CN115558035B - Gastrodia elata polysaccharide with immunoregulatory activity - Google Patents
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Abstract
The invention discloses a gastrodia elata polysaccharide with immunoregulatory activity, wherein the molecular weight of the peak position of RGCP-2 of the gastrodia elata polysaccharide is 5215 and Da, the weight average molecular weight is 5631 and Da, and the number average molecular weight is 4472 and Da; RGCP-3 has a peak molecular weight of 32769Da, a weight average molecular weight of 39991 Da and a number average molecular weight of 27487 Da; preliminary analysis of the structures of the gastrodia elata polysaccharide RGCP-2 and RGCP-3 by ultraviolet spectrophotometry, high performance liquid chromatography, infrared spectrometry and other methods shows that the gastrodia elata polysaccharide RGCP-2 and RGCP-3 are alpha-configuration pyranose and have NO triple helix structure, the gastrodia elata polysaccharide RGCP-2 and RGCP-3 can inhibit the release of NO by mouse macrophage RAW264.7, and inhibit the generation of ROS in RAW264.7 cells induced by LPS, and the gastrodia elata polysaccharide RGCP-2 and RGCP-3 can be used as important RAW materials for developing immunoregulation medicines and functional foods.
Description
Technical Field
The invention belongs to the technical field of preparation of active ingredients of natural products, and particularly relates to gastrodia elata polysaccharide with immunoregulatory activity.
Background
Rhizoma Gastrodiae is named as rhizoma Gastrodiae, herba Ardisiae Japonicae, herba Duchesneae Indicae, rhizoma Bistortae, rhizoma Gastrodiae of OrchidaceaeGastrodia elataBl. dried tubers. Tiantian (Chinese character of 'Tian')Hemp is a perennial symbiotic plant and must be symbiotic with the armillaria mellea belonging to the family Tricholomataceae. Gastrodia elata is mainly produced in Shaanxi, yunnan, guizhou, sichuan and other places and is a rare Chinese medicinal material in China. The traditional Chinese medicine in China considers that the gastrodia elata has the effects of calming liver, calming wind, tranquilizing and improving sleep.
Immunostimulatory polysaccharides are a relatively new class of bioactive compounds that can enhance the body's natural resistance to viral and bacterial infections or assist in the treatment of hypoimmunity diseases, as one of the important strategies for improving the defense mechanisms in elderly and cancer patients. However, the drugs for improving the immune function of human bodies can bring more side effects such as drug resistance, drug dependence, teratogenicity and the like while regulating the immune activity. Therefore, searching for a natural, efficient and nontoxic organism immunomodulator is a current hot research direction. With the gradual penetration of research on natural active substances, polysaccharide is paid more attention to by more researchers, and astragalus polysaccharide, maca polysaccharide and the like are proved to have good immunoregulatory efficacy in recent years.
The polysaccharide is a natural polymer compound, is an indispensable component of all living organisms, and is closely related to maintaining biological functions. At present, the study on the gastrodia elata is mainly focused on gastrodin, phenols and glycosides, and the gastrodia elata polysaccharide is also an important active ingredient in the gastrodia elata, and is a substance with the most abundant content except for the medicinal effect ingredients such as gastrodin, p-hydroxybenzyl alcohol and the like. Therefore, the polysaccharide in the gastrodia elata is separated, the active ingredients of the gastrodia elata are mined, and the gastrodia elata polysaccharide extractive has important significance in deep development of gastrodia elata medicinal materials, guarantee of life and health of people and promotion of industrial development.
Disclosure of Invention
The invention provides polysaccharide extracted and separated from gastrodia elata, wherein the polysaccharide is white or light yellow flocculent powder, the polysaccharide is RGCP-2 or RGCP-3, the molecular weight of the peak position of the polysaccharide RGCP-2 is 5215 and Da, the weight average molecular weight is 5631 and Da, the number average molecular weight is 4472 and Da, the molecular weight of the peak position of the polysaccharide RGCP-3 is 32769Da, the weight average molecular weight is 39991 Da, and the number average molecular weight is 27487 Da.
The gastrodia elata polysaccharide has immunoregulatory activity, and can remarkably inhibit mouse macrophage RAW264.7 from releasing NO and inhibit LPS-induced RAW264.7 intracellular ROS from generating when the concentration of polysaccharide RGCP-2 and RGCP-3 is 12.5-50 mug/mL.
The invention aims at realizing the following technical scheme:
(1) Cleaning fresh rhizoma Gastrodiae, steaming until there is no white core, oven drying at 40-50deg.C, and pulverizing;
(2) Adding distilled water into the gastrodia elata powder in the step (2) according to the mass-volume ratio g of mL=1:15-25, leaching in a water bath kettle at 60 ℃, centrifuging, repeatedly extracting filter residues for 2-4 times, collecting combined filtrate, and concentrating the filtrate under reduced pressure to 1/6 of the original volume;
(3) Adding 3-5 times of absolute ethyl alcohol into the concentrated solution in the step (2), standing at 4 ℃, filtering, collecting precipitate, placing the precipitate in a 37 ℃ oven, volatilizing the ethyl alcohol to obtain crude gastrodia elata polysaccharide extract;
(4) Adding distilled water into rhizoma Gastrodiae polysaccharide crude extract, water-bathing at 60deg.C for 2 h, centrifuging to remove insoluble substances, and repeating the above steps for 2 times;
(5) Adding sevage reagent (chloroform: n-butanol=4:1) into the supernatant obtained in the step (4) to remove proteins, putting into a shaking table to shake vigorously, taking out, pouring into a separating funnel, standing for layering, removing an intermediate phase and a lower phase, sequentially adding the sevage reagent to remove proteins, continuously adding the sevage reagent to remove proteins if white substances exist on the liquid surface between the two phases, and indicating that the proteins are basically removed if the white substances do not exist;
(6) Transferring the deproteinized sugar solution into a dialysis bag, and dialyzing with distilled water for 2-3 d every 6 h times;
(7) Placing the dialyzed sugar solution at-80deg.C, and lyophilizing to obtain rhizoma Gastrodiae crude polysaccharide GCP;
(8) Dissolving rhizoma Gastrodiae crude polysaccharide GCP in distilled water, loading onto DEAE-52 cellulose chromatographic column, continuously gradient eluting with 0, 0.1, 0.2 mol/L NaCl solution, collecting eluate with automatic part collector, tracking and monitoring eluting condition with phenol-sulfuric acid method and spectrophotometer, drawing eluting curve according to absorbance value, collecting liquid corresponding to eluting peak, dialyzing, and lyophilizing to obtain several polysaccharides;
(9) And (3) respectively loading the polysaccharide obtained in the step (8) on a Sephadex G-50 Sephadex chromatographic column for further purification, eluting with 0.1 mol/L NaCl solution, collecting the eluent by an automatic part collector, tracking and monitoring the elution condition by a phenol-sulfuric acid method and a spectrophotometry method, drawing a curve according to a light absorption value, collecting the liquid corresponding to an elution peak, dialyzing and freeze-drying to obtain the purified gastrodia elata polysaccharide.
The invention has the advantages and technical effects that:
(1) The polysaccharides RGCP-2 and RGCP-3 are natural extracts, are novel polysaccharides extracted from gastrodia elata, and have good safety; the peak molecular weight of the polysaccharide RGCP-2 is 5215 and Da, the weight average molecular weight is 5631 and Da, the number average molecular weight is 4472 and Da, the peak molecular weight of the polysaccharide RGCP-3 is 32769Da, the weight average molecular weight is 39991 Da, the number average molecular weight is 27487 Da, and the polysaccharide is high-purity and uniform;
(2) The gastrodia elata polysaccharide RGCP-2 and RGCP-3 have good immunoregulatory activity, and can be used as important raw materials for developing immunoregulatory medicines and functional foods;
(3) The gastrodia elata polysaccharide RGCP-2 and RGCP-3 provided by the invention have the advantages of simple preparation process and low cost, and are suitable for large-scale industrial production.
Drawings
FIG. 1 is a graph showing the elution of DEAE-52 anion exchange column chromatography of crude rhizoma Gastrodiae polysaccharide GCP;
FIG. 2 is a graph showing the elution of Sephadex G-50 gel column chromatography of crude rhizoma Gastrodiae polysaccharide GCP-2;
FIG. 3 is a graph showing the elution of Sephadex G-50 gel column chromatography of crude polysaccharide GCP-3 of rhizoma Gastrodiae;
FIG. 4 is a graph showing the elution of Sephadex G-50 gel column chromatography of crude polysaccharide GCP-4 of rhizoma Gastrodiae;
FIG. 5 ultraviolet spectrograms of rhizoma Gastrodiae polysaccharides RGCP-2, RGCP-3 and RGCP-4;
FIG. 6 shows a high-efficiency gel permeation chromatogram of rhizoma Gastrodiae polysaccharide RGCP-2;
FIG. 7 shows a high-efficiency gel permeation chromatogram of rhizoma Gastrodiae polysaccharide RGCP-3;
FIG. 8 shows a high-efficiency gel permeation chromatogram of rhizoma Gastrodiae polysaccharide RGCP-4;
FIG. 9 is a HPLC plot of a standard monosaccharide mixture;
FIG. 10 shows PMP derivative HPLC diagrams of rhizoma Gastrodiae polysaccharide RGCP-2 and RGCP-3;
FIG. 11 Congo red experimental diagrams of gastrodia elata polysaccharides RGCP-2 and RGCP-3;
FIG. 12 is an infrared spectrogram of rhizoma Gastrodiae polysaccharide RGCP-2;
FIG. 13 shows an infrared spectrogram of rhizoma Gastrodiae polysaccharide RGCP-3;
FIG. 14 effects of Gastrodia elata polysaccharides RGCP-2 and RGCP-3 on RAW264.7 cell proliferation;
FIG. 15 effects of the gastrodia elata polysaccharides RGCP-2 and RGCP-3 on LPS-induced production of NO in RAW264.7 cells;
FIG. 16 effects of Gastrodia elata polysaccharides RGCP-2 and RGCP-3 on LPS-induced RAW264.7 intracellular ROS production.
Detailed Description
The technical scheme of the invention is further described in detail by examples, but the content of the invention is not limited to the examples, and the methods in the examples are conventional methods unless otherwise specified, and materials, reagents and the like used are obtained from commercial sources unless otherwise specified;
the polysaccharide content was determined in the examples using the phenol-sulfuric acid method: taking polysaccharide 0.5 mL of each tube, adding distilled water 0.5 mL, adding the prepared 6% phenol solution 0.5 mL, adding concentrated sulfuric acid 2.5 mL, mixing uniformly, and standing; after the tube cooled, the absorbance was measured at 490 and nm.
Example 1: extraction, separation and purification of gastrodia polysaccharide
1. Cleaning fresh rhizoma Gastrodiae, steaming until there is no white core, oven drying at 40-50deg.C, and pulverizing;
2. adding 200 mL distilled water into rhizoma Gastrodiae dry powder 10 g at a mass volume ratio g: mL of 1:20, extracting in water bath at 60deg.C for 30 min, centrifuging at 4000 rpm for 10 min, extracting the residue with water again, extracting repeatedly for 3 times, collecting combined filtrates, and concentrating to about 100 mL at 50deg.C with rotary evaporator;
3. slowly adding 400 mL anhydrous ethanol into the concentrated solution, standing at 4deg.C for 24h to remove pigment and precipitate polysaccharide, filtering, collecting precipitate, and drying the precipitate in oven at 37deg.C to obtain crude rhizoma Gastrodiae polysaccharide extract;
4. adding distilled water into the crude gastrodia polysaccharide extract, centrifuging in a water bath at 60 ℃ for 2 h, removing insoluble substances, reserving supernatant, adding a proper amount of distilled water into the supernatant, centrifuging in a water bath at 60 ℃ for 2 h, taking supernatant, adding sevage reagent (chloroform: n-butanol=4:1), putting into a shaking table, shaking vigorously at 200 rpm for 30 min, taking out, pouring into a separating funnel, standing and layering for about 30 min, removing intermediate phase and lower phase, sequentially adding sevage reagent to remove protein, repeating for 10 times, continuously adding sevage reagent to remove protein if white substances exist on the liquid surface between the two phases, and basically removing protein if white substances do not exist;
5. transferring the deproteinized sugar solution into a 3500Da dialysis bag, and dialyzing with distilled water for 2 d every 6 h times;
6. subpackaging the dialyzed sugar solution, placing at-80 ℃, and obtaining crude gastrodia elata polysaccharide GCP through freeze drying;
7. dissolving rhizoma Gastrodiae crude polysaccharide GCP in distilled water, loading onto DEAE-52 cellulose chromatographic column, gradient eluting with 0, 0.1, 0.2 mol/L NaCl solution, eluting 4 column volumes per concentration, collecting eluate with automatic part collector, tracking and monitoring eluting condition with phenol-sulfuric acid method and spectrophotometer, drawing eluting curve according to absorbance value, see figure 1, collecting liquid corresponding to eluting peak in the figure, dialyzing with dialysis bag with molecular weight cutoff of 3500Da, desalting, concentrating, and lyophilizing to obtain rhizoma Gastrodiae polysaccharide GCP-1, GCP-2, GCP-3 and GCP-4 (GCP-2, GCP-3 and GCP-4 are mainly studied later due to lower GCP-1 yield);
8. dissolving rhizoma Gastrodiae polysaccharide GCP-2, GCP-3 and GCP-4 with 0.1 mol/L NaCl solution, respectively loading onto Sephadex G-50 Sephadex chromatographic column for further purification, eluting with 0.1 mol/L NaCl for 1 column volume, collecting eluate with automatic part collector, tracking and monitoring eluate by phenol-sulfuric acid method and spectrophotometry, drawing curve according to absorbance value, respectively collecting the liquids corresponding to the elution peaks in the figures, dialyzing the collected liquids with molecular weight cutoff 3500Da dialysis bag for desalting, concentrating, and lyophilizing to obtain purified rhizoma Gastrodiae polysaccharide RGCP-2, RGCP-3 and RGCP-4.
Example 2: structural characterization of gastrodia elata polysaccharide
1. Ultraviolet spectral analysis
Preparing solution of polysaccharides RGCP-2, RGCP-3 and RGCP-4 with mass concentration of 1.0 mg/mL, using distilled water as blank control, and performing full-band scanning at 800-200 nm with ultraviolet-visible spectrophotometer;
FIG. 5 is a full-band scan of polysaccharide samples with UV-Vis spectrophotometers 800-200 nm, from which no absorption was observed at 260 nm, 280 nm, 320 nm, indicating that polysaccharide separated by DEAE-52 cellulose column did not contain nucleic acid and protein.
2. Determination of molecular weight of polysaccharide
Chromatographic separation conditions: chromatographic column: BRT105-104-102 series gel column (8×300 mm); mobile phase: 0.05 mol/L NaCl solution; flow rate: 0.6 mL/min, column temperature: 40. the temperature is lower than the temperature; sample injection amount: 20. mu L; a detector: differential detector RI-10A; precisely weighing a polysaccharide sample and a standard substance respectively;
dextran standards 1152 Da, 5000 Da, 11600 Da, 23800 Da, 48600 Da, 80900 Da, 148000 Da, 273000 Da, 409800 Da, 667800 Da were prepared as 5mg/mL solutions, respectively, centrifuged at 12000 rpm for 10 min, the supernatant was filtered with a 0.22 μm microporous filter membrane, and the samples were transferred to a 1.8 mL sample-feeding vial, analyzed by HPLC, and plotted to give lgMp-RT (peak molecular weight), lgMw-RT (weight average molecular weight), lgMn-RT (number average molecular weight) calibration curves:
the lgMp-RT correction curve equation is: y= -0.1829 x+11.554, r 2 = 0.9965；
The lgMw-RT correction curve equation is: y= -0.1951 x+12.11, r 2 = 0.996；
The lgMn-RT correction curve equation is: y= -0.1807 x+11.393, r 2 = 0.9928。
Preparing polysaccharide sample into 5mg/mL solution, centrifuging at 12000 rpm for 10 min, filtering supernatant with microporous membrane of 0.22 μm, transferring sample into sample injection vial of 1.8 mL, and performing HPLC sample injection analysis;
the results are shown in the high-efficiency gel permeation chromatograms of gastrodia elata polysaccharides RGCP-2, RGCP-3 and RGCP-4 in the figures 6, 7 and 8, the molecular weight of each sample is calculated according to a correction curve equation, the calculated results are shown in the following table, and the unit of the molecular weight in the table is Da, namely Dalton (Dalton);
as can be seen from the high-efficiency gel permeation chromatograms, the gastrodia elata polysaccharide RGCP-2 and RGCP-3 respectively show peaks at 42.847 min and 38.483 min and show a single peak (the peak of which is a mobile phase at 45.6 min), the molecular weight of the RGCP-2 peak is 5215Da, the weight average molecular weight is 5631 Da, the number average molecular weight is 4472 and Da, and the polydispersity index (Mw/Mn) is 1.259, which indicates that the molecular weight distribution is narrower; RGCP-3 has a peak molecular weight of 32769Da, a weight average molecular weight of 39991 Da, a number average molecular weight of 27487 Da, and a polydispersity index (Mw/Mn) of 1.455, indicating a narrower molecular weight distribution; and the content of RGCP-2 and RGCP-3 is 100%, thus representing the high purity and uniformity of polysaccharide. Gastrodia elata polysaccharide RGCP-4 has peaks (45.6 min is the peak of mobile phase) at 31.733, 34.56, 37.507 and 40.855 min respectively, so RGCP-4 is not homogeneous polysaccharide (only RGCP-2 and RGCP-3 were studied later).
3. Analysis of monosaccharide composition
(1) Pre-column derivatization of monosaccharide standards
Respectively and accurately weighing mannose Man, rhamnose Rha, glucuronic acid GlcA, galacturonic acid GalA, glucose Glc, galactose Gal, xylose Xyl and arabinose Ara, respectively, dissolving 0.01 mmol in 8 mL ammonia water, vortex mixing, taking 800 mu L to 5 mL centrifuge tubes of the mixed sugar solution, adding 800 mu L PMP (0.5 mol/L), vortex mixing, carrying out water bath reaction at 70 ℃ for 30 min, taking out and cooling to room temperature, adding 3 mL water, placing in a 55 ℃ vacuum drying oven for volatilizing, and adding water for repeating twice. After volatilizing in a vacuum drying oven, adding 1 mL deionized water and 1 mL chloroform into a centrifuge tube, performing vortex extraction (the derivatized monosaccharide is dissolved in the water phase, residual PMP is dissolved in the chloroform phase, and centrifugation can be performed at 3500 rpm for 5 min to accelerate delamination), removing the lower chloroform phase, continuing to add 1 mL chloroform for extraction for several times until the chloroform phase is colorless (the chloroform phase without removing PMP is yellow green), transferring the upper water phase into another centrifuge tube, centrifuging at 13000 rpm for 5 min, taking the supernatant, passing through a 0.45 μm water-based filter membrane, and performing liquid phase detection analysis on the sample.
(2) Pre-column derivatization of polysaccharide samples
Accurately weighing 5mg polysaccharide in 15 mL tube sealer, adding 2 mL TFA (4 mol/L) into the tube sealer, placing into rotor, sealing with cover, placing into 121 deg.C oil bath, stirring for 6 h (polysaccharide hydrolysis is complete, solution is clear and transparent), taking out the tube sealer after hydrolysis, and cooling to room temperature. Transferring polysaccharide hydrolysate into a centrifuge tube of 5 mL, volatilizing in a vacuum drying oven at 55deg.C to remove TFA, adding 2 mL methanol, volatilizing in a vacuum drying oven at 45deg.C to remove residual TFA, and repeating the above steps for 3 times. 300. Mu.L of concentrated ammonia water was added to the centrifuge tube, vortexed, and the hydrolyzed sugar was dissolved in ammonia water. Then 300. Mu.L of PMP (0.5 mol/L) was added to the tube, and the mixture was vortexed and mixed well, and reacted in a water bath at 70℃for 30 minutes. Taking out, cooling to room temperature, adding 3 mL water into the centrifuge tube, mixing uniformly by vortex, volatilizing in a vacuum drying oven at 55 ℃, and repeating twice. After volatilizing in a vacuum drying oven, adding 1 mL deionized water and 1 mL chloroform into the centrifuge tube, performing vortex extraction, discarding the lower chloroform phase, and continuously adding 1 mL chloroform for extraction for several times until the chloroform phase is colorless. The upper aqueous phase was transferred to another centrifuge tube, centrifuged at 13000 rpm for 5 min, the supernatant was collected, filtered through a 0.45 μm aqueous filter, and the sample was subjected to liquid phase detection analysis.
(3) Liquid phase detection conditions
Chromatographic column model: c (C) 18 Column, flow rate: 1 mL/min, mobile phase: 83:17 (v/v,%) 0.1 mol/L phosphate buffer (pH 6.7) and acetonitrile, column temperature: 30. c, detecting wavelength: 245 nm, sample injection volume: 20. mu L.
The PMP derivative HPLC diagram of the gastrodia elata polysaccharide RGCP-2 and RGCP-3 is shown in figure 10, and the comparison of the mixed monosaccharide standard HPLC diagram of figure 9 shows that RGCP-2 consists of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, and the mole percentages of the monosaccharides are calculated according to peak areas to be 0.902%, 0.458%, 0.422%, 0.564%, 82.258%, 0.920%, 13.930% and 0.545% in sequence; RGCP-3 is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, and the mole percentages of the monosaccharides are 0.254%, 0.316%, 0.269%, 0.547%, 84.695%, 0.102%, 13.355% and 0.462% in order according to the peak area calculation.
4. Congo red test analysis
The gastrodia polysaccharide RGCP-2 and RGCP-3 are respectively weighed and prepared into polysaccharide solutions with the concentration of 0.5 mg/mL, a 2 mL sample is sucked and mixed with Congo red solution with the concentration of 2 mL of 50 mu mol/L in equal volume, after standing for a period of time, 1 mol/L NaOH solution is added into each test tube, so that the final concentration of sodium hydroxide in each tube reaches 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 and 0.50 mol/L respectively, and the reaction is carried out for 15 min under the condition of room temperature, and an ultraviolet-visible spectrometer is used for scanning (the wavelength range is 600-400 nm). The change of the maximum absorption wavelength of the mixed solution under NaOH solutions with different concentrations is measured, and the maximum absorption wavelength is plotted and analyzed by taking the concentration of NaOH as an abscissa (x) and taking the maximum absorption wavelength as an ordinate (Y).
As can be seen from the results of FIG. 11, as the concentration of NaOH increases, the maximum absorption wavelength of the complex formed by the gastrodia elata polysaccharides RGCP-2 and RGCP-3 and Congo red does not increase with the change of the concentration of NaOH, namely, no red shift occurs, and the trend of the complex is basically the same as that of the pure Congo red solution, namely, the gastrodia elata polysaccharides RGCP-2 and RGCP-3 have no triple helix structure.
5. Infrared spectroscopic analysis
Weighing polysaccharides RGCP-2 and RGCP-3, mixing 5mg with KBr powder, tabletting, and measuring 4000-400 cm by Fourier infrared spectrometer -1 Scanning is performed over a range of wavelengths.
The results are shown in FIGS. 12 and 13, and the infrared spectrum analysis results of the gastrodia elata polysaccharides RGCP-2 and RGCP-3 are as follows: 3421 cm -1 The absorption peak at the position is attributed to the stretching vibration of-OH; 2928 cm -1 The absorption peak at the position is attributed to asymmetric stretching vibration of C-H; 1637 cm -1 The absorption peak in the vicinity is generated by-c=o bond stretching vibration; 1414 cm -1 At the absorption peak-CH 2 C-H angular vibration in-or-CH-and 2928 cm -1 at-CH of 2 -or-the C-H telescopic vibration absorption peak in CH-is a characteristic absorption peak of the polysaccharide; 1381 cm -1 The absorption peak at this point may be caused by the deformation vibration of the c—h bond; in the range of 1000-1200 and 1200 cm -1 1153 and cm of (2) -1 , 1084 cm -1 ，1025 cm -1 Three characteristic absorption peaks, namely that the sugar ring configuration in the gastrodia elata polysaccharide is pyrane type (only two characteristic absorption peaks exist on the furan type sugar ring in the interval); 1025 cm -1 The characteristic absorption of glucose is the absorption peak, which shows that the polysaccharide mainly comprises monosaccharide which is glucose; 928 cm -1 The absorption peak is the characteristic absorption of-C-O-C, which is the characteristic absorption peak of glucose in the typical D-pyran form; 854 cm -1 The absorbance peak indicates that the glycosidic bond type is mainly alpha-configuration; not see 1616 cm -1 Of (2) NH 2 and-NH 3 The characteristic absorption peaks of (2) indicate no proteoglycan. As shown by comparison and analysis of FT-IR spectra, the gastrodia elata polysaccharide RGCP-2 and RGCP-3 are alpha-configuration pyranose.
Example 3: identification of immunoregulatory activity of gastrodia polysaccharide
1. Effect of Gastrodia elata polysaccharide on RAW264.7 cell proliferation
100. Mu.L of log phase cells were seeded in 96-well plates at a cell density of 4X 10 3 ~5×10 3 Individual wells/well placed in 5% CO 2 Incubating overnight in a 37 ℃ incubator, taking 100 mu L of RGCP-2 and RGCP-3 with different concentrations (12.5, 25, 50, 100 and 200 mu g/mL) to act on cells 24h respectively after the cells are attached, and setting a blank Control group (Control group) without adding gastrodia polysaccharide; after discarding the supernatant, the sterile PBS was washed once. Each well was charged with 110. Mu.L of a mixture of MTT (5 mg/mL) and serum-free DMEM (volume ratio 2:9) and placed in 5% CO 2 Incubation is finished by incubating 4h in a 37 ℃ incubator, the incubation is stopped, the supernatant is carefully removed, the bottom of the pore plate is not required to be touched, 150 micro L of DMSO is added into each pore, and the mixture is placed on a shaking table to oscillate for 10 min at a low speed, so that the crystals are fully dissolved. The absorbance of each well was measured at the enzyme-linked immunosorbent assay OD 490 nm, normalized to 100% for the blank, and the relative cellular activity (%) of each well was calculated, 3 times in biological parallel for each concentration.
The results are shown in fig. 14, which shows that the overall survival rate of RAW264.7 cells shows a trend of basically unchanged first and then declining as the polysaccharide concentration of gastrodia elata increases. Compared with a blank control group, after 12.5, 25 and 50 mug/mL of gastrodia elata polysaccharide RGCP-2 and RGCP-3 act on cells, the cell viability is not obviously affected (P is more than 0.05); as RGCP-2 and RGCP-3 concentrations increased, cell viability decreased in the concentration range of 100-200. Mu.g/mL (P < 0.01).
2. Influence of gastrodia polysaccharide on NO secretion of RAW264.7 cells
The change of the NO content is detected by adopting a classical Griess method, and the method is specifically as follows: taking RAW264.7 cells in logarithmic growth phase according to 5×10 4 The cells/well were added to 96-well cell culture plates for 24 h; adding 10 μl of rhizoma Gastrodiae polysaccharide (RGCP-2 and RGCP-3) with different concentrations (12.5, 25, 50 μg/mL) respectively, and acting on cell 24h respectively, then adding 10 μl of 0.1 μg/mL LPS respectively to rhizoma Gastrodiae polysaccharide group and LPS-induced group (LPS-induced cell 24h, no RGCP-2 and RGCP-3), setting blank control group (normal cell culture, no treatment), setting 3 multiple holes in each group, placing at 37deg.C, and 5% CO 2 Culturing in an incubator for 24h, taking 60 mu L of cell culture supernatant, adding 50 mu L of 1% sulfanilamide, uniformly mixing, and incubating for 10 min; then adding 50 mu L of 0.1% N-1-naphthyl ethylenediamine hydrochloride, mixing uniformly, and incubating for 10 min at normal temperature; the absorbance of the sample at OD540 nm was recorded with a microplate reader.
The results are shown in FIG. 15, which shows that LPS stimulated cells for 24 hours resulted in a very significant increase in intracellular NO content (P < 0.0001) compared to the blank, indicating that cellular inflammation was exacerbated; after RGCP-2 and RGCP-3 acted on cell 24h at concentrations of 12.5, 25, 50 μg/mL, LPS-induced production of NO in RAW264.7 cells was significantly inhibited (P < 0.05), and the inhibition of RGCP-3 was concentration-dependent; the results show that RGCP-2 and RGCP-3 (12.5, 25, 50 mug/mL) can inhibit LPS to stimulate abnormal generation of NO in RAW264.7 cells, and the results show that gastrodia elata polysaccharide RGCP-2 and RGCP-3 can inhibit generation of NO in macrophages under an inflammatory state to a certain extent, further inhibit generation and progress of inflammation in the macrophages, and can play an anti-inflammatory immunoregulation role on the macrophages under the inflammatory state.
3. Influence of gastrodia polysaccharide on RAW264.7 cells to produce ROS
At 1X 10 3 Inoculating RAW264.7 cells at the density of each hole on a 12-hole plate, culturing overnight until the cells are attached, respectively adding LPS, RGCP-2 and RGCP-3 into each group according to the experimental design of step 2, discarding the culture solution after 24 hours of action, dropwise adding 400 mu L of DCFH-DA (final concentration is 20 mu mol/L) probe into each hole, and incubating in a cell incubator for 30 minutes at 37 ℃ in a dark place; washing with PBS for 2 times, fixing with paraformaldehyde for 10 min, washing with PBS again for 2 times, staining with DAPI for 3-5 min, and washing with PBS for 3 times again; observations were made with an inverted fluorescence microscope and recorded with photographs.
The results are shown in FIG. 16, compared with the blank control group, the DCFH-DA green fluorescence intensity of LPS group cells is obviously enhanced, which indicates that after LPS stimulates RAW264.7 cells, the rapid generation of intracellular ROS is induced, and compared with the LPS group, the DCFH-DA green fluorescence intensity of gastrodia elata polysaccharide RGCP-2 and RGCP-3 groups is obviously reduced, thereby indicating that the gastrodia elata polysaccharide RGCP-2 and RGCP-3 can inhibit the generation of LPS-induced RAW264.7 intracellular ROS.
Claims (2)
1. A polysaccharide having immunomodulatory activity, characterized in that: the polysaccharide is separated from rhizoma Gastrodiae, and the polysaccharide is homogeneous polysaccharide RGCP-2 with peak molecular weight 5215Da, weight average molecular weight 5631 Da and number average molecular weight 4472 Da, or homogeneous polysaccharide RGCP-3 with peak molecular weight 32769Da, weight average molecular weight 39991 Da and number average molecular weight 27487 Da;
the polysaccharide RGCP-2 consists of mannose with the molar ratio of 0.902 percent, rhamnose with the molar ratio of 0.458 percent, glucuronic acid with the molar ratio of 0.422 percent, galacturonic acid with the molar ratio of 0.564 percent, glucose with the molar ratio of 82.258 percent, galactose with the molar ratio of 0.920 percent, xylose with the molar ratio of 13.930 percent and arabinose with the molar ratio of 0.545 percent; polysaccharide RGCP-3 consists of mannose with the molar ratio of 0.254 percent, rhamnose with the molar ratio of 0.316 percent, glucuronic acid with the molar ratio of 0.269 percent, galacturonic acid with the molar ratio of 0.547 percent, glucose with the molar ratio of 84.695 percent, galactose with the molar ratio of 0.102 percent, xylose with the molar ratio of 13.355 percent and arabinose with the molar ratio of 0.462 percent; the polysaccharide RGCP-2 and the polysaccharide RGCP-3 have no triple helix structure and are alpha-configuration pyranose.
2. The polysaccharide having immunomodulatory activity of claim 1, wherein the polysaccharide has immunomodulatory activity of: when the concentration of the polysaccharide RGCP-2 and the polysaccharide RGCP-3 is 12.5-50 mug/mL, the secretion of NO in the macrophages of the mice in an inflammatory state can be obviously inhibited, and the generation of ROS in the macrophages induced by LPS can be inhibited.
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| CN112920287A (en) * | 2021-01-29 | 2021-06-08 | 中山大学 | Amomum villosum polysaccharide with immunoregulation effect and preparation method and application thereof |
| CN114133461A (en) * | 2021-12-06 | 2022-03-04 | 中国科学院昆明植物研究所 | Extraction method and application of gastrodia elata oligosaccharide GEP-2 |
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| CN112920287A (en) * | 2021-01-29 | 2021-06-08 | 中山大学 | Amomum villosum polysaccharide with immunoregulation effect and preparation method and application thereof |
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