CN104119429B - Huai Er albumen and its preparation method and application - Google Patents
Huai Er albumen and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to Huai Er albumen and its preparation method and application, the monose composition of Huai Er albumen of the invention is fucose, arabinose, galactolipin, glucose, xylose and mannose, its mass ratio is 3.2：2.0：33.5：5.5：1.2：50.4, the weight average molecular weight of the polysaccharide protein is 7.0 × 105~5.0 × 106Da, is preferably 2.69 × 106Da.The polysaccharide protein of the present invention can be used for the medicine for preparing treatment tumour.
Description
Technical field
The present invention relates to a kind of Huai Er albumen and its preparation method and application.
Background technology
Trametes robinioplila is On Polyporaceae Chinese scholartree bolt bacterium（Trametes robiniophila Murr.）Fructification, be a kind of
Important Medicinal fungi, has the medication history in more than 1600 years in China.Ainsworth classification of fungi systems are belonged to
In Eumycota, Basidiomycotina, Hymenomycetes, Polyporaceae, Trametes.Trametes robinioplila fructification is medium to larger, suberin, sterile
Handle, is born on the trunk of the broad leaf trees such as Chinese scholartree, locust tree, is distributed in the ground such as Hebei, Shaanxi, Liaoning, Hunan, Guangxi, Fujian, is a kind of
It can cause the domestomycetes of trees heart-wood rot.It is this product bitter, pungent, it is mild-natured nontoxic, there are " controlling wind ", " blood-breaking ", the work(of " beneficial power "
Effect, is clinically used to treat a variety of diseases.Since Chinese scholar tree is increasingly rare in old-age group, cause trametes robinioplila resource very rare, it is difficult to full
Sufficient clinical application demand, it is more difficult to meet the industrial production demand of trametes robinioplila Related product.In order to solve the resource problem of trametes robinioplila,
Long-term research and practice are passed through by Qidong Gaitianli Pharmaceutical Co., Ltd., have captured trametes robinioplila mycelium large scale fermentation problem, real
Show mycelial industrialized production, and with locust tree microellobosporia（Trametes robinioplila mycelium fermentation thing）Extract develops trametes robinioplila for raw material
The medicine such as grain and Chinese scholartree Qihuang Granule, meets clinical application demand.
Substantial amounts of chemical composition and pharmacology activity research data confirm that the active ingredient of extractive of locust tree microellobosporia is in recent years
Huai Er albumen, its primary efficacy are treatment or adjuvant therapy of tumors disease.However, at present for Huai Er albumen
Composition, structure and preparation method research are seldom.In terms of chemical constitution study, only the monose composition of Thick many candies and proportion grading have
Document report, lack to its homogeneous polysaccharide isolate and purify and structural research, saccharide residue species as contained by homogeneous polysaccharide albumen,
Ratio, the ordering in main chain, the ratio shared by protein part, composition etc., and these information are for illustrating locust tree microellobosporia
Real effective component, the mechanism of action of Huai Er albumen and structure-activity relationship etc. are most important.Equally in Huai Er albumen
Bioactivity research in terms of, more using Thick many candies as research object, lack using homogeneous polysaccharide component acquisition it is internal, external
Pharmaceutical research data, lead to not to understand Huai Er albumen in depth related to human immunocyte or tumor cell surface more
The mechanism of saccharide acceptor, thus deeply can not illustrate its action principle.
The content of the invention
The defects of in order to overcome the prior art, the present invention provide a kind of Huai Er albumen and its preparation method and application.
The above-mentioned purpose of the present invention is achieved through the following technical solutions.
On the one hand, the present invention provides a kind of Huai Er albumen, and the monose composition of the polysaccharide protein is fucose, Arab
Sugar, galactolipin, glucose, xylose and mannose, its mass ratio are 3.2：2.0：33.5：5.5：1.2：50.4.
Preferably, the weight average molecular weight of the polysaccharide protein is 7.0 × 105~5.0 × 106Da, preferably 2.69 ×
106Da。
Preferably, the preparation method of the polysaccharide protein includes the following steps：
（1）It is 2%~20% by concentration（Mass/volume）, it is preferably 8%（Mass/volume）Extractive of locust tree microellobosporia it is water-soluble
Liquid removes floating preteins using Sevage methods, collects carbohydrate fraction；
（2）By step（1）Obtained carbohydrate fraction is added in absolute ethyl alcohol, forms it into determining alcohol as 30%~80%（Body
Product）, it is preferably 30%~50%（Volume）, more preferably 40%（Volume）Sugar juice, centrifugation, obtain sediment；
（3）By step（2）Obtained sediment is dissolved in water, filtering, concentration, dialysis collecting bag inside points；And
（4）Using ion exchange column chromatography to step（3）The bag inside points being collected into are separated, wherein the elution used
Liquid is water, 0.1Mol/L~1.0Mol/L NaCl aqueous solutions, is preferably 0.1Mol/L~0.3Mol/L NaCl aqueous solutions, more excellent
Elect the NaCl aqueous solutions of 0.1Mol/L as.
Preferably, the preparation method of the polysaccharide protein, which further includes the product afforded to NaCl solution and carries out dialysis, removes
The step of salt.
Preferably, the preparation method of the polysaccharide protein is further included using Sepharose CL-6B agarose Gel columns pair
The step of product after dialysis desalination is isolated and purified.
On the other hand, the present invention provides a kind of preparation method of above-mentioned polysaccharide protein, which includes the following steps：
（1）It is 2%-20% by concentration（Mass/volume）, it is preferably 8%（Mass/volume）Extractive of locust tree microellobosporia it is water-soluble
Liquid removes floating preteins using Sevage methods, collects carbohydrate fraction；
（2）By step（1）Obtained carbohydrate fraction is added in absolute ethyl alcohol, forms it into determining alcohol as 30%~80%（Body
Product）, it is preferably 30%~50%（Volume）, more preferably 40%（Volume）Sugar juice, centrifugation, obtain sediment；
（3）By step（2）Obtained sediment is dissolved in water, filtering, concentration, dialysis collecting bag inside points；And
（4）Using ion exchange column chromatography to step（3）The bag inside points being collected into are separated, wherein the elution used
Liquid is water, 0.1Mol/L~1.0Mol/L NaCl aqueous solutions, is preferably 0.1Mol/L~0.3Mol/L NaCl aqueous solutions, more excellent
Elect the NaCl aqueous solutions of 0.1Mol/L as, to obtain the final product.
Preferably, the preparation method further includes the step of product afforded to NaCl solution carries out dialysis desalination.
Preferably, the preparation method further include using Sepharose CL-6B agarose Gel columns to dialysis desalination after
Product the step of being isolated and purified.
In a specific embodiment, the preparation method includes the following steps：
（1）Extractive of locust tree microellobosporia trametes robiniophila 300g accurately is weighed, appropriate distilled water is added and is diluted to concentration about
For 8%（Mass/volume）Weak solution, floating preteins is removed using Sevage methods afterwards, repetitive operation 5 times, collects carbohydrate portion
Point；
（2）To step（1）Absolute ethyl alcohol is slowly added into obtained locust tree microellobosporia carbohydrate fraction becomes determining alcohol be
40%（Volume）Sugar juice, 4 DEG C stand 24 it is small when after, centrifuged 10 minutes with 3000 revs/min of speed, precipitation, is precipitated
Thing；
（3）Weigh appropriate step（2）Obtained sediment, is dissolved in 150ml distilled water, and filtering, is concentrated under reduced pressure
To volume 50ml；Dialysis collecting bag inside points, and
（4）Using DEAE-52 ion exchange column chromatographies to step（3）The bag inside points being collected into are separated, and first use water
Elution, then eluted using 0.1Mol/L NaCl solutions, 0.1Mol/L NaCl solutions elution fraction carries out after concentration
Desalination is analysed, dialysis procedure is to dialyse three days to flowing water, and distilled water is dialysed two days；And
（5）Using Sepharose CL-6B agarose Gel columns to step（4）Obtained product is isolated and purified, directly
Untill high performance liquid chromatography is detected as sterling.
Another aspect, the present invention provide purposes of the above-mentioned polysaccharide protein in the medicine for preparing treatment tumour.
Compared with prior art, the present invention at least has the advantages that：
The present invention is precipitated using the water extraction of system, alcohol, removes floating preteins, dialysis small molecule, ion-exchange chromatography first
And the separated means of gel molecular exclusion chromatography separate from locust tree microellobosporia and obtain a homogeneous polysaccharide protein component, and it is comprehensive
The analysis of application molecular weight, monose composition and the means such as amino acid composition analysis, infrared spectrum analysis and methylation analysis of conjunction are right
It has carried out the confirmation of chemical constitution feature, specify that its weight average molecular weight, monose composition and ratio, amino acid composition and ratio
Example, and the connection mode of contained saccharide residue.The locust tree microellobosporia polysaccharide group that such a design feature is apparent, molecular weight is homogeneous
Point, it is the preferable molecular model for studying Huai Er active ingredient and its bioactivity, to illustrate the immunological regulation of Huai Er
And antitumor active ingredient and mechanism of action are laid a good foundation, the present invention is by for locust tree microellobosporia deep level development and related preparations
Quality control provides scientific basis.
Brief description of the drawings
Hereinafter, the embodiment that the present invention will be described in detail is carried out with reference to attached drawing, wherein：
Fig. 1 is classified alcohol for extractive of locust tree microellobosporia trametes robiniophila and precipitates flow chart；
Fig. 2 purifies flow chart for 40% ethanol precipitation position TCP-40 column chromatographys of extractive of locust tree microellobosporia trametes robiniophila；
Fig. 3 is Huai Er albumen TP-1 molecular weight（Mw）Bioassay standard curve；
Fig. 4 is the chromatography of ions figure of reference substance solution in the analysis of Huai Er albumen TP-1 monosaccharide components；
Fig. 5 is the UPLC-GPC collection of illustrative plates of Huai Er albumen TP-1；
Fig. 6 is Huai Er albumen TP-1 monosaccharide composition analysis chromatography of ions figure；
Fig. 7 is3H-TdR incorporation methods detect mouse spleen cell proliferation, wherein, TCP：Thick many candies；TP-1：Huai Er egg
In vain -1；TP-2：Huai Er albumen -2；TP-3：Huai Er protein-3；TP-4：Huai Er protein-4；To-1：Trametes robinioplila is low
Molecular weight component -1；To-2：Trametes robinioplila lower-molecular-weight component -2；To-3：Trametes robinioplila lower-molecular-weight component -3；To-4：Trametes robinioplila low molecule
Measure component -4；TFP：Trametes robinioplila slightly total floating preteins part；
Fig. 8 is3H-TdR incorporation methods detection mouse T, B lymphocyte proliferation, wherein, figure A is CD19+ cells；Figure B is CD3
+ cell；Figure C is mouse T lymphocyte；Figure D is mouse bone-marrow-derived lymphocyte；Dex：Glucan；TCP：Thick many candies；TP-1：Trametes robinioplila is more
Glycoprotein -1；TP-2：Huai Er albumen -2；TP-3：Huai Er protein-3；TP-4：Huai Er protein-4；LPS：Fat is more
Sugar；
Mouse and people T, the change of bone-marrow-derived lymphocyte ratio after Fig. 9 stimulates for Huai Er, wherein, figure A is thin for mouse T lymphs
Born of the same parents；Figure B is mouse bone-marrow-derived lymphocyte；Figure C is human T-lymphocyte；Figure D is human B lymphocyte；Dex：Glucan
（Dextran）；TCP：Thick many candies；TP-1：Huai Er albumen -1；TP-2：Huai Er albumen -2；TP-3：Huai Er egg
In vain -3；TP-4：Huai Er protein-4；LPS：Lipopolysaccharides；
Figure 10 is the expression of T, bone-marrow-derived lymphocyte surface C D69 after Huai Er stimulates, and T drenches after figure A stimulates for Huai Er
The expression of bar cell surface CD69, figure B are the expression of bone-marrow-derived lymphocyte surface C D69 after Huai Er stimulates, wherein Dex：Gather Portugal
Sugar（Dextran）；TCP：Thick many candies；TP-1：Huai Er albumen -1；TP-2：Huai Er albumen -2；TP-3：Huai Er
Protein-3；TP-4：Huai Er protein-4；LPS：Lipopolysaccharides；
Turnover of Mouse Peritoneal Macrophages NO releases change, wherein Dex after Figure 11 stimulates for Huai Er：Glucan
（Dextran）；TCP：Thick many candies；TP-1：Huai Er albumen -1；TP-2：Huai Er albumen -2；TP-3：Huai Er egg
In vain -3；TP-4：Huai Er protein-4；To-1：Trametes robinioplila lower-molecular-weight component -1；To-2：Trametes robinioplila lower-molecular-weight component -2；To-
3：Trametes robinioplila lower-molecular-weight component -3；To-4：Trametes robinioplila lower-molecular-weight component -4；TFP：Trametes robinioplila slightly total floating preteins part；LPS：Fat
Polysaccharide；
Figure 12 Huai Ers albumen is to the inhibitory action of Tumor angiogenesis endothelial cell proliferation, wherein TP-1：Trametes robinioplila is more
Glycoprotein -1.
Embodiment
Below by embodiment, the present invention will be described in detail, it will be appreciated that and following embodiments are merely to illustrate the present invention, without
Limit the scope of the invention in any way.
Embodiment 1：Huai Er albumen（TP-1）Isolation and purification method
Accurately weigh extractive of locust tree microellobosporia trametes robiniophila（There is provided by Qidong Gaitianli Pharmaceutical Co., Ltd.）300g, adds
It is about 8% that appropriate distilled water, which is diluted to concentration,（Mass/volume）Weak solution, floating preteins is gone using Sevage methods afterwards,
Repetitive operation 5 times, collects carbohydrate fraction respectively（TCP）And trametes robinioplila slightly total floating preteins part（TFP）, trametes robinioplila slightly total floating preteins
Partial vacuum drying for standby, locust tree microellobosporia glucide part（TCP）After the chloroform and n-butanol of removal remaining that be concentrated under reduced pressure,
Carry out classification alcohol precipitation.Absolute ethyl alcohol is slowly added into TCP first and becomes alcohol volumetric concentration as 40%（Volume）It is sugared molten
Liquid, 4 DEG C stand 24 it is small when after, 3000 revs/min centrifuge 10 minutes, precipitation numbering be TCP-40, be dried under reduced pressure in case further
Isolate and purify, relaying the continuous appropriate absolute ethyl alcohol of addition to supernatant afterwards becomes containing alcohol 60%（Volume）Sugar juice, 4 DEG C
Stand 24 it is small when after, 3000 revs/min, centrifuge 10 minutes, precipitation numbering be TCP-60, be dried under reduced pressure in case further separating pure
Change, continue up after this in clear liquid and add appropriate absolute ethyl alcohol to concentration of alcohol and reach 80%（Volume）, 4 DEG C stand it is 24 small
Shi Hou, 3000 revs/min centrifuge 10 minutes, supernatant discarding, and precipitation numbering is TCP-80, is dried under reduced pressure in case further separating pure
Change, the specific alcohol precipitation flow chart that is classified is shown in Fig. 1.
Weigh suitable trametes robiniophila 40%（Volume）Ethanol precipitation position TCP-40, is dissolved in 150ml distilled water
In, after 0.45 μm of water system membrane filtration, it is concentrated under reduced pressure into volume 50ml, collecting bag inside points of dialysing and bag outer portion, outside bag
Part is trametes robinioplila lower-molecular-weight component -1（TO-1）, bag inside points are separated by DEAE-52 ion exchange column chromatographies, are respectively adopted
3.0L is collected at distilled water and 0.1MNaCl solution, 0.5M NaCl elutions, each position, and water elution part is directly thickened to do, chlorine
Change sodium elution fraction and carry out dialysis desalination after concentration, dialysis procedure is to dialyse three days to flowing water, and distilled water is dialysed two days.So
Sepharose CL-6B agarose Gel columns are respectively adopted are isolated and purified for each section afterwards, until high performance liquid chromatography detects
Untill for sterling, by above-mentioned pillar layer separation process, eluted respectively from TCP-40 ion exchange column chromatography 0.1Mol/L NaCl
Position purifying obtains UPLC（Ultra performance liquid chromatography）It is verified as the polysaccharide protein TP-1 of homogeneous components（397mg）And from
0.5Mol/L NaCl elutions position obtains homogeneous polysaccharide albumen TP-2（1212mg）, separation process figure is shown in Fig. 2.
Weigh suitable trametes robiniophila 60%（Volume）Ethanol precipitation position TCP-60, is dissolved in 150ml distilled water
In, filtering, through being concentrated under reduced pressure into volume 50ml, dialysis collecting bag inside points and bag outer portion, is divided into trametes robinioplila low molecule outside bag
Measure component -2（TO-2）, bag inside points are separated by DEAE-52 ion exchange column chromatographies, and distilled water and 1.0Mol/ is respectively adopted
LNaCl solution elutes, and 3.0L is collected at each position, and water elution part is directly thickened to do, and sodium chloride elution fraction is concentrating
After carry out dialysis desalination, dialysis procedure is to dialyse three days to flowing water, and distilled water is dialysed two days.Then it is respectively adopted for each section
Sepharose CL-6B agarose Gel columns are isolated and purified, and untill high performance liquid chromatography is detected as sterling, are passed through
Above-mentioned pillar layer separation process, obtains from TCP-60 ion exchange column chromatography 1.0Mol/L NaCl elutions position purifying respectively
UPLC（Ultra performance liquid chromatography）It is verified as the polysaccharide protein TP-3 of homogeneous components（12.1g）.
Weigh suitable trametes robiniophila 80%（Volume）Ethanol precipitation position TCP-80, is dissolved in 150ml distilled water
In, filtering, is concentrated under reduced pressure into volume 50ml, collecting bag inside points of dialysing and bag outer portion, is divided into trametes robinioplila low molecular weight outside bag
Component -3（TO-3）, bag inside points are separated by DEAE-52 ion exchange column chromatographies, and distilled water and 1.0Mol/L is respectively adopted
3.0L is collected at NaCl water elutions, each position, and water elution part is directly thickened to do, sodium chloride elution fraction after concentration into
Row dialysis desalination, dialysis procedure are to dialyse three days to flowing water, and distilled water is dialysed two days.Then Sepharose is respectively adopted for each section
CL-6B agarose Gel columns are isolated and purified, untill high performance liquid chromatography is detected as sterling, by above-mentioned column chromatography
Separation process, obtains UPLC from TCP-80 ion exchange column chromatographies 1.0Mol/LNaCl elutions position purifying respectively（Ultra high efficiency
Liquid chromatogram）It is verified as the polysaccharide protein TP-4 of homogeneous components（10.0g）, collect trametes robiniophila 80%（Volume）In ethanol precipitation
Supernatant fraction obtains trametes robinioplila lower-molecular-weight component -4（TO-4）（Fig. 1）.
Embodiment 2：Huai Er albumen（TP-1）The research of chemical constitution
The present embodiment have studied the chemical constitution of the Huai Er albumen TP-1 of the preparation of embodiment 1.
First, Huai Er albumen（TP-1）Chemical constitution research method
1st, purity and the measure of molecular weight
UPLC-GPC-ELSD instrument configurations and chromatographic condition：U.S. Waters UPLC, TSK-3000GPC chromatographic columns, from
Dynamic injector, Millipore ultra-pure water ion-exchangers prepare high purity water（0.45 μm of cellulose acetate membrane filtration）；Flow velocity
0.3ml/min。
The preparation of standard curve：Suitable dextran standard items are weighed respectively, are added deionized water, are configured to dense
The reference substance solution of 0.5mg/ml is spent, carries out UPLC detections one by one afterwards, the result is shown in Fig. 3.
It is prepared by sample solution：A certain amount of TP-1 is weighed respectively, adds appropriate amount of deionized water, being configured to concentration is
The solution of 1mg/ml, Millipore0.22 μm of water system membrane filtration, sample detection.
2nd, monosaccharide composition analysis
Complete sour water solution：Precision weighs TP-1 (10mg) and is put into heavy wall pressure bottle, adds the 2Mol/L trifluoroacetic acids of 4ml
（TFA）, inflated with nitrogen, after tube sealing, when 100 DEG C of hydrolysis 2 are small, evaporated under reduced pressure.
The ion chromatography of sample
Instrument configuration and chromatographic condition：3000 type ion chromatographies of Dionex ICS, CarboPac PA20 analytical columns, 150
× 3mm, S/N 002823, CarboPac PA20 guard columns, 50*3mm, S/N 002652, leacheate composition and flow velocity 1-
25min, 1m Mol/L KOH；25.1-32min 30m Mol/L KOH；32.1-35min 1m Mol/L KOH；0.45mL/
Min, 10 μ L of sample introduction.
The preparation of reference substance solution：Fucose, arabinose, galactolipin, glucose, xylose, mannose reference substance is taken to fit
Amount, is dissolved into the respectively reference substance solution containing 10.0mg/L with deionized water, shakes up, to obtain the final product.
It is prepared by standard curve：Precision absorption reference substance mixing stock solution is appropriate, is diluted to respectively with deionized water
The standard solution of 0.5mg/L, 1mg/L, 5mg/L, 10mg/L, 15mg/L, carry out successively after 0.45 μm of filtering with microporous membrane from
Sub- chromatographic determination, ion chromatography condition are：3000 type ion chromatographies of Dionex ICS, CarboPac PA20 analytical columns, 150 ×
3mm, S/N002823, CarboPac PA20 guard columns, 50*3mm, S/N 002652, leacheate composition and flow velocity 1-25min,
1m Mol/L KOH；25.1-32min 30m Mol/L KOH,；32.1-35min 1m Mol/L KOH；0.45mL/min, into
10 μ L of sample.Using integrating peak areas value as ordinate（Y）, using each standard concentration as abscissa（X）, it is bent to draw each reference substance standard
Line simultaneously calculates regression equation, the results are shown in Table 1 and Fig. 4.
1 linear relationship of table investigates result
It is prepared by test solution：The complete acid hydrolysis products of Huai Er albumen TP-1 are redissolved in 50ml deionized waters, are surpassed
Sound makes dissolving in 10 minutes, takes solution appropriate, crosses II solid phase extraction column of 0.22 μm of aperture water system filter membrane and DIONEX RP.
It is accurate respectively to draw reference substance solution and each 10 μ L of test solution, inject ion chromatograph, to obtain the final product.
3rd, methylation analysis
Polysaccharide protein exhaustive methylation：Huai Er albumen TP-1 (10mg) is put into vacuum drying chamber in reaction bulb
Dry 5h（50℃）, add through the processed DMSO 2ml of over-molecular sieve, sonic oscillation 5 minutes, after sample is completely dissolved, adds
Enter to be ground into powdery NaOH 20mg, while air in most bottle is caught up with nitrogen, at room temperature ultrasound 10 minutes, stand 90 minutes, treat anti-
After answering the DMSO frosts completely in bottle, 0.1ml iodomethane is added dropwise（This process takes around 15~20 minutes）, simultaneous reactions
Thing can slowly thaw, and gradually clarify, until becoming glassy yellow.Then ultrasound 10 minutes, stand 30 minutes.Reduced pressure at room temperature steams
Evaporate, go to use up the iodomethane of excess, dialysed one day with water, decompression steaming to 2ml or so.After freeze-drying dry 5 in drier
Hour, after repeating aforesaid operations 2 times, take a small amount of sample to carry out infrared detection, if infrared spectrum Central Plains 3300cm-1Place is strong and wide
Hydroxyl peak disappear, and 2900cm-1Place methyl peak significantly increases, and shows that the permethylated reaction of sample has been completed.
It is prepared by partial methylation Alday alcohol acetonyl ester：The sample of exhaustive methylation is dissolved in 3mL volume fractions is
In 90% formic acid solution, tube sealing, depolymerization 6h at 100 DEG C, 2~3mL methanol is added into reaction bulb, and be concentrated under reduced pressure steaming at 40 DEG C
It is dry, repeat above operation three times to eliminate excessive formic acid, 2Mol/L TFA solution 4mL are then added into the sample after depolymerization,
Sealing is after hydrolyzing 2h at 110 DEG C, the evaporated under reduced pressure at 40 DEG C of the solution in reaction bulb, adds 2~3mL methanol, be evaporated, weight
Operated more than multiple repeatedly to eliminate excessive TFA.After sample after hydrolysis is dissolved with 3~4mL distilled water, about 20mg is added
NaBH43h is reduced at room temperature, then with glacial acetic acid tune pH value to 5 or so, after adding 1~2mL methanol and a drop glacial acetic acid, then
Evaporated under reduced pressure, repeats aforesaid operations repeatedly to eliminate excessive acetic acid.Sample through above-mentioned processing is placed in P2O5In vacuum desiccator
It is dried under reduced pressure one day, after 110 DEG C of dry 10~15min, adds 3mL aceticanhydrides, 110 DEG C of reaction 1h, add into reaction solution
2mL toluene, decompression boils off unreacted aceticanhydride at 40 DEG C after vibration, so repeatedly to eliminate aceticanhydride.Then by acetylation
Sample afterwards is dissolved in chloroform, adds isometric distillation water washing chloroform layer 3 times, eliminates water layer, and chloroform layer adds anhydrous
Sodium sulphate dries 10min, and filtering, after chloroformic solution reduced pressure at room temperature is concentrated into 0.1mL or so, carries out Gc-mss（GC-
MS）.The condition of makings is：50 DEG C of initial temperature, heating schedule is 40 DEG C/min, to 215 DEG C, keeps 40min, detector temperature
250 DEG C, DB-5 Capillary GC-MS chromatography post detections.
4th, amino acid analysis：
Weigh appropriate Huai Er albumen TP-1（10mg）It is put into test tube, adds the 6Mol/L HCl of 4ml at 100 DEG C
Hydrolyze 6 it is small when, boil off HCl, centrifuge, filtering, filtrate analyzed with amino-acid analyzer.
5th, IR spectrum analyses：
1mg samples are weighed, are dried in vacuum overnight, secondary daily pellet technique carries out IR detections.
2nd, Huai Er albumen（TP-1）Chemical constitution result of study
The Huai Er albumen prepared according to the research method of Part I to embodiment 1（TP-1）Chemical constitution carry out
Research, result of study are as follows：
1st, purity and molecular weight
TP-1 is pale yellow powder shape material, soluble easily in water, DMSO, and methanol, ethanol insoluble in high concentration etc. are organic molten
A symmetrical narrow chromatographic peak is presented in agent, the UPLC-ESLD collection of illustrative plates of the polysaccharide protein, and it is a pure polysaccharide protein to prompt it
Material, contrasts with molecular weight standards, and the molecular weight of the polysaccharide protein is 2.69 × 106Da（See Fig. 5）.Lowry reactions are in sun
Property, has weak absorption at UV scanning 280nm, shows that the material contains albumen.The infrared spectrum of TP-1 is in 1735cm-1Place is without suction
Receive, prompt TP-1 to be free of uronic acid.
2nd, monosaccharide composition analysis
After the complete sour water solutions of TP-1, a hydrolysate part has directly carried out ion chromatography, with standard monose pair
According to analysis result shows that the polysaccharide is made of fucose, arabinose, galactolipin, glucose, xylose and mannose, is detecting
During without find shown with uronic acid and the relevant chromatographic peak of nitrogen acetylamino sugars, it is a neutral polysaccharide to prompt it
（It is shown in Table 2 and Fig. 6）.
2 Huai Er albumen TP-1 monose of table forms and mass ratio ion chromatography result
By monosaccharide composition analysis as can be seen that Huai Er albumen TP-1 contains 6 kinds of monose, wherein with galactolipin and sweet
Reveal the content highest of sugar, xylose, fucose, arabinose and glucose content are less.
3rd, methylation analysis
In order to confirm the saccharide residue connection mode in TP-1, methylated using Needs methods to TP-1, in first three times
After base, by its with 90% formic acid depolymerization, with the complete sour water solutions of 2 Mol/L TFA, NaBH4Reduction and aceticanhydride acetylation system
For into Alday alcohol acetic ester derivative, GC-MS analyses are carried out, the results are shown in Table 3.
Table 3TP-1 methylation analysis results
Shown by methylation analysis, the structure of Huai Er albumen TP-1 is extremely complex.Connected containing 10 kinds of differences
With 1,1,2 hydroxyl and other saccharide residues form glucosides key connection for saccharide residue, wherein arabinose, xylose with 1 hydroxyl with
Other saccharide residues form glucosides key connections, and glucose forms glucosides key connection with 1,2 hydroxyl and other saccharide residues, galactolipin with
1,3,1,6 and 1,2,6 hydroxyls form glucosides key connection with other saccharide residues, and mannose is then with 1,1,6 hydroxyls
Glucosides key connection is formed with other saccharide residues, the peak area percent between specific saccharide residue is shown in Table 3.
Above-mentioned methylation analysis results have differences the different saccharide residues with reaction process from monosaccharide composition analysis result and damage
Mistake degree difference is related.
4th, amino acid analysis：
In order to analyze the composition of the amino acid in Huai Er albumen TP-1 and ratio, using hydrochloric acid water solution combination, Hitachi is automatic
Amino-acid analyzer, has carried out it amino acid composition and proportion grading, the results are shown in Table 4.
4 Huai Er albumen TP-1 amino acid composition analysis results of table
5.IR spectrum analyses
The infrared spectrum of TP-1 is in 1735cm-1TP-1 is prompted to be free of uronic acid without absorption in place.
3 trametes robiniophila of embodiment and contained chemical composition vivo immunization activity research
1st, sample source
Trametes robiniophila is provided by Qidong Gaitianli Pharmaceutical Co., Ltd..Thick many candies TCP, homogeneous polysaccharide TP-1, TP-2, TP-
3rd, TP-4, lower-molecular-weight component TO-1, TO-2, TO-3, TO-4 are prepared by embodiment 1.
2nd, mouse spleen cell proliferation is promoted
1）Whether detection Huai Er can stimulate mouse spleen cell proliferation
Method：Trametes robiniophila each component（Thick many candies TCP；Huai Er TP-1, TP-2, TP-3, TP-4；Low molecular weight group
Divide TO-1, TO-2, TO-3, TO-4；Trametes robinioplila slightly total floating preteins part TFP）And negative control medicine glucan（Dextran）With
Mouse boosting cell co-cultures, after 42h, incorporation3Detected after H-TdR, 48h using cell harvestor Filtermate harvester
Its proliferation index.As a result such as Fig. 7.
Conclusion：From3H-TdR results are seen, by the polysaccharide component extracted in trametes robiniophila（Including Thick many candies TCP, polysaccharide component
TP-1, TP-2, TP-3, TP-4）Mouse spleen cell proliferation can be stimulated；Lower-molecular-weight component cannot stimulate spleen thin in addition to TO-3
Born of the same parents breed.
2）Huai Er can stimulate mouse B cell to breed, and non-T cell
Method：Thick many candies TCP, Huai Er TP-1, TP-2, TP-3, TP-4 and negative controls glucan（Dextran）
（Concentration gradient is 100 μ g/ml, 30 μ g/ml, 10 μ g/ml）, positive control lipopolysaccharides（LPS）T, bone-marrow-derived lymphocyte with purifying is common
Culture, 42h incorporations3Its proliferation index is detected using cell harvestor Filtermate harvester after H-TdR, 48h.As a result
See Fig. 8.
Conclusion：Magnetic bead sorting obtains the T of high-purity, B cell, after being incubated 48h altogether with Huai Er,3H-TdR results are shown
Show, what Huai Er component TP-1, TP-2, TP-3, TP-4, TCP were stimulated is B cell, and non-T cell.And this stimulation
In dosage correlation.
3）Huai Er stimulates mouse boosting cell and human peripheral blood single nucleus cell（PBMC）Afterwards, T, the change of B cell ratio
Method：Thick many candies TCP, Huai Er TP-1, TP-2, TP-3, TP-4 and glucan（Dextran）（Concentration is 50 μ
g/ml）, lipopolysaccharides（LPS）Co-cultured with mouse boosting cell and human peripheral PBMC, 24 it is small when after, flow cytometer detection T, B cell ratio
Example.The result is shown in Fig. 9.
Conclusion：After Huai Er TP-1, TP-2, TP-3, TP-4 stimulate mouse boosting cell or human peripheral PBMC, with compareing
Medicine glucan（Dextran）Compare, B cell ratio substantially raises, and T cell ratio is without significant change.
4）After Huai Er stimulates mouse boosting cell, T, the expression of CD69 in B cell
Method：Thick many candies TCP, Huai Er TP-1, TP-2, TP-3, TP-4 are with compareing polysaccharide glucan（Dextran）
（Concentration is 50 μ g/ml）, lipopolysaccharides（LPS）With mouse boosting cell co-culture, 6 it is small when after, flow cytometer detection T, B cell surface C D69
Expression.The result is shown in Figure 10.
Conclusion：After Huai Er stimulates mouse boosting cell 6h, B cell starts high expression CD69, and T cell becomes without obvious
Change.
3rd, macrophage release NO is promoted
Method：Turnover of Mouse Peritoneal Macrophages in vitro culture, add Thick many candies TCP, Huai Er TP-1, TP-2, TP-3,
TP-4, lower-molecular-weight component TO-1, TO-2, TO-3, TO-4, trametes robinioplila slightly total floating preteins part TFP with compareing polysaccharide glucan
（Dextran）, lipopolysaccharides（LPS）Co-culture, 48 it is small when after, with Griess reagent box（Griess Reagent kit）Detection is thin
The content of NO in born of the same parents' culture supernatant, the result is shown in Figure 11.
Conclusion：NO testing results show, Huai Er can Stimulated Macrophages secretion NO, and secretory volume and polysaccharide
Concentration is in dosage correlation.The secretion of NO does not have significant changes then after lower-molecular-weight component stimulates.
Inhibitory action of the 4 Huai Er albumen of embodiment to Tumor angiogenesis endothelial cell proliferation
First, experimental method and step：
1st, experiment material
Cell：The vascular endothelial cell in human tumor source
Reagent：M199 culture mediums, dual anti-, hyclone（FBS）, basic fibroblast growth factor（bFGF）, gelatin
（gelatin）.
Sample to be tested：TP-1：Huai Er albumen -1
2nd, experimental procedure：
（1）0.2% gelatin is added in 96 orifice plates with the volley of rifle fire, per 40 microlitres of hole, is shaken up.Incubator is put into be incubated 30 minutes
More than.
（2）96 orifice plates are taken out, gelatin is suctioned out with the volley of rifle fire, is washed one time with PBS, it is spare to be put in super-clean bench.
（3）20%FBS is prepared, adds dual anti-M199 nutrient solutions.
（4）The vascular endothelial cell in the human tumor source of recovery is taken, nutrient solution is suctioned out, is washed once with PBS, used after centrifugation
The M199 of 10% serum, which is resuspended, to be counted, and adjustment cell concentration is 40000-50000/ml.
（5）Cell is added into 96 orifice plates with the volley of rifle fire, per 100 microlitres of hole, edge hole adds equivalent PBS.It is put into incubator.
（6）12 it is small when after, preparation contain 1%-5%FBS, the M199 culture mediums of 10-100ng/ml bFGF.
（7）96 orifice plates are taken out, culture medium is suctioned out with the volley of rifle fire, adds M199 and medicine containing 1%-5%FBS and bFGF, often
100 microlitres of hole, if control group and zeroing group.And sample to be tested is added, it is respectively TCP, TP-1, TP-2, TP-3 and TP-4.It is each
Sample sets 4 concentration, is respectively 10 μ g/L, 100 μ g/L, 500 μ g/L and 1000 μ g/L.Then at 37 °C, 5% CO2Lower company
It is continuous to be incubated.
（8）72 it is small when after, suck culture medium, add MTT (concentration 0.5mg/ml, filtration sterilization), be incubated 4-6 it is small when.
（9）MTT is sucked, adds 100 microlitres of DMSO, 570nm to survey OD.
2nd, experiment conclusion：
Huai Er albumen -1（TP-1）There is significant inhibitory action to the propagation of tumor vascular endothelial cell.The result is shown in
Figure 12.Numerical value is shown as mean ± standard deviation in figure.Each administration group compared with control group relatively after, and Analysis of variance and subsequent T
Examine.*P ＜ 0.05, representing two groups relatively has significant difference.***P ＜ 0.001, representing two groups relatively has pole significant difference.
Claims (16)
1. a kind of Huai Er albumen, the monose composition of the polysaccharide protein is fucose, arabinose, galactolipin, glucose, wood
Sugar and mannose, its mass ratio are 3.2：2.0：33.5：5.5：1.2：50.4；The preparation method of the polysaccharide protein includes following step
Suddenly：
(1) the extractive of locust tree microellobosporia aqueous solution that mass concentration is 2%~20% is removed into floating preteins using Sevage methods, received
Collect carbohydrate fraction；
(2) carbohydrate fraction for obtaining step (1) is added in absolute ethyl alcohol, and it is 30%~80% to form it into alcohol volumetric concentration
Sugar juice, centrifugation, obtains sediment；
(3) sediment that step (2) obtains is dissolved in water, filtered, concentration, dialysis collecting bag inside points；
(4) the bag inside points being collected into using ion exchange column chromatography to step (3) are separated, wherein the eluent used is
Water, 0.1Mol/L~1.0Mol/L NaCl aqueous solutions；
(5) product afforded to step (4) NaCl aqueous solutions carries out dialysis desalination；And
(6) product after step (5) dialysis desalination is isolated and purified using Sepharose CL-6B agarose Gel columns.
2. polysaccharide protein according to claim 1, it is characterised in that the weight average molecular weight of the polysaccharide protein for 7.0 ×
105~5.0 × 106Da。
3. polysaccharide protein according to claim 2, it is characterised in that the weight average molecular weight of the polysaccharide protein for 2.69 ×
106Da。
4. polysaccharide protein according to claim 1, it is characterised in that in step (1), the extractive of locust tree microellobosporia water
The mass concentration of solution is 8%.
5. polysaccharide protein according to claim 1, it is characterised in that in step (2), the alcohol volume of the sugar juice is dense
Spend for 30%~50%.
6. polysaccharide protein according to claim 1, it is characterised in that in step (2), the alcohol volume of the sugar juice is dense
Spend for 40%.
7. polysaccharide protein according to claim 1, it is characterised in that in step (4), the eluent is 0.1Mol/L
The NaCl aqueous solutions of~0.3Mol/L.
8. polysaccharide protein according to claim 1, it is characterised in that in step (4), the eluent is 0.1Mol/L
NaCl aqueous solutions.
9. a kind of preparation method of polysaccharide protein described in any item of the claim 1 to 8, which includes following step
Suddenly：
(1) the extractive of locust tree microellobosporia aqueous solution that mass concentration is 2%~20% is removed into floating preteins using Sevage methods, received
Collect carbohydrate fraction；
(2) carbohydrate fraction for obtaining step (1) is added in absolute ethyl alcohol, and it is 30%~80% to form it into alcohol volumetric concentration
Sugar juice, centrifugation, obtains sediment；
(3) sediment that step (2) obtains is dissolved in water, filtered, concentration, dialysis collecting bag inside points；
(4) the bag inside points being collected into using ion exchange column chromatography to step (3) are separated, wherein the eluent used is
Water, 0.1Mol/L~1.0Mol/L NaCl aqueous solutions；
(5) product afforded to step (4) NaCl aqueous solutions carries out dialysis desalination；And
(6) product after step (5) dialysis desalination is isolated and purified using Sepharose CL-6B agarose Gel columns.
10. preparation method according to claim 9, it is characterised in that in step (1), the extractive of locust tree microellobosporia
The mass concentration of aqueous solution is 8%.
11. preparation method according to claim 9, it is characterised in that in step (2), the alcohol volume of the sugar juice
Concentration is 30%~50%.
12. preparation method according to claim 9, it is characterised in that in step (2), the alcohol volume of the sugar juice
Concentration is 40%.
13. preparation method according to claim 9, it is characterised in that in step (4), the eluent is 0.1Mol/
The NaCl aqueous solutions of L~0.3Mol/L.
14. preparation method according to claim 9, it is characterised in that in step (4), the eluent is 0.1Mol/
The NaCl aqueous solutions of L.
15. preparation method according to claim 9, it is characterised in that the preparation method includes the following steps：
(1) extractive of locust tree microellobosporia trametes robiniophila 300g accurately is weighed, it is 8% to add appropriate distilled water and be diluted to concentration
Weak solution, removes floating preteins, repetitive operation 5 times, collects carbohydrate fraction using Sevage methods afterwards；
(2) absolute ethyl alcohol is slowly added into the locust tree microellobosporia carbohydrate fraction obtained to step (1) and becomes determining alcohol as 40%
Sugar juice, 4 DEG C stand 24 it is small when after, centrifuged 10 minutes with 3000 revs/min of speed, precipitation, obtains sediment；
(3) sediment that appropriate step (2) obtains is weighed, is dissolved in 150ml distilled water, filters, is concentrated under reduced pressure into body
Product 50ml；Dialysis collecting bag inside points, and
(4) the bag inside points being collected into using DEAE-52 ion exchange column chromatographies to step (3) are separated, using 0.1Mol/
L NaCl solutions are eluted, and elution fraction carries out dialysis desalination after concentration, and dialysis procedure is to dialyse three days to flowing water, distillation
Water is dialysed two days；And
(5) product obtained using Sepharose CL-6B agarose Gel columns to step (4) is isolated and purified, until high
Untill effect liquid phase chromatogram method is detected as sterling.
16. purposes of the polysaccharide protein described in any item of the claim 1 to 8 in the medicine for preparing treatment tumour.
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| CN102621260A (en) * | 2011-01-31 | 2012-08-01 | 启东盖天力药业有限公司 | Sophora fungus mycoplasma extract identification and detection method |
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| CN101204405A (en) * | 2006-12-22 | 2008-06-25 | 启东盖天力药业有限公司 | Extractive of locust tree microellobosporia, preparation method and uses thereof |
| CN102621260A (en) * | 2011-01-31 | 2012-08-01 | 启东盖天力药业有限公司 | Sophora fungus mycoplasma extract identification and detection method |
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