarrhea. Modern pharmacological studies have further revealed that Lantern Festival calyx has a series of remarkable biological activities such as anti-inflammatory, antibacterial, anti-tumor, antioxidant, hypoglycemic and immunosuppressive. Some studies (Lv Chunping, Wang Hongfang, Li Jing, et al. Experimental study on the anti-inflammatory effect of Lantern Festival [J]. Modern Preventive Medicine, 2007, (12): 2213-2214) also found that Lantern Festival extract has potential anti-inflammatory effects. Its mechanism of action is mainly through the effective inhibition of the production and secretion of nitric oxide (NO), prostaglandin E2 (PGE2) and inflammatory factors, thereby achieving anti-inflammatory effects.

[0004] Based on the findings in the prior art, the existing methods for separating and purifying polysaccharides from the calyx of Colletotrichum chinense are mostly complicated and difficult to meet the needs of large-scale industrial production. The purity of the separated polysaccharides from the calyx of Colletotrichum chinense is low and cannot meet the needs of practical applications. In addition, the structural analysis of the polysaccharides from the calyx of Colletotrichum chinense has not been fully completed, and the structural characteristics of the polysaccharides from the calyx of Colletotrichum chinense cannot be accurately analyzed. Therefore, the present invention proposes a method for separating, purifying and structurally analyzing polysaccharides from the calyx of Colletotrichum chinense with anti-inflammatory activity and its application to solve the problems existing in the prior art. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to propose a method for separation, purification and structural analysis of Pleurotus eryngii calyx polysaccharide with anti-inflammatory activity and its application, so as to solve the problems that most of the existing methods for separation and purification of Pleurotus eryngii calyx polysaccharide are complicated and difficult to meet the needs of large-scale industrial production, and the purity of the separated Pleurotus eryngii calyx polysaccharide is low, and the structural analysis of Pleurotus eryngii calyx polysaccharide has not been fully completed.

[0006] In order to achieve the purpose of the present invention, the present invention is implemented by the following technical scheme: a method for separating, purifying and structurally analyzing polysaccharides of Lantern Festival calyx with anti-inflammatory activity, comprising the following steps:

[0007] Step 1: firstly extract the crude polysaccharide of Lantern Festival calyx from Lantern Festival calyx by water extraction and alcohol precipitation method, then prepare Sevag reagent and use Sevag method to deproteinize the crude polysaccharide of Lantern Festival calyx to obtain the deproteinized crude polysaccharide of Lantern Festival calyx;

[0008] Step 2: firstly, using DEAE-52 cellulose to separate the crude polysaccharide of Lanterna calyx to obtain Lanterna calyx polysaccharide powder, and then using polydextrose G-100 to purify the Lanterna calyx polysaccharide powder to obtain Lanterna calyx refined polysaccharide powder;

[0009] Step 3: Use UV-visible analysis technology, Fourier-infrared spectroscopy, high performance liquid chromatography technology, nuclear magnetic resonance technology, scanning electron microscope analysis technology and Congo red determination technology to detect the structural characteristics of the refined polysaccharide of Lantern Festival calyx.

[0010] The further improvement lies in that in the step one, the specific steps of extracting the crude polysaccharide of the calyx of Lantern Festival are as follows: first, the calyx of Lantern Festival is dried in the sun, and then placed in a drying oven for drying, and cut into pieces after drying, and then distilled water is added to the cut calyx of Lantern Festival at a solid-liquid ratio of 1:20, and refluxed with 80°C hot water for extraction for 2 hours, followed by filtering, repeating the above operation twice and combining the filtrate, and then adding the combined filtrate to a rotary evaporator to concentrate it to one-sixth of the original volume, and then standing to room temperature, and then adding 95% ethanol while the filtrate is constantly stirred, the volume ratio of the filtrate to ethanol is 1:4, and then standing at a temperature of 4°C for 12 hours. After standing, suction filtration and water dissolution are added, and then the ethanol is removed by evaporation, and finally freeze-drying is performed to obtain the crude polysaccharide of the calyx of Lantern Festival.

[0011] A further improvement is that in the step 1, the Sevag reagent is prepared by mixing chloroform and n-butanol, the mixing volume ratio of chloroform and n-butanol is 4:1, and the chloroform and n-butanol are evenly mixed and put into a brown bottle, sealed and stored for later use.

[0012] The further improvement lies in that: in the step 1, the specific steps of deproteinizing the crude polysaccharide of the calyx of Lantern Festival are as follows: first, the crude polysaccharide of the calyx of Lantern Festival is dissolved in distilled water at a solid-liquid ratio of 1:20 to obtain a polysaccharide solution, and then the prepared Sevag reagent is added to the polysaccharide solution and mixed, the volume ratio of the Sevag reagent to the polysaccharide solution is 1:4, the mixed product is taken out after shaking and centrifuged, and the lower layer of liquid is retained, and the above operation is repeated until no white precipitate is precipitated between the liquid surfaces, and then the lower layer of liquid is combined and concentrated, and then freeze-dried after dialysis for 72 hours.

[0013] The further improvement is that in the step 2, the specific steps of separating the crude polysaccharide of Lantern Festival calyx are as follows: first, immerse the DEAE-52 cellulose in distilled water for 24 hours, after the DEAE-52 cellulose is completely swollen, pour out the impurities floating on the upper layer, then add 0.5 mol / L NaOH solution and 0.5 mol / L HCl solution in sequence to neutralize the acidic impurities and alkaline impurities, and when adding the NaOH solution and the HCl solution, both are treated with distilled water until the pH is neutral, and then the completely swollen DEAE-52 cellulose is The column was loaded by wet method and equilibrated for 6 hours, then a crude polysaccharide solution of 50 mg / mL of P. cyrtonema was added, and gradient elution was performed with water and 1 mol / L NaCl after loading the column. After elution, 10 mL of the eluate was collected in each tube to obtain polysaccharide solutions of different components, and the absorbance of the polysaccharide solutions of each component was determined respectively, and a DEAE-52 elution curve was drawn. The polysaccharide solutions of each component were then combined and concentrated, and loaded into a dialysis bag of 8000-14000Da for flowing water dialysis. After dialysis, the solution was freeze-dried to obtain separated P. cyrtonema polysaccharide powder.

[0014] The further improvement lies in that in the step 2, the specific steps of purifying the polysaccharide powder of the calyx of Lantern Festival are as follows: first, immerse the dry powder of polysaccharide gel G-100 in ultrapure water overnight, pour off the impurities floating on the upper layer, cool to room temperature after slightly boiling for 2 hours, then wet-load the soaked polysaccharide gel G-100 onto the column, and equilibrate the chromatographic column for 6 hours, then add the separated polysaccharide powder of polysaccharide calyx of Lantern Festival with a concentration of 50 mg / mL, elute with ultrapure water after loading, collect 5 mL of the eluate per tube after elution, obtain polysaccharide solutions of different components, respectively determine the absorbance of the polysaccharide solutions of each component, and draw the polysaccharide gel elution curve, then combine and concentrate the polysaccharide solutions of each component, and obtain the refined polysaccharide powder of polysaccharide calyx of Lantern Festival after freeze-drying.

[0015] A further improvement is that in step three, an ultraviolet spectrophotometer is used to detect whether the refined polysaccharide of the calyx of Lantern Festival contains impurities including proteins and polyphenolic compounds, and Fourier-infrared spectroscopy technology is used to analyze the characteristic functional groups of the refined polysaccharide of the calyx of Lantern Festival.

[0016] The further improvement is that in the step three, the molecular weight and monosaccharide composition of the refined polysaccharide of the calyx of Lantern Festival are determined by high performance liquid chromatography technology, and the molecular structure information of the refined polysaccharide of the calyx of Lantern Festival is determined by nuclear magnetic resonance spectrometry.

[0017] A further improvement is that in the step three, a scanning electron microscope analysis technique is used to observe the appearance of the refined polysaccharide of the calyx of Lantern Festival, and a Congo red assay is used to detect the triple helix structure of the refined polysaccharide of the calyx of Lantern Festival.

[0018] An application of a Pleurotus eryngii calyx polysaccharide with anti-inflammatory activity, wherein the Pleurotus eryngii calyx polysaccharide with anti-inflammatory activity is applied to the research and development of anti-inflammatory drugs.

[0019] The beneficial effects of the present invention are as follows: the present invention uses water extraction and alcohol precipitation, deproteinization, dialysis and freeze-drying technology to extract polysaccharides from the calyx of Lantern Festival, and uses DEAE-52 cellulose and dextran gel columns to perform separation and purification operations, so that the purity of the obtained extract can reach more than 90%, thereby laying a solid scientific foundation for the comprehensive development and application of Lantern Festival, and effectively promoting its in-depth exploration and value realization in related fields;

[0020] The separation and purification method of the present invention is simple, easy, stable and reliable, highly operable, has a concise and clear process flow, excellent separation effect, can be recycled multiple times, has excellent chemical stability, and meets the stringent requirements of industrial large-scale production. It shows extremely broad prospects and potential in practical applications and is expected to become a key technical support for the efficient utilization of brocade lantern resources.

[0021] In addition, the present invention uses advanced instrumental analysis methods such as ultraviolet spectrophotometry, infrared spectroscopy, high performance liquid chromatography, nuclear magnetic resonance spectroscopy and scanning electron microscopy to accurately analyze the structural characteristics of Lantern Festival calyx polysaccharide, providing key data support for in-depth understanding of the structure and function relationship of Lantern Festival calyx polysaccharide;

[0022] At the same time, the polysaccharide extract of the calyx of Lantern Festival prepared by the present invention showed significant anti-inflammatory activity in the experiment, and could effectively reduce the secretion of nitric oxide (NO), inflammatory factors and prostaglandin E2 (PGE2) in RAW264.7 cells induced by lipopolysaccharide (LPS), opening up a new path for its application in the research and development of anti-inflammatory drugs or related health products, and further demonstrating the medicinal value and application potential of the calyx of Lantern Festival polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 Schematic diagram of DEAE-52 elution curve of crude polysaccharide of Lantern Festival calyx in the embodiment of the present invention;

[0025] Figure 2 It is a schematic diagram of the elution curve of the crude polysaccharide of Lantern Festival calyx in the embodiment of the present invention on Sephadex G-100 column;

[0026] Figure 3 is a schematic diagram of the ultraviolet-visible spectrum of the refined polysaccharide of Lantern Festival calyx in the embodiment of the present invention;

[0027] Figure 4 is a schematic diagram of Fourier transform infrared spectrum of the refined polysaccharide of Lantern Festival calyx in the embodiment of the present invention;

[0028] Figure 5 LS and RI spectra of the refined polysaccharide of Lanterna calyx in the embodiment of the present invention;

[0029] Figure 6 is a schematic diagram of the monosaccharide composition chromatogram of a mixed monosaccharide standard and a polysaccharide in an embodiment of the present invention;

[0030] Figure 7 It is the four groups of Lantern Festival calyx refined polysaccharides in the embodiments of the present invention. 1 H-NMR spectrum and 13 Schematic diagram of C-NMR spectrum;

[0031] Figure 8 is a schematic diagram of a scanning electron microscope image of the surface morphology characteristics of a polysaccharide in an embodiment of the present invention;

[0032] Fig. 9 It is a schematic diagram of the maximum absorption wavelengths of Congo red and four groups of Lantern Festival calyx refined polysaccharides in sodium hydroxide solution in the embodiments of the present invention;

[0033] Fig.10 Schematic diagram of the effects of four Lantern Festival calyx polysaccharides on RAW264.7 cell viability in the embodiments of the present invention;

[0034] Fig.11 Schematic diagram of the effects of four Lantern Festival calyx polysaccharides in the examples of the present invention on NO secretion by LPS-stimulated RAW264.7 cells;

[0035] Fig.12 It is a schematic diagram of the effect of S-PMCP-3 in the embodiment of the present invention on LPS-induced TNF-α secretion of RAW264.7 cells;

[0036] Fig.13 It is a schematic diagram of the effect of S-PMCP-3 in the embodiment of the present invention on LPS-induced IL-1β secretion of RAW264.7 cells;

[0037] Fig.14 It is a schematic diagram of the effect of S-PMCP-3 in the embodiment of the present invention on LPS-induced IL-6 secretion of RAW264.7 cells;

[0038] Fig.15 It is a schematic diagram of the effect of S-PMCP-3 in the examples of the present invention on LPS-induced PGE2 secretion in RAW264.7 cells. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] In the current field of enrichment and purification technology of polysaccharides from the calyx of Pleurotus eryngii, DEAE-52 ion exchange fiber column and sephadex G-100 gel column are widely used. Compared with many other methods, this method has significant advantages. Its process flow is relatively simple, it can achieve good separation effect, and it can be reused many times. It has excellent chemical stability. It occupies an extremely important position in the research and development process of polysaccharides from the calyx of Pleurotus eryngii, and has laid a solid and reliable technical foundation for the subsequent industrial production and in-depth application of related products.

[0041] Based on the findings in the prior art, the existing methods for separation, purification and structural analysis of P. chinensis calyx polysaccharides have the problems of low purity of the isolated P. chinensis calyx polysaccharides and complex processes during the separation and purification process. There is currently no relevant application of P. chinensis calyx polysaccharide extracts in the field of anti-inflammatory effects.

[0042] Embodiment 1

[0043] See also Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 , Fig.12 , Fig.13 , Fig.14 , Fig.15 This embodiment provides a method for separating, purifying and structurally analyzing polysaccharides from the calyx of Lantern Festival with anti-inflammatory activity, comprising the following steps:

[0044] Step 1: Extracting crude polysaccharides from the calyx of Lanterna truncatula

[0045] First, place the calyx of the Chinese lantern under the sun to dry, then place it in a drying oven and dry it at 60°C for 2h, cut it into pieces after drying, then add distilled water to the cut calyx of the Chinese lantern at a solid-liquid ratio of 1:20, and reflux extract it with 80°C hot water for 2h, then filter it, repeat the above operation twice and combine the filtrate, then add the combined filtrate to a rotary evaporator to concentrate it to one-sixth of the original volume, and then let it stand to room temperature, then add 95% ethanol while the filtrate is constantly stirred, the volume ratio of the filtrate to ethanol is 1:4, and then let it stand at 4°C for 12h, after standing, filter it and add water to dissolve it, then remove the ethanol by evaporation, and finally freeze-dry it to obtain the product, which is the crude polysaccharide of the calyx of the Chinese lantern, so as to realize the extraction of the crude polysaccharide of the calyx of the Chinese lantern based on the water extraction and alcohol precipitation method;

[0046] Chloroform and n-butanol are then mixed in a volume ratio of 4:1 to prepare Sevag reagent, and the mixture is sealed in a brown bottle for storage, the crude polysaccharide of Lantern Festival calyx is dissolved in distilled water at a solid-liquid ratio of 1:20 to obtain a polysaccharide solution, and then the prepared Sevag reagent is added to the polysaccharide solution for mixing, the volume of Sevag reagent and polysaccharide solution is 1:4, the mixed product is taken out after oscillation for 2 hours and centrifuged at a speed of 6000r / min for 10 minutes to absorb the denatured white protein in the middle, and the lower layer of liquid is retained, and the above operation is repeated until no white precipitate is precipitated between the liquid surfaces, and then the lower layer of liquid is combined and concentrated, and then freeze-dried after dialysis for 72 hours to achieve deproteinization of the crude polysaccharide of Lantern Festival calyx based on the Sevag method;

[0047] In this example, the polysaccharide content, protein content and uronic acid content of the deproteinized Lanterna calyx crude polysaccharide are determined, and the specific steps are as follows:

[0048] 1) Determination of polysaccharide content

[0049] Instrument: UV-Vis spectrophotometer

[0050] Reagents: Glucose reference, 4% phenol solution, concentrated sulfuric acid

[0051] Preparation of reference solution: weigh 20 mg of dry glucose, dissolve it in distilled water, mix well, and dilute to 100 mL in a volumetric flask for later use;

[0052] Preparation of 4% phenol solution: weigh 2 g of recrystallized phenol, dissolve in distilled water, mix well, and dilute to 50 mL in a volumetric flask for later use;

[0053] Standard curve drawing: accurately draw 0.2, 0.4, 0.6, 0.8, 1.0, 1.2mL of glucose standard, add 1.8, 1.6, 1.4, 1.2, 1.0, 0.8mL of distilled water respectively, then add 1.0mL of 4% phenol solution and 7mL of concentrated sulfuric acid, shake well, let stand for 5min, let stand in a 40℃ water bath for 30min, cool to room temperature, use distilled water as blank, use glucose standard as control, parallel three groups, measure the absorbance A of sample solution at 490nm wavelength by ultraviolet spectrophotometry, use absorbance as ordinate and concentration as abscissa, draw standard curve, and get linear regression equation: A=9.9063C+0.0691, R 2 =0.9991, where: A represents absorbance, C is the mass concentration of polysaccharide (mg / mL);

[0054] Sample determination: Accurately pipette 2 mL of sample solution, and measure the absorbance starting from "1.0 mL of 4% phenol solution" according to the method under the standard curve drawing item, substitute it into the regression equation, and calculate the polysaccharide concentration;

[0055] 2) Protein content determination

[0056] Instrument: UV-Vis spectrophotometer

[0057] Reagents: bovine serum albumin reference, Coomassie brilliant blue G-250 solution, 85% phosphoric acid

[0058] Preparation of reference solution: weigh 25 mg of bovine serum albumin, dissolve in distilled water, mix well, and dilute to 100 mL in a volumetric flask for later use;

[0059] Preparation of Coomassie Brilliant Blue G-250 solution: weigh 10 mg of Coomassie Brilliant Blue G-250, dissolve it at 90%, mix well, add 10 mL of 85% phosphoric acid, dilute to 100 mL volumetric flask with distilled water, and keep it away from light for later use;

[0060] Plotting the standard curve: accurately pipette 0.2, 0.4, 0.6, 0.8, 1.0 mL of bovine serum albumin standard, add 0.8, 0.6, 0.4, 0.2, 0 mL of distilled water, respectively, then add 5.0 mL of Coomassie Brilliant Blue G-250 solution, shake well, let stand for 10 min, use distilled water as blank, use bovine serum albumin standard as control, parallel three groups, measure the absorbance A of the sample solution at a wavelength of 595 nm by ultraviolet spectrophotometry, use absorbance as ordinate and concentration as abscissa, plot the standard curve, and get the linear regression equation: A = 3.595C-0.0573, R 2 =0.9994, where: A represents absorbance, C is protein mass concentration (mg / mL);

[0061] Sample determination: Accurately pipette 1 mL of sample solution, and measure the absorbance starting from "add 5.0 mL of Coomassie Brilliant Blue G-250 solution" according to the method under the standard curve drawing item, substitute it into the regression equation, and calculate the protein concentration;

[0062] 3) Determination of uronic acid content

[0063] Instrument: UV-Vis spectrophotometer

[0064] Reagents: D-galacturonic acid reference substance, sodium tetraborate, m-hydroxybiphenyl

[0065] Preparation of reference solution: weigh 1 mg of D-galacturonic acid, dissolve in distilled water, mix well, and dilute to 10 mL in a volumetric flask for later use;

[0066] Preparation of sodium tetraborate solution: weigh 0.478 g of sodium tetraborate, dissolve it in concentrated sulfuric acid, mix well, and dilute to 100 mL in a volumetric flask for later use;

[0067] Preparation of m-hydroxybiphenyl solution: weigh 0.015 g m-hydroxybiphenyl, dissolve it in 0.5% sodium hydroxide, mix well, and dilute to 10 mL in a volumetric flask for later use;

[0068] Standard curve drawing: first add sodium tetraborate to each test tube, then add 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mL of D-galacturonic acid standard respectively in ice water bath, then add 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 mL of distilled water respectively, shake well, heat to react for 4 min, cool to room temperature, add 0.08 mL of m-hydroxybiphenyl solution, heat with boiling water for 5 min, cool to room temperature, use distilled water as blank, use D-galacturonic acid standard as control, parallel three groups, measure the absorbance A of sample solution at 525 nm wavelength by ultraviolet spectrophotometry, use absorbance as ordinate and concentration as abscissa, draw standard curve, and get linear regression equation: A=9.8771C+0.0571, R 2=0.9992, where: A represents absorbance, C is the mass concentration of uronic acid (mg / mL);

[0069] Sample determination: Accurately pipette 1 mL of sample solution, and measure the absorbance starting from "add 0.08 mL of m-hydroxybiphenyl solution" according to the method under the standard curve drawing item, substitute it into the regression equation, and calculate the uronic acid concentration;

[0070] In this embodiment, the polysaccharide content of the crude polysaccharide in the Lantern Festival calyx polysaccharide before and after deproteinization is increased from 21.7% to 62.75%, the protein content is reduced from 27.87% to 13.76%, and the uronic acid content is increased from 6.87% to 12.55%;

[0071] Step 2: Separation and purification of crude polysaccharides from the calyx of Lantern Festival

[0072] First, soak the DEAE-52 cellulose in distilled water for 24 hours. After the DEAE-52 cellulose is completely swollen, the impurities floating on the upper layer are discarded, and then 0.5 mol / L NaOH solution and 0.5 mol / L HCl solution are added in sequence to neutralize the acidic impurities and alkaline impurities, and further remove the impurities. When adding the NaOH solution and the HCl solution, distilled water is used to treat the pH to neutral. Then, the completely swollen DEAE-52 cellulose is wet-loaded onto a column (3.0×45 cm), and the column is balanced for 6 hours. h, then add a 50 mg / mL crude polysaccharide solution of Lanterna calyx, and after loading, use water and 1 mol / L NaCl to perform gradient elution at a flow rate of 1 mL / min, collect 10 mL of the eluate in each tube after elution to obtain polysaccharide solutions of different components, measure the absorbance of each polysaccharide solution, and draw a DEAE-52 elution curve, then combine and concentrate the polysaccharide solutions, and put them into a dialysis bag of 8000-14000Da for 72 hours of running water dialysis, and freeze-dry after dialysis to obtain separated Lanterna calyx polysaccharide powder;

[0073] The dextran gel G-100 dry powder was immersed in ultrapure water for overnight, and the impurities floating on the upper layer were poured off. After being slightly boiled for 2 hours, it was cooled to room temperature. Then, the soaked dextran gel G-100 was wet-loaded on a column (1.5×25 cm), and the chromatographic column was balanced for 6 hours. Then, the polysaccharide powder of the calyx of Lanterna strychnifolia was added to a polysaccharide solution with a concentration of 20 mg / mL. After loading, ultrapure water was used to elute at a flow rate of 0.5 mL / min. After elution, 5 mL of the eluate was collected per tube to obtain polysaccharide solutions of different components. The absorbance of each polysaccharide solution was determined respectively, and the dextran gel elution curve was drawn. Then, the polysaccharide solutions were combined and concentrated, and the refined polysaccharide powder of the calyx of Lanterna strychnifolia was obtained after freeze-drying.

[0074] In this embodiment, the crude polysaccharide of Lantern Festival calyx is separated by DEAE-52 cellulose chromatography column to obtain 7 components, named D-PMCP-1, D-PMCP-2, D-PMCP-3, D-PMCP-4, D-PMCP-5, D-PMCP-6 and D-PMCP-7. Four major component peaks D-PMCP-1, D-PMCP-2, D-PMCP-3 and D-PMCP-4 are selected for further purification according to the polysaccharide content and separation effect. After purification by polysaccharide gel G-100, the protein content is significantly reduced (5%-9%), and the polysaccharide content reaches more than 90%, and its purity meets the basis of structural identification;

[0075] Step 3: Structural analysis of the refined polysaccharide from the calyx of Lanterna sphenanthera

[0076] Ultraviolet visible spectrometry (UV-VIS), Fourier transform infrared spectroscopy (FT-IR), high performance liquid chromatography, nuclear magnetic resonance, scanning electron microscopy (SEM) and Congo red assay were used to detect the structural characteristics of the refined polysaccharide of Lanterna calyx. The specific detection steps are as follows:

[0077] S1. Use an ultraviolet spectrophotometer to detect whether the refined polysaccharide of Lantern Festival contains impurities such as protein and polyphenolic compounds. Prepare 2 mg / mL solution of each component of the refined polysaccharide components of Lantern Festival with distilled water, and record the absorption spectrum of the sample in the wavelength range of 200-800 nm.

[0078] like Figure 3 As shown, the UV-visible spectrum of the refined polysaccharide of Lanterna calyx of this example shows strong absorption in the wavelength range of 210-230nm, which may be due to the presence of unsaturated carbonyl and carboxyl groups, indicating that the polysaccharide may contain uronic acid. The weak absorption peaks of S-PMCP-1 and S-PMCP-2 at 260-280nm are mainly attributed to the presence of trace proteins or nucleic acids (see Figure 3 The UV scanning spectra of S-PMCP-3 and S-PMCP-4 showed no obvious absorption at 260 nm and 280 nm, indicating that these samples contained no or little protein or nucleic acid (see Figure 3 c and d of the Regulations);

[0079] S2. Characteristic functional group analysis of four refined polysaccharides from the calyx of Lanterna sphenanthera was performed. 2.00 mg of dried sample was placed in a dry agate mortar, 200 mg of KBr powder was weighed and ground evenly, pressed into tablets, and spun at 4000-500 cm -1 Scan within the range and record the FT-IR spectrum absorption;

[0080] like Figure 4As shown in Figure 2, the differences and similarities of polysaccharide functional groups in the refined polysaccharides of P. cyrtonema were analyzed by FT-IR. Several peaks were common to all extracts. The vibration caused by the stretching of OH groups in the infrared spectra of the four components was between 3450 and 3350 cm -1 The stretched CH vibration shows a broad and strong absorption peak at 2900-2950cm -1 There is a weak absorption peak at 1650-1600cm -1 There is a strong absorption peak at 1080cm -1 The nearby stretching peak is the characteristic absorption peak of the pyranose ring. In summary, it is shown that the four polysaccharides are all polypyranose.

[0081] In addition, 890cm -1 The characteristic absorption peaks near 1400 cm-1 indicated the presence of β-glycosidic bonds in S-PMCP-2 and S-PMCP-4. However, the FT-IR analysis of the four kinds of polysaccharides from the calyx of P. quinata showed that the -1 and 1080cm -1 The peaks at 1558 cm-1 and 1567 cm-2 indicated that the polysaccharide content of these refined polysaccharides from Lantern Festival was very high. -1 The absorption peak is shown at , which corresponds to the secondary -CONH- group of the protein, which is consistent with the results of UV-visible spectroscopy analysis;

[0082] S3, using gel permeation chromatography (SEC), multi-angle laser light scattering (MALLS) and differential refractive index detector (RI) to measure the molecular weight of the refined polysaccharide of Lanterna calyx, the sample was dissolved in 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to 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;

[0083] Chromatographic conditions: gel exclusion chromatography columns Ohpak SB-805HQ (300×8 mm) and OhpakSB-803HQ (300×8 mm) were used in series, column temperature: 45°C, injection volume: 100 μL, mobile phase: A (0.02% NaN3, 0.1% NaNO3), flow rate: 0.6 mL / min, elution time: 75 min;

[0084] See also Figure 5, the homogeneity and average molecular weight of four refined polysaccharides of Lantern Festival calyx were determined by SEC-MALLS-RI analysis. The Mw, Mn and Mw / Mn values ​​of the four samples are shown in Table 1 below. The Mw of S-PMCP-4 is the highest at 299 kDa, and the Mw of S-PMCP-1, S-PMCP-2 and S-PMCP-3 are 16, 71 and 77 kDa, respectively. As shown in Table 1 below, the Mw / Mn value of S-PMCP-1 is 1.277, indicating that the molecular weight distribution of the homogeneous polysaccharide is narrow (e.g. Figure 5 As shown in part a of ), the molecular weight distributions of S-PMCP-2, S-PMCP-3, and S-PMCP-4 are relatively broad;

[0085] Table 1 Molecular weight and polydispersity index of different samples

[0086]

[0087] S4. Prepare polysaccharide samples by PMP pre-column derivatization, and analyze the monosaccharide composition of Lantern Festival calyx polysaccharide by high performance liquid chromatography. The specific steps are as follows

[0088] 1) Preparation of hydrolyzed samples of polysaccharides from the calyx of P. quinata

[0089] Add 5 mg of Lanterna calyx polysaccharide sample to the reaction kettle, add 3 mL of trifluoroacetic acid (2 mol / L) solution, mix well, seal, hydrolyze at 110°C for 6 h, take out, evaporate to dryness in an evaporating dish, rinse repeatedly with methanol to remove excess acid, evaporate to dryness, add 1 mL of ultrapure water to dissolve, and obtain a hydrolyzate;

[0090] 2) Preparation of monosaccharide standard solution

[0091] Prepare the monosaccharide standard with ultrapure water to 1 mg / mL and store for later use;

[0092] 3) Preparation of mixed monosaccharide standard solution

[0093] Accurately weigh 1 mg of each monosaccharide standard, mix them, add 1 mL of ultrapure water to dissolve, and store for later use;

[0094] 4) Preparation of 1-phenyl-3-methyl-5-pyrazolone (PMP) solution

[0095] Weigh an appropriate amount of PMP, dissolve it in methanol to prepare a 0.5 mol / L PMP methanol solution, and store it in a dark place;

[0096] 5) PMP derivatization method:

[0097] 0.2 mL of purified polysaccharide acid hydrolyzate of Lanterna calyx, 0.2 mL of monosaccharide standard solution and 0.2 mL of mixed standard solution were respectively taken into centrifuge tubes, and the same volume of PMP methanol solution (0.5 mol / L) and NaOH solution (0.3 mol / L) were added, and the mixture was shaken and mixed, and the mixture was placed in a 70°C water bath for 1 h. After cooling to room temperature, 0.2 mL of hydrochloric acid solution (0.3 mol / L) was added to each tube, and the mixture was shaken. Finally, 1 mL of chloroform was added to remove excess PMP, and the supernatant was obtained by centrifugation. The mixture was centrifuged three times, and filtered with a 0.45 μm microporous filter membrane to obtain the derivatized sample solution.

[0098] 6) Preparation of standard regression curve

[0099] The derivatized monosaccharide standard solution was diluted by half and injected for detection, and the monosaccharide standard curve was drawn with the monosaccharide concentration as the horizontal axis and the peak area as the vertical axis;

[0100] 7) Chromatographic conditions

[0101] The chromatographic column used was Agilent C 18 column (4.6×250mm), HPLC using Shimadzu Essentia LC-16 series, detector: UV detector SPD-16, chromatographic conditions: acetonitrile-0.05mol / mL phosphate buffer solution pH=6.8 (18:82) as mobile phase, flow rate: 0.8mL / min, column temperature: 30℃, detection wavelength: 245nm, time: 90min;

[0102] The monosaccharide composition of the polysaccharide was identified by HPLC using the PMP pre-column derivatization method, and a standard curve was drawn. The results are shown in Table 2 below:

[0103] Table 2 Standard curve of monosaccharide standards

[0104]

[0105] The monosaccharide composition chromatogram of the mixed monosaccharide standard and polysaccharide is as follows Figure 6 As shown, from Figure 6 It can be seen that the standard monosaccharides have peaks of Man, GluN, Rib, Rha, GluA, GalA, Glu, Gal, Sor, Xyl, Arab and Fuc, and the time is 22.764min, 28.863min, 30.219min, 35.527min, 40.897min, 47.538min, 53.795min, 55.800min, 58.093min, 63.777min, 66.108min and 75.713min respectively;

[0106] As shown in Table 3 below, the molar ratios are as follows: S-PMCP-1 is composed of Sor, Glu, GalA, GluN, Man and Rib, with a molar ratio of 15.78:13.16:9.75:5.27:5.15:1; S-PMCP-2 is composed of GluN, Sor, Glu, Rib, GalA and Rha, with a molar ratio of 9.96:5.19:4.31:1.28:1.27:1; S-PMCP-3 is composed of Glu, GalA, GluN, Rib, GlcA, Sor, Rha and Man, with a molar ratio of The molar ratio was 6.76:3.74:3.67:3.24:3.13:2.57:1.07:1, and S-PMCP-4 was composed of GlcA, Glu, GalA, Rib, Man, Rha, and Sor in a molar ratio of 6.99:2.79:2.67:2.13:1.28:1.05:1. The results showed that S-PMCP-1 and S-PMCP-2 contained galacturonic acid, and S-PMCP-3 and S-PMCP-4 contained both glucuronic acid and galacturonic acid, which was consistent with the results of UV and IR spectroscopy analysis;

[0107] Table 3 Monosaccharide composition and molar ratio of four samples

[0108]

[0109] S5. The structures of the four samples were further studied by using nuclear magnetic resonance spectroscopy. 25 mg of purified polysaccharide from the calyx of Lanterna sphenanthera was dissolved in D2O (0.5 mL, 99.9, atom % D) three times in succession. After dissolution, the polysaccharide was placed in a nuclear magnetic resonance tube and measured by nuclear magnetic resonance spectroscopy at 600 MHz. 1 H-NMR and 13 C-NMR to obtain more comprehensive molecular structure information;

[0110] See also Figure 7 In this example, nuclear magnetic resonance spectroscopy was used to further study the structures of four refined polysaccharides from the calyx of Lantern Festival. The chemical shift of the anomeric hydrogen proton was between 4.5 and 5.9 ppm, the chemical shift of the α-glycosidic bond H was greater than 4.95 ppm, the chemical shift of the β-glycosidic bond H was less than 4.95 ppm, and the chemical shift of the anomeric carbon proton was between 95 and 105 ppm. Generally, the chemical shift greater than 103 ppm was the β-glycosidic bond, and less than 103 ppm was the α-glycosidic bond. Figure 7 As shown in part a of 1 The H NMR spectrum showed resonance caused by anomeric hydrogen protons in the frequency range of 4.3 to 5.9 ppm, indicating that S-PMCP-1 is composed of α-glycosidic bonds and β-glycosidic bonds. 13C NMR data showed that the anomeric carbon signal was mainly concentrated in the 90-112 ppm region, indicating that the glycosidic bond of S-PMCP-1 included β-configuration and α-configuration. 1 The H NMR spectra are consistent with Figure 7 As shown in part c of 1 In the H NMR spectrum, S-PMCP-2 has 11 isomeric proton signals at δ 4.32, 4.33, 4.40, 4.42, 4.44, 4.70, 4.98, 4.99, 5.13, 5.14, and 5.15 ppm. Similarly, the signals of the isomeric protons at δ 110.92, 109.24, 108.11, 107.37, 103.19, 102.55, 101.36, and 100.68 ppm 13 There are also 8 anomeric carbon signals in the CNMR spectrum, which indicates that its structure is mainly composed of β-configuration. The NMR information of S-PMCP-3 is similar to that of S-PMCP-1. Except for several different glycosidic bonds, they have the same signal peaks at the same chemical shift. The telomere hydrogen signals appear in the range of 4.33 to 5.67 ppm and the carbon signals appear in the range of 98.32 to 100.64 ppm, indicating that the glycosidic bonds of S-PMCP-4 are mainly α-configuration.

[0111] In addition, the strong chemical shift at δ4.70 ppm in the hydrogen spectrum is attributed to the solvent D2O. The presence of rhamnose in S-PMCP-2, S-PMCP-3, and S-PMCP-4 is attributed to the hydrogen spectrum methyl proton signal at δ1.13 and the hydrogen spectrum signal at δ16.40. 13 C displacement signal support, 13 C NMR (such as Figure 7 As shown in parts b, d, f and h in the figure, the carbon signals of the four purified samples are mainly distributed in the range of 60-175 ppm. An obvious absorption peak is observed at δ175.09 ppm for S-PMCP-1 and S-PMCP-2, corresponding to the carbonyl signal produced by galacturonic acid. Two obvious absorption peaks are observed at δ175.09 ppm for S-PMCP-3 and S-PMCP-4, corresponding to the carbonyl signals produced by glucuronic acid and galacturonic acid. The NMR results are consistent with the monosaccharide composition analysis.

[0112] S6. Observe the appearance of the refined polysaccharide of the calyx of the genus Lanterna by scanning electron microscopy. Disperse each polysaccharide sample in anhydrous ethanol, stick it on the specimen holder with double-sided conductive tape, blow off the excess sample, and use a sputtering coating machine to sputter gold powder onto the sample. Observe at 500, 2000 and 5000 times magnification under high vacuum conditions with an accelerating potential of 5 kV.

[0113] See also Figure 8In this example, the surface morphology of polysaccharides was analyzed at 500 times, 2000 times and 5000 times magnification ( Figure 8 (a, d, g and j parts are 500 times magnified, b, e, h and k parts are 2000 times magnified, c, f, I and l parts are 5000 times magnified). After 500 times magnification, S-PMCP-1, S-PMCP-3 and S-PMCP-4 are block or sheet structures, S-PMCP-2 is a strip-shaped branched structure, and the surface of S-PMCP-1 is relatively rough compared with the other three polysaccharides. This may be due to the destruction of the polysaccharide structure by long-term high-temperature extraction. At high magnifications of 2000 and 5000 times, the surfaces of the four samples show a wrinkled structure, and there are a large number of protruding particles on the surface of S-PMCP-2, S-PMCP-3 and S-PMCP-4, indicating different molecular polymerization degrees, indicating that the polysaccharides contain fewer impurities and a higher degree of purification.

[0114] S7. Use Congo red assay to detect the triple helix structure of 4 refined polysaccharides from the calyx of Lantern Festival. Take 2 mL of refined polysaccharide solution (2 mg / mL) of Lantern Festival and mix it with 2 mL of Congo red solution (80 mol / L). Then add 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L of NaOH solution, respectively. After shaking and mixing, let it stand at room temperature for 5 min. Detect with UV spectrophotometer in the detection range of 200-800 nm, and record the maximum absorption wavelength.

[0115] Congo red complexes with the triple helix structure of polysaccharides to produce a red shift, so the polysaccharide and Congo red mixed experiment is used to identify whether the polysaccharide has a triple helix structure. Under low concentration NaOH conditions, each component polysaccharide forms a complex with the Congo red reagent, and the maximum absorption wavelength red shifts with the increase of the NaOH solution concentration. The Congo red experiment determines the conformational changes of polysaccharides under different NaOH concentrations, such as Fig. 9 As shown, in the range of NaOH concentration of 0-0.5 mol / L, the maximum absorption wavelength of the complex of purified polysaccharide of P. quinata calyx and Congo red shifted red-shifted. Therefore, S-PMCP-1, S-PMCP-2, S-PMCP-3 and S-PMCP-4 had a triple helical conformation.

[0116] This embodiment also provides an application of a polysaccharide of the calyx of Lanterna with anti-inflammatory activity, and the polysaccharide of the calyx of Lanterna with anti-inflammatory activity is applied to the research and development of anti-inflammatory drugs;

[0117] This example studies the anti-inflammatory effect of LPS-induced RAW264.7 cells of the polysaccharide of Pleurotus eryngii, and the specific steps are as follows:

[0118] 1) Cell culture: RAW264.7 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture conditions were: 37°C, 5% CO2;

[0119] 2) Cell survival rate experiment: RAW264.7 cells were cultured at 1×10 4 The cells were added into a 96-well plate at a density of 100 μg / well and incubated for 24 h. The supernatant was discarded and four samples of different concentrations (25, 50, 100 and 200 μg / mL) were added and incubated for 24 h. The control group used an equal amount of culture medium. After discarding the supernatant, 100 μL of diluted MTT (0.5 mg / mL) was injected into each well and placed in an incubator for incubation for 4 h. After discarding the MTT culture medium, 100 μL of DMSO was added to dissolve it. After shaking at room temperature for 5 min, ensure that all particles were dissolved, and use an ELISA reader to measure the absorption intensity at 570 nm.

[0120] 3) Griess method was used to determine the effect of polysaccharide from P. chinensis on NO level in RAW264.7 cells induced by LPS: RAW264.7 cells in the logarithmic growth phase were taken and the concentration was 2×10 5 100 cells / well were inoculated in a 96-well plate. After culturing in the culture medium for 24 hours, different concentrations (25, 50, 100 and 200 μg / mL) of polysaccharide sample solutions of P. quinata were added in three parallel groups for 24 hours. LPS (1 μg / mL) was then added and cultured for another 24 hours. The cell supernatant was collected (3000 r / min, 10 min). The NO level in the culture supernatant of RAW264.7 cells was evaluated by the Griess method.

[0121] 4) ELISA method was used to determine the effect of S-PMCP-3 on the levels of TNF-α, IL-1β, IL-6 and PGE2 in RAW264.7 cells induced by LPS: ELISA kits were used to detect the levels of PGE2, TNF-α, IL-1β and IL-6. RAW264.7 cells were cultured at 2×10 5 The cells were cultured at an average density of 100 μg / well, and the cells were pretreated with S-PMCP-3 (25, 50, 100, and 200 μg / mL) for 24 h, and then stimulated with LPS (1 μg / mL). The cell supernatant was then collected and the levels of inflammatory cytokines and PGE2 were determined according to the instructions of the kit.

[0122] The results of the study are as follows:

[0123] 1) Effects of polysaccharides from the calyx of Pleurotus eryngii on the activity of RAW264.7 cells

[0124] like Fig.10As shown, the MTT method was used to determine the effects of the four components on the viability of RAW264.7 cells. RAW264.7 cells were mixed with samples at a concentration of 25 to 200 μg / mL and incubated with S-PMCP-1, S-PMCP-2, S-PMCP-3, and S-PMCP-4 for 24 hours. Compared with the untreated group, the cell viability of the S-PMCP-1 and S-PMCP-3 polysaccharide-treated groups was significantly better than that of S-PMCP-2 and S-PMCP-4 (P < 0.05). The results showed that compared with the control group, the cell viability was more than 90%, indicating that the four polysaccharides had no obvious cytotoxicity to RAW264.7 cells within the range of 200 μg / mL.

[0125] 2) Effect of polysaccharide from Pleurotus eryngii on NO level in RAW264.7 cells

[0126] like Fig.11 As shown in the figure, compared with the blank control group, LPS induced a significant increase in the secretion of NO in RAW264.7 cells, indicating that LPS can significantly increase the production of NO in RAW264.7 cells; PMCPs pretreatment significantly reduced the production of NO in a concentration-dependent manner (P < 0.05), among which S-PMCP-3 showed a relatively strong inhibitory effect. At a concentration of 200 μg / mL, the NO production was reduced to 24.95 μM after S-PMCP-3 treatment; This indicates that the total flavonoids of the calyx of Lantern Festival have a significant anti-inflammatory effect on LPS-induced RAW264.7 cells. Therefore, S-PMCP-3 was selected as the object of further study;

[0127] 3) Inhibitory effect of S-PMCP-3 on TNF-α in RAW264.7 cells

[0128] The results are as follows Fig.12 As shown, compared with the control group, the TNF-α content of RAW264.7 cells was significantly increased after being induced by LPS. Compared with the LPS treatment group, the TNF-α levels in other drug treatment groups were significantly lower than that in the model group (P < 0.05). At a concentration of 200 μg / mL, S-PMCP-3 could reduce the production of TNF-α to 494.67 pg / mL.

[0129] 4) Inhibitory effect of S-PMCP-3 on IL-1β in RAW264.7 cells

[0130] The results are as follows Fig.13As shown, compared with the control group, the IL-1β content of RAW264.7 cells was significantly increased after being induced by LPS. Compared with the LPS treatment group, the IL-1β levels in other drug treatment groups were significantly lower than that in the model group (P < 0.05). At a concentration of 200 μg / mL, S-PMCP-3 could reduce the production of IL-1β to 514.89 pg / mL;

[0131] 5) Inhibitory effect of S-PMCP-3 on IL-6 in RAW264.7 cells

[0132] The results are as follows Fig.14 As shown, compared with the control group, the IL-6 content of RAW264.7 cells was significantly increased after being induced by LPS. Compared with the LPS group, the release of IL-6 in other drug-treated groups was significantly reduced (P < 0.05). At a concentration of 200 μg / mL, S-PMCP-3 could reduce the production of IL-6 to 471.85 pg / mL;

[0133] 6) Inhibitory effect of S-PMCP-3 on PGE2 in RAW264.7 cells

[0134] The results are as follows Fig.15 As shown, compared with the control group, the content of PGE2 in RAW264.7 cells induced by LPS was significantly increased. Compared with the LPS group, the release of PGE2 in other drug-treated groups was significantly reduced (P < 0.05). At a concentration of 200 μg / mL, S-PMCP-3 could reduce the production of PGE2 to 845.09 pg / mL.

[0135] The above results show that the polysaccharide of S. cerasifera is non-toxic to RAW264.7 cells in the concentration range of 25-200 μg / mL, and can reduce the secretion of NO, inflammatory factors and PGE2 in RAW264.7 cells induced by LPS, and has a good anti-inflammatory effect.

[0136] Embodiment 2

[0137] This embodiment provides a method for separation, purification and structural analysis of polysaccharides from the calyx of Lantern Festival with anti-inflammatory activity. Compared with the first embodiment, the difference is that:

[0138] First, place the calyx of the Lantern Festival in the sun to dry, then place it in a drying oven and dry it at 60°C for 2h. After drying, cut it into pieces, take 80g of dried Lantern Festival calyx, then add distilled water to the shredded Lantern Festival calyx at a solid-liquid ratio of 1:20, and reflux extract with 90°C hot water for 2h, then filter, repeat the above operation twice and combine the filtrate, then add the combined filtrate to a rotary evaporator to concentrate it to one-sixth of the original volume, and then let it stand to room temperature, then add 95% ethanol while constantly stirring the filtrate, the volume ratio of the filtrate to ethanol is 1:4, and then let it stand at 4°C for 12h, after standing, filter it and add water to dissolve it, then remove the ethanol by evaporation, and finally freeze-dry it to obtain the product, which is the Lantern Festival calyx crude polysaccharide, so as to realize the extraction of Lantern Festival calyx crude polysaccharide based on water extraction and alcohol precipitation method.

[0139] Embodiment 3

[0140] This embodiment provides a method for separation, purification and structural analysis of polysaccharides from the calyx of Lantern Festival with anti-inflammatory activity. Compared with the first embodiment, the difference is that:

[0141] First, place the calyx of the Lantern Festival in the sun to dry, then place it in a drying oven and dry it at 60°C for 2h. After drying, cut it into pieces, take 80g of dried Lantern Festival calyx, then add distilled water to the shredded Lantern Festival calyx at a solid-liquid ratio of 1:20, and reflux extract with 80°C hot water for 1h, then filter, repeat the above operation three times and combine the filtrate, then add the combined filtrate to a rotary evaporator to concentrate it to one-sixth of the original volume, and then let it stand to room temperature, then add 95% ethanol while constantly stirring the filtrate, the volume ratio of the filtrate to ethanol is 1:4, and then let it stand at 4°C for 12h, after standing, filter it and add water to dissolve it, then remove the ethanol by evaporation, and finally freeze-dry it to obtain the product, which is the Lantern Festival calyx crude polysaccharide, so as to realize the extraction of Lantern Festival calyx crude polysaccharide based on water extraction and alcohol precipitation method.

[0142] Embodiment 4

[0143] This embodiment provides a method for separation, purification and structural analysis of polysaccharides from the calyx of Lantern Festival with anti-inflammatory activity. Compared with the first embodiment, the difference is that:

[0144] First, place the calyx of the Lantern Festival in the sun to dry, then place it in a drying oven and dry it at 60°C for 2h. After drying, cut it into pieces, take 80g of dried Lantern Festival calyx, then add distilled water to the shredded Lantern Festival calyx at a solid-liquid ratio of 1:20, and reflux extract with 80°C hot water for 2h, then filter, repeat the above operation twice and combine the filtrate, then add the combined filtrate to a rotary evaporator to concentrate it to one-sixth of the original volume, and then let it stand to room temperature, then add 80% ethanol while constantly stirring the filtrate, the volume ratio of the filtrate to ethanol is 1:4, and then let it stand at 4°C for 12h, after standing, filter it and add water to dissolve it, then remove the ethanol by evaporation, and finally freeze-dry it to obtain the product, which is the Lantern Festival calyx crude polysaccharide, so as to realize the extraction of Lantern Festival calyx crude polysaccharide based on water extraction and alcohol precipitation method.

[0145] Embodiment 5

[0146] This embodiment provides a method for separation, purification and structural analysis of polysaccharides from the calyx of Lantern Festival with anti-inflammatory activity. Compared with the first embodiment, the difference is that:

[0147] First, place the calyx of the Lantern Festival in the sun to dry, then place it in a drying oven and dry it at 60°C for 2h. After drying, cut it into pieces, take 80g of dried Lantern Festival calyx, then add distilled water to the shredded Lantern Festival calyx at a solid-liquid ratio of 1:10, and reflux extract with 80°C hot water for 2h, then filter, repeat the above operation twice and combine the filtrate, then add the combined filtrate to a rotary evaporator to concentrate it to one-sixth of the original volume, and then let it stand to room temperature, then add 80% ethanol while constantly stirring the filtrate, the volume ratio of the filtrate to ethanol is 1:4, and then let it stand at 4°C for 12h, after standing, filter it and add water to dissolve it, then remove the ethanol by evaporation, and finally freeze-dry it to obtain the product, which is the Lantern Festival calyx crude polysaccharide, so as to realize the extraction of Lantern Festival calyx crude polysaccharide based on water extraction and alcohol precipitation method.

[0148] Embodiment 6

[0149] This embodiment provides a method for separation, purification and structural analysis of polysaccharides from the calyx of Lantern Festival with anti-inflammatory activity. Compared with the first embodiment, the difference is that:

[0150] First, place the calyx of the Lantern Festival in the sun to dry, then place it in a drying oven and dry it at 60°C for 2h. After drying, cut it into pieces, take 80g of dried Lantern Festival calyx, then add distilled water to the shredded Lantern Festival calyx at a solid-liquid ratio of 1:30, and reflux extract with 80°C hot water for 2h, then filter, repeat the above operation twice and combine the filtrate, then add the combined filtrate to a rotary evaporator to concentrate it to one-sixth of the original volume, and then let it stand to room temperature, then add 80% ethanol while constantly stirring the filtrate, the volume ratio of the filtrate to ethanol is 1:4, and then let it stand at 4°C for 12h, after standing, filter it and add water to dissolve it, then remove the ethanol by evaporation, and finally freeze-dry it to obtain the product, which is the Lantern Festival calyx crude polysaccharide, so as to realize the extraction of Lantern Festival calyx crude polysaccharide based on water extraction and alcohol precipitation method.

[0151] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for separating, purifying and structurally analyzing polysaccharides from the calyx of Lantern Festival with anti-inflammatory activity, characterized in that: The following steps are involved: Step 1: firstly extract the crude polysaccharide of Lantern Festival calyx from Lantern Festival calyx by water extraction and alcohol precipitation method, then prepare Sevag reagent and use Sevag method to deproteinize the crude polysaccharide of Lantern Festival calyx to obtain the deproteinized crude polysaccharide of Lantern Festival calyx; Step 2: firstly, using DEAE-52 cellulose to separate the crude polysaccharide of Lanterna calyx to obtain Lanterna calyx polysaccharide powder, and then using polydextrose G-100 to purify the Lanterna calyx polysaccharide powder to obtain Lanterna calyx refined polysaccharide powder; Step 3: Use UV-visible analysis technology, Fourier-infrared spectroscopy, high performance liquid chromatography technology, nuclear magnetic resonance technology, scanning electron microscope analysis technology and Congo red determination technology to detect the structural characteristics of the refined polysaccharide of Lantern Festival calyx.

2. The method for separation, purification and structural analysis of a polysaccharide from the calyx of Lantern Festival with anti-inflammatory activity according to claim 1, characterized in that: In the step 1, the specific steps of extracting crude polysaccharides from the calyx of Lantern Festival are as follows: first, sun-dry the calyx of Lantern Festival, then place it in a drying oven to dry, cut it into pieces after drying, then add distilled water to the cut calyx of Lantern Festival at a solid-liquid ratio of 1:20, and reflux extract it with 80°C hot water for 2 hours, then filter it, repeat the above operation twice and combine the filtrate, then add the combined filtrate to a rotary evaporator to concentrate it to one-sixth of the original volume, and then let it stand to room temperature, then add 95% ethanol while constantly stirring the filtrate, the volume ratio of the filtrate to ethanol is 1:4, and then let it stand at a temperature of 4°C for 12 hours, after standing, filter it with suction and add water to dissolve it, then remove the ethanol by evaporation, and finally freeze-dry it to obtain crude polysaccharides from the calyx of Lantern Festival.

3. The method for separation, purification and structural analysis of a polysaccharide from the calyx of Lantern Festival with anti-inflammatory activity according to claim 1, characterized in that: In the step 1, the Sevag reagent is prepared by mixing chloroform and n-butanol, the mixing volume ratio of chloroform and n-butanol is 4:1, and the chloroform and n-butanol are evenly mixed and put into a brown bottle, sealed and stored for later use.

4. The method for separation, purification and structural analysis of a polysaccharide from the calyx of Lantern Festival with anti-inflammatory activity according to claim 1, characterized in that: In the step 1, the specific steps of deproteinizing the crude polysaccharide of the calyx of Lantern Festival are as follows: first, dissolve the crude polysaccharide of the calyx of Lantern Festival in distilled water at a solid-liquid ratio of 1:20 to obtain a polysaccharide solution, then add the prepared Sevag reagent to the polysaccharide solution and mix, the volume ratio of the Sevag reagent to the polysaccharide solution is 1:4, the mixed product is taken out after shaking and centrifuged, and the lower layer of liquid is retained, and the above operation is repeated until no white precipitate is precipitated between the liquid surfaces, and then the lower layer of liquid is combined and concentrated, and then freeze-dried after dialysis for 72 hours.

5. The method for separation, purification and structural analysis of a polysaccharide from the calyx of Lantern Festival with anti-inflammatory activity according to claim 1, characterized in that: In the step 2, the specific steps of separating the crude polysaccharide of Lantern Festival calyx are as follows: first, immerse the DEAE-52 cellulose in distilled water for 24 hours, and after the DEAE-52 cellulose is completely swollen, pour out the impurities floating on the upper layer, and then add 0.5 mol / L NaOH solution and 0.5 mol / L HCl solution in sequence to neutralize acidic impurities and alkaline impurities, and when adding the NaOH solution and the HCl solution, both are treated with distilled water to a neutral pH, and then the completely swollen DEAE-52 cellulose is wet-column-loaded , and balance the chromatographic column for 6 hours, then add a 50 mg / mL crude polysaccharide solution of the calyx of Lantern Festival, and after loading on the column, use water and 1 mol / L NaCl for gradient elution, collect 10 mL of the eluate in each tube after elution, and obtain polysaccharide solutions of different components, respectively determine the absorbance of the polysaccharide solution of each component, and draw a DEAE-52 elution curve, then combine and concentrate the polysaccharide solutions of each component, and load them into a dialysis bag of 8000-14000Da for flowing water dialysis, and freeze-dry after dialysis to obtain separated calyx of Lantern Festival polysaccharide powder.

6. The method for separation, purification and structural analysis of a polysaccharide from the calyx of Lantern Festival with anti-inflammatory activity according to claim 1, characterized in that: In the step 2, the specific steps of purifying the polysaccharide powder of the calyx of Lantern Festival are as follows: first, immerse the dry powder of polysaccharide gel G-100 in ultrapure water overnight, pour off the impurities floating on the upper layer, cool to room temperature after slightly boiling for 2 hours, then wet-load the soaked polysaccharide gel G-100 onto the column, and balance the chromatographic column for 6 hours, then add the separated polysaccharide powder of polysaccharide calyx of Lantern Festival with a concentration of 50 mg / mL, elute with ultrapure water after loading, collect 5 mL of the eluate per tube after elution, obtain polysaccharide solutions of different components, respectively determine the absorbance of the polysaccharide solutions of each component, draw the polysaccharide gel elution curve, then combine and concentrate the polysaccharide solutions of each component, and obtain the refined polysaccharide powder of polysaccharide calyx of Lantern Festival after freeze-drying.

7. The method for separation, purification and structural analysis of a polysaccharide from the calyx of Lantern Festival with anti-inflammatory activity according to claim 1, characterized in that: In the step three, an ultraviolet spectrophotometer is used to detect whether the refined polysaccharide of Lantern Festival contains impurities including proteins and polyphenolic compounds, and Fourier-infrared spectroscopy is used to analyze the characteristic functional groups of the refined polysaccharide of Lantern Festival.

8. The method for separation, purification and structural analysis of a polysaccharide from the calyx of Lantern Festival with anti-inflammatory activity according to claim 1, characterized in that: In the step three, high performance liquid chromatography technology is used to determine the molecular weight and monosaccharide composition of the refined polysaccharide of the calyx of Lantern Festival, and nuclear magnetic resonance spectrometer is used to determine the molecular structure information of the refined polysaccharide of the calyx of Lantern Festival.

9. The method for separation, purification and structural analysis of a polysaccharide from the calyx of Lantern Festival with anti-inflammatory activity according to claim 1, characterized in that: In the step three, the appearance morphology of the refined polysaccharide of the calyx of Lantern Festival is observed by scanning electron microscope analysis technology, and the triple helix structure of the refined polysaccharide of the calyx of Lantern Festival is detected by Congo red assay.

10. An application of a polysaccharide from the calyx of Pleurotus eryngii with anti-inflammatory activity, characterized in that: The Lantern Festival calyx polysaccharide with anti-inflammatory activity is used in the research and development of anti-inflammatory drugs.

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