Dendrobium globosum neutral polysaccharide as well as preparation method and application thereof

Abstract

The invention provides Dendrobium spheruliforme neutral polysaccharide, a preparation method and application, the Dendrobium spheruliforme neutral polysaccharide has the following structure: # imgabs0. The Dendrobium spheruliforme neutral polysaccharide is extracted from Dendrobium spheruliforme for the first time, and the structure, molecular weight and components of the polysaccharide are analyzed. In addition, in-vitro cell experiments find that the polysaccharide can significantly improve the activity (plt; 0.01) in the GES-1 cells damaged by ethanol, and the level of ROS in the GES-1 cells damaged by ethanol and the level of inflammatory factors (TNF-alpha, IL-8 and IL-1beta) in the GES-1 cells (plt; 0.05), so that GES-1 cell oxidative damage caused by ethanol is remarkably reduced, and the dendrobium globosum neutral polysaccharide has an extremely good application prospect in being developed into a medicine for protecting gastric mucosal damage or a protective agent for reducing side effects of non-steroidal anti-inflammatory drugs.

Description

Technical Field

[0001] The invention relates to the technical field of extraction and application of traditional Chinese medicine components, and in particular to a neutral polysaccharide of Dendrobium sphaerocephalum, a preparation method and application thereof. Background Art

[0002] Inflammation and peptic ulcer caused by gastric mucosal damage are common global problems. Alcohol consumption, psychological stress, poor eating habits, smoking and excessive use of nonsteroidal anti-inflammatory drugs can lead to gastric mucosal damage. Among them, the increasing incidence of acute and chronic gastritis, gastric mucosal epithelial damage and gastric ulcer caused by drinking has become a serious health problem in the drinking culture. Therefore, finding safe and effective drugs to protect the gastric mucosa is an important task in the treatment of gastric damage.

[0003] Dendrobium, the medicinal use of which was first recorded in Shennong's Herbal Classic, has a long history of medicinal use and has the functions of promoting the production of body fluids and benefiting the stomach, nourishing yin and clearing away heat. my country is rich in Dendrobium resources, with a total of 76 species and 2 varieties of Dendrobium. Existing studies have found that Dendrobium plants are rich in polysaccharides, and most of these polysaccharides have strong biological activities, such as inhibiting inflammation, protecting the liver, anti-tumor, immunomodulation, and treating gastric diseases.

[0004] Dendrobium thyrsiflorum Rchb.f is one of the main sources of Chinese medicine Dendrobium. It can be used for viewing and is famous for its beautiful flowers and bright colors. Because Dendrobium thyrsiflorum combines medicinal and ornamental uses, it has received widespread attention in the pharmaceutical and horticultural industries. Research on the chemical components of Dendrobium thyrsiflorum has been reported at home and abroad. Currently, various types of compounds such as coumarins, phenanthrene, bibenzyl, fluorenone and flavonoids have been isolated from Dendrobium thyrsiflorum. It is currently reported that Dendrobium thyrsiflorum is also rich in polysaccharide compounds, but there are no reports on the identification of polysaccharides in Dendrobium thyrsiflorum and related applications. Summary of the invention

[0005] The present invention extracts and analyzes the polysaccharide in Dendrobium sphaerocephalum, and finally obtains a polysaccharide with a molecular weight of 9.18×10 3 The chemical structure of the polysaccharide with a molar ratio of Da, glucose and mannose of 0.415:0.585 was elucidated and its pharmacological activity in protecting the gastric mucosa was explored.

[0006] In order to achieve the above-mentioned object and other related objects, the present invention provides a neutral polysaccharide of Dendrobium sphaerocephalum in the first aspect, wherein the neutral polysaccharide of Dendrobium sphaerocephalum has the following structure:

[0007]

[0008] Preferably, the neutral polysaccharide of Dendrobium sphaerocephalum is composed of glucose and mannose in a molar ratio of 0.415:0.585, and the molecular weight is: Mw=9.18×103 Da.

[0009] The second aspect of the present invention provides a method for preparing the neutral polysaccharide of Dendrobium sphaerocephalum described in the first aspect, comprising:

[0010] Step S1, preparing crude polysaccharide of Dendrobium sphaerocephalum;

[0011] Step S2, separating and purifying the crude polysaccharide of Dendrobium sphaerocephalum, the purification process comprising: first purifying the crude polysaccharide by anion exchange resin, collecting the first eluate and concentrating it; dialysis of the concentrate and then purifying it by a dextran gel column, collecting the second eluate and freeze-drying it.

[0012] Preferably, the step S1 comprises:

[0013] Step S1-1, crushing and sieving the Dendrobium sphaerocephalum;

[0014] Step S1-2, performing ultrasonic extraction on the Dendrobium sphaerocephalum powder using a solvent, and collecting the extract;

[0015] Step S1-3, removing protein from the extract;

[0016] Step S1-4, reacting the protein-removed extract with ethanol to obtain crude polysaccharides from Dendrobium sphaerocephalum.

[0017] Further preferably, the step S1-2 comprises:

[0018] Step S1-2-1, extracting the Dendrobium spherical powder by heating at 50-56° C. with acetone for 1-2 hours, and collecting the powder;

[0019] Step S1-2-2, heating the powder with deionized water at 95-100°C for 2-3 hours, separating the liquid, which is the extract; more preferably, extracting three times;

[0020] More preferably, the extraction is carried out by ultrasonic extraction with an ultrasonic frequency of 200-400W.

[0021] Further preferably, in step S1-3, sevage reagent is used to remove proteins in the extract.

[0022] Further preferably, in step S1-4, the volume proportion of ethanol in the system is not less than 90%.

[0023] Preferably, in step S2, the crude polysaccharide is purified by an anion exchange resin, using DEAE-cellulose anion exchange resin, adjusting the flow rate to 15 ml / min, eluting with deionized water and NaCl solutions of different concentrations, collecting the deionized water eluate and concentrating it; and / or,

[0024] The concentrate was dialyzed using a 3500 Da dialysis bag.

[0025] The third aspect of the present invention provides the use of the neutral polysaccharide of Dendrobium sphaerocephalum described in the first aspect in preparing a gastric mucosal repair drug.

[0026] The fourth aspect of the present invention provides the use of the neutral polysaccharide of Dendrobium sphaerocephalum described in the first aspect in the preparation of a protective agent for reducing the side effects of non-steroidal anti-inflammatory drugs.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention extracts a neutral polysaccharide from Dendrobium officinale for the first time, and analyzes the structure, molecular weight and composition of the polysaccharide. In addition, the present invention also finds through in vitro cell experiments that the polysaccharide can significantly improve the vitality of GES-1 cells damaged by ethanol (p<0.01), and significantly reduce the levels of ROS in GES-1 cells damaged by ethanol and inflammatory factors (TNF-α, IL-8 and IL-1β) in GES-1 cells (p<0.05), thereby significantly reducing the oxidative damage of GES-1 cells caused by ethanol. Therefore, the neutral polysaccharide of Dendrobium officinale has excellent application prospects in developing drugs for protecting gastric mucosal damage or protective agents for reducing the side effects of non-steroidal anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is an ion chromatography (IC) analysis of the monosaccharide composition in the neutral polysaccharide of Dendrobium sphaerocephalum of the present invention.

[0030] Figure 2 The neutral polysaccharide of Dendrobium sphericalensis of the present invention 1 H NMR spectrum.

[0031] Figure 3 The neutral polysaccharide of Dendrobium sphericalensis of the present invention 13 C NMR spectrum.

[0032] Figure 4 The HH-COSY spectrum of the neutral polysaccharide of Dendrobium sphaerocephalum of the present invention.

[0033] Figure 5 It is the HSQC two-dimensional spectrum of the neutral polysaccharide of Dendrobium sphaerocephalum of the present invention.

[0034] Figure 6 It is the HMBC two-dimensional spectrum of the neutral polysaccharide of Dendrobium sphaerocephalum of the present invention.

[0035] Figure 7 It is the NOESY two-dimensional spectrum of the neutral polysaccharide of Dendrobium sphaerocephalum of the present invention.

[0036] Figure 8The present invention shows the influence of the neutral polysaccharide of Dendrobium sphaerocephalum on the activity of GES-1 cells.

[0037] Fig. 9 The invention discloses the influence of the neutral polysaccharide of Dendrobium sphaerocephalum on the inflammatory factors of GES-1 cells.

[0038] Fig.10 The present invention shows the influence of the neutral polysaccharide of Dendrobium sphaerocephalum on ROS of GES-1 cells. DETAILED DESCRIPTION

[0039] In the examples of the present invention, if no specific conditions are specified, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0040] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention but not to limit the scope of protection of the present invention.

[0041] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0042] Example 1

[0043] Preparation of crude polysaccharide from Dendrobium sphaerocephalum

[0044] Wash and dry the Dendrobium officinale to constant weight, crush it and pass it through a 60-mesh sieve. Take the powder and weigh it accurately. Extract it with acetone by micro-boiling ultrasonic method for 1.5 hours, filter it and discard the filtrate. Extract the residue with deionized water by micro-boiling ultrasonic method for 2.5 hours, filter it and discard the filtrate. Repeat the extraction three times and combine the extracts. Add an equal volume of sevage reagent (n-butanol: chloroform = 1:4), extract, collect the supernatant, repeat the extraction to remove the protein. Accurately measure the extract, add anhydrous ethanol dropwise under ultrasonic vibration to make the ethanol volume in the system reach 90%, filter and collect the precipitate to obtain crude polysaccharides of Dendrobium officinale. Through this process, the extraction rate of crude polysaccharides of Dendrobium officinale is 19.8%.

[0045] Example 2

[0046] Isolation and purification of crude polysaccharides

[0047] The crude polysaccharide was separated by DEAE-cellulose anion exchange resin. The crude polysaccharide was dissolved in deionized water, heated, vortexed, and centrifuged at 12000rpm. The supernatant was sampled. The flow rate of deionized water was adjusted to 15ml / min for elution. The deionized water eluate was collected, concentrated, dialyzed with a 3500Da dialysis bag, and further purified with a Sephadex-50 column. The deionized water eluate was collected and freeze-dried to obtain neutral polysaccharides from Dendrobium sphaerocephalum. The molecular weight of the polysaccharide was Mw=9.18×10 3 Da, polysaccharide purity is 99.8%.

[0048] Example 3

[0049] Ion chromatography analysis of monosaccharide composition of neutral polysaccharides from Dendrobium sphaerocephalum

[0050] Take appropriate amounts of 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid, mannuronic acid) and add 2 mL of 3M TFA, hydrolyze at 120 ° C for 3 h, blow dry with nitrogen, add deionized water and vortex to mix, and prepare standard mother solution. Take each monosaccharide standard solution to accurately prepare concentration standards as mixed standards. According to the absolute quantitative method, the mass of different monosaccharides is determined, and the molar ratio is calculated according to the molar mass of the monosaccharide. Accurately weigh 5 mg of polysaccharide sample and place it in an ampoule, add 2 mL of 3M TFA, and hydrolyze at 120 ° C for 3 h. Accurately transfer the acid hydrolysis solution to a tube and blow dry with nitrogen, add 5 mL of water and vortex to mix, take 50 μL and add 950 μL of deionized water, and centrifuge at 12000 rpm for 5 min. The supernatant was taken for ion chromatography (IC) analysis. The results showed that the polysaccharide was mainly composed of glucose and mannose in a molar ratio of 0.415:0.585 ( Figure 1 ).

[0051] Example 4

[0052] Methylation analysis of polysaccharides

[0053] Weigh the polysaccharide sample (2-3 mg) and place it in a glass reaction bottle. Add 1 mL of anhydrous DMSO, quickly add methylation reagent A solution, seal, dissolve under ultrasound, and then add methylation reagent B solution. React in a magnetic stirring water bath at 30°C for 60 minutes. Finally, add 2 mL of ultrapure water to the above mixture to terminate the methylation reaction.

[0054] Take the methylated polysaccharide, add 1ml of 2M trifluoroacetic acid (TFA) to hydrolyze for 90min, and evaporate to dryness on a rotary evaporator. Add 2ml of double distilled water to the residue, reduce it with 60mg of sodium borohydride for 8 hours, add glacial acetic acid to neutralize, rotary evaporate, dry in an oven at 101 degrees, then add 1ml of acetic anhydride to acetylate at 100℃ for 1h, and cool. Then add 3mL of toluene, concentrate and evaporate to dryness under reduced pressure, and repeat 4-5 times to remove excess acetic anhydride.

[0055] The acetylated product was washed with 3 mL of CH 2 Cl 2 After dissolving, transfer to a separatory funnel, add a small amount of distilled water and shake thoroughly, then remove the upper aqueous solution, and repeat this process 4 times. 2 Cl 2 The layer was dried with an appropriate amount of anhydrous sodium sulfate, concentrated to 1 mL, and placed in a liquid phase vial. The acetylation product samples were analyzed using a gas chromatography-mass spectrometer. The results are shown in Table 1.

[0056] Table 1 Results of polysaccharide methylation analysis

[0057]

[0058] Example 5

[0059] NMR analysis of polysaccharide structure

[0060] Heavy water (D 2 O, 99.9%), deuterated acetone as internal reference; freeze dryer, nuclear magnetic resonance instrument Bruker 600M (Nuclear Magnetic Resonance, NMR); weigh 50 mg of polysaccharide sample, dissolve it in 0.5 ml of heavy water and freeze dry it. Then dissolve the freeze-dried powder in 0.5 ml of heavy water again, continue freeze drying, and repeat the above process to fully exchange active hydrogen. Then dissolve the sample in 0.5 ml of heavy water and place it in a 600 MHz nuclear magnetic resonance instrument at room temperature 25 ° C. 1 HNMR spectrum, 13 C NMR spectrum, DEPT135 one-dimensional spectrum and two-dimensional spectrum ( Figure 2 to Figure 7 ).

[0061] like Figure 2 As shown, the hydrogen spectrum signals are mainly concentrated between 3.0 and 5.5 ppm. δ3.2-4.0 ppm is the sugar ring proton signal, and the main terminal proton peaks δ5.31, 5.26, 5.15, 4.91, 4.88, 4.67, 4.66, and 4.57 are concentrated in the 4.3-5.5 ppm region.

[0062] like Figure 3 As shown, carbon spectrum analysis was performed on 13C NMR (201 MHz, D2 O): The NMR carbon spectrum signals are mainly concentrated between 60-120ppm. By observing the carbon spectrum, we can see that the main anomeric carbon signal peaks are δ101.49, 101.39, 101.36, 100.89, 99.87, 99.10, and the anomeric carbon region is mainly between δ93 and 105. The main signal peaks of δ71.39, 72.81, 77.80, 76.20, 62.20, 72.89, 71.30, 77.60, 76.10, 62.42, 71.94, 74.03, 70.61, 71.68, 62.65, 72.91, 74.56, 78.31, 72.53, 61.89, 72.86, 74.02, 77.10, 70.69, 68.46, 72.73, 74.63, 70.87, 71.47, 66.80 are distributed in the 60-85ppm area. According to the monosaccharide composition results, the polysaccharide is composed of mannose and glucose, indicating that the polysaccharide is mainly mannoglucan. Dept135 spectrum analysis showed that the peaks at 62.20, 62.42, 62.65, 61.89, 68.46, and 66.80 ppm were inverted peaks, indicating chemical shifts of C6.

[0063] like Figure 5 As shown, through the HSQC spectrum, it can be observed that the anomeric carbon signal is δ100.89, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ5.31, such as Figure 4 As shown, by HH-COSY, the signal of H1-2 is 5.31 / 3.55; the signal of H2-3 is 3.55 / 3.90; the signal of H3-4 is 3.90 / 3.58; we can infer that H1, H2, H3, H4 are δ5.31, 3.55, 3.90, 3.58 respectively, and as Figure 7 As shown, the NOESY spectrum observed that δ5.31 has correlation peaks with 3.58, 3.78, and 3.90, and H5 is 3.78ppm. The corresponding C5 is 72.53; the chemical shift of C6 is δ61.89, and the corresponding H6a is δ3.78. Therefore, this signal should be attributed to the glycosidic bond →4)-α-Glcp-(1→.

[0064] like Figure 6As shown, in the HMBC spectrum, according to the one-dimensional and two-dimensional NMR spectra, we attribute the glycosidic bond signals of the polysaccharide; the anomeric hydrogen of the glycosidic bond →4)-β-D-Manp-(1→ has a related signal peak with its own C4, indicating the existence of a linking mode of →4)-β-D-Manp-(1→4)-β-D-Manp-(1→. The anomeric hydrogen of the glycosidic bond →4)-β-D-Manp-(1→ has a related signal peak with its own C4, indicating the existence of a linking mode of →4)-β-D-Manp-(1→4)-α-D-Glcp-(1→.

[0065] In addition, the anomeric hydrogen of →4)-α-D-Glcp-(1→ has a correlation peak with the C4 of the glycosidic bond →4,6)-α-D-Glcp-(1→, indicating the presence of a glycosidic bond →4)-α-D-Glcp-(1→4,6)-α-D-Glcp-(1→. In addition, the anomeric hydrogen of →4,6)-α-D-Glcp-(1→ has a correlation peak with the C4 of the glycosidic bond →4)-α-D-Glcp-(1→, indicating the presence of a glycosidic bond →4,6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→.

[0066] In the NOESY spectrum, there are correlation peaks in H6 of α-D-Glcp-(1→) and →4,6)-α-D-Glcp-(1→, indicating the existence of glycosidic bond α-D-Glcp-(1→4,6)-α-D-Glcp-(1→.

[0067] In summary, we can infer that the main glycosidic bond structure of the polysaccharide is: the main chain connection mode is the glycosidic bond of →4)-β-D-Manp-(1→4)-α-D-Glcp-(1→, and the terminal α-D-Glcp-(1→) is connected to the main chain through the O-6 bond of →4,6)-α-D-Glcp-(1→. The structural formula is as follows:

[0068]

[0069] Example 6

[0070] Application of neutral polysaccharides from Dendrobium sphaerocephalum in protecting gastric mucosal cells

[0071] A control group (normal GES-1 cell group), a model group (ethanol EtOH treatment), a low-concentration group of Dendrobium sphaerocephalum polysaccharide (50 μg / mL+EtOH, specifically: polysaccharide and ethanol were mixed, the final concentration of polysaccharide was 50 μg / mL, and the operation of other polysaccharide groups was the same), a medium-concentration group of polysaccharide (100 μg / mL+EtOH), and a high-concentration group of polysaccharide (200 μg / mL+EtOH) were set up. A 96-well plate was taken, cells were digested and counted routinely, and 5.0×10 4Cells were plated in 96-well plates, 100 μL per well. After 8 hours of culture, the supernatant was discarded, 20 μL CCK-8 solution was added to each well, and culture was continued for 4 hours. The culture medium was discarded, 150 μL DMSO was added, and the plate was shaken for 10 minutes. After 1 minute of rest, the 96-well plate was placed in an ELISA reader, which collected the absorbance values ​​of each well at a wavelength of 490 nm and calculated the activity of each group through a series of formulas (such as Figure 8 ELISA was used to analyze the concentrations of IL-8, TNF-α, and IL-1β in the supernatant of GES-1 cells in each group (as shown). Fig. 9 DCFH-DA method was used to analyze the ROS in each group of cells (as shown in Fig.10 shown).

[0072] Figure 8 The results showed that compared with the control group that was not damaged by ethanol, the model group could significantly reduce the viability of GES-1 cells (p<0.05). Compared with the model group, each treatment group of Dendrobium sphaerocephalum polysaccharide could improve the viability of damaged GES-1 cells to varying degrees, and showed concentration dependence. Among them, at a high concentration (200μg / mL), the polysaccharide could significantly improve the viability of damaged GES-1 cells compared with the model group (p<0.01), an increase of 112.7%, and there was no significant difference compared with the control group. This shows that 200μg / mL of neutral polysaccharides from Dendrobium sphaerocephalum can significantly reduce the damage of ethanol to GES-1 cells and significantly improve their activity.

[0073] at the same time, Fig. 9 The results showed that the level of inflammatory factors in the Model group (ethanol treatment) was significantly higher than that in the control group (not treated with ethanol) (P<0.01), indicating that ethanol treatment would cause GES-1 cell damage, thereby increasing the secretion of IL-8, TNF-α and IL-1β cytokines by inflammatory cells. Compared with the model group, the group treated with Dendrobium sphaerocephalum polysaccharide could reduce the level of inflammatory factors to varying degrees. Especially at high concentrations (200μg / mL), polysaccharides could significantly reduce the levels of IL-8 (P<0.01), TNF-α (P<0.01), and IL-1β (P<0.05) in damaged cells, reducing them by 53.3%, 65.07%, and 62.06%, respectively. This showed that polysaccharides at a concentration of 200μg / mL could inhibit the expression of gastric mucosal inflammatory factors, thereby reducing the further damage of these inflammatory cytokines to the gastric mucosa, which was beneficial to promote the repair of the gastric mucosa.

[0074] Fig.10The results showed that the concentration of ROS in GES-1 cells after ethanol treatment increased significantly compared with the control group (p<0.05), and each treatment group of Dendrobium sphaerocephalum polysaccharide could reduce the ROS content in cells, and showed concentration dependence. At medium concentration (100μg / mL) and high concentration (200μg / mL), compared with the model group, the polysaccharide could significantly reduce the ROS content in damaged cells (p<0.05), reducing by 41.06% and 78.59%, respectively. This shows that Dendrobium sphaerocephalum polysaccharide can effectively reduce ROS in GES-1 cells damaged by ethanol, thereby alleviating the degree of damage to GES-1 cells.

[0075] In summary, in the in vitro test of repairing damaged gastric mucosal cells, neutral polysaccharides from Dendrobium sphaerocephalum performed well. The results showed that neutral polysaccharides from Dendrobium sphaerocephalum at a concentration of 200 μg / mL can significantly increase the viability of GES-1 cells damaged by ethanol (p<0.01), and significantly reduce the levels of ROS and inflammatory factors in GES-1 cells damaged by ethanol (p<0.05), thereby significantly reducing the oxidative damage of GES-1 cells caused by ethanol. Therefore, it can be speculated that neutral polysaccharides from Dendrobium sphaerocephalum may be a potential drug for protecting gastric mucosal damage and reducing the side effects of non-steroidal anti-inflammatory drugs on gastric damage.

[0076] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A neutral polysaccharide from Dendrobium sphaerocephalum, characterized in that: The neutral polysaccharide of Dendrobium sphaerocephalum has the following structure:

2. The detection method according to claim 1, characterized in that: The neutral polysaccharide of Dendrobium sphaerocephalum is composed of glucose and mannose in a molar ratio of 0.415:0.585, and the molecular weight is: Mw=9.18×10 3 Da.

3. The method for preparing neutral polysaccharides from Dendrobium sphaerocephalum according to claim 1 or 2, characterized in that: include: Step S1, preparing crude polysaccharide of Dendrobium sphaerocephalum; Step S2, separating and purifying the crude polysaccharide of Dendrobium sphaerocephalum, wherein the purification process comprises: firstly purifying the crude polysaccharide by anion exchange resin, collecting the first eluate and concentrating it; The concentrate was dialyzed and purified by passing through a Sephadex column, and the second eluate was collected and freeze-dried.

4. The preparation method according to claim 3, characterized in that: The step S1 comprises: Step S1-1, crushing and sieving the Dendrobium sphaerocephalum; Step S1-2, performing ultrasonic extraction on the Dendrobium sphaerocephalum powder using a solvent, and collecting the extract; Step S1-3, removing protein from the extract; Step S1-4, reacting the protein-removed extract with ethanol to obtain crude polysaccharides from Dendrobium sphaerocephalum.

5. The preparation method according to claim 4, characterized in that: The step S1-2 comprises: Step S1-2-1, extracting the Dendrobium spherical powder by heating at 50-56° C. with acetone for 1-2 hours, and collecting the powder; Step S1-2-2, heating the powder with deionized water at 95-100° C. for 2-3 hours, separating the liquid, which is the extract; preferably, extracting three times; Preferably, the extraction is carried out by ultrasonic extraction with an ultrasonic frequency of 200-400W.

6. The preparation method according to claim 4, characterized in that: In the step S1-3, sevage reagent is used to remove proteins in the extract.

7. The preparation method according to claim 4, characterized in that: In the step S1-4, the volume proportion of ethanol in the system is not less than 90%.

8. The preparation method according to claim 3, characterized in that: In the step S2, the crude polysaccharide is purified by anion exchange resin, using DEAE-cellulose anion exchange resin, adjusting the flow rate to 15 ml / min, eluting with deionized water and NaCl solutions of different concentrations, collecting the deionized water eluate and concentrating it; and / or, The concentrate was dialyzed using a 3500 Da dialysis bag.

9. Use of the neutral polysaccharide of Dendrobium sphaerocephalum according to claim 1 or 2 in preparing a gastric mucosal repairing drug.

10. Use of the neutral polysaccharide of Dendrobium sphaerocephalum according to claim 1 or 2 in the preparation of a protective agent for reducing the side effects of non-steroidal anti-inflammatory drugs.

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