CN111484565B - Sweet wormwood polysaccharide, preparation method thereof and application thereof in preparation of anticomplement medicines - Google Patents

Sweet wormwood polysaccharide, preparation method thereof and application thereof in preparation of anticomplement medicines Download PDF

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CN111484565B
CN111484565B CN201910077041.3A CN201910077041A CN111484565B CN 111484565 B CN111484565 B CN 111484565B CN 201910077041 A CN201910077041 A CN 201910077041A CN 111484565 B CN111484565 B CN 111484565B
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陈道峰
霍江燕
卢燕
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Abstract

The invention belongs to the field of traditional Chinese medicines, and relates to sweet wormwood polysaccharide, in particular to seven natural homogeneous polysaccharides in sweet wormwood, a preparation method thereof and application thereof in preparation of anticomplement medicines. The invention separates seven homogeneous polysaccharides AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9 from Chinese medicine Artemisia apiacea which clears away heat and toxic material, experiments prove that the prepared homogeneous polysaccharide of Artemisia apiacea has obvious inhibiting effect on complement activation, and can be further used as an active ingredient to prepare novel anticomplement medicines.

Description

Sweet wormwood polysaccharide, preparation method thereof and application thereof in preparation of anticomplement medicines
Technical Field
The invention belongs to the field of traditional Chinese medicines, and relates to sweet wormwood polysaccharide, in particular to seven natural homogeneous polysaccharides in sweet wormwood, a preparation method thereof and application thereof in preparation of anticomplement medicines.
Background
The prior art discloses that the complement system is an important component of the human immune system, and its normal activation plays an important role in eliminating foreign microorganisms, removing 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 causes an excessive reaction of the human immune system, resulting in damage to normal tissues of the human body itself, such as rheumatoid and rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), Acute Respiratory Distress Syndrome (ARDS), acute pneumonia caused by influenza virus, and severe atypical pneumonia (SARS). It follows that inhibition of abnormal activation of the complement system is one of the important pathways for the treatment of the above-mentioned diseases.
At present, although the traditional immunosuppressant drugs such as glucocorticoid, cyclophosphamide, methotrexate and the like which are allowed to be used in clinical practice have a certain relieving effect on diseases related to excessive complement activation, the drugs are not specific complement inhibitors, and practice shows that the long-term application of the drugs can reduce the defense function of the body, so that the anti-infection ability is reduced, secondary infection is easy to occur, potential focus is diffused, and various complications and side effects are generated, so that the search for a novel complement inhibitor with high efficiency and low toxicity is still an urgent priority for clinical treatment of the diseases.
According to the data, the medicinal plants widely contain components with anti-complement activity, and most of the natural active components have low toxicity to the body and can be directly digested and absorbed by the body. Chinese medicine resources are rich, a plurality of Chinese medicinal materials have obvious regulating effect on an immune system, and valuable resources of anticomplementary prodrug are searched; research reports that Chinese medicine and traditional Chinese medicine have obvious effect on preventing and treating SARS during the period of resisting SARS; therefore, the search for novel and efficient anticomplement substances from medicinal plants is of great significance.
Artemisia annua L belonging to Compositae is Artemisia annua L belonging to Artemisia annua LBelongs to the dried overground part of an annual herb artemisia annua, is bitter in taste and cold in nature, and has the effects of clearing away heat and toxic materials, resisting malaria and the like. Modern researches find that the traditional Chinese medicine, namely the sweet wormwood herb, has the effects of resisting malaria, resisting tumors, inhibiting bacteria and killing insects, resisting inflammation, regulating immunity, resisting fibrosis, inhibiting steatosis and the like. The sweet wormwood is used as a traditional Chinese medicine, is a traditional Chinese medicine resource with advantages in China, is used for treating various diseases including malaria in China, and has been clinically applied for more than 2000 years. Artemisinin is an antimalarial drug extracted from artemisia annua, professor yoyo professor yobo-yo awarded the nobel medical or physiological prize in 2015, so that artemisinin and artemisia annua regained attention. The research finds that the anti-complement activity of the sweet wormwood crude polysaccharide is obvious, and CH50And AP5056 mu g/ml and 90 mu g/ml respectively, but the report about the separation, preparation and application of the homogeneous polysaccharide with the anti-complement activity in the sweet wormwood herb is not found.
Disclosure of Invention
The invention aims to provide active ingredients with anticomplementary effect in natural medicines, and particularly relates to artemisia apiacea polysaccharide, a preparation method thereof and application thereof in preparing anticomplementary medicines, in particular to seven artemisia apiacea homogeneous polysaccharides (AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9) in artemisia apiacea polysaccharide, a preparation method thereof and application thereof in preparing a complement inhibition medicine.
The invention separates the water extract of the traditional Chinese medicine Artemisia annua for clearing away heat and toxic materials to obtain seven homogeneous polysaccharides, and in vitro experiments prove that the prepared seven polysaccharides have anticomplementary activity and can be further developed and prepared as a complement inhibitor.
In the invention, the traditional Chinese medicine sweet wormwood is dried aerial parts of Artemisia annua L. belonging to the genus Artemisia of the family Compositae.
The sweet wormwood polysaccharide AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9 are prepared and obtained by the following method:
extracting herba Artemisiae Annuae with 95% ethanol, filtering, oven drying the residue, extracting with hot water, filtering the extractive solution, concentrating, adding 4 times of 95% ethanol, standing, centrifuging to remove supernatant, dissolving the precipitate with water, removing protein with trichloroacetic acid, centrifuging, adjusting the supernatant to neutral, concentrating, dialyzing, and freeze drying to obtain crude polysaccharide. Dissolving the crude polysaccharide with distilled water, and performing primary separation by using DEAE-cellulose column chromatography; eluting with distilled water, 0.1, 0.2, 0.4 and 0.8mol/L NaCl solution, collecting fractions, concentrating, dialyzing (molecular weight cut-off of 14000Da) and lyophilizing to obtain 5 secondary components: Water-AAP, 0.1M-AAP, 0.2M-AAP, 0.4M-AAP, and 0.8M-AAP;
dissolving each secondary component in appropriate amount of mobile phase, centrifuging, separating the supernatant with gel chromatography Sephacryl S-200 and S-300, and collecting each fraction; detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separating tube, combining flow parts according to HPGPC result, concentrating and freeze-drying to obtain homogeneous polysaccharide; namely the sweet wormwood polysaccharide AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9.
The sweet wormwood polysaccharide AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9 has the following structural characteristics:
(1) the sweet wormwood polysaccharide AAP-3 has the structural characteristics that: polysaccharides consisting of six monosaccharides, with a molecular weight of about 109 kDa; total sugar content about 91%; the protein content is lower than 1%; the uronic acid content is about 28%. The monosaccharide molar ratio is mannose: rhamnose: galacturonic acid: glucose: galactose: arabinose is 1.1:1.8:2.5:0.9:1.4: 1.1. The connection mode mainly comprises 1, 4-linked galacturonic acid, terminal glucose, 1, 4-linked rhamnose and 1,3, 6-linked galactose;
(2) the sweet wormwood polysaccharide AAP-4 has the structural characteristics that: a polysaccharide consisting of six monosaccharides, having a molecular weight of about 43 kDa; total sugar content about 92%; the protein content is lower than 1%; the uronic acid content is about 15%. The monosaccharide molar ratio is mannose: rhamnose: galacturonic acid: glucose: galactose: arabinose is 1.5:1.4:1.3:1.0:2.3: 1.3. The connection mode mainly comprises 1, 4-connection mannose, terminal glucose, terminal galactose and 1, 4-connection galactose;
(3) the sweet wormwood polysaccharide AAP-5 has the structural characteristics that: a polysaccharide consisting of five monosaccharides and having a molecular weight of approximately 286 kDa; total sugar content about 91%; the protein content is lower than 1%; the uronic acid content is about 13%. The monosaccharide molar ratio is mannose: rhamnose: glucuronic acid: glucose: galactose ═ 1.2:1.3:1.2:2.5: 3.4. The connection mode mainly comprises 1, 4-connection glucose, 1, 3-connection glucose, terminal mannose and 1, 3-connection rhamnose;
(4) the sweet wormwood polysaccharide AAP-6 has the structural characteristics that: a polysaccharide consisting of four monosaccharides, with a molecular weight of about 218 kDa; total sugar content about 92%; the protein content is less than 1%. The monosaccharide molar ratio is mannose: rhamnose: glucose: galactose ═ 1.2:1.0:1.9: 1.5. The connection mode mainly comprises 1, 4-connection glucose, 1, 3-connection rhamnose, 1, 3-connection galactose and 1,3, 4-connection galactose;
(5) the sweet wormwood polysaccharide AAP-7 has the structural characteristics that: a polysaccharide of seven monosaccharides, having a molecular weight of about 245 kDa; total sugar content about 93%; the protein content is lower than 1%; the uronic acid content is about 4%. The monosaccharide molar ratio is mannose: rhamnose: 2-amino-2-deoxyglucose: glucuronic acid: glucose: 2-amino-2-deoxygalactose: galactose ═ 1.2:1.2:2.4:0.7:1.6:2.4: 1.4. The connection mode mainly comprises 1, 4-connection amino-2-deoxyglucose, 1, 4-connection-2-amino-2-deoxygalactose, terminal rhamnose, 1, 6-connection galactose and 1,3, 6-connection glucose;
(6) the sweet wormwood polysaccharide AAP-8 has the structural characteristics that: polysaccharides composed of five monosaccharides and having a molecular weight of about 300 kDa; total sugar content about 92%; the protein content is less than 1%. The monosaccharide molar ratio is mannose: 2-amino-2-deoxyglucose: glucose: galactose: arabinose was 6.1:6.2:10.4:5.8: 5.6. The connection mode mainly comprises terminal glucose, 1, 4-connection-2-amino-2-deoxyglucose, 1, 3-connection rhamnose and 1, 4-connection galactose;
(7) the sweet wormwood polysaccharide AAP-9 has the structural characteristics that: polysaccharides consisting of five monosaccharides, with a molecular weight of about 174 kDa; total sugar content about 94%; the protein content is less than 1%. The monosaccharide molar ratio is mannose: rhamnose: glucose: galactose: arabinose 2.9:2.3:3.5:3.0: 1.2. The connection mode mainly comprises 1, 4-connection glucose, 1, 4-connection mannose, 1, 2-connection arabinose and 1, 3-connection rhamnose.
The invention carries out in vitro tests, and the results prove that sweet wormwood herb polysaccharides AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9 have obvious inhibition on cell hemolysis caused by activation of the classical pathway and the alternative pathway of complement, namely have obvious anticomplementary action;
the CH of the AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-950The values (concentration of test sample required for 50% inhibition of hemolysis by the classical pathway) were about 360. mu.g/mL, 1120. mu.g/mL, 204. mu.g/mL, 42. mu.g/mL, 163. mu.g/mL, 42. mu.g/mL and 42. mu.g/mL, respectively; AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8, and AAP-9 APs50The values (concentration of test article required for 50% inhibition of hemolysis by the alternative pathway) were approximately 547. mu.g/mL, 1283. mu.g/mL, 641. mu.g/mL, 95. mu.g/mL, 240. mu.g/mL, 56. mu.g/mL, and 76. mu.g/mL, respectively.
The invention provides active ingredients with anticomplementary effect in natural medicines, in particular seven artemisia apiacea homogeneous polysaccharides (AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9) in artemisia apiacea polysaccharides and a preparation method thereof.
Drawings
FIG. 1 shows the separation process of homogeneous sweet wormwood polysaccharide.
FIG. 2 shows HPGPC chromatograms of prepared AAP-3(A), AAP-4(B), AAP-5(C), AAP-6(D), AAP-7(E), AAP-8(F) and AAP-9(G), wherein TSK-GEL GMPWXLGel column (300X 7.6 mm); eluent: distilled water; flow rate: 0.8 ml/min.
Detailed Description
EXAMPLE 1 preparation of Artemisia annua polysaccharide
Pulverizing herba Artemisiae Annuae 10kg, extracting with 95% ethanol, filtering, extracting the residue with water solution for 3 times, concentrating, centrifuging, adding 4 times volume of 95% ethanol into the supernatant, standing, centrifuging to remove the supernatant, dissolving the precipitate with water, recovering under reduced pressure, and removing ethanol; removing free protein from the compound solution with 10% trichloroacetic acid, centrifuging, adjusting supernatant to neutral, dialyzing, concentrating, freeze drying to obtain crude polysaccharide, dissolving 100g of crude polysaccharide in distilled water, centrifuging, and separating supernatant with DEAE-cellulose column chromatography; eluting with distilled water, 0.1, 0.2, 0.4 and 0.8mol/L NaCl solution, eluting with volume more than 2 times column volume (about 10L), flow rate 25mL/min, collecting each fraction, and detecting absorbance value at 490nm (after sulfuric acid-phenol method color development) with separate tubes; according to the chromogenic reaction of sugar and the result of HPGPC detection, the fractions are combined, concentrated, dialyzed and freeze-dried to obtain 5 secondary components: Water-AAP, 0.1M-AAP, 0.2M-AAP, 0.4M-AAP, and 0.8M-AAP;
dissolving 0.1M-AAP (5.60g) in distilled water, centrifuging, and collecting supernatant with SephacrylTMSeparating with S-300 gel chromatographic column, eluting with distilled water solution at flow rate of 0.5mL/min, collecting each fraction, detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with separate tube, mixing the same fractions according to detection result, concentrating, dialyzing, and freeze drying to obtain homogeneous polysaccharides AAP-3(60mg) and AAP-4(90 mg);
dissolving 0.2M-AAP (4.04g) in distilled water, centrifuging, and fractionating the supernatant with SephacrylTMPerforming S-200 chromatographic separation, eluting with distilled water at flow rate of 0.5mL/min, collecting each fraction, detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separating tube, combining the same fractions according to the detection result, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharides AAP-5(300mg) and AAP-6(60 mg);
dissolving 0.4M-AAP (3.68g) in distilled water, centrifuging, and fractionating the supernatant with SephacrylTMS-300 chromatographic separation, eluting with distilled water at a flow rate of 0.5mL/min, collecting each fraction, detecting the absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separating tube, combining the same fractions according to the detection result, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharides AAP-7(500mg) and AAP-8(120 mg);
dissolving 0.8M-AAP (4.10g) in distilled water, centrifuging, and fractionating the supernatant with SephacrylTMSeparating by S-300 chromatography, eluting with distilled water at flow rate of 0.5mL/min, and collecting each fraction. Detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separating tube, mixing the same fractions according to the detection result, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharide AAP-9(300 mg);
the results of High Performance Gel Permeation Chromatography (HPGPC) tests confirmed that the prepared AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9 were all homogeneous components.
Example 2
Structural characterization of Artemisia apiacea polysaccharides (AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9)
(1) Determination of molecular weight
The method comprises the following steps of (1) measuring the relative molecular force of a polysaccharide sample by adopting a High Performance Gel Permeation Chromatography (HPGPC), wherein the basic principle is that homogeneous polysaccharide forms a symmetrical chromatographic peak through gel permeation chromatography, the peak-appearing 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 are as follows: by TSKGMPWXLSeparating with gel column at flow rate of 0.8mg/ml and sample amount of 20 μ l, with ultrapure water as mobile phase, column temperature of 25 deg.C, and Evaporative Light Scattering Detector (ELSD);
the experimental method comprises the following steps: accurately weighing 2.0mg of each of homogeneous polysaccharide and dextran series standard products, preparing a solution of 2.0mg/mL by using ultrapure water, filtering by using a 0.45-micron microporous filter membrane before sample injection, detecting, recording retention time, drawing a standard curve by using a logarithmic value (Lg) of the molecular weight of the standard polysaccharide as a vertical coordinate and the retention time as a horizontal coordinate, obtaining a corresponding linear regression equation, and calculating the relative molecular weight of the homogeneous polysaccharide; the relative molecular weights of AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9 are 109.2kDa, 43.4kDa, 286.4kDa, 218.3kDa, 245.3kDa, 300.1kDa and 174.1kDa, respectively;
(2) determination of total sugar, uronic acid, protein and sulfate group content
The content of AAP-3 total sugar is 91.65 +/-2.75 percent, the content of AAP-4 total sugar is 92.57 +/-2.78 percent, the content of AAP-5 total sugar is 91.20 +/-2.74 percent, the content of AAP-6 total sugar is 92.21 +/-2.77 percent, the content of AAP-7 total sugar is 93.51 +/-0.94 percent, the content of AAP-8 total sugar is 92.38 +/-2.77 percent, and the content of AAP-9 total sugar is 94.47 +/-2.83 percent;
the content of uronic acid is detected by m-hydroxy biphenyl method, the content of AAP-3 uronic acid is 28.61 + -0.86%, the content of AAP-4 uronic acid is 14.84 + -0.45%, the content of AAP-5 uronic acid is 12.64 + -0.38%, and the content of AAP-7 uronic acid is 4.41 + -0.13%;
protein content determination by Coomassie Brilliant blue method: 0.53 plus or minus 0.02 percent of AAP-3 protein, 0.93 plus or minus 0.04 percent of AAP-4 protein, 0.91 plus or minus 0.05 percent of AAP-5 protein, 0.76 plus or minus 0.04 percent of AAP-6 protein, 0.33 plus or minus 0.02 percent of AAP-7 protein, 0.45 plus or minus 0.02 percent of AAP-8 protein and 0.66 plus or minus 0.02 percent of AAP-9 protein;
(3) monosaccharide composition analysis
AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9 are respectively subjected to 2mol/L TFA at 110 ℃ for 5h for complete acid hydrolysis to obtain complete acid hydrolysis products, then the complete acid hydrolysis products are subjected to derivatization by reacting with 0.2mol/L PMP methanol solution for 150min under the alkaline condition of concentrated ammonia water, and the derivatization products are subjected to LC-MS analysis; the results show that:
AAP-3 is a polysaccharide composed of six monosaccharides, the molar ratio of the monosaccharides is mannose: rhamnose: galacturonic acid: glucose: galactose: arabinose 1.1:1.8:2.5:0.9:1.4: 1.1;
AAP-4 is a polysaccharide composed of six monosaccharides, the molar ratio of the monosaccharides is mannose: rhamnose: galacturonic acid: glucose: galactose: arabinose is 1.5:1.4:1.3:1.0:2.3: 1.3.
AAP-5 is a polysaccharide composed of five monosaccharides, the molar ratio of the monosaccharides is mannose: rhamnose: glucuronic acid: glucose: galactose ═ 1.2:1.3:1.2:2.5: 3.4;
AAP-6 is a polysaccharide composed of four monosaccharides, the molar ratio of the monosaccharides being mannose: rhamnose: glucose: galactose 1.2:1.0:1.9: 1.5;
AAP-7 is a polysaccharide composed of seven monosaccharides, the molar ratio of the monosaccharides being mannose: rhamnose: 2-amino-2-deoxyglucose: glucuronic acid: glucose: 2-amino-2-deoxygalactose: galactose ═ 1.2:1.2:2.4:0.7:1.6:2.4: 1.4;
AAP-8 is a polysaccharide composed of five monosaccharides, the molar ratio of the monosaccharides being mannose: 2-amino-2-deoxyglucose: glucose: galactose: arabinose 6.1:6.2:10.4:5.8: 5.6;
AAP-9 is a polysaccharide composed of five monosaccharides, the molar ratio of the monosaccharides being mannose: rhamnose: glucose: galactose: arabinose 2.9:2.3:3.5:3.0: 1.2;
(4) methylation analysis
Separately processing polysaccharides by modified Hakomori methodBased (AAP-3, AAP-4, AAP-5 and AAP-7 with uronic acid polysaccharides previously applied CMC-NaBH4Reduced and then methylated), the methylated product was perhydrolyzed with 2mol/L TFA, NaBD4Reducing and acetylating acetic anhydride to produce a partially methylated alditol acetate derivative, and performing GC-MS analysis;
combining with a standard map, AAP-3 methylation results show that the structure contains end-group-linked glucose, 1, 4-linked galacturonic acid, 1, 3-linked rhamnose, 1, 4-linked rhamnose, 1, 6-linked mannose, end-group-linked mannose, 1,3, 6-linked galactose, 1,3, 5-arabinose and 1, 5-arabinose in a molar ratio of 2.6:5.6:1.2:2.6:1.2:1.2:2.8:1.2: 1.2. Wherein, 1, 4-linked galacturonic acid, terminal glucose, 1, 4-linked rhamnose and 1,3, 6-linked galactose are mainly used;
AAP-4 methylation results show that the structure contains: 1, 4-linked mannose, end-linked glucose, 1, 4-linked galactose, end-linked rhamnose, end-linked galactose, 1, 3-linked rhamnose, 1, 3-linked galactose, 1,3,4, 6-linked galactose, 1,3, 4-linked mannose, 1,3, 6-linked galactose, end-linked arabinose, 1,3, 5-linked arabinose, in a molar ratio of 2.8:2.2:1.6:1.4:1.8:1.4:1.0:1.2:0.8:1.6:1.2: 1.4. Wherein 1, 4-linked mannose, terminal glucose, terminal galactose and 1, 4-linked galactose are mainly used;
AAP-5 methylation results show that the structure contains: 1, 4-linked glucose, 1, 3-linked glucuronic acid, terminal linked mannose, 1,4, 6-linked galactose, 1, 3-linked rhamnose, 1, 3-linked galactose, 1, 4-linked galactose, 1,3, 4-linked galactose, in a molar ratio of 5.7:2.8:2.7:1.0:1.8:2.8:0.9:0.9:1.8, wherein the 1, 4-linked glucose, 1, 3-linked glucose, terminal mannose and 1, 3-linked rhamnose are mainly used;
AAP-6 methylation results show that the structure contains: 1, 4-linked glucose, 1, 3-linked rhamnose, 1, 3-linked galactose, 1, 6-linked mannose, end-linked mannose, 1,3, 4-linked galactose in a molar ratio of 7.2:3.1:3.9:1.8:1.9:2.1, wherein 1, 4-linked glucose, 1, 3-linked rhamnose, 1, 3-linked galactose and 1,3, 4-linked galactose are mainly used;
the AAP-7 methylation result shows that the structure contains: terminal-linked rhamnose, terminal-linked glucose, 1, 3-linked mannose, 1, 3-linked galactose, 1, 4-linked-2-amino-2-deoxygalactose (or 1, 4-linked-2-amino-2-deoxyglucose), 1, 6-linked galactose, 1,3, 4-linked mannose, 1,3, 6-linked glucose in a molar ratio of 2.0:1.4:1.2:0.9:9.6:2.1:1.0:2.2, wherein the ratio of 1, 4-linked amino-2-deoxygalactose to 1, 4-linked-2-amino-2-deoxyglucose is about 1:1, and the ratio of 1, 4-linked amino-2-deoxyglucose, 1, 4-linked-2-amino-2-deoxygalactose, 1, 4-linked-2-amino-2-deoxyglucose, 1, 4-linked-2-amino-2-deoxygalactose, and the like, Terminal rhamnose, 1, 6-linked galactose and 1,3, 6-linked glucose are main components;
AAP-8 methylation results show that the structure contains: the end group is connected with glucose, 1, 4-connection-2-amino-2-deoxyglucose, 1, 3-connection rhamnose, 1,3, 4-connection mannose, 1, 4-connection galactose and arabinose, and the molar ratio of the end group to the arabinose is 5.9:4.1:3.2:1.0:2.9:3.2, wherein the end group is mainly connected with glucose, 1, 4-connection-2-amino-2-deoxyglucose, 1, 3-connection rhamnose and 1, 4-connection galactose;
the AAP-9 methylation result shows that the structure contains: 1, 4-linked glucose, 1, 4-linked mannose, 1, 2-linked arabinose, 1, 3-linked rhamnose, end-linked mannose, 1,3, 4-linked mannose, end-linked galactose, 1, 3-linked galactose, 1, 6-linked galactose, 1,3, 6-linked galactose, and the molar ratio of the 1, 4-linked glucose, 1, 4-linked mannose, 1, 2-linked arabinose, and 1, 3-linked rhamnose is 4.8:2.1:2.0:2.9:1.1:1.8:1.8:1.7:0.9:0.9, wherein the 1, 4-linked glucose, 1, 4-linked mannose, 1, 2-linked arabinose, and 1, 3-linked rhamnose are mainly used.
Example 3 classical pathway complement inhibition assay
Taking the serum of a guinea pig of 3 months age, sensitizing the serum by 2 percent sheep red blood cells, and diluting the serum to 1:100 by using a barbital buffer solution as a complement source of the current classical pathway; diluting rabbit anti-sheep erythrocyte antibody with Barbiturate Buffer Solution (BBS) to 1:1000 as hemolysin; sheep Red Blood Cells (SRBC) preserved in Alsever fluid were configured as 2% SRBC; polysaccharide 3mg is precisely weighed, added with BBS buffer solution for dissolution, and diluted into 8 concentrations in a double way. Polysaccharides at different concentrationsAfter 200. mu.L of the solution and 200. mu.L of complement which has been diluted to 1:100 are preincubated for 10min at 37 ℃, 100. mu.L of hemolysin (1:1000) and 100. mu.L of 2% SRBC are sequentially added, and the mixture is put into a low-temperature high-speed centrifuge after being put into a water bath at 37 ℃ for 30min and centrifuged for 10min at 5000rpm and 4 ℃. Taking 200 mu L of supernatant from each tube, placing the supernatant in a 96-well plate, and measuring the absorbance at 405 nm; the experiment was performed while setting a polysaccharide control group (200. mu.L of polysaccharide at a corresponding concentration plus 400. mu.L of BBS buffer), a complement control group (200. mu.L of BBS buffer instead of polysaccharide) and a whole hemolyzed group (100. mu.L of 2% SRBC was dissolved in 500. mu.L of triple distilled water), calculating the hemolysis inhibition ratio by subtracting the absorbance value of the polysaccharide group at each concentration from the absorbance value of the corresponding polysaccharide control group, plotting the logarithm of the polysaccharide concentration as the X-axis and the hemolysis inhibition ratio as the Y-axis, and calculating the Concentration (CH) of the sample required for 50% inhibition of hemolysis from the obtained fitting curve50Values), using heparin as a positive control, showed that the seven homopolysaccharides all had significant inhibitory activity on classical complement pathway activation (as shown in table 1).
Example 4 alternative pathway complement inhibition assay
Serum of healthy adult male volunteers was extracted with EGTA-BBS buffer (barbital buffer, pH 7.4, containing 5mM Mg)2+And 8mM EGTA) is diluted to 1:10, and is used as a complement source of a bypass route, rabbit red blood cells stored in a 3.8% sodium citrate solution are prepared into 0.5% rabbit red blood cells by EGTA-BBS buffer solution, polysaccharide is precisely weighed to be about 3mg, the EGTA-BBS buffer solution is added, the rabbit red blood cells are diluted to 8 concentrations in a double way, 150 mu L of polysaccharide solution with different concentrations and 150 mu L of complement with the concentration of 1:8 are preincubated for 10min at 37 ℃, 200 mu L of 0.5% rabbit red blood cells are added, the rabbit red blood cells are placed in a low-temperature high-speed centrifuge after being subjected to water bath at 37 ℃ for 30min, the rabbit red blood cells are centrifuged for 10min at 5000rpm and 4 ℃, 200 mu L of supernatant of each tube is taken in a 96 pore plate, and the absorbance is measured at 405 nm; the experiment was performed while setting a polysaccharide control group (150. mu.L of polysaccharide solution of corresponding concentration plus 350. mu.L of EGTA-BBS buffer solution), a complement control group (150. mu.L of EGTA-BBS buffer solution instead of polysaccharide solution) and a whole hemolyzed group (200. mu.L of 0.5% rabbit red blood cells were dissolved in 300. mu.L of triple distilled water), calculating the hemolysis inhibition ratio by subtracting the absorbance value of the polysaccharide group of each concentration from the absorbance value of the corresponding polysaccharide control group, plotting the logarithm of the polysaccharide concentration as the X-axis and the hemolysis inhibition ratio as the Y-axis to obtain a plotCalculating the concentration of the test substance required to inhibit hemolysis by 50% (AP)50Values) with heparin as a positive control, the results are shown in table 1.
TABLE 1 inhibition of complement activation by seven Artemisia apiacea homopolysaccharides
Figure BDA0001959212070000091
CH50And AP50The values are expressed as: mean ± SD (n ═ 3); NA: no active.

Claims (1)

1. Use of Artemisia apiacea polysaccharides AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9 having the following structural characteristics in the preparation of a medicament for inhibiting the complement, said Artemisia apiacea polysaccharides AAP-3, AAP-4, AAP-5, AAP-6, AAP-7, AAP-8 and AAP-9 being prepared by the following method:
pulverizing herba Artemisiae Annuae, extracting with 95% ethanol, filtering, extracting the residue with water solution for 3 times, concentrating, centrifuging, adding 4 times volume of 95% ethanol into the supernatant, standing, centrifuging to remove the supernatant, dissolving the precipitate with water, recovering ethanol under reduced pressure; removing free protein from the compound solution with 10% trichloroacetic acid, centrifuging, adjusting supernatant to neutral, dialyzing, concentrating, freeze drying to obtain crude polysaccharide, dissolving the crude polysaccharide with distilled water, centrifuging, and subjecting the supernatant to preliminary separation by DEAE-cellulose column chromatography; eluting with distilled water, 0.1, 0.2, 0.4 and 0.8mol/L NaCl solution, eluting with volume more than 2 times of column volume, flow rate 25mL/min, collecting each fraction, and detecting absorbance value at 490nm with a separation tube after color development by sulfuric acid-phenol method; according to the chromogenic reaction of sugar and the result of HPGPC detection, combining the flow parts, concentrating, dialyzing and freeze-drying to obtain 5 secondary components: Water-AAP, 0.1M-AAP, 0.2M-AAP, 0.4M-AAP, and 0.8M-AAP;
dissolving 0.1M-AAP with distilled water, centrifuging, separating the supernatant with Sephacryl S-300 gel chromatographic column, eluting with distilled water solution at flow rate of 0.5mL/min, collecting fractions, performing color development with sulfuric acid-phenol method, detecting absorbance value at 490nm with separate tubes, combining the same fractions according to the detection result, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharides AAP-3 and AAP-4;
dissolving 0.2M-AAP with distilled water, centrifuging, separating supernatant with Sephacryl S-200 chromatography, eluting with distilled water at flow rate of 0.5mL/min, collecting fractions, performing sulfuric acid-phenol color development, detecting absorbance value at 490nm with separate tubes, combining the same fractions according to detection results, concentrating, dialyzing, and freeze drying to obtain homogeneous polysaccharides AAP-5 and AAP-6;
dissolving 0.4M-AAP with distilled water, centrifuging, separating supernatant with Sephacryl S-300 chromatography, eluting with distilled water at flow rate of 0.5mL/min, collecting fractions, performing sulfuric acid-phenol color development, detecting absorbance value at 490nm with separate tubes, combining the same fractions according to detection results, concentrating, dialyzing, and freeze drying to obtain homogeneous polysaccharides AAP-7 and AAP-8;
dissolving 0.8M-AAP with distilled water, centrifuging, separating supernatant by Sephacryl S-300 chromatography, eluting with distilled water at flow rate of 0.5mL/min, collecting fractions, performing sulfuric acid-phenol method for color development, detecting absorbance value at 490nm with a separation tube, combining the same fractions according to detection results, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharide AAP-9.
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