CN116284295A - Commelina glycoprotein and preparation method and application thereof - Google Patents

Commelina glycoprotein and preparation method and application thereof Download PDF

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CN116284295A
CN116284295A CN202310163863.XA CN202310163863A CN116284295A CN 116284295 A CN116284295 A CN 116284295A CN 202310163863 A CN202310163863 A CN 202310163863A CN 116284295 A CN116284295 A CN 116284295A
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陈道峰
王小江
卢燕
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Abstract

The invention relates to dayflower glycoprotein and a preparation method and application thereof, wherein 8 dayflower glycoproteins CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5 are separated from dayflower, and experiments prove that the prepared dayflower glycoprotein has remarkable inhibition effect on complement activation and can be further used as an active ingredient for preparing novel anticomplement medicines.

Description

Commelina glycoprotein and preparation method and application thereof
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, and relates to glycoprotein, in particular to dayflower glycoprotein, and a preparation method and application thereof.
Background
The complement system is an important component of the human immune system and its normal activation plays an important role in destroying foreign microorganisms, clearing damaged or dead cells and tissues in the body, and maintaining the balance of the body. However, in some cases, abnormal activation of the complement system may cause an excessive reaction to the immune system of the human body, resulting in damage to normal tissues of the human body, such as rheumatoid and rheumatic arthritis, systemic Lupus Erythematosus (SLE), acute Respiratory Distress Syndrome (ARDS), influenza virus-induced acute pneumonia, severe atypical pneumonia (SARS), and the like. It follows that inhibition of overactivation of the complement system is a preferred approach for clinical treatment of respiratory viral infectious diseases.
At present, although traditional immunosuppressant drugs such as glucocorticoid, cyclophosphamide, methotrexate and the like widely used clinically have a certain therapeutic effect on certain diseases related to complement overactivation, the drugs are not specific complement inhibitors, and can reduce the defending function of organisms after long-term use, so that the anti-infective capability of human bodies is reduced, and even a plurality of complications and side effects are generated. Under the circumstance, the search for a novel complement inhibitor with high efficiency, specificity and low toxicity is an urgent need for clinically treating the diseases.
The herbal medicine, especially the heat-clearing and detoxicating traditional Chinese medicine, has the components with anticomplement activity, the traditional Chinese medicine resources in China are rich, a plurality of traditional Chinese medicines have obvious regulating effect on the immune system, and the herbal medicine is a precious resource for anticomplement medicine research. The traditional Chinese medicine and the traditional Chinese medicine have remarkable effects on the prevention and treatment of the COVID-19. Therefore, the search for novel and efficient anticomplement substances from medicinal plants is of great significance.
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 with fever, pyretic dysphoria with polydipsia, sore throat with swelling and pain, edema with oliguria, heat stranguria with pain, carbuncle and furuncle.
At present, patent CN102249862a discloses phenol compounds with anticomplement activity extracted from n-butanol extract of dayflower, and patent CN103083342a discloses flavans compounds with anticomplement activity extracted from n-butanol extract of dayflower.
Thus, it is expected to dig from dayflower to other different kinds of compounds having anticomplement activity, such as glycoprotein, etc.
Disclosure of Invention
The invention aims at providing dayflower glycoprotein, a preparation method and application thereof, in particular 8 dayflower glycoproteins CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5, a preparation method thereof and application thereof in preparing anticomplement medicines based on the current state of the art.
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 glycoprotein, which is dayflower glycoprotein CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5, and has the following structural characteristics:
CCW-4 is a glycoprotein consisting of 9 monosaccharides, with a molecular weight of 50.7kDa; total sugar content was 58.6%; uronic acid content 18.7%; protein content 14.3%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose=7.6:1.6:7.8:4.7:8.2:9.9:20.0:13.5:26.7; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine= 9.8:7.8:7.2:4.0:14.6:7.1:9.3:6.2:10.5:0.1:5.6:8.7:1.3:6.2:1.4:0.4; the linkage means includes 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, 4-linked glucuronic acid, 1, 2-linked galacturonic acid, terminal linked glucose, 1, 3-linked glucose, 1,2, 4-linked glucose, terminal linked galactose, 1, 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, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, 1, 4-linked arabinopyranose and 1,2, 3-linked arabinose, the molar ratio is 1.5:5.8:5.4:1.1:1.2:2.1:2.6:9.1:5.5:1.2:3.1:1.4:9.1:3.5:4.3:1.9:2.4:2.4:4.4:3.8:2.2:5.0:2.0:6.1:4.5:4.3:4.2;
CCW-5 is a glycoprotein consisting of 8 monosaccharides, with a molecular weight of 64.5kDa; the total sugar content was 55.4%; uronic acid content 7.5%; protein content 21.6%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=10.6:2.5:2.4:3.0:11.6:25.9:16.4:27.5; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine= 15.6:15.4:6.7:3.0:11.0:10.7:11.0:6.9:6.3:0.9:3.4:2.7:0.9:3.2:1.4:0.9; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, 1, 6-linked mannose, 1,3, 6-linked mannose, terminal-linked rhamnose, 1, 3-linked rhamnose, terminal-linked glucuronic acid, terminal-linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, 1,3, 4-linked glucose, terminal-linked galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal-linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal-linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose, the molar ratio is 2.3:3.1:4.2:1.9:1.8:0.6:3.3:5.4:1.7:2.9:1.7:7.6:1.4:5.5:11.0:1.0:2.9:4.3:2.6:4.5:2.1:9.0:2.7:6.2:7.0:3.3;
CCW-6 is a glycoprotein consisting of 8 monosaccharides with a molecular weight of 46.0kDa; the total sugar content was 50.6%; uronic acid content 6.3%; protein content 27.0%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=9.2:2.7:3.2:2.2:20.8:14.5:20.0:27.4; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine=9.3:9.1:6.3:4.6:11.7:7.6:9.0:5.7:12.3:8.0:5.0:2.2:6.4:2.5:0.3; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, terminal linked rhamnose, terminal linked glucuronic acid, terminal linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, terminal linked galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose, the molar ratio is 4.1:5.1:3.5:2.3:10.7:2.4:6.2:4.4:2.1:5.1:4.1:1.0:1.3:3.3:5.5:6.6:3.3:7.1:2.8:7.9:6.1:4.9;
CCW-7 is a glycoprotein consisting of 8 monosaccharides with a molecular weight of 69.2kDa; total sugar content was 57.4%; uronic acid content was 7.8%; protein content 0.4%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=11.5:2.9:2.7:3.4:10.9:20.1:19.8:28.7; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine= 14.9:17.0:8.9:3.6:9.2:9.9:11.5:7.2:6.1:0.7:3.0:1.7:0.7:3.2:1.4:0.9; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, 1, 6-linked mannose, 1,3, 6-linked mannose, terminal-linked rhamnose, terminal-linked glucuronic acid, terminal-linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, 1,3, 4-linked glucose, 1,2, 6-linked glucose, terminal-linked galactose, 1, 2-linked galactose, 1, 4-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, 1,2,3, 4-linked xylose, terminal-linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose, the molar ratio is 2.2:3.6:4.0:1.5:3.0:3.4:3.7:2.2:3.5:1.8:1.1:6.6:1.9:4.6:1.1:4.5:2.0:5.1:3.2:4.0:5.7:2.7:5.7:2.6:9.1:5.9:5.4;
CCW-8 is a glycoprotein consisting of 8 monosaccharides, with a molecular weight of 52.5kDa; the total sugar content was 53.6%; uronic acid content 4.3%; protein content 22.9%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=9.7:3.1:3.4:2.3:19.2:12.7:21.3:28.4; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine=9.7:9.5:7.0:4.6:15.4:7.8:9.1:5.0:10.7:6.2:4.5:1.6:6.4:2.1:0.4; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, 1, 6-linked mannose, terminal-linked rhamnose, terminal-linked glucuronic acid, terminal-linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, 1,3, 4-linked glucose, 1,4, 6-linked glucose, terminal-linked galactose, 1, 2-linked galactose, 1,4, 6-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 and 1,2, 3-linked arabinose, the molar ratio is 1.9:3.6:3.9:3.6:2.5:8.5:3.4:5.0:2.1:1.7:3.0:2.3:3.8:4.6:2.5:5.5:4.5:5.3:3.0:10.8:3.4:7.1:4.6:3.5;
CCW-9 is a glycoprotein consisting of 9 monosaccharides, with a molecular weight of 78.8kDa; total sugar content was 43.4%; uronic acid content 3.9%; protein content 31.8%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose=9.8:2.2:2.2:1.8:0.6:9.7:20.8:19.0:33.8; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine=9.4:9.3:7.1:5.4:12.0:7.4:12.3:6.2:11.1:6.0:3.7:1.8:5.7:1.9:0.5; the linkage means comprises 1, 3-linked mannose, 1, 6-linked mannose, 1,3, 6-linked mannose, terminal linked rhamnose, terminal linked glucuronic acid, terminal linked glucose, 1, 4-linked glucose, 1, 3-linked glucose, terminal linked galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose in a molar ratio of 3.3:1.2:1.3:2.0:1.6:6:2.8:2.5:7.7.1, 2:4:4:4.8:4.3:4.8:3.6:4.8:3.3.2:4.3:4.3:3.2:3.3:4.3:4.3:3.2:3.3;
CCB-4 is a glycoprotein consisting of 8 monosaccharides with a molecular weight of 55.2kDa; the total sugar content was 55.6%; uronic acid content 3.3%; protein content 27.7%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=10.1:4.0:2.8:1.7:20.0:17.5:20.6:23.4; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine= 17.9:11.5:8.0:3.1:8.4:9.8:12.1:6.8:7.4:1.1:4.1:2.8:0.8:3.8:2.3:0.3; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, terminal linked rhamnose, terminal linked glucuronic acid, terminal linked glucose, 1, 3-linked glucose, 1,4, 6-linked glucose, terminal linked galactose, 1, 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, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose composition, 1,2, 3-linked arabinose, the molar ratio 4.2:5.8:2.9:2.1:11.4:1.5:4.6:2.3:4.0:3.6:8.3:1.4:1.2:10.1:4.4:4.8:2.1:8.0:2.4:7.1:3.2:4.7;
CCB-5 is a glycoprotein consisting of 8 monosaccharides with a molecular weight of 69.3kDa; total sugar content 50.4%; uronic acid content 3.5%; protein content 24.7%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=7.6:2.8:1.9:1.5:14.1:13.3:34.6:24.1; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine= 16.3:12.7:7.8:3.8:12.2:8.8:11.1:6.3:6.6:1.1:3.9:2.6:0.8:3.4:2.2:0.4; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, terminal linked glucuronic acid, terminal linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, terminal linked galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose composition, 1,2, 3-linked arabinose, the molar ratio 3.8:4.2:1.6:8.0:1.8:3.4:4.3:1.3:6.2:3.9:1.2:5.0:10.8:8.4:7.9:4.1:12.6:1.7:5.0:2.5:2.5;
Wherein leucine denotes a mixture of leucine and its isomer isoleucine.
The second technical scheme of the invention is to provide the preparation method of dayflower glycoprotein 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 dayflower water extraction residues, adding ethanol into supernatant, standing and centrifuging again, adding water into the centrifuged precipitate for redissolution, recovering ethanol under reduced pressure, adding water for redissolution again, adding trichloroacetic acid, centrifuging, regulating pH of the centrifuged supernatant to be neutral, concentrating, dialyzing, and freeze-drying to obtain dayflower water extraction crude polysaccharide, which is named as CCW;
extracting Commelina communis water extraction residues with sodium hydroxide, centrifuging, regulating pH of supernatant after centrifuging to neutrality, concentrating, adding ethanol, standing, centrifuging, adding water for redissolution, recovering ethanol under reduced pressure, redissolving with water, adding trichloroacetic acid, centrifuging, regulating pH of supernatant after centrifuging to neutrality, concentrating, dialyzing, and lyophilizing to obtain Commelina communis alkali extraction polysaccharide, named CCB;
s2, dissolving the CCW obtained in the step S1 in water, performing preliminary separation by using DEAE-cellulose column chromatography, eluting by using sodium chloride solution with gradient concentration, collecting each fraction, concentrating, dialyzing, and freeze-drying to obtain 4 secondary components named as CCW-D-2, CCW-D-3, CCW-D-4 and CCW-D-5 respectively;
S3, adding water into the CCB obtained in the step S1 for dissolution, performing primary separation by using DEAE-cellulose column chromatography, eluting with sodium chloride solutions with different concentrations respectively, collecting each fraction, concentrating, dialyzing, and freeze-drying to obtain 2 secondary components which are named as CCB-D-3 and CCB-D-4 respectively;
s4, the CCW-D-2, the CCW-D-3, the CCW-D-4, the CCW-D-5 and the CCB-D-4 obtained in the S2 step are respectively treated with Sephacryl TM S200, separating by chromatography, eluting with ammonium bicarbonate solution, combining the same fractions according to the results of sugar color reaction, absorbance detection of protein and high performance gel permeation chromatography detection, and detecting anticomplement activity to obtain anticomplement active glycoprotein CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5.
Further, in step S1, dayflower is extracted with 95% ethanol by mass fraction.
Further, in step S1, ethanol of 95% by mass was added before two times of standing so that the ethanol concentration in the supernatant was 80%.
Further, in the step S1, the processes of adding trichloroacetic acid twice are as follows: after trichloroacetic acid was added, the mass fraction of trichloroacetic acid in the solution was 10%.
Further, in the S2 step, a sodium chloride solution of gradient concentration was set to 0.1mol/L, 0.2mol/L, 0.4mol/L and 0.8mol/L.
Further, in the S3 step, sodium chloride solutions of different concentrations were set to 0.2mol/L and 0.4mol/L.
Further, in the step S4, the concentration of the ammonium bicarbonate solution is 0.2mol/L.
The third technical scheme of the invention is to provide the application of the dayflower glycoprotein in one of the technical schemes, wherein the dayflower glycoprotein is used for preparing anticomplement medicines.
Further, the anticomplement drug is a drug that inhibits activation of the classical pathway of complement.
Compared with the prior art, the invention has the following beneficial effects:
the invention separates 8 dayflower glycoproteins CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5 from the dry aerial parts of dayflower, and the in vitro experiments prove that the dayflower glycoproteins have obvious complement inhibition activity and can be further used as active ingredients for preparing anticomplement medicines.
Drawings
FIG. 1, a flow chart for the isolation of dayflower glycoprotein.
FIG. 2, chromatograms of High Performance Gel Permeation Chromatography (HPGPC) of Commelina glycoproteins CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5, wherein the chromatographic column: 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.
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 glycoprotein
The separation flow is as shown in figure 1,
pulverizing 12kg Commelina communis, extracting with 95% ethanol, filtering, extracting the residue with hot water solution for 3 times, concentrating, centrifuging, and oven drying the centrifuged precipitate at 50deg.C to obtain Commelina communis 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 again, adding trichloroacetic acid with equal volume of 20% to obtain trichloroacetic acid with mass fraction of 10%, removing free protein, regulating pH of the centrifuged supernatant to neutrality, concentrating, dialyzing, and lyophilizing to obtain water-extracted crude polysaccharide CCW.
Taking 6kg of dayflower water extraction residues, adding 5% sodium hydroxide solution to soak for 1h at 4 ℃, centrifuging, taking supernatant, adjusting pH to be neutral by hydrochloric acid, concentrating, adding 95% ethanol with the mass fraction of 80% in the supernatant, standing for overnight at 4 ℃, centrifuging, re-dissolving the centrifuged precipitate by water, recovering ethanol under reduced pressure, re-dissolving by water again, adding trichloroacetic acid with the mass fraction of 20% with the same volume of trichloroacetic acid with the mass fraction of 10% in the solution, removing free protein, adjusting pH of the centrifuged supernatant to be neutral, concentrating, dialyzing, and freeze-drying to obtain the dayflower alkali extraction crude polysaccharide CCB.
80g of CCW was dissolved in distilled water and centrifuged, and the supernatant after centrifugation was subjected to preliminary separation by DEAE-cellulose column chromatography. Eluting with 0.1, 0.2, 0.4 and 0.8mol/L sodium chloride solution respectively, wherein the elution volume is more than 2 times of the column volume (about 10L), the flow rate is 25mL/min, collecting each fraction, and detecting absorbance values of 490nm (after the color development by the sulfuric acid-phenol method) and 280nm in a separation tube. According to the detection result, the fractions are combined, concentrated, dialyzed and freeze-dried to obtain 4 secondary components: CCW-D-2, CCW-D-3, CCW-D-4 and CCW-D-5.
30g of CCB was dissolved in distilled water and centrifuged, and the supernatant after centrifugation was subjected to preliminary separation by DEAE-cellulose column chromatography. Eluting with 0.2 and 0.4mol/L sodium chloride solution respectively, wherein the elution volume is more than 2 times of the column volume (about 10L), the flow rate is 25mL/min, collecting each fraction, and detecting absorbance values of 490nm (after the color development by the sulfuric acid-phenol method) and 280nm in a separation tube. According to the detection result, the fractions are combined, concentrated, dialyzed and freeze-dried to obtain 2 secondary components: CCB-D-3 and CCB-D-4.
Dissolving CCW-D-2 (26 g) 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. And detecting absorbance values of 490nm (after the color development by a sulfuric acid-phenol method) and 280nm by a separation tube, and combining the same fractions, concentrating, dialyzing and freeze-drying according to the detection result to obtain glycoprotein CCW-4 (860 mg).
Dissolving CCW-D-3 (6 g) 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) and 280nm were measured in a separate tube, and the same fractions were pooled, concentrated, dialyzed, and freeze-dried according to the measurement results to obtain glycoprotein CCW-5 (150 mg) and CCW-6 (420 mg).
Dissolving CCW-D-4 (4 g) 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 sulfuric acid-phenol method) and 280nm were measured in a separate tube, and the same fractions were pooled, concentrated, dialyzed, and freeze-dried according to the measurement results to obtain glycoprotein CCW-7 (200 mg) and CCW-8 (240 mg).
Dissolving CCW-D-5 (1.5 g) 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) and 280nm were measured in a separate tube, and the same fractions were combined according to the measurement result, concentrated, dialyzed and freeze-dried to obtain glycoprotein CCW-9 (110 mg).
Dissolving CCB-D-3 (1.5 g) 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. And detecting absorbance values of 490nm (after the color development by a sulfuric acid-phenol method) and 280nm by a separation tube, and combining the same fractions, concentrating, dialyzing and freeze-drying according to the detection result to obtain glycoprotein CCB-4 (90 mg).
Dissolving CCB-D-4 (1.0 g) 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. And detecting absorbance values of 490nm (after the color development by a sulfuric acid-phenol method) and 280nm by a separation tube, and combining the same fractions, concentrating, dialyzing and freeze-drying according to the detection result to obtain glycoprotein CCB-5 (80 mg).
CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5 were all molecular weight homogeneous fractions as measured by High Performance Gel Permeation Chromatography (HPGPC), as shown in FIG. 2.
Example 2: structural characterization of Commelina glycoprotein
(1) Determination of molecular weight
The relative molecular weight of glycoprotein sample is determined by HPGPC, and the basic principle is that glycoprotein is subjected to gel permeation chromatography to form symmetrical chromatographic peaks, the peak time is related to the molecular weight, and the calculation is carried out 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 glycoprotein and dextran series standard substances, preparing 3.0mg/mL of solution by using 0.002mol/L ammonium acetate, filtering by using a microporous filter membrane with the concentration 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 glycoprotein. The relative molecular weights of CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5 are 50.7kDa, 64.5kDa, 46.0kDa, 69.2kDa, 52.5kDa, 78.8kDa, 55.2kDa and 69.3kDa, respectively.
(2) Determination of total sugar, uronic acid, protein and sulfate group content
The total sugar content of the glycoprotein sample is measured by a sulfuric acid-phenol method, and the total sugar content of CCW-4 is 58.6 percent; the total sugar content of CCW-5 is 55.4%; the total sugar content of CCW-6 was 50.6%; CCW-7 total sugar content 57.4%; the total sugar content of CCW-8 was 53.6%; CCW-9 total sugar content 43.4%; the total sugar content of CCB-4 is 55.6%; the total sugar content of CCB-5 was 50.4%.
The uronic acid content of CCW-4 is 18.7% by measuring and detecting the uronic acid content by a m-hydroxybiphenyl method; the uronic acid content of CCW-5 was 7.5%; the uronic acid content of CCW-6 was 6.3%; the uronic acid content of CCW-7 was 7.8%; the uronic acid content of CCW-8 was 4.3%; the uronic acid content of CCW-9 was 3.9%; the uronic acid content of CCB-4 was 3.3%; the uronic acid content of CCB-5 was 3.5%.
The protein content was determined by Coomassie Brilliant blue method, and the protein content of CCW-4 was 14.3%; the protein content of CCW-5 was 21.6%; the protein content of CCW-6 was 27.0%; the protein content of CCW-7 was 20.4%; the protein content of CCW-8 was 22.9%; the protein content of CCW-9 was 31.8%; the protein content of CCB-4 was 27.7%; the protein content of CCB-5 was 24.7%.
CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5 were each free of sulfate groups as determined by the barium chloride turbidimetry.
(3) Analysis of monosaccharide composition
The products obtained by total hydrolysis of 2mol/L trifluoroacetic acid at 110 ℃ of CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5 are respectively subjected to derivatization of 1-phenyl-3-methyl-5-pyrazolone, and then are subjected to liquid phase analysis.
CCW-4 is a glycoprotein consisting of 9 monosaccharides in the molar ratio mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose=7.6:1.6:7.8:4.7:8.2:9.9:20.0:13.5:26.7.
CCW-5 is a glycoprotein consisting of 8 monosaccharides in the molar ratio mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=10.6:2.5:2.4:3.0:11.6:25.9:16.4:27.5.
CCW-6 is a glycoprotein consisting of 8 monosaccharides in the molar ratio mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=9.2:2.7:3.2:2.2:20.8:14.5:20.0:27.4.
CCW-7 is a glycoprotein consisting of 8 monosaccharides in the molar ratio mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=11.5:2.9:2.7:3.4:10.9:20.1:19.8:28.7.
CCW-8 is a glycoprotein consisting of 8 monosaccharides in the molar ratio mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=9.7:3.1:3.4:2.3:19.2:12.7:21.3:28.4.
CCW-9 is a glycoprotein consisting of 9 monosaccharides in the molar ratio mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose=9.8:2.2:2.2:1.8:0.6:9.7:20.8:19.0:33.8.
CCB-4 is a glycoprotein consisting of 8 monosaccharides in the molar ratio mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=10.1:4.0:2.8:1.7:20.0:17.5:20.6:23.4.
CCB-5 is a glycoprotein consisting of 8 monosaccharides in the molar ratio mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=7.6:2.8:1.9:1.5:14.1:13.3:34.6:24.1.
(4) Analysis of amino acid composition
The products obtained by total hydrolysis of CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5 with 6mol/L hydrochloric acid at 110deg.C were subjected to liquid chromatography.
CCW-4 is a glycoprotein consisting of 17 amino acids with the amino acid molar ratio aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine (containing isoleucine as an isomer with leucine): phenylalanine: histidine: lysine: tyrosine: cysteine= 9.8:7.8:7.2:4.0:14.6:7.1:9.3:6.2:10.5:0.1:5.6:8.7:1.3:6.2:1.4:0.4.
CCW-5 is a glycoprotein consisting of 17 amino acids with the amino acid molar ratio aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine (containing isoleucine as an isomer with leucine): phenylalanine: histidine: lysine: tyrosine: cysteine= 15.6:15.4:6.7:3.0:11.0:10.7:11.0:6.9:6.3:0.9:3.4:2.7:0.9:3.2:1.4:0.9.
CCW-6 is a glycoprotein consisting of 16 amino acids with the amino acid molar ratio aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: leucine (containing isoleucine as an isomer with leucine): phenylalanine: histidine: lysine: tyrosine: cysteine=9.3:9.1:6.3:4.6:11.7:7.6:9.0:5.7:12.3:8.0:5.0:2.2:6.4:2.5:0.3.
CCW-7 is a glycoprotein consisting of 17 amino acids with the amino acid molar ratio aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine (containing isoleucine as an isomer with leucine): phenylalanine: histidine: lysine: tyrosine: cysteine= 14.9:17.0:8.9:3.6:9.2:9.9:11.5:7.2:6.1:0.7:3.0:1.7:0.7:3.2:1.4:0.9.
CCW-8 is a glycoprotein consisting of 16 amino acids with the amino acid molar ratio aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: leucine (containing isoleucine as an isomer with leucine): phenylalanine: histidine: lysine: tyrosine: cysteine=9.7:9.5:7.0:4.6:15.4:7.8:9.1:5.0:10.7:6.2:4.5:1.6:6.4:2.1:0.4.
CCW-9 is a glycoprotein consisting of 16 amino acids with the amino acid molar ratio aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: leucine (containing isoleucine as an isomer with leucine): phenylalanine: histidine: lysine: tyrosine: cysteine=9.4:9.3:7.1:5.4:12.0:7.4:12.3:6.2:11.1:6.0:3.7:1.8:5.7:1.9:0.5.
CCB-4 is a glycoprotein consisting of 17 amino acids with the amino acid molar ratio aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine (containing isoleucine as an isomer with leucine): phenylalanine: histidine: lysine: tyrosine: cysteine= 17.9:11.5:8.0:3.1:8.4:9.8:12.1:6.8:7.4:1.1:4.1:2.8:0.8:3.8:2.3:0.3.
CCB-5 is a glycoprotein consisting of 17 amino acids with the amino acid molar ratio aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine (containing isoleucine as an isomer with leucine): phenylalanine: histidine: lysine: tyrosine: cysteine= 16.3:12.7:7.8:3.8:12.2:8.8:11.1:6.3:6.6:1.1:3.9:2.6:0.8:3.4:2.2:0.4.
(5) Methylation analysis
CMC-NaBH is used first 4 The uronic acid is reduced and then the glycoprotein is respectively methylated by adopting a modified Hakomori method, the methylated products are fully hydrolyzed by 2mol/L trifluoroacetic acid, sodium borodeuteride is reduced and acetic anhydride is acetylated to prepare partially methylated Aldi alcohol acetate derivatives, and then GC-MS analysis is carried out.
The CCW-4 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, 4-linked glucuronic acid, 1, 2-linked galacturonic acid, terminal linked glucose, 1, 3-linked glucose, 1,2, 4-linked glucose, terminal linked galactose, 1, 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, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, 1, 4-linked arabinopyranose and 1,2, 3-linked arabinose, the molar ratio is 1.5:5.8:5.4:1.1:1.2:2.1:2.6:9.1:5.5:1.2:3.1:1.4:9.1:3.5:4.3:1.9:2.4:2.4:4.4:3.8:2.2:5.0:2.0:6.1:4.5:4.3:4.2.
The CCW-5 structure contains: 1, 2-linked mannose, 1, 3-linked mannose, 1, 6-linked mannose, 1,3, 6-linked mannose, terminal rhamnose, 1, 3-linked rhamnose, terminal glucuronic acid, terminal glucose, 1, 3-linked glucose, 1, 4-linked glucose, 1,3, 4-linked glucose, terminal galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose, the molar ratio is 2.3:3.1:4.2:1.9:1.8:0.6:3.3:5.4:1.7:2.9:1.7:7.6:1.4:5.5:11.0:1.0:2.9:4.3:2.6:4.5:2.1:9.0:2.7:6.2:7.0:3.3.
The CCW-6 structure contains: 1, 2-linked mannose, 1, 3-linked mannose, terminal linked rhamnose, terminal linked glucuronic acid, terminal linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, terminal linked galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose, the molar ratio is 4.1:5.1:3.5:2.3:10.7:2.4:6.2:4.4:2.1:5.1:4.1:1.0:1.3:3.3:5.5:6.6:3.3:7.1:2.8:7.9:6.1:4.9.
The CCW-7 structure contains: 1, 2-linked mannose, 1, 3-linked mannose, 1, 6-linked mannose, 1,3, 6-linked mannose, terminal-linked rhamnose, terminal-linked glucuronic acid, terminal-linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, 1,3, 4-linked glucose, 1,2, 6-linked glucose, terminal-linked galactose, 1, 2-linked galactose, 1, 4-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, 1,2,3, 4-linked xylose, terminal-linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose, the molar ratio is 2.2:3.6:4.0:1.5:3.0:3.4:3.7:2.2:3.5:1.8:1.1:6.6:1.9:4.6:1.1:4.5:2.0:5.1:3.2:4.0:5.7:2.7:5.7:2.6:9.1:5.9:5.4.
The CCW-8 structure contains: 1, 2-linked mannose, 1, 3-linked mannose, 1, 6-linked mannose, terminal-linked rhamnose, terminal-linked glucuronic acid, terminal-linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, 1,3, 4-linked glucose, 1,4, 6-linked glucose, terminal-linked galactose, 1, 2-linked galactose, 1,4, 6-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 and 1,2, 3-linked arabinose, the molar ratio is 1.9:3.6:3.9:3.6:2.5:8.5:3.4:5.0:2.1:1.7:3.0:2.3:3.8:4.6:2.5:5.5:4.5:5.3:3.0:10.8:3.4:7.1:4.6:3.5.
The CCW-9 structure contains: 1, 3-linked mannose, 1, 6-linked mannose, 1,3, 6-linked mannose, terminal rhamnose, terminal linked glucuronic acid, terminal linked glucose, 1, 4-linked glucose, 1, 3-linked glucose, terminal linked galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose, the molar ratio is 3.3:1.2:1.3:2.0:1.6:6.8:2.6:2.5:7.0:1.9:3.5:8.8:1.2:2.2:4.3:4.4:6.2:3.2:13.7:3.3:8.6:5.8:4.6.
The CCB-4 structure comprises: 1, 2-linked mannose, 1, 3-linked mannose, terminal linked rhamnose, terminal linked glucuronic acid, terminal linked glucose, 1, 3-linked glucose, 1,4, 6-linked glucose, terminal linked galactose, 1, 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, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, 1,2, 3-linked arabinose, the molar ratio 4.2:5.8:2.9:2.1:11.4:1.5:4.6:2.3:4.0:3.6:8.3:1.4:1.2:10.1:4.4:4.8:2.1:8.0:2.4:7.1:3.2:4.7.
The CCB-5 structure comprises: 1, 2-linked mannose, 1, 3-linked mannose, terminal linked glucuronic acid, terminal linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, terminal linked galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, 1,2, 3-linked arabinose, the molar ratio 3.8:4.2:1.6:8.0:1.8:3.4:4.3:1.3:6.2:3.9:1.2:5.0:10.8:8.4:7.9:4.1:12.6:1.7:5.0:2.5:2.5.
Example 3: classical pathway anticomplement activity
Serum from 3 months old guinea pigs was taken and sensitized with 2% sheep erythrocytes and diluted 1:80 with Barbital Buffer (BBS) as the complement source for the classical pathway. The rabbit anti-sheep red blood cell antibody is diluted to a solution of 1:1000 in BBS as hemolysin; sheep Red Blood Cells (SRBC) stored in alserver fluid were configured as 2% SRBC. Glycoprotein samples were precisely weighed 3mg, dissolved in BBS buffer, and diluted to 8 concentrations. 200. Mu.L of glycoprotein solution of different concentrations was mixed with 200. Mu.L of complement diluted 1:80, followed by sequential addition of 100. Mu.L of hemolysin (1:1000) and 100. Mu.L of 2% SRBC, placed in a low temperature high speed centrifuge after 30min in a 37℃water bath, and centrifuged at 5000rpm for 10min. 200. Mu.L of the supernatant was taken per tube in a 96-well plate, and absorbance was measured at 405 nm. The experiments were performed simultaneously with glycoprotein control (200. Mu.L of glycoprotein plus 400. Mu.L BBS buffer), complement control (200. Mu.L BBS buffer instead of glycoprotein) and total hemolysis (100. Mu.L 2% SRBC in 500. Mu.L triple distilled water). The haemolysis inhibition rate was calculated by subtracting the absorbance value of the corresponding glycoprotein control group from the absorbance value of the glycoprotein group at each concentration. The 50% inhibition of the lysis was calculated from the fitted curve obtained by plotting the logarithm of the glycoprotein concentration as the X-axis and the inhibition of the haemolysis as the Y-axis Concentration of blood-required sample (CH) 50 Values). The results showed that 8 dayflower glycoproteins had significant inhibitory activity against complement classical pathway activation using heparin as a positive control (see table 1).
Table 1 inhibition of complement activation by eight dayflower glycoproteins
Figure SMS_1
Figure SMS_2
Wherein CH is 50 The values are expressed as: mean ± SD (n=3).
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. Dayflower glycoprotein, characterized in that the dayflower glycoprotein is dayflower glycoprotein CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5, and has the following structural characteristics:
CCW-4 is a glycoprotein consisting of 9 monosaccharides, with a molecular weight of 50.7kDa; total sugar content was 58.6%; uronic acid content 18.7%; protein content 14.3%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose=7.6:1.6:7.8:4.7:8.2:9.9:20.0:13.5:26.7; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine= 9.8:7.8:7.2:4.0:14.6:7.1:9.3:6.2:10.5:0.1:5.6:8.7:1.3:6.2:1.4:0.4; the linkage means includes 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, 4-linked glucuronic acid, 1, 2-linked galacturonic acid, terminal linked glucose, 1, 3-linked glucose, 1,2, 4-linked glucose, terminal linked galactose, 1, 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, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose, 1, 4-linked arabinopyranose and 1,2, 3-linked arabinose, the molar ratio is 1.5:5.8:5.4:1.1:1.2:2.1:2.6:9.1:5.5:1.2:3.1:1.4:9.1:3.5:4.3:1.9:2.4:2.4:4.4:3.8:2.2:5.0:2.0:6.1:4.5:4.3:4.2;
CCW-5 is a glycoprotein consisting of 8 monosaccharides, with a molecular weight of 64.5kDa; the total sugar content was 55.4%; uronic acid content 7.5%; protein content 21.6%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=10.6:2.5:2.4:3.0:11.6:25.9:16.4:27.5; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine= 15.6:15.4:6.7:3.0:11.0:10.7:11.0:6.9:6.3:0.9:3.4:2.7:0.9:3.2:1.4:0.9; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, 1, 6-linked mannose, 1,3, 6-linked mannose, terminal-linked rhamnose, 1, 3-linked rhamnose, terminal-linked glucuronic acid, terminal-linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, 1,3, 4-linked glucose, terminal-linked galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal-linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal-linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose, the molar ratio is 2.3:3.1:4.2:1.9:1.8:0.6:3.3:5.4:1.7:2.9:1.7:7.6:1.4:5.5:11.0:1.0:2.9:4.3:2.6:4.5:2.1:9.0:2.7:6.2:7.0:3.3;
CCW-6 is a glycoprotein consisting of 8 monosaccharides with a molecular weight of 46.0kDa; the total sugar content was 50.6%; uronic acid content 6.3%; protein content 27.0%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=9.2:2.7:3.2:2.2:20.8:14.5:20.0:27.4; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine=9.3:9.1:6.3:4.6:11.7:7.6:9.0:5.7:12.3:8.0:5.0:2.2:6.4:2.5:0.3; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, terminal linked rhamnose, terminal linked glucuronic acid, terminal linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, terminal linked galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose, the molar ratio is 4.1:5.1:3.5:2.3:10.7:2.4:6.2:4.4:2.1:5.1:4.1:1.0:1.3:3.3:5.5:6.6:3.3:7.1:2.8:7.9:6.1:4.9;
CCW-7 is a glycoprotein consisting of 8 monosaccharides with a molecular weight of 69.2kDa; total sugar content was 57.4%; uronic acid content was 7.8%; protein content 0.4%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=11.5:2.9:2.7:3.4:10.9:20.1:19.8:28.7; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine= 14.9:17.0:8.9:3.6:9.2:9.9:11.5:7.2:6.1:0.7:3.0:1.7:0.7:3.2:1.4:0.9; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, 1, 6-linked mannose, 1,3, 6-linked mannose, terminal-linked rhamnose, terminal-linked glucuronic acid, terminal-linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, 1,3, 4-linked glucose, 1,2, 6-linked glucose, terminal-linked galactose, 1, 2-linked galactose, 1, 4-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, 1,2,3, 4-linked xylose, terminal-linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose, the molar ratio is 2.2:3.6:4.0:1.5:3.0:3.4:3.7:2.2:3.5:1.8:1.1:6.6:1.9:4.6:1.1:4.5:2.0:5.1:3.2:4.0:5.7:2.7:5.7:2.6:9.1:5.9:5.4;
CCW-8 is a glycoprotein consisting of 8 monosaccharides, with a molecular weight of 52.5kDa; the total sugar content was 53.6%; uronic acid content 4.3%; protein content 22.9%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=9.7:3.1:3.4:2.3:19.2:12.7:21.3:28.4; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine=9.7:9.5:7.0:4.6:15.4:7.8:9.1:5.0:10.7:6.2:4.5:1.6:6.4:2.1:0.4; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, 1, 6-linked mannose, terminal-linked rhamnose, terminal-linked glucuronic acid, terminal-linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, 1,3, 4-linked glucose, 1,4, 6-linked glucose, terminal-linked galactose, 1, 2-linked galactose, 1,4, 6-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 and 1,2, 3-linked arabinose, the molar ratio is 1.9:3.6:3.9:3.6:2.5:8.5:3.4:5.0:2.1:1.7:3.0:2.3:3.8:4.6:2.5:5.5:4.5:5.3:3.0:10.8:3.4:7.1:4.6:3.5;
CCW-9 is a glycoprotein consisting of 9 monosaccharides, with a molecular weight of 78.8kDa; total sugar content was 43.4%; uronic acid content 3.9%; protein content 31.8%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: xylose: arabinose=9.8:2.2:2.2:1.8:0.6:9.7:20.8:19.0:33.8; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine=9.4:9.3:7.1:5.4:12.0:7.4:12.3:6.2:11.1:6.0:3.7:1.8:5.7:1.9:0.5; the linkage means comprises 1, 3-linked mannose, 1, 6-linked mannose, 1,3, 6-linked mannose, terminal linked rhamnose, terminal linked glucuronic acid, terminal linked glucose, 1, 4-linked glucose, 1, 3-linked glucose, terminal linked galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose and 1,2, 3-linked arabinose in a molar ratio of 3.3:1.2:1.3:2.0:1.6:6:2.8:2.5:7.7.1, 2:4:4:4.8:4.3:4.8:3.6:4.8:3.3.2:4.3:4.3:3.2:3.3:4.3:4.3:3.2:3.3;
CCB-4 is a glycoprotein consisting of 8 monosaccharides with a molecular weight of 55.2kDa; the total sugar content was 55.6%; uronic acid content 3.3%; protein content 27.7%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=10.1:4.0:2.8:1.7:20.0:17.5:20.6:23.4; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine= 17.9:11.5:8.0:3.1:8.4:9.8:12.1:6.8:7.4:1.1:4.1:2.8:0.8:3.8:2.3:0.3; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, terminal linked rhamnose, terminal linked glucuronic acid, terminal linked glucose, 1, 3-linked glucose, 1,4, 6-linked glucose, terminal linked galactose, 1, 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, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose composition, 1,2, 3-linked arabinose, the molar ratio 4.2:5.8:2.9:2.1:11.4:1.5:4.6:2.3:4.0:3.6:8.3:1.4:1.2:10.1:4.4:4.8:2.1:8.0:2.4:7.1:3.2:4.7;
CCB-5 is a glycoprotein consisting of 8 monosaccharides with a molecular weight of 69.3kDa; total sugar content 50.4%; uronic acid content 3.5%; protein content 24.7%; no sulfate group; the molar ratio of the monosaccharides is mannose: glucosamine: rhamnose: glucuronic acid: glucose: galactose: xylose: arabinose=7.6:2.8:1.9:1.5:14.1:13.3:34.6:24.1; the amino acid molar ratio is aspartic acid: glutamic acid: serine: arginine: glycine: proline: alanine: threonine: valine: methionine: leucine: phenylalanine: histidine: lysine: tyrosine: cysteine= 16.3:12.7:7.8:3.8:12.2:8.8:11.1:6.3:6.6:1.1:3.9:2.6:0.8:3.4:2.2:0.4; the linkage means includes 1, 2-linked mannose, 1, 3-linked mannose, terminal linked glucuronic acid, terminal linked glucose, 1, 3-linked glucose, 1, 4-linked glucose, terminal linked galactose, 1, 2-linked galactose, 1,4, 6-linked galactose, 1,3, 6-linked galactose, terminal linked xylose, 1, 4-linked xylose, 1,3, 4-linked xylose, 1,2,3, 4-linked xylose, terminal linked arabinose, 1, 2-linked arabinose, 1, 3-linked arabinose, 1, 5-linked arabinose composition, 1,2, 3-linked arabinose, the molar ratio 3.8:4.2:1.6:8.0:1.8:3.4:4.3:1.3:6.2:3.9:1.2:5.0:10.8:8.4:7.9:4.1:12.6:1.7:5.0:2.5:2.5;
Wherein leucine denotes a mixture of leucine and its isomer isoleucine.
2. A method for preparing dayflower glycoprotein 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 dayflower water extraction residues, adding ethanol into supernatant, standing and centrifuging again, adding water into the centrifuged precipitate for redissolution, recovering ethanol under reduced pressure, adding water for redissolution again, adding trichloroacetic acid, centrifuging, regulating pH of the centrifuged supernatant to be neutral, concentrating, dialyzing, and freeze-drying to obtain dayflower water extraction crude polysaccharide, which is named as CCW;
extracting Commelina communis water extraction residues with sodium hydroxide, centrifuging, regulating pH of supernatant after centrifuging to neutrality, concentrating, adding ethanol, standing, centrifuging, adding water for redissolution, recovering ethanol under reduced pressure, redissolving with water, adding trichloroacetic acid, centrifuging, regulating pH of supernatant after centrifuging to neutrality, concentrating, dialyzing, and lyophilizing to obtain Commelina communis alkali extraction polysaccharide, named CCB;
s2, dissolving the CCW obtained in the step S1 in water, performing preliminary separation by using DEAE-cellulose column chromatography, eluting by using sodium chloride solution with gradient concentration, collecting each fraction, concentrating, dialyzing, and freeze-drying to obtain 4 secondary components named as CCW-D-2, CCW-D-3, CCW-D-4 and CCW-D-5 respectively;
S3, adding water into the CCB obtained in the step S1 for dissolution, performing primary separation by using DEAE-cellulose column chromatography, eluting with sodium chloride solutions with different concentrations respectively, collecting each fraction, concentrating, dialyzing, and freeze-drying to obtain 2 secondary components which are named as CCB-D-3 and CCB-D-4 respectively;
s4, the CCW-D-2, the CCW-D-3, the CCW-D-4, the CCW-D-5 and the CCB-D-4 obtained in the S2 step are respectively treated with Sephacryl TM S200 chromatographic separation, eluting with ammonium bicarbonate solution, detecting protein absorbance according to sugar color reaction, and detecting by high performance gel permeation chromatographyAs a result, the same fractions were pooled and assayed for anticomplement activity to give anticomplement glycoproteins CCW-4, CCW-5, CCW-6, CCW-7, CCW-8, CCW-9, CCB-4 and CCB-5.
3. The method for producing dayflower glycoprotein according to claim 2, wherein in step S1, dayflower is extracted with 95% ethanol by mass.
4. The method for producing dayflower glycoprotein according to claim 2, wherein in step S1, 95% ethanol is added by mass before two standing so that the ethanol concentration in the supernatant is 80%.
5. The method for preparing dayflower glycoprotein 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%.
6. The method for producing dayflower glycoprotein according to claim 2, wherein in the step S2, a gradient concentration of sodium chloride solution is set to 0.1mol/L, 0.2mol/L, 0.4mol/L and 0.8mol/L.
7. The method for preparing dayflower glycoprotein according to claim 2, wherein in the step S3, sodium chloride solutions of different concentrations are set to 0.2mol/L and 0.4mol/L.
8. The method for preparing dayflower glycoprotein according to claim 2, wherein the concentration of the ammonium bicarbonate solution in the step S4 is 0.2mol/L.
9. Use of a dayflower glycoprotein according to claim 1 for the preparation of an anticomplement medicament.
10. The use of dayflower glycoprotein according to claim 9, wherein the anticomplement drug is a drug that inhibits activation of the classical pathway of complement.
CN202310163863.XA 2023-02-24 2023-02-24 Commelina glycoprotein and preparation method and application thereof Pending CN116284295A (en)

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