CN107090048A - Lasiosphaera fenzlii polysaccharide and its purposes in anticomplement medicament is prepared - Google Patents
Lasiosphaera fenzlii polysaccharide and its purposes in anticomplement medicament is prepared Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0087—Glucomannans or galactomannans; Tara or tara gum, i.e. D-mannose and D-galactose units, e.g. from Cesalpinia spinosa; Tamarind gum, i.e. D-galactose, D-glucose and D-xylose units, e.g. from Tamarindus indica; Gum Arabic, i.e. L-arabinose, L-rhamnose, D-galactose and D-glucuronic acid units, e.g. from Acacia Senegal or Acacia Seyal; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- C—CHEMISTRY; METALLURGY
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
The invention belongs to the field of Chinese medicines, it is related in Lasiosphaera fenzlii natural homogeneous polysaccharide and its purposes in anticomplement medicament is prepared, natural homogeneous polysaccharide includes Lasiosphaera fenzlii polysaccharide TFP 1, TFP 2, TFP 3 and TFP 4 in described Lasiosphaera fenzlii, the present invention carries out natural homogeneous polysaccharide in isolated Lasiosphaera fenzlii to the sodium hydroxide solution extract for commonly using heat-clearing and detoxifying herb Lasiosphaera fenzlii, confirmed through experiment in vitro, described natural homogeneous polysaccharide has significant inhibitory action to the classical pathway and alternative pathway of complement activation, active component can be further used as, anticomplement medicament is prepared.
Description
Technical field
The invention belongs to the field of Chinese medicines, it is related to polysaccharide, and in particular to four natural homogeneous polysaccharides and its anti-preparing in Lasiosphaera fenzlii
Purposes in complement medicine.
Background technology
Complement system is the important component of human immune system, its normal Activate eliminating external microorganism, it is clear
Except in body damage or death cell and tissue, maintain body balance in play an important role.But the mistake of the system
Degree activation can cause the overreaction of human immune system, cause the damage of human body itself normal structure, such as rheumatoid joint
Inflammation, senile dementia, systemic loupus erythematosus（SLE）And the rejection after organ transplant etc..In MOF
Syndrome, such as ischemic damage and reperfusion, acute myocardial infarction AMI, ARDS（ARDS）Deng in acute illness, complement
Excessive activation also play important function.
Although the immunodepressant such as current clinically widely used glucocorticoid, endoxan, the cry of certain animals of methylamine butterfly are to certain
The disease related to excess complement activation has certain therapeutic action a bit, but due to the not single-minded complement inhibitor of such medicine,
Prolonged application can reduce the defensive enginery of body, cause anti-infection ability to decline, and easy scabies secondary infection and spread hidden focus, produce
Raw multiple complications and side effect.Therefore clinically it is badly in need of efficient, low toxicity, single-minded new complement inhibitor.
The active component of anticomplementary action is widely present in nature, China's natural resources of Chinese medicinal materials enriches, and there are many Chinese medicines to exempting from
Epidemic disease system has obvious adjustment effect, is the precious resources for finding anticomplement class prodrug.During resist SARS, the traditional Chinese medical science,
Preventing and treating of the Chinese medicine to SARS obtains remarkable result.Lasiosphaera fenzlii is Basidiomycotina, Lycoperdon fungi Lasiosphaera fenzlii Lasiosphaera
Fenzlii Reich. fructification, is a kind of conventional heat-clearing and detoxifying herb, be usually used in treatment abscess of throat, aphonia chronic cough,
Accumulated heat haematemesis etc..Pharmacological research shows that Lasiosphaera fenzlii has the pharmacological action such as antibacterial, antiviral, analgesia, anti-oxidant, studies have found that
Lasiosphaera fenzlii Thick many candies anti-complement activity significantly, but there is not yet separates the report prepared to anti-complement activity homogeneous polysaccharide in Lasiosphaera fenzlii.
Present situation based on prior art, present inventor intends grinding by the anti-complement activity composition to related Chinese medicine
Study carefully there is provided four in Lasiosphaera fenzlii natural homogeneous polysaccharides and its purposes in anticomplement medicament is prepared, be the related disease of complement
Treatment provides material base.
The content of the invention
It is an object of the invention to provide the active component in natural drug with anticomplementary action, it is related in Lasiosphaera fenzlii natural homogeneous many
Natural homogeneous polysaccharide includes four Lasiosphaera fenzlii polysaccharide in sugar and its purposes in anticomplement medicament is prepared, described Lasiosphaera fenzlii（TFP-
1st, TFP-2, TFP-3 and TFP-4）.
The present invention carries out day in isolated Lasiosphaera fenzlii to the sodium hydroxide solution extract for commonly using heat-clearing and detoxifying herb Lasiosphaera fenzlii
Right homogeneous polysaccharide, the natural homogeneous polysaccharide includes four homogeneous polysaccharides, is respectively designated as TFP-1, TFP-2, TFP-3 and TFP-4；
Confirmed through experiment in vitro, four described Lasiosphaera fenzlii polysaccharide have significant complement inhibitory activity, can further prepare anticomplement medicine
Thing.
In the present invention, described Mabo is Lycoperdon fungi Lasiosphaera fenzlii Lasiosphaera Fenzlii Reich.
Fructification.
Lasiosphaera fenzlii polysaccharide TFP-1, TFP-2, TFP-3 and TFP-4 of the present invention following structural features are stated：
（1）The heteroglycan that TFP-1 is made up of three kinds of monose, molecular weight is about 512000Da；Total sugar content is 96.94%；Be free of
Protein and uronic acid；Without sulfate.Monose mol ratio is glucose（Glc）:Mannose（Man）:Galactolipin（Gal）=
6.30: 1.03: 2.67.The result that methylates shows the glucose connected in structure containing end（Terminal Glc）, 1,3 connect
The galactolipin connect（1, 3-linked Gal）, 1,2 connection mannoses（1, 2- linked Man）, 1,3 connection grapes
Sugar（1, 3-linked Glc）, 1,6 connection glucose（1, 6-linked Glc）With the glucose of 1,2,3,6 connections
(1,2,3,6-linked Glc), mol ratio is 2.848: 2.669: 1.031: 2.586: 0.821: 0.825.
（2）The heteroglycan that TFP-2 is made up of three kinds of monose, molecular weight is about 591000Da；Total sugar content is 97.24%；
Protein content 0.87%；Without uronic acid；Without sulfate.Complete sour water solution obtains three kinds of monose, and monose mol ratio is grape
Sugar（Glc）:Mannose（Man）:Galactolipin（Gal）= 8.68: 0.69: 0.63.The result that methylates, which is shown, contains end in structure
The glucose of connection（Terminal Glc）, 1,3 connection galactolipins（1, 3-linked Gal）, 1,2 connection mannoses
（1, 2- linked Man）, 1,3 connection glucose（1, 3-linked Glc）, 1,6 connection glucose（1, 6-
linked Glc）With 1, the glucose (1,2,3,6-linked Glc) of 2,3,6 connections, mol ratio is 1.442:
0.627: 0.971: 3.085: 3.403: 0.875。
（3）The heteroglycan that TFP-3 is made up of four kinds of monose, molecular weight is about 619000Da；Total sugar content is 95.20%；
Glucuronic acid content 8.89%；Without protein；Without sulfate.Complete sour water solution obtains four kinds of monose, and monose mol ratio is grape
Sugar（Glc）:Glucuronic acid（GlcA）:Mannose（Man）:Galactolipin（Gal）= 6.57: 0.89: 1.25: 1.29.
The result that methylates shows the glucose (Terminal Glc) connected in structure containing end, the glucuronic acid of end connection
(Terminal GlcA), the galactolipin of 1,3 connections（1, 3-linked Gal）, 1,2 connection mannoses（1, 2-
Linked Man), 1,3,6 connection glucose（1, 3, 6-linked Glc）With the glucose of 1,2,3,6 connections
（1, 2, 3, 6-linked Glc）, mol ratio is 3.12: 0.9: 1.32: 1.20: 2.61: 0.75.
（4）The heteroglycan that TFP-4 is made up of five kinds of monose, molecular weight is about 982000Da；Total sugar content is 56.94%；
Glucuronic acid content 47.95%, protein content is 34.89%.Sour water solution obtains what five kinds of monose were constituted, and saccharide residue mol ratio is Portugal
Grape sugar (Glc):Glucuronic acid（GlcA):Mannose（Man）:Galactolipin（Gal）:Galacturonic acid（GalA）=1.286:
4.60: 1.22: 0.594: 2.30。
Lasiosphaera fenzlii polysaccharide of the present invention（TFP-1, TFP-2, TFP-3 and TFP-4）Prepared by following methods：
Chinese medicine Lasiosphaera fenzlii is extracted with 95% ethanol, and filtration, residue is extracted with sodium hydroxide solution, is filtered, and filtrate is adjusted to neutrality,
Concentration, adds 95% ethanol of 4 times of volumes, stands, and supernatant is removed in centrifugation, and precipitation is redissolved with water, then removes removing protein with trichloroacetic acid,
Centrifugation, supernatant is adjusted to neutrality, concentrates, and dialysis is freeze-dried to obtain Thick many candies；Thick many candies add distilled water to dissolve, and use DEAE-
Cellulose column chromatography initial gross separations, are eluted with the mol/L of distilled water, 0.4,0.8,1.2 and 2.0 NaCl solution, collect each
Stream part, concentration dialyses and freezed to obtain 5 secondary components, numbering：WaterTFP, 0.4TFP, 0.8TFP, 1.2TFP and
2.0TFP；The anti-complement activity of component is surveyed, 0.4TFP and 1.2TFP significant to anti-complement activity carry out next step separation；
0.4TFP is dissolved with distilled water, and centrifugation, supernatant uses Sephacryl by several timesTMS-300 posts（100×4.5 cm）Separation,
0.1mol/L sodium chloride solution elution, collects each stream part；Merge phase according to according to the result of sugared chromogenic reaction and Activity determination
Same stream part, concentrates, dialyses and is freeze-dried and to obtain polysaccharide 0.4TFP-1 and 0.4TFP-2.0.4TFP-1 and 0.4TFP-2 are added respectively
Distilled water dissolves, centrifugation, supernatant SephacrylTMS-300 posts（100×2.5 cm）Chromatography, with 0.1mol/L's
Sodium chloride solution is eluted, according to sugared chromogenic reaction and the identical stream part of result merging of Activity determination, concentration, dialysis and freeze-drying
Obtain polysaccharide TFP-1 and TFP-2；
1.2TFP is dissolved with distilled water, centrifuged, supernatant uses Sephacryl by several timesTMS-300 posts（100×2.5 cm）Point
From 0.1 mol/L NaCl solution elution, according to sugared chromogenic reaction and the identical stream part of result merging of Activity determination, is concentrated, thoroughly
Analyse and be freeze-dried and to obtain polysaccharide TFP-4 and TFP-3.
In vitro test of further entering to pass through is confirmed, is as a result shown, Lasiosphaera fenzlii polysaccharide TFP-1, TFP-2, TFP-3 and TFP-4 couple
Complement is classical and alternative pathway activates triggered cell haemolysis and has obvious suppression,
CH50Value（The concentration of test sample needed for classical pathway 50% suppresses haemolysis）Respectively 181.7 ± 14.45 μ g/mL,
132.7± 13.70 µg/mL、 78.81 ± 3.21 µg/mL、136.1± 11.60 µg/mL；AP50Value（Alternative pathway
The concentration of test sample needed for 50% suppression haemolysis）Respectively 162.45 ± 23.90 μ g/mL, 142.76 ± 20.70 μ g/
mL、136.23 ± 10.60 µg/mL、139.1± 11.60 µg/mL。
Experimental result confirms that described Lasiosphaera fenzlii polysaccharide TFP-1, TFP-2, TFP-3 and TFP-4 has obvious anticomplement to make
With；Anticomplement medicament can further be prepared.
Brief description of the drawings
Fig. 1 is Lasiosphaera fenzlii polysaccharide TFP-1 (A), TFP-2 (B), TFP-3 (C) and the TFP-4 (D) of the present invention
HPGPC chromatograms, wherein, TSK-GEL GMPWXL gel columns (300 × 7.6mm)；Eluent：0.01 M NaCl；Flow velocity：
0.8 ml/min。
Embodiment
Embodiment 1 prepares Lasiosphaera fenzlii polysaccharide TFP-1, TFP-2, TFP-3 and TFP-4
Lasiosphaera fenzlii medicinal material 10Kg is crushed, and is extracted with 95% ethanol, filtration, and the dregs of a decoction are extracted 3 times with 0.1mol/L sodium hydroxide solution,
Filtration, merges extract solution, modulates neutral with 1mol/L hydrochloric acid, concentrates, centrifugation, supernatant adds 95% ethanol of 4 times of volumes,
Stand, supernatant is removed in centrifugation, precipitation is redissolved with water, is recovered under reduced pressure, and removes ethanol；Redissolve liquid and free egg is removed with trichloroacetic acid again
In vain, centrifuge, supernatant is adjusted to neutrality, dialyse, concentration, freeze-drying produces Thick many candies.Thick many candies 100g adds distilled water to dissolve, from
The heart, supernatant DEAE-cellulose posts（Cl-1 Type, 100 × 4.5 cm）Chromatography carries out initial gross separation.With distilled water, 0.4,
0.8th, 1.2 and 2.0 mol/L NaCl solution elution, elution volume is more than 2 times of column volumes（About 3 L）, flow velocity is 3.6 mL/
Min, collects each stream part, pipe detection 490nm（After sulfuric acid-phynol method colour developing）Under absorbance, according to sugared chromogenic reaction simultaneously
With reference to the result of ultraviolet detection, merge flow point, concentration is dialysed and is freeze-dried and to obtain 5 secondary components, numbers：：WaterTFP、
0.4TFP, 0.8TFP, 1.2TFP and 2.0TFP.Active tracking display：0.4TFP and 1.2TFP have preferable anticomplement to live
Property；
By 0.4TFP（10 g）Plus distilled water dissolving, centrifugation, supernatant is by several times with Sephacryl S-300 posts（100×4.5
cm）Chromatography, 0.1 mol/L NaCl solutions elution, flow velocity is 0.8 mL/min, collects each flow point.Pipe detects 490 nm
（After sulfuric acid-phynol method colour developing）Under absorbance, each pipe cut of activity tracking merges identical flow point according to testing result, dense
Contract, dialyse and be freeze-dried and to obtain polysaccharide 0.4TFP-1（2.3 g）And 0.4TFP-2（4.2 g）, by 0.4TFP-1 and 0.4TFP-2
Respectively plus distilled water dissolving, centrifugation, supernatant respectively by several times use Sephacryl S-300 posts（100×2.5 cm）Chromatography,
0.1 mol/L NaCl solutions are eluted, and flow velocity is 0.8 mL/min, collects each flow point.Pipe detects 490 nm（Sulfuric acid-phynol method
After colour developing）Under absorbance, each pipe cut of activity tracking merges identical flow point according to testing result, concentration and is freeze-dried
Obtain polysaccharide TFP-1（60 mg）And TFP-2（120 mg）；
By 1.2TFP（6.3 g）Plus distilled water dissolving, centrifuge, supernatant uses Sephacryl by several timesTMS-300 posts（100×2.5
cm）Chromatography, 0.1mol/L NaCl solution elution, flow velocity is 0.8 mL/min, collects each flow point, and pipe detects 490 nm
（After sulfuric acid-phynol method colour developing）Under absorbance, each pipe cut of activity tracking.Identical flow point is merged according to testing result, it is dense
Contract, dialyse and be freeze-dried and to obtain polysaccharide TFP-3（150 mg）And TFP-4（60 mg）；
Through High Performance Gel Permeation Chromatography（HPGPC）TFP-1, TFP-2, TFP-3 and TFP-4 are detected, as a result display is homogeneous
Composition.
The Lasiosphaera fenzlii polysaccharide of embodiment 2（TFP-1, TFP-2, TFP-3 and TFP-4）Structural characterization
（1）The measure of molecular weight
Using TSK-GEL GMPWXL gel columns（300×7.6 mm）, mobile phase is 0.01 mol/L NaCl, flow velocity 0.8
ML/min, 25 DEG C of column temperature, by standard curve（The Dextran of T series）Calculate, TFP-1, TFP-2, TFP-3 and TFP-4 molecule
Amount is respectively 512000Da, 591000Da, 619000Da and 982000Da；
（2）Total reducing sugar, uronic acid, albumen and sulfate assay
It is 96.94% that sulfuric acid-phynol method, which determines TFP-1 total sugar contents,；TFP-2 total sugar contents are 97.24%；TFP-3 total sugar contents
For 95.20%；TFP-4 total sugar contents are 56.94%；
Meta-hydroxydiphenyl method determines detection glucuronic acid content, and TFP-1 and TFP-2 are free of uronic acid composition；TFP-3 glucuronic acid content
8.89%, TFP-4 glucuronic acid content 47.95%；
Coomassie Brilliant Blue determines protein content：TFP-1 is free of albumen；TFP-2 protein contents 0.87%；TFP-3 is free of albumen；
TFP-4 protein contents 34.89%；
BaCl2Turbidimetry for Determination TFP-1, TFP-2, TFP-3 and TFP-4 are free of sulfate；
（3）Sugared composition analysis
The product that TFP-1, TFP-2, TFP-3 and TFP-4 are obtained through 2 mol/L TFA in 110 °C of all-hydrolytics respectively, is successively carried out
NaBH4Reduction, aceticanhydride acetylation is prepared into ALDI alcohol acetic ester derivative, carries out gas phase composition analysis（Blakeney AB,
Harris PJ, Henry RJ, et al. A simple and rapid preparation of alditol
acetates for monosaccharide analysis. Carbohydr Res. 1983, 113:291-299）；
TFP-1 is the main heteroglycan being made up of three kinds of monose, and saccharide residue mol ratio is glucose（Glc）:Mannose（Man）:
Galactolipin（Gal）= 6.30: 1.03: 2.67；
TFP-2 is the main heteroglycan being made up of three kinds of monose, and saccharide residue mol ratio is glucose（Glc）:Mannose（Man）:
Galactolipin（Gal）= 8.68: 0.69: 0.63；
TFP-3 is the main heteroglycan being made up of four kinds of monose, and saccharide residue mol ratio is glucose（Glc）:Glucuronic acid
（GlcA）Mannose（Man）:Galactolipin（Gal）= 6.57: 0.89: 1.25: 1.29；
TFP-4 is the main heteroglycan being made up of five kinds of monose, and saccharide residue mol ratio is glucose (Glc):Glucuronic acid
（GlcA):Mannose Man:Galactolipin（Gal）:Galacturonic acid (GalA)=1.286: 4.60: 1.22: 0.594:
2.30；
（4）Methylation analysis
Reference literature method（Needs PW, Selvendran RR. Avoiding oxidative degradation
during sodium hydroxyl/dimethyl-iodide mediated carbohydrate methylation in
dimethyl sulfoxide. Carbohydr Res. 1993, 245:1-10）TFP-1, TFP-2 and TFP-3 are entered respectively
Row methylates, the product after methylating 90% formic acid depolymerization, 2 mol/L TFA all-hydrolytics, NaBH4Reduction and aceticanhydride acetylation
The ALDI alcohol acetic ester derivative of partial methylation is made, GC-MS analyses are then carried out；
Combined standard collection of illustrative plates judges that the main connected modes of TFP-1 have the glucose that end is connected（Terminal Glc）, 1,3 connect
The galactolipin connect（1,3-linked Gal）, 1,2 connection mannoses（1,2- linked Man）, 1,3 connection glucose
（1,3-linked Glc）, 1,6 connection glucose（1,6-linked Glc）With 1,2,3,6 connection glucose (1,
2,3,6-linked Glc), the glucose connected with end（Terminal Glc）, 1,3 connection galactolipins（1,3-
linked Gal）, 1,3 connection glucose（1,3-linked Glc）Based on；
The main connected modes of TFP-2 have the glucose that end is connected（Terminal Glc）, 1,3 connection galactolipins（1, 3-
linked Gal）, 1,2 connection mannoses（1, 2- linked Man）, 1,3 connection glucose（1, 3-linked
Glc）, 1,6 connection glucose（1, 6-linked Glc）With the glucose (1,2,3,6- of 1,2,3,6 connections
Linked Glc), wherein with the glucose of 1,3 connection（1, 3-linked Glc）, 1,6 connection glucose（1, 6-
linked Glc）Based on；
The main connected modes of TFP-3 have glucose (Terminal Glc), the glucuronic acid of end connection that end is connected
(Terminal GlcA), the galactolipin of 1,3 connections（1, 3-linked Gal）, 1,2 connection mannoses（1, 2-
Linked Man), 1,3,6 connection glucose（1, 3, 6-linked Glc）With the glucose of 1,2,3,6 connections
（1, 2, 3, 6-linked Glc）.Wherein with the mannose of 1,2 connections（1,2- linked Man), 1,3,6 connection
Glucose（1, 3, 6-linked Glc）Based on.
The classical pathway Complement inhibition of embodiment 3 is tested
The serum of 3 monthly age cavys is taken, with VBS2+Buffer solution（Barbitol buffer solution, pH=7.4, containing 0.5 mM Mg2+With 0.15
mM Ca2+）It is diluted to 1:100, it is used as the Complement source of this classical pathway；By the antibody of rabbit-anti sheep red blood cell with VBS2+It is slow
Fliud flushing is diluted to 1:1000 are used as hemolysin；It is stored in the sheep red blood cell in Alsever liquid（SRBC）It is configured to 2% SRBC.Essence
The close weighing mg of polysaccharide 3, adds VBS2+Buffer solution, two-fold dilution is into 8 concentration；The μ L of polysaccharide solution 200 of various concentrations
With being diluted to 1:The 100 μ L of complement 200 sequentially add 100 μ L hemolysins after 37 DEG C of min of preincubate 10（1:
1000）With the SRBC of 100 μ L 2%, low-temperature and high-speed centrifuge is put into after the min of 37 oC water-baths 30, in 5000 rpm, 4 oC bars
10 min are centrifuged under part, often pipe takes 200 μ L of supernatant in 96 orifice plates, absorbance is determined in 405 nm；Experiment sets polysaccharide simultaneously
Control group（The polysaccharide of 200 μ L respective concentrations adds 400 μ L VBS2+Buffer solution）, complement control group（With 200 μ L VBS2+Buffering
Liquid replaces polysaccharide）With full haemolysis group（The SRBC of 100 μ L 2% are dissolved in 500 μ L tri-distilled waters）, by the polysaccharide group extinction of each concentration
Angle value, which is deducted, calculates haemolysis inhibiting rate after corresponding polysaccharide control group absorbance, using the logarithm of polysaccharide concentration as X-axis, haemolysis suppression
Rate processed is mapped as Y-axis, the concentration of test sample needed for obtained matched curve calculates 50% suppression haemolysis（CH50Value）, made with heparin
For positive control drug, as a result show, four homogeneous polysaccharides have significant inhibitory activity to classical pathway of complement activation（Such as
Shown in table 1）.
The alternative pathway Complement inhibition of embodiment 4 is tested
Healthy adult male volunteers sera is taken, with VBS-Mg-EGTA buffer solutions（Barbitol buffer solution, pH=7.4, containing 5 mM
Mg2+With 8 mM EGTA）It is diluted to 1:8, as the Complement source of alternative pathway, it is stored in the rabbit of 3.8% liquor sodii citratis
Red blood cell is configured to 0.5% rabbit erythrocyte with VBS-Mg-EGTA buffer solutions；The mg of precision weighing polysaccharide about 3, adds VBS-Mg-
EGTA buffer solutions, two-fold dilution is into 8 concentration, the μ L of polysaccharide solution 150 of various concentrations and 1:The 8 μ L of complement 150 are at 37 DEG C
After the min of preincubate 10, the rabbit erythrocytes of 200 μ L 0.5% are added, low-temperature and high-speed centrifugation is put into after the min of 37 oC water-baths 30
Machine, 10 min are centrifuged under the conditions of 5000 rpm, 4 oC, and often pipe takes 200 μ L of supernatant in 96 orifice plates, are determined and are inhaled in 405 nm
Luminosity；Experiment sets polysaccharide control group simultaneously（The polysaccharide solution of 150 μ L respective concentrations adds 350 μ L VBS-Mg-EGTA to buffer
Liquid）, complement control group（Polysaccharide solution is replaced with 150 μ L VBS-Mg-EGTA buffer solutions）With full haemolysis group（200 μL0.5%
Rabbit erythrocyte is dissolved in 300 μ L tri-distilled waters）；The polysaccharide group absorbance of each concentration is deducted into corresponding polysaccharide control group absorbance
Haemolysis inhibiting rate is calculated after value, using the logarithm of polysaccharide concentration as X-axis, haemolysis inhibiting rate is mapped as Y-axis, obtained fitting is bent
The concentration of test sample needed for line computation 50% suppresses haemolysis（AP50Value）, using heparin as positive control drug, as a result show, described four
Individual homogeneous polysaccharide has the cell haemolysis substantially suppressed caused by alternative pathway of complement activation（As shown in table 1）.
Table 1 is inhibitory action of four kinds of many carbohydrates and their derivatives of Lasiosphaera fenzlii to complement activation.
Table 1
Wherein, CH50And AP50Value is expressed as：Average value ± SD（n=3）.
Claims (4)
1. Lasiosphaera fenzlii polysaccharide, it is characterised in that described Lasiosphaera fenzlii polysaccharide is natural homogeneous many in natural homogeneous polysaccharide, described Lasiosphaera fenzlii
Sugar is respectively Lasiosphaera fenzlii polysaccharide TFP-1, TFP-2, TFP-3 and TFP-4 with following architectural feature：
Lasiosphaera fenzlii polysaccharide TFP-1：The main heteroglycan being made up of three kinds of monose, saccharide residue mol ratio is glucose（Glc）:Mannose
（Man）:Galactolipin（Gal）= 6.30: 1.03: 2.67；Molecular weight is 512000Da；Without albumen and uronic acid；Total reducing sugar contains
Amount 96.94%；Without sulfate；
Lasiosphaera fenzlii polysaccharide TFP-2：The main heteroglycan being made up of three kinds of monose, saccharide residue mol ratio is glucose（Glc）:Mannose
（Man）:Galactolipin（Gal）= 8.68: 0.69: 0.63；Molecular weight is 591000Da；Protein content：0.87%；Without alditol
Acid；Total sugar content 97.24%；Without sulfate；
Lasiosphaera fenzlii polysaccharide TFP-3：The main heteroglycan being made up of four kinds of monose, saccharide residue mol ratio is glucose（Glc）:Grape
Uronic acid（GlcA）Mannose（Man）:Galactolipin（Gal）= 6.57: 0.89: 1.25: 1.29；Molecular weight is 619000
Da；Without albumen；Glucuronic acid content：8.89%；Sugared content 95.20%；Without sulfate；
Lasiosphaera fenzlii polysaccharide TFP-4：The main heteroglycan being made up of five kinds of monose, saccharide residue mol ratio is glucose (Glc):Grape
Uronic acid（GlcA):Mannose（Man）:Galactolipin（Gal）:Galacturonic acid（GalA）=1.286: 4.60: 1.22:
0.594: 2.30；Molecular weight is 982000 Da；Protein content is 34.89%；Glucuronic acid content：47.95%；Sugared content
56.94%；Without sulfate.
2. purposes of the Lasiosphaera fenzlii polysaccharide in Complement inhibition medicine is prepared described in claim 1.
3. the purposes as described in claim 2, wherein many Glyco inhabiting classical pathway of complement activation of described Lasiosphaera fenzlii.
4. the purposes as described in claim 2, wherein thin caused by many Glyco inhabiting alternative pathway of complement activation of described Lasiosphaera fenzlii
Cell lysis blood.
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Cited By (1)
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| CN111773237A (en) * | 2019-04-03 | 2020-10-16 | 复旦大学 | Turkey knotweed polysaccharide, preparation method thereof and application thereof in preparation of anticomplement medicines |
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