CN115651089B - Gastrodia elata polysaccharide with antioxidant activity - Google Patents
Gastrodia elata polysaccharide with antioxidant activity Download PDFInfo
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Abstract
The invention discloses gastrodia elata polysaccharide RGCP-3 with antioxidant activity, which has a peak molecular weight of 32769 Da, a weight average molecular weight of 39991 Da and a number average molecular weight of 27487 Da; preliminary analysis of the structure of the gastrodia polysaccharide RGCP-3 by ultraviolet spectrophotometry, high performance liquid chromatography, infrared spectrometry and other methods shows that the gastrodia polysaccharide RGCP-3 is alpha-configuration pyranose, which consists of mannose with the molar ratio of 0.254%, rhamnose with the molar ratio of 0.316%, glucuronic acid with the molar ratio of 0.269%, galacturonic acid with the molar ratio of 0.547%, glucose with the molar ratio of 84.695%, galactose with the molar ratio of 0.102%, xylose with the molar ratio of 13.355% and arabinose with the molar ratio of 0.462%, has no triple helix structure, vc is used as a control, and the gastrodia polysaccharide RGCP-3 has higher DPPH free radical scavenging capability, hydroxyl free radical scavenging capability and reducing capability, and RGCP-3 has high H scavenging capability on the whole plant 2 O 2 The induced RAW264.7 cell oxidative damage has better protection effect, and the gastrodia elata polysaccharide RGCP-3 can provide a new choice for preparing antioxidant drugs or health care products.
Description
Technical Field
The invention relates to the technical field of active ingredients of natural products, in particular to gastrodia elata polysaccharide with antioxidant activity.
Background
Rhizoma Gastrodiae is named as rhizoma Gastrodiae, herba Ardisiae Japonicae, herba Duchesneae Indicae, rhizoma Bistortae, rhizoma Gastrodiae of OrchidaceaeGastrodia elataBl. dried tubers. Gastrodia elata is a perennial symbiotic plant and has the function of symbiosis with Armillariella mellea of 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.
The increase in free radicals is an important factor in the production of aging. The polysaccharide has inhibiting effect on oxygen free radical generation and erythrocyte lipid peroxidation, and has SOD-like substance activity. The excessive generation of free radicals can cause oxidative damage to biological membranes and DNA, and cause cancers, arteriosclerosis, aging and various senile chronic diseases, such as the use of biological antioxidants to cut off peroxidation chain reaction can inhibit free radical damage of organisms, thereby maintaining optimal health state and preventing and treating the diseases and delaying aging.
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 research on gastrodia elata is mainly focused on gastrodin, phenols and glycosides, and gastrodia elata polysaccharide is also an important active ingredient in gastrodia elata, is a substance with the most abundant content except for the medicinal components such as gastrodin, p-hydroxybenzyl alcohol and the like, and has a series of pharmacological activities, so that the gastrodia elata polysaccharide is separated, the active ingredient of the gastrodia elata polysaccharide is mined, and the gastrodia elata polysaccharide has important significance for deep development of gastrodia elata medicinal materials, guarantee of life health of people and promotion of industrial development.
Disclosure of Invention
The invention provides gastrodia polysaccharide RGCP-3 extracted from gastrodia elata, which is white or light yellow flocculent powder, wherein the peak molecular weight of RGCP-3 is 32769 Da, the weight average molecular weight is 39991 Da, and the number average molecular weight is 27487 Da.
The gastrodia elata polysaccharide RGCP-3 has higher DPPH free radical scavenging capability, hydroxyl free radical scavenging capability and reducing capability, andand pair H 2 O 2 The induced RAW264.7 cell oxidative damage has better protection effect.
The aim of the invention is achieved by 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 the crude extract of rhizoma Gastrodiae polysaccharide, water-bathing at 60deg.C for 2 hr, centrifuging, removing 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 2-3d every 6 h;
(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) The polysaccharide obtained in the step (8) is respectively loaded on a Sephadex G-50 Sephadex chromatographic column for further purification, 0.1 mol/L NaCl solution is used for eluting, an automatic part collector is used for collecting eluent, a phenol-sulfuric acid method and a spectrophotometry method are used for tracking and monitoring the eluting condition, a curve is drawn according to the light absorption value, liquid corresponding to an eluting peak is collected, and the purified gastrodia elata polysaccharide is obtained after dialysis and freeze-drying;
(10) Preliminary analysis is carried out on the structures of gastrodia polysaccharide RGCP-2 and RGCP-3 by ultraviolet spectroscopy, high performance liquid chromatography, infrared spectroscopy and other methods;
(11) Detecting the antioxidant activity of gastrodia elata polysaccharide RGCP-3 DPPH.
The invention has the advantages and technical effects that:
(1) The polysaccharide RGCP-3 is a natural extract, is a novel polysaccharide extracted from rhizoma gastrodiae, and has good safety; the polysaccharide RGCP-3 has a peak molecular weight of 32769 Da, a weight average molecular weight of 39991 Da and a number average molecular weight of 27487 Da, and is a high-purity and uniform polysaccharide;
(2) The gastrodia elata polysaccharide RGCP-3 has better antioxidant activity, and can provide a new choice for preparing antioxidant drugs or health care products;
(3) The gastrodia elata polysaccharide RGCP-3 has simple preparation process and low cost, and is suitable for large-scale industrial production.
Drawings
FIG. 1 is a DEAE-52 anion exchange chromatography elution diagram 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 shows the results of the ability of RGCP-2 and RGCP-3 of gastrodia elata polysaccharide to scavenge DPPH free radicals;
FIG. 15 shows the results of the ability of RGCP-2 and RGCP-3 of gastrodia elata polysaccharide to scavenge hydroxyl radicals;
FIG. 16 total reducing force results of Gastrodia elata polysaccharides RGCP-2 and RGCP-3;
FIG. 17 Gastrodia elata polysaccharide pair H 2 O 2 Inducing the protective effect of injury.
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 24 h 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 2h, removing insoluble substances, reserving supernatant, adding a proper amount of distilled water into the supernatant, centrifuging in a water bath at 60 ℃ for 2h, 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 3500 Da dialysis bag, and dialyzing with distilled water for 2 d every 6h 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 3500 Da, 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 liquid corresponding to eluting peak in the graph, dialyzing the collected liquid with molecular weight cutoff 3500 Da 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 5 mg/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 a polysaccharide sample into a 5 mg/mL solution, centrifuging at 12000 rpm for 10 min, filtering the supernatant with a microporous filter membrane of 0.22 mu m, transferring the sample into a 1.8 mL sample injection vial, 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 5215 Da, 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 32769 Da, 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 5 mg 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 6h (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 ℃, detection 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 sequence 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, 1mol/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 5 mg 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: 3421cm -1 The absorption peak at the position is attributed to the stretching vibration of-OH; 2928 cm -1 Where (a)The absorption peak 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 , 1084cm -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: rhizoma Gastrodiae crude polysaccharide RGCP-3 antioxidant activity research
1. DPPH radical scavenging Capacity determination
Preparing 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL of gastrodia elata polysaccharide RGCP-2 and gastrodia elata polysaccharide RGCP-3 solution respectively by deionized water, taking 500 mu L of the gastrodia elata polysaccharide RGCP-2 and gastrodia elata polysaccharide RGCP-3 solution, placing the 500 mu L of the gastrodia elata polysaccharide RGCP-2 and the gastrodia elata polysaccharide RGCP-3 in a 2 mL centrifuge tube, adding 500 mu L of DPPH solution (4 mg of DPPH is dissolved in 100 mL absolute ethyl alcohol) under the condition of avoiding light, carrying out vortex mixing, placing the mixture for 30 min at room temperature and avoiding light, centrifuging the mixture at 5000 rpm for 10 min, taking supernatant liquid to measure absorbance at 517 nm, taking ascorbic acid (Vc) +DPPH solution as a positive control, and taking deionized water+DPPH solution as a blank control; each sample is independently tested for 3 times under the same condition so as to ensure the repeatability of the test; DPPH radical scavenging was calculated as follows:
DPPH radical clearance (%) =Wherein A is 1 Absorbance of 500 μl sample solution or vc+500 μl LDPPH solution; a is that 2 Absorbance of 500 μl sample solution+500 μl absolute ethanol; a is that 0 Absorbance of 500 μl deionized water+500 μl DPPH solution;
as shown in FIG. 14, with increasing concentration of RGCP-2 and RGCP-3, the scavenging rate of DPPH free radicals increases, and at concentration of 5 mg/mL, the scavenging ability of RGCP-2 and RGCP-3 reaches the highest, namely 78.10 + -3.99%, 96.10 + -0.77%, respectively, and the result shows that RGCP-3 has higher scavenging ability of DPPH free radicals.
2. Determination of the scavenging ability of hydroxyl radicals
Preparing rhizoma Gastrodiae polysaccharide RGCP-2 and RGCP-3 solution with concentration of 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL respectively with deionized water, placing 500 μl into 5 mL centrifuge tube, sequentially adding 6 mmol/L FeSO 4 500. Mu.L of solution, 500. Mu.L of 6 mmol/L salicylic acid solution (0.083 g salicylic acid in 100 mL absolute ethanol) and finally 8 mmol/L H of solution were added 2 O 2 500. Mu.L of the solution was used to start the reaction, heated in a 37℃water bath for 60 min, absorbance was measured at 510 and nm, and V was measured at the same concentration C As a positive control, deionized water was used as a blank, and each sample was independently tested 3 times under the same conditions to ensure the repeatability of the test, and the hydroxyl radical clearance was calculated as follows:
hydroxyl radical clearance (%) =；
Wherein A is 1 500. Mu.L of sample solution or Vc+500. Mu.L of FeSO 4 Solution +500. Mu.L salicylic acid solution +500. Mu. L H 2 O 2 Absorbance of the solution; a is that 2 500. Mu.L of sample solution+500. Mu.L of FeSO 4 Solution +500. Mu.L absolute ethanol +500. Mu. L H 2 O 2 Absorbance of the solution; a is that 0 500. Mu.L deionized water+500. Mu.L FeSO 4 Solution +500. Mu.L salicylic acid solution +500. Mu. L H 2 O 2 Absorbance of the solution;
as shown in FIG. 15, as the concentration of the gastrodia elata polysaccharides RGCP-2 and RGCP-3 increases, the hydroxyl radical scavenging rate increases, and in the concentration range of 1-5 mg/mL, the hydroxyl radical scavenging rate of RGCP-2 gradually increases, and the hydroxyl radical scavenging rate of RGCP-3 increases sharply and finally approaches Vc.
3. Total reducing force measurement
Gastrodia elata polysaccharide RGCP-2 and RGCP-3 solutions with the concentration of 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL and 5 mg/mL are respectively prepared, and 500 mu L is taken and placed in a 5 mL centrifuge tube. Adding 500 mu L of 0.2 mol/L phosphate buffer (pH 6.6) and 500 mu L of 1% potassium ferricyanide solution, uniformly mixing, preserving heat at 50 ℃ for 20 min, adding 500 mu L of 10% trichloroacetic acid, fully uniformly mixing, centrifuging at 3000 rpm for 10 min, taking supernatant 1 mL, adding 1 mL% 0.1% ferric trichloride, and detecting absorbance at 700 nm wavelength; the absorbance obtained by detection is the total reducing force; each sample was independently tested 3 times under the same conditions to ensure test reproducibility.
As a result, as shown in FIG. 16, the polysaccharide samples RGCP-2 and RGCP-3 were in dose-response in the concentration range of 1-5 mg/mL, and the polysaccharide concentration increased and the reducing power increased. When the concentration is 5 mg/mL, the reduction capability of RGCP-2 and RGCP-3 reaches the highest, the corresponding absorbance is 0.704+/-0.011,0.749 +/-0.002 respectively, and the reduction capability of RGCP-3 is stronger than that of RGCP-2 in the measurement range.
4. Gastrodia elata polysaccharide pair H 2 O 2 Protective effect for inducing oxidative damage of RAW264.7 cells
RAW264.7 cells were cultured in 24. 24 h, then all the medium was carefully removed, RAW264.7 cells in logarithmic growth phase were taken, counted and inoculated with 4X 10 cells 3 ~5×10 3 Each well was performed in a 96-well plate. In incubator (37 ℃, 5% CO) 2 ) Medium culture 4H, removing supernatant, H 2 O 2 Model group was added with 100. Mu.L of H at a final concentration of 100. Mu. Mol/L 2 O 2 The administration was separately added with 100. Mu.L of polysaccharide RGCP-2 and RGCP-3 at different concentrations (12.5, 25, 50, 100, 200. Mu.g/mL) and incubated for 4. 4H, followed by H 2 O 2 (final concentration 100. Mu. Mol/L) treatment 20 h, control group (Control group) did not perform any treatmentAdding an equal volume of serum-free DMEM medium; 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 Incubating in an incubator at 37 ℃ for 4 h, stopping incubation, carefully removing the supernatant, avoiding touching the bottom of the pore plate, adding 150 mu L DMSO into each pore, and oscillating for 10 min at a low speed on a shaking table to fully dissolve the crystals; the absorbance of each well was measured at OD490 nm in an enzyme-linked immunosorbent assay, normalized with 100% of the blank, and the relative cell activity (%) of each well was calculated. Each concentration was performed 3 times in biological parallel.
The results are shown in FIG. 17, H compared with the blank group 2 O 2 After induction of oxidative damage to RAW264.7 cells, cell activity was significantly reduced (P<0.0001). And H is 2 O 2 Compared with the model group, after the gastrodia polysaccharide RGCP-3 is dried, the cell survival rate is improved along with the increase of the concentration of the gastrodia polysaccharide RGCP-3, and the cell survival rate is obviously improved when the concentration is 25 mug/mL (P<0.01 A) is provided; at concentrations of 50, 100, 200 μg/mL, cell viability was significantly improved (P<0.0001 A) is provided; and gastrodia polysaccharide RGCP-3 is opposite to H 2 O 2 The induced oxidative damage of RAW264.7 cells was concentration dependent, whereas RGCP-2 was not shown to increase cell viability.
Claims (2)
1. Polysaccharide RGCP-3 with antioxidant activity is derived from rhizoma Gastrodiae, and has molecular weight of 32769 Da at peak position, weight average molecular weight of 39991 Da, and number average molecular weight of 27487 Da, and is homogeneous polysaccharide;
the gastrodia elata polysaccharide RGCP-3 is alpha-configuration pyranose, and consists of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose in the molar ratio of 0.254%, 0.316%, 84.695%, 0.102%, 13.355% and 0.462%, and has no triple helix structure.
2. The polysaccharide RGCP-3 having antioxidant activity as claimed in claim 1, wherein: has DPPH free radical scavenging ability, hydroxyl free radical scavenging ability and reducing ability, forH 2 O 2 The induced oxidative damage of RAW264.7 cells has a protective effect.
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| CN102464725A (en) * | 2010-11-17 | 2012-05-23 | 中国科学院上海药物研究所 | Gastrodia elata BL. polysaccharide and degradation products thereof and preparation methods and applications of degradation products |
| CN114805629A (en) * | 2021-01-29 | 2022-07-29 | 中国科学院上海药物研究所 | Gastrodia elata homogeneous polysaccharide and preparation method and application thereof |
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| CN102464725A (en) * | 2010-11-17 | 2012-05-23 | 中国科学院上海药物研究所 | Gastrodia elata BL. polysaccharide and degradation products thereof and preparation methods and applications of degradation products |
| CN114805629A (en) * | 2021-01-29 | 2022-07-29 | 中国科学院上海药物研究所 | Gastrodia elata homogeneous polysaccharide and preparation method and application thereof |
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