Dendrobium huoshuangshanquan polysaccharide with gastric mucosa protection function and extraction method thereof

Abstract

This invention discloses a polysaccharide from *Dendrobium nobile* with gastric mucosal protective function and its extraction method, belonging to the field of food biotechnology and natural product development technology. This invention successfully separates polysaccharide components from *Dendrobium nobile* with different acetyl contents using ultrasound-assisted enzymatic hydrolysis combined with gradient temperature-controlled fractional alcohol precipitation. The gradient temperature-controlled fractional alcohol precipitation utilizes the difference in solubility of polysaccharides with different acetyl contents in aqueous solution with temperature changes, sequentially separating high, medium, and low acetyl content polysaccharide components under low, medium, and high temperature conditions. Animal experiments verified that the high acetyl content component (DDP-H) has a significantly better protective effect against alcoholic gastric mucosal damage than the crude polysaccharide (DDP-C) and other fractions (DDP-M, DDP-L), with an ulcer inhibition rate of up to 61.23%. This invention achieves precise enrichment of highly gastroprotective polysaccharide components from *Dendrobium nobile*, providing a new effective ingredient for developing functional foods or drugs to prevent or improve alcoholic gastric mucosal damage.

Description

Technical Field

[0001] This invention belongs to the field of food biotechnology and natural product development technology, and more specifically relates to a polysaccharide from Dendrobium officinale with gastric mucosa protection function and its extraction method. Background Technology

[0002] With the fast pace of modern life and the prevalence of drinking culture, alcohol-induced gastric mucosal damage (acute gastritis, gastric ulcers) is on the rise. Ethanol can directly damage gastric mucosal epithelial cells, dissolve the mucus layer, and cause microcirculatory disturbances and oxidative stress, leading to gastric mucosal bleeding, erosion, and even perforation. Currently used clinical treatments, such as proton pump inhibitors like omeprazole, are effective, but long-term use may cause side effects such as intestinal flora imbalance and indigestion, and lacks universality for daily intervention in sub-healthy populations. Therefore, developing safe, natural, and food-medicine homologous gastric protective factors has become a research hotspot.

[0003] Dendrobium nobile, a valuable plant used for both food and medicine, has similar efficacy to Dendrobium officinale and is rich in polysaccharides and other active ingredients. Current research on Dendrobium nobile polysaccharides largely focuses on the extraction and preliminary activity evaluation of crude polysaccharides. However, the complex composition and wide molecular weight distribution of crude polysaccharides lead to unstable biological activity and unclear dose-response relationships. There is currently a lack of systematic research on the differences in the protective effects of different molecular weight fractions of Dendrobium nobile polysaccharides on the gastric mucosa. Studies on further purifying Dendrobium nobile polysaccharides through fractional alcohol precipitation and screening for specific molecular weight fractions with optimal structure-activity relationships that can alleviate acute alcoholic gastric mucosal damage are rarely reported. How to obtain Dendrobium nobile polysaccharide components with higher activity, better safety, and controllable quality is a pressing technical problem that needs to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a polysaccharide from *Dendrobium nobile* with gastric mucosa protection function and its extraction method. This invention utilizes ultrasound-assisted enzymatic hydrolysis and gradient temperature-controlled fractional alcohol precipitation based on differences in acetyl content to obtain polysaccharide components with varying acetyl content. Furthermore, through structure-activity relationship and in vivo bioactivity screening, a specific component (DDP-H) with optimal gastric protective activity was identified, solving the problems of inconsistent activity and lack of specificity in existing *Dendrobium nobile* polysaccharide products.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is to provide a method for extracting polysaccharides from Dendrobium nobile with gastric mucosa protective function, the steps of which include: After being crushed and sieved, the purple-skinned dendrobium was pre-treated by defatting to obtain defatted purple-skinned dendrobium powder. The defatted Dendrobium officinale powder was mixed evenly with acetate buffer (pH=5.0) to obtain a mixed system. The mixed system was then subjected to ultrasound-assisted enzymatic hydrolysis extraction to obtain an extract. The extract was purified to prepare crude polysaccharide of Dendrobium nobile (DDP-C). The crude polysaccharide of Dendrobium nobile was fractionated based on acetyl content to obtain Dendrobium nobile polysaccharides with different acetyl contents; The crude polysaccharide of Dendrobium nobile was dissolved, stirred under the first temperature condition, centrifuged and filtered, the filtrate was collected, ethanol was added for alcohol precipitation, the first precipitate was collected and dried to obtain the polysaccharide component with high acetyl content (DDP-H). The first precipitate was dissolved, stirred under a second temperature condition, centrifuged and filtered, the filtrate was collected, ethanol was added for alcohol precipitation, the second precipitate was collected, and dried to obtain the polysaccharide component with medium acetyl content (DDP-M). The second precipitate was dissolved, stirred under a third temperature condition, centrifuged and filtered, the filtrate was collected, ethanol was added for alcohol precipitation, the precipitate was collected and dried to obtain the low acetyl polysaccharide fraction (DDP-L). Wherein, the first temperature is lower than the second temperature, and the second temperature is lower than the third temperature.

[0006] Furthermore, the pulverization and sieving process involves passing the material through a 20-mesh sieve.

[0007] Furthermore, the defatting pretreatment step includes: mixing Dendrobium nobile and ethanol solution at a material-to-liquid ratio of 1:10 (g / mL), shaking and stirring in a water bath at 70°C for 4 hours, centrifuging (4000g, 5min) to collect the precipitate, and then drying at 45°C to constant weight.

[0008] Optionally, the volume fraction of the ethanol solution is 95%.

[0009] Furthermore, the ratio of the defatted Dendrobium officinale powder to the acetate buffer solution is 1:50 (g / mL).

[0010] Furthermore, the ultrasound-assisted enzymatic extraction step includes: adding cellulase to the mixed system, stirring and enzymatically hydrolyzing at 50°C for 67 min, inactivating the enzyme in a boiling water bath for 10 min after enzymatic hydrolysis, cooling and then ultrasonically extracting at 50°C and ultrasonic power of 156W for 30 min, centrifuging, collecting the supernatant, and concentrating under reduced pressure at 55°C to a solid content of 80-90 wt% to obtain the extract.

[0011] Optionally, the cellulase has an enzyme activity of 700 U / g.

[0012] Optionally, the mass-to-volume ratio of the cellulase to the mixed system is 3-5%.

[0013] Further, the purification process includes: mixing the extract with Sevag reagent (chloroform: n-butanol = 4:1, v / v) at a volume ratio of 4:1, shaking and separating the liquid, taking the upper aqueous phase, adding AB-8 macroporous resin at a material-to-liquid ratio of 1:10 (g / mL), allowing it to stand at 4°C for adsorption for 12 hours, filtering, repeating the operation until the filtrate is clear and transparent, then adding ethanol to a final ethanol concentration of 80% (v / v), allowing it to stand at 4°C for 8-16 hours, centrifuging to collect the precipitate, redissolving it, and then freeze-drying it under vacuum to obtain crude polysaccharide of Dendrobium nobile (DDP-C).

[0014] Furthermore, in the step of grading based on acetyl content: the first temperature is 10°C; the second temperature is 25°C; and the third temperature is 45°C.

[0015] Furthermore, the stirring time under the first temperature condition, the second temperature condition, and the third temperature condition is 6 hours.

[0016] Furthermore, the addition of ethanol for alcohol precipitation is as follows: ethanol is added until the final concentration is 90%, the standing temperature is 4°C, and the standing time is 12 hours.

[0017] The second technical solution of the present invention provides a polysaccharide of Dendrobium officinale with the function of protecting the gastric mucosa, wherein the polysaccharide of Dendrobium officinale with the function of protecting the gastric mucosa is extracted by the above extraction method.

[0018] The third technical solution of the present invention provides the application of the above-mentioned Dendrobium officinale polysaccharide with gastric mucosa protection function in the preparation of functional foods or drugs for preventing or improving alcoholic gastric mucosal damage.

[0019] The present invention discloses the following technical effects: Innovative Separation Strategy: This invention is the first to discover and utilize the significant differences in water solubility of polysaccharides from *Dendrobium nobile* with different acetyl groups. Through a simple gradient temperature-controlled dissolution and fractional alcohol precipitation technique, it achieves effective separation of polysaccharide components with high, medium, and low acetyl groups. This method is simple to operate, operates under mild conditions, and avoids the use of complex and expensive chromatographic equipment, providing a new approach for polysaccharide activity screening based on acetyl content.

[0020] Precise enrichment of active ingredients: Animal experiments have verified that the high acetyl content fraction (DDP-H) exhibits significantly superior bioactivity compared to crude polysaccharide (DDP-C) and other fractions (DDP-M, DDP-L) in combating alcoholic gastric mucosal damage. This invention achieves precise enrichment of polysaccharide fractions with specific acetylation levels from Dendrobium nobile that possess optimal gastric protective activity.

[0021] Clear structure-activity relationship: This invention elucidates the intrinsic link between acetyl content and gastric protective activity. The unique high acetyl content of DDP-H endows it with strong mucosal adsorption capacity, which is not possessed by the low-acetyl and medium-acetyl content components.

[0022] Superior Mechanism of Action: The DDP-H extracted in this invention can significantly inhibit excessive gastric acid secretion caused by alcohol stimulation, effectively reducing the secondary corrosion of the damaged mucosa by the acidic environment. At the same time, its high acetyl content and spatial structure enable it to form a stable "biological protective film" on the gastric mucosa surface, physically blocking the direct contact between ethanol and gastric epithelial cells, and promoting the secretion and retention of gastric mucus. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a standard curve for the determination of total sugar content based on D-glucose.

[0024] Figure 2 This is a standard curve for BSA-based protein content determination.

[0025] Figure 3 This is a standard curve for the determination of uronic acid content based on galacturonic acid.

[0026] Figure 4 This is a standard curve for the determination of reducing sugar content based on D-glucose.

[0027] Figure 5 The maximum absorption wavelengths of the DDP-C, DDP-H, DDP-M, DDP-L, and Congo red complexes at different alkaline concentrations are given.

[0028] Figure 6 The growth and weight change curves for each group are shown.

[0029] Figure 7 The results for food intake and water intake for each group are shown in the left graph, and the right graph shows the water intake.

[0030] Figure 8 The macroscopic pathological condition of the gastric tissue of mice in each group was assessed. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Unless otherwise specified, all raw materials and reagents involved in the specific embodiments of this invention are commercially available products.

[0037] Unless otherwise specified, room temperature and ambient temperature in the specific embodiments of this invention refer to 20-30℃.

[0038] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0039] Example 1 Preparation of graded components of Dendrobium nobile polysaccharides: S1. Raw material pretreatment (defatting): Pulverize *Dendrobium nobile* to pass through a 20-mesh sieve. Add 95% ethanol solution at a material-to-liquid ratio of 1:10 (g / mL). Extract by shaking and stirring in a 70℃ water bath for 4 hours. Centrifuge (4000g, 5min) to collect the precipitate. Repeat the above operation once. Combine the precipitate residues and dry them in a 45℃ oven to constant weight to obtain defatted *Dendrobium nobile* powder.

[0040] S2. Ultrasonic-assisted enzymatic extraction: Take the defatted Dendrobium officinale powder obtained in step S1, add acetate buffer (pH=5.0) at a material-to-liquid ratio of 1:50 (g / mL), add cellulase (enzyme activity 700 U / g, cellulase to mixed system mass-to-volume ratio 4%), and stir at 50℃ for 67 min for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme in a boiling water bath for 10 min. After cooling, extract by ultrasound at 50℃ and ultrasonic power 156 W for 30 min. Centrifuge, collect the supernatant, and concentrate under reduced pressure at 55℃ to an appropriate volume (solid content 80wt%) to obtain the concentrated extract.

[0041] S3. Purification and Preparation of Crude Polysaccharide: Add Sevag reagent (chloroform: n-butanol = 4:1, v / v) to the extract concentrate obtained in step S2, with a volume ratio of extract concentrate to Sevag reagent of 4:1. Shake and separate the liquid to remove proteins, repeating the operation until no protein layer appears. Take the upper aqueous phase and add pretreated AB-8 macroporous resin at a solid-liquid ratio of 1:10 (g / mL). Incubate at 4℃ for 12 hours for adsorption, filter, and repeat the operation until the filtrate is clear and transparent. Slowly add anhydrous ethanol to the decolorized filtrate until the final ethanol concentration is 80% (v / v), and incubate at 4℃ overnight. Collect the precipitate by centrifugation, reconstitute, and freeze-dry under vacuum to obtain crude polysaccharide of Dendrobium nobile (DDP-C).

[0042] S4. Gradient temperature-controlled fractional alcohol precipitation based on acetyl content: The crude polysaccharide (DDP-C) of Dendrobium nobile obtained in step S3 was dissolved in deionized water to prepare an aqueous solution with a concentration of 50 wt%.

[0043] (a) Separation of the high acetyl content fraction (DDP-H): The crude polysaccharide solution was thoroughly stirred and dissolved at 10°C for 6 hours. Then, it was centrifuged and filtered, and the filtrate was collected. Anhydrous ethanol was slowly added to the filtrate until the final concentration was 90% (v / v), and the mixture was allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation, reconstituted, and then freeze-dried under vacuum to obtain the high acetyl content Dendrobium officinale polysaccharide (DDP-H).

[0044] (b) Separation of the acetyl-rich fraction (DDP-M): The precipitate obtained after centrifugation in step (a) was redissolved in deionized water and stirred thoroughly at 25°C for 6 hours. The solution was then centrifuged and filtered, and the filtrate was collected. Anhydrous ethanol was slowly added to the filtrate until a final concentration of 90% (v / v) was achieved, and the solution was allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation, reconstituted, and then freeze-dried under vacuum to obtain the acetyl-rich Dendrobium officinale polysaccharide (DDP-M).

[0045] (c) Separation of the low-acetyl content fraction (DDP-L): The precipitate obtained after centrifugation in step (b) was redissolved in deionized water and stirred thoroughly at 45°C for 6 hours. The solution was then centrifuged and filtered, and the filtrate was collected. Anhydrous ethanol was slowly added to the filtrate until a final concentration of 90% (v / v) was achieved, and the solution was allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation, reconstituted, and then freeze-dried under vacuum to obtain low-acetyl content Dendrobium officinale polysaccharide (DDP-L).

[0046] Comparative Example 1 Single ultrasound extraction: Crush *Dendrobium nobile* into powder and pass it through a 20-mesh sieve. Add 95% ethanol solution at a solid-liquid ratio of 1:10 (g / mL) and extract by shaking and stirring in a 70℃ water bath for 4 hours. Centrifuge (4000g, 5min, the same below) to collect the precipitate, and repeat the above operation once. Combine the precipitate residues and dry them in a 45℃ oven to constant weight to obtain defatted *Dendrobium nobile* powder. Add acetate buffer (pH=5.0) at a solid-liquid ratio of 1:50 (g / mL) and extract by ultrasonication at 50℃ and 156 W for 30 min. Centrifuge and collect the supernatant. Concentrate under reduced pressure at 55℃ to a solid content of 80wt% to obtain the extract concentrate. Add Sevag reagent (chloroform:n-butanol=4:1, v / v) to the extract concentrate, with a volume ratio of extract concentrate to Sevag reagent of 4:1. Shake and separate the liquid to remove protein. Repeat the operation until no protein layer appears. Take the upper aqueous phase and add pretreated AB-8 macroporous resin at a material-to-liquid ratio of 1:10 (g / mL). Incubate at 4°C for 12 hours for adsorption, filter, and repeat the process until the filtrate is clear and transparent. Slowly add anhydrous ethanol to the decolorized filtrate until the final ethanol concentration is 80% (v / v), and incubate overnight at 4°C. Collect the precipitate by centrifugation, reconstitute, and freeze-dry under vacuum to obtain the single ultrasonic extract, crude polysaccharide of Dendrobium nobile (DDP-U).

[0047] Comparative Example 2 Single enzymatic extraction: The purple-skinned Dendrobium officinale was pulverized and passed through a 20-mesh sieve. A 95% ethanol solution was added at a material-to-liquid ratio of 1:10 (g / mL), and the mixture was extracted by shaking and stirring in a 70℃ water bath for 4 hours. The precipitate was collected by centrifugation (4000g, 5 min), and the above operation was repeated once. The precipitate residues were combined and dried in a 45℃ oven to constant weight to obtain defatted purple-skinned Dendrobium officinale powder. Acetate buffer (pH=5.0) was added at a material-to-liquid ratio of 1:50 (g / mL), along with cellulase (700 U / g enzyme activity, 4% by volume). Enzymatic hydrolysis was carried out at 50℃ with stirring for 67 min. After enzymatic hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 min. After cooling, the mixture was centrifuged, and the supernatant was collected and concentrated under reduced pressure at 55℃ to a solid content of 80 wt% to obtain the concentrated extract. Sevag reagent (chloroform:n-butanol = 4:1, v / v) was added to the extract concentrate, with a volume ratio of extract concentrate to Sevag reagent of 4:1. The mixture was shaken and separated to remove proteins. This process was repeated until no protein layer appeared. The upper aqueous phase was collected and pretreated AB-8 macroporous resin was added at a solid-liquid ratio of 1:10 (g / mL). The mixture was allowed to stand at 4°C for 12 hours for adsorption. The mixture was then filtered, and the process was repeated until the filtrate was clear and transparent. Anhydrous ethanol was slowly added to the decolorized filtrate until the final ethanol concentration reached 80% (v / v). The mixture was allowed to stand overnight at 4°C. The precipitate was collected by centrifugation, reconstituted, and then freeze-dried under vacuum to obtain the single enzymatic hydrolysis extract, crude polysaccharide of Dendrobium nobile (DDP-E).

[0048] Comparative Example 3 Single hydrothermal extraction: Crush *Dendrobium nobile* into powder and pass it through a 20-mesh sieve. Add 95% ethanol solution at a solid-liquid ratio of 1:10 (g / mL) and extract by shaking and stirring in a 70℃ water bath for 4 hours. Centrifuge (4000g, 5min, the same below) to collect the precipitate, and repeat the above operation once. Combine the precipitate residues and dry them in a 45℃ oven to constant weight to obtain defatted *Dendrobium nobile* powder. Add acetate buffer (pH=5.0) at a solid-liquid ratio of 1:50 (g / mL) and extract by sonication at 50℃ for 30 min. Centrifuge and collect the supernatant. Concentrate under reduced pressure at 55℃ to a solid content of 80% to obtain the extract concentrate. Add Sevag reagent (chloroform:n-butanol=4:1, v / v) to the extract concentrate, with a volume ratio of extract concentrate to Sevag reagent of 4:1. Shake and separate the liquid to remove protein. Repeat the operation until no protein layer appears. Take the upper aqueous phase and add pretreated AB-8 macroporous resin at a material-to-liquid ratio of 1:10 (g / mL). Allow to stand at 4°C for 12 hours for adsorption, then filter. Repeat the process until the filtrate is clear and transparent. Slowly add anhydrous ethanol to the decolorized filtrate until the final ethanol concentration is 80% (v / v), and allow to stand at 4°C overnight. Collect the precipitate by centrifugation, reconstitute, and freeze-dry under vacuum to obtain the single ultrasonic extract, crude polysaccharide from Dendrobium officinale (DDP-H).

[0049] Comparative Examples 1-3 were extracted using single ultrasonic extraction, single enzymatic extraction, and traditional hot water extraction. The extraction rates and acetyl content (%) of the obtained products are shown in Table 1.

[0050] Table 1 Comparison of extraction rates and acetyl content of products using different extraction methods The results showed that the extraction rate and the acetyl content of the crude polysaccharide obtained by the ultrasound-assisted enzymatic hydrolysis extraction method (Example 1) were significantly better than those of single ultrasound, enzymatic hydrolysis or hydrothermal extraction methods.

[0051] Comparative Example 4 The traditional fractionation method based on "different ethanol concentrations" (i.e., fractionation based on molecular weight) employs a conventional ethanol fractionation precipitation method, with the following specific steps: S1-S3, same as in Example 1.

[0052] S4. Prepare an aqueous solution of the crude polysaccharide (DDP-C) from *Dendrobium nobile* (C-C) in step S3, using the same concentration as in Example 1. Slowly add anhydrous ethanol to the solution with stirring until the final ethanol concentration reaches 35%. Let the solution stand overnight at 4°C, centrifuge to collect the precipitate, and freeze-dry to obtain component (C-35). Continue adding anhydrous ethanol to the supernatant obtained after centrifugation until the final ethanol concentration reaches 65%. Let the solution stand overnight at 4°C, centrifuge to collect the precipitate, and freeze-dry to obtain component (C-65). Continue adding anhydrous ethanol to the supernatant obtained after centrifugation until the final ethanol concentration reaches 80%. Let the solution stand overnight at 4°C, centrifuge to collect the precipitate, and freeze-dry to obtain component (C-80).

[0053] The acetyl content and molecular weight of the three components were determined respectively.

[0054] Results: The main difference between the three components C-35, C-65, and C-80 is their molecular weight (high molecular weight, medium molecular weight, and low molecular weight). There is no obvious pattern or significant difference in their acetyl content, meaning that this method cannot achieve precise separation based on acetyl content.

[0055] Comparative Example 5 Single-temperature dissolution-stage alcohol precipitation (compared to gradient temperature control of this invention): S1-S3, same as in Example 1.

[0056] S4. Compared with Example 1, the difference is that the dissolution temperature is 25°C or 10°C. The exemplary steps are as follows: The crude polysaccharide (DDP-C) of Dendrobium nobile obtained in step S3 was dissolved in deionized water to prepare an aqueous solution with a concentration of 50 wt%.

[0057] (a) The above crude polysaccharide solution was thoroughly stirred and dissolved at 25°C for 6 hours. Then, it was centrifuged and filtered, and the filtrate was collected. Anhydrous ethanol was slowly added to the filtrate until the final concentration was 80% (v / v), and the solution was allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation, reconstituted, and then freeze-dried under vacuum to obtain Dendrobium officinale polysaccharide S-1.

[0058] (b) The precipitate obtained after centrifugation in step (a) was redissolved in deionized water and stirred thoroughly at 25°C for 6 hours. Then, it was centrifuged and filtered, and the filtrate was collected. Anhydrous ethanol was slowly added to the filtrate until the final concentration was 80% (v / v), and the mixture was allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation, reconstituted, and then freeze-dried under vacuum to obtain Dendrobium officinale polysaccharide S-2.

[0059] (c) The precipitate obtained after centrifugation in step (b) was redissolved in deionized water and stirred thoroughly at 25°C for 6 hours. Then, it was centrifuged and filtered, and the filtrate was collected. Anhydrous ethanol was slowly added to the filtrate until the final concentration was 80% (v / v), and the mixture was allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation, reconstituted, and then freeze-dried under vacuum to obtain Dendrobium officinale polysaccharide S-3.

[0060] Separation effect: Due to the constant temperature throughout the process, the core principle of the difference in water solubility of polysaccharides with different acetyl contents at different temperatures could not be utilized. Polysaccharides with different solubilities could not be dissolved and separated in stages. The acetyl content of the three components (S-1, S-2, S-3) showed no significant difference, and the yields of S-2 and S-3 were extremely low, making effective fractionation impossible.

[0061] This comparative example demonstrates that the gradient temperature control strategy adopted in this invention, which involves "preferentially dissolving high-acetyl components at low temperatures, re-dissolving medium-acetyl components at higher temperatures, and dissolving low-acetyl components at even higher temperatures," is a key technical feature for achieving efficient separation, and none of these features can be omitted.

[0062] Experimental Example 1: Determination of Chemical Composition and Structural Analysis of Polysaccharides from Dendrobium officinale of Different Molecular Weights Determination of total sugar content: A standard curve was prepared by using D-glucose as a standard to prepare a 100 μg / mL D-glucose standard solution. 0 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, and 1.0 mL were respectively placed in test tubes, and then water was added to a final volume of 1.0 mL. Subsequently, 0.5 mL of 6% (m / v) phenol solution and 2.5 mL of concentrated sulfuric acid were added to each tube. After thorough mixing, the mixture was allowed to stand for 20 min, and the absorbance was measured at 490 nm. A 100 μg / mL polysaccharide sample solution was prepared, and the absorbance was measured following the same steps. Finally, the total sugar content in the sample was calculated based on the glucose standard curve. Each experiment was performed in triplicate to ensure data reliability. The total sugar content results of DDPs are shown in Table 2.

[0063] Figure 1 This is a standard curve for the determination of total sugar content based on D-glucose.

[0064] Protein content determination: The quantitative analysis of protein content was performed using the Coomassie Brilliant Blue staining method. The specific operation procedure is as follows: First, prepare 0.1% (m / v) Coomassie Brilliant Blue G-250 staining solution and 50 μg / mL bovine serum albumin (BSA) standard solution.

[0065] During the standard curve preparation stage, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, and 1.0 mL of BSA standard solution were accurately transferred into 10 clean test tubes, respectively. Distilled water was then added to each tube to bring the volume to 1.0 mL. 5 mL of G-250 staining solution was added to each tube, and the mixture was thoroughly mixed using a vortex apparatus and then incubated in a dark environment for 10 min. Distilled water was used as a blank control group. The absorbance of each solution was measured at a wavelength of 595 nm, and each experiment was performed in triplicate to ensure data reliability. A 10 mg / mL polysaccharide sample solution was prepared, and the absorbance was measured following the same steps. The protein content results of DDPs are shown in Table 2.

[0066] Figure 2 This is a standard curve for BSA-based protein content determination.

[0067] Determination of uronic acid content: A standard curve was prepared using galacturonic acid as a standard. 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the solution were placed in test tubes, and water was added to a final volume of 1.0 mL. Then, 6.0 mL of sodium tetraborate-sulfuric acid solution (0.478 g sodium tetraborate dissolved in 100 mL concentrated sulfuric acid) was added, mixed well, and placed in a boiling water bath for 5 min. After complete cooling, 0.1 mL of m-hydroxybiphenyl solution (1.5 mg / mL) was added to the reaction system, mixed well, and allowed to stand for 5 min. The absorbance was measured at 525 nm using a microplate reader. A 60 μg / mL polysaccharide was prepared, and the absorbance was measured following the same steps. The uronic acid content of the DDPs is shown in Table 2.

[0068] Figure 3 This is a standard curve for the determination of uronic acid content based on galacturonic acid.

[0069] Determination of reducing sugar content: DNS solution was prepared in advance. A 1 mg / mL glucose stock solution was prepared using D-glucose as a standard. 0.1 mL, 0.15 mL, 0.2 mL, 0.25 mL, 0.3 mL, 0.35 mL, 0.4 mL, 0.45 mL, and 0.5 mL were respectively transferred to different test tubes, and water was added to each tube to a final volume of 0.5 mL. Then, 1 mL of DNS reagent was added to each tube, and the mixture was thoroughly mixed. The tubes were then heated in a boiling water bath for 5 min. After cooling to room temperature, 3.5 mL of distilled water was added to each tube, and the absorbance was measured at 540 nm using a microplate reader to plot a standard curve. The absorbance of a 2 mg / mL polysaccharide sample solution was measured following the same steps. The reducing sugar content of DDPs is shown in Table 2.

[0070] Figure 4 This is a standard curve for the determination of reducing sugar content based on D-glucose.

[0071] Molecular weight determination: The molecular weight of the DDP solution was determined using an Agilent 1260 HPLC system equipped with a refractive index detector (RID). The specific method is as follows: First, the sample was dissolved in 0.02 M potassium dihydrogen phosphate solution to prepare a sample solution with a concentration of 4 mg / mL. The solution was then filtered through a 0.22 μm aqueous filter before injection. Specific chromatographic conditions were as follows: A tandem TSK-gel G5000 PWXL (7.8 × 300 mm, 10 μm particle size) and TSK-gel G3000 PWXL (7.8 × 300 mm, 7 μm particle size) gel chromatography column were used for separation; 0.02 M potassium dihydrogen phosphate buffer solution was used as the mobile phase, with a flow rate set at 0.6 mL / min; the column oven temperature was controlled at 35 ± 0.1℃; and the detector temperature was maintained at 45 ± 0.1℃. The injection volume for each analysis was 20 μL. A calibration curve equation was established using a series of pullulan standards with known molecular weights (642,000, 337,000, 194,000, 21,100, 9,600, and 6,100 Da), and the molecular weight of the test samples was calculated based on this regression equation. The molecular weight results of DDPs are shown in Table 2.

[0072] Table 2 Acetyl content determination: The acetyl content and degree of substitution of DDPs were quantitatively analyzed by acid-base titration. The specific operation steps are detailed below: Accurately weigh 10 mg of DDPs sample into a reaction flask and add 10 mL of 0.01 mol / L sodium hydroxide standard solution. After the sample is fully dissolved, place the reaction flask in a 50°C constant-temperature shaker and react at a shaking rate of 250 rpm for 2 hours. After the reaction is complete, cool to room temperature and perform back titration using 1% phenolphthalein solution as a titration indicator and 0.01 mol / L hydrochloric acid standard solution until the red color in the solution completely disappears and remains unchanged for 15 seconds. At this point, the pH of the system is 7.0. Finally, calculate the percentage content of acetyl groups in the DDPs sample using the following formula: ; In the above calculation formula, V1 and C1 represent the volume (mL) and concentration (mol / L) of sodium hydroxide (NaOH) solution added during the experiment, respectively, and their specific values ​​are fixed at 10 mL and 0.01 mol / L; V2 and C2 correspond to the volume (mL) and concentration (mol / L) of HCl solution consumed in the back titration stage, respectively, with C2 fixed at 0.01 mol / L; m is the mass (g) of the polysaccharide sample to be tested, which is 0.01 g in the experiment.

[0073] Acetyl groups, as key substituents in polysaccharide molecules, can significantly alter the spatial conformation of polysaccharides, thereby affecting their physicochemical properties and biological activities. The percentage acetyl groups in each polysaccharide component is shown in Table 3.

[0074] Table 3 Acetyl content (%) of different polysaccharide components from Dendrobium nobile As shown in Table 3, the gradient temperature control grading method of the present invention successfully obtained polysaccharide components with significant differences in acetyl content.

[0075] Determination of the triple helix structure: Crude polysaccharide (DDP-C) and its fractionated components (DDP-H, DDP-M, DDP-L) from *Dendrobium nobile* were dissolved in deionized water to prepare 1.0 mg / mL test polysaccharide solutions. 2.0 mL of each test solution was mixed with an equal volume of 100 μmol / L Congo red reagent and vortexed until homogeneous. NaOH solution was added to the mixture to create a gradient of NaOH concentrations within the ranges of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L. After the mixtures were allowed to stand for equilibration for 10 min, a UV-Vis spectrophotometer was used to scan the samples across the wavelength range of 450–600 nm, recording the maximum absorption wavelengths of each component and the Congo red complex at different alkaline concentrations. The experimental results are shown below. Figure 5 .

[0076] Figure 5 The maximum absorption wavelengths of the DDP-C, DDP-H, DDP-M, DDP-L, and Congo red complexes at different alkaline concentrations are given.

[0077] The triple-helix conformation of polysaccharides is generally considered a crucial structural basis for their immunomodulatory, antioxidant, and gastric mucosal protective activities. Polysaccharides with a triple-helix structure can form complexes with Congo red, causing a red shift in its maximum absorption wavelength. After complexing with Congo red, triple-helix polysaccharides exhibit the following trends: in a low-concentration alkaline range, the triple-helix polysaccharide causes a significant and constant red shift in the maximum absorption wavelength (λmax) of Congo red; as the alkalinity increases, the complex enters the conformational transition region. This is because with increasing alkalinity, the hydrogen bonds within the structure gradually break, and the polysaccharide structure gradually transforms into a random coil. Since the random coil structure of the polysaccharide cannot continue to bind with Congo red, the complex gradually dissociates, eventually causing λmax to drop sharply back to near the absorption wavelength of Congo red itself. Figure 5As can be seen, within the low alkaline concentration range, the maximum absorption wavelength curves of the complexes of DDP-C, DDP-H, and DDP-M with Congo red are significantly higher than those of the Congo red control group. This "red shift" relative to the control group indicates that the above three components form a typical helical-Congo red complex with Congo red. As the alkalinity increases, the maximum absorption wavelength of the complex drops back to a level comparable to the control group. This typical trend suggests that DDP-C, DDP-H, and DDP-M may have an ordered triple helix structure. Although the maximum absorption wavelength of the complex formed by DDP-L and Congo red remains at a high level and hardly changes with the NaOH concentration, this does not necessarily mean that it has a triple helix structure. Some literature points out that dextran with a molecular weight less than 50 kDa generally cannot form an ordered helical structure. Furthermore, the literature also points out that the Congo red experiment has limitations; single helices and even some random coils may also bind to Congo red. The curve of DDP-L lacks the typical "high to low" unwinding process. Judging from its molecular weight (24.05 kDa), the conformation of DDP-L is more likely to be a stable short-chain bundle aggregate rather than a typical triple helix structure.

[0078] Experimental Example 2: Determination of the in vitro antioxidant activity of polysaccharides from Dendrobium officinale with different molecular weights DPPH free radical scavenging capacity determination A 0.15 mM DPPH working solution (using 70% methanol solution as solvent) was prepared. 100 μL of DDP solutions at various concentration gradients were measured and mixed thoroughly with an equal volume of DPPH working solution. The mixture was then placed in a dark environment at room temperature for 30 min. After the reaction, the absorbance of the sample at 517 nm was measured using a microplate reader. Deionized water was used as a blank control group, and 70% methanol solution was used as a sample control group. The DPPH free radical scavenging ability at different concentrations of DDPs is shown in Table 4. All experimental groups were performed in triplicate to ensure data stability. Low molecular weight polysaccharides have shorter chains, looser structures (no ordered structure), and relatively larger specific surface areas. This means that the active hydroxyl groups and hemiacetal ends on their molecular chains are more easily exposed and can collide with free radicals, resulting in higher in vitro free radical scavenging activity. High molecular weight polysaccharides (such as DDP-H) often form complex entangled or helical structures, encapsulating active groups internally and hindering contact and reaction with free radicals.

[0079] ; In the formula, A1, A2 and A3 represent the absorbance values ​​of the sample solution, the sample control and the blank control, respectively.

[0080] ABTS + Free radical scavenging ability determination: ABTS at a concentration of 7 mM + The mother liquor and 2.45 mM potassium persulfate solution were mixed evenly in an equal volume ratio and allowed to stand at room temperature in the dark for 16 hours. The activated solution was then serially diluted with phosphate buffer (pH 7.4) until its absorbance at 734 nm stabilized at 0.70 ± 0.02, thus obtaining ABTS. + Working fluid.

[0081] Take 50 μL of polysaccharide sample solutions of various concentration gradients and add them to 150 μL of ABTS. ＋ After thorough vortexing in the working solution, incubate for 6 min at room temperature in the dark. Measure the absorbance of the sample at 734 nm. Deionized water was used as a blank control group in this experiment. ABTS at different concentrations of DDPs... + The free radical scavenging capacity is shown in Table 4. All experimental groups were set up in triplicate to ensure data stability.

[0082] ; In the formula, A S A and B represent the absorbance values ​​of the sample solution and the blank control, respectively.

[0083] Table 4 Antioxidant capacity of different samples As can be seen from the data in Table 4, the DDP-H group exhibits excellent antioxidant capacity, which is significantly better than that of other components.

[0084] Experimental Example 3: Safety Evaluation of Different Molecular Weights of Dendrobium nobile Polysaccharides and Their Protective Effect on Alcoholic Gastric Injury Experimental Materials and Methods: Laboratory animals: SPF-grade male BALB / c mice, 6 weeks old; Grouping and administration: Mice were randomly divided into 7 groups: blank control group (NC), model group (MC), positive drug group (OME, 20 mg / kg), DDP-C group, DDP-H group, DDP-M group and DDP-L group; Modeling procedure: Each prophylactic drug group was administered the corresponding drug by gavage for 14 consecutive days (dose: 400 mg / kg). The control and model groups were administered the same volume of physiological saline by gavage. After the last administration, the patients were kept NPO (no food, but water was allowed) for 24 hours. Except for the control group, all other groups were administered 0.1 mL / 20 g body weight of 90% ethanol by gavage to induce acute gastric mucosal injury. The patients were sacrificed 1 hour later for tissue collection.

[0085] Preliminary safety assessment results: Body weight is the most direct indicator reflecting the growth, development, and health status of laboratory animals, such as... Figure 6 As shown.

[0086] Figure 6 The graph shows the growth and weight change curves for each group. As can be seen from the graph, the weight of mice in all experimental groups showed a steady increasing trend over time. Compared with the blank control group (NC), there were no significant differences in the weight gain curves of mice in each DDP treatment group (DDP-H, M, L, and DDP-C).

[0087] Figure 7 The results for food intake and water intake in each group are shown in the left and right graphs, respectively. As can be seen from the graphs, the daily intake fluctuations among the groups were within the normal physiological range. DDPs intake did not cause anorexia or water refusal in the mice, indicating that the sample had good palatability and did not negatively affect the mice's appetite. Throughout the prophylactic administration period, the mice in all groups were in good spirits, had glossy fur, and exhibited normal activity; no signs of poisoning or death were observed.

[0088] Normally, the intake of exogenous toxins leads to stress-induced hypertrophy or atrophy of the liver (detoxification organ) and kidneys (excretory organs), which can be directly reflected in changes in organ indices (organ weight / body weight). As shown in Table 5, compared with the NC group, there were no statistically significant differences in the indices of major organs such as the stomach, liver, and kidneys among the DDPs-treated groups. P >0.05). This indicates that within the set dosage range, prophylactic administration of Dendrobium officinale polysaccharides of different molecular weights did not cause swelling or pathological changes in the major organs of mice, demonstrating good safety and the potential to be developed into a dietary supplement for long-term consumption.

[0089] Table 5 Organ Index Note: Data in the table are expressed as mean ± standard deviation (mean ± SD) (n=6); different lowercase letters in the same column indicate significant differences. P <0.05).

[0090] Evaluation results of protective efficacy against alcohol-induced acute gastric mucosal injury: In macroscopic morphological observation of gastric tissue, the MC group showed severe gastric mucosal congestion and swelling, with numerous elongated hemorrhagic bands and the disappearance of gastric wall folds, exhibiting typical characteristics of acute alcohol-induced injury. The degree of gastric mucosal hemorrhage and edema was reduced to varying degrees in all polysaccharide groups. Among them, the DDP-M group showed the best protective effect, with a smooth gastric mucosal surface and only a few punctate hemorrhages, and its overall morphology was similar to that of the clinically commonly used positive drug OME group and the blank group. The macroscopic assessment of the pathological state of gastric tissue in each group of mice is shown below. Figure 8 .

[0091] Figure 8 The macroscopic pathological condition of the gastric tissue of mice in each group was assessed.

[0092] Based on the commonly used Ulcer Index (UI) scoring criteria in the literature, the ulcer area ratio, UI value, and ulcer inhibition rate were calculated for each group to assess gastric mucosal damage. Compared with the MC group, the ulcer area ratio and UI value of each treatment group were significantly reduced ( P <0.05). The DDP-H group had the lowest UI value, with an ulcer inhibition rate of 61.23%, significantly better than the DDP-M group (31.34%) and the DDP-L group (22.08%), and also significantly higher than the crude polysaccharide DDP-C group (21.77%). This indicates that the high acetyl content (DDP-H) obtained through graded alcohol precipitation enrichment of Dendrobium officinale polysaccharides has the strongest "stomach-protecting" ability. The morphological indicators of gastric tissue damage in each group of mice are shown in Table 6. The specific scoring criteria for UI value and the formula for calculating the ulcer inhibition rate are as follows: Scoring criteria: No damage (0 points); Point-like lesions or lesions with a length <1mm (1 point); The length is between 1-2mm (2 points); The length is between 2-3mm (3 points); The length is between 3-4mm (4 points); If the width is greater than 1 mm, the length damage score is doubled.

[0093] .

[0094] Table 6 Morphological indicators of gastric tissue damage in each group of mice Acute alcohol intake directly affects gastric parietal cell receptors (such as H2R and M3R), activating the proton pump and leading to excessive gastric acid, thus exacerbating mucosal damage. During dissection, to facilitate manipulation, 1 mL of physiological saline was precisely added to each mouse's gastric contents to dilute the gastric juice. After centrifugation (4℃, 3000g*10min), the pH of the supernatant was measured. Compared to the NC group, the gastric juice pH in the MC group was significantly lower (…). P <0.01) indicates a severely acidic gastric microenvironment. However, prophylactic administration of DDPs effectively reversed this trend (with statistically significant differences compared to the MC group). The DDP-H group exhibited optimal ability to regulate gastric pH at homeostatic levels, approaching those of the positive control drug OME group. This suggests that the mechanism of DDPs, particularly the high-acetyl-rich fraction, may involve inhibiting H+. + / K +- The activity of ATPase (proton pump) or its inherent buffering capacity neutralizes gastric acid, thereby playing a protective role for the gastric mucosa. The pH of gastric juice in each group is shown in Table 7.

[0095] Table 7. Gastric juice pH in each group Alsin blue staining solution can specifically adsorb and bind to acidic mucin in gastric mucus. Since alcohol damage leads to the degradation and loss of mucin in the gastric mucus layer, the amount of mucus can be reflected by observing the adsorption of staining solution on the tissue. The gastric mucus content in the MC group was significantly lower than that in the NC group. This is because high-concentration ethanol has strong dehydrating and lipid-soluble properties, rapidly dissolving the mucus gel layer on the gastric mucosa surface and directly damaging the epithelial cells that secrete mucus. This results in damage to the mucus-bicarbonate barrier, allowing gastric acid and proteases to directly erode the gastric wall, further exacerbating the damage and forming linear hemorrhages and ulcers that can be directly observed in macroscopic morphology.

[0096] This invention confirms that DDP-M can significantly reverse this pathological process. Among DDPs, the DDP-H group had the highest mucus content, comparable to the positive control drug OME group. Combined with the previously mentioned Congo red binding experiment and related structural analysis data, this may be because DDP-H has a high acetyl group content and a stable, ordered helical structure. This highly ordered conformation gives it excellent bioadhesion on the gastric mucosa surface, enabling it to cross-link with gastric mucin, thereby shielding epithelial cells from the hydrolytic erosion of gastric acid and pepsin. The gastric mucus content of each group is shown in Table 8.

[0097] Table 8. Gastric mucus content of mice in each group The above findings validated the application of different fractions of Dendrobium nobile polysaccharide in the preparation of products for the prevention and treatment of gastric ulcers in mice, particularly in the prevention and treatment of ethanol-induced acute gastric mucosal injury. The application of Dendrobium nobile polysaccharide in the preparation of products for the prevention and treatment of gastric ulcers is also applicable to other animals or humans.

[0098] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0099] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting polysaccharides from Dendrobium nobile with gastric mucosal protective function, characterized in that the steps include... include: After being crushed and sieved, the purple-skinned dendrobium was pre-treated by defatting to obtain defatted purple-skinned dendrobium powder. The defatted Dendrobium officinale powder was mixed evenly with acetate buffer to obtain a mixed system. The mixed system was then subjected to ultrasound-assisted enzymatic hydrolysis extraction to obtain an extract. The extract was purified to prepare crude polysaccharide from Dendrobium nobile. The crude polysaccharide of Dendrobium nobile was fractionated based on acetyl content to obtain Dendrobium nobile polysaccharides with different acetyl contents; The grading process based on acetyl content includes: The crude polysaccharide of Dendrobium nobile was dissolved, stirred under the first temperature condition, centrifuged and filtered, the filtrate was collected, ethanol was added for alcohol precipitation, the first precipitate was collected and dried to obtain the polysaccharide component with high acetyl content. The first precipitate was dissolved, stirred under a second temperature condition, centrifuged and filtered, the filtrate was collected, ethanol was added for alcohol precipitation, the second precipitate was collected, and dried to obtain a polysaccharide component with medium acetyl content. The second precipitate was dissolved, stirred under a third temperature condition, centrifuged and filtered, the filtrate was collected, ethanol was added for alcohol precipitation, the precipitate was collected and dried to obtain a polysaccharide component with low acetyl content. Wherein, the first temperature is lower than the second temperature, and the second temperature is lower than the third temperature.

2. The extraction method as described in claim 1, characterized in that, The pulverization and sieving process involves passing the material through a 20-mesh sieve.

3. The extraction method as described in claim 1, characterized in that, The defatting pretreatment steps include: mixing Dendrobium nobile and ethanol solution at a material-to-liquid ratio of 1:10 (g / mL), shaking and stirring in a water bath at 70℃ for 4 hours, collecting the precipitate by centrifugation, and then drying at 45℃ to constant weight.

4. The extraction method as described in claim 3, characterized in that, The volume fraction of the ethanol solution is 95%.

5. The extraction method as described in claim 1, characterized in that, The ratio of defatted Dendrobium officinale powder to acetate buffer solution is 1:50 (g / mL). And / or, the ultrasound-assisted enzymatic extraction step includes: adding cellulase to the mixed system, stirring and enzymatically hydrolyzing at 50°C for 67 min, inactivating the enzyme in a boiling water bath for 10 min after enzymatic hydrolysis, cooling and then ultrasonically extracting at 50°C and ultrasonic power of 156 W for 30 min, centrifuging, collecting the supernatant, and concentrating under reduced pressure at 55°C to a solid content of 80-90 wt% to obtain the extract.

6. The extraction method as described in claim 5, characterized in that, The cellulase has an enzyme activity of 700 U / g; And / or, the mass-to-volume ratio of the cellulase to the mixed system is 3%-5% (g / mL).

7. The extraction method as described in claim 1, characterized in that, The purification process includes: mixing the extract with Sevag reagent at a volume ratio of 4:1, shaking and separating the liquid, taking the upper aqueous phase, adding AB-8 macroporous resin at a material-to-liquid ratio of 1:10 (g / mL), allowing it to stand at 4℃ for 12 hours for adsorption, filtering, repeating the operation until the filtrate is clear and transparent, then adding ethanol to a final ethanol concentration of 80% (v / v), allowing it to stand at 4℃ for 8-16 hours, centrifuging to collect the precipitate, redissolving it, and then freeze-drying it under vacuum to obtain crude polysaccharide from Dendrobium nobile.

8. The extraction method as described in claim 1, characterized in that, In the step of the fractionation process based on acetyl content: The first temperature is 10°C; the second temperature is 25°C; and the third temperature is 45°C. And / or, the stirring time for stirring under the first temperature condition, stirring under the second temperature condition, and stirring under the third temperature condition is 6 hours; And / or, the addition of ethanol for alcohol precipitation is as follows: ethanol is added to a final concentration of 90%, the standing temperature is 4°C, and the standing time is 12 hours.

9. A polysaccharide from Dendrobium nobile with gastric mucosa protective function, characterized in that, The gastric mucosa-protecting Dendrobium officinale polysaccharide is obtained by the extraction method described in any one of claims 1-8.

10. The use of the Dendrobium officinale polysaccharide of claim 9, which has the function of protecting the gastric mucosa, in the preparation of functional foods or medicines for preventing or improving alcoholic gastric mucosal damage.

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