CN106986949A - Dogbane flower polysaccharide, extracting method and its application - Google Patents
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Abstract
The present invention relates to a kind of extracting method of dogbane flower polysaccharide, it comprises the following steps：1）Dogbane flower first uses petroleum ether degreasing, again fat-soluble ingredient is extracted with ethanol, take filter residue to add water to carry in 90 ± 5 DEG C of progress heat, heat carries rear alcohol precipitation, take precipitation to be washed successively with absolute ethyl alcohol, acetone, absolute ether, be freeze-dried, then dissolved with distilled water, using Sevag method deproteinations, the dialysis of obtained supernatant, freeze-drying are obtained into Thick many candies；2）Thick many candies obtain four polysaccharide component Vp1, Vp2a I, Vp2a II and Vp3 after ion-exchange chromatography and gel permeation column chromatogram purification.Result of the test shows：Dogbane flower refined polysaccharide component Vp1 has good rush coagulating effectiveness, can be individually used for preparing coagulant thing；Dogbane flower refined polysaccharide component Vp3 then has good anticoagulant effect, can be individually used for preparing anticoagulation medicine.
Description
Technical field
The invention belongs to technical field of extraction of Chinese traditional medicine, and in particular to a kind of dogbane flower polysaccharide, extracting method and its application.
Background technology
Bluish dogbane（Apocynum venetum L）It is Apocynaceae（Apocynaceae）Plant, is commonly called as wild flax, also known as
" apocynum cannabinum ", " camellia fiber crops ", " tea stalk " etc..Dogbane flower nature and flavor are sweet, hardship, are slightly cold, and are depressured with soothing the liver, mental-tranquilization,
The function of clearing away heat and promoting diuresis.Mainly containing flavonoids and cardiac glycoside etc. in dogbane flower, its active ingredient have reduce blood pressure, blood fat,
Cardiac stimulant diuresis, soothing the liver is soothing the liver, and anti-inflammatory is relieving cough and asthma, the function such as antiallergy.In Xinjiang, local resident is made tea guarantor with its flower and leaf
It is strong to use.Dogbane flower fragrant odour is pleasant, is always the traditional blood pressure reducing beverage of the Uygur nationality.In addition dogbane flower, which is decocted in water for oral dose, to control
Treat dizzy, filling pillow after drying can be with improving eyesight, hypotensive.Polysaccharide is connected by the monose and glycosidic bond of more than 10
High polymer.Polysaccharide acts not only as supporting tissue, energy source, also participate in biosynthesis reaction and intercellular identification,
Fertilization, embry ogenesis, neural cell development, hormone activation, cell propagation, infection, the Nasopharyngeal neoplasms of viral and bacterium etc.
Biological phenomena and physiology course.With the development of experimental technique, domestic and international researcher is further deepened to the understanding of polysaccharide.Document
Retrieval display：Plant polyose has anti-oxidant, hypoglycemic, reducing blood lipid, antithrombotic, antibacterial, anti-inflammatory and immunological regulation, antifatigue etc.
Effect.Pharmacological activity is closely related with chemical composition, have not yet to see dogbane flower polysaccharide structure and external thrombolysis (or
Fibrinolytic) effect relevant report, therefore research dogbane flower polysaccharide, for bluish dogbane deep development utilize have important meaning
Justice.
Blood clotting abbreviation blood coagulation, be in endogenous or（With）Thrombokinase is produced under exogenous cruor pathway,
Thrombokinase produces fibrin ferment in the presence of clotting factor, and last fibrin ferment makes fibrinogen be changed into fiber egg
In vain, blood is changed into gel state by collosol state.Coagulation process is generally divided into：1. intrinsic coagulation pathway；2. it is exogenous solidifying
Blood approach；3. common coagulation pathway.Exogenous cruor pathway is to start from tissue factor exposed to blood, is swashed to factor X
Process living；Clinically with prothrombin time（PT）Determine to reflect the situation of exogenous cruor pathway.Intrinsic coagulation way
Footpath refers to activate from factor XI, plasma thromboplastin antecedent I, to the process of tenase；Clinically with activated partial thromboplastin time（APTT）Come anti-
Reflect the situation of internal intrinsic coagulation pathway.Fibrin is activated to from factor X to be formed, and is the common coagulation pathway in inside and outside source；
Mainly include fibrin ferment generation and fibrin two stages of formation.
The content of the invention
Present invention aims at be to overcome prior art defect there is provided a kind of dogbane flower polysaccharide, extracting method and its
Application in terms of blood coagulation.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of extracting method of dogbane flower polysaccharide, it comprises the following steps：
1）Dogbane flower first uses petroleum ether degreasing, then extracts fat-soluble ingredient with ethanol, takes filter residue to add water in 90 ± 5 DEG C and carries out heat
Carry, heat carries rear alcohol precipitation, take precipitation to be washed successively with absolute ethyl alcohol, acetone, absolute ether, be freeze-dried, it is then water-soluble with distilling
Solution, using Sevag method deproteinations, Thick many candies are obtained by the dialysis of obtained supernatant, freeze-drying；
2）Thick many candies after ion-exchange chromatography and gel permeation column chromatogram purification, obtain four polysaccharide component Vp1, Vp2a-I,
Vp2a-II and Vp3.
Step 1）Specially：Refluxing extraction 1-3 times, each refluxing extraction 1- after dogbane flower and petroleum ether are mixed
3h, refluxing extraction is filtered after terminating, and filter residue dries, and is added 70 ± 5% ethanol in 50 ± 5 DEG C of heating extractions 2-4 time, is heated every time
1-3h is extracted, heating extraction is filtered after terminating, and filter residue dries acquisition degreasing dogbane flower, then with distilled water in 90 ± 5 DEG C of heat
Extract 3-4 time, heat extracts 3-4h every time, heat is extracted terminate after suction filtration while hot, filtrate decompression concentration acquisition concentrate, Ran Houxiang
Ethanol is added in concentrate makes final concentration of 70 ± 5%, stands, centrifuging to precipitate.
Step 2）Specifically include following steps：
a）Thick many candies distilled water is dissolved, 0.45 μm of filtering with microporous membrane is loaded to DEAE-52 chromatographic columns, successively with distillation
Water, 0.1 mol/L sodium chloride solutions and 0.2 mol/L sodium chloride solutions are eluted, and eluent is carried out using phend-sulphuric acid
Detection, the absorbance surveyed at 490 nm, to elute pipe number as abscissa, absorbance is ordinate, draws elution curve, merges same
The polysaccharide sample of one eluting peak, is concentrated under reduced pressure, and dialyses, freeze-drying；Wherein, the eluting peak of distilled water is the mol/ of component 1,0.1
The eluting peak of L sodium chloride solutions is that the eluting peak of the mol/L sodium chloride solutions of component 2,0.2 is component 3；
b）Component 1 is dissolved with distilled water, 0.22 μm of filtering with microporous membrane, Sephadex G-100 chromatographic columns is loaded to, with distillation
Water is eluted, and is detected using phend-sulphuric acid, surveys its absorbance at 490 nm, to elute pipe number as abscissa,
Absorbance is ordinate, draws elution curve, obtains single eluting peak, be concentrated under reduced pressure, be freeze-dried after obtained polysaccharide component
It is named as Vp1；
c）Component 2 is using step b）Same method is eluted, and obtains two groups of eluting peaks, be concentrated under reduced pressure, be freeze-dried after
To polysaccharide component be respectively designated as Vp2a-I, Vp2a-II；
d）Component 3 is using step b）Same method is eluted, and obtains single eluting peak, be concentrated under reduced pressure, be freeze-dried after
To polysaccharide component be named as Vp3；
Dogbane flower polysaccharide is Vp1 or Vp3.
Dogbane flower the polysaccharide Vp1 or Vp3 obtained using said extracted method.
Dogbane flower polysaccharide component Vp1 is preparing the application in coagulant object space face.
Applications of the dogbane flower polysaccharide component Vp3 in terms of anticoagulation medicine is prepared.
Compared to the prior art, beneficial effects of the present invention：
The present invention provides the plant dogbane flower of active polysaccharide origin, and extract from dogbane flower obtain four it is refined many
Saccharic composition Vp1, Vp2a-I, Vp2a-II and Vp3, and the external anticoagulant effect of dogbane flower refined polysaccharide is investigated.Examination
Result is tested to show：Dogbane flower refined polysaccharide component Vp1 has good rush coagulating effectiveness, and Vp1 promotees coagulating effectiveness and is better than cloud
Southern baiyao, it is seen that it can be individually used for preparing Procoagulants.Dogbane flower refined polysaccharide component Vp3 then has good anti-freezing
Blood effect, and Vp3 anticoagulant effects are better than Breviscapinun, it is seen that it can be individually used for preparing anticoagulant.
Brief description of the drawings
Fig. 1 is clotting mechanism figure；
Fig. 2 is the elution curve of the dogbane flower polysaccharide component 1, component 2 and the component 3 that are afforded through DEAE-52 chromatographic columns；
Fig. 3 is dogbane flower refined polysaccharide component Vp1, Vp2a-I, the Vp2a- afforded through SephadexG-100 chromatographic columns
II and Vp3 elution curve.
Embodiment
Technical scheme is further discussed in detail with reference to embodiments, but protection scope of the present invention
It is not limited thereto.
The flower of drying of bluish dogbane is collected in Aksu, Xinjiang in July, 2015.Via institute of Chinese materia medica of He'nan University
Professor Li Changqin be accredited as Apocynaceae perennial root plant bluish dogbane (Apocynum venetum L. flower) is dried.Implement
Concentration of alcohol unless otherwise specified, refers to volumetric concentration in example.
The extraction of the dogbane flower polysaccharide of embodiment 1
A kind of extracting method of dogbane flower polysaccharide, it comprises the following steps：
1）The g of dogbane flower 770 dried in the shade is taken, refluxing extraction 2 times at 60 DEG C in round-bottomed flask with 2 L petroleum ethers, every time backflow
Extract 1 h.Refluxing extraction is filtered after terminating, and filter residue dries, and adds the L of 70% ethanol 9, in 50 DEG C of heating extractions 3 times, every time heating
Extract 1 h.Heating extraction is filtered after terminating, and filter residue dries to obtain the g of degreasing dogbane flower 479.7.Whole degreasing dogbane flowers are taken,
Plus 10 times of weight distilled water in 90 DEG C heat extract 4 times, every time heat extract 4 h.Heat is extracted terminate after suction filtration while hot, filtrate decompression is dense
Contracting obtains concentrate, and 95% ethanol is then added into concentrate makes final concentration of 70%, stands 24 h, centrifuging to precipitate.It is heavy to take
Form sediment and then use absolute ethyl alcohol, acetone, absolute ether is washed twice successively.Freeze-drying（Carried out in freeze drier, -40 DEG C
Vacuum freeze drying 10-12 h, similarly hereinafter）, then dissolved with distilled water, using Sevag method deproteinations（Specially：Will dissolving
There are the distilled water and Sevag reagents of precipitation by 5：1 volume ratio mixing, concussion, centrifugation layering, by middle denatured protein with
Layer organic solvent is removed, and supernatant is repeated 5-10 times by above-mentioned steps, occurred until without denatured protein；Sevag is tried
Agent is volume ratio 5:1 chloroform, n-butanol mixed liquor）, obtained supernatant is dialysed（Dialyse 2 at 4 DEG C with bag filter
My god, bag filter molecular cut off is 8000-14000, and every 4 h changes first water, similarly hereinafter）To remove small-molecule substance, most
After be freeze-dried, obtain the g of Thick many candies 58.58；
2）About 250 mg Thick many candies are dissolved in 15 ml distilled water and centrifuged.Supernatant is taken with 0.45 μm of miillpore filter mistake
Filter, filtrate is loaded to DEAE-52 chromatographic columns（2.6×40 cm）, successively with distilled water, 0.1 mol/L sodium chloride solutions and 0.2
Mol/L sodium chloride solutions are eluted（The r/min of constant current flow rate pump 20, collects eluent, often the min of pipe 8 with automatic collector,
The every mL of pipe 6）, eluent is detected using phend-sulphuric acid, with the absorbance at ELIASA survey wavelength 490 nm, to elute
Pipe number is abscissa, and absorbance is ordinate, draws elution curve, merges the polysaccharide sample of same eluting peak, is concentrated under reduced pressure, thoroughly
Analysis（Condition is with above-mentioned steps 1）, freeze-drying；Wherein, the eluting peak of distilled water is the mol/L sodium chloride solutions of component 1,0.1
Eluting peak is that the eluting peak of the mol/L sodium chloride solutions of component 2,0.2 is component 3；
3）Component 1 is dissolved as the g/ml of concentration 0.04 solution with distilled water, centrifugation（TGL-16rG, 5000 r/min, 15
min）, 0.22 μm of filtering with microporous membrane, filtrate is loaded to Sephadex G-100 chromatographic columns（1.6×100cm）, use distilled water
Eluted（The r/min of constant current flow rate pump 4, collects eluent, often pipe 40 minutes, often the mL of pipe 6 with automatic collector）, using benzene
Phenol-sulfuric acid method is detected, surveys its absorbance at the nm of wavelength 490, to elute pipe number as abscissa, and absorbance is vertical seat
Mark, draws elution curve, obtains single eluting peak, be concentrated under reduced pressure, be freeze-dried after obtained polysaccharide component be named as Vp1；
4）Component 2 uses step 3）Same method is eluted, and obtains two groups of eluting peaks, be concentrated under reduced pressure, be freeze-dried after
To polysaccharide component be respectively designated as Vp2a-I, Vp2a-II；
5）Component 3 uses step 3）Same method is eluted, and obtains single eluting peak, be concentrated under reduced pressure, be freeze-dried after
To polysaccharide component be named as Vp3；
Dogbane flower polysaccharide is Vp1 or Vp3.
The anti-freezing blood test of the dogbane flower polysaccharide of embodiment 2.
Method：Four detections of the external blood plasma blood coagulation of rabbit.
Instrument and material：TGL-16gR high speed desktop refrigerated centrifuges（Anting Scientific Instrument Factory, Shanghai）；HF6000-4 half
Application of automated coagulation analyzer（Southern Chinese prescription Medical Devices Co., Ltd.）；LRH-150 biochemical cultivation cases（The permanent scientific and technological limited public affairs in Shanghai one
Department）；Sodium chloride injection；0.109 mol/L liquor sodii citratises, Breviscapine（Hu'nan Hengsheng Pharmaceutical Co., Ltd.,
20110202）；Activated partial thromboplastin time（APTT）Determine kit（1121911）, prothrombin time（PT）Determine examination
Agent box（105295）, thrombin time（TT）Determine kit（121168）, fibrinogen（FIB）Assay kit
（132107）Produced by Shanghai Sun Bio-Tech Co., Ltd..
Experimental animal：Beaver rabbit, male, the kg of body weight 2.0~2.5 is provided by institute of Chinese materia medica of pharmaceutical college of He'nan University（Doctor
Dynamic word 14-2-6）.Circulate and raise in 25 ± 2 DEG C and humidity 45-65%, 12/12 hour brightness environment, can freely obtain
Water and food, animal feeding is in standard cage.
Sample solution is prepared：
Weigh dogbane flower polysaccharide Vp1 6.2mg, V2pa-I 6mg, Vp2a-II 7.5mg, Vp3 4.6mg and be dissolved in appropriate life
Manage in salt solution, be configured to the solution of 5 mg/mL concentration；
Weigh Breviscapinun 8mg to be dissolved in 600 μ L physiological saline, the solution that concentration is 13.33 mg/mL is made；
Weigh Yunnan Baiyao 1mg to be dissolved in 200 μ L physiological saline, the solution that concentration is 5 mg/mL is made.
Experimental method.
(1) detection method influenceed on APTT
The preparation of blood plasma：Rabbit auricular vein takes the mL of blood 3.6, and it is 0.109 mol/L citric acids to be placed in containing 400 μ L concentration
In the centrifuge tube of sodium, gently overturn and mix, 3000 rpm centrifuge 15 min, take supernatant liquor, standby.
25 μ L sample solution are added in test cup, the μ of APTT reagents 100 of 100 μ L blood plasma and 37 DEG C of pre-temperatures is added
L, 37 DEG C are incubated the 0.025 mol/L CaCl that 37 DEG C of pre-temperatures are added after 5 min2The μ L of solution 100, record setting time, are
APTT values.
(2) detection method influenceed on PT
The preparation method of blood plasma is ibid.25 μ L sample solution is added in test cup, 100 μ L blood plasma, 37 DEG C is added
The μ L of PT reagents 200 that 37 DEG C of pre-temperatures are added after 3 min are incubated, setting time, as PT values is recorded.
(3) detection method influenceed on TT
The preparation method of blood plasma is ibid.50 μ L samples solution and 200 μ L blood plasma are added in test cup, is incubated after 3 min
The μ L of TT reagents 200 are added, setting time, as TT values is recorded.
(4) detection method influenceed on FIB
The preparation method of blood plasma is ibid.Standard curve is drawn to specifications.200 μ L blood plasma and 100 μ L sample solution are taken, so
After add 700 μ L FIB buffer solutions（It is contained in the kit of purchase）, mix.Take the blood plasma after foregoing dilution 200 μ L, 37 DEG C
The min of pre-temperature 3, adds 100 μ L FIB thrombin solutions（FIB thrombin solutions are coagulated with 2.5ml FIB buffer solutions dilution FIB
Obtained by hemase, FIB fibrin ferments are contained in the kit of purchase）, record the content of fibrinogen, as FIB values.
Data processing：The data processing of anticoagulant active
As a result represented using arithmetic mean of instantaneous value and standard deviation, numerical statistic uses SPSS19.0 software one-way analysis of variances
（One-Way ANOVA）Compare between being organized, P<0.05 explanation has statistical significance.Measurement result is shown in Table 1.
Note：Compared with blank group, * * *p<0.001,0.01<**p<0.001；
Compared with Yunnan Baiyao,$$$ p<0.001, compared with Breviscapinun,### p<0.001。
As can be seen from Table 1：Compared with blank group, Vp1 and Vp3 can pole be significantly reduced APTT（p<0.001）, wherein
Vp1 effects have exceeded positive control Yunnan Baiyao（p<0.001）, and Vp3 effects can not show a candle to positive control Yunnan Baiyao（p<
0.001）；Vp2a-I significantly extends APTT with Vp2a-II poles（p<0.001）, but effect is weaker than positive control Breviscapinun.
Compared with blank group, Vp3 significantly extends PT with Vp2a--II poles（p<0.001）, its effect and positive control Breviscapinun
It is similar；Vp1 is similar to blank group to Vp2a-I action effects.Compared with blank group, Vp1 can pole be significantly reduced TT（p<
0.001）, its effect can not show a candle to positive control Yunnan Baiyao（p<0.001）；Vp3 can pole significantly extend TT（p<0.001）, its
Effect can not show a candle to positive control Breviscapinun（p<0.001）；Vp2a-I is similar to blank group to Vp2a-II action effects.With sky
White group compares, Vp1 can pole significantly raise FIB（p<0.001）, Vp2a-I, Vp2a-II and Vp3 action effects and blank group phase
Seemingly.
Summary result can be seen that：Dogbane flower polysaccharide component Vp1 has certain external procoagulant activity, can use
In preparing coagulant thing；It is anti-available for preparing and dogbane flower polysaccharide component Vp3 then has certain external blood coagulation resisting function
Blood-clotting agent.
Claims (6)
1. a kind of extracting method of dogbane flower polysaccharide, it is characterised in that comprise the following steps：
1）Dogbane flower first uses petroleum ether degreasing, then extracts fat-soluble ingredient with ethanol, takes filter residue to add water in 90 ± 5 DEG C and carries out heat
Carry, heat carries rear alcohol precipitation, take precipitation to be washed successively with absolute ethyl alcohol, acetone, absolute ether, be freeze-dried, it is then water-soluble with distilling
Solution, using Sevag method deproteinations, Thick many candies are obtained by the dialysis of obtained supernatant, freeze-drying；
2）Thick many candies after ion-exchange chromatography and gel permeation column chromatogram purification, obtain four polysaccharide component Vp1, Vp2a-I,
Vp2a-II and Vp3.
2. the extracting method of dogbane flower polysaccharide as claimed in claim 1, it is characterised in that step 1）Specially：By bluish dogbane
Refluxing extraction 1-3 times after flower and petroleum ether mixing, each refluxing extraction 1-3h, refluxing extraction is filtered after terminating, and filter residue dries,
70 ± 5% ethanol are added in 50 ± 5 DEG C of heating extractions 2-4 time, each heating extraction 1-3h, heating extraction is filtered after terminating, and is filtered
Slag dries acquisition degreasing dogbane flower, is then extracted 3-4 times in 90 ± 5 DEG C of heat with distilled water, heat extracts 3-4h every time, heat is carried
Take suction filtration while hot after end, filtrate decompression concentration obtains concentrate, then added into concentrate ethanol make final concentration of 70 ±
5%, stand, centrifuging to precipitate.
3. the extracting method of dogbane flower polysaccharide as claimed in claim 1, it is characterised in that step 2）Specifically include following step
Suddenly：
a）Thick many candies distilled water is dissolved, 0.45 μm of filtering with microporous membrane is loaded to DEAE-52 chromatographic columns, successively with distillation
Water, 0.1 mol/L sodium chloride solutions and 0.2 mol/L sodium chloride solutions are eluted, and eluent is carried out using phend-sulphuric acid
Detection, the absorbance surveyed at 490 nm, to elute pipe number as abscissa, absorbance is ordinate, draws elution curve, merges same
The polysaccharide sample of one eluting peak, is concentrated under reduced pressure, and dialyses, freeze-drying；Wherein, the eluting peak of distilled water is the mol/ of component 1,0.1
The eluting peak of L sodium chloride solutions is that the eluting peak of the mol/L sodium chloride solutions of component 2,0.2 is component 3；
b）Component 1 is dissolved with distilled water, 0.22 μm of filtering with microporous membrane, Sephadex G-100 chromatographic columns is loaded to, with distillation
Water is eluted, and is detected using phend-sulphuric acid, surveys its absorbance at 490 nm, to elute pipe number as abscissa,
Absorbance is ordinate, draws elution curve, obtains single eluting peak, be concentrated under reduced pressure, be freeze-dried after obtained polysaccharide component
It is named as Vp1；
c）Component 2 is using step b）Same method is eluted, and obtains two groups of eluting peaks, be concentrated under reduced pressure, be freeze-dried after
To polysaccharide component be respectively designated as Vp2a-I, Vp2a-II；
d）Component 3 is using step b）Same method is eluted, and obtains single eluting peak, be concentrated under reduced pressure, be freeze-dried after
To polysaccharide component be named as Vp3；
Dogbane flower polysaccharide is Vp1 or Vp3.
4. the dogbane flower polysaccharide Vp1 or Vp3 that are obtained using any one of the claims 1 to 3 extracting method.
5. dogbane flower polysaccharide Vp1 described in claim 4 is preparing the application in coagulant object space face.
6. applications of the dogbane flower polysaccharide Vp3 in terms of anticoagulation medicine is prepared described in claim 4.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112159484A (en) * | 2020-10-10 | 2021-01-01 | 黄河科技学院 | Anticoagulant fructus polygoni multiflori polysaccharide and extraction and separation method and application thereof |
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| CN104107187A (en) * | 2013-04-18 | 2014-10-22 | 东北师范大学 | Application of apocynum venetum acidic polysaccharide to medicines for treating diabetes |
| CN105037578A (en) * | 2015-08-24 | 2015-11-11 | 河南大学 | Anticoagulant blackberry seed polysaccharides, and extraction separation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112159484A (en) * | 2020-10-10 | 2021-01-01 | 黄河科技学院 | Anticoagulant fructus polygoni multiflori polysaccharide and extraction and separation method and application thereof |
| 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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