CN116284476A - Commelina polysaccharide and preparation method and application thereof - Google Patents
Commelina polysaccharide and preparation method and application thereof Download PDFInfo
- Publication number
- CN116284476A CN116284476A CN202310163864.4A CN202310163864A CN116284476A CN 116284476 A CN116284476 A CN 116284476A CN 202310163864 A CN202310163864 A CN 202310163864A CN 116284476 A CN116284476 A CN 116284476A
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- Prior art keywords
- linked
- linkage
- arabinose
- galactose
- xylose
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- Pending
Links
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 90
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 90
- 241000233838 Commelina Species 0.000 title claims abstract description 71
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 230000000968 intestinal effect Effects 0.000 claims abstract description 32
- 239000006041 probiotic Substances 0.000 claims abstract description 22
- 235000018291 probiotics Nutrition 0.000 claims abstract description 22
- 235000013406 prebiotics Nutrition 0.000 claims abstract description 19
- 241001135228 Bacteroides ovatus Species 0.000 claims abstract description 16
- 241000606123 Bacteroides thetaiotaomicron Species 0.000 claims abstract description 9
- 241000606125 Bacteroides Species 0.000 claims abstract description 7
- 229920002678 cellulose Polymers 0.000 claims abstract description 5
- 235000010980 cellulose Nutrition 0.000 claims abstract description 5
- 238000002474 experimental method Methods 0.000 claims abstract description 4
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 156
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 156
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- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 84
- 229930182830 galactose Natural products 0.000 claims description 78
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- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 51
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- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 claims description 18
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- QGGOCWIJGWDKHC-UHFFFAOYSA-N O4-Methyl-D-glucuronsaeure Natural products OC(=O)C(O)C(OC)C(O)C(O)C=O QGGOCWIJGWDKHC-UHFFFAOYSA-N 0.000 claims description 3
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention relates to dayflower polysaccharide and a preparation method and application thereof, wherein 5 uniform polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 are separated from dayflower, and experiments prove that the dayflower uniform polysaccharide is not digested by gastrointestinal fluid in vitro, can promote proliferation of three intestinal probiotics of bacteroides ovatus (Bacteroides ovatus), bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron) and bacteroides celluloses (Bacteroides cellulosilyticus), and can generate short-chain fatty acid and 1, 2-propanediol, and can be further used for preparing prebiotics.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, and relates to dayflower polysaccharide, in particular to dayflower polysaccharide and a preparation method and application thereof.
Background
Prebiotics (probiotics) refers to substances that are selectively fermented by intestinal probiotics to improve the intestinal flora and benefit human health. Prebiotics have a number of physiological activities, such as non-digestibility, improving intestinal flora, promoting the growth of intestinal probiotics, lowering intestinal pH, increasing short chain fatty acid production, regulating the body's metabolism and immune system, alleviating constipation, etc. The currently internationally accepted prebiotics mainly include fructo-oligosaccharides (FOS), isomaltooligosaccharides (IMO), lactulose oligosaccharides (LACT), etc., which are mainly used for the proliferation of Lactobacillus enterica (Lactobacillus spp.) and Bifidobacterium spp. In recent years, with the improvement of the living standard and the enhancement of health care consciousness of people, the traditional prebiotic products cannot meet the increasing market demands. Bacteroides ovatus (Bacteroides ovatus), bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron) and Bacteroides celluloses (Bacteroides cellulosilyticus) have the ability to metabolize polysaccharides and choline salts, have physiological effects on the treatment of diabetes, cardiovascular diseases, inflammatory bowel diseases, cancers and the like, and are considered to be novel probiotics with potential probiotic effects. Thus, finding new prebiotics, especially those with the ability to promote proliferation of b.ovatus, b.theta iotaomomeron and b.cellosiryticus, is a major challenge.
Commelina communis is carried in Ben Cao Shi Yi and Ben Cao gang mu, and is derived from dry aerial parts of Commelina communis Commelina communis L of Commelinaceae, has sweet, light and cold taste, and has effects of clearing heat and purging fire, removing toxic substances, inducing diuresis and relieving swelling, and can be used for treating common cold and fever, pyretic dysphoria and polydipsia, sore throat, edema and oliguria, pyrexia and pain, carbuncle and furuncle. Commelina polysaccharide is one of the main components of Commelina, and research on Commelina polysaccharide mainly focuses on extraction process and detection method, and has few functional characteristics. The prior data show that the research on the biological activity of the dayflower aqueous extract has been widely carried out, but no research on improving the proliferation of probiotics, especially the proliferation of B.ovatus, B.theta iotaotaomicron and B.cellosis, by taking dayflower polysaccharide as a prebiotic is reported at present.
Disclosure of Invention
The invention aims to overcome the defect that proliferation of B.ovatus, B.theta iotaotaomicron and B.cellulosisiticus can not be improved in the related dayflower polysaccharide, and provides dayflower polysaccharide, a preparation method and application thereof. The present invention provides 5 homogeneous polysaccharides from Commelina communis for promoting proliferation of the 3 probiotics B.ovatus, B.theta iotaomomicron and B.cellosiryticus in the human intestinal tract.
The aim of the invention can be achieved by the following technical scheme:
one of the technical schemes of the invention is to provide dayflower polysaccharide which is dayflower uniform polysaccharide CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3, and has the following structural characteristics:
dayflower uniform sugar CCW-1: a polysaccharide consisting of 9 monosaccharides, having a molecular weight of about 56.9kDa; the total sugar content was 90.7%; uronic acid content 4.8%; protein content 1.7%; no sulfate group; monosaccharide molar ratio 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 linkage means includes terminal linkage mannose, 1, 2-linkage mannose, 1, 3-linkage mannose, 1, 6-linkage mannose, 1,3, 6-linkage mannose, terminal linkage rhamnose, 1, 2-linkage rhamnose, 1, 3-linkage rhamnose, 1,2, 3-linkage rhamnose, terminal linkage glucuronic acid, terminal linkage glucose, 1,2, 3-linkage glucose, 1,2, 4-linkage glucose, 1,2, 6-linkage glucose, terminal linkage galactose, 1, 2-linkage galactose, 1, 4-linkage galactose, 1, 3-linkage galactose, 1, 6-linkage galactose, 1,4, 6-linkage galactose, 1,3, 6-linkage galactose, terminal linkage xylose, 1, 4-linkage xylose, 1,3, 4-linkage xylose, 1,2,3, 4-linkage xylose, terminal linkage arabinose, 1, 2-linkage arabinose, 1, 3-linkage arabinose, 1,3, 5-linkage arabinose and 1, 3-linkage arabinose, the molar ratio 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;
dayflower uniform sugar CCW-2: a polysaccharide consisting of 9 monosaccharides, having a molecular weight of about 39.0kDa; the total sugar content was 85.5%; uronic acid content 15.6%; protein content 2.2%; no sulfate group; monosaccharide molar ratio 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 linkage means include 1, 2-linked mannose, 1, 3-linked mannose, terminal linked rhamnose, 1, 3-linked rhamnose, 1,3, 4-linked rhamnose, terminal linked glucuronic acid, 1, 2-linked galacturonic acid, terminal linked glucose, 1, 3-linked glucose, 1,3, 4-linked glucose, 1,2, 6-linked glucose, terminal 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, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2, 4-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, 1, 4-linked arabinose and 1, 3-linked arabinose, the molar ratio is 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;
dayflower uniform sugar CCB-1: a polysaccharide consisting of 7 monosaccharides, having a molecular weight of about 44.0kDa; the total sugar content was 92.6%; uronic acid content was 0.8%; protein content 1.0%; no sulfate group; monosaccharide molar ratio mannose: glucosamine: rhamnose: glucose: galactose: xylose: arabinose=10.2:0.5:0.9:22.5:7.9:45.3:12.6; the linkage means includes 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, 6-linked glucose, terminal-linked galactose, 1, 4-linked galactose, terminal-linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal-linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, the 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;
dayflower uniform sugar CCB-2: a polysaccharide consisting of 5 monosaccharides, having a molecular weight of about 22.6kDa; total sugar content 94.4%; uronic acid content 1.0%; protein content 1.1%; no sulfate group; monosaccharide molar ratio mannose: glucose: galactose: xylose: arabinose=8.3:40.8:4.5:37.9:8.5; the linkage means includes 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, 1,4, 6-linked glucose, 1, 4-linked galactose, 1, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, the 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;
dayflower uniform sugar CCB-3: a polysaccharide consisting of 10 monosaccharides, having a molecular weight of about 58.8kDa; the total sugar content was 86.7%; uronic acid content 11.5%; protein content 0.8%; no sulfate group; monosaccharide molar ratio 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 linkage pattern includes 1, 3-mannose, terminal linkage rhamnose, terminal linkage glucuronic acid, 1, 2-linkage galacturonic acid, terminal linkage glucose, terminal linkage galactose, 3, 6-linkage galactose, terminal linkage xylose, 1, 4-linkage xylose, 1,3, 4-linkage xylose, 1,2,3, 4-linkage xylose, terminal linkage arabinose, 1, 2-linkage arabinose, 1, 3-linkage arabinose, 1, 5-linkage arabinose and 1,2, 3-linkage arabinose in 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.
The second technical scheme of the invention is to provide a preparation method of dayflower polysaccharide according to the first technical scheme, which comprises the following steps:
s1, taking dayflower, sequentially extracting with ethanol, filtering, extracting filtered residues with hot water, concentrating, centrifuging, drying the centrifuged precipitate to obtain water-extracted residues, adding ethanol into supernatant, standing, centrifuging, adding water into the centrifuged precipitate for redissolution, recovering ethanol under reduced pressure, adding water for redissolution, adding trichloroacetic acid and centrifuging again, regulating pH of the centrifuged supernatant to be neutral, sequentially concentrating, dialyzing, and freeze-drying to obtain dayflower water-extracted crude polysaccharide, which is named as CCW;
sequentially extracting water extraction residues with sodium hydroxide, centrifuging, regulating pH of the supernatant after centrifuging to neutrality, concentrating, adding ethanol, standing, centrifuging, re-dissolving the precipitate after centrifuging with water, recovering ethanol under reduced pressure, re-dissolving with water again, adding trichloroacetic acid, centrifuging, regulating pH of the supernatant after centrifuging to neutrality, concentrating, dialyzing, and freeze-drying to obtain Commelina alkali crude polysaccharide named CCB;
s2, dissolving the CCW obtained in the step S1 by adding distilled water, performing primary separation by using DEAE-cellulose column chromatography, eluting by using distilled water, and sequentially concentrating, dialyzing and freeze-drying the collected fractions to obtain 1 secondary component named as CCW-D-1;
s3, dissolving the CCB obtained in the step S1 by adding distilled water, performing primary separation by using DEAE-cellulose column chromatography, eluting by using distilled water and 0.1mol/L sodium chloride solution in sequence, collecting each fraction, and sequentially concentrating, dialyzing and freeze-drying to obtain 2 secondary components named CCB-D-1 and CCB-D-2;
s4, adding water into the CCW-D-1 obtained in the step S2 and the CCB-D-1 and the CCB-D-2 obtained in the step S3 respectively for dissolving and centrifuging, and respectively using Sephacryl to obtain supernatant after centrifuging TM S200, separating by chromatography, eluting with ammonium bicarbonate solution, collecting each fraction, combining the fractions according to the ultraviolet detection result of the color reaction of sugar content of each fraction, sequentially concentrating, dialyzing, freeze-drying, and detecting by high-efficiency gel permeation chromatography to obtain dayflower uniform polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3, and performing an activity experiment.
Further, in the step S1, commelina communis is extracted by ethanol with a mass fraction of 95%.
Further, in the step S1, ethanol with a mass fraction of 95% was added before two times of standing so that the ethanol concentration in the supernatant was 80%.
Further, in the step S1, the water extraction residue is extracted by sodium hydroxide with a mass fraction of 5%.
Further, in the step S1, the process of adding trichloroacetic acid twice is as follows: after trichloroacetic acid was added, the mass fraction of trichloroacetic acid in the solution was 10%.
A third aspect of the present invention provides an application of the dayflower polysaccharide according to one of the above aspects, wherein the dayflower polysaccharide is used for preparing prebiotics.
Further, the prebiotic is a prebiotic food containing the dayflower polysaccharide.
Further, the prebiotic food is a prebiotic food for promoting the growth and reproduction of intestinal probiotics.
Still further, the intestinal probiotics are selected from any one or more of bacteroides ovatus, bacteroides thetaiotaomicron, or bacteroides cellulosus.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, 5 dayflower uniform polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 are separated from the dry aerial parts of dayflower, in vitro experiments prove that the dayflower polysaccharide is not digested by simulated stomach and intestinal juice, the growth and proliferation of intestinal beneficial bacteria bacteroides ovatus (B.ovatus), bacteroides thetaiotaomicron (B.thetaiotaomicron) and bacteroides celluloses (B.cellulolyticus) in 3 are improved, short chain fatty acid and 1, 2-propanediol are generated, and the dayflower polysaccharide can be further used for preparing prebiotics.
Drawings
FIG. 1 is a flow chart for the separation of dayflower uniform polysaccharide.
FIG. 2 is a chromatogram of High Performance Gel Permeation Chromatography (HPGPC) of Commelina communis homogeneous polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3, wherein the chromatographic columns: shodex SUGAR KS-804 (300X 8.0 mm) and Shodex SUGAR KS-802 gel column (300X 8.0 mm) were connected in series; eluent: 0.002mol/L ammonium acetate; flow rate: 0.6mL/min.
FIG. 3 is a chromatogram of HPGPC of Commelina communis homogeneous polysaccharide before and after simulated gastric and intestinal fluid digestion, wherein W0 refers to simulated gastric fluid digestion for 0h, W6 refers to simulated gastric fluid digestion for 6h, C0 refers to simulated intestinal fluid digestion for 0h, and C6 refers to simulated intestinal fluid digestion for 6h.
Figure 4, growth profile of dayflower uniform polysaccharide to increase proliferation of 3 probiotics in human intestinal tract.
Figure 5 is a bar graph of short chain fatty acids and 1,2 propanediol produced by degradation of dayflower uniform polysaccharide by 3 probiotics.
Where BO in fig. 4 and 5 represents b.ovatus, BC represents b.cellosilytic, and BT represents b.theta iotaomomicron.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
In the following examples and comparative examples, unless otherwise specified, the starting materials or processing techniques are all those which are conventional commercially available in the art.
Example 1: preparation of Commelina Costus Homoellendorffii Homoellendorfs polysaccharide CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3
As shown in fig. 1, a flow chart for separating dayflower uniform polysaccharide is shown:
crushing 12kg of Commelina communis medicinal material, extracting with 95% ethanol, filtering, extracting the filtered residue with hot water solution for 3 times, concentrating, centrifuging, drying the centrifuged precipitate at 50deg.C to obtain water extraction residue, adding 95% ethanol into the supernatant to obtain ethanol concentration of 80%, standing at 4deg.C overnight, centrifuging, re-dissolving the centrifuged precipitate with water, recovering ethanol under reduced pressure, re-dissolving with water, adding trichloroacetic acid with equal volume mass fraction of 20% to obtain trichloroacetic acid with mass fraction of 10%, removing free protein, and regulating the centrifuged supernatant to neutrality, concentrating, dialyzing, and lyophilizing to obtain Commelina communis water extraction crude polysaccharide CCW. Adding 6kg of water extraction residues, adding 5% sodium hydroxide solution, soaking for 1h at 4 ℃, centrifuging, taking supernatant, regulating to neutrality by hydrochloric acid, concentrating, adding 95% ethanol to the supernatant to reach an ethanol concentration of 80%, standing at 4 ℃ for overnight, centrifuging, adding water for redissolution, recovering ethanol under reduced pressure, adding water for redissolution, adding trichloroacetic acid with an equal volume mass fraction of 20%, so that the mass fraction of trichloroacetic acid in the solution is 10%, removing free protein, regulating the supernatant after centrifuging to neutrality, concentrating, dialyzing, and freeze-drying to obtain Commelina communis alkali extraction crude polysaccharide CCB.
Dissolving 80g Commelina communis water extracted crude polysaccharide CCW in distilled water, centrifuging, and primarily separating the supernatant by DEAE-cellulose column chromatography. Eluting with distilled water, wherein the eluting volume is greater than 2 times of column volume (about 10L), the flow rate is 25mL/min, collecting each fraction, and detecting absorbance at 490nm (after color development by sulfuric acid-phenol method) in a tube. According to sugar color reaction, the combined fractions, concentration, dialysis and freeze drying are carried out to obtain 1 secondary component: CCW-D-1.
30g of Commelina alkali crude polysaccharide CCB is dissolved in distilled water, and centrifuged, and the supernatant after centrifugation is subjected to preliminary separation by DEAE-cellulose column chromatography. Eluting with distilled water and 0.1mol/L sodium chloride, wherein the eluting volume is greater than 2 times of column volume (about 10L), the flow rate is 25mL/min, collecting each fraction, and detecting absorbance at 490nm (after color development by sulfuric acid-phenol method) in a separation tube. According to the sugar color reaction result, the fractions are combined, concentrated, dialyzed and freeze-dried to obtain 2 secondary components: CCB-D-1, CCB-D-2.
Dissolving 8g of CCW-D-1 in distilled water, centrifuging, and separating supernatant with Sephacryl TM S200 chromatography (molecular weight cut-off 2kDa-400 kDa). Eluting with 0.2mol/L ammonium bicarbonate solution at a flow rate of 0.5mL/min, and collecting the fractions. The absorbance at 490nm (after the color development by the sulfuric acid-phenol method) was measured in a tube, and the same fractions were combined, concentrated, dialyzed, and freeze-dried according to the measurement results to obtain uniform polysaccharides CCW-1 (520 mg) and CCW-2 (340 mg).
Dissolving 8g of CCB-D-1 in distilled water, centrifuging, and separating supernatant with Sephacryl TM S200 chromatography (molecular weight cut-off 2kDa-400 kDa). Eluting with 0.2mol/L ammonium bicarbonate solution at a flow rate of 0.5mL/min, and collecting the fractions. The absorbance at 490nm (after color development by sulfuric acid-phenol method) was detected in a tube, and the same fractions were combined, concentrated, dialyzed, and freeze-dried according to the detection result to obtain uniform polysaccharides CCB-1 (330 mg) and CCB-2 (290 mg).
Dissolving 11g of CCB-D-2 distilled water, centrifuging, and separating supernatant with Sephacryl TM S200 chromatography (molecular weight retention 2kDa-400 kDa). Eluting with 0.2mol/L ammonium bicarbonate solution at a flow rate of 0.5mL/min, and collecting the fractions. And detecting the absorbance value at 490nm (after the color development by an acid-phenol method) by a separation tube, and combining the same fractions, concentrating, dialyzing and freeze-drying according to the detection result to obtain uniform polysaccharide CCB-3 (820 mg).
CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 were all homogeneous components as measured by High Performance Gel Permeation Chromatography (HPGPC), as shown in FIG. 2.
Example 2: structural characterization of Commelina communis homogeneous polysaccharide CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3
(1) Determination of molecular weight
The relative molecular weight of polysaccharide sample is determined by HPGPC, and the basic principle is that homogeneous polysaccharide forms symmetrical chromatographic peaks by gel permeation chromatography, the peak time is related to the molecular weight, and the calculation is performed according to a calibration curve obtained by known molecular weight.
Chromatographic conditions: the separation was performed by using Shodex SUGAR KS-804 (300X 8.0 mm) and Shodex SUGAR KS-802 gel column (300X 8.0 mm) in series, the flow rate was 0.6mL/min, the sample injection amount was 20. Mu.L, 0.002mol/L ammonium acetate was used as mobile phase, the column temperature was 35℃and the detector was a differential refractive detector (RID).
The experimental method comprises the following steps: accurately weighing 2.0mg of each of the uniform polysaccharide and the dextran series standard substance, preparing 3.0mg/mL of solution by using 0.002mol/L ammonium acetate, filtering by using a microporous filter membrane with the thickness of 0.22 mu m before sample injection, detecting, recording retention time, drawing a standard curve by taking the logarithmic value (Lg) of the molecular weight of the standard polysaccharide as an ordinate and the retention time as an abscissa, obtaining a corresponding linear regression equation, and calculating the relative molecular weight of the uniform polysaccharide. The relative molecular weights of CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 are 56.9kDa, 39.0kDa, 44.0kDa, 22.6kDa and 58.8kDa, respectively.
(2) Determination of total sugar, uronic acid, protein and sulfate group content
The total sugar content of CCW-1 is 90.7% by the sulfuric acid-phenol method; the total sugar content of CCW-2 is 85.5%; the total sugar content of CCB-1 was 92.6%; the total sugar content of CCB-2 was 94.4%; the total sugar content of CCB-3 was 86.7%.
The uronic acid content of CCW-1 is measured and detected by a m-hydroxybiphenyl method and is 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%; the uronic acid content of CCB-3 was 11.5%.
The protein content was determined by Coomassie Brilliant blue method, and the protein content of CCW-1 was 1.7%; the protein content of CCW-2 was 2.2%; the protein content of CCB-1 is 1.0%; the protein content of CCB-2 is 1.1%; the protein content of CCB-3 was 0.8%.
The CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 were each free of sulfate groups as measured by the barium chloride turbidimetry.
(3) Analysis of monosaccharide composition
And (3) carrying out liquid phase analysis on products obtained by carrying out full hydrolysis on CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3 respectively by 2mol/L trifluoroacetic acid at 110 ℃, and carrying out derivatization on 1-phenyl-3-methyl-5-pyrazolone.
CCW-1 is a polysaccharide consisting of 9 monosaccharides, the molar ratio of monosaccharides being 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.
CCW-2 is a polysaccharide consisting of 9 monosaccharides, with the 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.
CCB-1 is a polysaccharide consisting of 7 monosaccharides in the molar ratio mannose: glucosamine: rhamnose: glucose: galactose: xylose: arabinose=10.2:0.5:0.9:22.5:7.9:45.3:12.6.
CCB-2 is a polysaccharide consisting of 5 monosaccharides in the molar ratio mannose: glucose: galactose: xylose: arabinose=8.3:40.8:4.5:37.9:8.5.
CCB-3 is a polysaccharide consisting of 10 monosaccharides in the molar ratio 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.
(4) Methylation analysis
CMC-NaBD is used first 4 The uronic acid is reduced and then the polysaccharide is respectively methylated by adopting a modified Hakomori method, the methylated product is fully hydrolyzed by 2mol/L trifluoroacetic acid, sodium borodeuteride is reduced and acetic anhydride is acetylated to prepare partially methylated Aldi alcohol acetate derivative, and then GC-MS analysis is carried out.
The CCW-1 structure contains: terminal linked mannose, 1, 2-linked mannose, 1, 3-linked mannose, 1, 6-linked mannose, 1,3, 6-linked mannose, terminal linked rhamnose, 1, 2-linked rhamnose, 1, 3-linked rhamnose, 1,2, 3-linked rhamnose, terminal linked glucuronic acid, terminal linked glucose, 1,2, 3-linked glucose, 1,2, 4-linked glucose, 1,2, 6-linked glucose, terminal 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, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1, 3-linked arabinose, the molar ratio 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.
The CCW-2 structure contains: 1, 2-linked mannose, 1, 3-linked mannose, terminal linked rhamnose, 1, 3-linked rhamnose, 1,3, 4-linked rhamnose, terminal linked glucuronic acid, 1, 2-linked galacturonic acid, terminal linked glucose, 1, 3-linked glucose, 1,3, 4-linked glucose, 1,2, 6-linked glucose, terminal 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, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2, 4-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, 1, 4-linked arabinose and 1, 3-linked arabinose, the molar ratio is 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.
The CCB-1 structure comprises: 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, 6-linked glucose, terminal-linked galactose, 1, 4-linked galactose, terminal-linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal-linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, the 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.
The CCB-2 structure comprises: 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, 1,4, 6-linked glucose, 1, 4-linked galactose, 1, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, the molar ratio is 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.
The CCB-3 structure comprises: 1, 3-mannose, terminal linkage rhamnose, terminal linkage glucuronic acid, 1, 2-linkage galacturonic acid, terminal linkage glucose, terminal linkage galactose, 3, 6-linkage galactose, terminal linkage xylose, 1, 4-linkage xylose, 1,3, 4-linkage xylose, 1,2,3, 4-linkage xylose, terminal linkage arabinose, 1, 2-linkage arabinose, 1, 3-linkage arabinose, 1, 5-linkage arabinose and 1,2, 3-linkage arabinose in a molar ratio of 1.9:1.3:5.0:1.2:3.5:3.6:2.1:33.3:7.4:10.6:2.8:13.6:1.2:5.6:1.0:3.4.
Example 3: prebiotic activity of dayflower uniform polysaccharide
(1) In vitro simulation of gastric and intestinal fluid digestion
The simulated gastric fluid is prepared mainly by referring to the United states pharmacopoeia method, and is prepared as follows: 2.0g of sodium chloride and 3.2g of pepsin are taken, 7.0mL of concentrated hydrochloric acid is added, the volume is fixed to 1L by water, and the pH value of the simulated gastric fluid is 1.2. 2mL of prepared simulated gastric fluid is taken in a glass test tube, preheated for 15min at 37 ℃,10 mg of dayflower uniform polysaccharide is added into the test tube for uniform mixing, and the shaking reaction is carried out at 37 ℃. And taking 100 mu L of reaction solution into a 1.5mL centrifuge tube when the reaction time is 0, 1,2,4 and 6 hours respectively, adding 30 mu L of 0.2mol/L sodium carbonate solution to terminate the reaction, boiling at 100 ℃ for 20 minutes, centrifuging, and freeze-drying supernatant. Wherein the sample for 0h is prepared by mixing pepsin-containing simulated gastric fluid with 0.2mol/L sodium carbonate solution, and adding herba Commelinae uniform polysaccharide.
The preparation of the simulated intestinal juice is mainly prepared by referring to the United states pharmacopoeia method, and comprises the following steps: taking 6.8g of monopotassium phosphate, adding 800mL of water for dissolution, adjusting the pH value to 6.8+/-0.1 by using 0.2mol/L sodium hydroxide solution, adding 10g of trypsin, and diluting to 1L by adding water after dissolution. And (3) performing enzymolysis on the pepsin for 6 hours to obtain a dayflower uniform polysaccharide, simulating gastric juice reactant, performing digestion on the dayflower uniform polysaccharide by a simulated intestinal juice system, sequentially performing reaction for 0, 1,2,4 and 6 hours, adding 20 mu L of 30% acetic acid solution to terminate the reaction, boiling for 20 minutes at 100 ℃, centrifuging, and freeze-drying supernatant. Wherein the 0h sample is prepared by mixing simulated intestinal fluid containing trypsin with 30% acetic acid solution, and adding polysaccharide substrate. After digestion, the lyophilized samples were reconstituted with 0.002mol/L sodium sulfate and analyzed by HPGPC. The molecular weight and distribution of the uniform polysaccharide before and after gastric and intestinal fluid digestion are simulated, which shows that the dayflower uniform polysaccharide can resist gastric and intestinal fluid digestion, as shown in fig. 3.
(2) Effect of Commelina homogeneous polysaccharide on proliferation of 3 probiotics
Anaerobic cultivation of 3 intestinal probiotics using Tryptone Soy Broth (TSB): oval bacteroides (b.ovatus), bacteroides thetaiotaomicron (b.theta) and bacteroides celluloses (b.cellosisiticus) to exponential growth phase, taking 1mL of the culture solution into a centrifuge tube, centrifuging at 3000rpm for 5min, re-suspending the bacterial pellet with sterile PBS, centrifuging again, and discarding the supernatant to collect the bacterial pellet. The bacterial cell precipitate was diluted to OD with Minimal Medium (MM) 600 =0.2, 100 μl was added to 96-well plates, and 100 μl of 5mg/mL dayflower uniform polysaccharide sample was added thereto, anaerobic culture was performed at 37 ℃, and OD was measured at 0, 10, 22, 26, 30, 34, 46, 60, 72h with a microplate reader 600 And drawing a growth curve.
As shown in FIG. 4, when the dayflower uniform polysaccharide was used as the sole carbon source, all of the 5 dayflower uniform polysaccharides increased proliferation of the above 3 intestinal probiotics, but the proliferation effect was different, and the proliferation activity was the strongest with CCB-3. H as a negative control 2 Group O, none of these 3 intestinal probiotics proliferated. This is within 22h of the CCW-1 and CCW-2 groups3 intestinal probiotics proliferated rapidly, then proliferated slowly and entered the plateau, 34-46h, b.s ovatus proliferated rapidly again, slowly after 46h, entered the plateau again, while b.theta-omomeron and b.c. celloside died largely at 46h, with a slow rate of death after 60 h. These 3 intestinal probiotics proliferate slowly over 10h, 10-46h, b.ovatus and b.celulosilastic proliferate slowly after which massive death occurred at 46h, death rate slowed down after 60h, entered plateau again, b.theotamicin entered plateau at 22h, and proliferated slowly at all times after that. These 3 intestinal probiotics proliferate slowly within 10h in CCB-2 group, 10-46h, b.ovatus and b.celulosidicus proliferate rapidly, followed by slow proliferation, 46h massive death, 60h slower rate of death, b.theotamicin entered the plateau at 22h, 46h again began to proliferate rapidly, and entered the plateau again. The 3 intestinal probiotics proliferated rapidly within 22h in CCB-3 group, followed by slow proliferation, entering plateau.
(3) Short chain fatty acid and 1, 2-propylene glycol content determination
Taking culture solution for anaerobic culture for 72 hours, centrifuging at 13000rpm for 10 minutes, filtering supernatant with a 0.22 mu m filter membrane, and measuring the short chain fatty acid and 1, 2-propylene glycol content by adopting a GC-MS method. And drawing a standard curve according to the peak areas of standard solutions of formic acid, acetic acid, propionic acid, butyric acid and 1, 2-propanediol with different concentrations, and quantifying the peak areas of the corresponding short-chain fatty acid and 1, 2-propanediol in the sample.
As shown in fig. 5, these 3 intestinal probiotics all degraded dayflower homogeneous polysaccharide and produced acetic acid and propionic acid, with the acid production capacity as b. The CCW-1, CCW-2 groups produced 1, 2-propanediol in addition to acetic acid, propionic acid, while the CCW-2, CCB-1, CCB-2 groups produced small amounts of formic acid. Short chain fatty acid is one of important metabolites produced by degrading polysaccharide by human intestinal microorganisms, and has important effects on maintaining intestinal pH value and improving intestinal flora.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. The dayflower polysaccharide is dayflower uniform polysaccharide CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3, and has the following structural characteristics:
dayflower uniform sugar CCW-1: a polysaccharide consisting of 9 monosaccharides, having a molecular weight of about 56.9kDa; the total sugar content was 90.7%; uronic acid content 4.8%; protein content 1.7%; no sulfate group; monosaccharide molar ratio 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 linkage means includes terminal linkage mannose, 1, 2-linkage mannose, 1, 3-linkage mannose, 1, 6-linkage mannose, 1,3, 6-linkage mannose, terminal linkage rhamnose, 1, 2-linkage rhamnose, 1, 3-linkage rhamnose, 1,2, 3-linkage rhamnose, terminal linkage glucuronic acid, terminal linkage glucose, 1,2, 3-linkage glucose, 1,2, 4-linkage glucose, 1,2, 6-linkage glucose, terminal linkage galactose, 1, 2-linkage galactose, 1, 4-linkage galactose, 1, 3-linkage galactose, 1, 6-linkage galactose, 1,4, 6-linkage galactose, 1,3, 6-linkage galactose, terminal linkage xylose, 1, 4-linkage xylose, 1,3, 4-linkage xylose, 1,2,3, 4-linkage xylose, terminal linkage arabinose, 1, 2-linkage arabinose, 1, 3-linkage arabinose, 1,3, 5-linkage arabinose and 1, 3-linkage arabinose, the molar ratio 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;
dayflower uniform sugar CCW-2: a polysaccharide consisting of 9 monosaccharides, having a molecular weight of about 39.0kDa; the total sugar content was 85.5%; uronic acid content 15.6%; protein content 2.2%; no sulfate group; monosaccharide molar ratio 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 linkage means include 1, 2-linked mannose, 1, 3-linked mannose, terminal linked rhamnose, 1, 3-linked rhamnose, 1,3, 4-linked rhamnose, terminal linked glucuronic acid, 1, 2-linked galacturonic acid, terminal linked glucose, 1, 3-linked glucose, 1,3, 4-linked glucose, 1,2, 6-linked glucose, terminal 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, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2, 4-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, 1, 4-linked arabinose and 1, 3-linked arabinose, the molar ratio is 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;
dayflower uniform sugar CCB-1: a polysaccharide consisting of 7 monosaccharides, having a molecular weight of about 44.0kDa; the total sugar content was 92.6%; uronic acid content was 0.8%; protein content 1.0%; no sulfate group; monosaccharide molar ratio mannose: glucosamine: rhamnose: glucose: galactose: xylose: arabinose=10.2:0.5:0.9:22.5:7.9:45.3:12.6; the linkage means includes 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, 6-linked glucose, terminal-linked galactose, 1, 4-linked galactose, terminal-linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal-linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, the 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;
dayflower uniform sugar CCB-2: a polysaccharide consisting of 5 monosaccharides, having a molecular weight of about 22.6kDa; total sugar content 94.4%; uronic acid content 1.0%; protein content 1.1%; no sulfate group; monosaccharide molar ratio mannose: glucose: galactose: xylose: arabinose=8.3:40.8:4.5:37.9:8.5; the linkage means includes 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, 1,4, 6-linked glucose, 1, 4-linked galactose, 1, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, the 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;
dayflower uniform sugar CCB-3: a polysaccharide consisting of 10 monosaccharides, having a molecular weight of about 58.8kDa; the total sugar content was 86.7%; uronic acid content 11.5%; protein content 0.8%; no sulfate group; monosaccharide molar ratio 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 linkage pattern includes 1, 3-mannose, terminal linkage rhamnose, terminal linkage glucuronic acid, 1, 2-linkage galacturonic acid, terminal linkage glucose, terminal linkage galactose, 3, 6-linkage galactose, terminal linkage xylose, 1, 4-linkage xylose, 1,3, 4-linkage xylose, 1,2,3, 4-linkage xylose, terminal linkage arabinose, 1, 2-linkage arabinose, 1, 3-linkage arabinose, 1, 5-linkage arabinose and 1,2, 3-linkage arabinose in 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 dayflower polysaccharide according to claim 1, comprising the steps of:
s1, taking dayflower, sequentially extracting with ethanol, filtering, extracting filtered residues with hot water, concentrating, centrifuging, drying the centrifuged precipitate to obtain water-extracted residues, adding ethanol into supernatant, standing, centrifuging, adding water into the centrifuged precipitate for redissolution, recovering ethanol under reduced pressure, adding water for redissolution, adding trichloroacetic acid and centrifuging again, regulating pH of the centrifuged supernatant to be neutral, sequentially concentrating, dialyzing, and freeze-drying to obtain dayflower water-extracted crude polysaccharide, which is named as CCW;
sequentially extracting water extraction residues with sodium hydroxide, centrifuging, regulating pH of the supernatant after centrifuging to neutrality, concentrating, adding ethanol, standing, centrifuging, re-dissolving the precipitate after centrifuging with water, recovering ethanol under reduced pressure, re-dissolving with water again, adding trichloroacetic acid, centrifuging, regulating pH of the supernatant after centrifuging to neutrality, concentrating, dialyzing, and freeze-drying to obtain Commelina alkali crude polysaccharide named CCB;
s2, dissolving the CCW obtained in the step S1 by adding distilled water, performing primary separation by using DEAE-cellulose column chromatography, eluting by using distilled water, and sequentially concentrating, dialyzing and freeze-drying the collected fractions to obtain 1 secondary component named as CCW-D-1;
s3, dissolving the CCB obtained in the step S1 by adding distilled water, performing primary separation by using DEAE-cellulose column chromatography, eluting by using distilled water and 0.1mol/L sodium chloride solution in sequence, collecting each fraction, and sequentially concentrating, dialyzing and freeze-drying to obtain 2 secondary components named CCB-D-1 and CCB-D-2;
s4, adding water into the CCW-D-1 obtained in the step S2 and the CCB-D-1 and the CCB-D-2 obtained in the step S3 respectively for dissolving and centrifuging, and respectively using Sephacryl to obtain supernatant after centrifuging TM S200, separating by chromatography, eluting with ammonium bicarbonate solution, collecting each fraction, combining the fractions according to the ultraviolet detection result of the color reaction of sugar content of each fraction, sequentially concentrating, dialyzing, freeze-drying, and detecting by high-efficiency gel permeation chromatography to obtain dayflower uniform polysaccharides CCW-1, CCW-2, CCB-1, CCB-2 and CCB-3, and performing an activity experiment.
3. The method for producing dayflower polysaccharide according to claim 2, wherein in step S1, dayflower is extracted by ethanol with a mass fraction of 95%.
4. The method for preparing dayflower polysaccharide according to claim 2, wherein in step S1, ethanol with a mass fraction of 95% is added before two standing so that the ethanol concentration in the supernatant is 80%.
5. The method for producing dayflower polysaccharide according to claim 2, wherein in step S1, the water extraction residue is extracted by sodium hydroxide with a mass fraction of 5%.
6. The method for preparing dayflower polysaccharide according to claim 2, wherein in step S1, the process of adding trichloroacetic acid twice is as follows: after trichloroacetic acid was added, the mass fraction of trichloroacetic acid in the solution was 10%.
7. Use of a dayflower polysaccharide according to claim 1 for the preparation of prebiotics.
8. The use of dayflower polysaccharide according to claim 7, wherein the prebiotic is a prebiotic food containing the dayflower polysaccharide.
9. The use of dayflower polysaccharide according to claim 8, wherein the prebiotic food is a prebiotic food that promotes the growth and propagation of intestinal probiotics.
10. Use of dayflower polysaccharide according to claim 9, wherein the intestinal probiotics are selected from any one or more of bacteroides ovatus, bacteroides thetaiotaomicron or bacteroides celluloses.
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