Preparation method and application of dried ginger polysaccharide

Abstract

The invention relates to a preparation method and application of dried ginger polysaccharide. The preparation method comprises the following steps: crushing dried ginger slices to obtain dried ginger powder; the obtained dry powder is subjected to degreasing treatment; mixing the degreased dried ginger powder with distilled water, stirring and extracting, collecting the obtained supernatant, and removing impurities to obtain a polysaccharide extract; carrying out concentration and alcohol precipitation on the obtained polysaccharide leach liquor, dissolving the obtained alcohol precipitation substance in distilled water, and carrying out centrifugation and freeze-drying to obtain dried ginger crude polysaccharide; and carrying out starch removal and fractionation purification on the obtained dried ginger crude polysaccharide to obtain the dried ginger polysaccharide. The prepared rhizoma zingiberis polysaccharide can promote inflammation through a TLR4 / IKKbeta / P100 signal channel, provides a basis for the rhizoma zingiberis polysaccharide as a potential immune preparation, and has wide development potential and broad market prospects.

Description

Technical Field

[0001] The invention relates to the technical field of processing dried ginger polysaccharide, in particular to a preparation method and application of dried ginger polysaccharide. Background Art

[0002] Polysaccharides are high-molecular carbohydrates formed by the polymerization of more than 10 monosaccharides connected by glycosidic bonds. They are widely distributed in nature as structural components, storage nutrients, and special active ingredients. Natural active polysaccharides show broad research and application prospects due to their unique pharmacological effects and less toxic side effects. Dried ginger is the dried rhizome of ginger, a plant of the ginger family. It has the effects of warming the middle and dispersing cold, restoring yang and unblocking meridians, warming the lungs and transforming fluid. It is mainly used to treat spleen and stomach cold syndrome, cold pain in the abdomen, vomiting and diarrhea, yang deficiency syndrome, cold fluid and cough, etc. It is mentioned in Shennong's Herbal Classic and Treatise on Febrile Diseases. According to research, the biological activity of dried ginger is somewhat different from that of fresh ginger. For example, the main component of fresh ginger is gingerol, while in dried ginger, gingerol is dehydrated to 6-shogaol. Due to the presence of electrophilic Michael acceptor molecules, 6-shogaol is more effective than gingerol in exerting anti-cancer, antioxidant and anti-inflammatory effects.

[0003] Studies have shown that there are four methods for extracting ginger polysaccharides, namely: hot water extraction, ultrasound-assisted extraction, alkaline solution extraction and enzyme-assisted extraction. Data show that compared with other extraction methods, enzyme-assisted extraction has the highest extraction rate. However, at present, there are few research reports on the preparation and immunomodulatory activity of dried ginger polysaccharides. Therefore, the present invention provides a method for preparing dried ginger polysaccharides, studying its fine structure and evaluating its immunomodulatory activity. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a preparation method and application of dried ginger polysaccharide. The present invention studies its fine structure and evaluates its immunomodulatory activity, providing a material basis for clinical and academic research.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide a method for preparing dried ginger polysaccharide, comprising the following steps:

[0007] (1) Pretreatment of dried ginger: crushing dried ginger slices to obtain dried ginger powder;

[0008] (2) Defatting the dried ginger powder: defatting the dried ginger powder obtained in step (1);

[0009] (3) polysaccharide extraction: mixing the defatted dried ginger powder obtained in step (2) with distilled water, stirring and extracting, collecting the obtained supernatant, and removing impurities to obtain a polysaccharide extract;

[0010] (4) concentrating and precipitating the polysaccharide extract obtained in step (3), dissolving the obtained precipitate in distilled water, centrifuging, and freeze-drying to obtain crude dried ginger polysaccharide;

[0011] (5) removing starch and fractionating and purifying the crude dried ginger polysaccharide obtained in step (4) to obtain dried ginger polysaccharide.

[0012] In one embodiment of the present invention, in step (2), the defatting treatment is: soaking the dried ginger powder in an acetone solution for defatting to obtain defatted dried ginger powder.

[0013] In one embodiment of the present invention, in step (3), the mass volume ratio of the defatted dried ginger powder to distilled water is 1:10-1:30.

[0014] In one embodiment of the present invention, in step (3), the leaching conditions are: 80°C-100°C leaching for 3h-5h;

[0015] And / or, the extraction is performed 1 to 3 times.

[0016] In one embodiment of the present invention, in step (4), the concentration is to concentrate the polysaccharide extract to 1 / 3-1 / 5 of the original volume;

[0017] And / or, the alcohol precipitation is performed by adding anhydrous ethanol to the concentrated polysaccharide extract, and after precipitation, centrifuging to separate the solid and liquid and obtain the solid phase.

[0018] In one embodiment of the present invention, in step (5), the destarch step comprises: mixing the crude dried ginger polysaccharide with α-amylase in a solvent; and the weight ratio of the crude dried ginger polysaccharide to the α-amylase is 25:1.

[0019] In one embodiment of the present invention, in step (5), the fractionation is: fractionating the crude dried ginger polysaccharide from which starch has been removed through a DEAE-cellulose column, and eluting with distilled water and NaCl, respectively.

[0020] The second object of the present invention is to provide dried ginger polysaccharide obtained by the preparation method, wherein the molecular weight of the dried ginger polysaccharide is 4022Da.

[0021] The third object of the present invention is to provide the use of the dried ginger polysaccharide in the preparation of immunosuppressants.

[0022] The fourth object of the present invention is to provide an immunosuppressant comprising the dried ginger polysaccharide.

[0023] The present invention uses enzyme-assisted extraction to obtain a polysaccharide with a molecular weight of 4022 Da. The polysaccharide prepared by the present invention can promote inflammation through the TLR4 / IKKβ / P100 signaling pathway and can be used as a potential immunosuppressant.

[0024] The above technical solution of the present invention has the following advantages compared with the prior art:

[0025] The present invention provides a preparation method and application of dried ginger polysaccharide. The present invention provides a dried ginger polysaccharide with a molecular weight of 4022Da, which can upregulate the expression of proinflammatory cytokines IL-6, IL-1β and TNF-α in Raw264.7 cells, and significantly enhance the production of reactive oxygen species. Protein immunoblotting detection showed that the expression of TLR4, the phosphorylation of IKKβ and the downstream expression of NF-κB2 were significantly upregulated. These results indicate that the dried ginger polysaccharide manufactured by the present invention can promote inflammation through the TLR4 / IKKβ / P100 signaling pathway, providing a basis for it as a potential immune preparation, with broad development potential and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0027] Figure 1 This is a process flow chart of the preparation of dried ginger polysaccharide of the present invention;

[0028] Figure 2 is the infrared spectrum of the dried ginger polysaccharide of the present invention;

[0029] Figure 3 is a gas chromatography-mass spectrogram of the dried ginger polysaccharide of the present invention;

[0030] Figure 4 This is the nuclear magnetic resonance spectrum of the dried ginger polysaccharide of the present invention;

[0031] Figure 5 This is a graph showing the intracellular reactive oxygen species (ROS) of dried ginger polysaccharides of different concentrations according to the present invention;

[0032] Figure 6 This is a Western blot analysis diagram of the dried ginger polysaccharide of the present invention. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0034] The present invention provides a preparation method and application of dried ginger polysaccharide, comprising the following steps:

[0035] Step 1: Pretreatment of dried ginger

[0036] Crush the dried ginger slices and store in a cool and dry place;

[0037] Step 2: Defatting of dried ginger powder

[0038] Soak the dried ginger powder in an acetone solution at room temperature for degreasing, replace the acetone solution every 12 hours, repeat three times, dry the defatted dried ginger powder naturally in a cool place, and store it in a dry environment for later use;

[0039] Step 3: Polysaccharide Extraction

[0040] 3.1 Extraction: Add distilled water to the defatted dried ginger powder at a mass volume ratio of 1:10-1:30 and stir, extract in a water bath at 80-100°C for 3 hours, then take out and cool to room temperature;

[0041] 3.2 Centrifugation: The extract was centrifuged at 4000r / min for 10min to separate the supernatant and precipitate;

[0042] 3.3 Re-extraction: Extract twice more according to the method in steps 3.1 and 3.2, and combine the supernatants of the three extractions;

[0043] 3.4 Impurity removal: The supernatant is filtered through vacuum paper to remove tiny particles and prepare the polysaccharide extraction clear solution;

[0044] 3.5 Precipitation of polysaccharides: Use a rotary evaporator to concentrate the clear liquid to 1 / 3 of the original volume, then add anhydrous ethanol to the solution until the final ethanol concentration is 60%. After precipitation at 4°C for 24 hours, centrifuge at 6000r / min for 15 minutes to separate the precipitate and solution, discard the supernatant and collect the precipitate, spread it on a plate, and allow the residual ethanol to completely evaporate in a ventilated place. Add an appropriate amount of distilled water to the crude polysaccharide to dissolve, centrifuge again (8000r / min, 10min) to remove residual impurities, and freeze-dry to obtain the crude polysaccharide of dried ginger;

[0045] Step 4: Polysaccharide purification

[0046] 4.1 Starch removal: α-amylase was added to the dried ginger aqueous solution, and the sample / amylase ratio was 25:1 (weight ratio). After no starch residue was detected in the sample, a crude dried ginger polysaccharide sample was obtained by dialysis and freeze-drying.

[0047] 4.2 Fractionation: The crude dried ginger polysaccharide sample after removing starch was fractionated by DEAE-cellulose column (Cl-, 120cm×6cm), eluted with distilled water, 0.1M NaCl, 0.2M NaCl and 0.4M NaCl respectively. The main fraction eluted with distilled water was collected, concentrated and freeze-dried to obtain the refined dried ginger polysaccharide. The chromatographic column was eluted with 0.2M NaCl at a flow rate of 0.3mL / min. Finally, the main peak fraction was concentrated, dialyzed and freeze-dried to obtain the purified dried ginger polysaccharide for further study.

[0048] 4.2 Drying.

[0049] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0050] Example 1

[0051] This embodiment provides a method for preparing dried ginger polysaccharide (schematic diagram as shown in Figure 1 As shown) and application, comprising the following steps:

[0052] Step 1: Pretreatment of dried ginger

[0053] Crush the dried ginger slices and store in a cool and dry place;

[0054] Step 2: Defatting of dried ginger powder

[0055] Soak the dried ginger powder in an acetone solution at room temperature for degreasing, replace the acetone solution every 12 hours, repeat three times, dry the defatted dried ginger powder naturally in a cool place, and store it in a dry environment for later use;

[0056] Step 3: Polysaccharide Extraction

[0057] 3.1 Extraction: Add distilled water to the defatted dried ginger powder at a mass volume ratio of 1:10 and stir, extract in a water bath at 80°C for 3 h, then take out and cool to room temperature;

[0058] 3.2 Centrifugation: The extract was centrifuged at 4000r / min for 10min to separate the supernatant and precipitate;

[0059] 3.3 Re-extraction: Extract twice more according to the method in steps 3.1 and 3.2, and combine the supernatants of the three extractions;

[0060] 3.4 Impurity removal: The supernatant is filtered through vacuum paper to remove tiny particles and prepare the polysaccharide extraction clear solution;

[0061] 3.5 Precipitation of polysaccharides: Use a rotary evaporator to concentrate the clear liquid to 1 / 3 of the original volume, then add anhydrous ethanol to the solution until the final ethanol concentration is 60%. After precipitation at 4°C for 24 hours, centrifuge at 6000r / min for 15 minutes to separate the precipitate and solution, discard the supernatant and collect the precipitate, spread it on a plate, and allow the residual ethanol to completely evaporate in a ventilated place. Add an appropriate amount of distilled water to the crude polysaccharide to dissolve, centrifuge again (8000r / min, 10min) to remove residual impurities, and freeze-dry to obtain the crude polysaccharide of dried ginger;

[0062] Step 4: Polysaccharide purification

[0063] 4.1 Starch removal: α-amylase was added to the dried ginger aqueous solution, and the sample / amylase ratio was 25:1 (weight ratio). After no starch residue was detected in the sample, a crude dried ginger polysaccharide sample was obtained by dialysis and freeze-drying.

[0064] 4.2 Fractionation: The crude dried ginger polysaccharide sample after removing starch was fractionated by DEAE-cellulose column (Cl-, 120cm×6cm), eluted with distilled water, 0.1M NaCl, 0.2MNaCl and 0.4MNaCl respectively. The main fraction eluted with distilled water was collected, concentrated and freeze-dried to obtain the refined dried ginger polysaccharide. The chromatographic column was eluted with 0.2MNaCl at a flow rate of 0.3mL / min. Finally, the main peak fraction was concentrated, dialyzed and freeze-dried to obtain the purified dried ginger polysaccharide.

[0065] 4.2 Drying.

[0066] Step 5: Fine structure and immune activity of dried ginger polysaccharides

[0067] 5.1 Molecular weight determination and monosaccharide composition analysis

[0068] Molecular weight determination: The purity and relative molecular weight of dried ginger polysaccharide were determined by high performance gel permeation chromatography (HPGPC). The chromatographic conditions were as follows: Waters 2414 high performance liquid chromatograph (Waters Company), TOSOH TSKPWXLG4000 and TSKPWXL G2500 were connected in series, the column oven temperature was 35°C, and the mobile phase was 0.15 mol / L NaNO 3 (Contains 0.02% NaN 3 , w / w), the flow rate was 0.5 mL / min, the injection volume was 20 μL, and the data acquisition time was 30 min.

[0069] Monosaccharide determination: First, 2 mg of dried ginger polysaccharide was hydrolyzed in 2M trifluoroacetic acid at 110°C for 4 h, and then methanol was added and evaporated several times to remove the trifluoroacetic acid. The hydrolyzed monosaccharide was reacted with an appropriate amount of PMP solution at 70°C for 1 h, and then the excess PMP solution was removed by chloroform extraction. Finally, the derivatized monosaccharide was detected by HPLC-UV detector.

[0070] 5.2 Fourier transform infrared spectrometer analysis

[0071] 1 mg of dried ginger polysaccharide sample was weighed and analyzed by Fourier transform infrared spectrometer Spotlight 400 (PerkinElmer, UK). The analysis range was 4000-600 cm -1 , the results are as follows Figure 2 As shown, through Figure 2 It can be seen that the dried ginger polysaccharide sample has a peak at 4000-400 cm-1 The typical polysaccharide characteristic absorption peaks are shown in the range.

[0072] 5.3 Methylation and gas chromatography-mass spectrometry analysis

[0073] Dissolve 20 mg of dried ginger polysaccharide in dimethyl sulfoxide, then quickly add anhydrous sodium hydroxide powder to the solution and stir until completely dissolved. Then put the methylation reaction mixture into an ice bath, add iodomethane dropwise, stir at room temperature for 30 minutes, and then add distilled water until the reaction stops. Dialyze the reaction solution with distilled water, and repeat this process three times. The sample is vacuum freeze-dried to obtain methylated polysaccharide. Infrared spectroscopy is used to detect whether the polysaccharide hydroxyl group is completely methylated. If not, repeat the above process. Finally, partially methylated alditol acetates (PMAAs) were prepared and analyzed by gas chromatography-mass spectrometry. The mass spectra of the derivatives were analyzed using the Complex Carbohydrate Structure Database of the Complex Carbohydrate Research Center (http: / / www.ccrc.uga.edu / ). The results are as shown in Figure 3 As shown, through Figure 3 It can be seen that the main chain of dried ginger polysaccharide is composed of (1→4)-Glcp, and the T-Glcp side chain replaces the C-6 position.

[0074] 5.4 Nuclear Magnetic Resonance Spectroscopy Analysis

[0075] After vacuum drying, 30 mg of dried ginger polysaccharide was suspended in 0.5 mL of D2O (atomic content 99%, Aldrich Company). 1H NMR and 13C NMR spectra were obtained using a Varian Mercury 600 NMR spectrometer. The chemical shifts of 13C and 1H NMR are reported in ppm with acetone (δ2.22 for 1H, δ30.89 and δ215.94 for 13C) as internal reference. 1D-NMR includes 1H NMR and 13C NMR spectra, and 2D-NMR includes 1H-1H-COSY, HSQC and MBC. All data were processed using MestReNova 14.2.1 software. The results are shown in Figure 4 As shown, through Figure 4 It can be seen that dried ginger polysaccharide is a neutral α-glucan, the main chain of which is composed of →4)-α-Glcp-(1→, and the side chain T-Glcp is connected to the main chain through C-6.

[0076] 5.5 RNA extraction and qPCR

[0077] Total RNA from cells was extracted using the EZ-press RNA purification kit and reverse transcribed into cDNA using the PrimeScriptTM 1st Strand cDNA Synthesis Kit according to the manufacturer's protocol. qPCR was performed using the V7PCR system. Each sample was run in duplicate and the Ct value of the target transcript was determined. GAPDH was used as an internal control.

[0078] 5.6 Determination of intracellular reactive oxygen species (ROS)

[0079] RAW264.7 cells were seeded into 96-well plates (3 × 10 5 Each group of cells was cultured overnight to allow them to adhere to the wall. Four parallel cultures were set up in each group, and then the concentrations of 0 and 50 μg / mL were respectively used. -1 , 100μg / mL -1 , 200μg / mL -1 , 300μg / mL -1 , 400μg / mL -1 , 800μg / mL -1 and 1,000 μg / mL -1 The old culture medium was replaced with the dried ginger polysaccharide culture medium, and the cells were cultured for 24 h. -1 ) was used as a positive control. LPS (1 μg / mL -1 ) was used as a positive control. The cells were stained with dichlorofluorescein diacetate (DCFH-DA, Beyotime) at 37°C for 30 min to determine the total intracellular ROS. The cells were then rinsed twice with PBS and analyzed using a fluorescence microscope. The cells were then rinsed twice with PBS and analyzed using a fluorescence microscope. The results are shown in Figure 5 As shown, through Figure 5 It can be seen that dried ginger polysaccharides increased the generation of ROS in a significant dose-dependent manner.

[0080] 5.7 Western blot analysis

[0081] RAW264.7 cells were cultured with different concentrations of LPS or dried ginger polysaccharide for 24 h, and then total protein was extracted with RIPA buffer containing protease inhibitors, and the protein content of each group was detected using a BCA detection kit. Subsequently, the protein samples were separated on an 8-12% SDS-PAGE gel and transferred to a PVDF membrane. The primary antibody and secondary antibody were incubated with the membrane in sequence, and the density of the resulting bands was quantified using KwikQuant image analyzer software. The detailed information of the primary antibodies used are as follows: IKKβ antibody, Phospho-IKKα / β-S176 / 180 antibody, TLR4 antibody, NF-κB2 p100 / p52 antibody. The results are shown in Figure 6 As shown, through Figure 6It can be seen that dried ginger polysaccharide can significantly enhance the phosphorylation of IKKβ. The expression of downstream NF-κB (p100 and p52) is also significantly upregulated. In addition, GJ0D can enhance the expression of TLR4.

[0082] Example 2

[0083] This embodiment provides a preparation method and application of dried ginger polysaccharide, comprising the following steps:

[0084] Step 1: Pretreatment of dried ginger

[0085] Crush the dried ginger slices and store in a cool and dry place;

[0086] Step 2: Defatting of dried ginger powder

[0087] Soak the dried ginger powder in an acetone solution at room temperature for degreasing, replace the acetone solution every 12 hours, repeat three times, dry the defatted dried ginger powder naturally in a cool place, and store it in a dry environment for later use;

[0088] Step 3: Polysaccharide Extraction

[0089] 3.1 Extraction: Add distilled water to the defatted dried ginger powder at a mass volume ratio of 1:20 and stir, extract in a water bath at 90°C for 3 h, then take out and cool to room temperature;

[0090] 3.2 Centrifugation: The extract was centrifuged at 4000r / min for 10min to separate the supernatant and precipitate;

[0091] 3.3 Re-extraction: Extract twice more according to the method in steps 3.1 and 3.2, and combine the supernatants of the three extractions;

[0092] 3.4 Impurity removal: The supernatant is filtered through vacuum paper to remove tiny particles and prepare the polysaccharide extraction clear solution;

[0093] 3.5 Precipitation of polysaccharides: Use a rotary evaporator to concentrate the clear liquid to 1 / 3 of the original volume, then add anhydrous ethanol to the solution until the final ethanol concentration is 60%. After precipitation at 4°C for 24 hours, centrifuge at 6000r / min for 15 minutes to separate the precipitate and solution, discard the supernatant and collect the precipitate, spread it on a plate, and allow the residual ethanol to completely evaporate in a ventilated place. Add an appropriate amount of distilled water to the crude polysaccharide to dissolve, centrifuge again (8000r / min, 10min) to remove residual impurities, and freeze-dry to obtain the crude polysaccharide of dried ginger;

[0094] Step 4: Polysaccharide purification

[0095] 4.1 Starch removal: α-amylase was added to the dried ginger aqueous solution, and the sample / amylase ratio was 25:1 (weight ratio). After no starch residue was detected in the sample, a crude dried ginger polysaccharide sample was obtained by dialysis and freeze-drying.

[0096] 4.2 Fractionation: The crude dried ginger polysaccharide sample after removing starch was fractionated by DEAE-cellulose column (Cl-, 120cm×6cm), eluted with distilled water, 0.1M NaCl, 0.2M NaCl and 0.4M NaCl respectively. The main fraction eluted with distilled water was collected, concentrated and freeze-dried to obtain the refined dried ginger polysaccharide. The chromatographic column was eluted with 0.2M NaCl at a flow rate of 0.3mL / min. Finally, the main peak fraction was concentrated, dialyzed and freeze-dried to obtain the purified dried ginger polysaccharide for further study.

[0097] 4.2 Drying.

[0098] Step 5: Fine structure and immune activity of dried ginger polysaccharides

[0099] 5.1 Molecular weight determination and monosaccharide composition analysis

[0100] First, 2 mg of dried ginger polysaccharide was hydrolyzed in 2M trifluoroacetic acid at 110°C for 4 h, and then methanol was added and evaporated several times to remove the trifluoroacetic acid. The hydrolyzed monosaccharide was reacted with an appropriate amount of PMP solution at 70°C for 1 h, and then the excess PMP solution was removed by chloroform extraction. Finally, the derivatized monosaccharide was detected by high performance liquid chromatography-ultraviolet detector.

[0101] 5.2 Fourier transform infrared spectrometer analysis

[0102] 1 mg of dried ginger polysaccharide sample was weighed and analyzed by Fourier transform infrared spectrometer Spotlight 400 (PerkinElmer, UK). The analysis range was 4000-600 cm -1 .

[0103] 5.3 Methylation and gas chromatography-mass spectrometry analysis

[0104] 20 mg of dried ginger polysaccharide was dissolved in dimethyl sulfoxide, and then anhydrous sodium hydroxide powder was quickly added to the solution and stirred until completely dissolved. The mixture of the methylation reaction was then placed in an ice bath, methyl iodide was added dropwise, stirred at room temperature for 30 minutes, and distilled water was added until the reaction was terminated. The reaction solution was dialyzed with distilled water, and this process was repeated three times. The sample was vacuum freeze-dried to obtain methylated polysaccharides. Infrared spectroscopy was used to detect whether the polysaccharide hydroxyl groups were completely methylated. If not, the above process was repeated. Finally, partially methylated alditol acetates (PMAAs) were prepared and analyzed by gas chromatography-mass spectrometry. The mass spectra of the derivatives were analyzed using the Complex Carbohydrate Structure Database of the Complex Carbohydrate Research Center (http: / / www.ccrc.uga.edu / ).

[0105] 5.4 Nuclear Magnetic Resonance Spectroscopy Analysis

[0106] After vacuum drying, 30 mg of dried ginger polysaccharide was suspended in 0.5 mL of D2O (atomic content 99%, Aldrich). 1HNMR and 13CNMR spectra were obtained using a Varian Mercury 600 NMR spectrometer. The chemical shifts of 13C and 1HNMR were reported in ppm with acetone (1H was δ2.22, 13C was δ30.89 and δ215.94) as internal reference. 1D-NMR includes 1HNMR and 13CNMR spectra, and 2D-NMR includes 1H-1H-COSY, HSQCandMBC. All data were processed using MestReNova14.2.1 software.

[0107] 5.5 RNA extraction and qPCR

[0108] Total RNA from cells was extracted using the EZ-press RNA purification kit and reverse transcribed into cDNA using the PrimeScriptTM 1st Strand cDNA synthesis kit according to the manufacturer's protocol. qPCR was performed using the V7 PCR system. Each sample was run in duplicate and the Ct value of the target transcript was determined. GAPDH was used as an internal control.

[0109] 5.6 Determination of intracellular reactive oxygen species (ROS)

[0110] RAW264.7 cells were seeded into 96-well plates (3 × 10 5 Each group of cells was cultured overnight to allow them to adhere to the wall. Four parallel cultures were set up in each group, and then the concentrations of 0 and 50 μg / mL were respectively used. -1 , 100μg / mL -1 , 200μg / mL -1 , 300μg / mL -1 , 400μg / mL -1 , 800μg / mL -1 and 1,000 μg / mL -1 The old culture medium was replaced with the dried ginger polysaccharide culture medium, and the cells were cultured for 24 h. -1 ) was used as a positive control. LPS (1 μg / mL -1 ) was used as a positive control. The cells were stained with dichlorofluorescein diacetate (DCFH-DA, Beyotime) at 37°C for 30 min to determine the total intracellular ROS. The cells were then rinsed twice with PBS and analyzed using a fluorescence microscope. The cells were then rinsed twice with PBS and analyzed using a fluorescence microscope.

[0111] 5.7 Western blot analysis

[0112] RAW264.7 cells were cultured with different concentrations of LPS or dried ginger polysaccharide for 24 h, and then total protein was extracted with RIPA buffer containing protease inhibitors, and the protein content of each group was detected using a BCA detection kit. Subsequently, the protein samples were separated on an 8-12% SDS-PAGE gel and transferred to a PVDF membrane. The primary and secondary antibodies were incubated with the membrane in sequence, and the density of the resulting bands was quantified using KwikQuant image analyzer software. The detailed information of the primary antibodies used is as follows: IKKβ antibody, Phospho-IKKα / β-S176 / 180 antibody, TLR4 antibody, and NF-κB2p100 / p52 antibody.

[0113] Example 3

[0114] This embodiment provides a preparation method and application of dried ginger polysaccharide, comprising the following steps:

[0115] Step 1: Pretreatment of dried ginger

[0116] Crush the dried ginger slices and store in a cool and dry place;

[0117] Step 2: Defatting of dried ginger powder

[0118] Soak the dried ginger powder in an acetone solution at room temperature for degreasing, replace the acetone solution every 12 hours, repeat three times, dry the defatted dried ginger powder naturally in a cool place, and store it in a dry environment for later use;

[0119] Step 3: Polysaccharide Extraction

[0120] 3.1 Extraction: Add distilled water to the defatted dried ginger powder at a mass volume ratio of 1:30 and stir, extract in a water bath at 100°C for 3 h, then take out and cool to room temperature;

[0121] 3.2 Centrifuge the extract at 4000r / min for 10min to separate the supernatant and precipitate;

[0122] 3.3 Re-extraction: Extract twice more according to the method in steps 3.1 and 3.2, and combine the supernatants of the three extractions;

[0123] 3.4 Impurity removal: The supernatant is filtered through vacuum paper to remove tiny particles and prepare a polysaccharide extraction supernatant;

[0124] 3.5 Precipitation of polysaccharides Use a rotary evaporator to concentrate the clear liquid to 1 / 3 of the original volume, then add anhydrous ethanol to the solution until the final ethanol concentration is 60%. After precipitation at 4°C for 24 hours, centrifuge at 6000r / min for 15 minutes to separate the precipitate and solution, discard the supernatant and collect the precipitate, spread it on a plate, and allow the residual ethanol to completely evaporate in a ventilated place. Add an appropriate amount of distilled water to the crude polysaccharide to dissolve, centrifuge again (8000r / min, 10min) to remove residual impurities, and freeze-dry to obtain the crude polysaccharide of dried ginger;

[0125] Step 4: Polysaccharide purification

[0126] 4.1 Destarchization α-amylase was added to the dried ginger aqueous solution, and the sample / amylase ratio was 25:1 (weight ratio). After no starch residue was detected in the sample, the crude dried ginger polysaccharide sample was obtained by dialysis and freeze-drying.

[0127] 4.2 Fractionation The crude dried ginger polysaccharide sample with starch removed was fractionated on a DEAE-cellulose column (Cl-, 120 cm × 6 cm) and eluted with distilled water, 0.1 M NaCl, 0.2 M NaCl and 0.4 M NaCl, respectively. The main fraction eluted with distilled water was collected, concentrated and freeze-dried to obtain the refined dried ginger polysaccharide. The chromatographic column was eluted with 0.2 M NaCl at a flow rate of 0.3 mL / min. Finally, the main peak fraction was concentrated, dialyzed and freeze-dried to obtain the purified dried ginger polysaccharide for further study.

[0128] 4.2 Drying

[0129] Step 5: Fine structure and immune activity of dried ginger polysaccharides

[0130] 5.1 Molecular weight determination and monosaccharide composition analysis

[0131] First, 2 mg of dried ginger polysaccharide was hydrolyzed in 2M trifluoroacetic acid at 110°C for 4 h, and then methanol was added and evaporated several times to remove the trifluoroacetic acid. The hydrolyzed monosaccharide was reacted with an appropriate amount of PMP solution at 70°C for 1 h, and then the excess PMP solution was removed by chloroform extraction. Finally, the derivatized monosaccharide was detected by high performance liquid chromatography-ultraviolet detector.

[0132] 5.2 Fourier transform infrared spectrometer analysis

[0133] 1 mg of dried ginger polysaccharide sample was weighed and analyzed by Fourier transform infrared spectrometer Spotlight 400 (PerkinElmer, UK). The analysis range was 4000-600 cm -1 .

[0134] 5.3 Methylation and gas chromatography-mass spectrometry analysis

[0135] 20 mg of dried ginger polysaccharide was dissolved in dimethyl sulfoxide, and then anhydrous sodium hydroxide powder was quickly added to the solution and stirred until completely dissolved. The mixture of the methylation reaction was then placed in an ice bath, methyl iodide was added dropwise, stirred at room temperature for 30 minutes, and distilled water was added until the reaction was terminated. The reaction solution was dialyzed with distilled water, and this process was repeated three times. The sample was vacuum freeze-dried to obtain methylated polysaccharides. Infrared spectroscopy was used to detect whether the polysaccharide hydroxyl groups were completely methylated. If not, the above process was repeated. Finally, partially methylated alditol acetates (PMAAs) were prepared and analyzed by gas chromatography-mass spectrometry. The mass spectra of the derivatives were analyzed using the Complex Carbohydrate Structure Database of the Complex Carbohydrate Research Center (http: / / www.ccrc.uga.edu / ).

[0136] 5.4 Nuclear Magnetic Resonance Spectroscopy Analysis

[0137] After vacuum drying, 30 mg of dried ginger polysaccharide was suspended in 0.5 mL of D2O (atomic content 99%, Aldrich). 1HNMR and 13CNMR spectra were obtained using a Varian Mercury 600 NMR spectrometer. The chemical shifts of 13C and 1HNMR were reported in ppm with acetone (1H was δ2.22, 13C was δ30.89 and δ215.94) as internal reference. 1D-NMR includes 1HNMR and 13CNMR spectra, and 2D-NMR includes 1H-1H-COSY, HSQCandMBC. All data were processed using MestReNova14.2.1 software.

[0138] 5.5 RNA extraction and qPCR

[0139] Total RNA from cells was extracted using the EZ-press RNA purification kit and reverse transcribed into cDNA using the PrimeScriptTM 1st Strand cDNA synthesis kit according to the manufacturer's protocol. qPCR was performed using the V7 PCR system. Each sample was run in duplicate and the Ct value of the target transcript was determined. GAPDH was used as an internal control.

[0140] 5.6 Determination of intracellular reactive oxygen species (ROS)

[0141] RAW264.7 cells were seeded into 96-well plates (3 × 10 5 Each group of cells was cultured overnight to allow them to adhere to the wall. Four parallel cultures were set up in each group, and then the concentrations of 0 and 50 μg / mL were respectively used. -1 , 100μg / mL -1 , 200μg / mL -1 , 300μg / mL -1 , 400μg / mL-1 , 800μg / mL -1 and 1,000 μg / mL -1 The old culture medium was replaced with the dried ginger polysaccharide culture medium, and the cells were cultured for 24 h. -1 ) was used as a positive control. LPS (1 μg / mL -1 ) was used as a positive control. The cells were stained with dichlorofluorescein diacetate (DCFH-DA, Beyotime) at 37°C for 30 min to determine the total intracellular ROS. The cells were then rinsed twice with PBS and analyzed using a fluorescence microscope. The cells were then rinsed twice with PBS and analyzed using a fluorescence microscope.

[0142] 5.7 Western blot analysis

[0143] RAW264.7 cells were cultured with different concentrations of LPS or dried ginger polysaccharide for 24 h, and then total protein was extracted with RIPA buffer containing protease inhibitors, and the protein content of each group was detected using a BCA detection kit. Subsequently, the protein samples were separated on an 8-12% SDS-PAGE gel and transferred to a PVDF membrane. The primary and secondary antibodies were incubated with the membrane in sequence, and the density of the resulting bands was quantified using KwikQuant image analyzer software. The detailed information of the primary antibodies used is as follows: IKKβ antibody, Phospho-IKKα / β-S176 / 180 antibody, TLR4 antibody, and NF-κB2p100 / p52 antibody.

[0144] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing dried ginger polysaccharide, characterized in that: The following steps are involved: (1) Pretreatment of dried ginger: crushing dried ginger slices to obtain dried ginger powder; (2) Defatting the dried ginger powder: defatting the dried ginger powder obtained in step (1); (3) polysaccharide extraction: mixing the defatted dried ginger powder obtained in step (2) with distilled water, stirring and extracting, collecting the obtained supernatant, and removing impurities to obtain a polysaccharide extract; (4) concentrating and precipitating the polysaccharide extract obtained in step (3), dissolving the obtained precipitate in distilled water, centrifuging, and freeze-drying to obtain crude dried ginger polysaccharide; (5) removing starch and fractionating and purifying the crude dried ginger polysaccharide obtained in step (4) to obtain dried ginger polysaccharide.

2. The preparation method according to claim 1, characterized in that: In step (2), the defatting treatment is: soaking the dried ginger powder in an acetone solution for defatting to obtain defatted dried ginger powder.

3. The preparation method according to claim 1, characterized in that: In step (3), the mass volume ratio of the defatted dried ginger powder to distilled water is 1:10-1:

30.

4. The preparation method according to claim 1, characterized in that: In step (3), the leaching conditions are: 80°C-100°C leaching for 3h-5h; And / or, the extraction is performed 1 to 3 times.

5. The preparation method according to claim 1, characterized in that: In step (4), the concentration is to concentrate the polysaccharide extract to 1 / 3-1 / 5 of the original volume; And / or, the alcohol precipitation is performed by adding anhydrous ethanol to the concentrated polysaccharide extract, and after precipitation, centrifuging to separate the solid and liquid and obtain the solid phase.

6. The preparation method according to claim 1, characterized in that: In step (5), the destarch step is to mix the crude dried ginger polysaccharide with α-amylase in a solvent.

7. The preparation method according to claim 1, characterized in that: In step (5), the fractionation is as follows: the crude dried ginger polysaccharide from which starch has been removed is fractionated through a DEAE-cellulose column and eluted with distilled water and NaCl, respectively.

8. The dried ginger polysaccharide obtained by the preparation method according to claims 1-7, characterized in that: The molecular weight of the dried ginger polysaccharide is 4022Da.

9. Use of the dried ginger polysaccharide according to claim 8 in the preparation of immunosuppressants.

10. An immunosuppressant, characterized in that Contains the dried ginger polysaccharide according to claim 8.

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