CN101390868B - Plant polysaccharide and preparation method and use thereof - Google Patents

Abstract

The invention belongs to the Chinese medicine field, in particular to a plant polysaccharide PS-1, the preparation method thereof and the application in the preparation of anti-complement medicine. The invention obtains PS-1 through separating and extracting from the water extract of Chinese herbal compound used for preventing severe acute respiratory syndrome (SARS) and composed of houttuynia cordata, chrysanthemum indicum L, artemisia capillaries, eupatorium fortune and Amomum tsao-ko in the proportion of 15:6:15:10:3; as proven in the experiment that PS-1 respectively has inhibition effect on the classical pathway and alternative pathway of the activation of complement, acts on the ingredients of C1q, C1r, C1s, C2, C3,C4,C5 and C9, and has no anticoagulative effect. The plant polysaccharide PS-1 can be further used as an active ingredient for the preparation of new anti-complement medicine.

Description

A kind of vegetable polysaccharides

Technical field

The invention belongs to the field of Chinese medicines, relate to vegetable polysaccharides, be specifically related to a kind of vegetable polysaccharides and preparation method thereof and the purposes in the preparation anticomplement medicament.

Background technology

Under normal physiological condition, the function of complement mainly is to attack external cause of disease and remove immune complex, and keeps the balance of body.Yet the improper activation of complement system can cause human immune system's overreaction, causes the damage and the inflammatory reaction of human body self normal structure.Research shows, the disease relevant with the complement excessive activation has rejection after rheumatoid arthritis, alzheimer disease, systemic lupus erythematosus (sle) (SLE), the organ transplantation, multiple organ dysfunction syndrome syndrome, adult respiratory distress syndrome (ARDS) etc.It is reported; Serious symptom severe acute respiratory syndrome (SARS) and the bird flu that causes by H5N1 type viral infection; Can find immune overreaction symptom clinically; Like ARDS, septic shock and Reyes syndrome etc. have research to think that above-mentioned reaction symptom and cellular immunization, humoral immunization excessive activation are relevant.Existing research confirms that the excessive activation of SARS and complement system is relevant; Though do not prove at present the excessive activation that can cause complement system behind the highly pathogenic H5N1 type avian influenza people as yet on evidence; Yet existing research shows influenza infection activating complement alternative pathway, in the generation of viral respiratory system disease, plays an important role.In view of the important function of complement excessive activation in causing human many critical illness, therefore be badly in need of seeking the novel complement inhibitor of high-efficiency low-toxicity.

Extensively there is active component in the natural drug with anticomplementary action; Part natural drug such as Herba Ephedrae have been accomplished both at home and abroad; Radix Morindae Officinalis, Radix Ginseng, the screening of the Cortex Eucommiae etc.; Finding that the chemical compounds such as some polysaccharide, protein, polypeptide, flavone, steroid class, terpenoid and alkaloid that occurring in nature extensively exists have significant ACA, is many with glycosylated protein and polysaccharide chemical compound wherein.Heparin is a sulfated polysaccharides that is polymerized by alduronic acid and glucosamine, is that research is early, more sophisticated complement inhibitor.Just have the bibliographical information heparin that complement system is had inhibitory action as far back as nineteen twenty-nine, and its mechanism of action is furtherd investigate, the discovery heparin can be had an effect with 13 kinds of compositions in the complement system.But because heparin has blood coagulation resisting function, need higher concentration competence exertion drug effect in vivo, and side effect is big, has limited its clinical practice.The low-molecular-weight sulfated fucan of from the Sargassum of brown, telling (Fucans), because of its ACA is 5 times of heparin, anticoagulant active but be merely heparin 5～10% and receive much attention.Fucans all has the obvious suppression effect, its IC at external classical pathway and alternative pathway to complement activation ₅₀And AP ₅₀Be respectively 0.8mg/mL and 0.54mg/mL.Its mechanism of action discovered that it can act on complement components such as C1, C4, C2.The fucoidan (Fucoidan) that other has bibliographical information from brown algae Ascophyllumnodosum, to tell also has stronger anticomplementary action (IC ₅₀＜0.5mg/mL), pharmacological research shows that this polysaccharide is to combine C1q through competing with the complement activation agent, reduces the ability that C1 splits branch C2, C4, thereby the inhibition complement activation.In addition, also from plants such as several plants of Malvaceae, Radix Ginseng, Rhizoma Curcumae Longae, Radix Paeoniae, Radix Bupleuri, Rhizoma Chuanxiong and Radix Glycyrrhizae, tell a series of acidic polysaccharose and neutral polysaccharides that ACA is arranged, and illustrated its structure.

Summary of the invention

The purpose of this invention is to provide the active component that has anticomplementary action in the natural drug, be specifically related to a kind of vegetable polysaccharides PS-1, and preparation method thereof and suppress the purposes in the medicine at the preparation complement.

In view of complement takes place at SARS and bird flu complication-ARDS, developing important function; The water extract that the present invention recommends to be used to prevent and treat the Chinese medicine compound of serious symptom severe acute respiratory syndrome (SARS) to State Administration of Traditional Chinese Medicine separates and obtains homogeneous polysaccharide, called after PS-1.Have significant complement through experiment in vitro confirmation PS-1 and suppress active, and similar with the mechanism of action of heparin, but do not possess the blood coagulation resisting function that does not limit its performance activity in vivo, indicating that the PS-1 side effect is little, have the interior ACA of body preferably.

The present invention obtains homogeneous polysaccharide PS-1 from Chinese medicine compound (State Administration of Traditional Chinese Medicine's recommendation) separation and Extraction that is used for preventing and treating serious symptom severe acute respiratory syndrome (SARS).

Described Chinese medicine compound is made up of Herba Houttuyniae, Flos Chrysanthemi Indici, Herba Artemisiae Scopariae, Herba Eupatorii, Fructus Tsaoko, and its part by weight is 15: 6: 15: 10: 3.

The architectural feature of vegetable polysaccharides PS-1 of the present invention is described as: the heteropolysaccharide that PS-1 is made up of five kinds of monosaccharide, saccharide residue mol ratio are rhamnose (Rha): arabinose (Ara): mannose (Man): glucose (Glc): galactose (Gal)=1.3: 1.9: 1.0: 8.8: 4.8; Its molecular weight is greater than 1 * 10 ⁶Da is less than 2 * 10 ⁶Da, specific optical rotation are [α] _D ²⁵+ 14.2 (c0.318, H ₂O); The sugared content of PS-1 is 90.9%, also has 4.16% alduronic acid, 2.79% albumen and 4.30% sulfate; Its connected mode comprises end, 1,4-, 1,6-and 1,3, the glucose that 6-connects, end, 1; 6-and 1,3, the galactose that 6-connects, end, 1, the arabinose that 5-connects; Terminal, 1, the rhamnose that 3-connects, 1,6-, 1, the mannose that 4-connects with some 1; 3-, 1,3,4-, 1,4, the hexose that 6-connects.Wherein with 1,4-and 1, the glucose that 6-connects is main.

Polysaccharide PS-1 of the present invention prepares through following method:

Herba Houttuyniae, Flos Chrysanthemi Indici, Herba Artemisiae Scopariae, Herba Eupatorii, Fructus Tsaoko Chinese medicine of the five flavours are 15: 6: 15 by its proportion of composing: 10: 3 compatibilities, extract with 95% ethanol merceration, and filter, with hot water extraction 3 times; Filter, merge extractive liquid, concentrates, and is centrifugal; Supernatant removes floating preteins with trichloroacetic acid, and is centrifugal, the dialysis of supernatant water; Dialysis solution adds ethanol to containing alcohol amount 80% after being concentrated into small size, and centrifugal, the deposition lyophilization promptly gets crude polysaccharides.The dissolving of crude polysaccharides adding distil water, centrifugal, the supernatant gradation is with DEAE-cellulose post (Cl ^-1Type, 30 * 2.5cm) chromatographies carry out initial gross separation.With distilled water, 0.4,0.6,1.0 and the NaCl eluant solution of 2.0mol/L, elution volume is greater than 2 times of column volumes, and flow velocity is 0.6mL/min, collects each stream part, and pipe detects the absorbance under 280nm and the 490nm (after the sulfuric acid-phynol method colour developing).Result according to sugared chromogenic reaction and ultraviolet detection merges identical flow point, concentrates, and dialysis and lyophilization get 5 secondary component: Fr1, Fr2, Fr3, Fr4 and Fr5.With Fr3, Fr4 and Fr5 merge the adding distil water dissolving according to active tracking results, and centrifugal, the supernatant gradation is with Sephacryl S-400 post (100 * 2.5cm) chromatography.The distilled water eluting, flow velocity is 0.6mL/min, collects each flow point.Pipe detects the absorbance under 280nm and the 490nm (sulfuric acid-phynol method colour developing back), and active the tracking respectively managed fraction.Merge identical flow point according to testing result, concentrated and lyophilization gets polysaccharide PS-1.Through efficient gel permeation chromatography (HPGPC) and high performance capillary electrophoresis (HPCE) detection PS-1 is the composition of homogeneous.

The architectural feature of PS-1:

1) mensuration of molecular weight

Employing TSK-GEL GMPWXL gel column (300 * 7.6mm), mobile phase is 0.01mol/L NaCl, flow velocity 0.8mL/min, and 25 ℃ of column temperatures, the Dextran serial with T compares, and the PS-1 molecular weight is greater than 1 * 10 ⁶Da, and less than 2 * 10 ⁶Da.

2) results of elemental analyses shows that PS-1 contains C, 34.91%; H, 4.95%; N, 2.55%.Specific optical rotation is [α] _D ²⁵+ 14.2 (c 0.318, H ₂O).

3) total sugar content of sulfuric acid-phynol method mensuration PS-1 is 90.9%, and the glucuronic acid content that a hydroxyl biphenyl method is measured PS-1 is 4.16%, and the protein content that the Coomassie brilliant blue method is measured PS-1 is 2.79%, BaCl ₂The sulfate content of turbidimetry for Determination PS-1 is 4.30%.

4) sugared composition analysis

The product that PS-1 obtains in 110 ℃ of all-hydrolytics through 2mol/L TFA successively carries out NaBH ₄Reduction; The acetic anhydride acetylation is prepared into ALDI alcohol acetic ester derivant; 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.1983,113:29-299).The result can know the heteropolysaccharide that PS-1 is made up of five kinds of monosaccharide, and the saccharide residue mol ratio is rhamnose (Rha): arabinose (Ara): mannose (Man): glucose (Glc): galactose (Gal)=1.3: 1.9: 1.0: 8.8: 4.8.

5) methylation analysis

PS-1 reference literature method (Needs PW; Selvendran RR.Avoiding oxidativedegradation during sodium hydroxyl/dimethyl-iodide mediatedcarbohydrate methylation in dimethyl sulfoxide.Carbohydr Res.1993; 245:1-10) methylate; Product after methylating is with 90% formic acid depolymerization, 2mol/L TFA all-hydrolytic, NaBH ₄The ALDI alcohol acetic ester derivant of part methylization is processed in reduction and acetic anhydride acetylation, carries out GC-MS then and analyzes.The combined standard collection of illustrative plates confirms that the PS-1 connected mode comprises end, 1,4-, 1,6-and 1,3, the glucose that 6-connects; End, 1,6-and 1,3, the galactose that 6-connects, end, 1, the arabinose that 5-connects; Terminal, 1, the rhamnose that 3-connects, 1,6-, 1, the mannose that 4-connects with some 1; 3-, 1,3,4-, 1,4, the hexose that 6-connects.Wherein with 1,4-and 1, the glucose that 6-connects is main.

Confirm that through in vitro tests PS-1 activates the cell haemolysis that is caused to classical and alternative pathway inhibition is all arranged.

1) anticomplementary activates classical pathway cell hemolytic test

1: 10 diluent of human serum is as complement; The antigenic activation classical pathway of complement causes sheep red blood cell haemolysis, reference literature method (Kabat, E.A.; Mayer; M.M..Complement and complementfixation in experimental immunology.Charles C.Thomas Publisher, Ilinois U.S.A.1964.pp.133-240.) records PS-1 and can suppress cell haemolysis.CH ₅₀?＝0.36mg/mL，n＝3。

2) anticomplementary activates 1: 10 diluent of alternative pathway cell hemolytic test human serum as complement, and the activating complement alternative pathway causes rabbit erythrocyte haemolysis, reference literature method (Klerx; J.P., Beukelman, C.J.; Van; D.H., Willers, J.M..Microassay for colorimetric estimationof complement activity in guinea pig; Human and mouse serum.Journalof Immunological Methods.1983.63 215-220) records PS-1 and can suppress cell haemolysis.AP ₅₀＝0.17mg/mL，n＝3。

The present invention utilizes complement disappearance serum, and the reference literature method (Zhou Jie, Zhang Yun is firm; Zhang Jianwen; Xu Han, Chen Daofeng. the Chinese medicine Cortex Eucommiae is to the effect of complement system. and Fudan Journal (medicine) .2006,33 (1): 10-106) research PS-1 suppresses the action target spot of complement system.The result shows that PS-1 acts on C1Q., C1r, C1s, C2, C3, C4, C5, C9 component, and is consistent with the mechanism of action of heparin.

Reference literature method of the present invention (Wang Xuefeng, Wang Hongli. thrombosis and hemostatic detect and use (front page). Shanghai: Shanghai world book publishing company, 2002; P:99); Measure recalcification time (recalcificationtime, RT) and thrombin time (thrombin time, TT); With the positive contrast of 5.2mg/L heparin, VBS ²⁺The negative contrast of buffer (Vehicle), PS-1 concentration is followed successively by 1500,750,375mg/L.But the result shows heparin significant prolongation RT and TT (P＜0.05) when 5.2mg/L, and the PS-1 of variable concentrations and the RT of negative control and TT do not have significant difference.Show that PS-1 does not have blood coagulation resisting function.

Homogeneous polysaccharide PS-1 according to the invention confirms to have strong ACA through experiment in vitro, acts on C1Q., C1r, C1s, C2, C3, C4, C5, C9 component, and does not have the blood coagulation resisting function that influences the activity in vivo performance.Can be used for preparing complement and suppress medicine.

The specific embodiment

Embodiment 1 preparation polysaccharide PS-1

Herba Houttuyniae, Flos Chrysanthemi Indici, Herba Artemisiae Scopariae, Herba Eupatorii, Fructus Tsaoko Chinese medicine of the five flavours are 15: 6: 15 by its proportion of composing: 10: 3 compatibilities amount to 9.6Kg.Extract with 95% ethanol merceration, filter, medicinal residues dry in room temperature underlying ventilation, use hot water extraction then 3 times; Filter, merge extractive liquid, concentrates, and is centrifugal; Supernatant removes floating preteins with trichloroacetic acid, and is centrifugal, supernatant water dialysis 3 days; Dialysis solution adds ethanol to containing alcohol amount 80% after being concentrated into small size, and centrifugal, the deposition lyophilization promptly gets crude polysaccharides.Crude polysaccharides adds an amount of dissolved in distilled water, and is centrifugal, and the supernatant gradation is with DEAE-cellulose post (Cl ^-1Type, 30 * 2.5cm) chromatographies carry out initial gross separation.With distilled water, 0.4,0.6,1.0 and the NaCl eluant solution of 2.0mol/L; The elution volume of every kind of eluent is slightly larger than 2 times of column volumes; Flow velocity is 0.6mL/min, collects each stream part respectively, and pipe detects the absorbance under 280nm and the 490nm (sulfuric acid-phynol method colour developing back).And the result according to sugared chromogenic reaction and ultraviolet detection merges identical flow point, and through concentrating, dialysis and lyophilization obtain 5 secondary component: Fr1, Fr2, Fr3, Fr4 and Fr5.Active tracking results shows Fr3, and three positions of Fr4 and Fr5 have ACA.With Fr3, Fr4 and Fr5 merge the back and add an amount of dissolved in distilled water, and centrifugal, (100 * 2.5cm) chromatographies separate with Sephacryl S-400 post in the supernatant gradation.As eluent, flow velocity is 0.6mL/min with distilled water, collects each flow point.Pipe detects the absorbance under 280nm and the 490nm (sulfuric acid-phynol method colour developing back), and active simultaneously the tracking respectively managed fraction.Merge identical flow point according to testing result, concentrated and lyophilization obtains PS-1.Through efficient gel permeation chromatography (HPGPC) and high performance capillary electrophoresis (HPCE) detection PS-1 is the composition of homogeneous.

The architectural feature of PS-1 is described as: the heteropolysaccharide that PS-1 is made up of five kinds of monosaccharide, saccharide residue mol ratio are rhamnose (Rha): arabinose (Ara): mannose (Man): glucose (Glc): galactose (Gal)=1.3: 1.9: 1.0: 8.8: 4.8.Its molecular weight is greater than 1 * 10 ⁶Da, and less than 2 * 10 ⁶Da, specific optical rotation [α] _D ²⁵+ 14.2 (c0.318, H ₂O).The sugared content of PS-1 is 90.9%, also has 4.16% alduronic acid, 2.79% albumen and 4.30% sulfate.Its connected mode comprises end, 1,4-, 1,6-and 1,3, the glucose that 6-connects, end, 1; 6-and 1,3, the galactose that 6-connects, end, 1, the arabinose that 5-connects; Terminal, 1, the rhamnose that 3-connects, 1,6-, 1, the mannose that 4-connects with some 1; 3-, 1,3,4-, 1,4, the hexose that 6-connects.Wherein with 1,4-and 1, the glucose that 6-connects is main.

Embodiment 2 classical pathway complement inhibition tests

Get the volunteer of NAM serum, with VBS ²⁺(barbitol buffer solution, pH=7.4 contain 0.5mM Mg to buffer ²⁺With 0.15mM Ca ²⁺) dilution is 1: 10, as the complement of classical pathway source.With the antibody of the anti-sheep red blood cell of rabbit with VBS ²⁺Buffer dilution be 1: 1000 as hemolysin; The sheep red blood cell (SRBC) that is stored in the Alsever liquid is configured to 2%SRBC.The about 1mg of precision weighing PS-1 adds VBS ²⁺The buffer dissolving is diluted to 8 concentration.The PS-1 solution 100 μ L of variable concentrations and 1: 10 complement 100 μ L add 200 μ L VBS successively behind 37 ℃ of preincubate 10min ²⁺Buffer, 100 μ L hemolysins (1: 1000) and 100 μ L2%SRBC put into the low-temperature and high-speed centrifuge behind 37 ℃ of water-bath 30min, centrifugal 10min under 5000rpm, 4 ℃ of conditions.Get every pipe supernatant 200 μ L respectively in 96 orifice plates, measure absorbance at 405nm.Experiment is provided with the PS-1 matched group simultaneously, and (the PS-1 solution of 100 μ L respective concentration adds 500 μ L VBS ²⁺Buffer), the complement control group is (with 100 μ L VBS ²⁺Buffer replaces PS-1 solution) and full haemolysis group (100 μ L 2%SRBC are dissolved in the 500 μ L tri-distilled waters).Calculate the haemolysis suppression ratio after the PS-1 of each concentration group absorbance deducted corresponding PS-1 matched group absorbance.As the X axle, the haemolysis suppression ratio is as the mapping of Y axle with the logarithm of PS-1 concentration, and the fitting a straight line that obtains is calculated CH ₅₀Value.As positive control drug, the result shows that PS-1 can suppress classical pathway of complement and activate the cell haemolysis (table 1) that is caused with heparin.Table 1 is the inhibitory action of PS-1 to complement.

Embodiment 3 alternative pathway complement inhibition tests

Get the volunteer of NAM serum, (barbitol buffer solution, pH=7.4 contain 5mM Mg with the VBS-Mg-EGTA buffer ²⁺With 8mM EGTA) dilution is 1: 10, as the complement of alternative pathway source.The rabbit erythrocyte that is stored in 3.8% liquor sodii citratis is configured to 2% rabbit erythrocyte with the VBS-Mg-EGTA buffer.The about 1mg of precision weighing PS-1 adds the VBS-Mg-EGTA buffer, is diluted to 8 concentration.The PS-1 solution 150 μ L of variable concentrations and 1: 10 complement 150 μ L add 200 μ L2% rabbit erythrocytes behind 37 ℃ of preincubate 10min, behind 37 ℃ of water-bath 30min, put into the low-temperature and high-speed centrifuge, centrifugal 10min under 5000rpm, 4 ℃ of conditions.Get every pipe supernatant 200 μ L respectively in 96 orifice plates, measure absorbance at 405nm.Experiment is provided with PS-1 matched group (the PS-1 solution of 150 μ L respective concentration adds 350 μ L VBS-Mg-EGTA buffer), complement control group (replacing PS-1 solution with 150 μ L VBS-Mg-EGTA buffer) and full haemolysis group (200 μ L, 2% rabbit erythrocyte is dissolved in the 300 μ L tri-distilled waters) simultaneously.Calculate the haemolysis suppression ratio after the PS-1 of each concentration group absorbance deducted corresponding PS-1 matched group absorbance.As the X axle, the haemolysis suppression ratio is as the mapping of Y axle with the logarithm of PS-1 concentration, and the fitting a straight line that obtains is calculated AP ₅₀Value.As positive control drug, the result shows that PS-1 can suppress alternative pathway of complement and activate the cell haemolysis (table 1) that is caused with heparin.

Table 1

?	CH 50(mg/mL)	AP 50(mg/mL)
			The PS-1 heparin	0.36±0.050.11±0.03	0.17±0.0160.047±0.002

CH ₅₀And AP ₅₀Value is expressed as: meansigma methods ± SD (n=3).

Embodiment 4 PS-1 are to the action target spot of complement system

People source complement (1: 10) 0.2mL respectively with C3, the C4 antiserum of 0.2mL 1: 1 dilution, C1r, C1s, the C5 antiserum of dilution in 1: 32, the C1q of dilution in 1: 64, C2, C9 antiserum mix, behind 37 ℃ of water-bath 15min, 5000rpm, centrifugal 10min.Supernatant is complement disappearance serum.The target spot test set: complement (1: 10) 100 μ L and concentration are PS-1 solution (suppressing the required least concentration of complement hemolysis near 100%) the 100 μ L mixings of 1.19mg/mL; Behind 37 ℃ of preparatory water-bath 10min; Add disappearance serum 200 μ L, 1: 1000 hemolysin 100 μ L and 2%SRBC100 μ L.Behind 37 ℃ of water-bath 30min, put into the low-temperature and high-speed centrifuge, centrifugal 10min under 5000rpm, 4 ℃ of conditions.Get every pipe supernatant 200 μ L respectively in 96 orifice plates, measure absorbance at 405nm.Experiment is provided with the PS-1 matched group simultaneously, and (the PS-1 solution of 100 μ L respective concentration adds 500 μ L VBS ²⁺Buffer), the complement control group is (with 300 μ L VBS ²⁺Buffer replaces PS-1 solution and disappearance serum), the disappearance serum group is (with 200 μ L VBS ²⁺Buffer replaces PS-1 solution and complement) and full haemolysis group (100 μ L2%SRBC are dissolved in the 500 μ L tri-distilled waters).Calculate hemolysis rate behind the deduction PS-1 matched group absorbance.Relatively lack the variation of serum group and target spot test set hemolysis rate,, judge that PS-1 has or not antagonism to C1Q., C1r, C1s, C2, C3, C4, C5 and C9 according to target spot test set haemolysis situation.The target spot test set lacks the serum group haematolysis ability more accordingly and has recovered, and explains that PS-1 does not act on this disappearance component; If haematolysis ability can not recover, explain that then PS-1 acts on this disappearance component.

Embodiment 5 PS-1 are to the influence of blood coagulation system

Recalcification time (recalcification time, mensuration RT):

The anesthesia of Cavia porcellus ip 1g/kg urethane is faced upward the position and is fixed.Separate common carotid artery, intubate blood-letting, (V/V) anticoagulant in 1: 9 of 3.8% sodium citrate.3000rpm, centrifugal 10min, platelet poor plasma (plateletpoor plasma, PPP).15 μ L PS-1 solution add 150 μ L PPP, and 37 ℃ of water-bath 5min add 0.025mol/L CaCl ₂150 μ L pick up counting, and every separated 5s detects with the little hook of special metal, and the time (s) of fiber protein yarn appears in record, measures three times, averages.Be terminal point with 600s writing time, exceeds terminal point and still do not solidify with 600s and calculate.With the positive contrast of 5.2mg/L heparin, VBS ²⁺The negative contrast of buffer, the concentration of PS-1 is followed successively by 1500,750,375mg/L.

Thrombin time (thrombin time, mensuration TT):

Freeze dried thrombin after (being equivalent to 5U/mL), can use behind 37 ℃ of water-bath 5min after the 2mL distilled water redissolves.15 μ L PS-1 solution add 150 μ L PPP, and 37 ℃ of water-bath 5min add thrombin 150 μ L, and the time (s) of fiber protein yarn appears in record, measures three times, averages.With the positive contrast of 5.2mg/L heparin, VBS ²⁺The negative contrast of buffer, the concentration of PS-1 is followed successively by 1500,750,375mg/L.

Claims (4)

1. a vegetable polysaccharides is characterized in that the heteropolysaccharide that said polysaccharide is made up of five kinds of monosaccharide, and its chemical constitution is: the saccharide residue mol ratio is a rhamnose: arabinose: mannose: glucose: galactose=1.3: 1.9: 1.0: 8.8: 4.8; Its molecular weight is greater than 1 * 10 ⁶Da is less than 2 * 10 ⁶Da, specific optical rotation [α] _D ²⁵+ 14.2 (c 0.318, H ₂O), sugared content is 90.9%, alduronic acid 4.16%, albumen 2.79%, sulfate 4.30%; Its connected mode comprises end, 1,4-, 1,6-and 1,3, the glucose that 6-connects, end, 1; 6-and 1,3, the galactose that 6-connects, end, 1, the arabinose that 5-connects, end, 1; The rhamnose that 3-connects, 1,6-, 1, the mannose and 1 that 4-connects, 3-, 1,3; 4-, 1,4, the hexose that 6-connects, the connected mode of said polysaccharide is with 1,4-and 1, the glucose that 6-connects is main; Called after polysaccharide PS-1.

2. by the described vegetable polysaccharides of claim 1, it is characterized in that said polysaccharide adopts water extract-alcohol precipitation, ion-exchange chromatography and gel filtration chromatography separation and Extraction from Chinese medicine compound to obtain; Said compound recipe is made up of Herba Houttuyniae, Flos Chrysanthemi Indici, Herba Artemisiae Scopariae, Herba Eupatorii, Fructus Tsaoko, and its part by weight is 15: 6: 15: 10: 3.

3. the method for preparing of the vegetable polysaccharides of claim 2 is characterized in that comprising the steps:

Pressed proportion of composing 15: 6: 15: 10: 3 compatibility Chinese medicine Herba Houttuyniae, Flos Chrysanthemi Indici, Herba Artemisiae Scopariae, Herba Eupatorii, Fructus Tsaoko, extract with 95% ethanol merceration, filter hot water extraction 3 times; Filter, merge extractive liquid, concentrates, and is centrifugal; Supernatant removes floating preteins with trichloroacetic acid, and is centrifugal, the supernatant dialysis; Add ethanol after dialysis solution concentrates to containing alcohol amount 80%, centrifugal, the deposition lyophilization gets crude polysaccharides; Crude polysaccharides is dissolved in water, and is centrifugal, and the supernatant gradation is with DEAE-cellulose post Cl ^-1Type, 30 * 2.5cm chromatography, with distilled water, 0.4,0.6,1.0 and the NaCl eluant solution of 2.0mol/L, flow velocity is 0.6mL/min, collects each stream part respectively, pipe detects the absorbance under 280nm and the 490nm; Merge same stream part according to testing result, concentrate, dialysis and lyophilization get 5 secondary components, and with being dissolved in water after the ACA component merging wherein, centrifugal, supernatant is with Sephacryl S-400 post 100 * 2.5cm chromatography; The distilled water eluting, flow velocity 0.6mL/min collects each stream part; Pipe detects the absorbance under 280nm and the 490nm, merges same stream part according to testing result, and concentrated and lyophilization gets vegetable polysaccharides PS-1.

4. the vegetable polysaccharides of claim 1 suppresses the purposes in the medicine at the preparation complement.