A kind of black skinned Alpinia thunbergii acidic polysaccharide ORP-2 and its application

Abstract

The invention discloses a kind of black-skinned Alpinia thunbergii acidic polysaccharide ORP-2 and its application, wherein the black-skinned Alpinia thunbergii acidic polysaccharide ORP-2 is an acidic polysaccharide obtained by separating and purifying the black-skinned Alpinia thunbergii polysaccharide as a raw material, and its molecular weight is 1.64×10 4 Da, uronic acid content is 11.49%, and its monosaccharide composition is mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: fucose = 13.89: 4.70: 1.03: 0.20: 33.83: 18.36: 6.61 in molar ratio. In the Caco-2 cell intestinal microbial adhesion experiment, ORP-2 can promote the growth and adhesion of intestinal probiotics Lactobacillus plantarum.

Description

Technical Field

[0001] The invention relates to a kind of Alpinia thunbergii acidic polysaccharide ORP-2 and application thereof, belonging to the technical field of active component extraction and separation. Technical Background

[0002] Black-skinned Alternaria raphanipes, scientific name Oudemansiella raphanipes, belongs to Basidiomycetes, Hymenomycetes, Agaricales, Trichodermaceae, also known as long root mushroom. Because of its dark brown body and chicken mushroom shape, its trade name is "black-skinned Alternaria raphanipes". It is docile and easy to cultivate, with crisp and tender flesh. It is a top-grade edible fungus, rich in fiber, vitamins, minerals, and proteins. Among them, polysaccharides are the most abundant biomacromolecules, with a wide range of nutritional and medicinal effects. However, due to its complex structure and difficulty in separation and purification, the current research on black-skinned Alternaria raphanipes is mostly focused on the optimization of artificial cultivation and mycelium fermentation technology, and there are few reports on the extraction and separation methods and activity research of acidic polysaccharides of black-skinned Alternaria raphanipes.

[0003] A wide variety of microorganisms live in the gastrointestinal tract of healthy people, which are called intestinal flora. Any disease is accompanied by the deterioration of the flora, an increase in the abundance of pathogenic bacteria, and a decrease in the abundance of beneficial bacteria. A large number of microorganisms gather in the mucus layer of the human colon. The mucus layer is constantly consumed and replenished. It is the first immune defense line of the intestine, limiting the contact between bacteria and the intestinal epithelium, and also providing nutrition and adhesion sites for the flora. Inside the mucus layer of the colon, there are a variety of glycoproteins, glycolipids and other sugar complexes (glycocalyx) on the surface of endothelial cells, which play a role in protecting the intestine and regulating immunity. There are many sugar chains in the food we eat every day, and there are a large number of sugar chains on the cell walls of many microorganisms in the human intestine. These sugar chains, sugar chains in food, and sugar chains on the intestinal mucus layer, these three sugar chains interact in our intestines and jointly regulate human health. However, how do these three sugar chains work? What is the connection between them? What role do polysaccharides play in the process of microbial adhesion? It remains to be explored in depth by humans.

[0004] Sugar chains - considered to be the "code of life", play an important role in maintaining human health. At present, the research on polysaccharides mainly focuses on anti-oxidation, hypoglycemic, lipid-lowering, and immune enhancement. Polysaccharides are a natural high-molecular polymer that is difficult to be directly absorbed through the gastrointestinal tract, but can be decomposed and utilized by microorganisms to produce short-chain fatty acids, which have a positive significance for maintaining the integrity of the intestinal mucosal barrier and stabilizing the intestinal microenvironment. At present, the interaction and mechanism between polysaccharides and intestinal flora and intestinal barriers are still unclear. Summary of the invention

[0005] The present invention aims to provide a kind of acidic polysaccharide ORP-2 of Alpinia thunbergii and its application. The present invention is the first to apply the single component polysaccharide ORP-2 obtained by separation and purification of Alpinia thunbergii polysaccharide to the experiment of adhesion of intestinal probiotics Lactobacillus plantarum to Caco-2 cells, which provides an opportunity to study the interaction between Alpinia thunbergii polysaccharide and intestinal flora and intestinal mucus layer, and has broad application prospects in the fields of medical care and so on.

[0006] The black-skinned Alpinia thunbergii acidic polysaccharide ORP-2 of the present invention is an acidic polysaccharide obtained by separating and purifying the black-skinned Alpinia thunbergii polysaccharide as a raw material, and its molecular weight is 1.64×10 4 Da, the uronic acid content is 11.49%, and its monosaccharide composition is mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: fucose = 13.89: 4.70: 1.03: 0.20: 33.83: 18.36: 6.61 in molar ratio.

[0007] The black-skinned Alpinia fasciata acidic polysaccharide ORP-2 of the present invention is obtained by a method comprising the following steps:

[0008] Step 1: freeze-dried black-skinned Alpinia chorii subtilis is crushed, extracted with water and precipitated with alcohol, deproteinized, dialyzed and freeze-dried to obtain crude polysaccharide of black-skinned Alpinia chorii subtilis, 200 mg of crude polysaccharide of black-skinned Alpinia chorii subtilis with a purity of not less than 80% is completely dissolved in 10 mL of purified water, centrifuged at 10000 r / min for 10 min until there is no precipitation, the supernatant is passed through a 0.45 μm water filter membrane, and then placed on a DEAE-52 cellulose chromatography column at a flow rate of 0.6 mL / min, washed with water until no polysaccharide flows out (removing the washed polysaccharide), then eluted with a 0.1 mol / L NaCl solution, concentrated, dialyzed and freeze-dried to obtain salt-washed polysaccharide;

[0009] Step 2: dissolve the salt-washed polysaccharide obtained in step 1 in purified water to prepare a 1 mg / L solution, pass through a filter membrane with a molecular weight cutoff of 50,000 at a flow rate of 1.5 mL / min, collect the components less than 50,000, concentrate to 10 mL and freeze-dry;

[0010] Step 3: The polysaccharide less than 50,000 obtained in step 2 is dissolved in purified water to prepare a 10 mg / L solution, and centrifuged at 10000 r / min for 10 min until there is no precipitation, and the supernatant is filtered through a 0.45 μm water filter membrane, and then placed on a superdex-75 molecular sieve gel chromatography column at a flow rate of 0.2 mL / min. After HPLC analysis, the polysaccharide solutions of the same components are combined, concentrated and freeze-dried to obtain the black-skinned Alpinia arborvitae acidic polysaccharide ORP-2.

[0011] The application of the black-skinned Alpinia tremula acidic polysaccharide ORP-2 of the present invention is to prepare an intestinal microbial adhesive by using the black-skinned Alpinia tremula acidic polysaccharide ORP-2.

[0012] The black-skinned Alpinia fasciata acidic polysaccharide ORP-2 of the present invention can enhance the activity of Caco-2 cells on the one hand, and can promote the adhesion of intestinal probiotics Lactobacillus plantarum to Caco-2 cells on the other hand, thereby protecting the intestinal barrier function.

[0013] The beneficial effects of the present invention are embodied in:

[0014] The invention uses crude polysaccharide of Alpinia thunbergii as raw material, performs DEAE-52 cellulose column chromatography, washes with water to remove neutral polysaccharide, elutes with 0.1 mol / l NaCl to obtain acidic polysaccharide, and then separates and purifies with ultrafiltration membrane with a molecular weight cutoff of 50,000 and superdex-75 molecular sieve gel column chromatography to obtain single component acidic polysaccharide ORP-2. Firstly, wash with water to remove neutral sugar, then elute with 0.1 mol / l Nacl, so that acidic sugar can be eluted to the maximum extent, and components with high sugar content can be obtained, so as to prepare for fine separation and purification of polysaccharide, and finally obtain high-purity acidic polysaccharide ORP-2 of Alpinia thunbergii.

[0015] The black-skinned Alpinia solani acidic polysaccharide ORP-2 obtained by the present invention can enhance the activity of Caco-2 cells on the one hand, and can promote the adhesion of intestinal probiotics Lactobacillus plantarum to Caco-2 cells on the other hand, thereby protecting the intestinal barrier function, and provides an opportunity for further studying the mechanism of action between the black-skinned Alpinia solani acidic polysaccharide ORP-2 and intestinal flora and intestinal barrier in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The HPLC chromatogram of the acidic polysaccharide ORP-2 of Alpinia fasciata in Example 1 of the present invention is shown in the figure. It can be seen from the figure that the retention time of ORP-2 is 18.869 min, and its molecular weight is 1.64×10 4 Da.

[0017] Figure 2 This is a result diagram of the monosaccharide composition of the acidic polysaccharide ORP-2 of Colletotrichum niger in Example 1 of the present invention. The monosaccharide composition of ORP-2 is mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: fucose = 13.89: 4.70: 1.03: 0.20: 33.83: 18.36: 6.61 in molar ratio.

[0018] Figure 3 This is the effect of the acidic polysaccharide ORP-2 of Alpinia cerana in Example 2 of the present invention on the activity of Caco-2 cells. The CCK-8 kit was used to detect that the acidic polysaccharide ORP-2 of Alpinia cerana can enhance the activity of Caco-2 cells, among which 600 μg / mL ORP-2 has the most significant effect.

[0019] Figure 4This is the effect of the acidic polysaccharide ORP-2 of Alpinia fasciata on the adhesion of Lactobacillus plantarum to Caco-2 in Example 3 of the present invention. The blank group A was not added with ORP-1 and ORP-2, the model group B was added with 600 μg / mL ORP-1, and the model group C was added with 600 μg / mL ORP-2. It can be seen from the figure that ORP-2 can significantly enhance the adhesion of Lactobacillus plantarum to Caco-2 cells. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further analyzed and explained below through specific embodiments.

[0021] Embodiment 1:

[0022] The acidic polysaccharide ORP-2 of Alpinia thunbergii in this embodiment is obtained by separation and purification of the polysaccharide of Alpinia thunbergii as raw material, and its molecular weight is 1.64×10 4 Da, uronic acid content: 11.49%, monosaccharide composition: mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: fucose = 13.89: 4.70: 1.03: 0.20: 33.83: 18.36: 6.61. The preparation steps are as follows:

[0023] The freeze-dried black-skinned Alpinia chorii multiflora is crushed, extracted with water and precipitated with alcohol, deproteinized, dialyzed and freeze-dried to obtain the crude polysaccharide of black-skinned Alpinia chorii multiflora. 200 mg of the crude polysaccharide of black-skinned Alpinia chorii multiflora with a purity of not less than 80% is used as a raw material, fully dissolved in purified water, centrifuged at 10000 r / min for 10 min, the supernatant is collected and placed on a DEAE-52 cellulose column chromatography at a flow rate of 0.6 mL / min, washed with water to remove neutral polysaccharides, and then 0.1 mol / l Nacl eluted components are collected, concentrated, freeze-dried, 100 mg is weighed and dissolved, and then passed through an ultrafiltration membrane with a molecular weight cutoff of 50,000 at a flow rate of 0.6 mL / min, and components less than 50,000 are collected, concentrated, freeze-dried, 100 mg is weighed and dissolved in 2 mL of purified water, and passed through a superdex-75 molecular sieve gel column chromatography at a flow rate of 0.2 mL / min, and the outflow components are collected, and the purified component acidic polysaccharide ORP-2 is obtained through concentration, freeze-drying and HPLC analysis.

[0024] Figure 1 This is the high performance liquid chromatogram of the acidic polysaccharide ORP-2 of Alpinia cerana obtained in this Example 1.

[0025] Figure 2 This is the monosaccharide composition diagram of the acidic polysaccharide ORP-2 of Alpinia fasciata obtained in this Example 1.

[0026] Embodiment 2:

[0027] The cultured Caco-2 cells were digested with trypsin to form single free cells, centrifuged at 1200 r / min for 5 minutes, the supernatant was removed, and the precipitate was added with blank culture medium and blown to 1×10 5 Take a 96-well plate and set up blank wells, control wells and experimental wells. Add 100 μL of culture medium (without cells) to the blank wells, and add 100 μL of cell suspension with the same cell concentration to the control wells and the drug-added wells. Set up six replicates for each group and culture them in a 37°C incubator for 24 hours.

[0028] Drug treatment: Use blank culture medium to prepare 1000 μg / mL ORP-2 stock solution, filter through 0.22um filter membrane, and then gradient dilution to prepare 200, 400, 600, 800, 1000 (μg / mL) ORP-2 solution. Take out the cell culture plate and wash it twice with PBS. Add 100 μL of cell culture medium containing different concentrations of ORP-2 to the experimental wells, add 100 μL of cell culture medium to the control wells, and culture in a 37°C incubator for 24 hours.

[0029] Add CCK-8 and detect: remove the culture plate, wash twice with PBS, add 10 μL of CCK-8 solution to each well, and add cell culture medium to 100 μL, mix well, continue incubation for 4 hours, and then measure the OD value at 450 nm with a microplate reader.

[0030] Cell survival rate = [(OD of experimental well - OD of blank well) / (OD of control well - OD of blank well)] × 100%

[0031] Figure 3 This is the effect of the acidic polysaccharide ORP-2 of Alpinia mellifera on the activity of Caco-2 cells in Example 2.

[0032] Example 3: ORP-2 promotes the adhesion of Lactobacillus plantarum to Caco-2 cells.

[0033] (1) Cultivation of Lactobacillus plantarum: MRS liquid medium was prepared, and a single clone of Lactobacillus plantarum purified twice was selected and placed in 100 mL MRS liquid medium. The cells were cultured in an anaerobic incubator at 37°C for 22 h, and then centrifuged at 8000 rpm for 5 min. The cells were washed twice with PBS and resuspended in basic DMEM medium to 1 × 10 9 CFU / mL.

[0034] (2) Caco-2 cell culture: The cultured Caco-2 cells were digested with trypsin to form single free cells, centrifuged at 1200 r / min for 5 min, the supernatant was removed, and the precipitate was added with blank culture medium and blown evenly to 1×10 5 Take a 24-well cell culture plate and place a cell slide at the bottom, add 0.5 mL of cell suspension to each well, and culture in a 37°C incubator for 48 hours until the cells are completely attached.

[0035] (3) Adhesion of Lactobacillus plantarum to Caco-2 cells: The cell culture plate was removed and washed twice with PBS. 0.5 mL of blank group A and 1×10 9 CFU / mL of plant lactobacillus suspension, model groups B and C were added with 0.5mL of the same concentration of bacterial suspension containing 600μg / mL ORP-1 and 600μg / mL ORP-2, respectively, and co-cultured in a 37°C incubator for 4h, washed twice with PBS, fixed with 4% paraformaldehyde for 2h, stained with a Gram staining kit, observed and photographed under a fluorescent inverted microscope. ORP-1 is the previous research result of this research group, and is provided here for comparison. For details, see CN110627919B.

[0036] Figure 4 This is the effect of the neutral polysaccharide ORP-1 of Alpinia mellifera and the acidic polysaccharide ORP-2 of Alpinia mellifera on the adhesion of Lactobacillus plantarum to Caco-2 cells in Example 3.

Claims

1. A kind of Acidic Polysaccharide ORP-2 of Alpinia thunbergii, characterized in that: The black-skinned Alpinia thunbergii acidic polysaccharide ORP-2 is an acidic polysaccharide obtained by separation and purification from the black-skinned Alpinia thunbergii polysaccharide as a raw material, and its molecular weight is 1.64×10 4 Da, uronic acid content is 11.49%, and its monosaccharide composition is mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: fucose = 13.89: 4.70: 1.03: 0.20: 33.83: 18.36: 6.61 in molar ratio; The black-skinned Alpinia thunbergii acidic polysaccharide ORP-2 is obtained by a method comprising the following steps: Step 1: 200 mg of crude polysaccharide of Alpinia cerevisiae with a purity of ≥80% was completely dissolved in 10 mL of purified water, centrifuged until there was no precipitation, the supernatant was filtered through a 0.45 μm water filter membrane, and then placed on a DEAE-52 cellulose chromatography column, washed with water until no polysaccharide flowed out, and then eluted with a 0.1 mol / L NaCl solution, concentrated, dialyzed, and freeze-dried to obtain salt-washed polysaccharide; Step 2: The salt-washed polysaccharide obtained in step 1 was dissolved in purified water to prepare a 1 mg / L solution, and passed through a filter membrane with a molecular weight cutoff of 50,000, and the components less than 50,000 were collected, concentrated to 10 mL, and freeze-dried; Step 3: Dissolve the polysaccharide less than 50,000 obtained in step 2 in purified water to prepare a 10 mg / L solution, centrifuge until there is no precipitation, take the supernatant and filter it through a 0.45 μm water filter membrane, and then place it on a superdex-75 molecular sieve gel chromatography column, combine the polysaccharide solutions of the same components, concentrate and freeze-dry to obtain the black-skinned Alpinia argentea acidic polysaccharide ORP-2.

2. An application of the Acidic Polysaccharide ORP-2 of Alpinia cerasifera according to claim 1, characterized in that: The intestinal microbial adhesive is prepared by using the black-skinned Alpinia fasciata acidic polysaccharide ORP-2.

3. The use according to claim 2, characterized in that: The black-skinned Alpinia thunbergii acidic polysaccharide ORP-2 can enhance the activity of Caco-2 cells.

4. The use according to claim 2, characterized in that: The black-skinned Alpinia fasciata acidic polysaccharide ORP-2 can promote the intestinal probiotic Lactobacillus plantarum to adhere to Caco-2 cells and protect the intestinal barrier function.

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