Preparation method and application of fresh dendrobium officinale refined polysaccharide

Abstract

The invention discloses a preparation method and application of fresh dendrobium officinale refined polysaccharide, and the preparation method of the fresh dendrobium officinale refined polysaccharide comprises the following steps: adding a proper amount of water into fresh dendrobium officinale, juicing, concentrating filtrate, adding ethanol for alcohol precipitation, dissolving precipitate with water, deproteinizing, and drying to obtain crude polysaccharide; the crude polysaccharide is subjected to graded alcohol precipitation and ion exchange column purification to obtain semi-refined polysaccharide, the semi-refined polysaccharide is purified by a gel column to obtain the fresh dendrobium officinale refined polysaccharide, and the polysaccharide is acidic glucomannan which is high in purity, uniform and moderate in molecular weight and contains beta-configuration pyranoside. In-vivo and in-vitro activity tests show that the compound polysaccharide has exact curative effects of preventing drunkenness, dispelling the effects of alcohol and preventing stomach injury and hepatocyte injury, is prepared by compounding the compound polysaccharide with traditional Chinese medicines of radix puerariae, hovenia dulcis thunb and tomato seed oil, and has the effect of obviously enhancing the immunity of liver functions.

Description

Technical Field

[0001] The invention relates to a preparation method and application of fresh Dendrobium officinale refined polysaccharide, belonging to the field of traditional Chinese medicine pharmacy. Background Art

[0002] As the main sites of alcohol metabolism, the liver and stomach are often stimulated and damaged, causing many diseases. Alcoholic liver disease (ALD) is the most common chronic liver disease and has become the second largest cause of liver damage in China after viral infection. Due to the complex pathological mechanism of ALD, there are currently no effective clinical treatments and drugs. Dendrobium candidum has a history of medicinal use in my country for more than 2,000 years. The pharmacopoeia records that it has the effects of benefiting the stomach and promoting fluid production, nourishing yin and clearing heat, and resisting fatigue. Modern research has shown that polysaccharides are the main active ingredients of Dendrobium candidum, with a water-soluble polysaccharide content of up to 22.7%, which has a variety of pharmacological activities such as treating gastrointestinal inflammation, improving liver function, lowering blood sugar, and anti-oxidation. In 2023, Dendrobium candidum was also officially included in the list of medicines and foods, and its safety has been widely recognized. It can be seen that Dendrobium candidum and its polysaccharide components have broad prospects in the development of health products and medicines for nourishing the stomach, protecting the liver, and relieving alcohol.

[0003] Fresh medicine refers to freshly harvested animal and plant Chinese medicine raw materials, which are directly used for treatment without processing or preparation. The active ingredients of fresh medicine have not changed or lost, and the original natural medicine properties of the medicine are retained, with better efficacy and a long history of clinical application. There are 7 kinds of fresh medicines included in the 2020 edition of the "Pharmacopoeia of the People's Republic of China", namely Rehmannia glutinosa, Dendrobium officinale, ginger, Phragmites australis, Leonurus japonicus, Houttuynia cordata and Vitex foetida. The document "Study on the Differences in the Composition and Antioxidant Activity of Fresh and Dried Dendrobium Officinale Polysaccharides" reported that the antioxidant activity of fresh Dendrobium polysaccharides in vitro and in vivo is stronger than that of dried Dendrobium polysaccharides.

[0004] The extraction methods of Dendrobium officinale polysaccharides mainly include hot water extraction, enzymatic extraction, ultrasonic assisted extraction, microwave assisted extraction, ultra-high pressure extraction, etc. For example, the Chinese patent "A method for extracting Dendrobium officinale polysaccharides" with patent application number "201510747301.5" and the Chinese patent "A method for extracting Dendrobium officinale polysaccharides by ultrasonic optimization" with patent application number "201510362041.X" both require the Dendrobium officinale to be dried first and use the dried Dendrobium officinale as raw material. Although the Chinese patent "A method for extracting Dendrobium officinale polysaccharides" with patent application number "201410010334.7" uses fresh Dendrobium officinale, a large amount of organic solvents and alkaline solutions are used in the extraction process, which is easy to cause environmental pollution.

[0005] Therefore, both the preparation method of fresh Dendrobium officinale polysaccharide and the active function of fresh Dendrobium officinale polysaccharide in organisms need further in-depth research. Summary of the invention

[0006] The first technical problem to be solved by the present invention is to provide a refined polysaccharide of fresh Dendrobium officinale.

[0007] The second technical problem to be solved by the present invention is to provide a method for rapidly preparing refined polysaccharides from fresh Dendrobium officinale.

[0008] The third technical problem to be solved by the present invention is to provide a refined fresh Dendrobium officinale polysaccharide having the effects of protecting the liver and sobering up.

[0009] The fourth technical problem to be solved by the present invention is to provide the application of the compound containing fresh Dendrobium officinale polysaccharide in the preparation of alcohol-relieving, liver-protecting and stomach-nourishing medicines.

[0010] The fifth technical problem to be solved by the present invention is to provide the application of the compound containing fresh Dendrobium officinale polysaccharide in the preparation of health food with the effect of enhancing liver function and immunity.

[0011] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0012] In the first aspect, the present invention provides a refined polysaccharide of fresh Dendrobium officinale, which is a glucomannan containing an O-acetyl structure, and its main chain is mainly connected by three glycosidic bonds: →4)-β-D-Glcp-(1→, →4)-β-D-Manp-(1→ and →4)-2-O-acetyl-β-D-Manp-(1→), and its repeating connection unit is:

[0013]

[0014] , where Manp is mannose and Glcp is glucose.

[0015] The fresh refined polysaccharide of Dendrobium candidum has a weight average molecular weight of 2.32×10 5 Da, the sugar content is 90.78%, the uronic acid content is 0.22%, the protein content is 0.09%, the proportion of mannose and glucose reaches 99.61%, and the ratio of mannose to glucose is 2.65.

[0016] In a second aspect, the present invention provides a method for preparing refined polysaccharides from fresh Dendrobium officinale, the preparation method being carried out according to the following steps:

[0017] (1) Take an appropriate amount of fresh Dendrobium candidum, add 30 times the amount of pure water, squeeze the juice, and take the filtrate;

[0018] (2) The filtrate is concentrated under reduced pressure at 60°C to 70°C to form a concentrated solution, which is precipitated with 80% ethanol, left to stand overnight, and centrifuged to obtain a precipitate. The precipitate is dissolved in pure water and centrifuged, and then precipitated with alcohol again, and this is repeated twice. The polysaccharide precipitate is freeze-dried to obtain crude polysaccharides of fresh Dendrobium officinale;

[0019] (3) Add an appropriate amount of pure water to the crude polysaccharide of fresh Dendrobium officinale to fully dissolve it and prepare a polysaccharide solution with a concentration of about 5 mg / mL, then add Sevag reagent (V chloroform: V n-butanol = 4:1) with a volume of one-third of the polysaccharide solution, and continue stirring for 30 minutes after mixing. Let it stand for a while, and centrifuge to remove the precipitate. Repeat the stirring and centrifugation operation until there is no obvious precipitation after stirring, and obtain the crude polysaccharide deproteinized sample;

[0020] (4) Dissolve the deproteinized polysaccharide sample in an appropriate amount of pure water and stir thoroughly to ensure complete dissolution. Then slowly add anhydrous ethanol while gradually increasing the stirring speed to make the ethanol concentration in the solution reach 40%. Separate the precipitate by centrifugation to obtain a 40% ethanol-precipitated polysaccharide (DOP40); continue to add anhydrous ethanol to the supernatant to an ethanol concentration of 60% and 80%, and centrifuge to obtain a 60% ethanol-precipitated polysaccharide (DOP60) and an 80% ethanol-precipitated polysaccharide (DOP80). Freeze-dry them separately to obtain polysaccharides of different alcohol precipitation components;

[0021] (5) The polysaccharide sample precipitated with 60% ethanol was dissolved in water, loaded on a DEAE-Sepharose Fast Flow column, eluted with ultrapure water, collected by an automatic collector, the flow rate of the peristaltic pump was 2.5 mL / min, and one tube was collected for every 10 mL. The absorbance was determined by the phenol-sulfuric acid method. Each gradient was washed until no sugar was detected. The same fractions were combined according to the absorption peaks, concentrated under reduced pressure at 50°C to 60°C, and freeze-dried to obtain fresh semi-refined polysaccharides of Dendrobium officinale.

[0022] (6) Dissolve the semi-refined polysaccharide of fresh Dendrobium officinale in water, load it on a Sephadex G-100 column, elute with ultrapure water, collect with an automatic collector, use a peristaltic pump at a flow rate of 0.2 mL / min, collect 2 mL in one tube, determine the absorbance by the phenol-sulfuric acid method, wash each gradient until no sugar is detected, merge the same fractions according to the absorption peak, concentrate under reduced pressure at 50°C to 60°C, and freeze-dry to obtain the refined polysaccharide DOP60-1A of fresh Dendrobium officinale.

[0023] In a third aspect, the present invention provides the use of the refined polysaccharide of fresh Dendrobium officinale prepared according to the above preparation method in the preparation of alcohol-relieving, liver-protecting and stomach-nourishing medicines.

[0024] An ethanol-induced AML-12 cell injury model and a mouse acute alcoholic liver injury model were established, and it was found that the refined polysaccharide of fresh Dendrobium officinale of the present invention can alleviate and protect alcoholic liver injury in mice by inhibiting inflammation and regulating lipid metabolism; in an acute drunkenness animal model, it was confirmed that the refined polysaccharide of fresh Dendrobium officinale can delay the drunkenness time of drunken mice, advance the sobering time, significantly increase the activities of liver ADH and ALDH, and effectively improve the state of gastric mucosal injury, with an effect similar to that of positive drugs.

[0025] The invention provides a fresh Dendrobium officinale polysaccharide compound preparation for preventing and treating alcohol-induced acute and chronic liver damage.

[0026] In a fourth aspect, the present invention provides the use of the compound containing fresh Dendrobium officinale polysaccharide in the preparation of a drug having the functions of sobering up, protecting the liver and nourishing the stomach.

[0027] In a fifth aspect, the present invention provides the use of the fresh Dendrobium officinale polysaccharide compound in the preparation of a health food having the effect of enhancing liver function and immunity.

[0028] The preparation steps of the fresh Dendrobium officinale polysaccharide compound preparation are as follows:

[0029] Take 1-3 parts of the above-mentioned refined polysaccharide of fresh Dendrobium officinale, 4-6 parts of Pueraria root extract, 1-3 parts of Hovenia dulcis extract, and 1-3 parts of tomato seed oil, mix them, add pharmaceutically acceptable excipients, flavoring agent mogroside and preservative potassium sorbate, and prepare an oral preparation according to a conventional method in pharmacy.

[0030] The dosage form of the Chinese medicine preparation is an oral preparation, which can be any one of hard capsules, soft capsules, tablets, granules, powders, pills, tea bags, soft candies, oral liquids and beverages, and is easy to use.

[0031] The preparation methods of Pueraria root extract and Hovenia dulcis fruit extract are as follows: take the medicinal material Pueraria root or Hovenia dulcis fruit, grind it with a grinder, heat and reflux extract with 10 times water for 2 to 3 times, each time for 1 to 1.5 hours, combine the decoctions, filter, and concentrate the filtrate under reduced pressure at 60°C to 70°C to form a concentrated solution, precipitate it with 65% ethanol concentration, let it stand overnight, centrifuge to obtain the supernatant, recover the ethanol, vacuum freeze-dry, and grind it into powder.

[0032] Tomato seed oil was purchased from Hebei Silk Road Chenguang Oil Co., Ltd., Pueraria root extract was purchased from Guangzhou Wenbosen Biotechnology Co., Ltd., in which the puerarin content was not less than 10%, and Hovenia dulcis extract was purchased from Chenguang Biotechnology Group Co., Ltd., in which the Hovenia dulcis flavonoids content was not less than 2%.

[0033] Explanation of the prescription: Traditional Chinese medicine believes that alcohol is a hot and toxic substance that is damp in nature. Long-term excessive drinking can lead to dysfunction of the spleen and stomach, followed by stagnation of liver qi and damage to the internal organs. The main raw materials of the compound fresh Dendrobium candidum polysaccharide preparation are composed of Dendrobium candidum polysaccharide, Pueraria root, Hovenia dulcis, and tomato seed oil. Alcohol helps fire to promote yang, but it is easy to consume qi and damage yin. The Dendrobium candidum in the prescription is good at nourishing stomach yin, which can relieve the intense heat and toxicity in the stomach, promote fluid production and quench thirst, and is the main medicine. The combination of Hovenia dulcis and Pueraria root has the effects of stopping vomiting and relieving restlessness, and detoxifying alcohol. Hovenia dulcis is diuretic, and Pueraria root raises yang. While detoxifying alcohol, it can also take into account the characteristics of dampness easily trapping the spleen, promote the spleen's clear qi, help the spleen and stomach to function well, and lead dampness and heat downward, and they are all ministerial medicines. The combination of the above medicines can strengthen the spleen and stomach, clear away heat and dampness, regulate qi and soothe the liver, and relieve dry throat and thirst caused by alcohol poisoning, as well as stomach irritation, as well as poor qi and blood stasis caused by damage to the spleen and stomach and liver and gallbladder. Tomato seed oil is a new food raw material rich in polyunsaturated fatty acids, lycopene, β-carotene, phytosterols and other active ingredients. It has the potential to improve lipid metabolism disorders and regulate intestinal flora. It can also be used as an excipient for oral preparations, which is conducive to molding and stable ingredients.

[0034] The present invention is beneficial in that:

[0035] (1) A method for preparing a refined polysaccharide of fresh Dendrobium officinale is provided, which has good effects of sobering up, protecting the liver and nourishing the stomach, and can be made into a corresponding preparation, which can be used for the drug treatment of alcoholic liver disease.

[0036] (2) Provide a fresh Dendrobium officinale polysaccharide compound preparation for preventing and treating alcohol-induced acute and chronic liver damage. The preparation is based on traditional Chinese medicine culture, is safe and non-toxic, and has clear alcohol-relieving and liver-protecting effects. The invention of this product is expected to alleviate the current severe liver and stomach damage situation, bring considerable economic benefits to the enterprise, and also promote the advantages of Chinese medicine in daily health care. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 PMP-HPLC chromatogram of refined polysaccharides from fresh Dendrobium officinale;

[0038] Figure 2 Gel chromatogram of refined polysaccharides from fresh Dendrobium officinale;

[0039] Figure 3 Infrared spectrum of refined polysaccharides from fresh Dendrobium officinale;

[0040] Figure 4 GC-MS total ion chromatogram of Smith degradation products of refined polysaccharides from fresh Dendrobium officinale;

[0041] Figure 5 Total ion chromatogram of methylation analysis of refined polysaccharides from fresh Dendrobium officinale;

[0042] Figure 6 Refined polysaccharide from fresh Dendrobium candidum 1H NMR spectrum (D2O, 500 MHz);

[0043] Figure 7 Refined polysaccharide from fresh Dendrobium candidum 13 C NMR spectrum (D2O, 125 MHz);

[0044] Figure 8 Refined polysaccharide from fresh Dendrobium candidum 1 H- 1 HCOSY spectrum (D2O, 500 MHz);

[0045] Fig. 9 HSQC spectrum of purified polysaccharides from fresh Dendrobium officinale (D2O, 500MHz);

[0046] Fig.10 HMBC spectrum of refined polysaccharides from fresh Dendrobium officinale (D2O, 500MHz);

[0047] Fig.11 Bodipy staining results of refined fresh Dendrobium officinale samples on alcohol-damaged AML12 cells (K, M, and DOP60-1A in the figure represent the blank control group, model group, and refined polysaccharide sample group of fresh Dendrobium officinale, respectively);

[0048] Fig.12 Effects of refined polysaccharides from Dendrobium officinale on gastric mucosal pathology in drunken mice (Ctrl, M, Positive, and DOP60-1A in the figure represent blank control group, model group, positive drug group, and refined polysaccharide sample group of fresh Dendrobium officinale, respectively). DETAILED DESCRIPTION

[0049] The essential content and beneficial effects of the present invention will be further described in detail below in conjunction with embodiments, and these embodiments are only used to illustrate the present invention rather than to limit the present invention. In addition, after reading the content taught by the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0050] Experimental Example 1: Preparation of refined polysaccharides from fresh Dendrobium officinale

[0051] (1) Take 500 g of fresh Dendrobium candidum, add 30 times the amount of pure water, squeeze the juice, and take 15 L of filtrate;

[0052] (2) The filtrate was concentrated under reduced pressure at 60°C to 70°C to form a concentrated solution, and precipitated with 80% ethanol, allowed to stand overnight, and centrifuged to obtain a precipitate. The precipitate was dissolved in pure water and centrifuged, and then precipitated with alcohol again, and repeated twice. The polysaccharide precipitate was freeze-dried to obtain 56.8 g of fresh Dendrobium officinale crude polysaccharide;

[0053] (3) Take 50g of fresh crude polysaccharide of Dendrobium candidum, add 10L of pure water to fully dissolve it, and prepare a polysaccharide solution with a concentration of about 5mg / mL, then add Sevag reagent (V chloroform: V n-butanol = 4:1) with a volume of one-third of the polysaccharide solution, and continue stirring for 30 minutes after mixing. Let it stand for a while, and centrifuge to remove the precipitate. Repeat the stirring and centrifugation operation until there is no obvious precipitation after stirring, and obtain 30.5g of crude polysaccharide deproteinized sample;

[0054] (4) Dissolve the deproteinized polysaccharide sample in 1000 mL of pure water and stir thoroughly to ensure complete dissolution. Then slowly add anhydrous ethanol while gradually increasing the stirring speed to make the ethanol concentration in the solution reach 40%. Separate the precipitate by centrifugation to obtain 17.3 g of 40% ethanol-precipitated polysaccharide (DOP40); continue to add anhydrous ethanol to the supernatant to 60% and 80% ethanol concentrations, and centrifuge to obtain 12.5 g of 60% ethanol-precipitated polysaccharide (DOP60) and 0.7 g of 80% ethanol-precipitated polysaccharide (DOP80). Freeze-dry them separately to obtain polysaccharides of different alcohol precipitation components;

[0055] (5) 10 g of the polysaccharide sample precipitated with 60% ethanol was dissolved in water, loaded on a DEAE-Sepharose Fast Flow column, eluted with ultrapure water, collected by an automatic collector, and the flow rate of the peristaltic pump was 2.5 mL / min. One tube was collected for every 10 mL. The absorbance was determined by the phenol-sulfuric acid method. Each gradient was washed until no sugar was detected. The same fractions were combined according to the absorption peaks, concentrated under reduced pressure at 50°C to 60°C, and freeze-dried to obtain 5.9 g of fresh semi-refined polysaccharide of Dendrobium officinale.

[0056] (6) Dissolve the semi-refined polysaccharide of fresh Dendrobium officinale in water, load it on a Sephadex G-100 column, elute with ultrapure water, collect with an automatic collector, use a peristaltic pump at a flow rate of 0.2 mL / min, collect 2 mL in one tube, measure the absorbance by the phenol-sulfuric acid method, wash each gradient until no sugar is detected, merge the same fractions according to the absorption peak, concentrate under reduced pressure at 50°C to 60°C, and freeze-dry to obtain 4.8 g of fresh Dendrobium officinale refined polysaccharide DOP60-1A, with a yield of 0.96%.

[0057] Experimental Example 2: Preparation of Refined Polysaccharides from Fresh Dendrobium Officinale

[0058] (1) Take 300 g of fresh Dendrobium candidum, add 30 times the amount of pure water, squeeze the juice, and take 9 L of filtrate;

[0059] (2) The filtrate was concentrated under reduced pressure at 60°C to 70°C to form a concentrated solution, which was precipitated with 80% ethanol, left to stand overnight, and centrifuged to obtain a precipitate. The precipitate was dissolved in pure water and centrifuged, and then precipitated with alcohol again, and this process was repeated twice. The polysaccharide precipitate was freeze-dried to obtain 34.1 g of fresh crude polysaccharide of Dendrobium officinale.

[0060] (3) Take 30g of fresh crude polysaccharide of Dendrobium candidum, add 6L of pure water to fully dissolve it, and prepare a polysaccharide solution with a concentration of about 5mg / mL, then add Sevag reagent (V chloroform: V n-butanol = 4:1) with a volume of one-third of the polysaccharide solution, and continue stirring for 30 minutes after mixing. Let it stand for a while, and centrifuge to remove the precipitate. Repeat the stirring and centrifugation operation until there is no obvious precipitation after stirring, and obtain 18.3g of crude polysaccharide deproteinized sample;

[0061] (4) Dissolve the deproteinized polysaccharide sample in 600 mL of pure water and stir thoroughly to ensure complete dissolution. Then slowly add anhydrous ethanol while gradually increasing the stirring speed to make the ethanol concentration in the solution reach 40%. Separate the precipitate by centrifugation to obtain 10.3 g of 40% ethanol-precipitated polysaccharide (DOP40); continue to add anhydrous ethanol to the supernatant to 60% and 80% ethanol concentrations, and centrifuge to obtain 7.5 g of 60% ethanol-precipitated polysaccharide (DOP60) and 0.4 g of 80% ethanol-precipitated polysaccharide (DOP80). Freeze-dry them separately to obtain polysaccharides of different alcohol precipitation components;

[0062] (5) 6 g of the polysaccharide sample precipitated with 60% ethanol was dissolved in water, loaded on a DEAE-Sepharose Fast Flow column, eluted with ultrapure water, collected by an automatic collector, and the peristaltic pump flow rate was 2.5 mL / min. One tube was collected for every 10 mL. The absorbance was determined by the phenol-sulfuric acid method. Each gradient was washed until no sugar was detected. The same fractions were combined according to the absorption peaks, concentrated under reduced pressure at 50°C to 60°C, and freeze-dried to obtain 3.5 g of fresh semi-refined polysaccharide of Dendrobium officinale.

[0063] (6) Dissolve the semi-refined polysaccharide of fresh Dendrobium officinale in water, load it on a Sephadex G-100 column, elute with ultrapure water, collect with an automatic collector, use a peristaltic pump at a flow rate of 0.2 mL / min, collect 2 mL in one tube, determine the absorbance by the phenol-sulfuric acid method, wash each gradient until no sugar is detected, merge the same fractions according to the absorption peak, concentrate under reduced pressure at 50°C to 60°C, and freeze-dry to obtain 2.8 g of refined polysaccharide DOP60-1A of fresh Dendrobium officinale, with a yield of 0.93%.

[0064] Experimental Example 3: Preparation of refined polysaccharides from fresh Dendrobium officinale

[0065] (1) Take 600 g of fresh Dendrobium candidum, add 30 times the amount of pure water, squeeze the juice, and take 18 L of filtrate;

[0066] (2) The filtrate was concentrated under reduced pressure at 60°C to 70°C to form a concentrated solution, and precipitated with 80% ethanol, allowed to stand overnight, and centrifuged to obtain a precipitate. The precipitate was dissolved in pure water and centrifuged, and then precipitated with alcohol again, and repeated twice. The polysaccharide precipitate was freeze-dried to obtain 68.2 g of fresh crude polysaccharide of Dendrobium officinale;

[0067] (3) Take 60g of fresh crude polysaccharide of Dendrobium candidum, add 12L of pure water to fully dissolve it, and prepare a polysaccharide solution with a concentration of about 5mg / mL, then add Sevag reagent (V chloroform: V n-butanol = 4:1) with a volume of one-third of the polysaccharide solution, and continue stirring for 30 minutes after mixing. Let it stand for a while, and centrifuge to remove the precipitate. Repeat the stirring and centrifugation operation until there is no obvious precipitation after stirring, and obtain 36.6g of crude polysaccharide deproteinized sample;

[0068] (4) Dissolve the deproteinized polysaccharide sample in 1200 mL of pure water and stir thoroughly to ensure complete dissolution. Then slowly add anhydrous ethanol while gradually increasing the stirring speed to make the ethanol concentration in the solution reach 40%. Separate the precipitate by centrifugation to obtain 20.7 g of 40% ethanol-precipitated polysaccharide (DOP40); continue to add anhydrous ethanol to the supernatant to 60% and 80% ethanol concentrations, and centrifuge to obtain 15.0 g of 60% ethanol-precipitated polysaccharide (DOP60) and 0.8 g of 80% ethanol-precipitated polysaccharide (DOP80). Freeze-dry them separately to obtain polysaccharides of different alcohol precipitation components;

[0069] (5) 12 g of the polysaccharide sample precipitated with 60% ethanol was dissolved in water, loaded on a DEAE-Sepharose Fast Flow column, eluted with ultrapure water, collected by an automatic collector, and the flow rate of the peristaltic pump was 2.5 mL / min. One tube was collected for every 10 mL. The absorbance was determined by the phenol-sulfuric acid method. Each gradient was washed until no sugar was detected. The same fractions were combined according to the absorption peaks, concentrated under reduced pressure at 50°C to 60°C, and freeze-dried to obtain 7.1 g of fresh semi-refined polysaccharide of Dendrobium officinale.

[0070] (6) Dissolve the semi-refined polysaccharide of fresh Dendrobium officinale in water, load it on a Sephadex G-100 column, elute with ultrapure water, collect with an automatic collector, use a peristaltic pump at a flow rate of 0.2 mL / min, collect 2 mL in one tube, determine the absorbance by the phenol-sulfuric acid method, wash each gradient until no sugar is detected, merge the same fractions according to the absorption peak, concentrate under reduced pressure at 50°C to 60°C, and freeze-dry to obtain 5.7 g of fresh Dendrobium officinale refined polysaccharide DOP60-1A, with a yield of 0.95%.

[0071] Experimental Example 4: Structural Characterization of Refined Polysaccharides from Fresh Dendrobium Officinale

[0072] The fresh refined polysaccharides of Dendrobium officinale used in this example are all the fresh refined polysaccharides of Dendrobium officinale DOP60-1A prepared in Example 1.

[0073] (1) Determination of total sugar content: The total sugar content of the refined polysaccharide of fresh Dendrobium officinale was determined by the phenol-sulfuric acid method and was 90.78%.

[0074] (2) Protein content determination: The protein content of fresh refined polysaccharide of Dendrobium officinale was determined by BCA kit to be 0.09%, indicating that the impurity content was low.

[0075] (3) Determination of uronic acid content: The uronic acid content of the purified polysaccharide of fresh Dendrobium officinale was determined by the m-hydroxybiphenyl method and was 0.22%, indicating that it was an acidic polysaccharide.

[0076] (4) Monosaccharide composition analysis

[0077] Acid hydrolysis: 15 mg of fresh refined polysaccharide from Dendrobium officinale was dissolved in 3 mL of trifluoroacetic acid (3 mol / L), and the mixture was reacted at 110 °C for 6 h to obtain a hydrolyzate. After evaporation under reduced pressure, methanol was repeatedly added 4 times to replace and remove the residual TFA.

[0078] Derivatization: Take 400 μL of each of the refined polysaccharide and monosaccharide standard (rhamnose, mannose, galactose, glucose arabinose, glucuronic acid, galacturonic acid) after acid hydrolysis, add 400 μL of 0.5 mol / L PMP methanol solution and 0.3 mol / L NaOH, mix and mix thoroughly, and heat in a 70°C water bath for 100 min. After the reaction is completed, add 500 μL of 0.3 mol / L HCL, mix well, extract with 2 mL of chloroform, remove the lower chloroform solution, repeat the operation 3 times, filter through a microporous filter membrane, and take 10 μL of the standard and sample test solution into the liquid phase.

[0079] Liquid chromatography conditions: C18 column (4.6 mm×250 mm, 5 μm); mobile phase: acetonitrile-0.05 mol / L potassium dihydrogen phosphate solution (20:80); detection wavelength: 250 nm; flow rate: 0.8 mL / min; injection volume: 10 μL.

[0080] The results of monosaccharide composition analysis are as follows Figure 1 As shown in the figure, the refined polysaccharides from fresh Dendrobium officinale are mainly composed of glucose and mannose, and the molar ratio of glucose to mannose is 2.65:1.

[0081] (5) Homogeneity and molecular weight determination: Fresh refined polysaccharide from Dendrobium officinale was prepared into a 5 mg / mL solution with a 0.05 mol / L NaCl-water solution, and the molecular weight was determined by high performance liquid gel permeation chromatography. The chromatographic column was a 105-103-101 tandem gel column (8 mm × 300 mm), the column temperature was set to 40 °C, the detector was a differential detector RID-20, the mobile phase was a 0.05 mol / L NaCl-water solution, the flow rate was 0.8 mL / min, and the injection volume was 25 uL.

[0082] The purified polysaccharide from fresh Dendrobium candidum showed a single symmetrical peak shape ( Figure 2 ), indicating that it is a uniform and refined polysaccharide, and its molecular weight is calculated to be 2.32×10 5 Da;

[0083] (6) Infrared analysis: Take an appropriate amount of freeze-dried fresh refined polysaccharide from Dendrobium officinale and mix it with KBr and grind it evenly. Use a high-throughput Fourier transform infrared (FT-IR) spectrometer to scan it in the range of 4000-400 cm -1 , record the infrared spectrum infrared analysis results such as Figure 3 As shown in the figure, the characteristics of polysaccharides correspond to the stretching vibration of OH and CH of polysaccharides; 1740cm -1 The absorption peaks near the acetoxy group or carbonyl group are attributed to the stretching vibration of C=O, indicating that it contains a small amount of uronic acid; 1200-1000cm -1 The three absorptions between them are caused by the stretching vibration of COC on the sugar ring, indicating that the sample contains a pyranoside structure; -1 The absorption peak near 810cm indicates that the glycosidic bonds in the sample are mainly β-type; -1 The absorption peak near β-D-mannopyranoside indicates that the sample contains D-Man. The results show that the refined polysaccharide of fresh Dendrobium officinale is an acidic polysaccharide containing β-D-mannopyranoside bonds.

[0084] (7) Smith degradation analysis: The reaction solution that had been completely oxidized by sodium periodate and treated with ethylene glycol was dialyzed, concentrated, and then placed in an ice-water bath. 50 mg of sodium borohydride was added, stirred in the dark, and reduced for 24 hours. 0.2 M acetic acid solution was added dropwise to the reduced product, and the pH was adjusted to 5.5. After dialyzation, it was freeze-dried. The freeze-dried sample was dissolved in 4 mL of 2 mol / L trifluoroacetic acid and sealed in a 110°C oven for hydrolysis for 3 hours. After the hydrolysis was completed, it was evaporated to dryness under reduced pressure, 7 mL of methanol was added, and evaporated to dryness under reduced pressure. This operation was repeated 5 times. The supernatant was taken and evaporated to dryness under reduced pressure. 1 mL of pyridine and 1 mL of acetic anhydride were added to the product, ultrasonically dissolved, filled with N2, sealed, reacted in a 90°C water bath for 30 minutes, concentrated to dryness under reduced pressure, added a small amount of anhydrous methanol, evaporated to dryness, and repeated three times. Chloroform and purified water were added in a ratio of 1:1 (v / v) to extract the product, retain the organic phase, evaporated to dryness under reduced pressure, and extracted three times. 0.75 mL of chloroform was added to the product obtained by evaporation to dissolve it, and then GC-MS detection was performed.

[0085] The ratio of glycosidic bond connection types of fresh Dendrobium officinale refined polysaccharides can be obtained by Smith degradation product analysis. The results are shown in Table 1 and Figure 4 GC-MS analysis ultimately showed that part of the mannose, acetylmannose and glucose residues in the refined polysaccharides of fresh Dendrobium officinale existed in the form of 1→3 linkages that were not oxidized by periodic acid, and there were also certain proportions of 1→2 or 1→6 linkages and 1→4 linkages.

[0086] Table 1 shows the GC-MS analysis results of Smith degradation products

[0087]

[0088] (8) Methylation analysis

[0089] Weigh 10 mg of dried refined Dendrobium officinale polysaccharide, add 5 mL of anhydrous dimethyl sulfoxide, seal with nitrogen, and fully dissolve. Then add 30 mg of ground NaOH powder and ultrasonicate for 1 hour. Slowly add 2 mL of iodomethane (shaking while adding) under ice bath conditions, ultrasonicate for 0.5 hours, stand in the dark for 0.5 hours, and add 2 mL of ultrapure water to terminate the reaction. Add 2 mL of dichloromethane, centrifuge to take the dichloromethane layer, and blow dry with nitrogen. Repeat the above steps 2-3 times. Add 4 mL of 2 mol / L trifluoroacetic acid to the dried product, react at 100 ° C for 3 hours, evaporate to dryness using a rotary evaporator, add 2 mL of methanol, blow dry with nitrogen, and repeat three times. Add 0.1 mol / L NaOH solution and 100 mg of sodium borohydride powder and react for 2 hours. Add acetic acid until the pH is acidic. Add 2 mL of methanol and 1 drop of acetic acid, concentrate to dryness under reduced pressure, and repeat four times. Add 1 mL of acetic anhydride and 1 mL of pyridine, react at 100°C for 2 h, add 2 mL of toluene and concentrate to dryness under reduced pressure. No hydroxyl absorption peak is found in infrared detection. Dissolve in 3 mL of chloroform and detect by GC-MS.

[0090] Through methylation analysis, the glycosidic bond types of fresh refined polysaccharides of Dendrobium officinale can be obtained. The results are shown in Table 2 and Figure 5 GC-MS analysis finally showed that the main glycosidic bond composition of the refined polysaccharide of fresh Dendrobium officinale included →4)-Manp-(1→, →4)-Glcp-(1→ and a small amount of →3,4)-Rhap-(1→, and the molar ratio of the three was 7.6:1.8:1.0. Combined with the presence of glycosides in the Smith degradation product and the absence of (1→3)-linked glycosidic bonds in the methylated product, it is speculated that the glycosides in the Smith degradation product come from the acetoxy-1,4-linked glycosidic bonds in DOP60-1A. In the strong alkaline environment of the methylation reaction, the acetoxy group is unstable and easy to fall off, and was not detected in the methylation results.

[0091] Table 2 is the analysis of the results of methylated sugar alcohol acetyl esters of fresh refined polysaccharides from Dendrobium candidum

[0092]

[0093] (9) Nuclear magnetic resonance analysis: 70 mg of the freeze-dried DOP60-1A sample was accurately weighed and dissolved in 1.6 mL of D2O to perform sufficient proton exchange, and the solution was freeze-dried, and the process was repeated three times. The final freeze-dried sample was dissolved in a small amount of D2O, and then the structure of DOP60-1A was analyzed in detail using nuclear magnetic resonance (NMR) technology.

[0094] The results of NMR are as follows Figure 6-10As shown. The obtained refined Dendrobium officinale polysaccharide was analyzed by nuclear magnetic resonance, and its main chain was mainly connected by three glycosidic bonds: →4)-β-D-Glcp-(1→, →4)-β-D-Manp-(1→ and →4)-2-O-acetyl-β-D-Manp-(1→, and its repeating connection unit was:

[0095]

[0096] , where Manp is mannose and Glcp is glucose.

[0097] Experimental Example 5: Protective effect of fresh refined polysaccharide of Dendrobium officinale on alcohol-stimulated AML12 cells:

[0098] Establishment of alcohol-induced AML12 cell injury model: AML12 cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% double antibody mixture, and cultured in a 37°C constant temperature cell culture incubator containing 5% CO2. When the density of cell adherence growth reached about 80%, 0.25% trypsin was added for digestion and passage. Samples were taken from AML12 cells in the logarithmic growth phase, treated with 0.25% trypsin to separate the cells, and the cell density was adjusted to 1×10 with DMEM complete medium. 5 / mL, the prepared cell suspension was inoculated into a 96-well plate at 100 μL per well, and incubated in an incubator for 24 hours. The DMEM medium containing 12.5, 25, 50, 100, 400, and 800 mmol / L ethanol was added to the solution, and 5 replicates were set for a single concentration and incubated for 24 hours. After the incubation, the DMEM medium with a 10% concentration of CCK-8 was added by changing the solution, and the culture was continued in the cell culture incubator for 1-2 hours. The wells with only blank culture medium were set as normal control wells, and the wells without cell inoculation were set as blank zeroing wells, and the absorbance value was measured at a wavelength of 450nm, and the survival rate of the cells was calculated to determine the modeling concentration of ethanol for AML12 cells.

[0099] Sample cytotoxicity screening: AML12 cells in the logarithmic growth phase were digested with 0.25% trypsin and prepared with DMEM complete medium to a density of 1×10 5 / mL cell suspension, inoculate 100μL per well into a 96-well plate, and place in an incubator for 24h until the cells adhere to the wall. Change the medium and add DMEM medium containing 0, 1, 2, 5, 10, 20, 50, 100, 200, 400μg / mL samples, repeat 3 replicates for each concentration, and incubate for 24h. After the incubation is completed, discard the drug-containing medium, add 100μL of DMEM medium containing 10% CCK-8, place in a cell culture incubator and continue to incubate for 2h, set the wells with only blank medium as normal control wells, set the wells without inoculated cells as blank zero wells, measure the absorbance at 450nm, and calculate the cell survival rate to determine the concentration of the sample on AML12 cells.

[0100] Bodipy staining analysis: The experimental group cells were divided into blank control group, model group, and polysaccharide administration group. After 24 hours of plating, except for the blank control group, the other groups were damaged with 100mmol / L ethanol culture medium for 24 hours to form a cell alcohol injury model. The culture medium was discarded, 100mmol / L ethanol culture medium was added to the model group, serum-free culture medium was added to the blank control group, and 200μg / mL of fresh refined polysaccharide DOP60-1A prepared by the method of Example 1 was given to the administration group. The culture medium was aspirated, washed with PBS 2 to 3 times, and the cells were stained with Bodipy dye for 30 minutes at room temperature and in the dark. The excess dye was aspirated, rinsed with PBS, and the effect of staining was observed using an optical microscope. At the same time, the stained cells were subjected to flow cytometry to quantify the fluorescence intensity of the cells.

[0101] The experimental results are as follows Fig.11 As shown, compared with the blank control group, the model group showed a significantly increased green fluorescence, indicating that obvious lipid accumulation occurred in the alcohol-induced AML12 cells; compared with the cells in the model group, the fluorescence intensity in the drug-treated group cells was significantly decreased, and the difference was statistically significant, which intuitively showed that the refined polysaccharide at a concentration of 200 μg / mL can significantly reduce the alcohol-induced AML12 cell lipid droplet accumulation and has a certain liver protective effect.

[0102] Experimental Example 6: Protective effect of fresh refined polysaccharides from Dendrobium officinale on alcoholic liver injury model in mice

[0103] In this Example 1, the fresh refined polysaccharide DOP60-1A of Dendrobium officinale prepared as described above was used to observe its liver protection effect in an alcoholic liver injury experiment in mice, as follows:

[0104] Experimental animals: 50 C57BL / 6J male mice, 8-10 weeks old, weighing 18-20 g.

[0105] Experimental drugs: blank control group K (normal saline 0.1 mL / 10g body weight, 10 rats), model group M (normal saline 0.1 mL / 10g body weight, 10 rats), positive control group P (silybin meglumine added with water to prepare 5 mg / mL solution, 50 mg / kg administration, 10 rats), high-dose DH and low-dose DL administration groups (fresh refined polysaccharide DOP60-1A prepared by the method of Example 1, 400 and 100 mg / kg, 10 rats each).

[0106] Experimental methods: All groups of mice were fed with Lieber-DeCarli liquid controlled diet (F1259SP; Bio-Serv, Flemington, NJ) for 5 days. From the 6th day, the mice in the blank group were still fed with F1259SP liquid diet, and the other groups were fed with F1258SP liquid diet containing ethanol for 10 days. Each group was gavaged with the corresponding drug from the first day of the experiment. After the end of the 15th day of administration, the mice were fasted for 12 hours and then killed to obtain liver tissue and blood.

[0107] Behavioral status observation: observe the mental state of each group of mice every day, whether the behavior is abnormal, whether the hair is falling off, record the daily weight of the mice, and observe whether the weight fluctuation is within a normal range.

[0108] Determination of ALT and AST levels in mouse serum and TG and TCHO levels in liver tissue: According to the instructions of the mouse ALT and AST kits, the ALT and AST levels in serum were detected; referring to the operating steps of the mouse TG and TCHO kits, 20 mg of mouse liver tissue was accurately weighed, 200 μL of anhydrous ethanol was added to homogenize on ice, centrifuged at 10000 r / min for 10 min, the supernatant was taken, and the TG and TCHO levels in the liver tissue were determined.

[0109] Determination of IL-1β, CXCL10, Srebp-1c, and CPT1 mRNA expression levels in mouse liver tissue: cut liver tissue of approximately equal weight from each mouse, add pre-cooled RNA lysis solution, add chloroform after homogenization, let stand for 5 minutes, centrifuge, take the supernatant, add isopropanol, and let stand for 30 minutes. Centrifuge at 12000r / min, 4℃ for 10 minutes, pour the supernatant to obtain a white precipitate. Add 75% ethanol for elution, let stand for 5 minutes, centrifuge at 12000r / min, 4℃ for 5 minutes, discard the ethanol, dry the white precipitate, add DEPC water to dissolve it, and determine the RNA concentration. Prepare the sample according to the concentration, perform reverse transcription, add DEPC water to dilute it in a certain proportion after transcription, and store it in a refrigerator at 4℃ after shaking. The sample is plated, and 4μL of sample and 6μL of primer: luciferase = 1:5 mixture are added to each grid of the plate, and then placed in a fluorescent quantitative PCR instrument for detection. The test results are shown in Tables 3 and 4.

[0110] Statistical methods: GraphPad Prism 8.0.1 software was used for data analysis, and all data were expressed as mean ± standard deviation (X ± S). When normality and homogeneity of variance were met, one-way analysis of variance was used to compare the means among multiple groups, and P < 0.05 was considered statistically significant.

[0111] Table 3 Effects of samples on ALT and AST activities in mouse serum and TG and TCHO contents in liver (X±SEM, n=6-8)

[0112] Group ALT(U / L) AST(U / L) TG(mmol / g) TC(mmol / g) Blank control group 46.21±0.22 104.45±12.50 0.13±0.01 0.12±0.003 Model Group <![CDATA[175.07±8.93 ## ]]> <![CDATA[289.50±15.05 ## ]]> <![CDATA[0.28±0.02 ## ]]> <![CDATA[0.20±0.01 ## ]]> Positive drug group 159.39±0.4* 223.90±15.70* 0.10±0.01* 0.12±0.01* DOP-L Group 37.03±3.20** 57.47±2.71** 0.13±0.01** 0.15±0.01** DOP-H Group 32.53±1.48** 41.25±6.95** 0.09±0.02** 0.13±0.001**

[0113] Table 4 Effects of samples on the expression of inflammation-related genes in mouse liver tissue (X±SEM, n=6-8)

[0114]

[0115]

[0116] The test results are shown in Tables 3 and 4. Compared with the blank control group, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of the model group mice increased significantly, which confirmed that the acute alcoholic liver injury model of mice was successfully established; compared with the model control group, after administration of different doses of fresh Dendrobium officinale refined polysaccharide DOP60-1A, the activity of ALT and AST in serum, the content of TG and TCHO in liver tissue were effectively reduced, and the gene expression levels of IL-1β, CXCL10, and Srebp-1c in the liver tissue of mice were significantly reduced, and the gene expression level of CPT1 was significantly increased. The above results show that fresh Dendrobium officinale refined polysaccharide can alleviate and protect alcoholic liver injury in mice by inhibiting inflammation and regulating lipid metabolism.

[0117] Experimental Example 7: Preventive and hepatoprotective effects of refined polysaccharides from fresh Dendrobium candidum on acute alcoholism model in mice

[0118] In this example, the fresh refined polysaccharide of Dendrobium officinale prepared by the method of the above-mentioned Example 1 was used to observe its alcohol sobering effect in an acute drunkenness experiment on mice, as follows:

[0119] Experimental animals: 24 KM male mice, 8-10 weeks old, weighing 28-30 g.

[0120] Experimental drugs: model group M (56° Red Star Erguotou 0.15 mL / 10 g body weight, 8 mice), positive control group Positive (Neptune Jinzun tablets 200 mg / kg, 8 mice), and drug administration group DOP60-1A (fresh Dendrobium officinale refined polysaccharide prepared by the method of Example 1 300 mg / kg, 8 mice).

[0121] Experimental method: After one week of adaptive feeding for all mice, the mice were fasted for 12 hours and given positive drugs or fresh refined polysaccharide DOP60-1A from Dendrobium officinale by gavage. 30 minutes later, they were given alcohol and the time of alcohol administration was recorded. After alcohol administration, the mice were placed in cages individually and their behavior was observed. When the righting reflex of the mice disappeared, the time of drunkenness was recorded; when the righting reflex of the mice recovered, the time of sobering up was recorded.

[0122] Calculation of the drunkenness and soberness time of mice: the drunkenness time of each mouse = drunkenness time - gavage time; the soberness time of each mouse = soberness time - drunkenness time.

[0123] Statistical methods: GraphPad Prism 8.0.1 software was used for data analysis, and all data were expressed as mean ± standard deviation (X ± S). When normality and homogeneity of variance were met, one-way analysis of variance was used to compare the means among multiple groups, and P < 0.05 was considered statistically significant.

[0124] The test results are shown in Table 5. Compared with the model group, the drunkenness time of mice in the drug group was significantly prolonged, and the sobering time was significantly shortened, which was similar to the results of the positive drug group. The above results show that the refined polysaccharide of fresh Dendrobium candidum can alleviate the drunkenness reaction of mice by delaying drunkenness and accelerating sobering up.

[0125] Table 5 The drunkenness and soberness time of mice in each group (X±SEM, n=6-8)

[0126] Group Drunk time / min Sobering time / min Model Group 36.50±7.046 451.8±36.89 DOP60-1A Group 80.50±12.71* 207.6±61.87** Positive drug group 86.86±19.84* 222.9±48.49**

[0127] Experimental Example 8: Protective effect of refined polysaccharides from fresh Dendrobium officinale on gastric mucosa in mice with acute alcohol intoxication model

[0128] In this example, the fresh refined polysaccharide of Dendrobium officinale prepared by the method of the above-mentioned Example 1 was used to observe its gastric mucosal protective effect in the acute drunkenness experiment of mice, as follows:

[0129] Experimental animals: 32 KM male mice, 8-10 weeks old, weighing 28-30 g.

[0130] Experimental drugs: blank control group Ctrl (normal saline 0.1 mL / 10g body weight, 8 rats), model group M (56° Red Star Erguotou 0.15 mL / 10g body weight, 8 rats), positive control group Positive (Neptune Jinzun tablets 200 mg / kg, 8 rats), drug group DOP60-1A (fresh Dendrobium officinale polysaccharide 300 mg / kg, 8 rats).

[0131] Experimental method: After one week of adaptive feeding, all mice were gavaged and administered drugs continuously for 7 days. Except for the normal control group, mice in other groups were gavaged with alcohol 30 minutes after the last administration. Mice were killed 2 hours after gavage, and the stomach tissues were removed, the contents were cleared, and then fixed in 4% paraformaldehyde.

[0132] Behavioral status observation: observe the mental state of each group of mice every day, whether the behavior is abnormal, whether the hair is falling off, record the daily weight of the mice, and observe whether the weight fluctuation is within a normal range.

[0133] HE staining of mouse gastric mucosa: After the mouse gastric tissue was fixed in 4% paraformaldehyde for one day, the fixed tissue was embedded in paraffin and sliced. The slices were stained with HE according to the instructions of the kit, and the stained slices were sealed with neutral gum and scanned panoramically in a slice scanner.

[0134] Statistical methods: GraphPad Prism 8.0.1 software was used for data analysis, and all data were expressed as mean ± standard deviation (X ± S). When normality and homogeneity of variance were met, one-way analysis of variance was used to compare the means among multiple groups, and P < 0.05 was considered statistically significant.

[0135] The test results are as follows Fig.12 As shown in the results, compared with the blank control group, the gastric mucosal epithelial cells in the model group were severely damaged and detached, and the glandular structure was disordered; compared with the model group, the gastric mucosal injury state in the drug-treated group was significantly improved, the gastric mucosal structure was relatively complete, without obvious damage, and the upper epithelial cells were closely arranged, similar to the positive drug group and the control group. The above results show that fresh refined polysaccharides from Dendrobium candidum can protect the stomach by reducing the damage of gastric mucosal epithelial cells.

[0136] Experimental Example 9: Preparation of a compound preparation of fresh Dendrobium candidum polysaccharide: Take 2 parts of refined fresh Dendrobium candidum polysaccharide prepared by the method of Example 1, 5 parts of Pueraria extract, 2 parts of Hovenia dulcis extract, and 2 parts of tomato seed oil, mix them, add starch, flavoring agent Mogroside and preservative potassium sorbate, granulate, and prepare into granules. Tomato seed oil was purchased from Hebei Silk Road Chenguang Oil Co., Ltd., Pueraria extract was purchased from Guangzhou Wenbosen Biotechnology Co., Ltd., in which the puerarin content accounted for not less than 10%, and Hovenia dulcis extract was purchased from Chenguang Biotechnology Group Co., Ltd., in which the Hovenia dulcis flavonoid content accounted for not less than 2%.

[0137] Experimental Example 10: Take 1 part of fresh refined polysaccharide of Dendrobium candidum prepared by the method of Example 1, 6 parts of Pueraria extract, 1 part of Hovenia dulcis extract, and 3 parts of tomato seed oil, add dextrin, flavoring agent mogroside and preservative potassium sorbate, granulate, press into tablets, and prepare into tablets. Tomato seed oil was purchased from Hebei Silk Road Chenguang Oil Co., Ltd., and Pueraria extract was purchased from Guangzhou Wenbosen Biomedicine Co., Ltd., in which the puerarin content was not less than 10%, and Hovenia dulcis extract was purchased from Chenguang Biotechnology Group Co., Ltd., in which the Hovenia dulcis flavonoid content was not less than 2%.

[0138] Experimental Example 11: Take 1-3 parts of fresh refined polysaccharide of Dendrobium candidum prepared by the method of Example 1, 4 parts of kudzu root extract, 3 parts of Hovenia dulcis extract, and 1 part of tomato seed oil, add starch, flavoring agent mogroside and preservative potassium sorbate, granulate, and prepare into capsules. Tomato seed oil was purchased from Hebei Silk Road Chenguang Oil Co., Ltd., and kudzu root extract was purchased from Guangzhou Wenbosen Biomedicine Co., Ltd., in which the puerarin content was not less than 10%, and Hovenia dulcis extract was purchased from Chenguang Biotechnology Group Co., Ltd., in which the Hovenia dulcis flavonoid content was not less than 2%.

[0139] Experimental Example 12: Protective effect of fresh Dendrobium officinale polysaccharide compound preparation on alcoholic liver injury model in mice

[0140] The fresh Dendrobium officinale polysaccharide compound preparation prepared according to the method of Example 9 was used to observe its liver protection effect in an alcoholic liver injury experiment in mice, as follows:

[0141] Experimental animals: 50 C57BL / 6J male mice, 8-10 weeks old, weighing 18-20 g.

[0142] Experimental drugs: blank control group K (normal saline 0.1 mL / 10g body weight, 10 rats), model group M (normal saline 0.1 mL / 10g body weight, 10 rats), positive control group P (silybin meglumine was added with water to prepare a 5 mg / mL solution, 50 mg / kg was administered, 10 rats), drug administration group high dose FH (fresh Dendrobium officinale polysaccharide compound preparation prepared by the method of Example 9, 400 mg / kg, 10 rats), low dose FL (fresh Dendrobium officinale polysaccharide compound preparation prepared by the method of Example 9, 100 mg / kg, 10 rats).

[0143] Experimental methods: All groups of mice were fed with Lieber-DeCarli liquid controlled diet (F1259SP; Bio-Serv, Flemington, NJ) for 5 days. From the 6th day, the mice in the blank group were still fed with F1259SP liquid diet, and the other groups were fed with F1258SP liquid diet containing ethanol for 10 days. Each group was gavaged with the corresponding drug from the first day of the experiment. After the end of the 15th day of administration, the mice were fasted for 12 hours and then killed to obtain liver tissue and blood.

[0144] Behavioral status observation: observe the mental state of each group of mice every day, whether the behavior is abnormal, whether the hair is falling off, record the daily weight of the mice, and observe whether the weight fluctuation is within a normal range.

[0145] Determination of ALT and AST levels in mouse serum and TG and TCHO levels in liver tissue: According to the instructions of the mouse ALT and AST kits, the ALT and AST levels in serum were detected; referring to the operating steps of the mouse TG and TCHO kits, 20 mg of mouse liver tissue was accurately weighed, 200 μL of anhydrous ethanol was added to homogenize on ice, centrifuged at 10000 r / min for 10 min, the supernatant was taken, and the TG and TCHO levels in the liver tissue were determined.

[0146] Determination of IL-1β, CXCL10, Srebp-1c, and CPT1 mRNA expression levels in mouse liver tissue: cut liver tissue of approximately equal weight from each mouse, add pre-cooled RNA lysis solution, add chloroform after homogenization, let stand for 5 minutes, centrifuge, take the supernatant, add isopropanol, and let stand for 30 minutes. Centrifuge at 12000r / min, 4℃ for 10 minutes, pour the supernatant to obtain a white precipitate. Add 75% ethanol for elution, let stand for 5 minutes, centrifuge at 12000r / min, 4℃ for 5 minutes, discard the ethanol, dry the white precipitate, add DEPC water to dissolve it, and determine the RNA concentration. Prepare the sample according to the concentration, perform reverse transcription, add DEPC water to dilute it in a certain proportion after transcription, and store it in a refrigerator at 4℃ after shaking. The sample is plated, and 4μL of sample and 6μL of primer: luciferase = 1:5 mixture are added to each grid of the plate, and then placed in a fluorescent quantitative PCR instrument for detection. The test results are shown in Tables 6 and 7.

[0147] Statistical methods: GraphPad Prism 8.0.1 software was used for data analysis, and all data were expressed as mean ± standard deviation (X ± S). When normality and homogeneity of variance were met, one-way analysis of variance was used to compare the means among multiple groups, and P < 0.05 was considered statistically significant.

[0148] Table 6 Effects of samples on ALT and AST activities in mouse serum and TG and TCHO contents in liver (X±SEM, n=6-8)

[0149] Group ALT(U / L) AST(U / L) TG(mmol / g) TC(mmol / g) Blank control group 46.21±0.22 104.45±12.50 0.13±0.01 0.12±0.003 Model Group 175.07±8.93 289.50±15.05 0.28±0.02 0.20±0.01 Positive drug group 161.56±1.90*** 286.31±10.42** 0.10±0.01*** 0.12±0.01*** FL Group 80.71±4.45*** 173.17±18.75*** 0.12±0.01*** 0.14±0.01*** FH group 26.07±1.63*** 127.86±7.46*** 0.10±0.01*** 0.12±0.01***

[0150] Table 7 Effects of samples on the expression of inflammation-related genes in mouse liver tissue (X±SEM, n=6-8)

[0151] Group IL-1β CXCL10 Srebp-1c CPT1 Blank control group 1.04±0.13 1.03±0.15 1.06±0.15 1.07±0.06 Model Group 6.51±1.25 6.25±0.95 2.84±0.15 0.47±0.06 Positive drug group 2.14±0.65* 1.91±0.54** 1.42±0.22* 1.02±0.07* FL Group 2.36±1.11 1.85±0.61** 2.16±0.73 1.37±0.28** FH group 1.75±0.49* 1.38±0.50** 1.80±0.54 1.74±0.10***

[0152] As shown in Tables 6 and 7, compared with the blank control group, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of the model group mice increased significantly, which confirmed that the acute alcoholic liver injury model of mice was successfully established; compared with the model control group, after administration of different doses of fresh Dendrobium officinale polysaccharide compound preparation, the ALT and AST activities in serum, the TG and TCHO contents in liver tissue were effectively reduced, and the gene expression levels of IL-1β, CXCL10, and Srebp-1c in the liver tissue of mice were significantly reduced, and the CPT1 gene expression level was significantly increased. The above results show that the Dendrobium officinale polysaccharide compound preparation can alleviate and protect alcoholic liver injury in mice by inhibiting inflammation and regulating lipid metabolism.

Claims

1. A refined polysaccharide from fresh Dendrobium officinale, characterized in that: The refined polysaccharide is a glucomannan containing an O-acetyl structure, and its main chain is mainly connected by three glycosidic bonds: →4)-2-O-acetyl-β-D-Manp-(1→, →4)-β-D-Manp-(1→ and →4)-β-D-Glcp-(1→, and its repeating connection unit is: Among them, Manp is mannose and Glcp is glucose.

2. The fresh refined polysaccharide from Dendrobium officinale according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Take an appropriate amount of fresh stems of Dendrobium officinale, add 30 times the amount of pure water, squeeze the juice, and take the filtrate; (2) the filtrate is concentrated under reduced pressure at 60° C. to 70° C. to form a concentrated solution, which is precipitated with 80% ethanol, left to stand overnight, and centrifuged to obtain a precipitate; The precipitate is dissolved in pure water and centrifuged, and then precipitated with alcohol again, and the process is repeated twice; the polysaccharide precipitate is freeze-dried to obtain crude polysaccharide of fresh Dendrobium officinale; (3) Add an appropriate amount of pure water to the crude polysaccharide of fresh Dendrobium officinale to fully dissolve it and prepare a polysaccharide solution with a concentration of about 5 mg / mL, then add Sevage reagent with a volume ratio of one-third of the polysaccharide solution, the Sevage reagent is composed of chloroform and n-butanol in a volume ratio of 4:1, and continue to stir for 30 minutes after mixing; let it stand for a while, and centrifuge to remove the precipitate; repeat the stirring and centrifugation operations until there is no obvious precipitate after stirring, and obtain a crude polysaccharide deproteinized sample; (4) Dissolve the deproteinized polysaccharide sample in an appropriate amount of pure water and stir thoroughly to ensure complete dissolution; Then, anhydrous ethanol was slowly added, and the stirring speed was gradually increased to make the ethanol concentration in the solution reach 40%; the precipitate was separated by centrifugation to obtain a polysaccharide DOP40 precipitated with 40% ethanol; anhydrous ethanol was continuously added to the supernatant to an ethanol concentration of 60% and 80%, and the polysaccharide DOP60 precipitated with 60% ethanol and the polysaccharide DOP80 precipitated with 80% ethanol were obtained by centrifugation; the polysaccharides were freeze-dried respectively to obtain polysaccharides of different alcohol precipitation components; (5) The polysaccharide sample precipitated with 60% ethanol was dissolved in water, loaded on a DEAE-Sepharose Fast Flow column, eluted with ultrapure water, collected by an automatic collector, the flow rate of the peristaltic pump was 2.5 mL / min, and one tube was collected for every 10 mL. The absorbance was determined by the phenol-sulfuric acid method. Each gradient was washed until no sugar was detected. The same fractions were combined according to the absorption peaks, concentrated under reduced pressure at 50°C to 60°C, and freeze-dried to obtain fresh semi-refined polysaccharides of Dendrobium officinale. (6) Dissolve the semi-refined polysaccharide of fresh Dendrobium officinale in water, load it on a Sephadex G-100 column, elute with ultrapure water, collect with an automatic collector, use a peristaltic pump at a flow rate of 0.2 mL / min, collect 2 mL in one tube, determine the absorbance by the phenol-sulfuric acid method, wash each gradient until no sugar is detected, merge the same fractions according to the absorption peak, concentrate under reduced pressure at 50°C to 60°C, and freeze-dry to obtain the refined polysaccharide DOP60-1A of fresh Dendrobium officinale.

3. Use of the fresh refined polysaccharide of Dendrobium candidum as claimed in claim 1 in the preparation of alcohol-relieving, liver-protecting and stomach-nourishing medicines.

4. A fresh Dendrobium officinale polysaccharide compound preparation for preventing and treating alcohol-induced acute and chronic liver damage, characterized in that: The invention is prepared from the following ingredients in parts by weight: 1 to 3 parts of the refined fresh Dendrobium officinale polysaccharide according to claim 1, 4 to 6 parts of Pueraria root extract, 1 to 3 parts of Hovenia dulcis fruit extract, and 1 to 3 parts of tomato seed oil.

5. A fresh Dendrobium officinale polysaccharide compound preparation for preventing and treating alcohol-induced acute and chronic liver damage as claimed in claim 4, characterized in that: The ingredients are mixed, and pharmaceutically acceptable excipients, a flavoring agent, mogroside and a preservative, potassium sorbate are added, and an oral preparation is prepared according to a conventional method in pharmacy.

6. A fresh Dendrobium officinale polysaccharide compound preparation for preventing and treating alcohol-induced acute and chronic liver damage according to claim 4, characterized in that: The preparation methods of Pueraria root extract and Hovenia dulcis fruit extract are as follows: the Pueraria root or Hovenia dulcis fruit is crushed by a mill, 10 times water is heated and refluxed for extraction 2 to 3 times, each time for 1 to 1.5 hours, the decoctions are combined, filtered, the filtrate is concentrated under reduced pressure at 60°C to 70°C to form a concentrated solution, precipitated with 65% ethanol concentration, allowed to stand overnight, centrifuged to obtain a supernatant, ethanol is recovered, vacuum freeze-dried, and ground into powder to obtain the extract.

7. A fresh Dendrobium officinale polysaccharide compound preparation for preventing and treating alcohol-induced acute and chronic liver damage according to claim 6, characterized in that: The puerarin content in the kudzu root extract is not less than 10%, and the Hovenia dulcis flavonoids content in the Hovenia dulcis flavonoids extract is not less than 2%.

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