Dendrobium loddigesii polysaccharide extract as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of a dendrobium loddigesii polysaccharide extract, which comprises the following steps: crushing cleaned dendrobium loddigesii into dendrobium loddigesii powder, extracting with ethanol, filtering, and drying to obtain dendrobium loddigesii powder to be extracted; mixing p-toluenesulfonic acid, n-butyl alcohol and water to obtain an extraction solution; adding to-be-extracted dendrobium loddigesii powder into the extracting solution, heating and extracting, separating, dialyzing to obtain a crude extracting solution, carrying out alcohol precipitation, dialyzing the precipitate, and freeze-drying to obtain the dendrobium loddigesii polysaccharide extract. The mixed solution is used for extracting the dendrobium loddigesii polysaccharide, extraction and separation of the dendrobium loddigesii polysaccharide extract can be achieved, deproteinization treatment is not needed, the purity of the obtained dendrobium loddigesii polysaccharide extract can reach 90% or above, the process is simple and convenient, reproducibility is good, cost is low, and large-scale production is easy. The invention further discloses application of the dendrobium loddigesii polysaccharide extract prepared by the preparation method and the pharmaceutical composition of the dendrobium loddigesii polysaccharide extract in preparation of drugs or health care products for preventing or treating liver injury.

Description

Technical Field

[0001] The invention relates to the technical field of plant polysaccharide extraction, and in particular to a Dendrobium officinale polysaccharide extract and a preparation method and application thereof. Background Art

[0002] In the field of global public health, liver injury, as a major and complex health problem, continues to attract extensive scientific research attention. Its causes are diverse, covering viral infections, excessive alcohol intake, and, of particular concern, damage caused by drugs and chemicals, namely drug-induced liver injury (DILI) and chemical liver injury. These forms of injury occupy an important position in clinical medical practice because they have the risk of inducing acute liver failure, resulting in significant morbidity and mortality. Given that many therapeutic drugs and industrial chemicals may cause damage to the liver through specific or dose-independent biological mechanisms, the accurate prediction and effective management of DILI constitute a major challenge in current medical research.

[0003] The pathophysiological mechanism of liver injury is extremely complex, involving a series of finely regulated biological events, which are centered on the initiation of inflammatory responses and oxidative stress. After the liver is exposed to hepatotoxic substances, a series of complex biological responses are triggered, including the activation of Kupffer cells and the release of cytokines and chemokines mediated by them. While these bioactive molecules promote the migration and infiltration of immune cells into liver tissue, they also aggravate tissue damage by inducing the production of reactive oxygen species (ROS). In addition, when the balance between the antioxidant defense system and pro-oxidant factors in the body is broken, an oxidative stress state will be induced, which will not only lead to the aggravation of lipid peroxidation, but also cause structural damage to proteins and nucleic acids, thereby further deteriorating liver function.

[0004] In view of the above challenges, the current scientific research community has shown great interest in developing new liver-protecting drugs, especially those derived from nature. With its long history and rich practical experience, traditional Chinese medicine has shown unique advantages in using herbs and natural substances to protect liver health. In particular, polysaccharides extracted from medicinal plants have become a hot topic of current research due to their excellent antioxidant and anti-inflammatory activities. These natural products show significant liver-protective effects by regulating the body's immune response and reducing oxidative stress. Therefore, polysaccharides are regarded as potential alternatives to traditional synthetic liver-protecting drugs, and their development and application are expected to provide patients with liver damage with safer, more effective and comprehensive treatment options, thereby opening up new research directions in the treatment of liver damage.

[0005] Dendrobium Loddigesii Rolfe belongs to the Orchidaceae family of the genus Dendrobium and was once included in the Chinese Pharmacopoeia as one of the herbal sources of the traditional Chinese medicine "Dendrobium". Pharmacologically, Dendrobium plants are known for their various bioactive compounds, including alkaloids, phenanthrene and polysaccharides. These substances contribute to a wide range of health benefits, such as anti-tumor, anti-inflammatory and immune-enhancing properties. At present, the pharmacological activity of polysaccharides from Dendrobium Loddigesii Rolfe has not been reported.

[0006] Publication No. CN105497059A discloses a compound of dendrobium extract and iron and its preparation method and application. The preparation method of dendrobium polysaccharide in the dendrobium extract is to grind the dendrobium medicinal material, depigment and defatting with solvent, evaporate the solvent, add 10-50 times of water to heat and extract for 0.5-4 hours, filter to obtain filtrate or centrifuge to obtain centrifuge, then add 4 times the volume of ethanol to the obtained filtrate or centrifuge, centrifuge, dissolve the obtained precipitate in water, and then remove protein by Sevage method or trichloroacetic acid method or hydrochloric acid method to obtain the dendrobium polysaccharide. This method requires rough extraction of precipitation and then deproteinization, and the polysaccharide content in the obtained dendrobium polysaccharide is less than 70%.

[0007] Publication No. CN114573726A discloses a method for extracting plant polysaccharides, comprising the following steps: first, different types of substances as hydrogen bond acceptors are selected to directly form a low eutectic system with polysaccharides in Dendrobium officinale to achieve one-step extraction of polysaccharides, and then the crude polysaccharide solution is precipitated by ethanol, the protein is removed by the Sevage method, and vacuum freeze-dried to obtain purified polysaccharide powder. This method is a low eutectic extraction method, in which there are a large number of hydrogen bond network structures, van der Waals forces and other electrostatic interactions between the components in the low eutectic solvent, and the fluidity is poor, which leads to poor extraction effect. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a method for preparing a polysaccharide extract of Dendrobium officinale. The method uses a mixed solution of p-toluenesulfonic acid, n-butanol and water to extract Dendrobium officinale polysaccharide, which can realize the extraction and separation of the Dendrobium officinale polysaccharide extract without the need for deproteinization. The purity of the obtained Dendrobium officinale polysaccharide extract can reach more than 90%. The method is simple in process, easy to operate, has good reproducibility, low cost, and is easy for large-scale production.

[0009] A method for preparing a polysaccharide extract of Dendrobium officinale, comprising the following steps:

[0010] (1) crushing the washed Dendrobium officinale into Dendrobium officinale powder, extracting with ethanol, filtering, and drying to obtain the Dendrobium officinale powder to be extracted;

[0011] (2) mixing p-toluenesulfonic acid, n-butanol and water to obtain an extraction solution;

[0012] (3) adding the Dendrobium officinale powder to be extracted obtained in step (1) to the extraction solution obtained in step (2), heating and extracting, separating, collecting the aqueous phase, and dialyzing the aqueous phase using a dialysis membrane of 8000 to 14000 Da to obtain a crude extract;

[0013] (4) After the crude extract obtained in step (3) is subjected to alcohol precipitation, the precipitate is dialyzed and freeze-dried to obtain a polysaccharide extract of Dendrobium officinale.

[0014] p-Toluenesulfonic acid (p-TsOH) shows unique advantages in polysaccharide extraction, especially in the separation of lignin. p-Toluenesulfonic acid dissolves the lignin component, which is usually insoluble in water, in water, and this dissolution helps to separate lignin from the polysaccharides in the cell wall. p-TsOH can also separate sugars in the wood structure, which are insoluble in water, so this method can be used to effectively separate polysaccharides from lignin. p-TsOH can be separated from the waste liquid by recrystallization and reused, which reduces the cost of solvent recovery and reduces the impact on the environment.

[0015] n-Butanol is usually used as an extractant or precipitant in polysaccharide extraction to separate polysaccharides from other impurities by shaking. By adding n-butanol to the polysaccharide extract, the solubility of the polysaccharide can be reduced, causing it to precipitate. This precipitation effect helps to obtain a polysaccharide product with higher purity. During the polysaccharide extraction process, n-butanol can also remove impurities such as proteins in the extract.

[0016] In the present invention, a mixed solution of p-toluenesulfonic acid, n-butanol and water is used to extract the polysaccharide of Dendrobium officinale, so that it forms three phases, the upper phase is an organic layer, which mainly extracts substances with lower polarity such as pigments and lipids; the middle phase is a protein extraction layer; the lower phase is an aqueous layer, which mainly extracts some water-soluble substances. p-Toluenesulfonic acid mainly separates polysaccharides from lignin through its hydrophilic and hydrophobic properties and recyclability; while n-butanol plays an important role in polysaccharide extraction mainly through extraction and precipitation and protein removal function. The three-phase separation method can selectively extract water-soluble components such as the polysaccharide extract of Dendrobium officinale into the bottom aqueous phase, distribute the remaining components into other phases, collect the aqueous phase and perform dialysis treatment.

[0017] The present invention combines a three-phase separation method with a dialysis membrane with a specific molecular weight cutoff for dialysis treatment, and can obtain a high-purity polysaccharide extract from Dendrobium officinale without protein removal.

[0018] Preferably, in step (1), the extraction step is to soak the Dendrobium officinale powder in ethanol, which is repeated at least twice.

[0019] The present invention carries out an ethanol defatting step before extraction, and this step can remove most of the free proteins and alcohol-soluble components, so that the purity of the finally prepared Dendrobium candidum polysaccharide extract is improved.

[0020] Preferably, in step (2), in the extraction solution, the amount of p-toluenesulfonic acid added is 0.01-0.02 wt%, the amount of n-butanol added is 20-40 wt%, and the balance is water.

[0021] Preferably, in step (3), the weight ratio of the Dendrobium officinale powder to be extracted to the extraction solution is 1:20-40.

[0022] Preferably, in step (3), the temperature of the heating extraction is 60-100° C., and the time is 1-3 hours.

[0023] Preferably, in step (3), the separation step is to pass the mixture after heating and extraction through at least three layers of filter paper and centrifuge to separate the lower aqueous phase.

[0024] Preferably, in step (3), the alcohol precipitation step is to add at least 4 times the volume of ethanol to the crude extract, let it stand, and centrifuge to separate the precipitate.

[0025] The present invention also provides a polysaccharide extract of Dendrobium officinale prepared by the above preparation method, wherein the polysaccharide content of the extract is above 90%.

[0026] The present invention also provides a pharmaceutical composition comprising the above-mentioned Dendrobium officinale polysaccharide extract, which can better prevent and protect chemical liver damage.

[0027] Preferably, the pharmaceutical composition comprises: the above-mentioned Dendrobium officinale polysaccharide extract, Poria cocos polysaccharide, Lycium barbarum polysaccharide and Ganoderma lucidum polysaccharide, and the mass ratio of the Dendrobium officinale polysaccharide extract, Poria cocos polysaccharide, Lycium barbarum polysaccharide and Ganoderma lucidum polysaccharide is 1:1 to 2:1 to 2:1.

[0028] The composition composed of Dendrobium officinale polysaccharide extract, Poria cocos polysaccharide, Lycium barbarum polysaccharide and Ganoderma lucidum polysaccharide has an important characteristic of synergistic effect between its various polysaccharide components. These polysaccharide components are all from natural plants and have multiple pharmacological effects such as antiviral, anti-inflammatory, and liver protection. When they are combined in a certain mass ratio (such as 1:1:1:1 or 1:2:2:1), they can produce obvious liver protection effects. Compared with the Dendrobium officinale polysaccharide extract group, the pharmaceutical composition can more significantly restore the vitality of liver cells in oxidative stress injury models and alcohol injury models. This composition can provide comprehensive protection for human health and is expected to become an important direction for the development of health products and drugs in the future.

[0029] The present invention also provides the use of the above-mentioned Dendrobium officinale polysaccharide extract and pharmaceutical composition in the preparation of drugs or health products for preventing or treating liver damage.

[0030] Liver tissue with liver damage will release a large number of inflammatory factors, causing liver cell damage. The polysaccharide extract of Dendrobium officinale can inhibit the expression of inflammatory factors in the serum of mice with liver damage model. The polysaccharide extract of Dendrobium officinale can enhance the activity of superoxide dismutase, catalase and glutathione peroxidase in the liver, reduce the content of malondialdehyde, alleviate oxidative stress response, and play a liver protective role.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention uses a mixed solution of p-toluenesulfonic acid, n-butanol and water to extract Dendrobium officinale polysaccharides, thereby realizing the extraction and separation of the Dendrobium officinale polysaccharide extract, and then performs dialysis treatment with a dialysis membrane with a specific molecular weight cutoff, without the need for protein removal. The purity of the obtained Dendrobium officinale polysaccharide extract can reach more than 95%. The method is simple in process, easy to operate, has good reproducibility, low cost, and is easy for large-scale production.

[0033] (2) The polysaccharide extract of Dendrobium officinale in the present invention can inhibit the expression of inflammatory factors in the serum of mice with liver injury model. The polysaccharide extract of Dendrobium officinale can enhance the activity of superoxide dismutase, catalase and glutathione peroxidase in the liver, reduce the content of malondialdehyde, alleviate oxidative stress, and play a liver protective role.

[0034] (3) In the present invention, four polysaccharides with liver protection effects are mixed in proportion to prepare a pharmaceutical composition. Compared with the polysaccharide extract group of Dendrobium officinale, the pharmaceutical composition can more significantly restore the vitality of liver cells in the oxidative stress injury model and the alcohol injury model, and can play a better liver protection effect. The pharmaceutical composition provides comprehensive protection for human health and is expected to become an important direction for the development of health products and drugs in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the monosaccharide composition spectrum of the Dendrobium officinale polysaccharide extract prepared in Example 1.

[0036] Figure 2 The contents of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase in the serum of mice in Example 1, Comparative Example 1 and the control group, wherein * is p<0.05, ** is p<0.01, *** is p<0.001, indicating the comparison results between the model group and the control group; # is p<0.05, ## is p<0.01, and ### is p<0.001, indicating the comparison results between each drug administration group and the model group.

[0037] Figure 3 The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) in the liver tissues of mice in Example 1, Comparative Example 1 and the control group, wherein * is p<0.05, ** is p<0.01, *** is p<0.001, indicating the comparison results between the model group and the control group; # is p<0.05, ## is p<0.01, and ### is p<0.001, indicating the comparison results between each drug administration group and the model group.

[0038] Figure 4 The expression levels of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum of mice in Example 1, Comparative Example 1 and the control group, where * is p<0.05, ** is p<0.01, and *** is p<0.001, indicating the comparison results between the model group and the control group; # is p<0.05, ## is p<0.01, and ### is p<0.001, indicating the comparison results between each medication group and the model group.

[0039] Figure 5 The morphology, hematoxylin-eosin staining and Masson's staining results of the liver tissues of mice in Example 1, Comparative Example 1 and the control group are shown, with magnifications of 100 and 200, and a scale bar of 100 μm.

[0040] Figure 6 The contents of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase in the serum of mice in Example 1, Application Example 1 and the control group, wherein * is p<0.05, ** is p<0.01, *** is p<0.001, indicating the comparison results between the model group and the control group; # is p<0.05, ## is p<0.01, ### is p<0.001, indicating the comparison results between each medication group and the model group.

[0041] Figure 7 The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) in the liver tissues of mice in Example 1, Application Example 1 and the control group, wherein * is p<0.05, ** is p<0.01, *** is p<0.001, indicating the comparison results between the model group and the control group; # is p<0.05, ## is p<0.01, and ### is p<0.001, indicating the comparison results between each drug administration group and the model group.

[0042] Figure 8The expression levels of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum of mice in Example 1, Application Example 1 and the control group, where * is p<0.05, ** is p<0.01, and *** is p<0.001, indicating the comparison results between the model group and the control group; # is p<0.05, ## is p<0.01, and ### is p<0.001, indicating the comparison results between each medication group and the model group.

[0043] Fig. 9 The morphology of liver tissues of mice in Example 1, Application Example 1 and the control group, as well as the results of hematoxylin-eosin staining and Masson's staining, are shown at magnifications of 100 and 200, and the scale bar is 100 μm.

[0044] Fig.10 These are the cell viability results of anti-oxidative stress injury of mouse hepatocytes in vitro in Example 1, Application Example 1 and the control group, where * is p<0.05, ** is p<0.01, *** is p<0.001, indicating the comparison results between the model group and the control group; # is p<0.05, ## is p<0.01, and ### is p<0.001, indicating the comparison results between each drug-administered group and the model group.

[0045] Fig.11 The cell viability results of in vitro mouse hepatocytes against alcohol damage in Example 1, Application Example 1 and the control group, where * is p<0.05, ** is p<0.01, *** is p<0.001, indicating the comparison results between the model group and the control group; # is p<0.05, ## is p<0.01, ### is p<0.001, indicating the comparison results between each drug administration group and the model group. DETAILED DESCRIPTION

[0046] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited to the following examples.

[0047] The raw materials used in the present invention are all commercially available.

[0048] Example 1

[0049] (1) Pretreatment: Wash the Dendrobium officinale and crush it in a grinder to obtain Dendrobium officinale powder. Soak the Dendrobium officinale powder (1000 g) in 4 L of 90% ethanol, repeat twice to remove its alcohol-soluble components, each time for 4 hours. After filtering, collect the solid powder and dry it in a vacuum drying oven at 50°C to obtain the Dendrobium officinale powder to be extracted.

[0050] (2) Preparation of extraction solution: 0.01 wt% p-toluenesulfonic acid, 30.00 wt% n-butanol and 69.99 wt% deionized water.

[0051] (3) Accurately weigh 500 g of the processed Dendrobium officinale powder in the drying step (1), put the Dendrobium officinale powder to be extracted and the extraction solution into a round-bottom flask at a weight ratio of 1:30, and extract at 90° C. for 90 min. The extracted mixture is passed through three layers of filter paper and centrifuged at 10,000 rpm for 15 min to separate, collect the lower aqueous phase and dialyze with deionized water for 72 h, the dialysis membrane is 8 kDa, and obtain a crude extract.

[0052] (4) Add four times the volume of 95% ethanol to the crude extract obtained in step (3), and after standing at 4°C for 24 hours, centrifuge at 10,000 rpm for 15 minutes to obtain a precipitate of Dendrobium officinale polysaccharide extract, and wash it with 95% ethanol. The precipitate is dialyzed with deionized water for 72 hours, and the dialysis membrane is 3.5 kDa. The retentate after dialysis is collected and freeze-dried to obtain a Dendrobium officinale polysaccharide extract.

[0053] Example 2

[0054] (1) Pretreatment: Wash the Dendrobium officinale and crush it in a grinder to obtain Dendrobium officinale powder. Soak the Dendrobium officinale powder (1000 g) in 4 L of 90% ethanol, repeat twice to remove its alcohol-soluble components, each time for 4 hours. After filtering, collect the solid powder and dry it in a vacuum drying oven at 50°C to obtain the Dendrobium officinale powder to be extracted.

[0055] (2) Preparation of extraction solution: 0.02 wt % p-toluenesulfonic acid, 25.00 wt % n-butanol and 74.98 wt % deionized water.

[0056] (3) Accurately weigh 500 g of the processed Dendrobium officinale powder in the drying step (1), put the Dendrobium officinale powder to be extracted and the extraction solution into a round-bottom flask at a weight ratio of 1:30, and extract at 90° C. for 90 min. The extracted mixture is passed through three layers of filter paper and centrifuged at 10,000 rpm for 15 min to separate, collect the lower aqueous phase and dialyze with deionized water for 72 h, the dialysis membrane is 8 kDa, and obtain a crude extract.

[0057] (4) Add four times the volume of 95% ethanol to the crude extract obtained in step (3), and after standing at 4°C for 24 hours, centrifuge at 10,000 rpm for 15 minutes to obtain a precipitate of Dendrobium officinale polysaccharide extract, and wash it with 95% ethanol. The precipitate is dialyzed with deionized water for 72 hours, and the dialysis membrane is 3.5 kDa. The retentate after dialysis is collected and freeze-dried to obtain a Dendrobium officinale polysaccharide extract.

[0058] Example 3

[0059] (1) Pretreatment: Wash the Dendrobium officinale and crush it in a grinder to obtain Dendrobium officinale powder. Soak the Dendrobium officinale powder (1000 g) in 4 L of 90% ethanol, repeat twice to remove its alcohol-soluble components, each time for 4 hours. After filtering, collect the solid powder and dry it in a vacuum drying oven at 50°C to obtain the Dendrobium officinale powder to be extracted.

[0060] (2) Preparation of extraction solution: 0.015 wt% p-toluenesulfonic acid, 30.00 wt% n-butanol and 69.985 wt% deionized water.

[0061] (3) Accurately weigh 500 g of the processed Dendrobium officinale powder in the drying step (1), put the Dendrobium officinale powder to be extracted and the extraction solution into a round-bottom flask at a weight ratio of 1:30, and extract at 90° C. for 90 min. The extracted mixture is passed through three layers of filter paper and centrifuged at 10,000 rpm for 15 min to separate, collect the lower aqueous phase and dialyze with deionized water for 72 h, the dialysis membrane is 8 kDa, and obtain a crude extract.

[0062] (4) Add four times the volume of 95% ethanol to the crude extract obtained in step (3), and after standing at 4°C for 24 hours, centrifuge at 10,000 rpm for 15 minutes to obtain a precipitate of Dendrobium officinale polysaccharide extract, and wash it with 95% ethanol. The precipitate is dialyzed with deionized water for 72 hours, and the dialysis membrane is 3.5 kDa. The retentate after dialysis is collected and freeze-dried to obtain a Dendrobium officinale polysaccharide extract.

[0063] Comparative Example 1

[0064] The preparation method adopts the traditional water extraction and alcohol precipitation method to extract the polysaccharide from Dendrobium officinale.

[0065] (1) cleaning the Dendrobium officinale and crushing it with a grinder to obtain Dendrobium officinale powder;

[0066] (2) Weighing 500 g of Dendrobium officinale powder, adding 10 times the mass of water, heating and refluxing extraction twice, each extraction for 2 h, filtering the obtained water extract and combining them to obtain the Dendrobium officinale water extract;

[0067] (3) concentrating the water extract of Dendrobium officinale obtained in step (2) to a volume of 1:1 with the powder, adding anhydrous ethanol to a concentration of 80% to precipitate polysaccharides, centrifuging, and re-dissolving the precipitate with distilled water;

[0068] (4) Sevage reagent was added to remove protein, and then the organic reagent was removed, and the aqueous solution was freeze-dried to obtain the Dendrobium officinale polysaccharide-reference substance.

[0069] Sample analysis

[0070] 1. Determination of polysaccharide content and uronic acid content

[0071] The total polysaccharide content in the samples prepared in Example 1, Example 2, Example 3 and Comparative Example 1 was determined by the phenol-sulfuric acid method; the uronic acid content in the samples was determined by the carbazole sulfate method using galacturonic acid as the standard.

[0072] The total polysaccharide content of Dendrobium candidum in the sample of comparative example 1 was calculated by the phenol-sulfuric acid method to be 55.32%, and the uronic acid content in the polysaccharide of Dendrobium candidum was 5.50%; the total polysaccharide content of Dendrobium candidum in the sample of embodiment 1 was 95.32%, and the uronic acid content in the polysaccharide of Dendrobium candidum was 2.50%; the total polysaccharide content of Dendrobium candidum in the sample of embodiment 2 was 90.13%, and the uronic acid content in the polysaccharide of Dendrobium candidum was 3.70%; the total polysaccharide content of Dendrobium candidum in the sample of embodiment 3 was 92.78%, and the uronic acid content in the polysaccharide of Dendrobium candidum was 4.70%.

[0073] 2. Determination of monosaccharide composition of polysaccharide extracts from Dendrobium officinale

[0074] The polysaccharide extract of Dendrobium officinale prepared in Example 1 was prepared into a 2 mg / mL solution sample, placed in a sample bottle, 4 M TFA was added, nitrogen was filled in, and the sample was sealed and hydrolyzed at 110° C. for 10 h. After the hydrolysis was completed and cooled, the reaction solution was concentrated and evaporated to dryness under reduced pressure, and an appropriate amount of methanol was added, and the evaporation was repeated several times to remove the residual TFA (until there was no sour taste). The sample was re-dissolved with 6 mL of deionized water and directly subjected to liquid chromatography analysis.

[0075] Prepare standard stock solutions containing mannose, glucose, galactose, arabinose, fructose, xylose, rhamnose, fucose, galacturonic acid and glucuronic acid. Dilute the mixed monosaccharides to different concentrations, and perform linear regression analysis with the ratio of the peak area of ​​the monosaccharide reference substance to the peak area of ​​the internal standard at different concentrations as the ordinate and the concentration of each monosaccharide as the abscissa.

[0076] Figure 1 This is the monosaccharide composition spectrum of the Dendrobium officinale polysaccharide extract obtained in Example 1, wherein the proportion of each monosaccharide is mannose: glucose: galactose: arabinose: fructose: xylose: rhamnose: fucose: galacturonic acid: glucuronic acid = 29.8: 27.6: 10.4: 9.1: 6.8: 5.8: 5.3: 3.2: 1.2: 0.9, and mannose has the highest proportion.

[0077] 3. Evaluation of the hepatoprotective effect of polysaccharide extracts from Dendrobium officinale

[0078] 45 healthy SPF male BALB / c mice were randomly divided into 5 groups, 9 mice in each group, namely, a control group (10 mL / kg of normal saline); a positive drug group (silymarin, 50 mg / kg); a model group (10 mL / kg of normal saline); a control group (200 mg / kg) of the Dendrobium officinale polysaccharide prepared in Comparative Example 1; and a Dendrobium officinale polysaccharide extract group (200 mg / kg) prepared in Example 1. Each group was administered with drugs for 14 consecutive days. Three hours after administration on the last day, except for the empty control group, the rest of the mice were injected with 10 mL / kg of corn oil solution of 0.3% CCl4 into the peritoneal cavity, and the control group was injected with 10 mL / kg of corn oil. The mice were fasted but not water-deprived. After 18 hours, blood was collected from the eye sockets of the mice in each group, and the blood was centrifuged at 2500 rpm for 10 min after being placed at room temperature for 2 hours. The serum was collected to determine the activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AKP), as well as the expression of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum. After blood collection, the mice were killed by dislocation, dissected and the liver was removed and fixed in 4% paraformaldehyde solution, and hematoxylin-eosin staining and Masson staining were performed to observe the pathological sections of liver injury tissue. The rest of the liver was used to determine the activities of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in the liver. The experimental data were expressed as Mean ± SD, and the data were analyzed by one-way analysis of variance, and the t test was used between groups. The Graphpad statistical software was used for analysis, and p < 0.05 was considered statistically significant.

[0079] 1. Determination of AST, ALT, AKP transaminase content

[0080] Strictly follow the steps in the kit instructions to determine the activities of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase in serum.

[0081] Figure 2 The contents of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase in the serum of mice in Example 1, Comparative Example 1 and the control group, among which, compared with the control group, the levels of AST, ALT and AKP in the serum of mice in the CCl4-induced chemical liver injury model group were significantly increased, with significant statistical differences. Compared with the model group, the levels of AST, ALT and AKP in each drug administration group were significantly reduced and restored to a level similar to that of the control group. The therapeutic effect of the polysaccharide extract of Dendrobium officinale prepared in Example 1 was better than that of the positive drug group and the comparative example 1 group.

[0082] 2. Determination of SOD, CAT, MDA and GSH-Px levels

[0083] Weigh an appropriate amount of liver and prepare a 10% tissue homogenate according to the sample pretreatment method required by the kit at a ratio of liver: physiological saline = 1:9 g / mL. Follow the steps in the kit instructions to determine the activities of SOD, CAT, MDA, and GSH-Px in the combined homogenate.

[0084] Figure 3 The activities of superoxide dismutase, catalase and glutathione peroxidase in the liver tissue of mice in Example 1, Comparative Example 1 and the control group, as well as the content of malondialdehyde, wherein, compared with the control group, the levels of SOD, CAT and GSH-Px in the liver tissue of the model group were significantly reduced; compared with the model group, the activity levels of SOD, CAT and GSH-Px in each administration group were significantly increased, and had significant significance. At the same time, compared with the control group, the level of MDA in the liver tissue of the model group was significantly increased; compared with the model group, the level of MDA in the liver tissue of mice in each administration group was significantly reduced. Among them, the therapeutic effect of the polysaccharide extract of Dendrobium officinale prepared in Example 1 is better than that of the positive drug group and the comparative example 1 group.

[0085] 3. Determination of IL-1β, IL-6 and TNF-α inflammatory factors

[0086] Strictly follow the steps in the kit instructions to determine the expression levels of inflammatory factors IL-1β, IL-6 and TNF-α in serum.

[0087] Figure 4 The expression levels of inflammatory factors interleukin-1β, interleukin-6 and tumor necrosis factor-α in the serum of mice in each group, among which, compared with the control group, the levels of inflammatory factors IL-1β, IL-6 and TNF-α in the serum of mice in the model group were significantly increased. Compared with the model group, the levels of inflammatory factors in each administration group were significantly reduced and restored to a level similar to that of the control group. The therapeutic effect of the polysaccharide extract of Dendrobium officinale prepared in Example 1 was better than that of the positive drug group and the comparative example 1 group.

[0088] 4. Effects of polysaccharide extracts from Dendrobium officinale on liver tissue pathological changes in mice with CCl4-induced liver injury model

[0089] The liver tissues fixed with 4% paraformaldehyde were routinely dehydrated, wrapped in paraffin, and sectioned. Then, the liver tissues were stained with hematoxylin-eosin (HE) and Masson to observe the pathological changes of liver tissues.

[0090] Figure 5The morphology of liver tissues of mice in each group and the results of HE staining and Masson staining. The liver tissue sections were stained with hematoxylin-eosin and Masson staining and observed under a light microscope. The liver tissues of mice in the control group were clear, the liver lobule structure was intact, the portal area was clear, and the hepatocyte morphology was basically normal and arranged radially. In the livers of mice in the model group, partial necrosis of hepatocytes and vesicular changes in hepatocytes were observed, and scattered lipid vacuoles were partially observed; the degree of liver lesions in the mice in the groups of the polysaccharide extract of Dendrobium officinale prepared in Example 1 and Comparative Example 1 were significantly improved compared with those in the model group, among which the improvement in the group of the polysaccharide extract of Dendrobium officinale prepared in Example 1 was particularly obvious.

[0091] Application Example 1

[0092] The polysaccharide extract of Dendrobium officinale prepared in Example 1 was used to form a pharmaceutical composition, and the formula of the pharmaceutical composition was as follows: Dendrobium officinale polysaccharide, Poria cocos polysaccharide, Lycium barbarum polysaccharide and Ganoderma lucidum polysaccharide.

[0093] formula Dosage Dendrobium officinale polysaccharide 50mg Poria polysaccharide 50mg Lycium barbarum polysaccharide 50mg Ganoderma lucidum polysaccharide 50mg

[0094] 1. Evaluation of the hepatoprotective effect of the pharmaceutical composition

[0095] Twenty-eight healthy SPF male BALB / c mice were randomly divided into 4 groups, with 9 mice in each group, namely a control group (10 mL / kg of normal saline); a model group (10 mL / kg of normal saline); a group of the polysaccharide extract of Dendrobium candidum prepared in Example 1 (200 mg / kg); and a group of the pharmaceutical composition of Application Example 1 (200 mg / kg). Each group was administered with medication for 14 consecutive days. Three hours after administration on the last day, except for the empty control group, the rest of the mice were injected with 10 mL / kg of corn oil solution of 0.3% CCl4 into the peritoneal cavity, and the control group was injected with 10 mL / kg of corn oil. The mice were fasted but not water-deprived. After 18 hours, blood was collected from the eye sockets of the mice in each group, and the serum was centrifuged at 2500 rpm for 10 min after being placed at room temperature for 2 hours to obtain serum. The activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AKP) were measured, as well as the expression of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum. After blood collection, the mice were killed by dislocation, dissected and the liver was removed and fixed in 4% paraformaldehyde solution, and hematoxylin-eosin staining and Masson staining were performed to observe the pathological sections of liver injury tissue. The rest of the liver was used to determine the activities of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in the liver. The experimental data were expressed as Mean ± SD, and the data were analyzed by one-way analysis of variance, and the t test was used between groups. The Graphpad statistical software was used for analysis, and p < 0.05 was considered statistically significant.

[0096] 1. Determination of AST, ALT, AKP transaminase content

[0097] Strictly follow the steps in the kit instructions to determine the activities of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase in serum.

[0098] Figure 6 The contents of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase in the serum of mice in Example 1, Application Example 1 and the control group, among which, compared with the control group, the serum AST, ALT and AKP levels of mice in the CCl4-induced chemical liver injury model group were significantly increased, with significant statistical differences. Compared with the model group, the AST, ALT and AKP levels of each administration group were significantly reduced and returned to a level similar to that of the control group.

[0099] 2. Determination of SOD, CAT, MDA and GSH-Px levels

[0100] Weigh an appropriate amount of liver and prepare a 10% tissue homogenate according to the sample pretreatment method required by the kit at a ratio of liver: physiological saline = 1:9 g / mL. Follow the steps in the kit instructions to determine the activities of SOD, CAT, MDA, and GSH-Px in the combined homogenate.

[0101] Figure 7 The activities of superoxide dismutase, catalase and glutathione peroxidase in the liver tissue of mice in Example 1, Application Example 1 and the control group, as well as the content of malondialdehyde, among which, compared with the control group, the levels of SOD, CAT and GSH-Px in the liver tissue of the model group were significantly reduced; compared with the model group, the activity levels of SOD, CAT and GSH-Px in each administration group were significantly increased, and had significant significance. At the same time, compared with the control group, the level of MDA in the liver tissue of the model group was significantly increased; compared with the model group, the level of MDA in the liver tissue of mice in each administration group was significantly reduced.

[0102] 3. Determination of IL-1β, IL-6 and TNF-α inflammatory factors

[0103] Strictly follow the steps in the kit instructions to determine the expression levels of inflammatory factors IL-1β, IL-6 and TNF-α in serum.

[0104] Figure 8 The expression levels of inflammatory factors interleukin-1β, interleukin-6 and tumor necrosis factor-α in the serum of mice in each group, among which, compared with the control group, the levels of inflammatory factors IL-1β, IL-6 and TNF-α in the serum of mice in the model group were significantly increased. Compared with the model group, the levels of inflammatory factors in each drug-treated group were significantly reduced and returned to a level similar to that of the control group.

[0105] 4. Effect of the drug combination on liver tissue pathological changes in mice with CCl4-induced liver injury model

[0106] The liver tissues fixed with 4% paraformaldehyde were routinely dehydrated, wrapped in paraffin, and sectioned. Then, the liver tissues were stained with hematoxylin-eosin (HE) and Masson to observe the pathological changes of liver tissues.

[0107] Fig. 9 The morphology of liver tissues of mice in each group and the results of HE staining and Masson staining. The liver tissue sections were observed under a light microscope after hematoxylin-eosin staining and Masson staining. The liver tissues of mice in the control group were clear, the liver lobule structure was intact, the portal area was clear, and the hepatocyte morphology was basically normal and arranged radially. In the livers of mice in the model group, partial necrosis of hepatocytes and vesicular changes of hepatocytes were observed, and scattered lipid vacuoles were observed in some of the livers; the degree of liver lesions in the extract groups prepared in Example 1 and Application Example 1 was significantly improved compared with that in the model group.

[0108] II. Evaluation of the in vitro hepatocyte protective effect of the drug composition

[0109] 1. NCTC1469 cell culture

[0110] NCTC1469 cells were purchased from Shanghai ATCC Cell Bank. The cells were cultured in DMEM medium containing 10% FBS. The cells were placed in an environment of 5% CO2 and a constant temperature of 37°C.

[0111] 2. CCK-8 method to detect the effects of Example 1 and Application Example 1 on the viability of NCTC1469 cells induced by hydrogen peroxide and alcohol

[0112] (1) NCTC1469 cells were grouped according to experimental groups.

[0113] (2) When the cell density in the culture dish is about 80%, 4 / well were inoculated into 96-well plates and transferred to a cell culture incubator for further incubation. When the cells in the well plate grew to about 60%, the 96-well plate was removed. The culture medium was discarded, and the culture medium and drug solution were added according to the experimental groups.

[0114] (3) When the drug action reached the endpoint (24 h), 10% CCK-8 was added to each well and the cells were incubated in a cell culture incubator for 30 min. The OD value was measured using an ELISA reader.

[0115] (4) Calculation of cell viability: Cell survival rate (%) = (OD of drug group - OD of blank group / OD of control group - OD of blank group) × 100%.

[0116] Fig.10In Example 1, Application Example 1 and the control group, the viability of in vitro hepatocyte oxidative stress injury in mice was measured. Compared with the control group, the cell viability in the model group was significantly decreased due to hydrogen peroxide-induced damage; compared with the model group, the cell viability level of each administration group increased with significant significance. Among them, the therapeutic effect of the pharmaceutical composition group prepared in Application Example 1 was better than that of the Example 1 group, and the cell viability was significantly restored.

[0117] Fig.11 In Example 1, Application Example 1 and the control group, the viability of in vitro hepatocytes damaged by alcohol in mice was measured. Compared with the control group, the cell viability in the model group was significantly decreased due to alcohol-induced damage; compared with the model group, the cell viability level of each administration group increased with significant significance. Among them, the therapeutic effect of the pharmaceutical composition group prepared in Application Example 1 was better than that of the Example 1 group, and the cell viability was significantly restored.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a polysaccharide extract from Dendrobium officinale, characterized in that: The following steps are involved: (1) crushing the washed Dendrobium officinale into Dendrobium officinale powder, extracting with ethanol, filtering, and drying to obtain the Dendrobium officinale powder to be extracted; (2) mixing p-toluenesulfonic acid, n-butanol and water to obtain an extraction solution; (3) adding the Dendrobium officinale powder to be extracted obtained in step (1) to the extraction solution obtained in step (2), heating and extracting, separating, collecting the aqueous phase, and dialyzing the aqueous phase using a dialysis membrane of 8000 to 14000 Da to obtain a crude extract; (4) After the crude extract obtained in step (3) is subjected to alcohol precipitation, the precipitate is dialyzed and freeze-dried to obtain a polysaccharide extract of Dendrobium officinale.

2. The method for preparing the polysaccharide extract of Dendrobium officinale according to claim 1, characterized in that: In step (1), the extraction step is to soak the Dendrobium officinale powder in ethanol, which is repeated at least twice.

3. The method for preparing the polysaccharide extract of Dendrobium officinale according to claim 1, characterized in that: In step (2), in the extraction solution, the added amount of p-toluenesulfonic acid is 0.01-0.02 wt%, the added amount of n-butanol is 20-40 wt%, and the balance is water.

4. The method for preparing the polysaccharide extract of Dendrobium officinale according to claim 1, characterized in that: In step (3), the weight ratio of the Dendrobium officinale powder to be extracted to the extraction solution is 1:20-40.

5. The method for preparing the polysaccharide extract of Dendrobium officinale according to claim 1, characterized in that: In step (3), the temperature of the heating extraction is 60 to 100° C., and the time is 1 to 3 hours.

6. The method for preparing the polysaccharide extract of Dendrobium officinale according to claim 1, characterized in that: In step (3), the separation step is to pass the mixture after heating and extraction through at least three layers of filter paper and centrifuge to separate the lower aqueous phase.

7. The method for preparing the polysaccharide extract of Dendrobium officinale according to claim 1, characterized in that: In step (3), the alcohol precipitation step is to add at least 4 times the volume of ethanol to the crude extract, let it stand, and centrifuge to separate the precipitate. 8 . The Dendrobium officinale polysaccharide extract prepared according to the method for preparing the Dendrobium officinale polysaccharide extract according to any one of claims 1 to 7 .

9. The pharmaceutical composition prepared from the polysaccharide extract of Dendrobium officinale according to claim 8, characterized in that: The pharmaceutical composition comprises: the Dendrobium officinale polysaccharide extract, Poria cocos polysaccharide, Lycium barbarum polysaccharide and Ganoderma lucidum polysaccharide as claimed in claim 7, and the mass ratio of the Dendrobium officinale polysaccharide extract, Poria cocos polysaccharide, Lycium barbarum polysaccharide and Ganoderma lucidum polysaccharide is 1:1 to 2:1 to 2:

1.

10. Use of the polysaccharide extract of Dendrobium officinale according to claim 8 or the pharmaceutical composition according to claim 9 in the preparation of a drug for preventing or treating liver damage.

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