CN103626880A - Prunella vulgaris polysaccharide, and preparation method and purpose thereof - Google Patents
Prunella vulgaris polysaccharide, and preparation method and purpose thereof Download PDFInfo
- Publication number
- CN103626880A CN103626880A CN201210306881.0A CN201210306881A CN103626880A CN 103626880 A CN103626880 A CN 103626880A CN 201210306881 A CN201210306881 A CN 201210306881A CN 103626880 A CN103626880 A CN 103626880A
- Authority
- CN
- China
- Prior art keywords
- connects
- pectinose
- wood sugar
- polysaccharide
- complement
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the field of Chinese medicine, relates to Prunella vulgaris polysaccharide, and a preparation method and purpose thereof in preparing anticomplement medicines. The Prunella vulgaris polysaccharide including uniform polysaccharide PW-PS1 and PW-PS2 is prepared from a dry fruit aqueous extract of a labiatae plant Prunella vulgaris Linn. Through in vitro tests, the uniform polysaccharide is proved to have strong anticomplement activity and inhibition effects on classical pathway and alternative pathway of a complement system; besides, the polysaccharide does not have the anticoagulant effect influencing in vivo activity and can be used for the preparation of complement inhibition medicaments. The PW-PS1 effects on C1q, C3 and C9 components of the complement system, and the PW-PS2 effects on the C1q, C2, C3, C5 and C9 components of the 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 the purposes in preparing anticomplement medicament.
Background technology
Known complement system is one of immune defense system of wanting of body weight for humans, the normal activation of complement system eliminating external microorganism, remove damage in body or dead cell and tissue, maintain in the balance of body and play an important role; Yet, described complement system excessive activation can cause damage and the inflammatory reaction of human body self healthy tissues there are some researches show, the disease relevant to complement excessive 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's study of pharmacy always, yet at present this type of disease is still lacked to comparatively desirable medicine, is therefore badly in need of clinically efficiently, low toxicity, single-minded novel complement inhibitor.From natural product, research and develop complement inhibitor and be in recent years one and be subject to more and more important research field of paying close attention to, it has the features such as cost is low, toxicity is low.Current, have that Chinese scholars is separated from comprise the multiple natural products of marine organisms obtains a large amount of inhibiting monomeric compounds of complement system that have, for the research and development of anticomplement medicament provide wide prospect.
Chinese medicine Spica Prunellae is the dry fruit ear of labiate Spica Prunellae (Prunella vulgaris Linn), because of " withered after the Summer Solstice ", gains the name, and is one of conventional traditional Chinese medicine.Modern chemistry composition Study shows, Spica Prunellae mainly contains the chemical compositions such as triterpenes, sterols, flavonoid, coumarins, organic acid, volatile oil and carbohydrate, there is step-down, hypoglycemic, antibacterial, anti-inflammatory, antianaphylaxis and the pharmacological action such as antiviral, be mainly used in clinically treating that conjunctival congestion with pain and swelling of the eye, photophobia are shed tears, mazoitis, breast cancer, hypertension, lymphoid tuberculosis, infiltrative pulmonary tuberculosis, simple goiter, parotitis, acute icterohepatitis etc.But there is not yet up to now the report of finding to have complement restraining effect polysaccharide from Spica Prunellae.
Summary of the invention
The object of this invention is to provide the activeconstituents in natural drug with anticomplementary action, be specifically related to Spica Prunellae homogeneous polysaccharide and preparation method thereof and the purposes in preparing anticomplement medicament.
The present invention's application modern pharmacology screening method, the material that separation is obtained carries out anticomplementary activity evaluation study, from the dry fruit ear aqueous extract of labiate Spica Prunellae (Prunella vulgaris Linn), separation obtains homogeneous polysaccharide, and confirms that it all has restraining effect to complement system classical pathway and alternative pathway; Though its complement inhibitory activity is weaker than heparin, do not possess the blood coagulation resisting function that limits its performance 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 (c 0.3, H
2o); Total sugar content is 96.94%, the uronic acid containing 17.87% and 0.53% albumen;
Described PW-PS1 is by pectinose Ara, 3 kinds of monose of wood sugar Xyl and 4-methoxyl group glucuronic acid 4-methox-glcA form, 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, 1,3 that 3-connects, the pectinose that 5-connects is 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 (c 0.3, H
2o); Total sugar content is 98.12%, still containing 58.20% uronic acid and 0.22% albumen;
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 wood sugar that the semi-lactosi that 4,6-connects and 1,3-connect is main.
Homogeneous polysaccharide described in the present invention is prepared by following method:
The dry fruit ear medicinal material of labiate Spica Prunellae (Prunella vulgaris Linn) is through pulverizing, under room temperature with 95% ethanol percolate extraction to after colourless, residue is flung 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; By extract the water extraction liquid merging of filtering gained at every turn, be concentrated into small volume, by 4 times of volume dehydrated alcohols precipitations, precipitation, after a small amount of water dissolution, stirs deproteinated with 15% Tricholroacetic Acid (TFA) under 4 ° of C; Solution was through the centrifugal precipitation of going, and supernatant liquor is adjusted to neutrality with 1mol/L NaOH, with flowing water dialysis 3 days; 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 is carried out initial gross separation by DEAE-cellulose column chromatography; With distilled water, 0.4,0.8,1.2 and the NaCl eluant solution 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); According to the result of sugared color reaction and ultraviolet detection, merge same stream part, concentrated, after dialysis and lyophilize, obtain 5 secondary component: PWD1, PWD2, PWD3, PWD4 and PWD5; According to complement inhibitory activity tracking results, PWD3, PWD4 and PWD5 are merged to adding distil water dissolving, centrifugal, Sephacryl S-400 column chromatography (100 * 2.5cm) separation for supernatant liquor gradation, the NaCl eluant solution 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), active each pipe stream part of following the tracks of; According to detected result, merge same stream part, concentrated and lyophilize obtains respectively homogeneous polysaccharide PW-PS1 and PW-PS2; Through High Performance Gel Permeation Chromatography (HPGPC) and high performance capillary electrophoresis (HPCE), detect the composition that PW-PS1 and PW-PS2 are homogeneous;
Adopt efficient gel post TSK-GEL GMPWXL (300 * 7.6mm), moving phase is 0.01mol/LNaCl, flow velocity 0.8ml/min, and 25 ° of C of column temperature, compare mensuration with the Dextran of T series, result demonstration, 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 is containing C, 43.38%; H, 7.242%; N, 0.29%; Specific optical rotation is [α]
d 25=-102.2 (c 0.3, H
2o); PW-PS2 is containing C, 36.47%; H, 6.205%; N, 0.50%; Specific optical rotation is [α]
d 25=-132.4 (c 0.3, H
2o);
The total sugar content that sulfuric acid-phynol method is measured PW-PS1 and PW-PS2 is respectively 96.94% and 98.12%, between the hydroxyl biphenyl method glucuronic acid content of measuring PW-PS1 and PW-PS2 be respectively 17.87% and 58.20%, the protein content that Xylene Brilliant Cyanine G method is measured 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, the PW-PS1 after uronic acid reduction and PW-PS2 in 110 ° of C complete hydrolysis, successively carry out NaBH through 2mol/L TFA
4reduction, aceticanhydride acetylize is prepared into ALDI alcohol acetic ester derivative, carries out gas phase compositional analysis (method referring to: Blakeney AB, Harris PJ, Henry RJ, et al.1983,113:291-299); Result shows, PW-PS1 is comprised 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 comprised 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 PW-PS2 reference literature (Needs PW, Selvendran RR.1993,245:1-10) after uronic acid reduction methylate, 90% formic acid depolymerization for the product after methylating, 2mol/L TFA complete hydrolysis, NaBH
4the ALDI alcohol acetic ester derivative that part methyl is made in reduction and aceticanhydride acetylize, then carries out GC-MS analysis; Combined standard collection of illustrative plates, determine the wood sugar of PW-PS1 mode of connection for comprising that end and 1,3-connect, 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, 1,3 that 3-connects, the pectinose that 5-connects is connected 4-methoxyl group glucuronic acid with end be main; PW-PS2 mode of connection is the pectinose that comprises that end connects, the wood sugar that 1,3-connects, and the rhamnosyl that 1,3-connects, end and Isosorbide-5-Nitrae, the semi-lactosi that 6-connects, wherein with end and Isosorbide-5-Nitrae, the wood sugar that the semi-lactosi that 6-connects and 1,3-connect is main;
PW-PS1 and the PW-PS2 effect to complement system:
1) anticomplement activates classical pathway cell hemolytic test: guinea pig serum 1:80 dilutes as complement, antigen activates classical pathway of complement causes sheep red blood cell haemolysis, reference literature method (Kabat EA, Mayer MM.Complement and complement fixation in experimental immunology.Charles C.Thomas Publisher, Illinois, USA.1964, pp.133-240) record PW-PS1 and PW-PS2 all can suppress 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 (Klerx JP, Beukelman CJ, Van DH, et al.Microassay for colorimetric estimation of complement activity in guinea pig, human and mouse serum.Immunological Methods, 1983,63 (2): 215-220) record PW-PS1 and PW-PS2 and all can suppress cell haemolysis, AP
50be respectively 0.40 ± 0.02mg/ml and 0.35 ± 0.03mg/ml (n=3);
3) research to complement system action target spot: the present invention utilizes complement disappearance serum, reference literature method (Zhou Jie, Zhang Yun is firm, Zhang Jianwen, Deng. the effect of the Chinese medicine bark of eucommia to complement system. Fudan Journal (medicine), 2006,33 (1): 101-106) research PW-PS1 and PW-PS2 suppress the action target spot of complement system.Result shows that PW-PS1 acts on the C1q of complement system, C3, C9 component, and PW-PS2 acts on C1q, C2, C3, C5 and the C9 component of complement system;
4) impact on 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 (recalcification time, RT) and thrombin time (thrombin time, TT), with the positive contrast of 3.4mg/L heparin, VBS
2+the negative contrast of damping fluid (Vehicle), PW-PS1 concentration is followed successively by 1700,850,425mg/L and PW-PS2 concentration are followed successively by 1500,750,375mg/L; Result shows, heparin can significant prolongation RT and TT (P ﹤ 0.05) when 3.4mg/L, the PW-PS1 of different concns, and the RT of PW-PS2 and negative control and TT are without significant difference.Show that PW-PS1 and PW-PS2 are all without blood coagulation resisting function.
The PW-PS1 of homogeneous polysaccharide described in the present invention and PW-PS2 are through experiment in vitro, and result shows to have stronger anticomplementary activity, and PW-PS1 acts on C1q, C3, the C9 component of complement system; PW-PS2 acts on C1q, C2, C3, C5 and the C9 component of complement system, and does not have the blood coagulation resisting function that affects activity in vivo performance, can be used for preparing complement and suppresses medicine.
Embodiment
Embodiment 1. preparation homogeneous polysaccharide PW-PS1 and PW-PS2
Spica Prunellae medicinal material is through pulverizing, under room temperature with 95% ethanol percolate extraction to after colourless, residue is flung to alcohol taste, after drying, boiling water boiling 3 times adds 4 times of volume water at every turn, each extraction time is 2h.By extract the water extraction liquid merging of filtering gained at every turn, be concentrated into small volume, by 4 times of volume dehydrated alcohols precipitations, precipitation, after a small amount of water dissolution, stirs deproteinated with 15% Tricholroacetic Acid under 4 ° of C.Solution was through the centrifugal precipitation of going, and supernatant liquor is adjusted to neutrality with lmol/L NaOH, to flowing water dialysis 3 days.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 is carried out initial gross separation by DEAE-cellulose column chromatography.With distilled water, 0.4,0.8,1.2 and the NaCl eluant solution 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 (after sulfuric acid-phynol method colour developing).According to the result of sugared color reaction and ultraviolet detection, merge same stream part, concentrated, after dialysis and lyophilize, obtain 5 secondary component: PWD1, PWD2, PWD3, PWD4 and PWD5.According to active tracking results, PWD3, PWD4 and PWD5 are merged to adding distil water dissolving, centrifugal, Sephacryl S-400 column chromatography (100 * 2.5cm) separation for supernatant liquor gradation, the NaCl eluant solution 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), active each pipe stream part of following the tracks of.According to detected result, merge same stream part, concentrated and lyophilize obtains polysaccharide PW-PS1 and PW-PS2.Through High Performance Gel Permeation Chromatography (HPGPC) and high performance capillary electrophoresis (HPCE), detect the composition that PW-PS1 and PW-PS2 are homogeneous.
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 (c 0.3, H
2o); Total sugar content is 96.94%, still containing 17.87% uronic acid and 0.53% albumen; PW-PS1 is comprised 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, the wood sugar, 1 wherein connecting with 1,3-, 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, its molecular weight is about 8 * 10
3da, specific optical rotation [α]
d 25=-132.4 (c 0.3, H
2o); Total sugar content is 98.12%, still containing 58.20% uronic acid and 0.22% albumen; PW-PS2 is comprised 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 that comprises 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 wood sugar that the semi-lactosi that 4,6-connects and 1,3-connect is main.
Embodiment 2. In Vitro Anti classical pathway of complement tests
Get complement (guinea pig serum) 0.1ml, add barbitol buffer solution (BBS) to be mixed with the solution of 1:5, with BBS two-fold dilution, become the solution of 1:10,1:20,1:40,1:80,1:160,1:320 and 1:640.Get 1:1000 hemolysin, each concentration complement and each 0.1ml of 2% sheep red blood cell (SRBC) and be dissolved in 0.3ml BBS, mix, after 37 ° of C water-bath 30min, put into low-temperature and high-speed whizzer, centrifugal 10min under 5000rpm, 4 ° of C conditions.Get respectively every pipe supernatant 0.2ml in 96 orifice plates, at 405nm, measure its absorbancy.Experiment arranges full haemolysis group (0.1ml 2%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.Take complement extent of dilution as X-axis, and the percentage of hemolysis that each weaker concn complement causes is Y-axis mapping.Selection reaches the minimum complement concentration of similar high hemolysis rate as guaranteeing that system can the normal required critical complement concentration of haemolysis.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.To after 37 ° of C water-bath 30min of every pipe, put into low-temperature and high-speed whizzer, under 5000rpm, 4 ° of C conditions, after centrifugal 10min, get respectively every pipe supernatant 0.2ml in 96 orifice plates, under 405nm, measure absorbancy.Experiment arranges trial-product control group, complement group and full haemolysis group simultaneously.After being deducted to corresponding trial-product control group absorbance, trial-product absorbance calculates hemolysis rate.Using trial-product concentration as X-axis, and haemolysis inhibiting rate is mapped as Y-axis, calculates the concentration (CH of the required trial-product of 50% inhibition haemolysis
50).
Embodiment 3. In Vitro Anti alternative pathway of complement tests
Get complement (human serum) 0.2ml, (barbitol buffer solution, pH=7.4, containing 5mM Mg to add AP dilution
2+, 8mM EGTA) 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, mix, 37 ° of C water-bath 30min are placed on low-temperature and high-speed whizzer, centrifugal 10min under 5000rpm, 4 ° of C conditions.Get respectively every pipe supernatant 0.2ml in 96 orifice plates, at 405nm, measure absorbancy.Experiment arranges full haemolysis group (0.20ml 0.5%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.Take complement extent of dilution as X-axis, and the percentage of hemolysis that each weaker concn complement causes is Y-axis mapping.Selection reaches the minimum complement concentration of similar high hemolysis rate as guaranteeing that system can the normal required critical complement concentration of haemolysis.Complement and the trial-product of getting definite threshold concentration mix, and after 37 ° of pre-water-bath 10min of C, add 0.2ml 0.5%RE.37 ° of C water-bath 30min of every pipe are placed on to low-temperature and high-speed whizzer, under 5000rpm, 4 ° of C conditions, after centrifugal 10min, get respectively every pipe supernatant 0.2ml in 96 orifice plates, under 405nm, measure its absorbancy.Experiment arranges trial-product control group, complement group and full haemolysis group simultaneously.After being deducted to corresponding trial-product control group absorbance, trial-product absorbance calculates hemolysis rate.Using trial-product concentration as X-axis, and haemolysis inhibiting rate is mapped as Y-axis, calculates the concentration (AP of the required trial-product of 50% inhibition haemolysis
50).
Embodiment 4.PW-PS1 and the PW-PS2 action target spot to complement system
People source complement (1:10) 0.2ml respectively with C3, the C4 antiserum(antisera) of 0.2ml 1:1 dilution, the C5 antiserum(antisera) of 1:32 dilution, C1q, C2, the C9 antiserum(antisera) of 1:64 dilution, after 37 ℃ of water-bath 15min, be placed in low-temperature and high-speed whizzer, centrifugal 10min under 5000rpm, 4 ° of C conditions.Supernatant is complement disappearance serum.Target spot test set: the PW-PS1 that complement (1:10) 0.1ml and concentration are 0.55mg/ml and the PW-PS2 solution of 0.43mg/ml (approaching 100% the required minimum concentration of inhibition complement hemolysis) 0.1ml mixes, after 37 ° of C water-bath 10min, add disappearance serum 0.2ml, 1:1000 hemolysin 0.1ml and 2%SRBC 0.1ml.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 respectively every pipe supernatant 0.2ml in 96 orifice plates, at 405nm, measure absorbancy.Experiment arranges PW-PS1 and PW-PS2 control group simultaneously, and (PW-PS1 of 0.1ml respective concentration and PW-PS2 solution add 0.5ml VBS
2+damping fluid), complement control group is (with 0.3ml VBS
2+damping fluid replaces PW-PS1 and PW-PS2 solution and disappearance serum), disappearance serologic group is (with 0.2ml VBS
2+damping fluid replaces PW-PS1 and PW-PS2 solution and complement) and full haemolysis group (0.1ml 2%SRBC is dissolved in 0.5ml tri-distilled water).Deduction PW-PS1 and PW-PS2 control group absorbance calculate hemolysis rate.Relatively lack the variation of serologic group and target spot test set hemolysis rate, according to target spot test set haemolysis situation, judgement PW-PS1 and PW-PS2 have or not antagonistic action to the C1q of complement system, C2, C3, C4, C5, C9.Target spot test set lacks more accordingly serologic group haemolysis and has recovered, and illustrates that PW-PS1 and PW-PS2 do not act on this disappearance component, if haemolysis can recover, illustrates that PW-PS1 and PW-PS2 act on this disappearance component.
The impact of embodiment 5.PW-PS1 on blood coagulation system
1. the mensuration of recalcification time (recalcification time, RT): the anesthesia of cavy ip 1g/kg urethane, face upward position fixing.Separated arteria carotis communis, intubate bloodletting, 3.8% Sodium Citrate 1:9 (V/V) anti-freezing.3000rpm, centrifugal 10min, obtains platelet poor plasma (platelet poor plasma, PPP).15 μ l liquids add 150 μ lPPP, and 37 ° of C water-bath 5min, add 0.025M CaCl
2150 μ l, start timing, every 5s, with the little hook of special metal, detect, and the time (s) of fiber protein yarn appears in record, measures three times, averages.600s take writing time as terminal, exceed terminal and still do not solidify with 600s and calculate.With the positive contrast of 3.4mg/L heparin, VBS
2+the negative contrast of damping fluid (Vehicle), PW-PS1 concentration is followed successively by 1700,850,425mg/L and PW-PS2 concentration are followed successively by 1500,750,375mg/L.
2. the mensuration of thrombin time (thrombin time, TT): the zymoplasm of freeze-drying after (being equivalent to 5 μ l/ml), can be used after 37 ° of C water-bath 5min after 2ml distilled water redissolves.15 μ l liquids add 150 μ l PPP, and 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+the negative contrast of damping fluid (Vehicle), PW-PS1 concentration is followed successively by 1700,850,425mg/L and PW-PS2 concentration are followed successively by 1500,750,375mg/L.
Claims (6)
1. Polysaccharides from Prunella vulgaris L, is characterized in that, described polysaccharide is homogeneous polysaccharide, comprises PW-PS1 and PW-PS2; Wherein,
The constitutional features of PW-PS1 is: faint yellow loose powder, molecular weight is 3 * 10
5da, specific optical rotation [α]
d 25=-102.2 (c 0.3, H
2o); Total sugar content is 96.94%, the uronic acid containing 17.87% and 0.53% albumen;
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 (c 0.3, H
2o); Total sugar content is 98.12%, the uronic acid containing 58.20% and 0.22% albumen.
2. by Polysaccharides from Prunella vulgaris L claimed in claim 1, it is characterized in that, described PW-PS1 is by pectinose Ara, wood sugar Xyl and 4-methoxyl group glucuronic acid 4-methox-glcA monose form, 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, 3, the pectinose that 5-connects, the 4-methoxyl group glucuronic acid that end connects, wherein with 1, the wood sugar that 3-connects, 1, 3, the pectinose that 5-connects is connected 4-methoxyl group glucuronic acid with end be main.
3. by Polysaccharides from Prunella vulgaris L claimed in claim 1, it is characterized in that, described PW-PS2 is by monosaccharides rhamnose Rha, pectinose Ara, wood sugar Xyl, semi-lactosi Gal and galacturonic acid GalA form, 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, 1, the wood sugar that 3-connects, 1, the rhamnosyl that 3-connects, end and 1, 4, the semi-lactosi that 6-connects, wherein with end and 1, 4, the semi-lactosi and 1 that 6-connects, the wood sugar that 3-connects is main.
4. 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 is flung to alcohol taste, dries, and boiling water boiling 3 times adds 4 times of volume water at every turn, and each extraction time is 2h;
Merge water extraction liquid, concentrated, by 4 times of volume dehydrated alcohol precipitations, after water dissolution, with 15% Tricholroacetic Acid, under 4 ° of C, stir deproteinated;
Solution is through the centrifugal precipitation of going, and supernatant liquor is adjusted to neutrality, dialysis, and solution is concentrated, and lyophilize obtains Spica Prunellae Crude polysaccharides;
Crude polysaccharides distilled water dissolves, centrifugal, and supernatant liquor is separated by DEAE-cellulose column chromatography, with distilled water, NaCl eluant solution, 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 column chromatography is separated, collects each stream part, surveys the absorbance under 280nm and 490nm, and concentrated, lyophilize obtains homogeneous polysaccharide PW-PS1 and PW-PS2.
5. the purposes of the Polysaccharides from Prunella vulgaris L of claim 1 in preparing anticomplement medicament.
6. by the purposes of claim 5, it is characterized in that, described polysaccharide PW-PS1 acts on C1q, C3 and the C9 component of complement system, and described polysaccharide PW-PS2 acts on C1q, C2, C3, C5 and the 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 |
Applications Claiming Priority (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 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103626880A true CN103626880A (en) | 2014-03-12 |
| CN103626880B CN103626880B (en) | 2016-03-30 |
Family
ID=50208355
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210306881.0A Expired - Fee Related CN103626880B (en) | 2012-08-24 | 2012-08-24 | Polysaccharides from Prunella vulgaris L and its production and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103626880B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104031171A (en) * | 2014-05-22 | 2014-09-10 | 华南理工大学 | High molecular weight selfheal polysaccharide, and preparation method and application thereof in immune medicaments |
| CN105754003A (en) * | 2016-05-03 | 2016-07-13 | 湖南中医药大学 | Preparation method and application of spica prunellae polysaccharide extracts |
| CN110498863A (en) * | 2018-05-17 | 2019-11-26 | 复旦大学 | Climbing fig leaf polyose and preparation method thereof and preparing the purposes in anticomplement medicament |
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2012
- 2012-08-24 CN CN201210306881.0A patent/CN103626880B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| LIANG FENG ET AL.: "Identification of Two Polysaccharides from Prunella vulgaris L. and Evaluation on Their Anti-Lung Adenocarcinoma Activity", 《MOLECULES》, 27 July 2010 (2010-07-27) * |
| 席与斌等: "夏枯草多糖的分离及抗氧化活性研究", 《广东药学院学报》, vol. 26, no. 6, 31 December 2010 (2010-12-31) * |
| 郝桂堂等: "夏枯草多糖的分离、纯化及结构初步分析", 《天然产物研究与开发》, vol. 19, 31 August 2007 (2007-08-31) * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104031171A (en) * | 2014-05-22 | 2014-09-10 | 华南理工大学 | High molecular weight selfheal polysaccharide, and preparation method and application thereof in immune medicaments |
| 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 |
| CN105754003A (en) * | 2016-05-03 | 2016-07-13 | 湖南中医药大学 | Preparation method and application of spica prunellae polysaccharide extracts |
| 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 |
| CN110498863A (en) * | 2018-05-17 | 2019-11-26 | 复旦大学 | Climbing fig leaf polyose and preparation method thereof and preparing the purposes in anticomplement medicament |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103626880B (en) | 2016-03-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ivanovska et al. | Study on the anti-inflammatory action of Berberis vulgaris root extract, alkaloid fractions and pure alkaloids | |
| Ammon et al. | Inhibition of Ileal Water Absorption by Intraluminal Fatty Acids INFLUENCE OF CHAIN LENGTH, HYDROXYLATION, AND CONJUGATION OF FATTY ACIDS | |
| CN102653568B (en) | Herba taraxaci homogeneous polysaccharide and preparation method and application thereof | |
| CN103626880B (en) | Polysaccharides from Prunella vulgaris L and its production and use | |
| AU2003260985B2 (en) | Extraction and purification method of active constituents from stem of Lonicera japonica Thunb., its usage for anti-inflammatory and analgesic drug | |
| Huang et al. | A pectic polysaccharide from water decoction of Xinjiang Lycium barbarum fruit protects against intestinal endoplasmic reticulum stress | |
| CN103508922B (en) | Dipeptide compound and use of the same in preparation of anti-complement drugs | |
| CN102558278B (en) | Triterpene compound and application thereof in preparation of anti-complementary medicament | |
| CN109771503A (en) | A kind of pepper extract and its application | |
| JPH05504785A (en) | Novel heparin derivative | |
| CN101390868B (en) | Plant polysaccharide and preparation method and use thereof | |
| CN107625800A (en) | A kind of purposes of the white phoenix dish extractive of general flavone and preparation method thereof with anti-inflammatory and antalgic | |
| Nanumala et al. | Evaluations of diuretic activity of methanolic extract of Physalis angulata L. leaves | |
| CN109620824A (en) | Quassinoids compound is preparing the purposes in anticomplement medicament | |
| CN101428020B (en) | Freeze-dried powder preparation of kuh-seng native, preparation method thereof | |
| CN104877037A (en) | Separation and purification method, products and application of Christia vespertilionis polysaccharides | |
| CN103087020B (en) | Boraginaceae phenol compound and purpose thereof in preparing anticomplement medicines | |
| CN105708851B (en) | The more carbohydrates and their derivatives of cordate houttuynia are preparing the purposes in Complement inhibition drug | |
| CN103417565B (en) | The application of depolymerization glycosaminoglycan extracted from sea cucumber in preparation control thrombotic diseases medicine | |
| CN113264975B (en) | An extract with antiinflammatory activity extracted from fructus Rosae Normalis rhizome and its application | |
| Nerdy et al. | Mucolytic activity of roselle (Hibiscus sabdariffa L.) Calyces extract on cow intestinal mucus | |
| CN110183413A (en) | Diphenylethylene compounds in two color jackfruits and its purposes in preparation treatment diseases associated with inflammation drug | |
| CN104095833B (en) | Diphenyl ether compound is preparing the purposes in beta-amyloid aggregation inhibitor | |
| CN107090050B (en) | Screwtree root polysaccharide and preparation method thereof and preparing the purposes in anticomplement medicament | |
| CN109091495A (en) | Fieldbalm polysaccharide is preparing the purposes and preparation method thereof in anticomplement medicament |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160330 Termination date: 20210824 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |