CN1844160A - Sulfonated changium root polysaccharides and preparing method and use thereof - Google Patents
Sulfonated changium root polysaccharides and preparing method and use thereof Download PDFInfo
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Abstract
In this invention, we extract raw polysaccharides from changium root named A B C respectively, and purify to get 6 polysaccharides I,II,III,IV,V,VI. After sulfation, we get sulfated polysaccharides A1, B1,C1. A1 has two sulfate groups on each monosaccharide at the average while B1 and C1 have three. The sulfated polysaccharides' molecular weight is more than 2 million D. The glycoside link is alpha type. The whole procedure of changium root sulfated polysaccharides is as followings: extract raw polysaccharides from changium root, and then get the polysaccharides sulfated. It is advantageous in simplied-manipulation and low cost. The pharmacological experiments prove its obvious suppression on rat transplanting tumor(S-180) and the reduction of platelet aggregation ratio and whole blood viscosity. It also reduces coagulant time.
Description
One, technical field
The present invention relates to a kind of natural polysaccharide sulphating thing, specifically relate to a kind of polysaccharide that from Root of Medicinal Changium, extracts, sulfonated changium root polysaccharides of making through sulphating and its production and application.
Two, background technology
Root of Medicinal Changium is the dry root of umbelliferae autogenus plant Root of Medicinal Changium Changium smyrnioides Wolff.Being used as medicine, it is bright, clear to start from, and is used for " tonifying lung gas is logical descending " (" new compilation of materia medica "); " control dizzy nausea, apoplexy dusk falls forward " (" medicine materical crude slice is newly joined "); " Chinese pharmacopoeia is recorded, meaning: " moistening the lung and resolving the phlegm, yin-nourishing and stomach, flat liver, detoxifcation ".Root of Medicinal Changium for " Chinese pharmacopoeia is recorded conventional Chinese medicine, and special product Jiangsu is the authentic medicinal herbs producing region, and the good reputation of " solely the bright party in peak " is arranged, and exports states such as Vietnam 50～sixties in a large number, its strong tonic effect in South East Asia one be with famous.
The overview of natural polysaccharide and sulphating thing thereof:
Along with molecular biological develop rapidly, the biological function of sugar is progressively disclosed and is familiar with.The sugar chain of polysaccharide can be controlled the division and the differentiation of cell, regulates the growth and aging of cell, energy enhancing body's immunological function etc.The seventies in 20th century, extensive day by day to the bioactive research field of polysaccharide, find that in succession glucide has anti-inflammatory, antiviral, radioprotective, reducing blood-fat, Ivy extract, anti-peptic ulcer, inducing interferon and produces, promotes functions such as protein, biological nucleic acid is synthetic.After the eighties, find that some Sulfate of polysaccharide natural or synthetic have the obvious suppression effect to hiv virus, this has caused that people pay close attention to and interest greatly.At present, the whole world is unprecedentedly active with exploitation to the development of carbohydrate medicine, and some medicine is put on market, has isolated saccharide compound more than 300 kinds from natural product, and has carried out the detection of biological function.Existing several polyose medicaments are used for clinical.
Polysaccharide is that a class non-specific immunity strengthens compound, can improve body's immunological function, strengthens resistance against diseases, and effects such as anti-old, health care, anti-cancer, anticancer, anti-hepatitis are arranged, and is subjected to people's generally attention, has obtained corresponding research and development.The sulfur-bearing polysaccharide also has anticoagulation, other physiological function such as antiviral as chondroitin sulfate, heparin etc.Can make the toxicity of these Sulfate of polysaccharide compounds lower by chemical improvement, activity is higher.Found successively since the sixties in 20th century, some semi-synthetic sulfated polysaccharides, sulfated derivative as lentinan, dextran, Achyranthan, it is the powerful inhibitor of multiple after birth virus, especially the activity that has anti-HIV-1, also have dextran also to obtain the anticoagulant active result identical, cause widely and pay attention to heparin through sulfation.In recent years, also the sulfation by some natural polysaccharides had obtained purposes polysaccharide derivates more widely.In vivo, in the outer experiment, sulfated polysaccharides all shows good antivirus action, that yet the immunological enhancement activity of sulfated polysaccharides is considered to is antiviral under the overall condition, one of the important mechanisms of antitumor action, so, the Japan scholar has studied the natural sulphur acidifying polysaccharide in the marine alga, find that it has antitumor action in vivo, and external invalid (Tian Gengyuan, Li Shoutong, Song Maili, Deng. the synthetic and antiviral activity [J] of achyranthes polysaccharide sulphate. Acta Pharmaceutica Sinica, 1995,30 (2): 107-111).
Natural heparin has the anti-HIV-1 activity, and activity is very low, though can remove its anticoagulant active by the chemical structure modification, but be helpless to resist/01 active raising, this is because SO
4 2-Contain quantity not sufficient, it has been generally acknowledged that each monosaccharide units needs 2 ~ 3 SO
4 2-, good anti-HIV-1 activity just can be arranged, caused medicine scholar and microbiologist's very big interest.Equally, as long as enough SO are arranged
4 2-, dextran also can have the active effect in inhibition/01.Except from known natural sulfur-bearing polysaccharide, filtering out the active medicine of anti-HIV-1, also by preparation sulfated polysaccharides such as some natural polysaccharide such as lentinan, dextran, Achyranthan, lichenstarch, laminarans, and seek and develop new sulfur-bearing polysaccharide constantly, as from blue algae, extracting sulfur acid fat body that obtains or the like.Simultaneously, except the antiviral application of Sulfate of polysaccharide, on the treatment cardiovascular diseases, also obtained corresponding exploitation.Therefore, adopt the Sulfate of polysaccharide of suitable polysaccharide parent (it is big to overcome most of polysaccharide molecular weights, is insoluble in water, is difficult to by defectives such as hemato encephalic barriers) preparation high substitution value and be a significant job to its biological activity research.
The pharmacologically active of sulfated polysaccharides now, the product of polysaccharide after sulfation has how better pharmacologically active, is subjected to people's attention always, the pharmacologically active of following several sulfated polysaccharides is its representative.Lentinan sulfate has good HIV (human immunodeficiency virus)-resistant activity.When concentration is 100mg/L, 10mg/L, its to the inhibiting rate of the reverse transcriptase of AMB virus respectively greater than 90% and 70%.Achyranthes polysaccharide sulphate (Tian Gengyuan, Li Shoutong, Song Maili, etc. the synthetic and antiviral activity [J] of achyranthes polysaccharide sulphate. Acta Pharmaceutica Sinica, 1995,30 (2): 107-111) in antiviral experiment, show good inhibition HBsAg, HBeAg virus function.The Radix Angelicae Sinensis polysaccharide sulfuric ester has promoter action preferably to splenocyte propagation, promoter action become with dosage good positive correlation (Yang Tiehong. Radix Angelicae Sinensis polysaccharide sulfuric ester synthetic and to the effect [J] of splenocyte propagation. The Fourth Military Medical University's journal, 2001,22 (5): 432-434).Water-solublely after the modification of tiger milk Sulfate of polysaccharide well improved, to Fe
2+The mouse hepatic mitochondria lipid peroxidation that causes has stronger restraining effect, to O
2-Has scavenging(action) efficiently; the dna damage that OH is caused has the certain protection effect simultaneously; show that it has better antioxidation; (Deng Chenghua just might play a role at aspects such as antitumor, anti-aging, anti-inflammatory, reducing blood-fat; Deng. the preparation and the Study on antioxidation [J] thereof of sulphating tiger milk polysaccharide. Chinese biochemical drug magazine; 2001,22 (1): 1-3).Water-solublely after the modification of shark suft bone Sulfate of polysaccharide obtain fine improvement, and have strong antitumour activity.Laminarin sulfate
[5]Can increase the phagocytic function of Turnover of Mouse Peritoneal Macrophages; Can increase hemolysin content in the mice serum, have the function that increases humoral immunization; The LT effect of certain promotion is arranged; The oligoleukocythemia that endoxan is caused has tangible antagonistic action; Can obviously prolong clotting time and prothrombin time; Can obviously shorten euglobulin lysis time, show to strengthen plasmin activity; The effect of obvious reduction serum cholesterol is arranged; in a word; it has body's immunity promoter action and body injury provide protection; also to preventing and treating atherosclerosis and the thrombosis (Li Dongxia that has great significance; preparation and spectroscopic identification [J] thereof Deng .SCAMP (shark suft bone polysaccharide) controlling sulfate polyose. spectroscopy and spectroscopic analysis; 2002,22 (1): 59-62).The astragalus polysaccharides sulfuric ester is by studies show that to the experiment of bhk cell toxic action and to the HSV-1 biosynthesizing is inhibiting, the effect of external anti-HSV-1 is better than acyclovir and toxicity is lower, show that also its clinical treatment to virus infection has application prospect (Feng Xiumei, Chen Bangyin, Zhang Hanping. the synthetic and antiviral activity research [J] of astragalus polysaccharides sulfuric ester. China Medicine University's journal, 2002,33 (2): 146-148).
Controlling sulfate polyose potential applicability in clinical practice controlling sulfate polyose is a class chemical structure complexity, biological activity is various, the biologically active substance that therapeutic effect relationship is clear and definite, anticoagulation wherein, antithrombotic, hypolipidemic activity research is more, relevant medicine have or be about to enter clinical application, and it is antiviral, the research of anti-tumor activity is less relatively, what have just just begins, believe that it will have good clinical application prospect (Wei Jingjian, Deng. controlling sulfate polyose chemistry and clinical application research progress [J]. Chinese biochemical drug magazine, 1999,20 (5): 260-262.; Dong Qun, the side is for many years. the application [J] of polysaccharide in field of medicaments. and Chinese Pharmaceutical Journal, 2001,36 (10): 649-651.).
The method of the method Sulfate of polysaccharideization of Sulfate of polysaccharideization is a lot, general pyranose form polysaccharide adopts Wolfrom (Ueno Y, et al.An antitumor activity of the Sclertia of Griforaum bellate (Fr.) PILAT[J] .Carbohydr Res, 1982,101:106-107.) method, furan type adopts Nagasawa (Adachi Y, et al.Physicochemical properties and antitumoractivities of chemical modified derivatives antitumor glucan " GriforaLE " from grifola frondasa[J] .Chem Pharm Bull, 1989,37:1838-1843.) method.Common method comprises: vitriol oil method, sulphur trioxide-pyridine method, chlorsulfonic acid-pyridine method
[1], sulphur trioxide-dimethyl formamide method etc.The most frequently used method has three kinds, and wherein the third method product reclaims conveniently, and yield is higher, so adopt the third method in a lot of research.(1) vitriol oil method: measure vitriol oil 7.5ml, propyl carbinol 2.5ml put in the three-necked bottle of band drying tube and whipping appts, adds vitriol oil 0.125g again, stirs, and ice bath is cooled to 0 ℃, adds polysaccharide powder 0.5g slowly, in 0 ℃ of reaction about 30 minutes.Reaction solution through neutralization, centrifugal after, use distill water dialysis 24h, be evaporated to about 20ml, add ethanol, leave standstill afterwards centrifugally, the collecting precipitation thing is dissolved in water with throw out, the 24h that dialyses again, dialyzate obtain sulfating product after lyophilize.(2) sulphur trioxide-pyridine method: pyridine 30ml is added in the 100ml three-necked bottle of having prolong and whipping appts, add sulphur trioxide-pyridine 2.5～3.0g while stirring and finish, be heated to 90 ℃ in hot water bath, add polysaccharide powder 0.5g again, constant temperature stirs, and is chilled to room temperature, reaction solution transfers to neutrality with the NaOH solution of 3mol/l, adds ethanol, separates out Sulfate of polysaccharide, centrifugal, collecting precipitation, will precipitate water-soluble, the dialysis 72h, filter, lyophilize gets polysaccharide sulfated product.(3) chlorsulfonic acid-pyridine method: will put in salt solution-ice bath with the three-necked bottle of prolong and whipping appts, add pyridine, stir, make it abundant cooling, slowly add chlorsulfonic acid 2～4ml with dropping funnel, about 30～40min dropwises, a large amount of faint yellow solids appear in the flask, add polysaccharide powder 0.9g then, rapidly three-necked bottle is moved in the boiling water bath, constant temperature stirs about 1h, be chilled to room temperature, in reaction solution impouring frozen water, the neutralization back adds ethanol, separates out precipitation, to precipitate water-soluble, dialysis 72h filters, and filtrate gets polysaccharide sulfated product after lyophilize.The sulphating condition of the lacquer polysaccharide of reports such as Jianhong Yang is suspended in the 16.2mL methyl-sulphoxide for the 162mg polysaccharide, 60 ℃ or 70 ℃ were reacted 1～5 hour down, violent (the Jianhong Yang etal.Biological Macromolecules of degraded under comparatively high temps, 2002,31:55-62).
Three, summary of the invention
1, goal of the invention:
The object of the present invention is to provide a kind of sulfonated changium root polysaccharides and preparation method and preparing the application for the treatment of in antitumor and the activating blood and removing stasis drug.
2, technical scheme:
The present invention from getting Root of Medicinal Changium Crude polysaccharides A, B, C, first obtains six homogeneous polysaccharide I, IIs, III, IV, V, VI through being further purified from Root of Medicinal Changium (Changium smyrnioides Wolff) root portion, gets sulfated polysaccharide A, B, C, A through sulphating
1, B
1, C
1, and to A
1Part has been carried out separation and purification, gets polysaccharide VII.Rheology test shows that each polysaccharide all has blood circulation invigorating efficacies; Esterificated polysaccharide A wherein
1, B
1Than nonesterified Polysaccharide A, B effect by force; And B1 is stronger than the A1 effect in the esterificated polysaccharide.The transplanted tumor test shows Polysaccharide A, B, A
1, B
1Remarkable antitumor effect is all arranged, wherein with B
1Tumor-inhibiting action is the strongest.
Sulfonated changium root polysaccharides of the present invention comprises A
1, B
1, C
1, its sulfate content is respectively A
1: 52.62%, B
1: 62.35%, C
1: 40.16%, A
1Two sulfates have been connected, B on average each monose
1Three sulfates have been connected, C on average each monose
1Three sulfates have been connected, Sulfate of polysaccharide thing A on average each monose
1Molecular weight surpass 2,000,000, form by sulfate glucose, this sugared glycosidic bond is configured as the α configuration.
Preparation method's step of sulfonated changium root polysaccharides of the present invention is as follows:
(1) extraction of Root of Medicinal Changium polysaccharide:
Get the Root of Medicinal Changium medicinal material, the knock segment takes by weighing 475-525g and places flask, adds the alcohol immersion 1-2h of 3800-4300ml 95%, 2-4h then refluxes on water-bath, filter, the same operation of filter residue 1 time refilters, filter residue dries, add 3800-4300ml distilled water and boil 1-2h, inclining extracting solution, centrifugal; Filter residue adds 2000ml distilled water again and boils 2h, and inclining extracting solution, centrifugal (3000rmin
-1) 20min, discard throw out, merge supernatant liquor, be put in refrigerator overnight;
With the extracting solution suction filtration in the refrigerator, filtrate is placed in two 5000ml large beakers, in water-bath, heat, concentrate, stop heating when being about to solid occur, add 0.5-0.8 95% ethanolic soln doubly that concentrates liquid measure, standing over night, centrifugal, get centrifugal sediment and successively use dehydrated alcohol, acetone, the ether washing, dry in 40 ℃ of vacuum drying ovens again, promptly get 30% alcohol precipitation Polysaccharide A powder, with the method operation supernatant liquor is transferred to 50% respectively, the ethanolic soln of 70% concentration can obtain 50% respectively then as stated above, 70% alcohol precipitation Polysaccharide B powder and C powder;
(2) in the three-necked bottle of three 500ml band drying tubes and whipping appts, each adds the 65-80ml vitriol oil, propyl carbinol 20-28ml respectively, add 1.10-1.30g ammonium sulfate again, stir, ice bath is cooled to 0 ℃, and each adds Root of Medicinal Changium Polysaccharide A, B, C, each 9-11g of powder respectively slowly;
(3) respectively respectively at 0 ℃ of reaction 120min, get reaction solution respectively, then each reaction solution being neutralized to pH value with alkali respectively is 7, and distill water dialysis 24 hours are used in centrifugal respectively back;
(4) difference concentrating under reduced pressure, alcohol precipitation leaves standstill centrifugal collecting precipitate, obtains being sulphating thing A respectively
1, B
1, C
1
Alkali described in the above-mentioned steps (3) is NaOH or KOH.
Above-mentioned sulfonated changium root polysaccharides, the application in preparation treatment antitumor drug.
Above-mentioned sulfonated changium root polysaccharides, the application in preparation treatment activating blood and removing stasis drug.
3, beneficial effect:
The present invention from getting Root of Medicinal Changium Polysaccharide A, B, C, first obtains six homogeneous polysaccharide I, IIs, III, IV, V, VI through being further purified from Root of Medicinal Changium (Changium smyrnioides Wolff) root portion, gets sulfated polysaccharide A through sulphating
1, B
1, C
1, its sulfate content is respectively A
1: 52.62%, B
1: 62.35%, C
1: 40.16%, A
1Two sulfates have been connected, B on average each monose
1Three sulfates have been connected, C on average each monose
1Connected three sulfates on average each monose, the molecular weight of Sulfate of polysaccharide thing surpasses 2,000,000, and it is made up of sulfate glucose, and this sugared glycosidic bond is configured as the α configuration.
The preparation method of sulfonated changium root polysaccharides at first carries the limit polysaccharide from the Root of Medicinal Changium medicinal material, then Sulfate of polysaccharideization is made.This preparation technology is novel simply, cost is low, show through pharmacological activity experiment results, sulfonated changium root polysaccharides can significantly reduce platelet aggregation rate and whole blood viscosity and the maximum aggregate index of stasis syndrome rat, clotting time obviously descends, plasma fibrin obviously raises, restraining effect to mouse transplanting tumor (S-180) is remarkable, sulfonated changium root polysaccharides A
1, B
1Inhibiting rate to tumour is respectively 52.89% and 56.25%.
Four, embodiment
Embodiment 1: the preparation method of sulfonated changium root polysaccharides, and the step of its preparation method is as follows:
(1) extraction of Root of Medicinal Changium polysaccharide:
Get the Root of Medicinal Changium medicinal material, the knock segment takes by weighing 500g and places flask, adds the alcohol immersion 1.5h of 4000ml 95%, the 3h that refluxes on water-bath then filters, and the same operation of filter residue 1 time refilters, filter residue dries, and adds 4000ml distilled water and boils 2h, and inclining extracting solution, centrifugal; Filter residue adds 2000ml distilled water again and boils 2h, and inclining extracting solution, centrifugal (3000rmin
-1) 20min, discard throw out, merge supernatant liquor, be put in refrigerator overnight;
With the extracting solution suction filtration in the refrigerator, filtrate is placed in two 5000ml large beakers, in water-bath, heat, concentrate, stop heating when being about to solid occur, add 0.6 times 95% ethanolic soln that concentrates liquid measure, standing over night, centrifugal, get centrifugal sediment successively with dehydrated alcohol, acetone, ether washing, dry in 40 ℃ of vacuum drying ovens again, promptly get 30% alcohol precipitation Polysaccharide A powder, operate the ethanolic soln that supernatant liquor is transferred to 50%, 70% concentration respectively with method, can obtain 50%, 70% alcohol precipitation Polysaccharide B powder and C powder respectively as stated above then;
(2) sulphating of polysaccharide: measure the 75ml vitriol oil, propyl carbinol 25ml places 500ml to be with the three-necked bottle of drying tube and whipping appts, adds 1.25g ammonium sulfate again, stir, ice bath was cooled to 0 ℃, adds 30% alcohol precipitation polysaccharide (A) powder 10g slowly, in 0 ℃ of reaction 120 minutes, reaction solution is neutralized to PH7, centrifugal back distill water dialysis 24 hours of using with NaOH, concentrating under reduced pressure, alcohol precipitation leaves standstill centrifugal collecting precipitate and is Sulfate of polysaccharide thing (A1); Through the sulfate Determination on content is 52.62%;
Embodiment 2: the preparation method of sulfonated changium root polysaccharides, and the step of its preparation method is as follows:
(1) extraction of Root of Medicinal Changium polysaccharide is with embodiment 1;
(2) sulphating of polysaccharide: measure the 75ml vitriol oil, propyl carbinol 25ml places 500ml to be with the three-necked bottle of drying tube and whipping appts, add 1.25g ammonium sulfate again, stir, ice bath is cooled to 0 ℃, adds 50% alcohol precipitation polysaccharide (B) powder 10g slowly, in 0 ℃ of reaction 120 minutes, reaction solution is neutralized to PH7, centrifugal back distill water dialysis 24 hours, concentrating under reduced pressure, the alcohol precipitation used with NaOH, leaving standstill centrifugal collecting precipitate and be sulfating product (B1), is 62.35% through the sulfate Determination on content.
Embodiment 3: the preparation method of sulfonated changium root polysaccharides, and the step of its preparation method is as follows:
(1) extraction of Root of Medicinal Changium polysaccharide is with embodiment 1;
(2) sulphating of polysaccharide: measure the 75ml vitriol oil, propyl carbinol 25ml places 500ml to be with the three-necked bottle of drying tube and whipping appts, add 1.25g ammonium sulfate again, stir, ice bath is cooled to 0 ℃, adds 70% alcohol precipitation polysaccharide (C) powder 10g slowly, in 0 ℃ of reaction 120 minutes, reaction solution is through neutralization, centrifugal back usefulness distill water dialysis 24 hours, concentrating under reduced pressure, alcohol precipitation, leaving standstill centrifugal collecting precipitate and be sulfating product (C1), is 40.16% through the sulfate Determination on content.
Embodiment 4: the pharmacologically active of sulfonated changium root polysaccharides
Method 1: each organized the continuous irrigation stomach 7 days, every day 1 time, irritated stomach after 30 minutes at the 7th day, except that the blank group, all the other each treated animal subcutaneous injection adrenalin hydrochlorides, subcutaneous injection adrenalin hydrochloride once more after 4 hours, animal was placed frozen water 5 minutes therebetween, duplicate the stasis syndrome rat model.After the rat modeling 16 hours, with 10% Chloral Hydrate (300mg/kg, ip), it is fixing to face upward the position, the bloodletting of carotid artery intubate, the liquor sodii citratis with 3.8% is with the 1:9 anti-freezing, every index of detection hemorheology blood.
Influence to rat platelet aggregation:
(1) with the blood of 3.8% Sodium Citrate anti-freezing with the centrifugal 10min of 800r/min, get platelet rich plasma (RPP), remainder is with the centrifugal 10min of 3000/min, get platelet poor plasma (PPP), utilize LG-PABER type platelet aggregation and thrombin analyser record MA and calculate inhibiting rate by following formula.
Assemble inhibiting rate (%)={ (model group MA-administration group MA)/model group MA } * 100%
Table 1 Root of Medicinal Changium polysaccharide and sulphating thing thereof are to the influence of stasis syndrome rat model platelet aggregation rate
| Group | Dose(g/kg) | Aggregation rate(％) | Inhibition rate(％) |
| NS MODEL DANSEN A B A1 B1 | ------- ------- 2 0.2 0.2 0.2 0.2 | 18.46±2.36 28.82±10.85* 22.90±13.17* 13.34±9.59** 13.44±7.52* 20.08±6.37** 12.86±5.33** | ------- ------- 20.54 53.71 53.37 30.33 55.38 |
Annotate: compare with negative control group: * P＜0.05.
Compare with positive controls: * P＜0.05, * * P＜0.01.
The result shows: the platelet aggregation rate of model group rat obviously raises (comparing P＜0.05 with the physiological saline group).Each Root of Medicinal Changium polysaccharide all can significantly reduce the platelet aggregation rate of acute blood stasis rat, A
1Effect weak slightly (comparing P＜0.01) with model group
(2) blood viscosity detects: a whole blood viscosity: adopt LG-R-80 series blood viscosity instrument to measure the blood of anticoagulant heparin.
Table 2 Root of Medicinal Changium polysaccharide and sulphating thing thereof are to the influence of stasis syndrome rat model whole blood viscosity
| Group Group | Dosage Dose | Blood viscosity Blood viscosity (mPa) | |||
| 11/s | 51/s | 301/s | 2001/s | ||
| Blank group model group red sage root group A B A 1 B 1 | -- -- 2 0.2 0.2 0.2 0.2 | 20.30±2.34 27.25±5.10 ** 26.42±9.62 22.89±5.11 22.85±10.96 28.16±6.61 29.22±3.28 | 10.58±1.12 12.34±1.89 * 11.67±3.21 10.47±1.63 10.63±4.02 11.47±2.52 * 12.70±1.20 | 6.94±0.37 7.04±0.79 * 6.77±1.19 6.34±0.68 6.48±1.81 6.95±1.26 7.42±0.60 * | 5.44±0.26 5.75±0.48 * 5.12±0.57 ** 4.87±0.42 * 4.81±0.92 * 5.09±0.74 ** 5.38±0.30 ** |
Annotate: compare with negative control group: * P＜0.05, * * P＜0.01.
Compare with positive controls: * P＜0.05, * * P＜0.01.
The result shows: the whole blood viscosity of stasis syndrome rat model obviously raises (comparing P＜0.01 with the physiological saline group); Root of Medicinal Changium Polysaccharide A, B can reduce the whole blood viscosity (200l/s) (comparing P＜0.05 with model group) of stasis syndrome rat model; A1, B1 all can significantly reduce the whole blood viscosity (5,30, or 200l/s) (comparing P＜0.05 or 0.01 with model group) of stasis syndrome rat model.
(3) erythrodegeneration ability, erythrocyte aggregation ability detect: adopt LG-R-190 type red blood cell deformation/ability of aggregation determinator to measure the blood of anticoagulant heparin.
Table 3 Root of Medicinal Changium polysaccharide and sulphating thing thereof are to stasis syndrome rat model red blood cell deformation and ability of aggregation
Influence
| Group | Dosage (g/kg) | MAXDI | MAXD |
| Physiological saline group model group red sage root group A B A1 B1 | ------- ------- 2 0.2 0.2 0.2 0.2 | 48.16±2.46 46.92±1.30 46.28±2.48 44.93±2.90 45.69±3.62 47.88±4.56 47.11±4.83 | 0.33±0.05 2.35±0.81** 1.44±0.32* 1.35±0.38* 1.15±0.28** 1.01±0.20** 1.07±0.10** |
Annotate: compare with negative control group: * * P＜0.01.
Compare with positive controls: * P＜0.05, * * P＜0.01.
Experimental result shows: the red corpuscle maximum distortion index of stasis syndrome rat model obviously descends (comparing P＜0.05 with the physiological saline group), and maximum aggregate index obviously raises (comparing P＜0.05 with the physiological saline group); Root of Medicinal Changium Polysaccharide B, A1, B1 all can significantly reduce the maximum aggregate index of stasis syndrome rat model (comparing P＜0.01 with model group), and Polysaccharide A then acts on weak (P＜0.05) slightly; And each polysaccharide does not have remarkable effect to the red corpuscle maximum distortion index of stasis syndrome rat model.
(4) detection of blood coagulation system:
The blood of the Sodium Citrate anti-freezing with 3.8% separates platelet poor plasma with the centrifugal 10min of 3000r/min, adopts LG-PAPER type platelet aggregation and thrombin analysis-e/or determining activated partial prothrombin time (PT), plasma fibrinogen (FIB).
Table 4 Root of Medicinal Changium polysaccharide and sulphating thing thereof are to the influence of stasis syndrome rat model blood coagulation system
| Group | Dosage (g/kg) | Prothrombin time PT (S) | Blood plasma fibrinogen FIB (g/l) |
| Physiological saline group model group red sage root group A B A1 B1 | ------- ------- 2 0.2 0.2 0.2 0.2 | 21.33±4.02 11.14±4.19* 16.44±3.23* 17.70±6.86* 17.86±7.02* 14.22±3.58** 18.06±4.36* | 2.02±0.45 3.27±0.74** 2.11±0.85** 3.35±1.79 2.56±0.54* 3.39±1.27 2.56±0.27** |
Annotate: compare with negative control group: * P＜0.05, * * P＜0.01.
Compare with positive controls: * P＜0.05, * * P＜0.01.
Experimental result shows: the PT of stasis syndrome rat model obviously descends, and FIB obviously raises (comparing P＜0.05 with the physiological saline group); Root of Medicinal Changium Polysaccharide A, B, A1, B1 all can increase the PT (comparing P＜0.05 with model group) of stasis syndrome rat model, and B, B1 can reduce FIB (compare with model group, be respectively P＜0.05,0.01); Make good use of with B1.
Method 2 is got 72 of ICR mouse, and is male, is divided into 6 groups at random.The tumor-bearing mice (ascitic type) of getting the strain of well-grown S-180 sarcoma extracts ascites under aseptic condition, with (V/V) physiological saline dilution in 1: 3, collect 0.2ml (about 107) and be inoculated in every subcutaneous place of right side of mice armpit, is placed in the laminar-flow rack and raises.According to the form below dosage gastric infusion every day is once observed mouse oxter tumour growing state every day, and successive administration is after eight days, and the cervical vertebra dislocation is put to death, and carefully peels off tumor tissue and weighs, and presses calculating.
Tumour inhibiting rate=(1-administration group knurl weight/model group knurl is heavy) * 100%
Table 5 Root of Medicinal Changium polysaccharide and sulphating thing thereof are to mice transplanted tumor (S-180) restraining effect
| Group | Dosage (g/kg) | Knurl heavy (g) | Inhibiting rate (%) |
| Model group | ----- | 1.12±0.26 | ----- |
| Endoxan | 0.01 | 0.30±0.14** | 73.19 |
| The Root of Medicinal Changium Polysaccharide A | 0.2 | 0.64±0.25** | 42.85 |
| The Root of Medicinal Changium Polysaccharide B | 0.2 | 0.60±0.19** | 46.43 |
| Root of Medicinal Changium controlling sulfate polyose A1 | 0.2 | 0.53±0.23** | 52.89 |
| Root of Medicinal Changium controlling sulfate polyose B1 | 0.2 | 0.49±0.24** | 56.25 |
Annotate: compare with positive controls: * * P＜0.001.
Table 5 Root of Medicinal Changium controlling sulfate polyose to the mice transplanted tumor restraining effect (X ± S, n=10)
| Group | Dosage (g/kg) | Knurl heavy (g) | Inhibiting rate (%) |
| Model group | ----- | 1.12±0.26 | ----- |
| Endoxan | 0.01 | 0.30±0.14** | 73.19 |
| The Root of Medicinal Changium Polysaccharide A | 0.2 | 0.64±0.25** | 42.85 |
| The Root of Medicinal Changium Polysaccharide B | 0.2 | 0.60±0.19** | 46.43 |
| Root of Medicinal Changium controlling sulfate polyose A1 | 0.2 | 0.53±0.23** | 52.89 |
| Root of Medicinal Changium controlling sulfate polyose B1 | 0.2 | 0.49±0.24** | 56.25 |
Claims (5)
1, a kind of sulfonated changium root polysaccharides is characterized in that it comprises A
1, B
1, C
1, its sulfate content is respectively A
1: 52.62%, B
1: 62.35%, C
1: 40.16%, A
1Two sulfates have been connected, B on average each monose
1Three sulfates have been connected, C on average each monose
1Connected three sulfates on average each monose, the molecular weight of Sulfate of polysaccharide thing surpasses 2,000,000, is made up of sulfate glucose, and this sugared glycosidic bond is configured as the α configuration.
2, the preparation method of sulfonated changium root polysaccharides according to claim 1 is characterized in that the step of this method is as follows:
(1) extraction of Root of Medicinal Changium polysaccharide:
Get the Root of Medicinal Changium medicinal material, the knock segment takes by weighing 475-525g and places flask, adds the alcohol immersion 1-2h of 3800-4300ml 95%, 2-4h then refluxes on water-bath, filter, the same operation of filter residue 1 time refilters, filter residue dries, add 3800-4300ml distilled water and boil 1-2h, inclining extracting solution, centrifugal; Filter residue adds 2000ml distilled water again and boils 2h, and inclining extracting solution, centrifugal (3000rmin
-1) 20min, discard throw out, merge supernatant liquor, be put in refrigerator overnight;
With the extracting solution suction filtration in the refrigerator, filtrate is placed in two 5000ml large beakers, in water-bath, heat, concentrate, stop heating when being about to solid occur, add 0.5-0.8 95% ethanolic soln doubly that concentrates liquid measure, standing over night, centrifugal, get centrifugal sediment and successively use dehydrated alcohol, acetone, the ether washing, dry in 40 ℃ of vacuum drying ovens again, promptly get 30% alcohol precipitation Polysaccharide A powder, with the method operation supernatant liquor is transferred to 50% respectively, the ethanolic soln of 70% concentration can obtain 50% respectively then as stated above, 70% alcohol precipitation Polysaccharide B powder and C powder;
(2) in the three-necked bottle of three 500ml band drying tubes and whipping appts, each adds the 65-80ml vitriol oil, propyl carbinol 20-28ml respectively, add 1.10-1.30g ammonium sulfate again, stir, ice bath is cooled to 0 ℃, and each adds Root of Medicinal Changium Polysaccharide A, B, C, each 9-11g of powder respectively slowly;
(3) respectively respectively at 0 ℃ of reaction 120min, get reaction solution respectively, then each reaction solution being neutralized to pH value with alkali respectively is 7, and distill water dialysis 24 hours are used in centrifugal respectively back;
(4) difference concentrating under reduced pressure, alcohol precipitation leaves standstill centrifugal collecting precipitate, obtains sulfonated changium root polysaccharides A respectively
1, B
1, C
1
3, the preparation method of sulfonated changium root polysaccharides according to claim 2 is characterized in that at the alkali described in the step (3) be NaOH or KOH.
4, the described sulfonated changium root polysaccharides of claim 1, the application in preparation treatment antitumor drug.
5, the described sulfonated changium root polysaccharides of claim 1, the application in preparation treatment activating blood and removing stasis drug.
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