Dendrobium polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to dendrobium polysaccharide as well as a preparation method and application thereof. The method comprises the following steps: preparing dendrobium into dendrobium powder; performing carbon dioxide supercritical extraction on the dendrobium nobile powder by using ethanol as an entrainer to obtain dendrobium nobile powder; performing water extraction on the dendrobium nobile extract powder to obtain a dendrobium nobile water extract; and drying the dendrobe water extract to obtain the dendrobe polysaccharide. The dendrobium polysaccharide obtained by the method is high in purity and large in molecular weight, and has the effects of dispelling the effects of alcohol and protecting liver and intestinal mucosa; the method is simple and convenient to operate, toxic reagents are not introduced in the whole process, the preparation process is green, natural and safe, and high yield, high purity and food safety are considered.

Description

Technical Field

[0001] This application relates to the field of Dendrobium active substance extraction technology, and in particular to a Dendrobium polysaccharide, its preparation method and application. Background Technology

[0002] Dendrobium, a precious traditional Chinese medicine, has been officially included in the list of substances with both medicinal and edible properties, possessing high medicinal value and health benefits. Currently, Dendrobium products on the market are mainly in the form of dried Dendrobium or Dendrobium officinale capsules, with limited deep processing and utilization. Traditional consumption methods cannot fully leverage the active substances in Dendrobium. Dendrobium polysaccharides are one of the recognized effective components of Dendrobium, possessing broad biological activity. Their extraction and purification processes, structural analysis, and efficacy research have become current research hotspots. However, current extraction processes for Dendrobium polysaccharides either yield polysaccharides with low purity, involve complex and energy-intensive extraction steps, or require the use of toxic reagents such as chloroform or n-butanol. Summary of the Invention

[0003] Therefore, it is necessary to provide a Dendrobium polysaccharide, its preparation method, and its application. The Dendrobium polysaccharide prepared by this method has high yield, high purity, and uses simple and safe reagents.

[0004] The first aspect of this application provides a method for preparing Dendrobium polysaccharides, comprising the following steps:

[0005] Dendrobium is processed into Dendrobium powder;

[0006] Ethanol was used as an entrainer to perform supercritical carbon dioxide extraction on the Dendrobium powder to obtain Dendrobium powder.

[0007] The Dendrobium powder is subjected to water extraction to obtain Dendrobium water extract, wherein the water extraction temperature is 60℃~80℃;

[0008] The Dendrobium water extract was dried to obtain the Dendrobium polysaccharide.

[0009] The above method involves preparing Dendrobium officinale powder and then using ethanol as an entrainer. First, supercritical carbon dioxide extraction effectively removes water-soluble polar components such as flavonoids, polyphenols, and pigments from the Dendrobium officinale, allowing them to enter the ethanol extract and preventing their dissolution in the subsequent water extraction step. Simultaneously, supercritical carbon dioxide extraction disrupts the cellulose network structure of the Dendrobium officinale cell walls, promoting the dissolution of Dendrobium officinale polysaccharides in the subsequent water extraction step, creating favorable conditions for the water extraction. Then, water extraction is used to extract the Dendrobium officinale polysaccharides from the powder. Controlling the extraction temperature at 60℃~80℃ not only reduces damage to the polysaccharide structure and effectively preserves the bioactivity of the polysaccharides but also increases the polysaccharide yield. This results in a final Dendrobium officinale polysaccharide with high purity, a high proportion of high molecular weight polysaccharides, and excellent quality. The method is simple to operate, introduces no toxic reagents throughout the process, and is green, natural, and safe, achieving both high yield, high purity, and food safety.

[0010] The Dendrobium polysaccharides prepared by the above method have the effects of relieving hangovers and protecting the liver and intestinal mucosa.

[0011] In some embodiments, the Dendrobium polysaccharide satisfies at least one of the following conditions:

[0012] (1) The purity of the Dendrobium polysaccharide is ≥90%;

[0013] (2) The Dendrobium polysaccharide includes mannose and glucose, and the molar ratio of mannose and glucose is 1 to 5;

[0014] (3) The weight-average molecular weight of the Dendrobium polysaccharide is 8 kDa ~ 2000 kDa;

[0015] (4) The mass percentage of high molecular weight polysaccharides with a weight average molecular weight of 500kDa to 2000kDa in the Dendrobium polysaccharide is 85% to 95%.

[0016] In some embodiments, the water extraction satisfies at least one of the following conditions:

[0017] (1) The temperature of the water extraction is 70℃~80℃;

[0018] (2) The mass ratio of the material to the liquid in the water extraction is 1:30~60;

[0019] (3) The water extraction time is 3h to 4h.

[0020] In some embodiments, after the water extraction and before obtaining the Dendrobium water extract, the following centrifugation step is further included:

[0021] The product extracted by water is centrifuged and the liquid phase after centrifugation is collected to obtain the Dendrobium water extract.

[0022] Optionally, the centrifugal factor for the centrifugal separation is 2000 g to 4000 g;

[0023] Optionally, the centrifugation time is 10 min to 15 min.

[0024] In some embodiments, the supercritical carbon dioxide extraction satisfies at least one of the following conditions:

[0025] (1) The mass of the ethanol accounts for 10% to 20% of the total mass of the ethanol and carbon dioxide;

[0026] (2) The extraction temperature of the supercritical carbon dioxide extraction is 45℃~60℃;

[0027] (3) The extraction pressure of the supercritical carbon dioxide extraction is 15MPa~30MPa;

[0028] (4) The extraction time of the supercritical carbon dioxide extraction is 1h to 2h.

[0029] In some embodiments, the ethanol accounts for 14% to 20% of the total mass of the ethanol and carbon dioxide; and / or,

[0030] The extraction temperature for the supercritical carbon dioxide extraction is 50℃~55℃; and / or,

[0031] The extraction pressure of the supercritical carbon dioxide extraction is 25 MPa to 30 MPa; and / or,

[0032] The extraction time for the supercritical carbon dioxide extraction is 1.2 h to 1.8 h.

[0033] In some embodiments, the particle size of the Dendrobium powder is 0.05 mm to 0.25 mm.

[0034] In some embodiments, the pre-drying step of the Dendrobium extract powder further includes the following drying step:

[0035] The Dendrobium extract was dried at 30℃~40℃ for 2h~4h.

[0036] In a second aspect of this application, a Dendrobium polysaccharide is provided, which is prepared using the method for preparing Dendrobium polysaccharide as described in the first aspect.

[0037] A third aspect of this application provides the use of the Dendrobium polysaccharide described in the second aspect in the preparation of food, health products, or drugs with effects of relieving hangovers, protecting the liver, or protecting the intestinal mucosa. Attached Figure Description

[0038] Figure 1Evaluation of the alcohol-relieving effect of the sample during the excitement period - typical diagram of the total movement distance of zebrafish after treatment (black line is slow movement distance, green line is medium movement distance, red line is fast movement distance).

[0039] Figure 2 Typical image of total movement distance of zebrafish after treatment, used to evaluate the hangover relief efficacy of the sample.

[0040] Figure 3 Typical image of liver fat staining intensity in zebrafish after treatment for evaluating the auxiliary protective efficacy against alcoholic fatty liver (yellow dashed box indicates the analysis area).

[0041] Figure 4 This is a typical pathological section of zebrafish liver after sample processing (red arrows indicate fat-like vacuoles).

[0042] Figure 5 A typical diagram of the number of goblet cells in the zebrafish intestine after sample treatment, which is a model of intestinal mucosal injury (the red dashed box represents the analysis area, and the blue particles represent goblet cells).

[0043] Figure 6 Evaluation of the protective effect of intestinal mucosal secretion - Typical diagram of the number of goblet cells in the zebrafish intestine after sample treatment.

[0044] Figure 7 This is a typical diagram of the number of neutrophils in the zebrafish intestine after sample treatment (the red dashed box represents the analysis area, and the green particles represent neutrophils).

[0045] Figure 8 This is a typical diagram of the zebrafish intestinal lumen area after sample processing (the red dashed box represents the analysis area).

[0046] Figure 9 This is a typical pathological section of zebrafish intestine after sample processing (red arrows indicate intestinal lumen diameter). Detailed Implementation

[0047] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0049] The main nutrients in Dendrobium officinale include polysaccharides, amino acids, alkaloids, flavonoids, phenolic compounds, and some trace elements. Among these, polysaccharides are the core active ingredient. Currently, many extraction processes for Dendrobium officinale polysaccharides involve ultrasonic-assisted negative pressure cavitation, which uses organic solvents such as petroleum ether and cyclohexane, posing potential health risks to consumers. Furthermore, the use of ultrasound accelerates the degradation of polysaccharides during extraction, damaging their structure and resulting in low yields and purity. Other methods employ microwave-assisted solvent extraction and cellulase co-processing, achieving higher polysaccharide yields. However, these methods use toxic reagents such as chloroform and n-butanol in defatting and protein removal steps, and are cumbersome, energy-intensive, and unsuitable for industrial production. Current Dendrobium polysaccharide extraction processes struggle to balance polysaccharide yield, purity, and food safety.

[0050] Based on this, the first aspect of this application provides a method for preparing Dendrobium polysaccharides, comprising the following steps:

[0051] Dendrobium is processed into Dendrobium powder;

[0052] Ethanol was used as an entrainer to perform supercritical carbon dioxide extraction on the Dendrobium powder to obtain Dendrobium powder.

[0053] The Dendrobium powder is subjected to water extraction to obtain Dendrobium water extract, wherein the water extraction temperature is 60℃~80℃;

[0054] The Dendrobium water extract was dried to obtain the Dendrobium polysaccharide.

[0055] The above method involves preparing Dendrobium officinale powder and then using ethanol as an entrainer. First, supercritical carbon dioxide extraction effectively removes water-soluble polar components such as flavonoids, polyphenols, and pigments from the Dendrobium officinale, preventing these components from dissolving in the subsequent water extraction step. Simultaneously, supercritical carbon dioxide extraction disrupts the cellulose network structure of the Dendrobium officinale cell walls, promoting the dissolution of Dendrobium officinale polysaccharides in the subsequent water extraction step, creating favorable conditions for the water extraction process. Then, water extraction is used to extract the Dendrobium officinale polysaccharides from the powder. Controlling the extraction temperature to 60℃~80℃ not only reduces damage to the polysaccharide structure and effectively preserves the bioactivity of the polysaccharides but also increases the polysaccharide yield. This results in a final Dendrobium officinale polysaccharide with high purity, a high proportion of high molecular weight polysaccharides, and excellent quality. The method is simple to operate, introduces no toxic reagents throughout the process, and is green, natural, and safe, achieving a balance between high yield, high purity, and food safety.

[0056] The Dendrobium polysaccharides prepared by the above method have the effects of relieving hangovers and protecting the liver and intestinal mucosa.

[0057] Understandably, Dendrobium includes, but is not limited to, Dendrobium officinale, Dendrobium huoshanense, Dendrobium nobile, Dendrobium chrysanthum, and Dendrobium fimbriatum. Dendrobium polysaccharides include, but are not limited to, Dendrobium officinale polysaccharides, Dendrobium huoshanense polysaccharides, Dendrobium nobile polysaccharides, Dendrobium chrysanthum polysaccharides, and Dendrobium fimbriatum polysaccharides.

[0058] As an example, the water extraction temperature can be 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, and 80℃, or it can be within the range formed by any two of the above point values ​​as endpoints.

[0059] Furthermore, the water extraction temperature is 70℃~80℃. Controlling the water extraction temperature at 70℃~80℃ not only further improves the polysaccharide yield through cell swelling, but also helps protect the integrity of β-glycosidic bonds, resulting in a high proportion of high molecular weight polysaccharides. It can also selectively inhibit the dissolution of impurities such as proteins and pectin, further improving the purity of Dendrobium polysaccharides.

[0060] In some embodiments, the particle size of the Dendrobium powder is ≤0.25 mm.

[0061] Furthermore, the particle size of the Dendrobium powder is 0.05 mm to 0.25 mm, preferably 0.10 mm to 0.18 mm.

[0062] In some of these implementations, edible ethanol is used as an entrainer.

[0063] In some of these embodiments, the purity of Dendrobium polysaccharides is ≥90%.

[0064] In some embodiments, Dendrobium polysaccharides include mannose and glucose, with a molar ratio of mannose to glucose of 1 to 5.

[0065] Furthermore, Dendrobium polysaccharides include mannose and glucose, with a molar ratio of mannose to glucose of 2 to 4.

[0066] In some embodiments, the weight-average molecular weight of Dendrobium polysaccharides is 8 kDa to 2000 kDa.

[0067] In some embodiments, the mass percentage of high molecular weight polysaccharides (500kDa~2000kDa) in Dendrobium polysaccharides is 85%~95%. The high proportion of high molecular weight polysaccharides in Dendrobium polysaccharides ensures the complete preservation of the polysaccharide structure and effective retention of biological activity, resulting in higher quality Dendrobium polysaccharides.

[0068] In some embodiments, the material-to-liquid mass ratio for water extraction is 1:30~60. This mass ratio can improve the dissolution efficiency of Dendrobium polysaccharides and promote the maintenance of good molecular state and structural integrity of Dendrobium polysaccharides during extraction, while maintaining suitable fluid viscosity, thus achieving a proper balance between the dissolution efficiency of Dendrobium polysaccharides and the quality of the obtained polysaccharides.

[0069] Understandably, the material-liquid mass ratio in water extraction refers to the mass ratio of Dendrobium officinale extract powder to water.

[0070] As an example, the mass ratio of feed to liquid in water extraction can be 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, and 1:60, or any two of the above values ​​can be used as endpoints within the range.

[0071] Furthermore, the material-to-liquid mass ratio in water extraction can be 1:30~45. At this mass ratio, the yield and purity of Dendrobium polysaccharides are further improved.

[0072] In some of these embodiments, the water extraction time is 3 to 4 hours.

[0073] Furthermore, the water extraction time is 3.2 h to 4 h.

[0074] In some embodiments, after water extraction and before obtaining the Dendrobium aqueous extract, the following centrifugation separation step is also included:

[0075] The product extracted by water is centrifuged and the liquid phase after centrifugation is collected to obtain the Dendrobium water extract.

[0076] Furthermore, the centrifugal factor for centrifugal separation is 2000 g to 4000 g.

[0077] Furthermore, the centrifugation time is 10 min to 15 min.

[0078] In some embodiments, during supercritical carbon dioxide extraction, the mass of ethanol accounts for 10% to 30% of the total mass of ethanol and carbon dioxide.

[0079] As an example, the mass of ethanol can be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, and 30% of the total mass of ethanol and carbon dioxide, or it can be within the range formed by any two of the above point values ​​as endpoints.

[0080] Furthermore, in supercritical carbon dioxide extraction, the mass of ethanol accounts for 10% to 20% of the total mass of ethanol and carbon dioxide. Controlling the mass ratio of ethanol at 10% to 20% not only allows for precise dissolution of polyphenols and pigments, further improving the purity of Dendrobium polysaccharides, but also maintains a low-viscosity fluid environment to prevent polysaccharide degradation. In addition, it can optimize the swelling degree of cellulose and further improve the water extraction yield.

[0081] Furthermore, the mass of ethanol accounts for 14% to 20% of the total mass of ethanol and carbon dioxide. Controlling the mass of ethanol to 14% to 20% of the total mass of ethanol and carbon dioxide can simultaneously improve the purity and yield of Dendrobium polysaccharides.

[0082] Furthermore, the mass of ethanol accounts for 14% to 18% of the total mass of ethanol and carbon dioxide. At this ratio, the purity and yield of Dendrobium polysaccharides can be further improved.

[0083] In some embodiments, the extraction temperature of supercritical carbon dioxide extraction is 45°C to 60°C.

[0084] Furthermore, the extraction temperature for supercritical carbon dioxide extraction is 50℃~55℃.

[0085] In some embodiments, the extraction pressure of supercritical carbon dioxide extraction is 15 MPa to 30 MPa.

[0086] Furthermore, the extraction pressure for supercritical carbon dioxide extraction is 25 MPa to 30 MPa.

[0087] In some embodiments, the extraction time for supercritical carbon dioxide extraction is 1 to 2 hours.

[0088] Furthermore, the extraction time for supercritical carbon dioxide extraction is 1.2 h to 1.8 h.

[0089] In some embodiments, the pre-treatment of Dendrobium powder with water also includes the following drying step:

[0090] The Dendrobium powder was dried at 30℃~40℃ for 2h~4h.

[0091] A second aspect of this application provides a Dendrobium polysaccharide prepared using the method for preparing Dendrobium polysaccharide as described in the first aspect.

[0092] A third aspect of this application provides the use of Dendrobium polysaccharide from the second aspect in the preparation of food, health products, or pharmaceuticals with effects of relieving hangovers, protecting the liver, or protecting the intestinal mucosa.

[0093] To make the objectives, technical solutions, and advantages of this application clearer and more concise, the following specific embodiments are used for illustration, but this application is by no means limited to these embodiments. The embodiments described below are merely preferred embodiments of this application and can be used to describe this application, but should not be construed as limiting the scope of this application. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

[0094] To better illustrate this application, the following description, in conjunction with specific embodiments, further explains its content. The following are specific embodiments.

[0095] The main raw materials in the following examples are from the following sources:

[0096] The edible ethanol is a commercially available product of COFCO Biochemical (Anhui) Co., Ltd., with a volume content of 95%.

[0097] Unless otherwise specified, all raw materials and reagents used in the following examples can be purchased commercially.

[0098] Example 1

[0099] The dried Dendrobium officinale strips were crushed and sieved to obtain 100g of Dendrobium officinale powder with a particle size of 0.05mm~0.25mm;

[0100] Dendrobium powder was placed in an extraction vessel, and edible ethanol was used as an entrainer. The edible ethanol was pumped into the carbon dioxide fluid and then into the extraction vessel. The mass ratio of edible ethanol to the total mass of edible ethanol and carbon dioxide was 14 wt%. This allowed the Dendrobium powder to fully contact, dissolve, and extract with supercritical carbon dioxide and edible ethanol. The extraction temperature was set at 45℃ and the extraction pressure at 20 MPa. After supercritical carbon dioxide extraction for 2 hours, an alcohol extract and Dendrobium powder were obtained. The alcohol extract contained water-soluble polar components such as flavonoids, polyphenols, and pigments, while the Dendrobium powder contained effective components such as Dendrobium polysaccharides.

[0101] After drying the Dendrobium ethanol extract powder at 30℃ for 4 hours to remove ethanol, it was put into an extraction vessel for polysaccharide water extraction. The mass ratio of material to liquid during water extraction was 1:60, the water extraction temperature was 70℃, and the water extraction time was 3 hours. After water extraction, the Dendrobium water extract was obtained by centrifugation at a centrifugation factor of 2000g for 15 minutes.

[0102] High-purity Dendrobium officinale polysaccharide was obtained by freeze-drying the aqueous extract of Dendrobium officinale.

[0103] Example 2

[0104] The dried Dendrobium officinale strips were crushed and sieved to obtain 100g of Dendrobium officinale powder with a particle size of 0.05mm~0.22mm;

[0105] Dendrobium powder was placed in an extraction vessel, and edible ethanol was used as an entrainer. The mass ratio of edible ethanol to the total mass of ethanol and carbon dioxide was 10 wt%. The extraction temperature was set to 50℃ and the pressure to 15 MPa. After supercritical carbon dioxide extraction for 1 hour, Dendrobium ethanol powder was obtained.

[0106] After drying the Dendrobium ethanol extract powder at 35℃ for 3.5h to remove ethanol, it was put into the extraction vessel for polysaccharide water extraction. The mass ratio of material to liquid during water extraction was 1:50, the water extraction temperature was 75℃, and the water extraction time was 3.5h. After water extraction, the Dendrobium water extract was obtained by centrifugation at a centrifugation factor of 2500g and a centrifugation time of 13min.

[0107] High-purity Dendrobium officinale polysaccharide was obtained by vacuum drying of the Dendrobium officinale aqueous extract.

[0108] Example 3

[0109] The dried Dendrobium officinale strips were crushed and sieved to obtain 100g of Dendrobium officinale powder with a particle size of 0.10mm~0.18mm;

[0110] Dendrobium powder was placed in an extraction vessel, and edible ethanol was used as an entrainer. The mass ratio of edible ethanol to the total mass of ethanol and carbon dioxide was 16 wt%. The extraction temperature was set at 50℃ and the pressure at 25 MPa. After supercritical carbon dioxide extraction for 1.5 h, Dendrobium ethanol powder was obtained.

[0111] After drying the Dendrobium ethanol extract powder at 35℃ for 3 hours to remove ethanol, it was put into the extraction vessel for polysaccharide water extraction. The material-to-liquid mass ratio during water extraction was 1:40, the water extraction temperature was 80℃, and the water extraction time was 4 hours. After the extraction, the Dendrobium water extract was obtained by centrifugation at a centrifugation factor of 3500g and a centrifugation time of 12 minutes.

[0112] High-purity Dendrobium officinale polysaccharide was obtained by freeze-drying the aqueous extract of Dendrobium officinale.

[0113] Example 4

[0114] The dried Dendrobium officinale strips were crushed and sieved to obtain 100g of Dendrobium officinale powder with a particle size of 0.10mm~0.15mm;

[0115] Dendrobium powder was placed in an extraction vessel, and edible ethanol was used as an entrainer. The mass ratio of edible ethanol to the total mass of ethanol and carbon dioxide was 18 wt%. The extraction temperature was set at 55℃ and the pressure at 25 MPa. After supercritical carbon dioxide extraction for 1.2 h, Dendrobium ethanol powder was obtained.

[0116] After drying the Dendrobium officinale ethanol extract powder at 30℃ for 2 hours to remove ethanol, it was put into an extraction vessel for polysaccharide water extraction. The material-to-liquid ratio during water extraction was 1:30, the temperature was 72℃, and the time was 3.5 hours. After the extraction, the Dendrobium officinale water extract was obtained by centrifugation at a factor of 3000g for 10 minutes. The Dendrobium officinale water extract was then dried at low temperature to obtain high-purity Dendrobium officinale polysaccharide.

[0117] Example 5

[0118] The dried Dendrobium officinale strips were crushed and sieved to obtain 100g of Dendrobium officinale powder with a particle size of 0.05mm~0.18mm;

[0119] Dendrobium powder was placed in an extraction vessel, and edible ethanol was used as an entrainer. The mass ratio of edible ethanol to the total mass of ethanol and carbon dioxide was 20 wt%. The extraction temperature was set at 55℃ and the pressure at 30 MPa. After supercritical carbon dioxide extraction for 1.8 h, Dendrobium ethanol powder was obtained.

[0120] After drying the Dendrobium ethanol extract powder at 30℃ for 3 hours to remove ethanol, it was put into the extraction vessel for polysaccharide water extraction. The mass ratio of material to liquid during water extraction was 1:45, the water extraction temperature was 78℃, and the water extraction time was 3.2 hours. After water extraction, the Dendrobium water extract was obtained by centrifugation at a centrifugation factor of 3700g and a centrifugation time of 12 minutes.

[0121] High-purity Dendrobium officinale polysaccharide was obtained by vacuum drying of the Dendrobium officinale aqueous extract.

[0122] Example 6

[0123] The dried Dendrobium officinale strips were crushed and sieved to obtain 100g of Dendrobium officinale powder with a particle size of 0.05mm~0.11mm;

[0124] Dendrobium powder was placed in an extraction vessel, and edible ethanol was used as an entrainer. The mass ratio of edible ethanol to the total mass of ethanol and carbon dioxide was 12 wt%. The extraction temperature was set to 60℃ and the pressure to 27 MPa. After supercritical carbon dioxide extraction for 2 hours, Dendrobium ethanol powder was obtained.

[0125] After drying the Dendrobium ethanol extract powder at 33℃ for 3 hours to remove ethanol, it was put into an extraction vessel for polysaccharide water extraction. The material-to-liquid mass ratio during water extraction was 1:45, the water extraction temperature was 80℃, and the water extraction time was 4 hours. After water extraction, the Dendrobium water extract was obtained by centrifugation at a centrifugation factor of 4000g and a centrifugation time of 15 minutes.

[0126] High-purity Dendrobium officinale polysaccharide is obtained by drying the water extract of Dendrobium officinale at low temperature.

[0127] Example 7

[0128] Example 7 is basically the same as Example 5, except that the water extraction temperature is 60°C.

[0129] Example 8

[0130] Example 8 is basically the same as Example 5, except that the mass of edible ethanol accounts for 30 wt% of the total mass of edible ethanol and carbon dioxide.

[0131] Comparative Example 1

[0132] Comparative Example 1 is basically the same as Example 5, except that the supercritical carbon dioxide extraction step is omitted and the Dendrobium officinale powder is directly extracted with water.

[0133] Comparative Example 2

[0134] Comparative Example 2 is basically the same as Example 5, except that the water extraction temperature is 25°C.

[0135] Comparative Example 3

[0136] Comparative Example 3 is basically the same as Example 5, except that the water extraction temperature is 90°C.

[0137] Comparative Example 4

[0138] Comparative Example 4 is basically the same as Example 5, except that water extraction is performed first, followed by supercritical carbon dioxide extraction. The specific process is as follows:

[0139] The dried Dendrobium officinale strips were pulverized and sieved to obtain 100g of Dendrobium officinale powder with a particle size of 0.05mm~0.18mm. The Dendrobium officinale powder was then subjected to polysaccharide water extraction. The material-to-liquid mass ratio during water extraction was 1:45, the extraction temperature was 78℃, and the extraction time was 3.2h. After water extraction, the Dendrobium officinale water extract was obtained by centrifugation at a centrifugation factor of 3700g for 12min. The Dendrobium officinale water extract was then vacuum dried and placed in an extraction vessel. Edible ethanol was used as an entrainer, with the mass ratio of edible ethanol to the total mass of ethanol and Dendrobium officinale powder being 20wt%. The extraction temperature was set at 55℃, the pressure at 30MPa, and supercritical carbon dioxide extraction was performed for 1.8h to obtain Dendrobium officinale polysaccharide.

[0140] The yield, purity, and weight-average molecular weight of the Dendrobium officinale polysaccharides prepared in each embodiment and comparative example were tested, and the test results are shown in Table 1 below.

[0141] The yield of Dendrobium officinale polysaccharide refers to the mass percentage of the obtained Dendrobium officinale polysaccharide to Dendrobium officinale powder.

[0142] The purity test method for Dendrobium officinale polysaccharides is based on the content determination of polysaccharides under the Dendrobium officinale section of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0143] The molar ratio of mannose to glucose in Dendrobium officinale polysaccharides was determined by high performance ion exchange chromatography. Sample preparation: 5.00 mg of Dendrobium officinale polysaccharide sample was weighed into a stoppered test tube, and 0.5 mL of 12 M H2SO4 was slowly added under ice bath conditions. The mixture was magnetically stirred for 30 min, followed by the addition of 2.5 mL of deionized water. After mixing, the mixture was hydrolyzed in an oil bath at 100℃ for 2 h. After cooling to room temperature, the volume was adjusted to 50 mL, and a certain amount of the solution was diluted 5 times. The solution was then filtered through a 0.22 µm aqueous filter membrane before injection for analysis. Chromatographic analysis conditions: Thermo Fisher ion chromatograph, Carbo Pac-10 ... TM PA20 analytical column, integrating amperometric detector, mobile phase: a mixture of 94.2% water, 0.8% sodium hydroxide (250mM molar concentration) and 5% sodium acetate (1M molar concentration), column temperature 30℃;

[0144] The weight-average molecular weight of Dendrobium polysaccharides was determined by high-performance gel permeation chromatography. The chromatographic conditions were as follows: Waterse2695 chromatograph, Waters Ultrahydrogel™ Linear (7.8×300 mm) gel column, differential detector, mobile phase of 0.1M NaNO3 solution, and column temperature of 35℃.

[0145] Table 1

[0146]

[0147] Note: The mass percentage of high molecular weight Dendrobium in Table 1 refers to the mass percentage of Dendrobium polysaccharides with a molecular weight of 500kDa≤Mw≤2000kDa.

[0148] As shown in Table 1 above, the examples used Dendrobium officinale powder as raw material and ethanol as an entrainer. First, supercritical carbon dioxide extraction was used to effectively remove water-soluble polar components such as flavonoids, polyphenols, and pigments from Dendrobium officinale under mild conditions. Then, Dendrobium officinale polysaccharides were extracted by water extraction. Without adding any additional organic solvents (such as petroleum ether and chloroform commonly used in the industry), not only was the yield of Dendrobium officinale polysaccharides high, but the purity of the obtained Dendrobium officinale polysaccharides also reached over 90%, with high molecular weight Dendrobium officinale polysaccharides accounting for 85% to 95%. This indicates that the polysaccharide structure was intact, the biological activity was effectively preserved, and the obtained Dendrobium officinale polysaccharides were of good quality.

[0149] Moreover, when the water extraction temperature is 70℃~80℃ and the mass of ethanol accounts for 10%~20% of the total mass of ethanol and carbon dioxide, the yield and purity of Dendrobium polysaccharides are higher.

[0150] The high-purity Dendrobium polysaccharide prepared in Example 3 was tested for its effects on relieving hangovers, protecting the liver, and protecting the intestinal mucosa.

[0151] (i) Study on the effects of high-purity Dendrobium officinale polysaccharide (named SHD) prepared in Example 3 on hangover relief and liver protection.

[0152] Experimental animals: Zebrafish were raised in aquarium water at 28℃ (water quality: 200 mg of quick-dissolving sea salt was added to every 1L of reverse osmosis water, with an electrical conductivity of 450 μS / cm ~ 550 μS / cm; pH of 6.5 ~ 8.5; and hardness of 50 ~ 100 mg / L CaCO3).

[0153] Experimental plan:

[0154] 1. Determination of Maximum Detectable Concentration (MTC)

[0155] Wild-type AB strain zebrafish, 5 days post-fertilization (5 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The samples were administered in water solution, and a normal control group was also included. The volume per well was 3 mL. After treatment at 28℃ for 1 day, the MTC of the samples relative to normal zebrafish was measured.

[0156] 2. Evaluation of the efficacy of alcohol detoxification during the excitement phase

[0157] Wild-type AB strain zebrafish with a 5-day pf (dpf) count were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The samples were administered in water-soluble form, with a positive control of 1000 µg / mL of milk thistle, kudzu root, and danshen tablets. A normal control group and a model control group were also included, with a volume of 3 mL per well. After treatment at 28℃ for one day, 10 zebrafish from each experimental group were randomly transferred to 96-well plates. Except for the normal control group, all other experimental groups were given anhydrous ethanol in water-soluble form to establish a zebrafish intoxication model during the excitation phase. The zebrafish were immediately placed in a behavioral analyzer, and the total movement distance within 1 hour was measured. The statistical analysis results of this index were used to evaluate the efficacy of the samples in alleviating intoxication during the excitation phase. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 was considered statistically significant.

[0158] Note: p-value is a statistical concept used to quantify the probability of observed data occurring under the condition that the null hypothesis is true; a small p-value (e.g., p < 0.05) indicates that the observed difference is unlikely to be caused by random error alone, thus rejecting the null hypothesis and considering the difference to be statistically significant.

[0159] 3. Evaluation of the efficacy of hangover remedy

[0160] Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). A normal control group and a model control group were also set up. Except for the normal control group, all experimental groups were first treated with anhydrous ethanol for 1 hour to establish a zebrafish hangover model, with a well volume of 3 mL. After removing the ethanol, the samples were treated with a 1000 µg / mL concentration of *Smilax glabra*, *Pueraria lobata*, and *Salvia miltiorrhiza* tablets (a positive control), at 28℃ for 1 hour. Ten zebrafish from each experimental group were then randomly transferred to 96-well plates and immediately placed in a behavioral analyzer. The total distance the zebrafish moved within 1 hour was measured using the behavioral analyzer. The hangover relief efficacy of the samples was evaluated based on the statistical analysis results of this index. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance.

[0161] 4. Evaluation of the effects of alcohol detoxification and liver protection (ADH, ALDH)

[0162] Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). A normal control group and a model control group were also set up. Except for the normal control group, all experimental groups were first treated with anhydrous ethanol for 1 h to establish a zebrafish hangover model, with a well volume of 3 mL. After removing the ethanol, the samples were treated with water, and a positive control of 1000 µg / mL of milk thistle, kudzu root, and danshen tablets was administered. Six experiments were conducted in parallel. After treatment at 28℃ for 2 h, ADH and ALDH assay kits were used, and data were collected using a multi-functional microplate reader to analyze the activities of ADH (alcohol dehydrogenase) and ALDH (acetaldehyde dehydrogenase) in zebrafish. The statistical analysis results of these indicators were used to evaluate the hangover relief and liver protection efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0163] 5. Evaluation of the adjuvant protective effect against alcoholic fatty liver disease

[0164] Wild-type AB strain zebrafish with a 5-day pf (dpf) count were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered via water-soluble solution. A positive control (Giant's Milk Thistle, Puerariae Radix et Rhizoma, and Salviae Miltiorrhizae Radix et Rhizoma) at a concentration of 1000 µg / mL was used. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were administered anhydrous ethanol to establish a zebrafish alcoholic fatty liver model. After treatment at 28℃ for 30 h, samples were collected, stained with Oil Red O, and bleached. Ten zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the intensity of liver fat staining was analyzed. The statistical analysis results of this index were used to evaluate the auxiliary protective efficacy against alcoholic fatty liver. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance.

[0165] 6. Evaluation of the effects of alcohol detoxification and liver protection (liver pathology slides)

[0166] Wild-type AB strain zebrafish with a 5-day pf (dpf) count were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered via water-soluble solution. A positive control (Giant's Milk Thistle, Puerariae Radix et Rhizoma, and Salviae Miltiorrhizae Radix et Rhizoma) at a concentration of 1000 µg / mL was used. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were administered anhydrous ethanol to establish a zebrafish alcoholic fatty liver model. After treatment at 28℃ for 30 h, the zebrafish were fixed with 4% histocellular fixative. Following a series of steps including dehydration, embedding, sectioning, and staining, histopathological H&E staining analysis was performed on the zebrafish. The hepatoprotective and alcohol-detoxifying effects of the samples were evaluated through histopathological analysis of the liver tissue.

[0167] Experimental conclusion:

[0168] 1. Determination of Maximum Detectable Concentration (MTC)

[0169] Under the conditions of this experiment, the MTC of the alcohol-relieving efficacy of SHD during the excitement phase was 1000 μg / mL. See Table 2 for the results of the concentration exploratory experiment on the alcohol-relieving efficacy of the samples during the excitement phase (n is the number of zebrafish, n = 30).

[0170] Table 2

[0171]

[0172] 2. Evaluation of the efficacy of alcohol detoxification during the excitement phase

[0173] Under the conditions of this experiment, SHD demonstrated the effect of relieving hangovers during the excitement phase. See Table 3 for details of the experimental results evaluating the hangover-relieving effect of the samples during the excitement phase (n = 10). Figure 1Compared with the normal control group, the total movement distance of the model group increased significantly, indicating that the model of intoxication during the excitement period was successfully established; the total movement distance of the zebrafish after SHD treatment was reduced compared with the model group, indicating that SHD has the effect of relieving intoxication during the excitement period.

[0174] Table 3

[0175]

[0176] Note: * represents p < 0.05; *** represents p < 0.001; the smaller the p value, the more significant the difference.

[0177] 3. Evaluation of the efficacy of hangover remedy

[0178] Under the experimental conditions, SHD demonstrated hangover relief effects. See Table 4 for details of the hangover relief efficacy evaluation results (n = 10). Figure 2 (The black line represents the distance of slow movement, the green line represents the distance of medium movement, and the red line represents the distance of fast movement.) Compared with the normal control group, the total movement distance of the model group was significantly reduced, indicating that the hangover model was successfully established. The total movement distance of the zebrafish after SHD treatment was increased compared with the model group, indicating that SHD has the effect of relieving hangovers.

[0179] Table 4

[0180]

[0181] 4. Evaluation of the effects of alcohol detoxification and liver protection (ADH, ALDH)

[0182] Under the experimental conditions, SHD increased the activities of ALDH and ADH. See Table 5 for the experimental results of the hangover relief and liver protection efficacy evaluation (ADH, ALDH) of the samples (n = 3). Compared with the normal control group, the ALDH and ADH activities in the model group were significantly decreased, indicating that the hangover model was successfully established. The ALDH and ADH activities after SHD treatment increased compared with the model group, indicating that SHD can increase the activities of ALDH and ADH and has the effect of relieving hangover and protecting the liver.

[0183] Table 5

[0184]

[0185] 5. Evaluation of the adjuvant protective effect against alcoholic fatty liver disease

[0186] Under the experimental conditions, SHD showed adjunctive protective efficacy against alcoholic fatty liver disease. See Table 6 for the experimental results evaluating the adjunctive protective efficacy against alcoholic fatty liver disease (n = 10). Figure 3Compared with the normal control group, the liver fat staining intensity in the model group was significantly increased, indicating that the alcoholic fatty liver model was successfully established; the liver fat staining intensity after SHD treatment was reduced compared with the model group, indicating that SHD has an auxiliary protective effect against alcoholic fatty liver.

[0187] Table 6

[0188]

[0189] 6. Evaluation of the effects of alcohol detoxification and liver protection (liver pathology slides)

[0190] Under the experimental conditions, histopathological examination of the liver in the normal control group showed that the zebrafish liver cells had clear outlines, no visible swelling or vacuoles, uniform cytoplasm distribution, no visible foreign bodies, and regular-sized, interlaced nuclei. Histopathological examination of the liver in the model control group showed a clear alcoholic fatty liver phenotype, characterized by unclear outlines of the nuclei and cytoplasm, and obvious fat-like vacuoles (indicated by red arrows). Histopathological examination of the liver in the positive control group treated with Jian An Shi milk thistle, kudzu root, and danshen tablets showed a significant improvement in the alcoholic fatty liver phenotype, suggesting that Jian An Shi milk thistle, kudzu root, and danshen tablets have an auxiliary effect in improving alcoholic fatty liver in zebrafish.

[0191] Histopathological examination of zebrafish livers at SHD concentrations of 500–1000 μg / mL showed improvement in the phenotype of alcoholic fatty liver, suggesting that the above concentration groups all had an auxiliary protective effect against alcoholic fatty liver. The livers of zebrafish at SHD concentrations of 250 μg / mL were similar to those in the model control group, suggesting that this concentration group did not have a significant auxiliary protective effect against alcoholic fatty liver.

[0192] In conclusion, under the experimental conditions, SHD has an adjunctive protective effect against alcoholic fatty liver disease. See details. Figure 4 .

[0193] (ii) Study on the intestinal mucosal protective efficacy of the high-purity Dendrobium officinale polysaccharide (named SHD) prepared in Example 3.

[0194] Experimental animals: Zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of quick-dissolving sea salt was added to every 1L of reverse osmosis water, conductivity was 450~550 μS / cm; pH was 6.5~8.5; hardness was 50~100 mg / L CaCO3).

[0195] Experimental plan:

[0196] 1. Model establishment and positive validation

[0197] Zebrafish with a 5 dpf melanin allele mutation (albino) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, an intestinal mucosal injury model was established by water-soluble administration of ethanol. Positive controls were administered mesalazine at concentrations of 125, 250, and 500 μg / mL (1.5% ethanol). After treatment at 28℃ for 1 day, the zebrafish were stained with Alcian blue. After staining, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Images were saved, and data were collected using NIS-Elements D 3.20 advanced image processing software. The number of goblet cells in the zebrafish intestine was analyzed. Statistical analysis of this index was used to evaluate the establishment of the ethanol-induced intestinal mucosal injury model and the protective effect of mesalazine on intestinal mucosal secretion. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 was considered statistically significant.

[0198] 2. Determination of Maximum Detectable Concentration (MTC)

[0199] Zebrafish (albino) with a melanin allele mutation, 5 days post-fertilization, were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water solution. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were treated with water solution of ethanol to establish a zebrafish intestinal mucosal injury model. After treatment at 28℃ for 1 day, the MTC of the samples in the model zebrafish was measured.

[0200] 3. Evaluation of the protective effect of intestinal mucosal secretion (goblet cells)

[0201] Zebrafish with a 5 dpf melanin allele mutation (albino) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered via water-soluble solution, with a positive control of mesalazine at a concentration of 250 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were treated with water-soluble ethanol to establish a zebrafish intestinal mucosal injury model. After treatment at 28℃ for 1 day, the samples were stained with Alcian blue. After staining, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Images were saved, and data were collected using NIS-Elements D 3.20 advanced image processing software. The number of goblet cells in the zebrafish intestine was analyzed, and the statistical analysis results of this index were used to evaluate the protective efficacy of intestinal mucosal secretion. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0202] 4. Evaluation of the protective effect of intestinal mucosal secretion (neutrophils)

[0203] Five-day-first-flush (dpf) transgenic green fluorescent zebrafish (MPX) with neutrophils were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered via water-soluble solution, with a positive control of mesalazine at a concentration of 250 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were treated with water-soluble ethanol to establish a zebrafish intestinal mucosal injury model. After treatment at 28℃ for one day, 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the number of neutrophils in the zebrafish intestine. The statistical analysis results of this index were used to evaluate the protective efficacy of intestinal mucosal secretion. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0204] 5. Evaluation of the protective effect of intestinal mucosal secretion (intestinal lumen area)

[0205] Five-day-first-flush (dpf) transgenic neutrophil-positive green fluorescent zebrafish (MPX) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered via water-soluble solution, with a positive control of mesalazine at a concentration of 250 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were treated with water-soluble ethanol to establish a zebrafish intestinal mucosal injury model. After treatment at 28℃ for one day, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the intestinal lumen area of ​​the zebrafish was analyzed. The statistical analysis results of this index were used to evaluate the protective efficacy of intestinal mucosal secretion. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0206] 6. Evaluation of the protective effect of intestinal mucosal secretion (intestinal pathological sections)

[0207] Wild-type AB strain zebrafish with a 5-day pf (dpf) count were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water, with a positive control of mesalazine at a concentration of 250 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were treated with water-soluble ethanol to establish a zebrafish intestinal mucosal injury model. After treatment at 28℃ for 1 day, the zebrafish were fixed with 4% histocellular fixative. Following a series of steps including dehydration, embedding, sectioning, and staining, the zebrafish underwent histopathological H&E staining analysis. The hepatoprotective and alcohol-detoxifying effects of the samples were evaluated through liver histopathological analysis.

[0208] Experimental conclusion:

[0209] 1. Model establishment and positive validation

[0210] Under the experimental conditions, 1.5% ethanol induced an intestinal mucosal injury model, specifically manifested as a decrease in the number of intestinal goblet cells; mesalazine 250 μg / mL exhibited protective efficacy against intestinal mucosal secretion, specifically manifested as an increase in the number of intestinal goblet cells compared to the model group. See Table 7 for details of the model establishment and positive validation experimental results (n = 10). Figure 5 .

[0211] Table 7

[0212]

[0213] 2. Determination of Maximum Detectable Concentration (MTC)

[0214] Under the conditions of this experiment, the MTC of SHD's protective effect against intestinal mucosal secretion was 1000 μg / mL. See Table 8 for the results of the concentration exploratory experiment on the protective effect against intestinal mucosal secretion in samples (n = 30).

[0215] Table 8

[0216]

[0217] 3. Evaluation of the protective effect of intestinal mucosal secretion (goblet cells)

[0218] Under the experimental conditions, SHD exhibited a protective effect against intestinal mucosal secretion, specifically by increasing the number of intestinal goblet cells. See Table 9 for the experimental results of the evaluation of the protective effect against intestinal mucosal secretion (goblet cells) (n = 10). Figure 6 (The red dashed box represents the analysis area, and the blue particles represent goblet cells.)

[0219] Table 9

[0220]

[0221] 4. Evaluation of the protective effect of intestinal mucosal secretion (neutrophils)

[0222] Under the experimental conditions, SHD exhibited a protective effect against intestinal mucosal secretion, specifically by reducing the number of intestinal neutrophils. See Table 10 for the experimental results of the evaluation of the protective effect against intestinal mucosal secretion (neutrophil count) (n=10). Figure 7 .

[0223] Table 10

[0224]

[0225] 5. Evaluation of the protective effect of intestinal mucosal secretion (intestinal lumen area)

[0226] Under the experimental conditions, SHD exhibited a protective effect against intestinal mucosal secretion, specifically by improving intestinal lumen area atrophy. See Table 11 for the experimental results of the evaluation of the protective effect against intestinal mucosal secretion (intestinal lumen area) (n = 10). Figure 8 .

[0227] Table 11

[0228]

[0229] 6. Evaluation of the protective effect of intestinal mucosal secretion (intestinal pathological sections)

[0230] Under the experimental conditions, histopathological examination of the intestines of the normal control group showed that the zebrafish had numerous and obvious gastrointestinal folds, and the intestinal epithelial cells were tightly connected with cilia and mucosa. Histopathological examination of the intestines of the model control group showed obvious intestinal mucosal damage phenotype, manifested as a significant reduction in the intestinal lumen (red arrows indicate the diameter of the intestinal lumen) and destruction of the intestinal mucosa. Histopathological examination of the intestines of the zebrafish in the positive control group treated with mesalazine showed a significant improvement in the intestinal mucosal damage phenotype, suggesting that mesalazine has an auxiliary effect in improving intestinal mucosal damage in zebrafish.

[0231] Histopathological examination of the zebrafish intestines at SHD concentrations of 500–1000 μg / mL showed significant improvement in the intestinal mucosal damage phenotype, suggesting that all concentrations had a protective effect against intestinal mucosal secretion. The zebrafish intestines at SHD concentrations of 250 μg / mL were similar to those in the model control group, suggesting that this concentration did not have a significant protective effect against intestinal mucosal damage.

[0232] In summary, under the experimental conditions, SHD has a protective effect on intestinal mucosal secretion. (See details...) Figure 9 .

[0233] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0234] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for preparing Dendrobium polysaccharide, characterized in that, Includes the following steps: Dendrobium is processed into Dendrobium powder; Ethanol was used as an entrainer to perform supercritical carbon dioxide extraction on the Dendrobium powder to obtain Dendrobium powder. The Dendrobium powder is subjected to water extraction to obtain Dendrobium water extract, wherein the water extraction temperature is 60℃~80℃; The Dendrobium water extract was dried to obtain the Dendrobium polysaccharide.

2. The method for preparing Dendrobium polysaccharides as described in claim 1, characterized in that, The Dendrobium polysaccharide satisfies at least one of the following conditions: (1) The purity of the Dendrobium polysaccharide is ≥90%; (2) The Dendrobium polysaccharide includes mannose and glucose, and the molar ratio of mannose and glucose is 1 to 5; (3) The weight-average molecular weight of the Dendrobium polysaccharide is 8 kDa ~ 2000 kDa; (4) The mass percentage of high molecular weight polysaccharides with a weight average molecular weight of 500kDa to 2000kDa in the Dendrobium polysaccharide is 85% to 95%.

3. The method for preparing Dendrobium polysaccharides as described in claim 1, characterized in that, The water extraction satisfies at least one of the following conditions: (1) The temperature of the water extraction is 70℃~80℃; (2) The mass ratio of the material to the liquid in the water extraction is 1:30~60; (3) The water extraction time is 3h to 4h.

4. The method for preparing Dendrobium polysaccharides as described in claim 1, characterized in that, After the water extraction and before obtaining the Dendrobium water extract, the following centrifugation separation step is also included: The product extracted by water is centrifuged and the liquid phase after centrifugation is collected to obtain the Dendrobium water extract. Optionally, the centrifugal factor for the centrifugal separation is 2000 g to 4000 g; Optionally, the centrifugation time is 10 min to 15 min.

5. The method for preparing Dendrobium polysaccharide according to any one of claims 1 to 4, characterized in that, The supercritical carbon dioxide extraction satisfies at least one of the following conditions: (1) The mass of the ethanol accounts for 10% to 20% of the total mass of the ethanol and carbon dioxide; (2) The extraction temperature of the supercritical carbon dioxide extraction is 45℃~60℃; (3) The extraction pressure of the supercritical carbon dioxide extraction is 15MPa~30MPa; (4) The extraction time of the supercritical carbon dioxide extraction is 1h to 2h.

6. The method for preparing Dendrobium polysaccharide as described in claim 5, characterized in that, The ethanol accounts for 14% to 20% of the total mass of the ethanol and carbon dioxide; and / or, The extraction temperature for the supercritical carbon dioxide extraction is 50℃~55℃; and / or, The extraction pressure of the supercritical carbon dioxide extraction is 25 MPa to 30 MPa; and / or, The extraction time for the supercritical carbon dioxide extraction is 1.2 h to 1.8 h.

7. The method for preparing Dendrobium polysaccharides according to any one of claims 1 to 4 and 6, characterized in that, The particle size of the Dendrobium powder is 0.05mm~0.25mm.

8. The method for preparing Dendrobium polysaccharides according to any one of claims 1 to 4 and 6, characterized in that, The process of pre-drying the Dendrobium powder also includes the following drying step: The Dendrobium extract was dried at 30℃~40℃ for 2h~4h.

9. A Dendrobium polysaccharide, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 8.

10. The use of the Dendrobium polysaccharide according to claim 9 in the preparation of food, health products, or drugs with the effects of relieving hangovers, protecting the liver, or protecting the intestinal mucosa.

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