CN115558035B - Gastrodia polysaccharide with immunoregulatory activity - Google Patents

Abstract

The invention discloses a Gastrodia polysaccharide with immunomodulatory activity, in which the peak molecular weight of Gastrodia polysaccharide RGCP-2 is 5215 Da, the weight average molecular weight is 5631 Da, and the number average molecular weight is 4472 Da; 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. Preliminary analysis of the structures of Gastrodia polysaccharides RGCP‑2 and RGCP‑3 was carried out by UV spectrometry, high performance liquid chromatography and infrared spectroscopy. The results showed that Gastrodia elata polysaccharides RGCP‑2 and RGCP‑3 The polysaccharides RGCP‑2 and RGCP‑3 are α-configuration pyranose and do not have a triple helix structure. Gastrodia polysaccharides RGCP‑2 and RGCP‑3 can inhibit the release of NO by mouse macrophages RAW264.7 and simultaneously inhibit LPS-induced RAW264. 7 Intracellular ROS production, gastrodia polysaccharides RGCP‑2 and RGCP‑3 can be used as important raw materials for the development of immunomodulatory drugs and functional foods.

Description

Gastrodia polysaccharide with immunoregulatory activity

Technical field

The invention belongs to the technical field of preparation of natural product active ingredients, and specifically relates to a gastrodia polysaccharide with immunomodulatory activity.

Background technique

Gastrodia elata, also known as red arrow, Dingfengcao, Duyaolan, Limu, and Helicao, is the dried tuber of the orchid plant Gastrodia elata Bl. Gastrodia elata is a perennial symbiotic plant that must live in symbiosis with the fungus Myraceae. Gastrodia elata is mainly produced in Shaanxi, Yunnan, Guizhou, Sichuan and other places, and is a valuable Chinese medicinal material in my country. Traditional Chinese medicine in my country believes that Gastrodia elata has the effects of calming the liver, calming wind, and calming sleep.

Immune stimulation is considered one of the important strategies to improve the body's defense mechanism in the elderly and cancer patients. Immunostimulating polysaccharides are a relatively new class of bioactive compounds that can enhance the body's natural resistance to viral and bacterial infections, or assist Treats diseases that weaken the immune system. However, drugs that improve human immune function will also bring many side effects, such as drug resistance, drug dependence, teratogenicity and carcinogenesis, etc. while regulating immune activity. Therefore, finding a natural, highly efficient, non-toxic immune modulator is a current hot research direction. As people's research on natural active substances gradually deepens, polysaccharides have attracted more and more attention from researchers. In recent years, astragalus polysaccharides, maca polysaccharides, etc. have been proven to have good immunomodulatory effects.

Polysaccharides are a type of natural polymer compounds that are essential components of all living organisms and are closely related to maintaining biological functions. Current research on Gastrodia elata mainly focuses on gastrodin, phenols and glycosides. Gastrodia polysaccharide is also an important active ingredient in Gastrodia elata. It is the most abundant substance besides gastrodin, p-hydroxybenzyl alcohol and other medicinal ingredients. Therefore, isolating the polysaccharides in Gastrodia elata and discovering its active ingredients is of great significance for the in-depth development of Gastrodia elata medicinal materials, protecting people's life and health, and promoting industrial development.

Contents of the invention

The invention provides a polysaccharide extracted and separated from Gastrodia elata. The polysaccharide is a white or light yellow flocculent powder, which is RGCP-2 or RGCP-3. The peak molecular weight of the polysaccharide RGCP-2 is 5215 Da and the weight average molecular weight is 5631. Da, the number average molecular weight is 4472 Da, the peak molecular weight of polysaccharide 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 of the present invention has immunomodulatory activity. When the concentration of the polysaccharides RGCP-2 and RGCP-3 is 12.5-50 μg/mL, it can significantly inhibit the release of NO by mouse macrophages RAW264.7 and simultaneously inhibit the LPS-induced RAW264.7 Intracellular ROS production.

The object of the present invention is achieved through the following technical solutions:

(1) Clean the fresh Gastrodia elata, steam until the core is clear and there is no white core, place it in an oven at 40-50 ℃ to dry, and then crush it after drying;

(2) According to the ratio of mass to volume ratio g:mL=1:15-25, add distilled water to the Gastrodia elata powder in step (2), extract in a 60°C water bath, centrifuge, and extract the filter residue 2-4 times repeatedly, and collect Combine the filtrate, and concentrate the filtrate under reduced pressure to 1/6 of the original volume;

(3) Add 3-5 times the volume of absolute ethanol to the concentrated solution in step (2), let it stand at 4°C, filter, collect the precipitate, and place the precipitate in a 37°C oven to evaporate the ethanol. Dry to obtain the crude extract of gastrodia polysaccharide;

(4) Add distilled water to the crude extract of Gastrodia polysaccharide, incubate in a 60°C water bath for 2 hours, centrifuge, remove insoluble matter, and retain the supernatant. Repeat this step twice;

(5) Take the supernatant obtained in step (4), add sevage reagent (chloroform: n-butanol = 4:1) to remove protein, place it in a shaker and shake vigorously, take it out and pour it into a separatory funnel, and let it stand for separation. layer, discard the middle phase and the lower phase, and continue to add Sevege reagent to the upper phase to remove protein. If there is white substance on the liquid surface between the two phases, continue to add Sevege reagent to remove protein. If there is no white substance, it means that the protein has been basically removed;

(6) Transfer the protein-removed sugar solution to a dialysis bag, replace it with distilled water every 6 hours and dialyze for 2-3 days;

(7) Place the dialyzed sugar solution at -80°C and freeze-dry it to obtain Gastrodia elata crude polysaccharide GCP;

(8) Dissolve Gastrodia elata crude polysaccharide GCP in distilled water, load it on DEAE-52 cellulose chromatography column, use 0, 0.1, 0.2 mol/L NaCl solution for continuous gradient elution, and use an automatic fraction collector to collect the eluate , use the phenol-sulfuric acid method and a spectrophotometer to track and monitor the elution, draw an elution curve based on the absorbance value, collect the liquid corresponding to the elution peak, freeze-dry after dialysis, and obtain several polysaccharides;

(9) Load the polysaccharides obtained in step (8) onto a Sephadex G-50 dextran gel chromatography column for further purification, elute with 0.1 mol/L NaCl solution, and collect the eluate with an automatic fraction collector. The phenol-sulfuric acid method and spectrophotometer method were used to track and monitor the elution situation, draw a curve based on the absorbance value, collect the liquid corresponding to the elution peak, and freeze-dry it after dialysis to obtain the purified gastrodia polysaccharide.

Advantages and technical effects of the present invention:

(1) The polysaccharides RGCP-2 and RGCP-3 of the present invention are natural extracts, new polysaccharides extracted from Gastrodia elata, and have good safety; the peak molecular weight of polysaccharide RGCP-2 is 5215 Da, and the weight average molecular weight It is 5631 Da, the number average molecular weight is 4472 Da, the peak molecular weight of polysaccharide RGCP-3 is 32769 Da, the weight average molecular weight is 39991 Da, and the number average molecular weight is 27487 Da. They are all high purity and homogeneous polysaccharides;

(2) The gastrodia polysaccharides RGCP-2 and RGCP-3 of the present invention have good immunomodulatory activity and can be used as important raw materials for the development of immunomodulatory drugs and functional foods;

(3) The preparation process of the gastrodia polysaccharides RGCP-2 and RGCP-3 of the present invention is simple, the cost is low, and it is suitable for large-scale industrial production.

Description of the drawings

Figure 1 DEAE-52 anion exchange column chromatography elution curve of Gastrodia elata crude polysaccharide GCP;

Figure 2 Sephadex G-50 gel column chromatography elution curve of Gastrodia elata crude polysaccharide GCP-2;

Figure 3 Sephadex G-50 gel column chromatography elution curve of Gastrodia elata crude polysaccharide GCP-3;

Figure 4 Sephadex G-50 gel column chromatography elution curve of Gastrodia elata crude polysaccharide GCP-4;

Figure 5 UV spectra of Gastrodia polysaccharides RGCP-2, RGCP-3 and RGCP-4;

Figure 6 High performance gel permeation chromatogram of Gastrodia polysaccharide RGCP-2;

Figure 7 High performance gel permeation chromatogram of Gastrodia polysaccharide RGCP-3;

Figure 8 High performance gel permeation chromatogram of Gastrodia polysaccharide RGCP-4;

Figure 9 HPLC chart of standard monosaccharide mixture;

Figure 10 HPLC diagram of PMP derivatives of Gastrodia polysaccharide RGCP-2 and RGCP-3;

Figure 11 Congo red experimental diagram of Gastrodia polysaccharides RGCP-2 and RGCP-3;

Figure 12 Infrared spectrum of Gastrodia polysaccharide RGCP-2;

Figure 13 Infrared spectrum of Gastrodia polysaccharide RGCP-3;

Figure 14 Effects of Gastrodia polysaccharides RGCP-2 and RGCP-3 on RAW 264.7 cell proliferation;

Figure 15 Effects of Gastrodia polysaccharides RGCP-2 and RGCP-3 on LPS-induced NO production in RAW 264.7 cells;

Figure 16 Effects of Gastrodia polysaccharides RGCP-2 and RGCP-3 on ROS production in LPS-induced RAW 264.7 cells.

Detailed ways

The technical solutions of the present invention will be further described in detail through examples below, but the content of the present invention is not limited thereto. The methods in this example are all conventional methods unless otherwise specified. The materials, reagents, etc. used are all conventional methods unless otherwise specified. Obtained from commercial channels;

In the examples, the polysaccharide content was measured using the phenol-sulfuric acid method: take 0.5 mL of polysaccharide from each tube, then add 0.5 mL of distilled water, add 0.5 mL of freshly prepared 6% phenol solution, then add 2.5 mL of concentrated sulfuric acid, mix well and let stand; After the test tube is cooled, the absorbance is measured at 490 nm.

Example 1: Extraction, separation and purification of gastrodia polysaccharide

1. Clean the fresh Gastrodia elata, steam until the core is clear and no white core, place it in an oven at 40-50°C to dry, and then crush it after drying;

2. According to the mass volume ratio g:mL of 1:20, add 200 mL distilled water to 10 g of Gastrodia elata dry powder, extract in a 60°C water bath for 30 min, centrifuge at 4000 rpm for 10 min, add water to the filter residue for extraction, and repeat the extraction 3 times. Collect the combined filtrate and concentrate to about 100 mL using a rotary evaporator at 50°C;

3. Slowly add 400 mL of absolute ethanol to the concentrated solution and let it stand for 24 hours at 4°C to remove the pigment and precipitate the polysaccharide. Filter and collect the precipitate. The precipitate is dried in an oven at 37°C to obtain the crude gastrodia polysaccharide extract. thing;

4. Add distilled water to the crude extract of Gastrodia polysaccharide, incubate in a 60°C water bath for 2 hours, centrifuge, remove insoluble matter, and leave the supernatant. Add an appropriate amount of distilled water to the supernatant, incubate in a 60°C water bath for 2 hours, centrifuge, and add the supernatant. sevage reagent (chloroform: n-butanol = 4:1), place it in a shaker and shake vigorously at 200 rpm for 30 minutes. Take it out and pour it into a separatory funnel. Leave it to separate for about 30 minutes. Discard the middle phase and the lower phase. Continue to add Sevege reagent to the upper phase to remove protein, repeat 10 times. If there is white substance on the liquid surface between the two phases, continue to add Sevege reagent to remove protein. If there is no white substance, it means that the protein has been basically removed;

5. Transfer the protein-removed sugar solution to a 3500 Da dialysis bag, replace it with distilled water every 6 hours and dialyze for 2 days;

6. Divide the dialyzed sugar solution into packages, place it at -80°C, and obtain Gastrodia elata crude polysaccharide GCP by freeze-drying;

7. Dissolve the Gastrodia elata crude polysaccharide GCP in distilled water, load it on the DEAE-52 cellulose chromatography column, and sequentially elute with 0, 0.1, and 0.2 mol/L NaCl solution gradient, each concentration elutes 4 column volumes. Use an automatic partial collector to collect the eluate, use the phenol-sulfuric acid method and a spectrophotometer to track and monitor the elution, and draw an elution curve based on the absorbance value, as shown in Figure 1. Collect the liquid corresponding to the elution peak in the figure, and determine the molecular weight cutoff The 3500Da dialysis bag was dialyzed to remove salt, concentrated, and freeze-dried to obtain Gastrodia polysaccharides GCP-1, GCP-2, GCP-3, and GCP-4 (due to the low yield of GCP-1, subsequent research on GCP-2 and GCP- 3 and GCP-4);

8. Dissolve the gastrodia polysaccharides GCP-2, GCP-3 and GCP-4 in 0.1 mol/L NaCl solution respectively, load them on Sephadex G-50 dextran gel chromatography column for further purification, and use 0.1 mol/L NaCl solution. NaCl elutes 1 column volume, and uses an automatic partial collector to collect the eluate. Track and monitor the elution using the phenol-sulfuric acid method and the spectrophotometer method. Draw a curve based on the absorbance value, as shown in Figure 2-4. Collect the eluate in the figure respectively. For the liquid corresponding to the off-peak, the collected liquid was desalted by dialysis with a dialysis bag with a molecular weight cutoff of 3500Da, then concentrated and freeze-dried to obtain purified gastrodia polysaccharides RGCP-2, RGCP-3 and RGCP-4.

Example 2: Structural characterization of Gastrodia polysaccharide

1. Ultraviolet spectrum analysis

Prepare polysaccharide RGCP-2, RGCP-3 and RGCP-4 solutions with a mass concentration of 1.0 mg/mL, use distilled water as a blank control, and use a UV-visible spectrophotometer to perform a full-band scan at 800-200 nm;

Figure 5 is a full-band scan of the polysaccharide sample under UV-visible spectrophotometer 800-200 nm. From the scanning curve, it can be seen that there is no absorption at 260 nm, 280 nm, and 320 nm, indicating that after separation by DEAE-52 cellulose column, Polysaccharides do not contain nucleic acids and proteins.

2. Determination of molecular weight of polysaccharides

Chromatographic separation conditions: Chromatographic column: BRT105-104-102 series gel column (8×300 mm); mobile phase: 0.05mol/L NaCl solution; flow rate: 0.6 mL/min, column temperature: 40°C; injection volume: 20 μL; detector: differential detector RI-10A; accurately weigh polysaccharide samples and standards respectively;

Prepare 5 mg/mL solutions with dextran standards of 1152 Da, 5000 Da, 11600 Da, 23800 Da, 48600 Da, 80900 Da, 148000 Da, 273000 Da, 409800 Da, and 667800 Da respectively, and centrifuge at 12000 rpm for 10 minutes. The supernatant was filtered with a 0.22 μm microporous filter, and then the sample was transferred to a 1.8 mL injection vial, injected and analyzed by HPLC, and lgMp-RT (peak molecular weight) and lgMw-RT (weight average molecular weight) were plotted. , lgMn-RT (number average molecular weight) calibration curve:

The lgMp-RT calibration curve equation is: y = -0.1829x + 11.554, R ² = 0.9965;

The lgMw-RT calibration curve equation is: y = -0.1951x + 12.11, R ² = 0.996;

The lgMn-RT calibration curve equation is: y = -0.1807x + 11.393, R ² = 0.9928.

The polysaccharide sample was prepared into a 5 mg/mL solution, centrifuged at 12000 rpm for 10 min, the supernatant was filtered with a 0.22 μm microporous filter, and then the sample was transferred to a 1.8 mL injection vial for HPLC injection analysis;

The results are shown in the high-efficiency gel permeation chromatograms of Gastrodia polysaccharides RGCP-2, RGCP-3 and RGCP-4 in Figures 6, 7 and 8. The molecular weight of each sample was calculated according to the calibration curve equation. The calculation results are shown in the table below. The unit of molecular weight in the table is Da, the full name is Dalton;

It can be seen from the high-performance gel permeation chromatogram that Gastrodia polysaccharide RGCP-2 and RGCP-3 peak at 42.847 min and 38.483 min respectively and present a single peak (45.6 min is the peak of the mobile phase). The peak molecular weight of RGCP-2 is 5215 Da, the weight average molecular weight is 5631 Da, the number average molecular weight is 4472 Da, and the polydispersity index (Mw/Mn) is 1.259, indicating a narrow molecular weight distribution; the peak molecular weight of RGCP-3 is 32769 Da, and the weight average molecular weight is 39991 Da. The number average molecular weight is 27487 Da, and the polydispersity index (Mw/Mn) is 1.455, indicating a narrow molecular weight distribution; and the RGCP-2 and RGCP-3 content both reach 100%, reflecting the high purity and uniformity of the polysaccharide. Gastrodia polysaccharide RGCP-4 has peaks at 31.733, 34.56, 37.507 and 40.855 min respectively (45.6 min is the peak of the mobile phase), so RGCP-4 is not a homogeneous polysaccharide (only RGCP-2 and RGCP-3 will be studied in the future) .

3. Analysis of monosaccharide composition

(1) Pre-column derivatization of monosaccharide standards

Accurately weigh 0.01 mmol each of mannose Man, rhamnose Rha, glucuronic acid GlcA, galacturonic acid GalA, glucose Glc, galactose Gal, xylose Xyl, and arabinose Ara, and dissolve them in 8 mL of ammonia water, vortex Mix well, take 800 μL of the mixed sugar solution into a 5 mL centrifuge tube, and add 800 μL of PMP (0.5 mol/L), vortex to mix, react in a 70°C water bath for 30 minutes, take out and cool to room temperature, add 3 mL of water , place it in a 55 ℃ vacuum drying oven to evaporate to dryness, add water and repeat twice. After evaporation in the vacuum drying oven, add 1 mL of deionized water and 1 mL of chloroform into the centrifuge tube, and perform vortex extraction (the derivatized monosaccharides are dissolved in the water phase, and the residual PMP is dissolved in the chloroform phase, which can be performed at 3500 rpm Centrifuge for 5 minutes to accelerate stratification), discard the lower chloroform phase, continue to add 1 mL of chloroform for extraction several times, until the chloroform phase is colorless (the chloroform phase that has not removed PMP is yellow-green), transfer the upper aqueous phase to another In a centrifuge tube, centrifuge at 13,000 rpm for 5 minutes, take the supernatant, pass it through a 0.45 μm water filter, and perform liquid phase detection and analysis on the sample.

(2) Pre-column derivatization of polysaccharide samples

Accurately weigh 5 mg of polysaccharide into a 15 mL sealed tube, add 2 mL of TFA (4 mol/L), put it into the rotor, seal it with a lid, and place it in a 121 °C oil bath for stirring for 6 h (the polysaccharide is completely hydrolyzed and the solution Clear and transparent), after hydrolysis is completed, take out the sealed tube and cool it to room temperature. The polysaccharide hydrolyzate was transferred to a 5 mL centrifuge tube, placed in a 55°C vacuum drying oven to evaporate to remove TFA, then 2 mL of methanol was added, and 45°C vacuum drying oven was evaporated to remove residual TFA. Add methanol and repeat this step three times. Add 300 μL of concentrated ammonia water into the centrifuge tube, vortex, and dissolve the hydrolyzed sugar in the ammonia water. Then add 300 μL of PMP (0.5 mol/L) into the centrifuge tube, vortex to mix, and react in a water bath at 70°C for 30 minutes. Remove and cool to room temperature, add 3 mL of water to the centrifuge tube, vortex to mix, place in a 55°C vacuum drying oven to evaporate, repeat twice. After evaporation in the vacuum drying oven, add 1 mL of deionized water and 1 mL of chloroform into the centrifuge tube, perform vortex extraction, discard the lower chloroform phase, and continue to add 1 mL of chloroform for extraction several times until the chloroform phase becomes colorless. Transfer the upper aqueous phase to another centrifuge tube, centrifuge at 13,000 rpm for 5 minutes, take the supernatant, pass it through a 0.45 μm water-based filter membrane, and perform liquid phase detection and analysis on the sample.

(3) Liquid phase detection conditions

Column model: 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, detection Wavelength: 245 nm, injection volume: 20 μL.

The HPLC diagram of the PMP derivatives of Gastrodia polysaccharide RGCP-2 and RGCP-3 is shown in Figure 10. Compared with the HPLC diagram of the mixed monosaccharide standard in Figure 9, the results show that RGCP-2 is composed of mannose, rhamnose, glucuronic acid, It is composed of lacturonic acid, glucose, galactose, xylose and arabinose. According to the peak area calculation, the molar percentage of each monosaccharide is 0.902%, 0.458%, 0.422%, 0.564%, 82.258%, 0.920%, 13.930%. and 0.545%; RGCP-3 is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose. The molar percentage of each monosaccharide is calculated according to the peak area. are 0.254%, 0.316%, 0.269%, 0.547%, 84.695%, 0.102%, 13.355% and 0.462%.

4. Congo red test analysis

Weigh Gastrodia polysaccharides RGCP-2 and RGCP-3 respectively and prepare them into a polysaccharide solution with a concentration of 0.5 mg/mL. Take 2 mL of the sample and mix it with 2 mL of Congo red solution with a concentration of 50 μmol/L in equal volumes. After standing for a period of time, add 1 mol/L NaOH solution to 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, 0.50 respectively. mol/L, react at room temperature for 15 minutes, and scan with a UV-visible spectrometer (wavelength range: 600-400 nm). Determine the changes in the maximum absorption wavelength of the mixed solution under different concentrations of NaOH solutions, and conduct graph analysis with the NaOH concentration as the abscissa (x) and the maximum absorption wavelength as the ordinate (Y).

It can be seen from the results in Figure 11 that as the NaOH concentration increases, the maximum absorption wavelength of the complex formed by Gastrodia polysaccharides RGCP-2 and RGCP-3 and Congo red does not increase with the change of NaOH concentration, that is, no red shift occurs. , its change trend is basically the same as that of pure Congo red solution, that is, neither Gastrodia polysaccharide RGCP-2 nor RGCP-3 has a triple helix structure.

5. Infrared spectrum analysis

Weigh 5 mg each of polysaccharides RGCP-2 and RGCP-3, mix with KBr powder and press into tablets, and scan using a Fourier transform infrared spectrometer in the wavelength range of 4000-400 cm ^-1 .

The results are shown in Figures 12 and 13. The infrared spectrum analysis results of Gastrodia polysaccharides RGCP-2 and RGCP-3 are as follows: the absorption peak at 3421 cm ^-1 is attributed to the stretching vibration of -OH; the absorption peak at 2928 cm ^-1 Attributed to the asymmetric stretching vibration of CH; the absorption peak near 1637 cm ^-1 is generated by the stretching vibration of the -C=O bond; the CH change angle in -CH ₂ - or -CH- at the absorption peak at 1414 cm ^-1 The vibration and the CH stretching vibration absorption peak in -CH ₂ ^- or -CH- at 2928 cm -1 are the characteristic absorption peaks of polysaccharides; the absorption peak at 1381 cm ^-1 may be caused by the deformation vibration of the CH bond; in There are three characteristic absorption peaks of 1153 cm ^{-1 ,} 1084 cm ^-1 , and 1025 cm ^-1 in 1000-1200 cm -1, indicating that the sugar ring configuration in gastrodia polysaccharide is pyran type (furan type sugar ring has only Two characteristic absorption peaks); the absorption peak at 1025 cm ^-1 is the characteristic absorption of glucose, indicating that the main monosaccharide of the polysaccharide is glucose; the absorption peak at 928 cm ^-1 is the characteristic absorption of -COC, which is a typical D-pyran The characteristic absorption peak of glucose in the form of glucose; the absorption peak at 854 cm ^-1 indicates that the glycosidic bond type is mainly α-configuration; the characteristic absorption peaks of -NH ₂ and -NH ₃ at 1616 cm ^-1 are not found, indicating that it does not contain proteoglycans. Through comparison and analysis of FT-IR spectra, it can be seen that both Gastrodia polysaccharides RGCP-2 and RGCP-3 are α-configuration pyranose.

Example 3: Identification of immunomodulatory activity of Gastrodia polysaccharide

1. Effect of Gastrodia polysaccharide on RAW264.7 cell proliferation

Take 100 μL logarithmic phase cells and inoculate them into a 96-well plate at a cell density of 4×10 ³ to 5×10 ³ cells/well. Place them in a 5% CO ₂ and 37 ℃ incubator and incubate overnight until the cells adhere to the wall. , take 100 μL of RGCP-2 and RGCP-3 of different concentrations (12.5, 25, 50, 100, 200 μg/mL) to act on the cells for 24 h respectively, and set a blank control group (Control group) without adding gastrodia polysaccharide; discard After supernatant, wash once with sterile PBS. Add 110 μL of a mixed solution of MTT (5 mg/mL) and serum-free DMEM (volume ratio 2:9) to each well, place it in a 5% CO ₂ , 37 °C incubator and incubate for 4 h. Stop the incubation and carefully remove the supernatant. Clean without touching the bottom of the well plate. Add 150 µL DMSO to each well and shake on a shaker at low speed for 10 minutes to fully dissolve the crystals. The absorbance value of each well was measured at OD 490 nm of the enzyme-linked immunoassay instrument, and normalized with the blank control group as 100%. The relative cell activity (%) of each well was calculated, and three biological parallels were performed for each concentration.

The results are shown in Figure 14. It can be seen from the figure that as the concentration of gastrodia polysaccharide increases, the overall survival rate of RAW264.7 cells shows a basically unchanged and then declining trend. Compared with the blank control group, 12.5, 25, and 50 μg/mL gastrodia polysaccharides RGCP-2 and RGCP-3 had no significant effect on cell survival rate after acting on cells (P>0.05); with the increase of RGCP-2 and RGCP- 3 As the concentration increases, the cell survival rate decreases in the concentration range of 100-200 μg/mL (P<0.01).

2. Effect of Gastrodia polysaccharide on NO secretion in RAW264.7 cells

The classic Griess method was used to detect changes in NO content. The details are as follows: Take RAW264.7 cells in the logarithmic growth phase, add 5×10 ⁴ cells/well into a 96-well cell culture plate and culture for 24 h; add 10 μL of different concentrations ( Gastrodia polysaccharide (RGCP-2 and RGCP-3) at 12.5, 25, 50 μg/mL) and acted on the cells for 24 h respectively, then Gastrodia polysaccharide group and LPS induction group (LPS induced cells for 24 h, without adding RGCP-2 and RGCP- 3) Add 10 μL of 0.1 μg/mL LPS respectively and set up a blank control group (cells are cultured normally without any treatment). Each group is set up with 3 duplicate wells and placed in a 37°C, 5% CO ₂ incubator for 24 hours. , then take 60 μL of cell culture supernatant, add 50 μL of 1% sulfonamide, mix well, and incubate for 10 min; then add 50 μL of 0.1% N-1-naphthylethylenediamine hydrochloride, mix well, and incubate at room temperature. 10 min; use a microplate reader to record the absorbance value of the sample at OD540 nm.

The results are shown in Figure 15. The figure shows that compared with the blank control group, LPS stimulated the cells for 24 hours, which significantly increased the intracellular NO content (P<0.0001), indicating that cellular inflammation was aggravated; when RGCP-2 and RGCP-3 After treating cells at concentrations of 12.5, 25, and 50 μg/mL for 24 h, LPS-induced NO production in RAW264.7 cells was significantly inhibited (P <0.05), and the inhibitory effect of RGCP-3 was concentration-dependent; It shows that RGCP-2 and RGCP-3 (12.5, 25, 50 μg/mL) can inhibit the abnormal production of NO in RAW264.7 cells stimulated by LPS. The above results show that Gastrodia polysaccharide RGCP-2 and RGCP-3 can inhibit the abnormal production of NO in RAW264.7 cells stimulated by LPS to a certain extent. Inhibiting the production of NO in macrophages under inflammatory conditions, thereby inhibiting the occurrence and progression of inflammation in macrophages, can exert an anti-inflammatory and immunoregulatory effect on macrophages under inflammatory conditions.

3. Effect of Gastrodia polysaccharide on ROS production in RAW264.7 cells

RAW264.7 cells were seeded in a 12-well plate at a density of 1 × 10 ³ cells/well, and cultured overnight until they adhered. Then, LPS, RGCP-2, and RGCP-3 were added to each group according to the experimental design in step 2, and the cells were incubated for 24 hours. Afterwards, discard the culture medium, drop 400 μL of DCFH-DA (final concentration: 20 μmol/L) probe into each well, and incubate in a cell culture incubator at 37°C in the dark for 30 min; wash twice with PBS and rinse with paraformaldehyde. Fix for 10 minutes, wash twice with PBS again, stain with DAPI for 3-5 minutes, wash again with PBS three times, observe with an inverted fluorescence microscope, and take photos and records.

The results are shown in Figure 16. Compared with the blank control group, the DCFH-DA green fluorescence intensity of cells in the LPS group was significantly enhanced, indicating that LPS stimulated RAW264.7 cells, inducing the rapid production of intracellular ROS. Compared with the LPS group, Gastrodia polysaccharide RGCP The green fluorescence intensity of DCFH-DA in the -2 and RGCP-3 groups was significantly reduced, which shows that both Gastrodia polysaccharide RGCP-2 and RGCP-3 can inhibit the production of ROS in RAW264.7 cells induced by LPS.

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.