Commelina communis polysaccharide and its preparation method and application

Abstract

The invention relates to a Commelina communis polysaccharide and a preparation method and application thereof. The invention separates five uniform polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 from Commelina communis. Experiments confirm that the Commelina communis uniform polysaccharides are not digested by gastrointestinal fluid in vitro, can promote the proliferation of three intestinal probiotics, namely Bacteroides ovatus, Bacteroides thetaiotaomicron and Bacteroides cellulosilyticus, and produce short-chain fatty acids and 1,2-propylene glycol, and can be further used for preparing prebiotics.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and relates to Commelina communis polysaccharide, in particular to Commelina communis polysaccharide and a preparation method and application thereof. Background Art

[0002] Prebiotics refer to substances that are selectively fermented by beneficial intestinal bacteria to improve intestinal flora and are beneficial to human health. Prebiotics have many physiological activities, such as non-digestibility, improving intestinal flora, promoting the growth of intestinal probiotics, reducing intestinal pH, increasing the production of short-chain fatty acids, regulating body metabolism and immune system, alleviating constipation, etc. At present, the prebiotics recognized internationally mainly include oligofructose (FOS), oligomalto-oligosaccharide (IMO), oligolactose (LACT), etc., which are mainly used to proliferate intestinal lactobacillus (Lactobacillus spp.) and bifidobacterium (Bifidobacteriumspp.). In recent years, with the improvement of people's living standards and the enhancement of health awareness, traditional prebiotic products cannot meet the growing market demand. Bacteroides ovatus, Bacteroides thetaiotaomicron and Bacteroides cellulosilyticus have the ability to metabolize polysaccharides and choline salts, have physiological effects in the treatment of diabetes, cardiovascular disease, inflammatory bowel disease and cancer, and are considered to be new probiotics with potential probiotic effects. Therefore, finding new prebiotics, especially prebiotics that promote the proliferation of B. ovatus, B. thetaiotaomicron and B. cellulosilyticus, is an urgent problem to be solved.

[0003] Commelina communis, recorded in "Supplement to Materia Medica" and "Compendium of Materia Medica", is derived from the dry aerial parts of Commelina communis L., a plant of the Commelina genus of the Commelinaceae family. It tastes sweet, light and cold, and enters the lung, stomach and small intestine meridians. It has the effects of clearing away heat and purging fire, detoxifying, promoting diuresis and reducing swelling. It can be used to treat colds and fever, fever and thirst, sore throat, edema and oliguria, hot stranguria, carbuncle, furuncle and toxicity. Commelina polysaccharide is one of the main components of Commelina. The research on Commelina polysaccharide mainly focuses on extraction technology and detection methods, and there is little research on its functional properties. According to existing data, the research on the biological activity of Commelina water extract has been widely carried out, but there is no research report on Commelina polysaccharide as a prebiotic to improve the proliferation of probiotics, especially to improve the proliferation of B. ovatus, B. thetaiotaomicron and B. cellulosilyticus. Summary of the invention

[0004] The purpose of the present invention is to overcome the problem that the existing coleus polysaccharides cannot increase the proliferation of B.ovatus, B.thetaiotaomicron and B.cellulosilyticus, and to provide coleus polysaccharides and their preparation method and application. The present invention provides five uniform polysaccharides in coleus for promoting the proliferation of three probiotics, B.ovatus, B.thetaiotaomicron and B.cellulosilyticus, in the human intestine.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] One of the technical solutions of the present invention is to provide Commelina polysaccharides, wherein the Commelina polysaccharides are Commelina uniform polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3, each having the following structural characteristics:

[0007] Commelina communis homomonosaccharide CCW-1: a polysaccharide composed of 9 monosaccharides, with a molecular weight of about 56.9 kDa; total sugar content of 90.7%; uronic acid content of 4.8%; protein content of 1.7%; no sulfate group; monosaccharide molar ratio of mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose = 17.7: 2.3: 5.4: 2.2: 0.5: 6.1: 25.0: 12.3 :28.5; the connection modes include terminal connection mannose, 1,2-connected mannose, 1,3-connected mannose, 1,6-connected mannose, 1,3,6-connected mannose, terminal connection rhamnose, 1,2-connected rhamnose, 1,3-connected rhamnose, 1,2,3-connected rhamnose, terminal connection glucuronic acid, terminal connection glucose, 1,2,3-connected glucose, 1,2,4-connected glucose, 1,2,6-connected glucose Sugar, terminally linked galactose, 1,2-linked galactose, 1,4-linked galactose, 1,3-linked galactose, 1,6-linked galactose, 1,4,6-linked galactose, 1,3,6-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, 1,2,3,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose sugar, 1,5-linked arabinose, and 1,2,3-linked arabinose in a molar ratio of 3.0:4.9:5.5:1.1:1.3:2.4:1.0:1.8:1.0:2.3:2.5:1.7:1.0:1.3:4.2:2.7:3.0:3.3:2.8:1.5:9.7:2.3:3.7:2.4:2.9:2.6:14.4:1.2:4.6:6.0:2.0;

[0008] Commelina communis homomonosaccharide CCW-2: a polysaccharide composed of 9 monosaccharides, with a molecular weight of about 39.0 kDa; total sugar content of 85.5%; uronic acid content of 15.6%; protein content of 2.2%; no sulfate group; monosaccharide molar ratio of mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose = 7.2:1.0:7.5:4.8:7.4:6.0:24.1:13 .2:28.8; the connection modes include 1,2-linked mannose, 1,3-linked mannose, terminally linked rhamnose, 1,3-linked rhamnose, 1,3,4-linked rhamnose, terminally linked glucuronic acid, 1,2-linked galacturonic acid, terminally linked glucose, 1,3-linked glucose, 1,3,4-linked glucose, 1,2,6-linked glucose, terminally linked galactose, 1,4-linked galactose, 1,3-linked Galactose, 1,2-linked galactose, 1,6-linked galactose, 1,2,4-linked galactose, 1,4,6-linked galactose, 1,3,6-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, 1,2,3,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose, 1,5-linked arabinose , 1,4-linked arabinopyranose and 1,2,3-linked arabinose, with a molar ratio of 1.7:2.8:5.1:1.1:1.5:4.7:7.5:1.8:1.3:2.2:1.3:7.8:2.4:1.5:2.0:1.7:2.6:1.5:4.5:2.9:3.3:3.5:4.0:3.2:10.4:1.1:4.4:4.3:4.7:3.1;

[0009] Commelina monosaccharide CCB-1: a polysaccharide composed of 7 monosaccharides, with a molecular weight of about 44.0 kDa; total sugar content of 92.6%; uronic acid content of 0.8%; protein content of 1.0%; no sulfate group; monosaccharide molar ratio of mannose: glucosamine: rhamnose: glucose: galactose: xylose: arabinose = 10.2:0.5:0.9:22.5:7.9:45.3:12.6; the connection modes include 1,3-linked mannose, 1,4,6-linked mannose, 1,3,6-linked mannose, terminal-linked rhamnose, 1,3-linked rhamnose, terminal-linked glucose, 1,4 -linked glucose, 1,4,6-linked glucose, terminally linked galactose, 1,4-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, 1,2,3,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose, 1,5-linked arabinose, molar ratio 3.1:5.4:2.9:0.6:0.5:6.5:9.8:5.7:4.8:3.5:4.3:31.4:5.9:1.8:1.0:7.2:1.6:2.4:1.4;

[0010] Commelina monosaccharide CCB-2: a polysaccharide composed of 5 monosaccharides, with a molecular weight of about 22.6kDa; total sugar content of 94.4%; uronic acid content of 1.0%; protein content of 1.1%; no sulfate group; monosaccharide molar ratio of mannose: glucose: galactose: xylose: arabinose = 8.3: 40.8: 4.5: 37.9: 8.5; the connection modes include 1,6-linked mannose, 1,4,6-linked mannose, terminal-linked glucose, 1,4-linked glucose, 1,6-linked glucose, 1,3,4-linked glucose Sugars, 1,4,6-linked glucose, 1,4-linked galactose, 1,6-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose, 1,5-linked arabinose, molar ratio 1.5:5.6:6.7:20.0:1.7:1.0:9.8:1.9:2.6:7.4:27.1:4.2:1.1:6.8:0.6:1.1:0.7;

[0011] Commelina monosaccharide CCB-3: a polysaccharide composed of 10 monosaccharides, with a molecular weight of about 58.8kDa; total sugar content of 86.7%; uronic acid content of 11.5%; protein content of 0.8%; no sulfate group; monosaccharide molar ratio of mannose: glucosamine: rhamnose: 4-O-methyl glucuronic acid: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose = 1.9:0.5:1.2:3.1:1.0:1.2:3.4:6.6:55.2:24.3; the connection modes include 1,3-mannose, terminal rhamnose, terminal glucuronic acid, 1,2- Linked to galacturonic acid, terminally linked to grape, terminally linked to galactose, 3,6-linked galactose, terminally linked to xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, 1,2,3,4-linked xylose, terminally linked to arabinose, 1,2-linked arabinose, 1,3-linked arabinose, 1,5-linked arabinose and 1,2,3-linked arabinose, with a molar ratio of 1.9:1.3:5.0:1.2:3.5:3.6:2.6:2.1:33.3:7.4:10.6:2.8:13.6:1.2:5.6:1.0:3.4.

[0012] The second technical solution of the present invention is to provide a method for preparing Commelina communis polysaccharide as described in one of the above technical solutions, comprising the following steps:

[0013] S1. Take Commelina communis, extract with ethanol, filter in sequence, extract the residue with hot water, concentrate, centrifuge, dry the precipitate after centrifugation to obtain a water-extracted residue, add ethanol to the supernatant, let it stand, centrifuge, add water to the precipitate after centrifugation, re-dissolve the precipitate, remove the ethanol under reduced pressure, add water to re-dissolve, add trichloroacetic acid, centrifuge, adjust the pH of the supernatant after centrifugation to neutral, concentrate, dialyze, and freeze-dry in sequence to obtain Commelina communis water-extracted crude polysaccharide, named CCW;

[0014] The water extraction residue is extracted with sodium hydroxide and centrifuged in sequence. The pH of the supernatant after centrifugation is adjusted to neutral, and then concentrated, ethanol is added, and then allowed to stand and centrifuged. The precipitate after centrifugation is re-dissolved in water, the ethanol is recovered under reduced pressure, re-dissolved in water again, trichloroacetic acid is added, and centrifuged. The pH of the supernatant after centrifugation is adjusted to neutral, and then concentrated, dialyzed, and freeze-dried to obtain a crude polysaccharide extracted from commelinine, named CCB.

[0015] S2, dissolving the CCW obtained in step S1 in distilled water, performing preliminary separation by DEAE-cellulose column chromatography, eluting with distilled water, and sequentially concentrating, dialyzing and freeze-drying the collected fractions to obtain a secondary component named CCW-D-1;

[0016] S3, dissolving the CCB obtained in step S1 in distilled water, performing preliminary separation by DEAE-cellulose column chromatography, eluting with distilled water and 0.1 mol / L sodium chloride solution in turn, collecting each fraction, and performing concentration, dialyzing and freeze-drying in turn to obtain two secondary components, named CCB-D-1 and CCB-D-2;

[0017] S4, CCW-D-1 obtained in step S2 and CCB-D-1 and CCB-D-2 obtained in step S3 were dissolved in water and centrifuged, and the supernatants after centrifugation were respectively treated with Sephacryl TM The samples were separated by S200 chromatography and eluted with ammonium bicarbonate solution. The fractions were collected and combined according to the ultraviolet detection results of the colorimetric reaction of the sugar content in each fraction. The fractions were concentrated, dialyzed and freeze-dried in sequence. The homogeneity was tested by high performance gel permeation chromatography to obtain the uniform polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 from Commelina communis, which were then subjected to activity experiments.

[0018] Furthermore, in step S1, Commelina communis is extracted by using 95% by mass ethanol.

[0019] Furthermore, in step S1, 95% by mass ethanol is added before the two standing steps so that the ethanol concentration in the supernatant is 80%.

[0020] Furthermore, in step S1, the water extraction residue is extracted by sodium hydroxide with a mass fraction of 5%.

[0021] Furthermore, in step S1, the processes of adding trichloroacetic acid twice are both as follows: after adding trichloroacetic acid, the mass fraction of trichloroacetic acid in the solution is 10%.

[0022] The third technical solution of the present invention is to provide an application of the Commelina polysaccharide as described in one of the above technical solutions, wherein the Commelina polysaccharide is used for preparing prebiotics.

[0023] Furthermore, the prebiotic is a prebiotic food containing the Commelina Commelina polysaccharide.

[0024] Furthermore, the prebiotic food is a prebiotic food that promotes the growth and reproduction of intestinal probiotics.

[0025] Furthermore, the intestinal probiotics are selected from any one or more of Bacteroides ovatus, Bacteroides thetaiotaomicron or Bacteroides cellulosus.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention separates and obtains five uniform Commelina polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 from the dry aerial parts of Commelina, and in vitro experiments confirm that the Commelina polysaccharides are not digested by simulated gastric and intestinal fluids, and improve the growth and proliferation of three beneficial intestinal bacteria, Bacteroides ovatus, Bacteroides thetaiotaomicron and Bacteroides cellulosilyticus, and produce short-chain fatty acids and 1,2-propylene glycol, and can be further used for preparing prebiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the flow chart for separation of homogeneous polysaccharides from Commelina communis.

[0029] Figure 2 The chromatograms of high performance gel permeation chromatography (HPGPC) of Commelina communis uniform polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 are shown in Figure 1, wherein the chromatographic columns are Shodex SUGAR KS-804 (300×8.0 mm) and Shodex SUGAR KS-802 gel columns (300×8.0 mm) connected in series; the eluent is 0.002 mol / L ammonium acetate; and the flow rate is 0.6 mL / min.

[0030] Figure 3 , Chromatograms of HPGPC of homogeneous polysaccharide from Commelina communis before and after digestion with simulated gastric and intestinal fluids, where W0 refers to 0h of simulated gastric fluid digestion, W6 refers to 6h of simulated gastric fluid digestion, C0 refers to 0h of simulated intestinal fluid digestion, and C6 refers to 6h of simulated intestinal fluid digestion.

[0031] Figure 4 , Growth curve of Commelina communis homogeneous polysaccharide improving the proliferation of three probiotics in the human intestine.

[0032] Figure 5 , Bar graph of the content of short-chain fatty acids and 1,2-propylene glycol produced after the homogeneous polysaccharide of Commelina communis was degraded by three kinds of probiotics.

[0033] in Figure 4 and Figure 5 BO stands for B.ovatus, BC stands for B.cellulosilyticus, and BT stands for B.thetaiotaomicron. DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] In the following examples and comparative examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0036] Example 1: Preparation of Commelina communis homogeneous polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3

[0037] like Figure 1 The following is a flow chart of separation of homogeneous polysaccharides from Commelina communis:

[0038] 12 kg of Commelina communis herbs were crushed, extracted with 95% ethanol and filtered. The filtered residue was extracted with a hot water solution for 3 times, concentrated and centrifuged. The precipitate after centrifugation was dried at 50°C to obtain a water-extracted residue. 95% ethanol was added to the supernatant until the ethanol concentration in the supernatant was 80%. The supernatant was allowed to stand overnight at 4°C and then centrifuged. The precipitate after centrifugation was re-dissolved with water, the ethanol was recovered under reduced pressure, and then re-dissolved with water. An equal volume of trichloroacetic acid with a mass fraction of 20% was added so that the mass fraction of trichloroacetic acid in the solution was 10% to remove free protein. The supernatant after centrifugation was adjusted to neutral, concentrated, dialyzed, and freeze-dried to obtain Commelina communis water-extracted crude polysaccharide CCW. 6 kg of water-extracted residue was added, soaked at 4°C for 1 hour in 5% sodium hydroxide solution, centrifuged and the supernatant was taken, adjusted to neutrality with hydrochloric acid and concentrated, 95% ethanol was added until the ethanol concentration in the supernatant was 80%, and the mixture was allowed to stand overnight at 4°C and then centrifuged. The precipitate after centrifugation was re-dissolved in water, the ethanol was recovered under reduced pressure, and then re-dissolved in water, and an equal volume of 20% trichloroacetic acid was added so that the mass fraction of trichloroacetic acid in the solution was 10% to remove free protein. The supernatant after centrifugation was adjusted to neutrality, concentrated, dialyzed, and freeze-dried to obtain the crude polysaccharide CCB extracted from commelinine.

[0039] 80g of crude polysaccharide CCW extracted from Commelina communis was dissolved in distilled water and centrifuged. The supernatant after centrifugation was initially separated by DEAE-cellulose column chromatography. The fractions were eluted with distilled water, the elution volume was greater than 2 times the column volume (about 10L), the flow rate was 25mL / min, and each fraction was collected. The absorbance value at 490nm (after sulfuric acid-phenol color development) was detected by a septum. According to the sugar color development reaction, the fractions were combined, concentrated, dialyzed, and freeze-dried to obtain a secondary component: CCW-D-1.

[0040] 30g of CCB extracted from commelinae was dissolved in distilled water and centrifuged. The supernatant after centrifugation was initially separated by DEAE-cellulose column chromatography. The elution was performed with distilled water and 0.1mol / L sodium chloride, and the elution volume was greater than 2 times the column volume (about 10L). The flow rate was 25mL / min, and each fraction was collected. The absorbance value at 490nm (after sulfuric acid-phenol color development) was detected by a septum. According to the results of the sugar color development reaction, the fractions were combined, concentrated, dialyzed, and freeze-dried to obtain two secondary components: CCB-D-1 and CCB-D-2.

[0041] 8 g of CCW-D-1 was dissolved in distilled water and centrifuged. The supernatant was washed with Sephacryl TM S200 chromatography (molecular weight cutoff 2kDa-400kDa) was used for separation. The fractions were eluted with 0.2mol / L ammonium bicarbonate solution at a flow rate of 0.5mL / min and collected. The absorbance value at 490nm (after color development by sulfuric acid-phenol method) was detected by a septum tube. The same fractions were combined, concentrated, dialyzed, and freeze-dried according to the test results to obtain uniform polysaccharides CCW-1 (520mg) and CCW-2 (340mg).

[0042] 8 g of CCB-D-1 was dissolved in distilled water and centrifuged. The supernatant was washed with Sephacryl TM S200 chromatography (molecular weight cutoff 2kDa-400kDa) was used for separation. The fractions were eluted with 0.2mol / L ammonium bicarbonate solution at a flow rate of 0.5mL / min and collected. The absorbance value at 490nm (after color development by sulfuric acid-phenol method) was detected by a septum tube. The same fractions were combined, concentrated, dialyzed, and freeze-dried according to the test results to obtain uniform polysaccharides CCB-1 (330mg) and CCB-2 (290mg).

[0043] Dissolve 11 g of CCB-D-2 in distilled water and centrifuge. The supernatant after centrifugation is washed with Sephacryl TM Separate by S200 chromatography (retention molecular weight 2kDa-400kDa). Elute with 0.2mol / L ammonium bicarbonate solution at a flow rate of 0.5mL / min and collect each fraction. Detect the absorbance value at 490nm (after acid-phenol color development) with a septum tube. Combine the same fractions according to the test results, concentrate, dialyze, and freeze-dry to obtain uniform polysaccharide CCB-3 (820mg).

[0044] High performance gel permeation chromatography (HPGPC) showed that CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 were all homogeneous components. Figure 2 shown.

[0045] Example 2: Structural characterization of the homogeneous polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 from Commelina communis

[0046] (1) Determination of molecular weight

[0047] HPGPC is used to determine the relative molecular weight of polysaccharide samples. The basic principle is that uniform polysaccharides pass through gel permeation chromatography to form symmetrical chromatographic peaks. The peak time is related to the molecular weight and is calculated based on the calibration curve obtained from the known molecular weight.

[0048] Chromatographic conditions: Shodex SUGAR KS-804 (300×8.0 mm) and Shodex SUGAR KS-802 gel columns (300×8.0 mm) were connected in series for separation, with a flow rate of 0.6 mL / min, an injection volume of 20 μL, 0.002 mol / L ammonium acetate as the mobile phase, a column temperature of 35°C, and a differential refractive index detector (RID) as the detector.

[0049] Experimental method: Weigh 2.0 mg of homogeneous polysaccharide and Dextrans series standard products accurately, and prepare a 3.0 mg / mL solution with 0.002 mol / L ammonium acetate. Filter with a 0.22 μm microporous filter membrane before injection and detect. Record the retention time. Draw a standard curve with the logarithm value (Lg) of the molecular weight of the standard polysaccharide as the ordinate and the retention time as the abscissa. Obtain the corresponding linear regression equation and calculate the relative molecular weight of the homogeneous polysaccharide. The relative molecular weights of CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 are 56.9 kDa, 39.0 kDa, 44.0 kDa, 22.6 kDa and 58.8 kDa, respectively.

[0050] (2) Determination of total sugar, uronic acid, protein and sulfate content

[0051] The total sugar content of CCW-1 was 90.7% determined by sulfuric acid-phenol method; the total sugar content of CCW-2 was 85.5%; the total sugar content of CCB-1 was 92.6%; the total sugar content of CCB-2 was 94.4%; and the total sugar content of CCB-3 was 86.7%.

[0052] The uronic acid content was detected by the m-hydroxybiphenyl method. The uronic acid content of CCW-1 was 4.8%; the uronic acid content of CCW-2 was 15.6%; the uronic acid content of CCB-1 was 0.8%; the uronic acid content of CCB-2 was 1.0%; and the uronic acid content of CCB-3 was 11.5%.

[0053] The protein content was determined by the Coomassie Brilliant Blue method. The protein content of CCW-1 was 1.7%, the protein content of CCW-2 was 2.2%, the protein content of CCB-1 was 1.0%, the protein content of CCB-2 was 1.1%, and the protein content of CCB-3 was 0.8%.

[0054] The barium chloride turbidimetry assay showed that CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 did not contain sulfate groups.

[0055] (3) Monosaccharide composition analysis

[0056] The products of CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 were fully hydrolyzed at 110°C with 2 mol / L trifluoroacetic acid, and then derivatized with 1-phenyl-3-methyl-5-pyrazolone and subjected to liquid phase analysis.

[0057] CCW-1 is a polysaccharide composed of 9 monosaccharides, and the molar ratio of monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose = 17.7:2.3:5.4:2.2:0.5:6.1:25.0:12.3:28.5.

[0058] CCW-2 is a polysaccharide composed of 9 monosaccharides, and the molar ratio of monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose = 7.2:1.0:7.5:4.8:7.4:6.0:24.1:13.2:28.8.

[0059] CCB-1 is a polysaccharide composed of 7 monosaccharides, and the molar ratio of monosaccharides is mannose: glucosamine: rhamnose: glucose: galactose: xylose: arabinose = 10.2: 0.5: 0.9: 22.5: 7.9: 45.3: 12.6.

[0060] CCB-2 is a polysaccharide composed of five monosaccharides, and the molar ratio of monosaccharides is mannose: glucose: galactose: xylose: arabinose = 8.3: 40.8: 4.5: 37.9: 8.5.

[0061] CCB-3 is a polysaccharide composed of 10 monosaccharides, and the molar ratio of monosaccharides is mannose: glucosamine: rhamnose: 4-O-methyl glucuronic acid: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose = 1.9:0.5:1.2:3.1:1.0:1.2:3.4:6.6:55.2:24.3.

[0062] (4) Methylation analysis

[0063] The uronic acid was first reduced with CMC-NaBD4 and then the polysaccharides were methylated using the modified Hakomori method. The methylated products were fully hydrolyzed with 2 mol / L trifluoroacetic acid, reduced with sodium borodeuteride and acetylated with acetic anhydride to prepare partially methylated alditol acetate derivatives, which were then analyzed by GC-MS.

[0064] The structure of CCW-1 contains: terminally linked mannose, 1,2-linked mannose, 1,3-linked mannose, 1,6-linked mannose, 1,3,6-linked mannose, terminally linked rhamnose, 1,2-linked rhamnose, 1,3-linked rhamnose, 1,2,3-linked rhamnose, terminally linked glucuronic acid, terminally linked glucose, 1,2,3-linked glucose, 1,2,4-linked glucose, 1,2,6-linked glucose, terminally linked galactose, 1,2-linked galactose, 1,4-linked galactose, 1,3-linked galactose, 1,6-linked galactose, 1,4,6-linked galactose, 1,3,6-linked galactose, terminally linked xylose The molar ratio of the oligosaccharide is 3.0:4.9:5.5:1.1:1.3:2.4:1.0:1.8:1.0:2.3:2.5:1.7:1.0:1.3:4.2:2.7:3.0:3.3:2.8:1.5:9.7:2.3:3.7:2.4:2.9:2.6:14.4:1.2:4.6:6.0:2.0.

[0065] The CCW-2 structure contains: 1,2-linked mannose, 1,3-linked mannose, terminally linked rhamnose, 1,3-linked rhamnose, 1,3,4-linked rhamnose, terminally linked glucuronic acid, 1,2-linked galacturonic acid, terminally linked glucose, 1,3-linked glucose, 1,3,4-linked glucose, 1,2,6-linked glucose, terminally linked galactose, 1,4-linked galactose, 1,3-linked galactose, 1,2-linked galactose, 1,6-linked galactose, 1,2,4-linked galactose, 1,4,6-linked galactose, 1,3,6-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3 ,4-linked xylose, 1,2,4-linked xylose, 1,2,3,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose, 1,5-linked arabinose, 1,4-linked arabinopyranose and 1,2,3-linked arabinose, with a molar ratio of 1.7:2.8:5.1:1.1:1.5:4.7:7.5:1.8:1.3:2.2:1.3:7.8:2.4:1.5:2.0:1.7:2.6:1.5:4.5:2.9:3.3:3.5:4.0:3.2:10.4:1.1:4.4:4.3:4.7:3.1.

[0066] The structure of CCB-1 contains: 1,3-linked mannose, 1,4,6-linked mannose, 1,3,6-linked mannose, terminally linked rhamnose, 1,3-linked rhamnose, terminally linked glucose, 1,4-linked glucose, 1,4,6-linked glucose, terminally linked galactose, 1,4-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, 1,2,3,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose, and 1,5-linked arabinose, with a molar ratio of 3.1:5.4:2.9:0.6:0.5:6.5:9.8:5.7:4.8:3.5:4.3:31.4:5.9:1.8:1.0:7.2:1.6:2.4:1.4.

[0067] The structure of CCB-2 contains: 1,6-linked mannose, 1,4,6-linked mannose, terminally linked glucose, 1,4-linked glucose, 1,6-linked glucose, 1,3,4-linked glucose, 1,4,6-linked glucose, 1,4-linked galactose, 1,6-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose, and 1,5-linked arabinose, with a molar ratio of 1.5:5.6:6.7:20.0:1.7:1.0:9.8:1.9:2.6:7.4:27.1:4.2:1.1:6.8:0.6:1.1:0.7.

[0068] The structure of CCB-3 contains: 1,3-mannose, terminally linked rhamnose, terminally linked glucuronic acid, 1,2-linked galacturonic acid, terminally linked grape, terminally linked galactose, 3,6-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, 1,2,3,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose, 1,5-linked arabinose and 1,2,3-linked arabinose, with a molar ratio of 1.9:1.3:5.0:1.2:3.5:3.6:2.6:2.1:33.3:7.4:10.6:2.8:13.6:1.2:5.6:1.0:3.4.

[0069] Example 3: Prebiotic activity of homogeneous polysaccharide from Commelina communis

[0070] (1) In vitro simulation of gastric and intestinal digestion

[0071] The preparation of simulated gastric fluid mainly refers to the United States Pharmacopoeia method, and the preparation is as follows: take 2.0g sodium chloride and 3.2g pepsin, add 7.0mL concentrated hydrochloric acid, and dilute to 1L with water. The pH value of the simulated gastric fluid is 1.2. Take 2mL of the prepared simulated gastric fluid in a glass test tube, preheat at 37℃ for 15min, add 10mg of Commelina communis homogeneous polysaccharide to the test tube and mix well, and oscillate at 37℃ for reaction. Take 100μL of the reaction solution in a 1.5mL centrifuge tube at the reaction time of 0, 1, 2, 4, and 6h, add 30μL of 0.2mol / L sodium carbonate solution to terminate the reaction, boil at 100℃ for 20min, centrifuge, and freeze-dry the supernatant. Among them, the 0h sample is the simulated gastric fluid containing pepsin first mixed with 0.2mol / L sodium carbonate solution, and then add Commelina communis homogeneous polysaccharide.

[0072] The preparation of simulated intestinal fluid mainly refers to the United States Pharmacopoeia method, and the preparation is as follows: take 6.8g potassium dihydrogen phosphate, add 800mL water to dissolve, adjust the pH value to 6.8±0.1 with 0.2mol / L sodium hydroxide solution, add 10g trypsin, dissolve, and then dilute with water to 1L. The homogeneous polysaccharide simulated gastric fluid reactant of Commelina communis obtained by pepsin hydrolysis for 6h is digested by the simulated intestinal fluid system, and the reaction time is 0, 1, 2, 4, and 6h, respectively. 20μL 30% acetic acid solution is added to terminate the reaction, boiled at 100℃ for 20min, centrifuged, and the supernatant is freeze-dried. Among them, the 0h sample is the simulated intestinal fluid containing trypsin first mixed with 30% acetic acid solution, and then the polysaccharide substrate is added. The freeze-dried sample after digestion is re-dissolved with 0.002mol / L sodium sulfate for HPGPC analysis. The molecular weight and distribution of the homogeneous polysaccharide did not change before and after simulated gastric and intestinal digestion, indicating that the homogeneous polysaccharide of Commelina communis can resist digestion by gastric and intestinal fluids. Figure 3 shown.

[0073] (2) Effects of Commelina communis polysaccharide on the proliferation of three probiotics

[0074] Three intestinal probiotics: Bacteroides ovatus, Bacteroides thetaiotaomicron and Bacteroides cellulosilyticus were cultured anaerobically in tryptone soy broth (TSB) until the exponential growth phase. 1 mL of culture solution was taken into a centrifuge tube and centrifuged at 3000 rpm for 5 min. The bacterial precipitate was resuspended with sterile PBS and centrifuged again. The supernatant was discarded to collect the bacteria. The bacterial precipitate was diluted with minimal medium (MM) to OD 600 =0.2, 100 μL was added to a 96-well plate, and then 100 μL of 5 mg / mL Commelina communis homogeneous polysaccharide sample was added, and cultured anaerobically at 37°C. The OD at 0, 10, 22, 26, 30, 34, 46, 60, and 72 h was measured using an ELISA reader. 600 , draw a growth curve.

[0075] The results are as follows Figure 4 As shown in the figure, when the homogeneous polysaccharide of Commelina was used as the only carbon source, the five homogeneous polysaccharides of Commelina could increase the proliferation of the above three intestinal probiotics, but the proliferation effects were different. CCB-3 had the strongest proliferation activity. In the negative control H2O group, none of the three intestinal probiotics could proliferate. In the CCW-1 and CCW-2 groups, the three intestinal probiotics proliferated rapidly within 22 hours, then slowly proliferated and entered the plateau phase. At 34-46 hours, Bs ovatus proliferated rapidly again, and slowly proliferated after 46 hours, and entered the plateau phase again. B. thetaiotaomicron and B. cellulosilyticus died in large numbers at 46 hours, and the death rate slowed down after 60 hours. In the CCB-1 group, the three intestinal probiotics proliferated slowly within 10 hours, and B.ovatus and B.cellulosilyticus proliferated rapidly within 10–46 hours, then proliferated slowly, and a large number of deaths occurred at 46 hours. The death rate slowed down after 60 hours and entered the plateau phase again. B.thetaiotaomicron entered the plateau phase at 22 hours and then proliferated slowly. In the CCB-2 group, the three intestinal probiotics proliferated slowly within 10 hours, and B.ovatus and B.cellulosilyticus proliferated rapidly within 10–46 hours, then proliferated slowly, and a large number of deaths occurred at 46 hours. The death rate slowed down after 60 hours, and B.thetaiotaomicron entered the plateau phase at 22 hours, and then began to proliferate rapidly again at 46 hours and entered the plateau phase again. In the CCB-3 group, the three intestinal probiotics proliferated rapidly within 22 hours, then proliferated slowly and entered the plateau phase.

[0076] (3) Determination of short-chain fatty acids and 1,2-propylene glycol content

[0077] The culture fluid of anaerobic culture for 72 hours was centrifuged at 13000rpm for 10 minutes, and the supernatant was filtered through a 0.22μm filter membrane. The content of short-chain fatty acids and 1,2-propylene glycol was determined by GC-MS. A standard curve was drawn based on the peak areas of formic acid, acetic acid, propionic acid, butyric acid, and 1,2-propylene glycol standard solutions of different concentrations, and the peak areas of the corresponding short-chain fatty acids and 1,2-propylene glycol in the sample were used for quantification.

[0078] The results are as follows Figure 5As shown in the results, these three intestinal probiotics can degrade the homogeneous polysaccharide of Commelina communis and produce acetic acid and propionic acid. Among the different polysaccharide groups, B. thetaiotaomicron has the weakest acid production ability. In addition to acetic acid and propionic acid, the CCW-1 and CCW-2 groups also produce 1,2-propylene glycol, while the CCW-2, CCB-1, and CCB-2 groups also produce a small amount of formic acid. Short-chain fatty acids are one of the important metabolites produced by the degradation of polysaccharides by human intestinal microorganisms, and play an important role in maintaining intestinal pH and improving intestinal flora.

[0079] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Commelina communis polysaccharide, characterized in that: The Commelina polysaccharides are Commelina uniform polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3, which respectively have the following structural characteristics: Commelina communis homogeneous sugar CCW-1: a polysaccharide composed of 9 monosaccharides, with a molecular weight of 56.9kDa; total sugar content of 90.7%; uronic acid content of 4.8%; protein content of 1.7%; no sulfate group; monosaccharide molar ratio of mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose = 17.7: 2.3: 5.4: 2.2: 0.5: 6.1: 25.0: 12.3: 28.5; the connection modes include terminal connection mannose, 1,2-connected mannose, 1,3-connected mannose, 1,6-connected mannose, 1,3,6-connected mannose, terminal connection rhamnose, 1,2-connected rhamnose, 1,3-connected rhamnose, 1,2,3-connected rhamnose, terminal connection glucuronic acid, terminal connection glucose, 1,2,3-connected glucose, 1,2,4-connected glucose, 1,2,6-connected glucose , terminally linked galactose, 1,2-linked galactose, 1,4-linked galactose, 1,3-linked galactose, 1,6-linked galactose, 1,4,6-linked galactose, 1,3,6-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, 1,2,3,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose , 1,5-linked arabinose and 1,2,3-linked arabinose, with a molar ratio of 3.0:4.9:5.5:1.1:1.3:2.4:1.0:1.8:1.0:2.3:2.5:1.7:1.0:1.3:4.2:2.7:3.0:3.3:2.8:1.5:9.7:2.3:3.7:2.4:2.9:2.6:14.4:1.2:4.6:6.0:2.0; Commelina communis homomonosaccharide CCW-2: a polysaccharide composed of 9 monosaccharides, with a molecular weight of 39.0 kDa; total sugar content of 85.5%; uronic acid content of 15.6%; protein content of 2.2%; no sulfate group; monosaccharide molar ratio of mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose = 7.2:1.0:7.5:4.8:7.4:6.0:24.1:13 .2:28.8; the connection modes include 1,2-linked mannose, 1,3-linked mannose, terminally linked rhamnose, 1,3-linked rhamnose, 1,3,4-linked rhamnose, terminally linked glucuronic acid, 1,2-linked galacturonic acid, terminally linked glucose, 1,3-linked glucose, 1,3,4-linked glucose, 1,2,6-linked glucose, terminally linked galactose, 1,4-linked galactose, 1,3-linked Galactose, 1,2-linked galactose, 1,6-linked galactose, 1,2,4-linked galactose, 1,4,6-linked galactose, 1,3,6-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, 1,2,3,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose, 1,5-linked arabinose , 1,4-linked arabinopyranose and 1,2,3-linked arabinose, with a molar ratio of 1.7:2.8:5.1:1.1:1.5:4.7:7.5:1.8:1.3:2.2:1.3:7.8:2.4:1.5:2.0:1.7:2.6:1.5:4.5:2.9:3.3:3.5:4.0:3.2:10.4:1.1:4.4:4.3:4.7:3.1; Commelina monosaccharide CCB-1: a polysaccharide composed of 7 monosaccharides, with a molecular weight of 44.0 kDa; total sugar content of 92.6%; uronic acid content of 0.8%; protein content of 1.0%; no sulfate group; monosaccharide molar ratio of mannose: glucosamine: rhamnose: glucose: galactose: xylose: arabinose = 10.2: 0.5: 0.9: 22.5: 7.9: 45.3: 12.6; the connection modes include 1,3-linked mannose, 1,4,6-linked mannose, 1,3,6-linked mannose, terminal-linked rhamnose, 1,3-linked rhamnose, terminal-linked glucose, 1,4- linked glucose, 1,4,6-linked glucose, terminally linked galactose, 1,4-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, 1,2,3,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose, 1,5-linked arabinose, molar ratio 3.1:5.4:2.9:0.6:0.5:6.5:9.8:5.7:4.8:3.5:4.3:31.4:5.9:1.8:1.0:7.2:1.6:2.4:1.4; Commelina monosaccharide CCB-2: a polysaccharide composed of 5 monosaccharides, with a molecular weight of 22.6kDa; total sugar content of 94.4%; uronic acid content of 1.0%; protein content of 1.1%; no sulfate group; monosaccharide molar ratio of mannose: glucose: galactose: xylose: arabinose = 8.3: 40.8: 4.5: 37.9: 8.5; the connection modes include 1,6-linked mannose, 1,4,6-linked mannose, terminal-linked glucose, 1,4-linked glucose, 1,6-linked glucose, 1,3,4-linked glucose Sugars, 1,4,6-linked glucose, 1,4-linked galactose, 1,6-linked galactose, terminally linked xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, terminally linked arabinose, 1,2-linked arabinose, 1,3-linked arabinose, 1,5-linked arabinose, molar ratio 1.5:5.6:6.7:20.0:1.7:1.0:9.8:1.9:2.6:7.4:27.1:4.2:1.1:6.8:0.6:1.1:0.7; Commelina monosaccharide CCB-3: a polysaccharide composed of 10 monosaccharides, with a molecular weight of 58.8kDa; total sugar content of 86.7%; uronic acid content of 11.5%; protein content of 0.8%; no sulfate group; monosaccharide molar ratio of mannose: glucosamine: rhamnose: 4-O-methyl glucuronic acid: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose = 1.9:0.5:1.2:3.1:1.0:1.2:3.4:6.6:55.2:24.3; the connection modes include 1,3-mannose, terminal rhamnose, terminal glucuronic acid, 1,2- It is connected to galacturonic acid, terminally connected to grape, terminally connected to galactose, 3,6-linked galactose, terminally connected to xylose, 1,4-linked xylose, 1,3,4-linked xylose, 1,2,4-linked xylose, 1,2,3,4-linked xylose, terminally connected to arabinose, 1,2-linked arabinose, 1,3-linked arabinose, 1,5-linked arabinose and 1,2,3-linked arabinose, with a molar ratio of 1.9:1.3:5.0:1.2:3.5:3.6:2.6:2.1:33.3:7.4:10.6:2.8:13.6:1.2:5.6:1.0:3.

4.

2. A method for preparing Commelina Commelina polysaccharide according to claim 1, characterized in that: The steps include: S1. Take Commelina communis, extract with ethanol, filter in sequence, extract the residue with hot water, concentrate, centrifuge, dry the precipitate after centrifugation to obtain a water-extracted residue, add ethanol to the supernatant, let it stand, centrifuge, add water to the precipitate after centrifugation, re-dissolve the precipitate, remove the ethanol under reduced pressure, add water to re-dissolve, add trichloroacetic acid, centrifuge, adjust the pH of the supernatant after centrifugation to neutral, concentrate, dialyze, and freeze-dry in sequence to obtain Commelina communis water-extracted crude polysaccharide, named CCW; The water extraction residue is extracted with sodium hydroxide and centrifuged in sequence. The pH of the supernatant after centrifugation is adjusted to neutral, and then concentrated, ethanol is added, and then allowed to stand and centrifuged. The precipitate after centrifugation is re-dissolved in water, the ethanol is recovered under reduced pressure, re-dissolved in water again, trichloroacetic acid is added, and centrifuged. The pH of the supernatant after centrifugation is adjusted to neutral, and then concentrated, dialyzed, and freeze-dried to obtain a crude polysaccharide extracted from commelinine, named CCB. S2, dissolving the CCW obtained in step S1 in distilled water, performing preliminary separation by DEAE-cellulose column chromatography, eluting with distilled water, and sequentially concentrating, dialyzing and freeze-drying the collected fractions to obtain a secondary component named CCW-D-1; S3, dissolving the CCB obtained in step S1 in distilled water, performing preliminary separation by DEAE-cellulose column chromatography, eluting with distilled water and 0.1 mol / L sodium chloride solution in turn, collecting each fraction, and performing concentration, dialyzing and freeze-drying in turn to obtain two secondary components, named CCB-D-1 and CCB-D-2; S4, CCW-D-1 obtained in step S2 and CCB-D-1 and CCB-D-2 obtained in step S3 were dissolved in water and centrifuged, and the supernatants after centrifugation were respectively treated with Sephacryl TM The samples were separated by S200 chromatography and eluted with ammonium bicarbonate solution. The fractions were collected and combined according to the ultraviolet detection results of the colorimetric reaction of the sugar content in each fraction. The fractions were concentrated, dialyzed and freeze-dried in sequence. The homogeneity was tested by high performance gel permeation chromatography to obtain the uniform polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 from Commelina communis, which were then subjected to activity experiments.

3. The method for preparing Commelina Commelina polysaccharide according to claim 2, characterized in that: In step S1, the Commelina communis is extracted by using 95% by mass ethanol.

4. The method for preparing Commelina Commelina polysaccharide according to claim 2, characterized in that: In step S1, 95% by mass ethanol was added before both standing to make the ethanol concentration in the supernatant reach 80%.

5. The method for preparing Commelina Commelina polysaccharide according to claim 2, characterized in that: In step S1, the water extraction residue is extracted by sodium hydroxide with a mass fraction of 5%.

6. The method for preparing Commelina Commelina polysaccharide according to claim 2, characterized in that: In step S1, the process of adding trichloroacetic acid twice is: after adding trichloroacetic acid, the mass fraction of trichloroacetic acid in the solution is 10%.

7. A use of the Commelina Commelina polysaccharide as claimed in claim 1, characterized in that: The Commelina Commelina polysaccharide is used for preparing prebiotics.

8. The use of Commelina Commelina polysaccharide according to claim 7, characterized in that: The prebiotics are prebiotic foods containing the Commelina communis polysaccharide.

9. The use of Commelina Commelina polysaccharide according to claim 8, characterized in that: The prebiotic food is a prebiotic food that promotes the growth and reproduction of intestinal probiotics.

10. The use of Commelina Commelina polysaccharide according to claim 9, characterized in that: The intestinal probiotics are selected from any one or more of Bacteroides ovatus, Bacteroides thetaiotaomicron or Bacteroides cellulosus.

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