CN105037578B - A kind of anticoagulation blackberry seed polysaccharide and its extraction separation method, application - Google Patents
A kind of anticoagulation blackberry seed polysaccharide and its extraction separation method, application Download PDFInfo
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Abstract
The invention belongs to technical field of plant extraction, and in particular to a kind of anticoagulation blackberry seed polysaccharide and its extraction separation method, application.After blackberry seed is crushed by the extraction separation method, crude polysaccharides are obtained with the method for water extract-alcohol precipitation, then three polysaccharide are obtained by 52 cellulose column chromatographies of DEAE and 100 gel column chromatographys of SephadexG respectively.The present invention adopts special extraction separation method isolated three polysaccharide from blackberry seed, and the external coagulating effectiveness to these three polysaccharide to investigate, and as a result shows, these three polysaccharide have good anticoagulant effect.
Description
Technical field
The invention belongs to technical field of plant extraction, and in particular to a kind of anticoagulation blackberry seed polysaccharide and its extraction separation side
Method, application.
Background technology
BlackberryRubusSpp.Blackberry is rose family rubus polymerization fruit plant, does not make medicinal, is the world
One of upper 4 kinds of emerging fruitlet fruit trees, originate in North America, its fruit sweet and sour taste, with very high nutritive value and medicinal valency
Value, in addition to eating raw, also major part makes the food such as quick-frozen fruit, fruit juice, fruit wine and fruit jam.Literature search finds to lead blackberry research
Concentrate on flavone, anthocyanin and content of vitamin E in fruit volatile oil, blackberry seed oil fatty acid composition and blackberry leaf, seed
In terms of analysis.
Blackberry seed is the by-product of the converted productss such as blackberry fruit wine, blackberry juice, containing about 27.14% functional grease, its
Middle unsaturated fatty acid total amount is up to 93.55%, in addition with aminoacid needed by human, to atherosclerosiss, anti-sugar
The aspects such as urine disease, immunomodulating, promotion growth and intelligent development have important effect.
The essence of blood coagulation is the process for being changed into undissolved solid fabric albumen in water in water miscible Fibrinogen,
Be in endogenouss or（With）Thrombokinase is produced under exogenous cruor pathway, blood coagulation is produced in the presence of thrombin
Enzyme, finally makes Fibrinogen be changed into fibrin in the presence of thrombin.What PT mainly reflected is extrinsic coagulation way
The activity of factor I, II, V, VII, X in footpath；APTT mainly reflects intrinsic coagulation system situation, with VIII, X, XI, XII
It is relevant Deng intrinsic coagulation factor active；TT values are mainly to reflect that Fibrinogen is changed into the important finger of fibrin degree
Mark；FIB mainly reflects the content of Fibrinogen.
Thrombosiss are the basic pathology processes of numerous disease such as dead, deep venous thrombosis of myocardial infarction, ischemic soldier etc., it
Severely compromise the health of the mankind, its formation and hematoblastic aggregation, adhesion, endogenouss and extrinsic coagulation system and
It is fibrinous to form closely related.According to the investigation report of World Health Organization (WHO), due to thrombosis caused by atherosclerosiss
The death that disease causes has been discharged to first.Arterial thrombus are that speckle is unstable to there occurs speckle due to atherosclerosiss
The rupture of block, makes the platelet in blood that aggregation to occur, have activated internal blood coagulation system, finally result in thrombosiss.Once
Ischemia and the necrosis of tissue will occur after blocking, serious consequence is produced.In the acute or special of arterial thrombotic disease
Under state, need auxiliary to use anticoagulation medicine, can be only achieved more preferable antithrombotic effect.
The content of the invention
It is an object of the invention to provide a kind of anticoagulation blackberry seed polysaccharide extracted from blackberry seed, while it is more to provide this
The extraction separation method of sugar, application.
For achieving the above object, the present invention is employed the following technical solutions：
The extraction separation method of anticoagulation blackberry seed polysaccharide, comprises the following steps：（1）Blackberry seed is crushed, is used under room temperature
Petroleum ether degreasing 3 times, after volatilizing solvent petroleum ether, medicinal residues are extracted 3 times with 70 % ethanol, are filtered, and dry in the shade residue, then to residue
In add distilled water extraction in ratio that feed liquid mass volume ratio is 20 mL/g（Extract 3 times at 80 DEG C, 3 h）, while hot
Sucking filtration, concentrating under reduced pressure after merging filtrate, then in concentrated solution, add 95% ethanol to be adjusted to final concentration of 70 % of ethanol, 4 DEG C are quiet
It is centrifuged after putting 24 h（10000 r/min, 10 min）, the precipitation for obtaining adds distills water dissolution dialysis（48 h, every 4 h are changed once
Distilled water）To remove small molecular weight impurity, concentrating under reduced pressure, lyophilization obtain final product blackberry seed crude polysaccharides；
（2）The blackberry seed crude polysaccharides for taking 300 mg are dissolved in 10mL distilled water, are filtered, are added to DEAE- after discarding impurity
52 cellulose chromatography posts (2.5*60 cm), it is molten with distilled water, 0.1mol/L sodium chloride solutions and 0.2 mol/L Sodium Chloride successively
Liquid carries out eluting, and eluent is detected using phend-sulphuric acid, the absorbance surveyed at 490 nm, with eluting pipe number as horizontal seat
Mark, absorbance is vertical coordinate, draws elution curve (Fig. 2), merges the polysaccharide sample of same eluting peak, freezes after dialysis, concentration
It is dried；Wherein, distilled water eluting peak is 2,0.2 mol/L chlorine of component for the eluting peak of 1,0.1 mol/L sodium chloride solutions of component
The eluting peak for changing sodium solution is component 3；
（3）The component 1 for weighing 70 mg is dissolved in 5mL distilled water, filter and discard impurity, is added to Sephadex G-100
Gel chromatographic columnses (1.5*100cm), carry out eluting with distilled water（Elution flow rate is 0.5 mL/min, often 2 mL of pipe）, using benzene
Phenol-sulfuric acid method is detected that at 490 nm of wavelength survey its absorbance, with eluting pipe number as abscissa, absorbance is vertical coordinate,
Draw elution curve（Fig. 3）, two groups of eluting peaks are obtained, the 2nd group of eluting peak is concentrated, the polysaccharide that lyophilization is obtained is named as BSP-
2；Component 2 and 3 carries out eluting using the method for component 1, obtains single eluting peak, is respectively designated as BSP-3 and BSP-4；
BSP-2, BSP-3 and BSP-4 are anticoagulation blackberry seed polysaccharide.
Wherein DEAE-52 cellulose chromatographies post is：Diethyllaminoethyl fiber type element, is a kind of anion-exchange fibre
Element, is usually used in polysaccharide purification and separation.After crude polysaccharides solution is adsorbed in DEAE-52, eluting is carried out with the salt of different ionic strength,
Separated according to the ion-exchange capacity difference of polysaccharide.
Wherein Sephadex G-100 gel chromatographic columnses are：It is a kind of semisynthetic gel, basic framework is with glucose
The polysaccharide of (dextran) for residue, between chain structure, with the chloro- 1,2 epoxy prapanes of 3- as cross-linking agent, is mutually interconnected with ether bridge
It is connected into as three-dimensional net high-polymer compound.
The anticoagulation blackberry seed polysaccharide obtained using said extracted separation method.
Application of the anticoagulation blackberry seed polysaccharide in terms of anticoagulation medicine is prepared.
The present invention adopts special extraction separation method isolated Polysaccharide B SP-2, BSP-3 and BSP-4 from blackberry seed,
And the external coagulating effectiveness to these three polysaccharide is investigated, and is as a result shown, these three polysaccharide have good anticoagulant effect,
And the anticoagulant effect of BSP-2 and BSP-4 is better than positive control breviscapine（P<0.001）, anticoagulant effect and the sun of BSP-3
Property control breviscapine quite (P>0.05), it is seen then that they can be used singly or in combination to prepare anticoagulant.
Description of the drawings
Fig. 1 is clotting mechanism figure；
Fig. 2 blackberry seed crude polysaccharides DEAE-52 elution curves；
The Sephadex G-100 gel chromatographic columnses elution curves of Fig. 3 components 1；
The Sephadex G-100 gel chromatographic columnses elution curves of Fig. 4 components 2；
The Sephadex G-100 gel chromatographic columnses elution curves of Fig. 5 components 3；
The GC chromatograms of Fig. 6 standard mixture of monosaccharides；
Fig. 7 BSP-2 hydrolysate GC chromatograms；
Fig. 8 BSP-3 hydrolysate GC chromatograms；
Fig. 9 BSP-4 hydrolysate GC chromatograms；
Specific embodiment
Below by way of specific embodiment, the above of the present invention is described in further detail again.
Blackberry seed is purchased from Henan Province Fengqiu County blackberry planting base, is rose family rubus plant blackberryRubus
Spp.Blackberry seeds.
Concentration of alcohol herein is volumetric concentration.
1st, the extraction separation method of anticoagulation blackberry seed polysaccharide, comprises the following steps：（1）Blackberry seed is crushed, under room temperature
With petroleum ether degreasing 3 times, after volatilizing solvent petroleum ether, medicinal residues are extracted 3 times with 70 % ethanol, are filtered, and dry in the shade residue, then to residual
Distilled water extraction is added in the ratio that feed liquid mass volume ratio is 20 mL/g in slag, is extracted 3 times at 80 DEG C, 3 h, while hot
Sucking filtration, concentrating under reduced pressure after merging filtrate, then in concentrated solution, add 95% ethanol to be adjusted to final concentration of 70 % of ethanol, 4 DEG C are quiet
It is centrifuged after putting 24 h（10000 r/min, 10 min）, the precipitation for obtaining adds distills water dissolution dialysis（48 h, every 4 h are changed once
Distilled water）To remove small molecular weight impurity, concentrating under reduced pressure, lyophilization obtain final product blackberry seed crude polysaccharides；
（2）The blackberry seed crude polysaccharides for taking 300 mg are dissolved in 10mL distilled water, are filtered, are added to DEAE- after discarding impurity
52 cellulose chromatography posts (2.5*60 cm), it is molten with distilled water, 0.1mol/L sodium chloride solutions and 0.2 mol/L Sodium Chloride successively
Liquid carries out eluting, and eluent is detected using phend-sulphuric acid, the absorbance surveyed at 490 nm, with eluting pipe number as horizontal seat
Mark, absorbance is vertical coordinate, draws elution curve (Fig. 2), merges the polysaccharide sample of same eluting peak, freezes after dialysis, concentration
It is dried；Wherein, distilled water eluting peak is 2,0.2 mol/L chlorine of component for the eluting peak of 1,0.1 mol/L sodium chloride solutions of component
The eluting peak for changing sodium solution is component 3；
（3）The component 1 for weighing 70 mg is dissolved in 5mL distilled water, filter and discard impurity, is added to Sephadex G-100
Gel chromatographic columnses (1.5*100cm), carry out eluting with distilled water（Elution flow rate is 0.5 mL/min, often 2 mL of pipe）, using benzene
Phenol-sulfuric acid method is detected that at 490 nm of wavelength survey its absorbance, with eluting pipe number as abscissa, absorbance is vertical coordinate,
Draw elution curve（Fig. 3）, two groups of eluting peaks are obtained, the 2nd group of eluting peak is concentrated, the composition that lyophilization is obtained is named as BSP-
2；Component 2 and 3 carries out eluting using the method for component 1, obtains single eluting peak（Figure 4 and 5）, be respectively designated as BSP-3 and
BSP-4；BSP-2, BSP-3 and BSP-4 are anticoagulation blackberry seed polysaccharide.
2 anticoagulation blackberry seed polysaccharides（BSP-2、BSP-3、BSP-4）Molecular weight determination and component analyses：
2.1st, polysaccharide sample BSP-2, BSP-3 and BSP-4 deliver to Beijing Physichemistry Analysis ＆ Measurment Centre's detection, according to China
People's republic's pharmacopeia（Version in 2010）Two annex VH（Molecular exclusion chromatography）It is measured.BSP-2、BSP-3、BSP-4
Mw be respectively 2.696e+5,6.089e+4,3.406e+5.
2.2 monosaccharide composition analysis
2.2.1 the hydrolysis of polysaccharide
BSP-2, BSP-3, the BSP-4 for accurately weighing 10 mg is dissolved in 2 mL, the trifluoroacetic acid of 2 mol/L respectively, then is turned
Move on in 5 mL ampoule bottles, inflated with nitrogen tube sealing, 3 h are hydrolyzed at 110 DEG C, then trifluoroacetic acid solution is removed with rotary evaporation, it is residual
The dissolving of a small amount of methanol, then rotary evaporation are added in excess to doing, so repeatedly 3 times, hydrolyzate is obtained standby.
2.2.2 the derivatization of monosaccharide
10 mg oxammonium hydrochloride .s and 0.5 mL pyridines are sequentially added in hydrolyzate, vibration is mixed, is put in 90 DEG C of water-baths
30 min of reacting by heating.Room temperature is cooled to after taking-up, 0.5 mL acetic anhydride is added, continuing 30 min of reaction at 90 DEG C carries out second
Acylated, product is injected gas chromatogram Jing after 0.22 μm of membrane filtration and is analyzed；The same procedure process of standard monosaccharide,
And make standard monosaccharide derivatives mixed liquor.
2.2.3 GC conditions
Chromatographic column：Thermo TG-Waxms（30 m × 0.32 mm, 0.5 μm)；Injector temperature：250℃；Fid detector
Temperature：280℃；Chromatographic column heating schedule：100 DEG C of 1 min of holding of initial temperature, are then risen by 100 DEG C with the speed of 4 DEG C/min
To 230 DEG C, 10min is kept；Carrier gas：High pure nitrogen, 2 mL/min of flow velocity；Sample size is 2 L.
2.2.4 monosaccharide composition analysis result
The GC chromatograms of standard mixture of monosaccharides are shown in Fig. 6, and Fig. 7,8,9 point are BSP-2, BSP-3, BSP-4 Jing after hydrolysis
Monosaccharide GC chromatograms, can determine the monosaccharide composition of sample by the comparison with retention time in standard monosaccharide collection of illustrative plates.Can by Fig. 7
Know, BSP-2 is at least made up of rhamnose, arabinose and xylose, its mol ratio is rhamnose：Arabinose：Xylose=0.236:
0.433:0.140, as shown in Figure 8, BSP-3 is at least made up of rhamnose, Fructose, arabinose, xylose, and mol ratio is Fructus rhamni (Rhamnus davurica Pall.)
Sugar：Fructose：Arabinose：Xylose=0.203:0.366:1.605:0.593, as shown in Figure 9, BSP-4 at least rhamnose and
Arabinose is constituted, its mol ratio=0.851:0.733.
3 anticoagulation blackberry seed polysaccharides（BSP-2、BSP-3、BSP-4）Activity analysiss
Method：Four detections of the external blood plasma blood coagulation of rabbit
3.1 instruments and material
TGL-16gR high speed desktop refrigerated centrifugers（Anting Scientific Instrument Factory, Shanghai）；LRH-150 biochemical cultivation cases (Shanghai
One permanent Science and Technology Ltd.)；Sodium chloride injection（Hebei Tiancheng Pharmaceutical Co., Ltd., A14091701）；0.109 mol/
L liquor sodii citratises（Self-control）, vitamin K1Injection (Tianjin Pharmaceutical Group Xinzheng Co., Ltd., 1403112)；Injection
With breviscapine (Hu'nan Hengsheng Pharmaceutical Co., Ltd., 20110202)；Prothrombin time（PT）Determine test kit（105241）；
Activated partial thromboplastin time（APTT）Determine test kit（112175）；Thrombin time（TT）Determine test kit（121132）；
Fibrinogen（FIB）Assay test kit（1320701）Produced by Shanghai Sun Bio-Tech Co., Ltd..
3.2 laboratory animal
Rex rabbit, male, 2.0～2.5 kg of body weight provide (the dynamic word 14-2-6 of doctor by institute of Chinese materia medica of pharmaceutical college of He'nan University
Number).
3.3 sample solutions are prepared
The sample of 1 mg（BSP-2、BSP-3、BSP-4）With 200 μ L solvents dissolving 5 mg/mL solution of system.Take Herba Erigerontiss
8 mg of element are obtained 13.33 mg/mL, vitamin K with 600 μ L solvents1Injection is obtained vitamin K with solvent dissolving1Concentration 5
The solution of mg/mL.Solvent（Also serve as blank solvent）For：Dehydrated alcohol:1,2- Propylene Glycol:Normal saline=1:1:3（Volume
Than）.
3.4 experimental technique
3.4.1 the detection method for APTT being affected
The preparation of blood plasma：Rabbit auricular vein takes 3.6 mL of blood, is placed in containing 0.109 mol/L sodium citrate, 400 μ L's
In 4 mL centrifuge tubes, gently overturn and mix, 3000 rpm are centrifuged 15 min, take the supernatant standby.
Add 50 μ L each sample solution respectively in different test cups, add 100 μ L blood plasma and 37 DEG C of pre-temperatures
100 μ L of APTT reagents, add 0.025 mol/L CaCl of 37 DEG C of pre-temperatures after 37 DEG C of 5 min of incubation2100 μ L of solution, record
Setting time.
3.4.2 the detection method for PT being affected
The same 3.4.1 of blood plasma preparation method, adds 50 μ L each sample solution respectively in different test cups, adds 100
The blood plasma of μ L, adds the 200 μ L of PT reagents of 37 DEG C of pre-temperatures, records setting time, as PT values after 37 DEG C of 3 min of incubation.
3.4.3 the detection method for TT being affected
The same 3.4.1 of blood plasma preparation method, adds 50 each sample solution of μ L and 200 μ L respectively in different test cups
Blood plasma, adds 200 μ L of TT reagents, records setting time after incubation 3min.
3.4.4 the detection method for FIB being affected
The same 3.4.1 of blood plasma preparation method, calibrates directrix curve according to reagent description.Take 200 μ L blood plasma and 200 μ L samples
Product, are subsequently adding 700 μ L buffer.After mixing, 200 μ L of blood plasma, 37 DEG C of 3 min of pre-temperature after dilution are taken, add thrombin molten
100 μ L of liquid, record the content of Fibrinogen.
3.4.5 data processing
As a result arithmetic mean of instantaneous value and standard deviation is adopted to represent that numerical statistic adopts SPSS19.0 software one factor analysis of variance
Method (One-Way ANOVA) compares its significant difference.Measurement result is shown in Table 1.
Table 1 external blood coagulation, four results
Note:Compare with blank### P＜ 0.001,0.01 ＜## p＜ 0.05,# P＞ 0.05；
With vitamin K1Relatively,△△△ P＜ 0.001,0.01 ＜△△ P＜ 0.05.
As shown in Table 1, the effect of BSP-2, BSP-3, BSP-4 prolongation TT has pole significant difference compared with blank group（P<
0.001）, illustrate that these three polysaccharide have certain blood coagulation resisting function, and the anticoagulant effect of BSP-3 and positive control Herba Erigerontiss
It is plain suitable（P>0.05）, and the anticoagulant effect of BSP-2 and BSP-4 is better than positive control breviscapine（P<0.001）.
Claims (3)
1. the extraction separation method of anticoagulation blackberry seed polysaccharide, it is characterised in that comprise the following steps：
（1）Blackberry seed is crushed, petroleum ether degreasing under room temperature, is used, after volatilizing petroleum ether, medicinal residues are extracted with ethanol, are filtered, are dried in the shade
Residue, then adds distilled water extraction, while hot sucking filtration in residue, concentrating under reduced pressure after merging filtrate, then adds in concentrated solution
Ethanol, stands centrifugation, and the precipitation for obtaining adds distillation water dissolution to dialyse to remove small molecular weight impurity, concentrating under reduced pressure, and lyophilization is
Obtain blackberry seed crude polysaccharides；
（2）Take blackberry seed crude polysaccharides to be dissolved in distilled water, filter, after discarding impurity, be added to DEAE-52 cellulose chromatography posts, according to
Secondary distilled water, 0.1mol/L sodium chloride solutions and 0.2 mol/L sodium chloride solutions carry out eluting, and eluent adopts phenol-sulfur
Acid system detected, surveys the absorbance at 490 nm places, and with eluting pipe number as abscissa, absorbance is vertical coordinate, draws eluting song
Line, merges the polysaccharide sample of same eluting peak, lyophilization after dialysis, concentration；Wherein, distilled water eluting peak is component 1,0.1
The eluting peak of mol/L sodium chloride solutions is component 3 for the eluting peak of 2,0.2 mol/L sodium chloride solutions of component；
（3）Component 1 is dissolved in distilled water, filter and discard impurity, is added to Sephadex G-100 gel chromatographic columnses, is used distilled water
Eluting is carried out, is detected using phend-sulphuric acid, its absorbance is surveyed at 490 nm of wavelength, with eluting pipe number as abscissa,
Absorbance is vertical coordinate, draws elution curve, obtains two groups of eluting peaks, concentrates the 2nd group of eluting peak, the polysaccharide that lyophilization is obtained
It is named as BSP-2；
（4）Component 2 and 3 adopts step（3）The method of component 1 carries out eluting, obtains single eluting peak, is respectively designated as BSP-3
And BSP-4；
BSP-2, BSP-3 and BSP-4 are anticoagulation blackberry seed polysaccharide.
2. the anticoagulation blackberry seed polysaccharide for being prepared using the method for claim 1.
3. application of the anticoagulation blackberry seed polysaccharide in terms of anticoagulation medicine is prepared described in claim 2.
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| CN105542023A (en) * | 2015-12-24 | 2016-05-04 | 河南大学 | Application of blackberry seed polysaccharide in aspect of preparing antithrombotic drugs |
| CN106986949B (en) * | 2017-05-12 | 2019-05-10 | 河南大学 | Dogbane flower polysaccharide, extracting method and its application |
| CN107540756B (en) * | 2017-09-15 | 2020-06-02 | 黄河科技学院 | Blood coagulation promoting privet flower polysaccharide and extraction and separation method and application thereof |
| CN107652369B (en) * | 2017-09-15 | 2020-07-31 | 黄河科技学院 | Anticoagulation glossy privet flower polysaccharide and extraction separation method and application thereof |
| CN107540759A (en) * | 2017-09-29 | 2018-01-05 | 贵州中科健生物医药有限公司 | A kind of method that polysaccharide is extracted from the fruit of Chinese wolfberry |
| CN112159484B (en) * | 2020-10-10 | 2022-03-22 | 黄河科技学院 | Anticoagulant fructus polygoni multiflori polysaccharide and extraction and separation method and application thereof |
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