CN105708851B - The more carbohydrates and their derivatives of cordate houttuynia are preparing the purposes in Complement inhibition drug - Google Patents
The more carbohydrates and their derivatives of cordate houttuynia are preparing the purposes in Complement inhibition drug Download PDFInfo
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Abstract
The invention belongs to the field of Chinese medicines, it is related to homogeneous polysaccharide and its derivative in cordate houttuynia and preparation method thereof and medicinal usage.The present invention isolated homogeneous polysaccharide HC-PS1, HC-PS2 and HC-PS3 from heat-clearing and detoxifying herb cordate houttuynia, and sulphation, hydroxyethylation and the carboxyl methylation derivant of these homogeneous polysaccharides are obtained by the method for chemical modification；It is verified by experiments, the cordate houttuynia homogeneous polysaccharide and its derivative have significant inhibiting effect to the classical pathway and alternative pathway of complement activation, can be further used as active constituent, prepare new anti-complement medicine.
Description
Technical field
The invention belongs to the field of Chinese medicines, are related to cordate houttuynia polysaccharide, and in particular to natural homogeneous polysaccharide and its derivative in cordate houttuynia
Object, and preparation method thereof and preparing the purposes in anticomplement medicament.
Background technique
The normal Activate of complement system eliminate external microorganism, remove damaged in human body or the cell of death and tissue,
It maintains to play an important role in the balance of body.However the improper activation of the system can cause that human immune system's is excessive
Reaction, causes the damage of human body itself normal tissue, participates in the pathologic process of a variety of diseases.Studies have shown that and excess complement swash
After relevant chronic disease living has rheumatoid arthritis, senile dementia, systemic loupus erythematosus (SLE) and organ transplant
Rejection etc.；And in multiple organ failure syndrome, as ischemic damage and reperfusion, acute myocardial infarction AMI, acute respiration are embarrassed
Compel in acute illness such as syndrome (ARDS), the excessive activation of complement also plays important function.
Although the immunosuppressor such as current clinically widely used glucocorticoid, cyclophosphamide, the cry of certain animals of methylamine butterfly are to certain
Disease relevant to excess complement activation has certain therapeutic effect a bit, but since such drug is not single-minded complement inhibitor,
Poor selectivity, prolonged application can reduce the defensive enginery of body, anti-infection ability caused to decline, and easy secondary infection and make potential disease
Stove diffusion, generates multiple complications and side effect.Therefore clinically it is badly in need of efficient, low toxicity, single-minded novel complement inhibitor.
It is widely present the active constituent with anticomplementary action in natural drug, part natural drug is completed both at home and abroad such as
The screening of Chinese ephedra, Morinda officinalis, ginseng, Cortex Eucommiae etc. finds certain polysaccharide, protein, polypeptide, the Huang being widely present in nature
The compounds such as ketone, steroid, terpene and alkaloid have significant anti-complement activity, from ginseng, turmeric, Chinese herbaceous peony, radix bupleuri, Rhizoma Chuanxiong
A series of acidic polysaccharoses and neutral polysaccharide for having anti-complement activity are separated in the plants such as Radix Glycyrrhizae.
Cordate houttuynia is the whole herb with root of saururaceae plant houttuynia cordata Houttuynia cordata Thunb., is a kind of normal
Heat-clearing and detoxifying herb, the treatment for the disease such as pneumonia, the infection of the upper respiratory tract, influenza, hepatitis.Pharmacological research shows fish raw meat
Grass has the pharmacological actions such as antibacterial, antiviral, analgesia, antitumor, immunological regulation, anti-oxidant, antiallergy, diuresis, myocardial preservation.
The study found that cordate houttuynia Thick many candies anti-complement activity is significant, and has prevention and treatment acute lung injury and alexipyretic effect, but so far, still
Have no the separation preparation to anti-complement activity homogeneous polysaccharide in cordate houttuynia and active report.
Summary of the invention
The object of the present invention is to provide the active constituents in natural drug with anticomplementary action, and in particular to cordate houttuynia is more
Carbohydrates and their derivative and preparation method thereof and preparing the purposes in Complement inhibition drug.
The present invention carries out isolated three homogeneous polysaccharides to the water extract of common heat-clearing and detoxifying herb cordate houttuynia, orders respectively
Entitled HC-PS1, HC-PS2 and HC-PS3, and sulphation, hydroxyethylation have been carried out to three polysaccharide respectively and carboxymethylated spread out
Biochemical analysis, through the experiment in vitro confirmation more carbohydrates and their derivatives of cordate houttuynia with significant complement inhibitory activity, and with
The activity of heparin quite, can be used as anticomplement medicament and be developed.
In the present invention, the Chinese Medicinal Houttuynia Cordata Thunb is Saururaceae Saururaceae heartleaf houttuynia platymiscium heartleaf houttuynia
The herb of Houttuynia cordata Thunb..
The structure feature of cordate houttuynia polysaccharide HC-PS1, HC-PS2 and HC-PS3 of the present invention are described as follows:
(1) HC-PS1 is the heteroglycan being mainly made of seven kinds of monosaccharide, and saccharide residue molar ratio is rhamnose (Rha): I
Uncle is sugared (Ara): mannose (Man): glucose (Glc): glucuronic acid (GlcA): galactolipin (Gal): galacturonic acid
(GalA)=1.0:3.0:3.9:2.5:2.4:6.1:3.0；Molecular weight is 274530Da；Specific rotation [α]D 25=-30.0 (c
0.3,H2O)；Protein content: 1.41%；Glucuronic acid content: 24.08%；Sugared content 94.60%；Without sulfate；It is main
Connection type has: end, arabinose of 1,5- connection etc., 1,3,6- connection and Isosorbide-5-Nitrae, the mannose etc. of 6- connection, end,
Isosorbide-5-Nitrae-connection, 1,3- connection, 1,3,6- connections, Isosorbide-5-Nitrae, the glucose etc. of 6- connection, end, Isosorbide-5-Nitrae-connection and 1, the half of 6- connection
Lactose etc., Isosorbide-5-Nitrae-connection galacturonic acid etc., the rhamnose additionally containing the connection of a small amount of end, wherein with end connection
Based on arabinose, Isosorbide-5-Nitrae, the mannose of 6- connection and end, Isosorbide-5-Nitrae-connection galactolipin (or galacturonic acid)；
(2) HC-PS2 is the heteroglycan being mainly made of five kinds of monosaccharide, and saccharide residue molar ratio is Man:Glc:GlcA:
Gal:GalA=1.0:0.3:1.7:0.7:4.6；Molecular weight is 7928Da；Specific rotation [α]D 25=-12.8 (c 0.3, H2O)；Egg
Bai Hanliang: 1.89%；Glucuronic acid content: 65.64%；Sugared content 93.10%；Without sulfate；Its main connection type has: end
Hold the arabinose etc. of connection；End, 1,6- connection and Isosorbide-5-Nitrae, the mannose etc. of 6- connection, Isosorbide-5-Nitrae-connection, 1,3- connection and 1,
The glucose etc. of 6- connection, Isosorbide-5-Nitrae-connection glucuronic acid etc., end, Isosorbide-5-Nitrae-connection, 1,3,4- connections, Isosorbide-5-Nitrae, 6- connection,
The galactolipin etc. of 1,3,6- connection, end, Isosorbide-5-Nitrae-connection, 1, the galacturonic acid etc. of 3,4- connections, additionally containing a small amount of end
The rhamnose of connection is held, wherein connecting with Isosorbide-5-Nitrae-connection glucose (or glucuronic acid) and end, Isosorbide-5-Nitrae-connection, 1,3,4-
Based on the galactolipin (or galacturonic acid) connect；
(3) HC-PS3 is the heteroglycan being mainly made of five kinds of monosaccharide, and saccharide residue molar ratio is Man:Glc:GlcA:
Gal:GalA=1.0:0.5:0.4:1.2:0.4；Molecular weight is 216384Da；Specific rotation [α]D 25=-62.8 (c 0.3,
H2O)；Protein content: 1.53%；Glucuronic acid content: 20.19%；Sugared content 93.73%；Without sulfate；Its main connection side
Formula has: end, arabinose of 1,5- connection etc.；The mannose etc. that 1,3,4- connection, 1,4,6- connection are connected with 1,3,6-；End
The glucose etc. that end, 1,4- connection, 1,3- connection are connected with 1,4,6-；End, 1,4- connection, galactolipin of 1,6- connection etc.；
Rhamnose additionally containing the connection of a small amount of end.The mannose wherein connected with 1,3,4-, Isosorbide-5-Nitrae-connection glucose and 1,
Based on the galactolipin of 4- connection；
The cordate houttuynia polysaccharide HC-PS1, cordate houttuynia polysaccharide HC-PS2, cordate houttuynia polysaccharide HC-PS3 pass through following steps
Preparation:
Cordate houttuynia is extracted with 95% ethyl alcohol cold soaking, and filtration is extracted with hot water, is filtered, and is concentrated, and centrifugation, supernatant is added 4 times
95% ethyl alcohol of volume is stood, and supernatant is removed in centrifugation, and precipitating is redissolved with water, is recovered under reduced pressure, and removes ethyl alcohol；Liquid is redissolved again with trichlorine
Acetic acid removes floating preteins, and centrifugation, supernatant is adjusted to neutrality, is concentrated, and dialysis is freeze-dried up to Thick many candies；Thick many candies add distillation
Water dissolution, centrifugation, supernatant use DEAE-cellulose column (Cl by several times-1Type) chromatography carry out initial gross separation, with distilled water,
0.4, the NaCl solution elution of 0.8,1.2 and 2.0 mol/L, collects each fraction, according to the knot of sugared chromogenic reaction and ultraviolet detection
Fruit merges identical flow point, carries out anti-complement activity measurement respectively after concentrated, dialysis and freeze-drying；
The wherein significant component of external anti-complement activity is taken, the NaCl solution of distilled water or 0.15mol/L is added to dissolve, from
The heart, supernatant use Sephacryl S-400 column, Sephacryl S-300 column or Superdex-200 post separation by several times, to steam
Distilled water or the elution of the NaCl solution of 0.15mol/L, collect each fraction, merge phase according to the result of sugared chromogenic reaction and ultraviolet detection
Same flow point, and anti-complement activity tracking is carried out, it is final to obtain the strong homogeneous polysaccharide of anti-complement activity.
Cordate houttuynia polysaccharide (HC-PS1, HC-PS2 and HC-PS3) of the present invention is prepared by following methods:
Cordate houttuynia is extracted with 95% ethyl alcohol cold soaking, and filtration is extracted with hot water, is filtered, and is concentrated, and centrifugation, supernatant is added 4 times
95% ethyl alcohol of volume is stood, and supernatant is removed in centrifugation, and precipitating is redissolved with water, is recovered under reduced pressure, and removes ethyl alcohol；Liquid is redissolved again with trichlorine
Acetic acid removes floating preteins, and centrifugation, supernatant is adjusted to neutrality, is concentrated, and dialysis is freeze-dried up to Thick many candies.Thick many candies add distillation
Water dissolution, centrifugation, supernatant use DEAE-cellulose column (Cl by several times-1Type) chromatography progress initial gross separation.With distilled water,
0.4, the NaCl solution elution of 0.8,1.2 and 2.0 mol/L, collects each fraction, according to the knot of sugared chromogenic reaction and ultraviolet detection
Fruit merges identical flow point, and 5 secondary components: H1, H2, H3, H4 and H5 are dialysed and be freeze-dried and to obtain in concentration.Taking has significant resist
The H3 and H4 of complement activity are further separated；
H3 plus distilled water are dissolved, centrifugation, supernatant uses Sephacryl S-400 column (100 × 2.5cm) to chromatograph by several times
Separation.Water elution is distilled, each flow point is collected；Merge identical flow point according to absorbance and anti-complement activity testing result, concentration and
It is freeze-dried to obtain polysaccharide H3-PS1 and H3-PS2；Add distilled water to dissolve respectively H3-PS1 and H3-PS2, is centrifuged, supernatant difference
Sephacryl S-300 column (100 × 1.6cm) chromatography is used by several times.Water elution is distilled, according to absorbance and anti-complement activity
Testing result merges identical flow point, is concentrated and is freeze-dried and to obtain polysaccharide HC-PS1 and HC-PS2；
The NaCl solution of H4 plus 0.15mol/L is dissolved, centrifugation, supernatant gradation Superdex-200 column (100 ×
1.6cm) chromatography, the NaCl solution elution of 0.15mol/L merge identical according to absorbance and anti-complement activity testing result
Flow point is concentrated and is freeze-dried and to obtain polysaccharide HC-PS1, HC-PS2 and HC-PS3.
The present invention be prepared for respectively the sulphation of cordate houttuynia polysaccharide (HC-PS1, HC-PS2 and HC-PS3), hydroxyethylation and
Carboxyl methylation derivant, and the structure of each derivatization product is relatively demonstrated by IR spectrum；It is confirmed through in vitro test, cordate houttuynia
Polysaccharide HC-PS1, HC-PS2 and HC-PS3 and its derivative are equal to the cell haemolysis that complement is classical and alternative pathway activation is caused
There is apparent inhibition, that is, has apparent anticomplementary action.
In the present invention, the CH of HC-PS1 and its sulphation, hydroxyethylation and carboxyl methylation derivant50It is worth (classical pathway
The concentration of test sample needed for 50% inhibition haemolysis) it is respectively 0.295 ± 0.032mg/mL, 0.128 ± 0.024mg/mL, 0.243
± 0.044mg/mL and 0.241 ± 0.038mg/mL；AP50Value (concentration of test sample needed for alternative pathway 50% inhibits haemolysis) point
It Wei not 0.318 ± 0.023mg/mL, 0.084 ± 0.012 mg/mL, 0.186 ± 0.013mg/mL and 0.085 ± 0.006mg/
mL；
In the present invention, the CH of HC-PS2 and its sulphation, hydroxyethylation and carboxyl methylation derivant50Value is respectively 0.183
± 0.017mg/mL, 0.091 ± 0.020mg/mL, 0.248 ± 0.034mg/mL and 0.202 ± 0.022mg/mL；AP50Value point
It Wei not 0.219 ± 0.018mg/mL, 0.068 ± 0.008mg/mL, 0.118 ± 0.011mg/mL and 0.075 ± 0.008mg/
mL；
In the present invention, the CH of HC-PS3 and its sulphation, hydroxyethylation and carboxyl methylation derivant50Value is respectively 0.271
± 0.026mg/mL, 0.137 ± 0.029mg/mL, 0.299 ± 0.040mg/mL and 0.260 ± 0.014mg/mL；AP50Value point
It Wei not 0.314 ± 0.025mg/mL, 0.080 ± 0.007mg/mL, 0.213 ± 0.010mg/mL and 0.073 ± 0.002mg/mL；
Detailed description of the invention
Fig. 1 is the HPGPC chromatogram of HC-PS1 (A), HC-PS2 (B) and HC-PS3 (C), wherein
300 × 7.6mm of TSK-GEL GMPWXL gel column；Eluent: 0.2M NaCl；Flow velocity: 0.8ml/min.
Fig. 2 is the HPCE chromatogram of HC-PS1 (A), HC-PS2 (B) and HC-PS2 (C), wherein
Efficient capillary tubing string 60cm × 75 μm；Buffer: 0.01mol/L boric acid-KOH pH of buffer 10.
Fig. 3 be sulphation (A), hydroxyethylated (B), carboxymethylated (C) HC-PS1 IR spectrogram.
Fig. 4 be sulphation (A), hydroxyethylated (B), carboxymethylated (C) HC-PS2 IR spectrogram.
Fig. 5 be sulphation (A), hydroxyethylated (B), carboxymethylated (C) HC-PS3 IR spectrogram.
Specific embodiment
Embodiment 1 prepares cordate houttuynia polysaccharide HC-PS1, HC-PS2 and HC-PS3
Herba Houttuyniae 5Kg is crushed, and with the extraction of 95% ethyl alcohol cold soaking, filtration is extracted 3 times with hot water, and filtration merges and extracts
Liquid is concentrated, and 95% ethyl alcohol of 4 times of volumes is added in centrifugation, supernatant, stands, and supernatant is removed in centrifugation, and precipitating is redissolved with water, depressurized back
It receives, removes ethyl alcohol；It redissolves liquid and floating preteins is gone with trichloroacetic acid again, be centrifuged, supernatant is adjusted to neutrality, dialyses, and concentration freezes dry
Dry Thick many candies to obtain the final product.Thick many candies 0.5g adds distilled water to dissolve, and centrifugation, supernatant uses DEAE-cellulose column (Cl by several times-1Type,
30 × 2.5 cm) chromatography progress initial gross separation.With the NaCl solution elution of distilled water, 0.4,0.8,1.2 and 2.0mol/L, elution
Volume is greater than 2 times of column volumes (about 300mL), and flow velocity 0.8mL/min collects each fraction, and pipe detects 490nm (sulfuric acid-phynol
Method colour developing after) under absorbance value.Merge identical flow point according to the result of sugared chromogenic reaction and ultraviolet detection, concentration, dialysis and
It is freeze-dried to obtain 5 secondary components: H1, H2, H3, H4 and H5.Active tracking structure shows that H3 and H4 has good anticomplement living
Property (the CH of H350Value is 0.05 ± 0.011mg/mL；The CH of H450Value is 0.06 ± 0.012mg/mL)；
By H3 (100mg) plus distilled water dissolution, it is centrifuged, supernatant gradation Sephacryl S-400 column (100 ×
2.5cm) chromatography.Water elution is distilled, flow velocity 0.8mL/min collects each flow point, and pipe detects 490nm (sulfuric acid-phynol
After method colour developing) under absorbance value, activity tracks each pipe fraction.Merge identical flow point according to testing result, concentration and freezing are dry
It is dry to obtain polysaccharide H3-PS1 (14.21mg) and H3-PS2 (12.48mg), add distilled water to dissolve respectively H3-PS1 and H3-PS2, from
The heart, supernatant use Sephacryl S-300 column (100 × 1.6cm) chromatography by several times respectively.Water elution is distilled, flow velocity is
0.8mL/min collects each flow point, and pipe detects the absorbance value under 490nm (after sulfuric acid-phynol method colour developing), and activity tracking is each
Pipe fraction merges identical flow point according to testing result, is concentrated and is freeze-dried and to obtain polysaccharide HC-PS1 (3.42 mg) and HC-PS2
(3.11mg)；
By the dissolution of the NaCl solution of H4 (50mg) plus 0.15mol/L, centrifugation, supernatant uses Superdex-200 column by several times
(100 × 1.6cm) chromatography, the NaCl solution elution of 0.15mol/L, flow velocity 0.8mL/min collect each flow point.Pipe
The absorbance value under 490nm (after sulfuric acid-phynol method colour developing) is detected, activity tracks each pipe fraction, merges phase according to testing result
Same flow point is concentrated and is freeze-dried and to obtain polysaccharide HC-PS1 (2.53mg), HC-PS2 (2.48mg) and HC-PS3 (2.56mg)；
HC-PS1, HC-PS2 are detected through High Performance Gel Permeation Chromatography (HPGPC) and high performance capillary electrophoresis (HPCE)
It is uniform ingredient with HC-PS3 (as shown in Fig. 1~2).
The structural characterization of 2 cordate houttuynia polysaccharide (HC-PS1, HC-PS2 and HC-PS3) of embodiment
(1) measurement of molecular weight
Using TSK-GEL GMPWXL gel column (300 × 7.6mm), mobile phase is 0.01mol/L NaCl, flow velocity
0.8mL/min, is calculated by standard curve (Dextran of T series) by 25 DEG C of column temperature, and HC-PS1, HC-PS2 and HC-PS3 points
Son amount is respectively 274530Da；7928Da and 216384Da；
(2) elemental analysis result
HC-PS1:C, 31.99%；H, 5.17%；N, 1.39%,
HC-PS2:C, 39.17%；H, 5.35%；N, 1.87%,
HC-PS3:C, 36.23%；H, 5.21%；N, 1.47%；
(3) specific rotation
HC-PS1:[α]D 20-30.0(c 0.3,H2O)；HC-PS2:[α]D 20-12.8(c 0.3,H2O)；HC-PS3:
[α]D 20-62.8(c 0.3,H2O)；
(4) total reducing sugar, uronic acid, albumen and sulfate assay
It is 94.60% that sulfuric acid-phynol method, which measures HC-PS1 total sugar content,；HC-PS2 total sugar content is 93.10%； HC-
PS3 total sugar content is 93.73%；
The glucuronic acid content that meta-hydroxydiphenyl method measures HC-PS1 is 24.08%；The glucuronic acid content of HC-PS2 is
65.64%；The glucuronic acid content 20.19% of HC-PS3；
The protein content that Coomassie Brilliant Blue measures HC-PS1 is 1.41%；The protein content of HC-PS2 is 1.89%；HC-
The protein content of PS3 is 1.53%；
BaCl2Turbidimetry for Determination HC-PS1, HC-PS2 and HC-PS3 are free of sulfate；
(5) sugared composition analysis
The product that HC-PS1, HC-PS2 and HC-PS3 are obtained through 2mol/L TFA in 110 DEG C of all-hydrolytics respectively successively carries out
NaBH4Reduction, aceticanhydride acetylation are prepared into Alday alcohol acetic ester derivative, carry 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)；
HC-PS1 is the heteroglycan being mainly made of seven kinds of monosaccharide, and saccharide residue molar ratio is Rha:Ara:Man:Glc:
GlcA:Gal:GalA=1.0:3.0:3.9:2.5:2.4:6.1:3.0 additionally contains a small amount of Xyl；
HC-PS2 is the heteroglycan being mainly made of five kinds of monosaccharide, and saccharide residue molar ratio is Man:Glc:GlcA:Gal:
GalA=1.0:0.3:1.7:0.7:4.6 additionally contains a small amount of Rha, Ara and Xyl；
HC-PS3 is the heteroglycan being mainly made of five kinds of monosaccharide, and saccharide residue molar ratio is Man:Glc:GlcA:Gal:
GalA=1.0:0.5:0.4:1.2:0.4 additionally contains a small amount of Rha, Ara and Xyl；
(6) 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) respectively to HC-PS1, HC-PS2 and HC-PS3 into
Row methylation, the 90% formic acid depolymerization of product after methylation, 2mol/L TFA all-hydrolytic, NaBH4Reduction and aceticanhydride acetylation
The Alday alcohol acetic ester derivative of partial methylation is made, then carries out GC-MS analysis；
The judgement of combined standard map, the main connection type of HC-PS1 have: end, arabinose of 1,5- connection etc., and 1,3,
6- connection and Isosorbide-5-Nitrae, the mannose etc. of 6- connection, end, Isosorbide-5-Nitrae-connection, 1,3- connection, 1,3,6- connections, Isosorbide-5-Nitrae, 6- connection
Glucose etc., there is end, Isosorbide-5-Nitrae-connection and 1, the galactolipin etc. of 6- connection, and Isosorbide-5-Nitrae-connection galacturonic acid etc. additionally contains
There is the rhamnose of a small amount of end connection, wherein with the arabinose that end connects, Isosorbide-5-Nitrae, the mannose of 6- connection and end, 1,
Based on the galactolipin (or galacturonic acid) of 4- connection；
The main connection type of HC-PS2 has: the arabinose etc. of end connection, end, 1,6- connection and Isosorbide-5-Nitrae, 6- connection
Mannose etc., Isosorbide-5-Nitrae-connection, 1,3- connection and glucose of 1,6- connection etc., Isosorbide-5-Nitrae-connection glucuronic acid etc., end,
Isosorbide-5-Nitrae-connection, 1,3,4- connections, Isosorbide-5-Nitrae, 6- connection, galactolipin of 1,3,6- connections etc., end, Isosorbide-5-Nitrae-connection, 1,3,4- connections
Galacturonic acid etc., the rhamnose additionally containing the connection of a small amount of end, wherein with Isosorbide-5-Nitrae-connection glucose (or glucose
Aldehydic acid) it is connected with end, 1,4-, based on the galactolipin (or galacturonic acid) of 1,3,4- connection；
The main connection type of HC-PS3 has: end, arabinose of 1,5- connection etc., 1,3,4- connection, Isosorbide-5-Nitrae, 6- connection
With 1, the mannoses etc. of 3,6- connections, end, Isosorbide-5-Nitrae-connection, 1,3- connection and Isosorbide-5-Nitrae, the glucose etc. of 6- connection, end, Isosorbide-5-Nitrae-
Connection, galactolipin of 1,6- connection etc., the rhamnose additionally containing the connection of a small amount of end, wherein with 1, the sweet dew of 3,4- connections
Based on sugar, Isosorbide-5-Nitrae-connection glucose and Isosorbide-5-Nitrae-connection galactolipin.
Embodiment 3 prepares polysaccharide derivates
(1) preparation of sulfated derivative: concentrated sulfuric acid 7.5mL, n-butanol 2.5mL are placed in equipped with condenser pipe and stirring dress
In the three-neck flask set, the ammonium sulfate of 15mg is added, is stirred, ice-water bath is cooled to 0 DEG C；It is slowly added to 50mg polysaccharide powder,
In 0 DEG C of conditioned response 30min.Reaction solution is neutralized through 10%NaOH, centrifuging and taking supernatant, and distilled water dialysis 48h is concentrated under reduced pressure into
Small size is added 4 times of 95% ethyl alcohol of volume, stands overnight, and sediment is collected in centrifugation, and it is derivative that sulphation is obtained after freeze-drying
Object；
(2) preparation of hydroxyethylation product: taking polysaccharide powder 50mg, and 0.5mol/L sodium hydroxide solution 1mL, room temperature is added
Stirring and dissolving, polysaccharide concentration 50mg/mL.Solution is first cooled with an ice bath to 0 DEG C, and the 30%NaOH solution of 3mL is added, then uses ice
Then plus the ethylene oxide of 10mL salt bath is cooled further to -15 DEG C~-10 DEG C, continues at same temperature and is stirred to react 2h,
Then mixture is stood overnight in 4 DEG C of refrigerators.Next day, mixture with 1mol/L HCl neutralize, make solution pH value closely in
Property, distilled water dialysis 3 days, reduced pressure is freeze-dried to obtain hydroxyethylation derivative；
(3) preparation of carboxymethylated product: taking polysaccharide 20mg to be suspended in 1mL isopropanol, and it is equal that 15min dispersion is stirred at room temperature
After even, 0.1mL 30%NaOH solution is slowly added dropwise in 5min, is vigorously stirred 1h, then adjusts mixed system to suitable 70 DEG C,
After 48mg monoxone is added, continue to be stirred to react 3h at a temperature of control.End of reaction is added appropriate distilled water dilution, uses
1mol/L glacial acetic acid adjusts the pH value of mixed liquor to 7~8, and distilled water is dialysed 3 days, is concentrated under reduced pressure, is freeze-dried to obtain carboxy methylation
Derivative；
(4) infrared spectrum measurement: each 1mg of polysaccharide derivates sample is taken, carries out IR detection with pressing potassium bromide troche respectively.Knot
Fruit (attached drawing 3~5) is as it can be seen that the IR spectrum of sulfating product is not only located at 3400cm compared with former polysaccharide-1The O-H key of left and right
Stretching vibration peak narrows diminution, while in 818cm-1And 1232cm-1Occur being attributed to the stretching of C-S-O and S=O key respectively
Vibration performance absorbs, and shows there is sulfate in polysaccharide, by Partial sulphation；Hydroxyethylation product IR spectrum and former polysaccharide phase
Than being located at 3200~3600cm-1The stretching vibration peak of the O-H key in region, which significantly increases, to broaden, while nearly 2900cm-1The C-H at place
Stretching vibration peak also greatly enhances, and shows that ethoxy has been substituted in the part of hydroxyl of saccharide ring；Carboxyl methylation derivant IR figure
Show that is located at a 1736cm in spectrum-1The characteristic absorption for locating carboxyl, shows that carboxymethyl is partially substituted in saccharide ring.
The test of 4 classical pathway Complement inhibition of embodiment
Healthy adult male volunteers sera is taken, with VBS2+(barbitol buffer solution, pH=7.4 contain 0.5mM to buffer
Mg2+With 0.15mM Ca2+) it is diluted to 1:10, as the Complement source of classical pathway, by the antibody of rabbit-anti sheep red blood cell with VBS2+
Buffer is diluted to 1:1000 as hemolysin；The sheep red blood cell (SRBC) being stored in Alsever liquid is configured to 2%SRBC；
VBS is added in precision weighing polysaccharide or derivatives thereof about 2mg2+Buffer solution, is diluted to 8 concentration, the polysaccharide of various concentration or
The 100 μ L of complement of its derivative solution 100 μ L and 1:10 sequentially add 200 μ LVBS after 37 DEG C of preincubate 10min2+Buffering
Liquid, 100 μ L hemolysins (1:1000) and 100 μ L 2%SRBC, are put into low-temperature and high-speed centrifuge after 37 DEG C of water-bath 30min,
5000rpm, 10min is centrifuged under the conditions of 4 DEG C, takes every 200 μ L of pipe supernatant in 96 orifice plates respectively, measure absorbance in 405nm, it is real
Testing while polysaccharide is arranged or derivatives thereof control group, (polysaccharide of 100 μ L respective concentrations or derivatives thereof solution adds 500 μ L VBS2+
Buffer), complement control group is (with 100 μ L VBS2+Buffer replaces polysaccharide or derivatives thereof solution) and full haemolysis group (100 μ L
2%SRBC is dissolved in 500 μ L tri-distilled waters), by polysaccharide of each concentration or derivatives thereof group absorbance value deduct corresponding polysaccharide or its
Haemolysis inhibiting rate is calculated after derivative control group absorbance value, using the logarithm of polysaccharide or derivatives thereof concentration as X-axis, haemolysis suppression
Rate processed is mapped as Y-axis, the concentration (CH of test sample needed for obtained fitting a straight line calculates 50% inhibition haemolysis50Value), with heparin
As positive control drug, three homogeneous polysaccharides and its derivative have significant suppression for classical pathway of complement activation as the result is shown
Make active (as shown in table 1).
The test of 5 alternative pathway Complement inhibition of embodiment
Healthy adult male volunteers sera is taken, (barbitol buffer solution, pH=7.4 contain with VBS-Mg-EGTA buffer
5mM Mg2+It is diluted to 1:10 with 8mM EGTA), as the Complement source of alternative pathway, is stored in 3.8% liquor sodii citratis
Rabbit erythrocyte is configured to 2% rabbit erythrocyte with VBS-Mg-EGTA buffer, and precision weighing polysaccharide or derivatives thereof about 2mg is added
VBS-Mg-EGTA buffer is diluted to 8 concentration, the complement of polysaccharide of various concentration or derivatives thereof solution 150 μ L and 1:10
150 μ L are added 200 μ L, 2% rabbit erythrocyte, are put into low-temperature and high-speed after 37 DEG C of water-bath 30min after 37 DEG C of preincubate 10min
Centrifuge is centrifuged 10min under the conditions of 5000rpm, 4 DEG C.It takes every 200 μ L of pipe supernatant in 96 orifice plates respectively, is measured in 405nm
(polysaccharide of 150 μ L respective concentrations or derivatives thereof solution adds for absorbance, experiment while the control group that polysaccharide is arranged or derivatives thereof
350 μ L VBS-Mg-EGTA buffers), complement control group (with 150 μ L VBS-Mg-EGTA buffers replace polysaccharide or its derivative
Object solution) and full haemolysis group (200 μ L, 2% rabbit erythrocyte is dissolved in 300 μ L tri-distilled waters), by the polysaccharide of each concentration or its derivative
Object group absorbance value calculates haemolysis inhibiting rate after deducting corresponding polysaccharide or derivatives thereof control group absorbance value, with polysaccharide or its spread out
The logarithm of biological concentration is mapped as X-axis, haemolysis inhibiting rate as Y-axis, and obtained fitting a straight line calculates 50% and inhibits haemolysis institute
Need the concentration (AP of test sample50Value).Using heparin as positive control drug, more carbohydrates and their derivatives can obviously inhibit to mend as the result is shown
Cell haemolysis (as shown in table 1) caused by the activation of body alternative pathway.
Inhibiting effect of the 1 three kinds of more carbohydrates and their derivatives of cordate houttuynia of table to complement activation
CH50And AP50Value expression are as follows: average value ± SD (n=3).
Claims (2)
1. cordate houttuynia polysaccharide is preparing the purposes in Complement inhibition drug；
The cordate houttuynia polysaccharide and its structure are respectively as follows:
Cordate houttuynia polysaccharide HC-PS1, structure feature are as follows: the heteroglycan being mainly made of seven kinds of monosaccharide, saccharide residue molar ratio are mouse
Lee's sugar: arabinose: mannose: glucose: glucuronic acid: galactolipin: galacturonic acid=1.0:3.0:3.9:2.5:
2.4: 6.1: 3.0；The arabinose that connection type is connected with end, Isosorbide-5-Nitrae, the mannose of 6- connection and end, Isosorbide-5-Nitrae-connection
Galactolipin or galacturonic acid based on；Molecular weight is 274530 Da；Specific rotation [α]D 25= -30.0 (c 0.3, H2O)；Egg
Bai Hanliang: 1.41%；Glucuronic acid content: 24.08%；Sugared content 94.60%；Without sulfate；
Cordate houttuynia polysaccharide HC-PS2, structure feature are as follows: the heteroglycan being mainly made of five kinds of monosaccharide, saccharide residue molar ratio are sweet
Dew sugar: glucose: glucuronic acid: galactolipin: galacturonic acid=1.0:0.3:1.7:0.7:4.6；Connection type with 1,
The glucose or glucuronic acid of 4- connection are connected with end, 1,4-, based on the galactolipin or galacturonic acid of 1,3,4- connection；
Molecular weight is 7928 Da；Specific rotation [α]D 25= -12.8 (c 0.3, H2O)；Protein content: 1.89%；Glucuronic acid content:
65.64%；Sugared content 93.10%；Without sulfate；
Cordate houttuynia polysaccharide HC-PS3, structure feature are as follows: the heteroglycan being mainly made of five kinds of monosaccharide, saccharide residue molar ratio are sweet
Dew sugar: glucose: glucuronic acid: galactolipin: galacturonic acid=1.0:0.5:0.4:1.2:0.4；Connection type with 1,
The mannoses of 3,4- connections, based on Isosorbide-5-Nitrae-connection glucose and Isosorbide-5-Nitrae-connection galactolipin；Molecular weight is 216384 Da；Than
Curl [α]D 25= -62.8 (c 0.3, H2O)；Protein content: 1.53%；Glucuronic acid content: 20.19%；Sugared content 93.73%；
Without sulfate；
The cordate houttuynia polysaccharide is prepared by following steps:
Cordate houttuynia is extracted with 95% ethyl alcohol cold soaking, and filtration is extracted with hot water, is filtered, and is concentrated, and 4 times of volumes are added in centrifugation, supernatant
95% ethyl alcohol, stand, supernatant is removed in centrifugation, and precipitating is redissolved with water, is recovered under reduced pressure, and removes ethyl alcohol；Liquid is redissolved to go again with trichloroacetic acid
Floating preteins, centrifugation, supernatant are adjusted to neutrality, are concentrated, and dialysis is freeze-dried up to Thick many candies；Thick many candies add distilled water to dissolve,
Centrifugation, supernatant use DEAE-cellulose column (Cl by several times-1 Type) chromatography carry out initial gross separation, with distilled water, 0.4,0.8,
The NaCl solution of 1.2 and 2.0 mol/L elutes, and collects each fraction, merges phase according to the result of sugared chromogenic reaction and ultraviolet detection
Same flow point carries out anti-complement activity measurement after concentrated, dialysis and freeze-drying respectively；
The wherein significant component of external anti-complement activity is taken, the NaCl solution of distilled water or 0.15 mol/L is added to dissolve, centrifugation, on
Clear liquid uses Sephacryl S-400 column, Sephacryl S-300 column or Superdex-200 post separation by several times, with distilled water or
The NaCl solution of 0.15 mol/L elutes, and collects each fraction, merges phase cocurrent flow according to the result of sugared chromogenic reaction and ultraviolet detection
Point, and anti-complement activity tracking is carried out, it is final to obtain the strong homogeneous polysaccharide of anti-complement activity.
2. purposes according to claim 1, which is characterized in that sulphation, hydroxyethylation or the carboxylic of the cordate houttuynia polysaccharide
Methylated derivative is preparing the purposes in Complement inhibition drug.
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Non-Patent Citations (2)
| Title |
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| Structural characterization and immunomodulatory effect of a polysaccharide HCP-2 from Houttuynia cordata;Cheng等;《Carbohydrate Polymers》;20140315;第103卷;摘要，第245页左栏第8段-右栏第1段 |
| 鱼腥草抗补体活性多糖的制备工艺研究;张娟娟等;《中国中药杂志》;20120715;第37卷(第14期);摘要 |
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