CN103626880B - Polysaccharides from Prunella vulgaris L and its production and use - Google Patents
Polysaccharides from Prunella vulgaris L and its production and use Download PDFInfo
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Abstract
The invention belongs to field of traditional Chinese medicine pharmacy, relate to Polysaccharides from Prunella vulgaris L and preparation method thereof and preparing the purposes in anticomplement medicament.Described Polysaccharides from Prunella vulgaris L comprises homogeneous polysaccharide PW-PS1 and PW-PS2, by from labiate Spica Prunellae Prunella? vulgaris? the dry fruit ear aqueous extract of Linn obtains, confirm through in vitro tests, described homogeneous polysaccharide has stronger anticomplementary activity, all restraining effect is had to complement system classical pathway and alternative pathway, and not there is the blood coagulation resisting function affecting activity in vivo and play, can be used for preparing Complement inhibition medicine.Wherein said PW-PS1 acts on C1q, C3 and C9 component of complement system, and PW-PS2 acts on C1q, C2, C3, C5 and C9 component of complement system.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, relate to Polysaccharides from Prunella vulgaris L and its production and use, be specifically related to Spica Prunellae homogeneous polysaccharide and preparation method thereof and preparing the purposes in anticomplement medicament.
Background technology
Known complement system is one of immune defense system of wanting of body weight for humans, the normal Activate of complement system eliminating external microorganism, remove the biological cells and tissues of damage or death in body, maintain in the balance of body and play an important role; But, described complement system excessive activation can cause the damage of human body self healthy tissues and inflammatory reaction there are some researches show, the disease relevant to excess complement activation has the multiple major diseases such as systemic lupus erythematous, rheumatoid arthritis, adult respiratory distress syndrome.For many years, anticomplement medicament research is the focus and emphasis of world pharmaceutical research always, but still lacks ideal medicine to this type of disease at present, is therefore badly in need of efficient, low toxicity, single-minded novel complement inhibitor clinically.Research and develop from natural product complement inhibitor be in recent years one be subject to more and more important research field paid close attention to, it has the features such as cost is low, toxicity is low.Current, have Chinese scholars to be separated from the multiple natural product comprising marine organisms to obtain and a large amount of have the inhibiting monomeric compound of complement system, the research and development for anticomplement medicament provide wide prospect.
Chinese medicine Spica Prunellae is the dry fruit ear of labiate Spica Prunellae (PrunellavulgarisLinn), because " namely withered after the Summer Solstice " is gained the name, is one of conventional traditional Chinese medicine.Modern chemical composition research shows, Spica Prunellae is mainly containing chemical compositions such as triterpenes, sterols, flavonoid, coumarins, organic acid, volatile oil and carbohydrates, there is step-down, hypoglycemic, antibacterial, anti-inflammatory, antianaphylaxis and the pharmacological action such as antiviral, be mainly used in treatment conjunctival congestion with pain and swelling of the eye clinically, photophobia is shed tears, mazoitis, breast cancer, hypertension, lymphoid tuberculosis, infiltrative pulmonary tuberculosis, simple goiter, parotitis, acute icterohepatitis etc.But there is not yet the report finding to have Complement inhibition effect polysaccharide from Spica Prunellae up to now.
Summary of the invention
The object of this invention is to provide in natural drug the activeconstituents with anticomplementary action, be specifically related to Spica Prunellae homogeneous polysaccharide and preparation method thereof and preparing the purposes in anticomplement medicament.
The present invention's application modern pharmacology screening method, anticomplementary activity evaluation study is carried out to being separated the material obtained, be separated from the dry fruit ear aqueous extract of labiate Spica Prunellae (PrunellavulgarisLinn) and obtain homogeneous polysaccharide, and confirm that it all has restraining effect to complement system classical pathway and alternative pathway; Though its complement inhibitory activity is weaker than heparin, does not possess and limit the blood coagulation resisting function that it plays activity in vivo.
In the present invention, described homogeneous polysaccharide comprises PW-PS1 and PW-PS2.
The constitutional features of described vegetable polysaccharides PW-PS1 is: faint yellow loose powder, molecular weight is about 3 × 10
5da, specific optical rotation [α]
d 25=-102.2 (c0.3, H
2o); Total sugar content is 96.94%, containing the uronic acid of 17.87% and the albumen of 0.53%;
Described PW-PS1 is by pectinose Ara, wood sugar Xyl and 4-methoxyl group glucuronic acid 4-methox-glcA3 kind monose composition, saccharide residue mol ratio is wherein: pectinose Ara: wood sugar Xyl:4-methoxyl group glucuronic acid 4-methox-glcA=1:2.6:0.8, its mode of connection comprises end and 1, the wood sugar that 3-connects, 1,5-and 1, the pectinose that 3,5-connects, the 4-methoxyl group glucuronic acid that end connects, wherein with 1, the wood sugar that 3-connects, 1,3, the 5-pectinose connected are connected 4-methoxyl group glucuronic acid with end be main;
In the present invention, the constitutional features of described vegetable polysaccharides PW-PS2 is: brown loose powder, molecular weight is about 8 × 10
3da, specific optical rotation [α]
d 25=-132.4 (c0.3, H
2o); Total sugar content is 98.12%, still containing the uronic acid of 58.20% and the albumen of 0.22%;
Described PW-PS2 is by 5 kinds of monose, comprise rhamnosyl Rha, pectinose Ara, wood sugar Xyl, semi-lactosi Gal and galacturonic acid GalA form, and saccharide residue mol ratio is wherein: rhamnosyl Rha: pectinose Ara: wood sugar Xyl: semi-lactosi Gal: galacturonic acid GalA=0.56:1:1.30:1.78:3.37, mode of connection wherein comprises: the pectinose that end connects, the wood sugar that 1,3-connects, 1, the rhamnosyl that 3-connects, end and Isosorbide-5-Nitrae, the semi-lactosi that 6-connects, wherein with end and 1, the semi-lactosi that 4,6-connects and the wood sugar that 1,3-connects are main.
Homogeneous polysaccharide described in the present invention is prepared by following method:
The dry fruit ear medicinal material of labiate Spica Prunellae (PrunellavulgarisLinn) is through pulverizing, and after extremely colourless with 95% ethanol percolate extraction under room temperature, residue flings to alcohol taste, after drying, boiling water boiling 3 times, adds 4 times of volume water at every turn, and each extraction time is 2h; Merge extracting the Aqueous extracts filtering gained at every turn, be concentrated into small volume, by 4 times of volume dehydrated alcohol precipitations, precipitate after a small amount of water dissolution, under 4 ° of C, stir deproteinated with 15% Tricholroacetic Acid (TFA); Solution goes precipitation through centrifugal, and supernatant liquor is adjusted to neutrality with 1mol/LNaOH, dialyses 3 days with flowing water; In bag, solution is concentrated into proper volume, and lyophilize obtains Spica Prunellae Crude polysaccharides; Described Crude polysaccharides adding distil water dissolves, and centrifugal, supernatant liquor gradation DEAE-cellulose column chromatography carries out initial gross separation; With distilled water, 0.4,0.8,1.2 and the NaCl solution wash-out of 2.0mol/L, elution volume is greater than 2 times of column volumes, and flow velocity is 0.8ml/min, collects each stream part, and pipe detects the absorbance under 280nm and 490nm (after sulfuric acid-phynol method colour developing); Result according to sugared color reaction and ultraviolet detection merges same stream part, concentrated, obtains 5 secondary components: PWD1, PWD2, PWD3, PWD4 and PWD5 after dialysis and lyophilize; According to complement inhibitory activity tracking results, PWD3, PWD4 and PWD5 are merged adding distil water to dissolve, centrifugal, supernatant liquor gradation SephacrylS-400 column chromatography (100 × 2.5cm) is separated, the NaCl solution wash-out of 0.1mol/L, flow velocity is 0.8ml/min, collects each stream part; Pipe detects the absorbance under 280nm and 490nm (after sulfuric acid-phynol method colour developing), each pipe stream part of active tracking; Merge same stream part according to detected result, concentrated and lyophilize obtains homogeneous polysaccharide PW-PS1 and PW-PS2 respectively; Detecting PW-PS1 and PW-PS2 through High Performance Gel Permeation Chromatography (HPGPC) and high performance capillary electrophoresis (HPCE) is homogeneous composition;
Adopt efficient gel post TSK-GELGMPWXL (300 × 7.6mm), moving phase is 0.01mol/LNaCl, flow velocity 0.8ml/min, and column temperature 25 ° of C, compare mensuration with the Dextran of T series, and result shows, and PW-PS1 molecular weight is about 3 × 10
5da, PW-PS2 molecular weight is about 8 × 10
3da;
Results of elemental analyses shows, PW-PS1 contains C, 43.38%; H, 7.242%; N, 0.29%; Specific optical rotation is [α]
d 25=-102.2 (c0.3, H
2o); PW-PS2 contains C, and 36.47%; H, 6.205%; N, 0.50%; Specific optical rotation is [α]
d 25=-132.4 (c0.3, H
2o);
The total sugar content that sulfuric acid-phynol method measures PW-PS1 and PW-PS2 is respectively 96.94% and 98.12%, the glucuronic acid content that meta-hydroxydiphenyl method measures PW-PS1 and PW-PS2 is respectively 17.87% and 58.20%, the protein content that Coomassie Brilliant Blue measures PW-PS1 and PW-PS2 is respectively 0.53% and 0.22%, BaCl
2the sulfate content of turbidimetry for Determination PW-PS1 and PW-PS2 is respectively 0.16% and 0.33%;
Sugar compositional analysis: PW-PS1, PW-PS2, through uronic acid reduction after PW-PS1 and PW-PS2 through 2mol/LTFA in 110 ° of C complete hydrolysis, successively carry out NaBH
4reduction, aceticanhydride acetylize is prepared into ALDI alcohol acetic ester derivative, carries out gas phase compositional analysis (method see: BlakeneyAB, HarrisPJ, HenryRJ, etal.1983,113:291-299); Result shows, PW-PS1 is made up of 3 kinds of monose, and saccharide residue mol ratio is: pectinose Ara: wood sugar Xyl:4-methoxyl group glucuronic acid 4-methox-glcA=1:2.6:0.8; PW-PS2 is made up of 5 kinds of monose, and saccharide residue mol ratio is: rhamnosyl Rha: pectinose Ara: wood sugar Xyl: semi-lactosi Gal: galacturonic acid GalA=0.56:1:1.30:1.78:3.37;
Methylation analysis: PW-PS1 and the PW-PS2 reference literature (NeedsPW, SelvendranRR.1993,245:1-10) after uronic acid reduction methylates, the 90% formic acid depolymerization of the product after methylating, 2mol/LTFA complete hydrolysis, NaBH
4the ALDI alcohol acetic ester derivative of partial methylation is made in reduction and aceticanhydride acetylize, then carries out GC-MS analysis; Combined standard collection of illustrative plates, determine that PW-PS1 mode of connection is for comprising the wood sugar of end and 1,3-connection, 1,5-and 1, the pectinose that 3,5-connects, the 4-methoxyl group glucuronic acid that end connects, wherein with 1, the wood sugar that 3-connects, 1,3, the 5-pectinose connected are connected 4-methoxyl group glucuronic acid with end be main; PW-PS2 mode of connection is the pectinose comprising end connection, the wood sugar that 1,3-connects, the rhamnosyl that 1,3-connects, end and Isosorbide-5-Nitrae, the semi-lactosi that 6-connects, and wherein with end and Isosorbide-5-Nitrae, the wood sugar of the semi-lactosi that 6-connects and 1,3-connection is main;
PW-PS1 and PW-PS2 is to the effect of complement system:
1) anticomplement activates classical pathway cell hemolytic test: guinea pig serum 1:80 dilution is as complement, antigen activates classical pathway of complement causes sheep red blood cell haemolysis, reference literature method (KabatEA, MayerMM.Complementandcomplementfixationinexperimentalimm unology.CharlesC.ThomasPublisher, Illinois, USA.1964, pp.133-240) record PW-PS1 and PW-PS2 all energy T suppression cell haemolysis, CH
50be respectively 0.28 ± 0.03mg/ml and 0.13 ± 0.01mg/ml (n=3);
2) anticomplement activates alternative pathway cell hemolytic test: human serum 1:10 dilutes as complement, activating complement alternative pathway causes rabbit erythrocyte haemolysis, reference literature method (KlerxJP, BeukelmanCJ, VanDH, etal.Microassayforcolorimetricestimationofcomplementacti vityinguineapig, humanandmouseserum.ImmunologicalMethods, 1983,63 (2): 215-220) PW-PS1 and PW-PS2 all energy T suppression cell haemolysis is recorded, AP
50be respectively 0.40 ± 0.02mg/ml and 0.35 ± 0.03mg/ml (n=3);
3) to the research of complement system action target spot: the present invention utilizes complement depletion serum, reference literature method (Zhou Jie, Zhang Yun is firm, Zhang Jianwen, Deng. Traditional Chinese medicine eucommia bark is to the effect of complement system. Fudan Journal (medicine), 2006,33 (1): 101-106) action target spot that PW-PS1 and PW-PS2 suppresses complement system is studied.Result shows that PW-PS1 acts on C1q, C3, C9 component of complement system, and PW-PS2 acts on C1q, C2, C3, C5 and C9 component of complement system;
4) on the impact of blood coagulation system: reference literature method (Wang Xuefeng of the present invention, Wang Hongli. the detection of Thrombosis and hemostasis and application (first version). Shanghai: Shanghai world book press, 2002, p.99), measure recalcification time (recalcificationtime, RT) and thrombin time (thrombintime, TT), with 3.4mg/L heparin for positive control, VBS
2+damping fluid is negative control (Vehicle), PW-PS1 concentration is followed successively by 1700,850,425mg/L and PW-PS2 concentration is followed successively by 1500,750,375mg/L; Result shows, heparin can significant prolongation RT and TT (P ﹤ 0.05) when 3.4mg/L, RT and TT of the PW-PS1 of different concns, PW-PS2 and negative control is without significant difference.Show PW-PS1 and PW-PS2 all without blood coagulation resisting function.
Homogeneous polysaccharide PW-PS1 described in the present invention and PW-PS2 is through experiment in vitro, and result shows to have stronger anticomplementary activity, and PW-PS1 acts on C1q, C3, C9 component of complement system; PW-PS2 acts on C1q, C2, C3, C5 and C9 component of complement system, and does not have the blood coagulation resisting function affecting activity in vivo and play, and can be used for preparing Complement inhibition medicine.
Embodiment
Embodiment 1. prepares homogeneous polysaccharide PW-PS1 and PW-PS2
Spica Prunellae medicinal material is through pulverizing, and after extremely colourless with 95% ethanol percolate extraction under room temperature, residue flings to alcohol taste, and after drying, boiling water boiling 3 times, adds 4 times of volume water at every turn, and each extraction time is 2h.Merge extracting the Aqueous extracts filtering gained at every turn, be concentrated into small volume, by 4 times of volume dehydrated alcohol precipitations, precipitate after a small amount of water dissolution, under 4 ° of C, stir deproteinated with 15% Tricholroacetic Acid.Solution goes precipitation through centrifugal, and supernatant liquor is adjusted to neutrality with lmol/LNaOH, dialyses 3 days to flowing water.In bag, solution is concentrated into proper volume, and lyophilize obtains Spica Prunellae Crude polysaccharides.Crude polysaccharides adding distil water dissolves, and centrifugal, supernatant liquor gradation DEAE-cellulose column chromatography carries out initial gross separation.With distilled water, 0.4,0.8,1.2 and the NaCl solution wash-out of 2.0mol/L, elution volume is greater than 2 times and lives volumes, and flow velocity is 0.8ml/min, collects each stream part, and pipe detects the absorbance under 280nm and 490nm (sulfuric acid-phynol method colour developing afterwards).Result according to sugared color reaction and ultraviolet detection merges same stream part, concentrated, obtains 5 secondary components: PWD1, PWD2, PWD3, PWD4 and PWD5 after dialysis and lyophilize.According to active tracking results, PWD3, PWD4 and PWD5 are merged adding distil water to dissolve, centrifugal, supernatant liquor gradation SephacrylS-400 column chromatography (100 × 2.5cm) is separated, and the NaCl solution wash-out of 0.1mol/L, flow velocity is 0.8ml/min.Pipe detects the absorbance under 280nm and 490nm (after sulfuric acid-phynol method colour developing), each pipe stream part of active tracking.Merge same stream part according to detected result, concentrated and lyophilize obtains polysaccharide PW-PS1 and PW-PS2.Detecting PW-PS1 and PW-PS2 through High Performance Gel Permeation Chromatography (HPGPC) and high performance capillary electrophoresis (HPCE) is homogeneous composition.
The constitutional features of vegetable polysaccharides PW-PS1 of the present invention is described as: faint yellow loose powder, molecular weight is about 3 × 10
5da, specific optical rotation [α]
d 25=-102.2 (c0.3, H
2o); Total sugar content is 96.94%, still containing the uronic acid of 17.87% and the albumen of 0.53%; PW-PS1 is made up of 3 kinds of monose, and saccharide residue mol ratio is: pectinose Ara: wood sugar Xyl:4-methoxyl group glucuronic acid 4-methox-glcA=1:2.6:0.8, and its mode of connection comprises end and 1, the wood sugar that 3-connects, 1,5-and 1,3, the pectinose that 5-connects, the 4-methoxyl group glucuronic acid that end connects, wherein with the wood sugar, 1 that 1,3-connects, the pectinose that 3,5-connects is connected 4-methoxyl group glucuronic acid with end be main.
The constitutional features of vegetable polysaccharides PW-PS2 of the present invention is described as: brown loose powder, and its molecular weight is about 8 × 10
3da, specific optical rotation [α]
d 25=-132.4 (c0.3, H
2o); Total sugar content is 98.12%, still containing the uronic acid of 58.20% and the albumen of 0.22%; PW-PS2 is made up of 5 kinds of monose, and saccharide residue mol ratio is: rhamnosyl Rha: pectinose Ara: wood sugar Xyl: semi-lactosi Gal: galacturonic acid GalA=0.56:1:1.30:1.78:3.37, and its mode of connection is the pectinose comprising end connection, the wood sugar that 1,3-connects, 1, the rhamnosyl that 3-connects, end and Isosorbide-5-Nitrae, the semi-lactosi that 6-connects, wherein with end and 1, the semi-lactosi that 4,6-connects and the wood sugar that 1,3-connects are main.
Embodiment 2. In Vitro Anti classical pathway of complement is tested
Get complement (guinea pig serum) 0.1ml, add the solution that barbitol buffer solution (BBS) is mixed with 1:5, become the solution of 1:10,1:20,1:40,1:80,1:160,1:320 and 1:640 with BBS two-fold dilution.Getting 1:1000 hemolysin, each concentration complement and 2% sheep red blood cell (SRBC) each 0.1ml is dissolved in 0.3mlBBS, and mixing, puts into low-temperature and high-speed whizzer after 37 ° of C water-bath 30min, centrifugal 10min under 5000rpm, 4 ° of C conditions.Get often pipe supernatant 0.2ml respectively and, in 96 orifice plates, measure its absorbancy at 405nm.Experiment arranges full haemolysis group (0.1ml2%SRBC is dissolved in 0.5ml tri-distilled water) simultaneously.Using the absorbancy of tri-distilled water haemolysis pipe as full haemolysis standard, calculate hemolysis rate.With complement extent of dilution for X-axis, the percentage of hemolysis that each weaker concn complement causes is Y-axis mapping.Select the minimum complement concentration reaching similar high hemolysis rate as the critical complement concentration guaranteed needed for the normal haemolysis of system energy.Complement and the trial-product of getting threshold concentration mix, and after 37 ° of pre-water-bath 10min of C, add appropriate BBS, hemolysin and 2%SRBC.Put into low-temperature and high-speed whizzer by after every pipe 37 ° of C water-bath 30min, get often pipe supernatant 0.2ml under 5000rpm, 4 ° of C conditions after centrifugal 10min respectively and, in 96 orifice plates, under 405nm, measure absorbancy.Experiment arranges trial-product control group, complement group and full haemolysis group simultaneously.Hemolysis rate is calculated after trial-product absorbance being deducted corresponding trial-product control group absorbance.Using trial-product concentration as X-axis, haemolysis inhibiting rate is mapped as Y-axis, calculates the concentration (CH that 50% suppresses trial-product needed for haemolysis
50).
Embodiment 3. In Vitro Anti alternative pathway of complement is tested
Get complement (human serum) 0.2ml, (barbitol buffer solution, pH=7.4, containing 5mMMg to add AP dilution
2+, 8mMEGTA) and liquid is mixed with the solution of 1:5, and two-fold dilution becomes the solution of 1:10,1:20,1:40,1:80,1:160,1:320 and 1:640.Get each concentration complement 0.15ml, AP diluent 0.15ml and 0.5% rabbit erythrocyte (RE) 0.20ml, mixing, 37 ° of C water-bath 30min are placed on low-temperature and high-speed whizzer, centrifugal 10min under 5000rpm, 4 ° of C conditions.Get often pipe supernatant 0.2ml respectively and, in 96 orifice plates, measure absorbancy at 405nm.Experiment arranges full haemolysis group (0.20ml0.5%RE is dissolved in 0.3ml tri-distilled water) simultaneously.Using the absorbancy of tri-distilled water haemolysis pipe as full haemolysis standard, calculate hemolysis rate.With complement extent of dilution for X-axis, the percentage of hemolysis that each weaker concn complement causes is Y-axis mapping.Select the minimum complement concentration reaching similar high hemolysis rate as the critical complement concentration guaranteed needed for the normal haemolysis of system energy.Complement and the trial-product of getting the threshold concentration determined mix, and after 37 ° of pre-water-bath 10min of C, add 0.2ml0.5%RE.Every pipe 37 ° of C water-bath 30min are placed on low-temperature and high-speed whizzer, under 5000rpm, 4 ° of C conditions after centrifugal 10min, get often pipe supernatant 0.2ml respectively and, in 96 orifice plates, under 405nm, measure its absorbancy.Experiment arranges trial-product control group, complement group and full haemolysis group simultaneously.Hemolysis rate is calculated after trial-product absorbance being deducted corresponding trial-product control group absorbance.Using trial-product concentration as X-axis, haemolysis inhibiting rate is mapped as Y-axis, calculates the concentration (AP that 50% suppresses trial-product needed for haemolysis
50).
Embodiment 4.PW-PS1 and PW-PS2 is to the action target spot of complement system
C3, C4 antiserum(antisera) that people source complement (1:10) 0.2ml dilutes with 0.2ml1:1 respectively, the C5 antiserum(antisera) of 1:32 dilution, C1q, C2, C9 antiserum(antisera) of 1:64 dilution, after 37 DEG C of water-bath 15min, be placed in low-temperature and high-speed whizzer, centrifugal 10min under 5000rpm, 4 ° of C conditions.Supernatant is complement depletion serum.Target spot test set: PW-PS2 solution (minimum concentration needed for the suppression complement hemolysis close to the 100%) 0.1ml of PW-PS1 and 0.43mg/ml that complement (1:10) 0.1ml and concentration are 0.55mg/ml mixes, after 37 ° of C water-bath 10min, add disappearance serum 0.2ml, 1:1000 hemolysin 0.1ml and 2%SRBC0.1ml.After 37 ° of C water-bath 30min, be placed in low-temperature and high-speed whizzer, centrifugal 10min under 5000rpm, 4 ° of C conditions.Get often pipe supernatant 0.2ml respectively and, in 96 orifice plates, measure absorbancy at 405nm.Experiment arranges PW-PS1 and PW-PS2 control group simultaneously, and (PW-PS1 and the PW-PS2 solution of 0.1ml respective concentration adds 0.5mlVBS
2+damping fluid), complement control group is (with 0.3mlVBS
2+damping fluid replaces PW-PS1 and PW-PS2 solution and disappearance serum), disappearance serologic group is (with 0.2mlVBS
2+damping fluid replaces PW-PS1 and PW-PS2 solution and complement) and full haemolysis group (0.1ml2%SRBC is dissolved in 0.5ml tri-distilled water).Deduction PW-PS1 and PW-PS2 control group absorbance calculates hemolysis rate.Relatively the change of disappearance serologic group and target spot test set hemolysis rate, according to target spot test set haemolysis situation, judges that PW-PS1 and PW-PS2 C1q, C2, C3, C4, C5, C9 to complement system is with or without antagonistic action.Target spot test set lacks serologic group haemolysis more accordingly and has recovered, and illustrates that PW-PS1 and PW-PS2 does not act on this disappearance component, if haemolysis can recover, then illustrates that PW-PS1 and PW-PS2 acts on this disappearance component.
Embodiment 5.PW-PS1 is on the impact of blood coagulation system
1. the mensuration of recalcification time (recalcificationtime, RT): cavy ip1g/kg urethane is anaesthetized, and faces upward position and fixes.Be separated arteria carotis communis, intubate bloodletting, 3.8% Sodium Citrate 1:9 (V/V) anti-freezing.3000rpm, centrifugal 10min, obtain platelet poor plasma (plateletpoorplasma, PPP).15 μ l liquids add 150 μ lPPP, and 37 ° of C water-bath 5min, add 0.025MCaCl
2150 μ l, start timing, and detect every the 5s little hook of special metal, the time (s) of fiber protein yarn appears in record, measures three times, averages.Writing time is terminal with 600s, exceeds terminal and does not still solidify and calculate with 600s.With 3.4mg/L heparin for positive control, VBS
2+damping fluid is negative control (Vehicle), PW-PS1 concentration is followed successively by 1700,850,425mg/L and PW-PS2 concentration is followed successively by 1500,750,375mg/L.
2. the mensuration of thrombin time (thrombintime, TT): the zymoplasm of freeze-drying after (being equivalent to 5 μ l/ml), can use after 37 ° of C water-bath 5min after 2ml distilled water redissolves.15 μ l liquids add 150 μ lPPP, 37 ° of C water-bath 5min, add zymoplasm 150 μ l, and the time (s) of fiber protein yarn appears in record, measures three times, averages.VBS
2+damping fluid is negative control (Vehicle), PW-PS1 concentration is followed successively by 1700,850,425mg/L and PW-PS2 concentration is followed successively by 1500,750,375mg/L.
Claims (4)
1. Polysaccharides from Prunella vulgaris L, is characterized in that, described polysaccharide comprises PW-PS1 and PW-PS2; Wherein, the constitutional features of PW-PS1 is: faint yellow loose powder, molecular weight is about 3 × 10
5da, specific optical rotation [α]
d 25=-102.2 (c0.3, H
2o); Total sugar content is 96.94%, containing the uronic acid of 17.87% and the albumen of 0.53%;
Described PW-PS1 is by pectinose Ara, wood sugar Xyl and 4-methoxyl group glucuronic acid 4-methox-glcA monose composition, saccharide residue mol ratio is wherein: pectinose Ara: wood sugar Xyl:4-methoxyl group glucuronic acid 4-methox-glcA=1:2.6:0.8, its mode of connection comprises end and 1, the wood sugar that 3-connects, 1,5-and 1, the pectinose that 3,5-connects, the 4-methoxyl group glucuronic acid that end connects, wherein with 1, the wood sugar that 3-connects, 1,3, the 5-pectinose connected are connected 4-methoxyl group glucuronic acid with end be main;
The constitutional features of PW-PS2 is: brown loose powder, molecular weight is about 8 × 10
3da, specific optical rotation [α]
d 25=-132.4 (c0.3, H
2o); Total sugar content is 98.12%, containing the uronic acid of 58.20% and the albumen of 0.22%;
Described PW-PS2 is by monosaccharides rhamnose Rha, pectinose Ara, wood sugar Xyl, semi-lactosi Gal and galacturonic acid GalA forms, saccharide residue mol ratio is wherein: rhamnosyl Rha: pectinose Ara: wood sugar Xyl: semi-lactosi Gal: galacturonic acid GalA=0.56:1:1.30:1.78:3.37, and mode of connection wherein comprises: the pectinose that end connects, 1, the wood sugar that 3-connects, the rhamnosyl that 1,3-connects, end and 1,4, the semi-lactosi that 6-connects, wherein with end and Isosorbide-5-Nitrae, the semi-lactosi that 6-connects and the wood sugar that 1,3-connects are main.
2. the preparation method of Polysaccharides from Prunella vulgaris L described in claim 1, is characterized in that, it comprises step:
Spica Prunellae medicinal material is through pulverizing, and under room temperature, 95% ethanol percolate extraction is to after colourless, and residue flings to alcohol taste, dries, boiling water boiling 3 times, adds 4 times of volume water at every turn, and each extraction time is 2h;
Merge Aqueous extracts, concentrated, by 4 times of volume dehydrated alcohol precipitations, after water dissolution, at 4 DEG C, stir deproteinated with 15% Tricholroacetic Acid;
Solution goes precipitation through centrifugal, and supernatant liquor is adjusted to neutrality, dialysis, and solution concentrates, and lyophilize obtains Spica Prunellae Crude polysaccharides;
Crude polysaccharides distilled water dissolves, centrifugal, supernatant liquor DEAE-cellulose pillar layer separation, with distilled water, NaCl solution wash-out, flow velocity is 0.8ml/min, collects each stream part, survey the absorbance under 280nm and 490nm, obtain secondary component: PWD1, PWD2, PWD3, PWD4 and PWD5;
Further pillar layer separation, collects each stream part, surveys the absorbance under 280nm and 490nm, and concentrated, lyophilize obtains polysaccharide PW-PS1 and PW-PS2.
3. the Polysaccharides from Prunella vulgaris L of claim 1 is preparing the purposes in anticomplement medicament.
4., by the purposes of claim 3, it is characterized in that, described polysaccharide PW-PS1 acts on C1q, C3 and C9 component of complement system, and described polysaccharide PW-PS2 acts on C1q, C2, C3, C5 and C9 component of complement system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210306881.0A CN103626880B (en) | 2012-08-24 | 2012-08-24 | Polysaccharides from Prunella vulgaris L and its production and use |
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| CN104031171B (en) * | 2014-05-22 | 2016-04-13 | 华南理工大学 | Polysaccharides from Prunella vulgaris L of high molecular and preparation method thereof and the application in immune drug |
| CN105754003B (en) * | 2016-05-03 | 2018-02-27 | 湖南中医药大学 | A kind of preparation method and applications of Polysaccharides from Prunella vulgaris L extract |
| CN110498863B (en) * | 2018-05-17 | 2021-09-07 | 复旦大学 | Ficus pumila leaf polysaccharide, preparation method thereof and application thereof in preparation of anticomplement medicines |
| CN111773237A (en) * | 2019-04-03 | 2020-10-16 | 复旦大学 | Turkey knotweed polysaccharide, preparation method thereof and application thereof in preparation of anticomplement medicines |
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| CN1965894A (en) * | 2006-05-12 | 2007-05-23 | 上海中医药大学 | Prunella vulgaris polysaccharide having immune activity, preparation method and application thereof |
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Non-Patent Citations (3)
| Title |
|---|
| Identification of Two Polysaccharides from Prunella vulgaris L. and Evaluation on Their Anti-Lung Adenocarcinoma Activity;Liang Feng et al.;《Molecules》;20100727;第5093-5103页 * |
| 夏枯草多糖的分离、纯化及结构初步分析;郝桂堂等;《天然产物研究与开发》;20070831;第19卷;第591-594页 * |
| 夏枯草多糖的分离及抗氧化活性研究;席与斌等;《广东药学院学报》;20101231;第26卷(第6期);第594-598页 * |
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