Preparation method of dendrobium huoshanense oligosaccharide with hepatoprotective activity

Abstract

The present application relates to the technical fields of oligosaccharide preparation, and discloses a preparation method of dendrobium huoshanense oligosaccharide with liver protection activity, and the specific steps are as follows: first, fresh dendrobium huoshanense stems are selected, impurities are removed, and low-temperature drying is performed, then deionized water is added and heated to extract dendrobium crude polysaccharide; then the crude polysaccharide solution is refined to obtain dendrobium refined polysaccharide; then beta-mannanase is added to the dendrobium refined polysaccharide extraction solution for enzymolysis, the polysaccharide solution is removed through a molecular cut-off membrane, and dendrobium huoshanense oligosaccharide stock solution concentrate is obtained; finally, the finished product is obtained through freeze-drying. The preparation method is simple, efficient and pollution-free, and the prepared dendrobium huoshanense oligosaccharide has significant effects in liver protection.

Description

Technical Field

[0001] This invention relates to the field of oligosaccharide preparation technology, and more specifically, to a method for preparing Dendrobium huoshanense oligosaccharides with hepatoprotective activity. Background Technology

[0002] With changes in modern lifestyles, the incidence of liver diseases such as fatty liver and liver fibrosis is rising year by year, posing a serious threat to human health. Currently, there are many types of drugs on the market for treating liver diseases, but they generally suffer from problems such as insignificant efficacy and significant side effects. Therefore, developing novel, highly effective, and low-toxicity liver-protective drugs or foods is of great importance.

[0003] Dendrobium huoshanense, a precious traditional Chinese medicine, possesses extensive medicinal value and health benefits. In recent years, with the deepening research on the active ingredients of traditional Chinese medicine, the oligosaccharide components in Dendrobium huoshanense have attracted widespread attention from researchers. Oligosaccharides, also known as oligosaccharides, are low-density polymers composed of 2-10 monosaccharides linked by glycosidic bonds. Compared with monosaccharides and polysaccharides, oligosaccharides possess unique biological activities and physiological functions. As an important component, the biological activity of Dendrobium huoshanense oligosaccharides still requires further research. Currently, there are no reports on the hepatoprotective activity of Dendrobium huoshanense oligosaccharides, which to some extent limits a comprehensive understanding of its pharmacological effects. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing hepatoprotective oligosaccharides from Dendrobium huoshanense, comprising the following steps:

[0005] (1) Preparation of crude polysaccharides from Dendrobium huoshanense

[0006] First, fresh Dendrobium huoshanense stems are selected and treated with impurities removal and low-temperature drying. Then, ethanol is used to remove a large number of fat-soluble small molecule components, followed by heating and extraction with deionized water to obtain crude Dendrobium huoshanense polysaccharides. During the polysaccharide extraction process, the extraction temperature of the polysaccharide solution should be controlled at 50-95℃, the extraction time at 30-180 minutes, the solid-liquid ratio at 5:1 to 50:1, and the number of extractions at 1-5 times.

[0007] (2) Polysaccharide refining

[0008] The crude polysaccharide obtained above was purified. First, ethanol precipitation was used to remove lipid-soluble components, then Seavge reagent was used to remove proteins and nucleic acids. Finally, purified polysaccharide was obtained through steps such as vacuum concentration and freeze centrifugation.

[0009] (3) Polysaccharide enzymatic hydrolysis

[0010] Specific β-mannanase was selected for enzymatic hydrolysis of the purified polysaccharide. The hydrolysis time was set to 4-12 hours, the enzyme concentration was controlled at 0.15u / mL-10u / mL, the hydrolysis was performed 2-4 times, and the hydrolysis temperature was maintained at 37℃-60℃.

[0011] (4) Oligosaccharide collection

[0012] The enzymatically hydrolyzed polysaccharide solution was subjected to ultrafiltration and centrifugation through a molecular weight cutoff membrane with a molecular weight of 3000 Da to collect oligosaccharides with a molecular weight ≤3000 Da, thereby removing polysaccharide components that were not completely hydrolyzed and obtaining a high-purity Dendrobium huoshanense oligosaccharide solution.

[0013] (5) Finished product preparation

[0014] The oligosaccharide solution of Dendrobium huoshanense collected after enzymatic hydrolysis was concentrated and freeze-dried to finally obtain the finished product of Dendrobium huoshanense oligosaccharide.

[0015] The beneficial effects of this invention are as follows: the preparation method of this invention can efficiently extract oligosaccharide components with significant hepatoprotective activity from Dendrobium huoshanense. By controlling the parameters of each preparation step, the quality and activity of the product are guaranteed. The obtained Dendrobium huoshanense oligosaccharide product has good application prospects in hepatoprotection, providing new raw materials and preparation methods for the development of hepatoprotective drugs or health products. Attached Figure Description

[0016] Figure 1 This is a flowchart of the distribution process of this invention;

[0017] Figure 2 This is a photograph of the hepatoprotective oligosaccharides from Dendrobium huoshanense prepared according to this invention;

[0018] Figure 3 Infrared detection of the hepatoprotective oligosaccharides from Dendrobium huoshanense prepared in this invention;

[0019] Figure 4 These are photographs of cell viability experiments of the present invention. Detailed Implementation

[0020] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0021] Example 1

[0022] This embodiment presents a method for preparing hepatoprotective oligosaccharides from Dendrobium huoshanense, comprising the following steps:

[0023] (1) Preparation of crude polysaccharides from Dendrobium huoshanense

[0024] First, fresh Dendrobium huoshanense stems were selected and treated with impurities removal and low-temperature drying. Then, ethanol was used to remove a large number of fat-soluble small molecule components, followed by heating and extraction with deionized water to obtain crude Dendrobium huoshanense polysaccharides. During the polysaccharide extraction process, the extraction temperature of the polysaccharide solution was controlled at 75℃, the extraction time was 105 minutes, the solid-liquid ratio was 30:1, and the extraction was performed three times.

[0025] (2) Polysaccharide refining

[0026] The crude polysaccharide obtained above was purified. First, ethanol precipitation was used to remove lipid-soluble components, then Seavge reagent was used to remove proteins and nucleic acids. Finally, purified polysaccharide was obtained through steps such as vacuum concentration and freeze centrifugation.

[0027] (3) Polysaccharide enzymatic hydrolysis

[0028] Specific β-mannanase was selected to enzymatically hydrolyze the purified polysaccharide. The hydrolysis time was set to 8 hours, the enzyme concentration was controlled at 0.55 u / mL, the hydrolysis was performed 3 times, and the hydrolysis temperature was maintained at 48℃.

[0029] (4) Oligosaccharide collection

[0030] The enzymatically hydrolyzed polysaccharide solution was subjected to ultrafiltration and centrifugation through a molecular weight cutoff membrane with a molecular weight of 3000 Da to collect oligosaccharides with a molecular weight ≤3000 Da, thereby removing polysaccharide components that were not completely hydrolyzed and obtaining a high-purity Dendrobium huoshanense oligosaccharide solution.

[0031] (5) Finished product preparation

[0032] The oligosaccharide solution of Dendrobium huoshanense collected after enzymatic hydrolysis was concentrated and freeze-dried to obtain the final product of Dendrobium huoshanense oligosaccharide. This product showed significant hepatoprotective activity in an acetaminophen (APAP)-induced HepG2 cell injury model, with a low IC50 value.

[0033] Example 2

[0034] The difference between this embodiment and Embodiment 1 is that:

[0035] During the extraction of polysaccharide samples, the extraction temperature of the polysaccharide solution needs to be controlled at 50℃, the extraction time is 30 minutes, the material-to-liquid ratio is 5:1, and the extraction is performed once.

[0036] The enzymatic hydrolysis time was set to 4 hours, the enzyme concentration was controlled at 0.15 u / mL, the enzymatic hydrolysis was performed twice, and the enzymatic hydrolysis temperature was maintained at 37℃.

[0037] Example 3

[0038] The difference between this embodiment and Embodiment 1 is that:

[0039] During the extraction of polysaccharide samples, the extraction temperature of the polysaccharide solution needs to be controlled at 95℃, the extraction time is 180 minutes, the material-to-liquid ratio is 50:1, and the extraction is performed 5 times.

[0040] The enzymatic hydrolysis time was set to 12 hours, the enzyme concentration was controlled at 10 u / mL, the enzymatic hydrolysis was performed 4 times, and the enzymatic hydrolysis temperature was maintained at 60℃.

[0041] Example 4

[0042] This embodiment presents a method for preparing hepatoprotective oligosaccharides from Dendrobium huoshanense, comprising:

[0043] Material pretreatment: Select fresh Dendrobium officinale stems and strips, remove impurities and dry at low temperature. Then crush the dried Dendrobium officinale stems and strips and pass them through a 24-mesh sieve to collect uniform Dendrobium officinale powder.

[0044] Ethanol extraction: Place 100g of Dendrobium powder in a round-bottom flask, add 2000mL of 80% ethanol solution at a solid-liquid ratio of 1:20, and extract twice to remove lipid-soluble small molecule components from Dendrobium. For the first extraction, add 1000mL of 80% ethanol solution and extract in an 80℃ water bath for 1 hour, then filter. For the second extraction, add another 1000mL of 80% ethanol solution to the residue from the first extraction, and similarly extract in an 80℃ water bath for 1 hour, then filter.

[0045] Water extraction and concentration: The remaining ethanol solution in the round-bottom flask after two filtrations was evaporated to dryness, then 1000 mL of double-distilled water was added, and the mixture was extracted in a 90°C water bath for 2 hours. The liquid was then filtered off. The filtrates were combined and concentrated under reduced pressure using a rotary evaporator until no significant water was evaporated.

[0046] Ethanol precipitation: Anhydrous ethanol was added to the concentrate to adjust the ethanol concentration to 76%. The mixture was then frozen at 4°C and allowed to stand for 12 hours. After standing, it was centrifuged at 4000 rpm for 20 minutes to obtain crude polysaccharide precipitate from Dendrobium huoshanense.

[0047] Protein removal treatment: The crude polysaccharide precipitate was dissolved in a small amount of double-distilled water. The solution was then mixed with the polysaccharide solution using the Sevage method (a mixture of chloroform and n-butanol, volume ratio 4:1) and shaken to denature and precipitate the protein. The precipitated protein was removed by centrifugation to obtain a purified polysaccharide solution after protein removal.

[0048] Freeze-drying: The purified polysaccharide solution after protein removal is freeze-dried to obtain purified polysaccharide powder.

[0049] Enzymatic hydrolysis: Weigh 10 mg of refined polysaccharide and add double-distilled water to prepare a 0.1 mg / mL solution of refined polysaccharide from Dendrobium huoshanense. Take 10 mL of the refined polysaccharide solution and add 2 mL of 2 U / mL β-mannanase. Hydrolyze at 37℃ for 12 hours.

[0050] Ultrafiltration centrifugation: After enzymatic hydrolysis, the solution is centrifuged using a 3000 Da ultrafiltration centrifuge tube to remove residual polysaccharide components that have not been completely hydrolyzed, thus obtaining a Dendrobium huoshanense oligosaccharide solution.

[0051] Activity assay: The obtained oligosaccharide solution was lyophilized, and then the protective effect of Dendrobium huoshanense oligosaccharides in the acetaminophen (APAP)-induced HepG2 cell injury model was determined by the MTT assay.

[0052] Example 5: Preparation of Dendrobium huoshanense oligosaccharides and their application in a cell damage model.

[0053] 1. Preparation of Dendrobium huoshanense powder

[0054] The Dendrobium officinale material from Huoshan was dried and impurities removed, then pulverized and passed through a 24-mesh sieve to collect uniform Dendrobium officinale powder.

[0055] 2. Removal of fat-soluble small molecule components

[0056] Weigh 100g of Dendrobium powder and place it in a round-bottom flask. Add 2000mL of 80% ethanol solution at a material-to-liquid ratio of 1:20 for extraction. First, add 1000mL of 80% ethanol solution and extract in an 80℃ water bath for 1 hour, then filter. Next, add another 1000mL of 80% ethanol solution to the residue from the first step, extract in an 80℃ water bath for 1 hour, and then filter.

[0057] 3. Extraction of crude polysaccharides from Dendrobium huoshanense

[0058] The remaining ethanol solution in the round-bottom flask was evaporated, and 1000 mL of double-distilled water was added. The mixture was extracted in a 95℃ water bath for 2 hours and the liquid was filtered out. The filtrates were combined and concentrated under reduced pressure using a rotary evaporator until no obvious water was evaporated. Then, anhydrous ethanol was added to the concentrate to adjust the ethanol concentration to 76%. The concentrate was then frozen and stored at 4℃. After standing for 12 hours, the mixture was centrifuged at 4000 rpm for 20 minutes to obtain the crude polysaccharide precipitate of Dendrobium huoshanense.

[0059] 4. Preparation of purified polysaccharide solution

[0060] The crude polysaccharide precipitate of Dendrobium huoshanense was dissolved in a small amount of double-distilled water. The Sevage method (a mixed solution of chloroform and n-butanol) was used to mix the polysaccharide solution at a volume ratio of 4:1 and shaken to denature and precipitate the protein. The precipitated protein was removed by centrifugation or separation to obtain a purified polysaccharide solution with the protein removed.

[0061] 5. Preparation of refined polysaccharide powder

[0062] The purified polysaccharide solution after removing proteins is freeze-dried to obtain purified polysaccharide powder.

[0063] 6. Preparation of refined polysaccharide solution from Dendrobium huoshanense

[0064] Weigh 10 mg of refined polysaccharide and add it to double-distilled water to prepare a Dendrobium officinale refined polysaccharide solution with a concentration of 0.1 mg / mL.

[0065] 7. Preparation of Oligosaccharides from Dendrobium huoshanense

[0066] Take 10 mL of purified polysaccharide solution and add 2 mL of 2 u / mL β-mannanase. Enzymatically hydrolyze at 37℃ for 12 h. Select an ultrafiltration centrifuge tube with a molecular weight of 3000 Da for centrifugation to remove residual polysaccharide components that have not been completely hydrolyzed. Freeze-dry the obtained oligosaccharide solution to obtain Dendrobium huoshanense oligosaccharides.

[0067] 8. Application of Dendrobium huoshanense oligosaccharides in cell damage models

[0068] (1) Effect of the sample on cell viability

[0069] After HepG2 cells reached the logarithmic growth phase, they were seeded in 96-well plates (100 µL, 1 × 10⁴ cells / well) and cultured at 37°C for 24 h. Then, 100 µL of samples at different dilutions (complete medium dilutions) were added to the cells, and the cells were incubated at 37°C for 24 h to establish experimental groups. After incubation, cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

[0070] (2) Establishment of an acetaminophen (APAP)-induced HepG2 cell damage model

[0071] HepG2 cell suspension was added to 96-well plates (100 µL, 1 × 10⁴ cells / well) and incubated at 37°C for 24 h, after which the original culture medium was removed. Different doses of APAP were dissolved in fresh culture medium and added to the cells, and incubated at 37°C for 24 h. Similarly, fresh culture medium without APAP was added to the cells as a blank control group. Cell viability was determined using the MTT assay, and the maximum half-inhibitory concentration (IC50) was selected. 50 Further experiments were conducted using APAP.

[0072] (4) Protective effect of Dendrobium huoshanense oligosaccharide samples on acetaminophen-induced HepG2 cells

[0073] HepG2 cell suspension was added to 96-well plates (100 µL, 1 × 10⁴ cells / well) and incubated at 37°C for 24 h. The original culture medium was then removed, and the cells were washed once with PBS. The experiment was divided into four groups: normal control group (100 µL of complete culture medium per well, no drug treatment); model group (100 µL of culture medium containing APAP per well); positive control group (100 µL of culture medium containing silymarin / APAP per well); and experimental group (100 µL of culture medium containing sample (5 µg, 10 µg, 20 µg, 40 µg, 80 µg) / APAP per well). After 24 h of incubation, cell viability was determined using the MTT assay.

[0074] Cell viability = (OD) 测定组 / OD 正常组 )*100%

[0075] Oligosaccharides with a molecular weight <3000 Da were measured, and their IC50 values ​​were [not specified]. 50 120.9 ug / mL

[0076] This experiment successfully prepared oligosaccharides from *Dendrobium huoshanense* with a molecular weight <3000 Da. The feasibility of this preparation was verified by a process combining β-mannan enzymatic hydrolysis and ultrafiltration purification, making it suitable for subsequent functional studies. Experimental results showed that the oligosaccharides had a significant protective effect against APAP-induced HepG2 cell damage (IC50 = 120.9 μg / mL), and its protective mechanism may involve antioxidant and anti-apoptotic pathways. *Dendrobium huoshanense* oligosaccharides demonstrate potential for development as novel hepatoprotective agents and have good application value.

[0077] Example 6: Preparation of Dendrobium huoshanense oligosaccharides and their application in a cell damage model.

[0078] 1. Preparation of Dendrobium huoshanense powder

[0079] The Dendrobium officinale material from Huoshan was dried and impurities removed, then pulverized and passed through a 10-mesh sieve to collect uniform Dendrobium officinale powder.

[0080] 2. Removal of fat-soluble small molecule components

[0081] Weigh 150g of Dendrobium powder and place it in a round-bottom flask. Add 1500mL of 80% ethanol solution at a material-to-liquid ratio of 1:10 for extraction. For the first extraction, add 1500mL of 80% ethanol solution and extract in an 85℃ water bath for 1 hour, then filter. Add another 1500mL of 80% ethanol solution to the residue from the first extraction, extract in an 80℃ water bath for 1 hour, and then filter.

[0082] 3. Extraction of crude polysaccharides from Dendrobium huoshanense

[0083] The remaining ethanol solution in the round-bottom flask was evaporated, and 1500 mL of double-distilled water was added. The mixture was extracted in a 95℃ water bath for 4 hours and the liquid was filtered out. The filtrates were combined and concentrated under reduced pressure using a rotary evaporator until no obvious water was evaporated. Then, anhydrous ethanol was added to the concentrate to adjust the ethanol concentration to 76%. The concentrate was then frozen and stored at 4℃. After standing for 12 hours, the mixture was centrifuged at 5000 rpm for 20 minutes to obtain the crude polysaccharide precipitate of Dendrobium huoshanense.

[0084] 4. Preparation of purified polysaccharide solution

[0085] The crude polysaccharide precipitate of Dendrobium huoshanense was dissolved in a small amount of double-distilled water. The Sevage method (a mixed solution of chloroform and n-butanol) was used to mix the polysaccharide solution at a volume ratio of 4:1 and shaken to denature and precipitate the protein. The precipitated protein was removed by centrifugation or separation to obtain a purified polysaccharide solution with the protein removed.

[0086] 5. Preparation of refined polysaccharide powder

[0087] The purified polysaccharide solution after removing proteins is freeze-dried to obtain purified polysaccharide powder.

[0088] 6. Preparation of refined polysaccharide solution from Dendrobium huoshanense

[0089] Weigh 20 mg of refined polysaccharide and add it to double-distilled water to prepare a Dendrobium officinale refined polysaccharide solution with a concentration of 0.2 mg / mL.

[0090] 7. Preparation of Oligosaccharides from Dendrobium huoshanense

[0091] Take 10 mL of purified polysaccharide solution and add 2 mL of 4 u / mL β-mannanase. Enzymatically hydrolyze at 37℃ for 12 h. Select an ultrafiltration centrifuge tube with a molecular weight of 3000 Da for centrifugation to remove residual polysaccharide components that have not been completely hydrolyzed. Freeze-dry the obtained oligosaccharide solution to obtain Dendrobium huoshanense oligosaccharides.

[0092] 8. Application of Dendrobium huoshanense oligosaccharides in cell damage models

[0093] (1) Effect of the sample on cell viability

[0094] After HepG2 cells reached the logarithmic growth phase, they were seeded in 96-well plates (100 µL, 1 × 10⁴ cells / well) and cultured at 37°C for 24 h. Then, 100 µL of samples at different dilutions (complete medium dilutions) were added to the cells, and the cells were incubated at 37°C for 24 h to establish experimental groups. After incubation, cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

[0095] (2) Establishment of an acetaminophen (APAP)-induced HepG2 cell damage model

[0096] Add HepG2 cell suspension to 96-well plates (100µL, 1×10⁶ cells / well). 4 Cells / well), incubated at 37°C for 24 h, then the original culture medium was removed. Different doses of APAP were dissolved in fresh culture medium and added to the cells, then incubated at 37°C for 24 h. Similarly, fresh culture medium without APAP was added to the cells as a blank control group. Cell viability was determined using the MTT assay, and the maximum half-inhibitory concentration (IC50) was selected. 50 Further experiments were conducted using APAP.

[0097] (4) Protective effect of Dendrobium huoshanense oligosaccharide samples on acetaminophen-induced HepG2 cells

[0098] Add HepG2 cell suspension to 96-well plates (100µL, 1×10⁶ cells / well). 4 Cells were incubated in 100 μL of complete culture medium per well at 37°C for 24 h, then the original culture medium was removed and the cells were washed once with PBS. The experiment was divided into four groups: normal control group (100 μL of complete culture medium per well, without drug treatment); model group (100 μL of culture medium containing APAP per well); positive control group (100 μL of culture medium containing silymarin / APAP per well); and experimental group (100 μL of culture medium containing sample (5 μg, 10 μg, 20 μg, 40 μg, 80 μg) / APAP per well). After 24 h of incubation, cell viability was determined using the MTT assay.

[0099] Cell viability = (OD) 测定组 / OD 正常组 )*100%

[0100] Measure its IC 50 It was 117.2 ug / mL.

[0101] Oligosaccharides from *Dendrobium huoshanense* were successfully prepared through a series of steps, and their protective effect in an APAP-induced HepG2 cell injury model was verified. First, *Dendrobium huoshanense* powder was dried, pulverized, and sieved. Then, lipid-soluble small molecule components were removed by extraction with 80% ethanol solution. Next, crude polysaccharides were obtained by water extraction, and proteins were removed by the Sevage method to obtain a purified polysaccharide solution. Finally, the purified polysaccharide powder was obtained by freeze-drying. The purified polysaccharide solution was then subjected to β-mannanase enzymatic hydrolysis and ultrafiltration centrifugation to obtain *Dendrobium huoshanense* oligosaccharides with a molecular weight <3000 Da. In cell experiments, cell viability was measured by the MTT assay, verifying that *Dendrobium huoshanense* oligosaccharides had a significant protective effect against APAP-induced HepG2 cell injury, with an IC50 value of 117.2 μg / mL. Experimental results show that the preparation process of Dendrobium huoshanense oligosaccharides is feasible, and that it exhibits potential hepatoprotective effects in cell injury models, providing experimental evidence for further research on its mechanism of action and the development of novel hepatoprotective agents.

[0102] The oligosaccharide product of Dendrobium huoshanense prepared by this invention is as follows: Figure 2 As shown in the figure. Mid-infrared spectroscopy revealed characteristic absorption peaks at 3420 cm⁻¹, 2934 cm⁻¹, 1624 cm⁻¹, 1416 cm⁻¹, 1073 cm⁻¹, and 544 cm⁻¹, respectively. These peaks are attributed to stretching vibrations of the OH bond, stretching vibrations of the CH bond, bending / deformation vibrations of the COH or CC bond, and stretching vibrations of the CO and CC bond, fully demonstrating the typical structural features of polyhydroxyl and glycosidic bonds in oligosaccharide molecules. Figure 4 The cell activity results of the Dendrobium oligosaccharide of this invention show that in the acetaminophen (APAP)-induced HepG2 cell injury model, the treatment group with Dendrobium oligosaccharide significantly improved cell survival rate, and its protective effect was comparable to that of the positive control drug. These results indicate that the oligosaccharide product has significant hepatoprotective activity in the in vitro model, and its IC50 value is low, suggesting that it has strong biological activity.

[0103] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A preparation method of Dendrobium huoshanense oligosaccharide having hepatoprotective activity, characterized in that, include: First, fresh Dendrobium huoshanense stems were cleaned and dried at low temperature. Then, ethanol was used to remove lipid-soluble small molecule components, followed by heating and extraction with deionized water to obtain crude polysaccharides. Subsequently, the crude polysaccharides were refined. The specific operations included: alcohol precipitation with ethanol solution to remove lipid-soluble components, followed by removal of proteins and nucleic acids with Seavge reagent, followed by concentration under reduced pressure and freeze centrifugation to obtain refined polysaccharides, followed by enzymatic hydrolysis with specific enzymes, and collection of oligosaccharides with a molecular weight ≤3000 Da by ultrafiltration centrifugation for hepatoprotective activity testing. In the enzymatic hydrolysis step of refined polysaccharides, β-mannanase is selected for enzymatic hydrolysis, the hydrolysis time is 4-12 hours, the enzyme concentration is 0.15u / mL-10u / mL, and the number of enzymatic hydrolysis cycles is 2-4. Finally, the enzymatically hydrolyzed Dendrobium huoshanense oligosaccharide solution was concentrated and freeze-dried to obtain the final Dendrobium huoshanense oligosaccharide product. This product showed significant hepatoprotective activity in an acetaminophen-induced HepG2 cell injury model, with an IC50 value of [missing information]. 50 The value is low, IC 50 The value was 117.2 μg / mL or IC50. 50 It was 120.9 ug / mL.

2. The method of claim 1, wherein: In the preparation of refined polysaccharide samples, the extraction temperature of the polysaccharide solution is controlled at 50-95℃, the extraction time is 30-180 minutes, the material-liquid ratio is 5:1 to 50:1, and the number of extractions is 1-5 times.

3. The method of claim 1, wherein: The enzymatically hydrolyzed polysaccharide solution was subjected to ultrafiltration and centrifugation through a molecular weight cutoff membrane with a molecular weight of 3000 Da to remove incompletely hydrolyzed polysaccharide components, thereby obtaining a high-purity Dendrobium huoshanense oligosaccharide solution.

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