CN104877037B - Separation and purification method, products and application of Christia vespertilionis polysaccharides - Google Patents
Separation and purification method, products and application of Christia vespertilionis polysaccharides Download PDFInfo
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Abstract
The invention provides a separation and purification method of Christia vespertilionis polysaccharides. The separation and purification method comprises the two steps: extraction of Christia vespertilionis polysaccharides and purification of Christia vespertilionis polysaccharides. Specifically, the separation and purification method comprises the following steps: removing impurities with absolute ethyl alcohol firstly; then, extracting the residues of whole Christia vespertilionis plants with boiling water; cooling the extraction liquid, centrifuging and taking supernate; concentrating the supernate under reduced pressure; precipitating with absolute ethyl alcohol and standing to obtain precipitate; centrifuging the precipitate further and then taking the supernate; precipitating with ethyl alcohol; repeating the operations round and round to obtain final precipitate; removing protein and freeze drying to obtain crude Christia vespertilionis polysaccharides; sequentially treating the crude Christia vespertilionis polysaccharides through separation and purification by ion exchange chromatography, dialysis, separation and purification by a sepharose gel column; and finally drying to obtain Christia vespertilionis polysaccharides. By the method, Christia vespertilionis polysaccharides are constant in composition and have the functions of removing free radicals and improving immunity; in addition, the extraction method is simple, pollution-free and suitable for industrial production.
Description
Technical field
The present invention relates to Separation of Natural Products purification art, have and be related to a kind of Vespertilio grass polysaccharide isolation and purification method.
Background technology
Polysaccharide（polysaccharides）It is that the macromolecular substances being formed are connected by monosaccharide, different biological internal extractions
Polysaccharide has different physiological actions.Vespertilio grass is the traditional national medical herbs of China's Zhuang, from pulse family Vespertilio grass platymiscium
Vespertilio grass（Christia vespertilionis (L. f.) Bahn. f.）, it is distributed mainly on China Guangxi, Guangdong, Hainan
Island.Locality is among the people with its herb on behalf of the tea, also with all herbal medicine, is decocted in water for oral dose and cures mainly pulmonary tuberculosis, carbuncle sore tumefacting virus etc..At present, to bat
The Study on Modernization report of bat grass seldom, and is limited only to resource distribution, species discrimination, plantation purposes, and Vespertilio grass chemistry is become
The research dividing is not reported so far, and the research of Vespertilio grass polysaccharide also falls within blank.
Content of the invention
It is an object of the invention to provide a kind of method isolating and purifying Vespertilio grass polysaccharide is it is desirable to be able to extract a kind of new
Type polysaccharide, this polysaccharide has functions that uniqueness, can provide technological guidance for the exploitation of Vespertilio grass resource.The mesh of the present invention
Be achieved through the following technical solutions：
A kind of isolation and purification method of Vespertilio grass polysaccharide, including the purification two extracting with Vespertilio grass polysaccharide of Vespertilio grass polysaccharide
Individual step,
The extraction of described Vespertilio grass polysaccharide comprises the following steps：
A. water extraction：Vespertilio grass herb obtains residue with after dehydrated alcohol reflux, extract, 1 ~ 3 time, and described residue is with boiling water extraction
20 ~ 60min obtains boiling water extraction mixture, with 2500 ~ 3500r/min centrifuging and taking supernatant after described boiling water extraction mixture cooling
Liquid, described 50 DEG C of concentrating under reduced pressure of supernatant obtain concentrated extracting solution；
B. precipitate with ethanol：The concentrated extracting solution of step a gained is cooled to 20 DEG C of addition dehydrated alcohol, the ethanol to extracting solution
Volume fraction is 80%, takes out, abandoning supernatant obtains precipitate after extracting solution is placed in 0 ~ 4 DEG C of placement 10 ~ 14 hours；Will be described
Precipitate volatilizes after ethanol with water dissolution recentrifuge, takes supernatant again with 80% ethanol precipitation, place 10 for 0 ~ 4 DEG C after centrifugation
Abandon supernatant after ~ 14 hours and obtain secondary precipitate, repeat this process 2 ~ 5 times, the final precipitate obtaining add water be prepared into water-soluble
Liquid；
C. removing protein：The aqueous solution of step b gained is concentrated water layer for 10 ~ 15 times afterwards with Sevag method removing protein and obtains crude polysaccharides
Solution；
D. lyophilization：The crude polysaccharides solution lyophilization of step c gained is obtained Vespertilio grass crude polysaccharides, respectively with anhydrous second
After the careless crude polysaccharides of alcohol, washing with acetone Vespertilio 2 ~ 5 times, vacuum lyophilization, obtain final product Vespertilio grass polysaccharide first product；
The purification of described Vespertilio grass polysaccharide is described Vespertilio grass polysaccharide first product to be sequentially passed through ion-exchange chromatography separate
Purification, dialysis, it is dried to obtain Vespertilio grass polysaccharide after agarose gel column separating purification.
As the improvement to purification step, the purification of described Vespertilio grass polysaccharide comprises the following steps:
A. ion-exchange chromatography isolates and purifies：Described Vespertilio grass polysaccharide first product is added distillation water dissolution, solid-liquid ratio be 5 ~
7g/100ml, is separated with ion-exchange chromatography, is respectively adopted distilled water and 0.18 ~ 0.22mol/L NaCl solution eluting；
B. dialyse：Carried out with the bag filter of molecular cut off 2000Da after NaCl solution elution fraction in step a is concentrated
Dialysis, first with tap water dialysis, then with distilled water dialysis；
C. agarose gel column separating purification：After dialysing in step b, retention material passes through agarose gel column separating purification,
To distill water elution, discard the eluent of the 1.5 times of gel column volumes in front end, the eluent collecting follow-up 1 times of gel column volume is done
Dry.
Further, weigh Vespertilio grass polysaccharide first product described in 5 ~ 7g in the purification step a of Vespertilio grass polysaccharide and be dissolved in 100ml steaming
In distilled water, ion-exchange chromatography column volume 500mL, NaCl solution concentration is 0.2mol/L；Tap water dialysis time in step b
For 2 days, distilled water dialysis time was 1 day；Agar gel post column volume 200mL in step c, discards front 300mL eluent, collects
Follow-up 200mL eluent.
It is another object of the present invention to realized by below scheme：
A kind of Vespertilio grass polysaccharide being obtained using above-mentioned isolation and purification method.
Further, described Vespertilio grass polysaccharide is homogeneous components.
Further, the molecular weight of described Vespertilio grass polysaccharide is 220000Da.
Further, the optical rotation of described Vespertilio grass polysaccharide is+38.5 °.
Further, described Vespertilio grass polysaccharide is made up of arabinose, xylose, glucose and galactose, the amount of its material
Ratio be 2.44:1:6.54:1.28.
It is another object of the present invention to realized by below scheme：
A kind of Vespertilio grass polysaccharide answering in terms of antioxidation and enhance immunity being obtained using above-mentioned isolation and purification method
With.
Beneficial effects of the present invention：
The invention provides a kind of extraction separation and purification Vespertilio grass easy and simple to handle, pollution-free, being suitable for industrialized production is many
The method of sugar, the blank filled up current Vespertilio grass component analyses, extracting；The Vespertilio grass polysaccharide being extracted by the method for the present invention
For homogeneous components, this polysaccharide has certain effect in terms of removing DPPH free radical, providing immunity of organisms, potential is developed
It is applied to medical, healthcare field, be prepared into antioxidation, anti-inflammatory, treatment immune disease class medicine.
Brief description
Fig. 1 is Vespertilio grass polysaccharide high-efficient liquid phase chromatogram；
Fig. 2 is the impact to RAW264.7 mouse macrophage multiplication capacity for the Vespertilio grass polysaccharide；
Fig. 3 is the impact to RAW264.7 mouse macrophage phagocytic activity for the Vespertilio grass polysaccharide；
Fig. 4 is that Vespertilio grass polysaccharide produces the impact of NO to mouse macrophage.
Specific embodiment
Embodiment 1
A kind of isolation and purification method of Vespertilio grass polysaccharide, including the purification two extracting with Vespertilio grass polysaccharide of Vespertilio grass polysaccharide
Individual step,
The extraction of described Vespertilio grass polysaccharide comprises the following steps：
A. water extraction：Vespertilio grass herb obtains residue with after dehydrated alcohol reflux, extract, 1 ~ 3 time, and described residue is with boiling water extraction
20 ~ 60min obtains boiling water extraction mixture, with 2500 ~ 3500r/min centrifuging and taking supernatant after described boiling water extraction mixture cooling
Liquid, described 50 DEG C of concentrating under reduced pressure of supernatant obtain concentrated extracting solution；
B. precipitate with ethanol：The concentrated extracting solution of step a gained is cooled to 20 DEG C of addition dehydrated alcohol, the ethanol to extracting solution
Volume fraction is 80%, takes out, abandoning supernatant obtains precipitate after extracting solution is placed in 0 ~ 4 DEG C of placement 10 ~ 14 hours；Will be described
Precipitate volatilizes after ethanol with water dissolution recentrifuge, takes supernatant again with 80% ethanol precipitation, place 10 for 0 ~ 4 DEG C after centrifugation
Abandon supernatant after ~ 14 hours and obtain secondary precipitate, repeat this process 2 ~ 5 times, the final precipitate obtaining add water be prepared into water-soluble
Liquid；
C. removing protein：The aqueous solution of step b gained is concentrated water layer for 10 ~ 15 times afterwards with Sevag method removing protein and obtains crude polysaccharides
Solution；
D. lyophilization：The crude polysaccharides solution lyophilization of step c gained is obtained Vespertilio grass crude polysaccharides, respectively with anhydrous second
After the careless crude polysaccharides of alcohol, washing with acetone Vespertilio 2 ~ 5 times, vacuum lyophilization, obtain final product Vespertilio grass polysaccharide first product；
The purification of described Vespertilio grass polysaccharide is described Vespertilio grass polysaccharide first product to be sequentially passed through ion-exchange chromatography separate
Purification, dialysis, it is dried to obtain Vespertilio grass polysaccharide after agarose gel column separating purification.The purification of described Vespertilio grass polysaccharide include with
Lower step:
A. ion-exchange chromatography isolates and purifies：Described Vespertilio grass polysaccharide first product is added distillation water dissolution, solid-liquid ratio be 5 ~
7g/100ml, is separated with ion-exchange chromatography, is respectively adopted distilled water and 0.18 ~ 0.22mol/L NaCl solution eluting；
B. dialyse：Dialysed with the bag filter of molecular cut off 2000Da after NaCl solution elution fraction in step a is concentrated,
First with tap water dialysis, then with distilled water dialysis；
C. agarose gel column separating purification：After dialysing in step b, retention material passes through agarose gel column separating purification,
To distill water elution, discard the eluent of the 1.5 times of gel column volumes in front end, the eluent collecting follow-up 1 times of gel column volume is done
Dry.
Embodiment 2
Optimize further on the basis of embodiment 1.
The extraction of described Vespertilio grass polysaccharide comprises the following steps：
A. water extraction：Vespertilio grass herb 750g, is cut into 3cm, obtains residue with after dehydrated alcohol reflux, extract, 2 times, described residual
Slag obtains boiling water extraction mixture with boiling water extraction 30min, with 2000r/min centrifuging and taking after described boiling water extraction mixture cooling
Supernatant, described 50 DEG C of concentrating under reduced pressure of supernatant obtain concentrated extracting solution 150mL；
B. precipitate with ethanol：The concentrated extracting solution of step a gained is cooled to 20 DEG C of addition dehydrated alcohol, the ethanol to extracting solution
Volume fraction is 80%, takes out, abandoning supernatant obtains precipitate after extracting solution is placed in 4 DEG C of placements 12 hours；By described precipitate
Volatilize after ethanol with water dissolution recentrifuge, after centrifugation, take supernatant again with 80% ethanol precipitation, 4 DEG C place 12 hours after abandon
Supernatant obtains secondary precipitate, repeats this and processes 3 times, the final precipitate obtaining adds water and is prepared into aqueous solution；
C. removing protein：The aqueous solution of step b gained is concentrated water layer for 12 times afterwards with Sevag method removing protein obtain slightly to 100ml
Polysaccharide solution；
D. lyophilization：The crude polysaccharides solution lyophilization of step c gained is obtained Vespertilio grass crude polysaccharides, respectively with 200mL
After the careless crude polysaccharides of dehydrated alcohol, washing with acetone Vespertilio 3 times, vacuum lyophilization, obtain final product Vespertilio grass polysaccharide first product；
The purification of described Vespertilio grass polysaccharide is described Vespertilio grass polysaccharide first product to be sequentially passed through ion-exchange chromatography separate
Purification, dialysis, it is dried to obtain Vespertilio grass polysaccharide after agarose gel column separating purification.The purification of described Vespertilio grass polysaccharide include with
Lower step:
A. ion-exchange chromatography isolates and purifies：Described Vespertilio grass polysaccharide first product 6.08g is added distilled water 100ml dissolving,
Separated with DEAE DE-52 ion-exchange chromatography, column volume 500ml, be respectively adopted distilled water and 0.2mol/L NaCl solution
Eluting, each gradient collects 3.0L；
B. dialyse：Carried out with the bag filter of molecular cut off 2000Da after NaCl solution elution fraction in step a is concentrated
Dialysis, is first dialysed 2 days with tap water, then is dialysed 1 day with distilled water；
C. agarose gel column separating purification：After dialysing in step b, retention material passes through Sepharose CL-68 agarose
Gel column separating purification, column volume 200ml, to distill water elution, discard front end 300ml eluent, next collect eluent
200ml, dry Vespertilio grass polysaccharide.
High performance liquid chromatography detection result is unimodal it was demonstrated that being homogeneous components by gained Vespertilio grass polysaccharide.
Efficient liquid phase testing conditions are：TSK G5000PWXL gel chromatographic columnses, 40 DEG C of column temperature, pure water is mobile phase, flow velocity
0.8 mL/min, RID detect.Chromatogram such as Fig. 1.
Embodiment 3
Characteristic measurement is carried out to the Vespertilio grass polysaccharide of the homogeneous components described in embodiment 2.
1）Angle-of-rotation measuring
Weigh Vespertilio grass polysaccharide 10.0 mg, with water dissolution, be settled to 25 mL, with water as blank, automatic polarimeter measures
Optical rotation, is computed, and obtains []20℃ D=+38.5°.
2）The mensure of molecular weight distribution
With HPLC, serial many saccharide and Vespertilio grass polysaccharide are measured, TSK G5000PWXL gel chromatographic columnses, post
40 DEG C of temperature, pure water is mobile phase, and flow velocity 0.8 mL/min, RID detect.
Weigh respectively appropriate molecular weight be 12000Da, 25000Da, 50000Da, 80000Da, 270000Da, 460000
The Dextran standard substance of Da, add deionized water, are made into the reference substance solution of concentration about 2 mg/ml, 0.45 μm of water system
Membrane filtration, carries out HPLC detection, afterwards one by one with retention time（RT）For abscissa x, the log10 value of molecular weight is vertical coordinate
Y, obtains standard curve y=- 0.332x+10.224（r = 0.998）.
Weigh a certain amount of Vespertilio grass polysaccharide, detected as stated above, with Vespertilio grass retention time according to above-mentioned standard
Curve equation is calculated, and the Mw obtaining Vespertilio grass polysaccharide is 220000 Da.
3）The mensure of monosaccharide composition
Vespertilio grass polysaccharide is carried out all-hydrolytic reductive derivazation, with standard monosaccharide as reference, list is carried out to Vespertilio grass polysaccharide
The mensure of sugar composition, gas phase condition is, chromatographic column：DB-5 （30 m × 0.25 mm × 0.25 μm）；Detector：Mass spectrum
Detector；Injector temperature：250 ℃；Detector temperature：280 ℃；Nitrogen flow rate：0.6 mL/min；Split ratio：20:1；Enter
Sample amount：5 μL；Heating schedule：200 DEG C of holding 2min, rise to 245 DEG C with the speed of 3 DEG C/min, then with 10 DEG C/min's
Speed rises to 270 DEG C, keeps 2 min.Record in the present embodiment Vespertilio grass polysaccharide by arabinose, xylose, glucose and half
Lactose forms, and the amount of wherein each monosaccharide material ratio is for 2.44: 1 : 6.54 : 1.28.
Embodiment 4
Experiment in vitro mensure is carried out to the Vespertilio grass polysaccharide of the homogeneous components described in embodiment 2.
1）The free-radical scavenging activity of Vespertilio grass polysaccharide measures
Take 0.5mg/mL Vespertilio grass polysaccharide aqueous solution 1.0mL, put in 10mL color comparison tube, add 3.0mL concentration to be 0.
The DPPH solution of 1mmol/L, 30 DEG C of lucifuges of water-bath react 30min, with distilled water as blank, measure extinction at 517nm wavelength
Value.Calculate DPPH free radical scavenging activity by following equation.
Experiment is repeated six times, the DPPH free radical scavenging activity trying to achieve Vespertilio grass polysaccharide is 28.99%
Using sample to the clearance rate of DPPH free radical as evaluation index.Vespertilio grass polysaccharide is molten to the DPPH of 0.1 mmol/L
The free radical scavenging activity of liquid is 28.99 % (RSD=2.28 %).
2）The impact that Vespertilio grass polysaccharide is bred to mouse macrophage
With DMEM culture medium culturing mouse macrophage Raw264.7, cell is transferred to 96 orifice plates, concentration be about 2 ×
104Individual/ml, adds Vespertilio grass polysaccharide solution according to variable concentrations to each hole, and after cultivating 12 hours, each hole adds MTT, cultivates 4
Hour, pour out culture fluid, each hole adds DMSO, stand 1 hour, with microplate reader mensuration absorbance, statistical result.
Measure the impact to RAW264.7 mouse macrophage multiplication capacity for the Vespertilio grass polysaccharide, variable concentrations administration result is shown in
Fig. 2.
Compare with blank control group in Fig. 2, * P< 0.05；** P < 0.01.Other each figures are same.
3）The impact to mouse macrophage phagocytic activity for the Vespertilio grass polysaccharide
With DMEM culture medium culturing mouse macrophage Raw264.7, cell is transferred to 96 orifice plates, concentration be about 2 ×
104Individual/ml, adds Vespertilio grass polysaccharide solution according to variable concentrations to each hole, and after cultivating 12 hours, each hole adds dimethyl diaminophenazine chloride molten
Liquid, cultivates 4 hours, pours out culture fluid, and each hole adds DMSO, stands 1 hour, with microplate reader mensuration absorbance, statistical result.
Using dimethyl diaminophenazine chloride as target alien material, measure the shadow to RAW264.7 mouse macrophage phagocytic activity for the Vespertilio grass polysaccharide
Ring, variable concentrations administration result is shown in Fig. 3.
4）Vespertilio grass polysaccharide produces the impact of NO to mouse macrophage
NO in cell culture supernatant is measured using nitrate reductase method2 -Reflection generates the amount of NO indirectly, according to NO inspection
Test agent box description is operated, and the result that variable concentrations administration produces total NO to mouse macrophage is shown in Fig. 4.
Vespertilio grass polysaccharide has the ability of certain removing DPPH free radical, right being investigated with variable concentrations Vespertilio grass polysaccharide
In the experiment of mouse macrophage impact, with the increase of administration concentration, Vespertilio grass polysaccharide promotes the propagation of mouse macrophage
Ability, promote cell phagocytosis and produce NO ability effect strengthen.But when the concentration is too high, accordingly above index is on the contrary
Occur declining and disturb cellular metabolism or create cytotoxicity and have thus it is speculated that acting on cell with heavy dose of Vespertilio grass polysaccharide
Close.
Claims (8)
1. a kind of isolation and purification method of Vespertilio grass polysaccharide it is characterised in that：Extraction including Vespertilio grass polysaccharide and Vespertilio grass are many
Two steps of purification of sugar,
The extraction of described Vespertilio grass polysaccharide comprises the following steps：
A. water extraction：Vespertilio grass herb obtains residue with after dehydrated alcohol reflux, extract, 1 ~ 3 time, described residue with boiling water extraction 20 ~
60min obtains boiling water extraction mixture, with 2500 ~ 3500r/min centrifuging and taking supernatant after described boiling water extraction mixture cooling,
Described 50 DEG C of concentrating under reduced pressure of supernatant obtain concentrated extracting solution；
B. precipitate with ethanol：The concentrated extracting solution of step a gained is cooled to 20 DEG C of addition dehydrated alcohol, the body of ethanol to extracting solution
Fraction is 80%, and after extracting solution is placed in 0 ~ 4 DEG C of placement 10 ~ 14 hours, abandoning supernatant obtains precipitate；By described precipitate
Volatilize after ethanol with water dissolution recentrifuge, after centrifugation, take supernatant again with 80% ethanol precipitation, 0 ~ 4 DEG C to place 10 ~ 14 little
When after abandon supernatant and obtain secondary precipitate, repeat this and process 2 ~ 5 times, the final precipitate obtaining adds water and is prepared into aqueous solution；
C. removing protein：The aqueous solution of step b gained is concentrated water layer afterwards with Sevag method removing protein 10 ~ 15 times, and to obtain crude polysaccharides molten
Liquid；
D. lyophilization：By the crude polysaccharides solution lyophilization of step c gained obtain Vespertilio grass crude polysaccharides, respectively with dehydrated alcohol,
After washing with acetone Vespertilio grass crude polysaccharides 2 ~ 5 times, vacuum lyophilization, obtain final product Vespertilio grass polysaccharide first product；
The purification of described Vespertilio grass polysaccharide be by described Vespertilio grass polysaccharide first product sequentially pass through ion-exchange chromatography isolate and purify,
It is dried to obtain Vespertilio grass polysaccharide after dialysis, agarose gel column separating purification.
2. isolation and purification method according to claim 1 it is characterised in that：The purification of described Vespertilio grass polysaccharide specifically includes
Following steps:
A. ion-exchange chromatography isolates and purifies：Described Vespertilio grass polysaccharide first product is added distillation water dissolution, solid-liquid ratio is 5 ~ 7g/
100ml, is separated with ion-exchange chromatography, is respectively adopted distilled water and 0.18 ~ 0.22mol/L NaCl solution eluting；
B. dialyse：Carried out with the bag filter of molecular cut off 2000Da after NaCl solution elution fraction in step a is concentrated
Analysis, first with tap water dialysis, then with distilled water dialysis；
C. agarose gel column separating purification：After dialysing in step b, retention material passes through agarose gel column separating purification, with
Distillation water elution, discards the eluent of the 1.5 times of gel column volumes in front end, and the eluent collecting follow-up 1 times of gel column volume is dried
Obtain Vespertilio grass polysaccharide.
3. isolation and purification method according to claim 2 it is characterised in that：5 are weighed in the purification step a of Vespertilio grass polysaccharide
Described in ~ 7g, Vespertilio grass polysaccharide first product is dissolved in 100ml distilled water, ion-exchange chromatography column volume 500mL, NaCl solution concentration
For 0.2mol/L；In step b, tap water dialysis time is 2 days, and distilled water dialysis time is 1 day；Agar gel post post in step c
Volume 200mL, discards front 300mL eluent, collects follow-up 200mL eluent and is dried to obtain Vespertilio grass polysaccharide.
4. a kind of Vespertilio grass polysaccharide being obtained using any one isolation and purification method in claim 1 ~ 3.
5. Vespertilio grass polysaccharide according to claim 4 it is characterised in that：Described Vespertilio grass polysaccharide is homogeneous components.
6. Vespertilio grass polysaccharide according to claim 5 it is characterised in that：The molecular weight of described Vespertilio grass polysaccharide is
220000Da.
7. Vespertilio grass polysaccharide according to claim 5 it is characterised in that：The optical rotation of described Vespertilio grass polysaccharide be+
38.5°.
8. Vespertilio grass polysaccharide according to claim 5 it is characterised in that：Described Vespertilio grass polysaccharide by arabinose, xylose,
Glucose and galactose composition, the ratio of the amount of its material is 2.44:1:6.54:1.28.
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