CN104119428B - A kind of Huai Er albumen and its production and use - Google Patents
A kind of Huai Er albumen and its production and use Download PDFInfo
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Abstract
The present invention relates to a kind of Huai Er albumen and its production and use, the monose composition of Huai Er albumen of the invention is arabinose, galactolipin, glucose, xylose and mannose, and its mass ratio is 8.9：1.6：3.4：7.4：1.3, the weight average molecular weight of the GL-PP is 7.0 × 105‑2.0×106Da, preferably 1.41 × 106Da.The GL-PP 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 production and use.
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
On handle, the trunk for being born in the broad leaf trees such as Chinese scholartree, locust tree, the ground such as Hebei, Shaanxi, Liaoning, Hunan, Guangxi, Fujian are distributed in, are a kind of
The domestomycetes of trees heart-wood rot can be caused.It is this product bitter, pungent, it is mild-natured nontoxic, there are " controlling wind ", " blood-breaking ", the work(of " beneficial power "
Effect, clinically for treating a variety of diseases.Because Chinese scholar tree is increasingly rare in old-age group, trametes robinioplila herb resource is on the brink of exhaustion, it is difficult to full
Sufficient clinical application demand, can not more meet industrial production demand.In order to solve the resource problem of trametes robinioplila, Qidong lid day power medicine company
Long-term research and practice are passed through by Co., Ltd, have captured trametes robinioplila mycelium large scale fermentation problem, have realized mycelial work
Industry metaplasia is produced, and with locust tree microellobosporia（Trametes robinioplila mycelium fermentation thing）Extract is development of raw materials huaier granules and Chinese scholartree Qihuang Granule
Deng medicine, clinical application demand is met.
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 is treatment or adjuvant therapy of tumors disease.However, at present for Huai Er albumen
Composition, structure and preparation method thereof research are seldom, the monose composition and ratio point for only having Thick many candies in terms of chemical constitution study
The document report of analysis, lacks isolating and purifying and structural research to its homogeneous polysaccharide, the saccharide residue as contained by homogeneous polysaccharide albumen
The information such as species, ratio, ordering in main chain, and these information for illustrate the real effective component of locust tree microellobosporia,
The mechanism of action and structure-activity relationship of Huai Er albumen etc. are most important.Equally in the bioactivity research side of Huai Er albumen
Face, more using Thick many candies as research object, lacks internal, the external pharmaceutical research data that application homogeneous polysaccharide component is obtained,
Make used so as to can not understand Huai Er albumen and human immunocyte or tumor cell surface related polysaccharides acceptor in depth
Journey, thus deeply can not illustrate its action principle.
The content of the invention
In order to overcome the defect of prior art, the present invention provides a kind of Huai Er albumen and its production and use.
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 GL-PP is arabinose, gala
Sugar, glucose, xylose and mannose, its mass ratio are 8.9：1.6：3.4：7.4：1.3.
Preferably, the weight average molecular weight of the GL-PP is 7.0 × 105-2.0×106Da, preferably 1.41 ×
106Da。
Preferably, the preparation method of the GL-PP comprises the following steps：
（1）It is 2%-20% by concentration（Mass/volume）, preferably 8%（Mass/volume）Extractive of locust tree microellobosporia（For example
Trametes robiniophila）The aqueous solution 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 for 75%-80%（Body
Product）, preferably 80%（Volume）Sugar juice, centrifugation, obtain sediment；
（3）By step（2）Obtained sediment is dissolved in water, filtering, part in concentration, dialysis collecting bag；And
（4）Using ion exchange column chromatography to step（3）Part is separated in the bag being collected into, wherein the elution used
Liquid is the 0.85Mol/L-1.5Mol/L NaCl aqueous solution, the preferably 1.0Mol/L NaCl aqueous solution.
Preferably, the preparation method of the GL-PP also includes dialysing to the product that the NaCl aqueous solution is afforded
The step of desalination.
Preferably, the preparation method of the GL-PP also includes 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 GL-PP, and the preparation method comprises the following steps：
（1）It is 2%-20% by concentration（Mass/volume）, preferably 8%（Mass/volume）Extractive of locust tree microellobosporia（For example
Trametes robiniophila）The aqueous solution 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 for 75%-80%（Body
Product）, preferably 80%（Volume）Sugar juice, centrifugation, obtain sediment；
（3）By step（2）Obtained sediment is dissolved in water, filtering, part in concentration, dialysis collecting bag；And
（4）Using ion exchange column chromatography to step（3）Part is separated in the bag being collected into, wherein the elution used
Liquid is the 0.85Mol/L-1.5Mol/L NaCl aqueous solution, and the preferably 1.0Mol/L NaCl aqueous solution is produced.
Preferably, the preparation method also includes the step of the product progress dialysis desalination afforded to the NaCl aqueous solution
Suddenly.
Preferably, after the preparation method also includes using Sepharose CL-6B agarose Gel columns to dialysis desalination
Product the step of isolated and purified.
In a specific embodiment, the preparation method comprises the following steps：
（1）It is accurate to weigh extractive of locust tree microellobosporia trametes robiniophila 300g, add appropriate distilled water and be diluted to concentration about
For 8%（Mass/volume）Weak solution, floating preteins is removed using Sevage methods afterwards, repeated 5 times, carbohydrate portion is collected
Point；
（2）To step（1）Absolute ethyl alcohol is slowly added into obtained locust tree microellobosporia carbohydrate fraction becomes determining alcohol be
80%（Volume）Sugar juice, after 4 DEG C stand 24 hours, with 3000 revs/min of centrifugation 10 minutes, precipitation is precipitated
Thing；
（3）Weigh appropriate step（2）Obtained sediment, is dissolved in 150ml distilled water, filtering, is concentrated under reduced pressure
To volume 50ml；Part in dialysis collecting bag, and
（4）Using DEAE-52 ion exchange column chromatographies to step（3）Part is separated in the bag being collected into, and is used
The 1.0M NaCl aqueous solution is eluted, and elution fraction carries out dialysis desalination after concentration, and dialysis procedure is to flowing water dialysis three
My god, 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 provides purposes of the above-mentioned GL-PP in the medicine for preparing treatment tumour.
Compared with prior art, the present invention at least has the advantages that：
The present invention using the water extraction of system, alcohol precipitation, deproteinized, dialysis small molecule, ion-exchange chromatography and coagulates first
The means of xanthan molecule amount exclusion chromatography separation are separated from locust tree microellobosporia obtains a homogeneous GL-PP component, and comprehensive
Analyzed using molecular weight, monose composition and the means such as amino acid composition analysis, infrared spectrum analysis and methylation analysis, it is entered
Go the confirmation of chemical constitution feature, specify that its weight average molecular weight, monose composition and ratio, amino acid composition and ratio, with
And the connected mode of contained saccharide residue.The locust tree microellobosporia polysaccharide component that such a design feature is apparent, molecular weight is homogeneous, is to grind
The preferable molecular model of Huai Er active ingredient and its bioactivity is studied carefully, to illustrate the immunological regulation of Huai Er and antitumor
Active ingredient and its mechanism of action lay a good foundation, the present invention will further be developed and related preparations for locust tree microellobosporia
Quality control provides scientific basis.
Brief description of the drawings
Hereinafter, embodiment of the present invention is described in detail with reference to accompanying drawing, wherein：
Fig. 1 is extractive of locust tree microellobosporia trametes robiniophila classification alcohol precipitation flow chart；
Fig. 2 is that the ethanol precipitation position TCP-80 column chromatographys of extractive of locust tree microellobosporia trametes robiniophila 80% are pure
Change flow chart；
Fig. 3 is Huai Er albumen TP-4 molecular weight（Mw）Bioassay standard curve；
Fig. 4 is the chromatography of ions figure of reference substance solution during Huai Er albumen TP-4 monosaccharide components are analyzed；
Fig. 5 is Huai Er albumen TP-4 ultra high efficiency liquid phase-gel chromatography（UPLC-GPC）Collection of illustrative plates；
Fig. 6 is Huai Er albumen TP-4 monosaccharide composition analysis chromatography of ions figure；
Fig. 7 is that 3H-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：The thick total floating preteins part of trametes robinioplila；
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；
Fig. 9 is mouse and people T, the change of bone-marrow-derived lymphocyte ratio after Huai Er is stimulated, wherein, figure A is that mouse T lymphs are thin
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 T, bone-marrow-derived lymphocyte surface C D69 expression after Huai Er is stimulated, and figure A is that T drenches after Huai Er is stimulated
Bar cell surface CD69 expression, figure B is the expression of bone-marrow-derived lymphocyte surface C D69 after Huai Er is stimulated, 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；
Figure 11 is Turnover of Mouse Peritoneal Macrophages NO releases change, wherein Dex after Huai Er is stimulated：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：The thick total floating preteins part of trametes robinioplila；LPS：Fat
Polysaccharide；
Figure 12 Huai Ers albumen is to the inhibitory action of Tumor angiogenesis endothelial cell proliferation, wherein TP-4：Trametes robinioplila is more
Glycoprotein -4.
Embodiment
The present invention is described in detail below by embodiment, it will be appreciated that following embodiments are merely to illustrate the present invention, without
The scope of the present invention is limited in any way.
Embodiment 1：Huai Er albumen（TP-4）Isolation and purification method
Accurately weigh extractive of locust tree microellobosporia trametes robiniophila（There is provided by Qidong Gaitianli Pharmaceutical Co., Ltd.）300g, is added
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,
Repeat 5 times, carbohydrate fraction is collected respectively（TCP）And the thick total floating preteins part of trametes robinioplila（TFP）, the thick total floating preteins of trametes robinioplila
Partial vacuum drying for standby, locust tree microellobosporia glucide part（TCP）After the chloroform and n-butanol for the removal remaining that is concentrated under reduced pressure,
Carry out classification alcohol precipitation.It is 40% to be slowly added into absolute ethyl alcohol into TCP first and become alcohol volumetric concentration（Volume）It is sugared molten
Liquid, after 4 DEG C stand 24 hours, 3000 revs/min centrifuge 10 minutes, and precipitation numbering is TCP-40, are dried under reduced pressure in case further
Isolate and purify, become containing alcohol 60% to the continuous appropriate absolute ethyl alcohol of addition of supernatant relaying afterwards（Volume）Sugar juice, 4 DEG C
After standing 24 hours, 3000 revs/min, centrifuge 10 minutes, precipitation numbering is TCP-60, is 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, and precipitation numbering is TCP-80, are dried under reduced pressure in case further isolating and purifying, specific point
Level alcohol precipitation flow chart is shown in Fig. 1.
Weigh appropriate trametes robiniophila 40%（Volume）Ethanol precipitation position TCP-40, is dissolved in 150ml distilled water
In, filtering is concentrated under reduced pressure into outside part and bag outer portion in volume 50ml, dialysis collecting bag, bag and is divided into trametes robinioplila low molecule amount
Component -1（TO-1）, bag is interior partly to be separated by DEAE-52 ion exchange column chromatographies, and distilled water and 0.1M NaCl is respectively adopted
3.0L is collected at solution, 0.5M NaCl elutions, each position, and water elution part is directly thickened to do, and sodium chloride elution fraction exists
Dialysis desalination is carried out after concentration, dialysis procedure is that flowing water is dialysed three days, and distilled water is dialysed two days.Then each section is respectively adopted
Sepharose CL-6B agarose Gel columns are isolated and purified, untill high performance liquid chromatography is detected as sterling, are passed through
Above-mentioned pillar layer separation process, is obtained from TCP-40 ion exchange column chromatography 0.1Mol/L NaCl elutions position purifying respectively
UPLC（Ultra performance liquid chromatography）It is verified as the GL-PP TP-1 of homogeneous components（397mg）And washed from 0.5Mol/L NaCl
De- position obtains homogeneous polysaccharide albumen TP-2（1212mg）, separation process figure is shown in Fig. 2.
Weigh appropriate trametes robiniophila 60%（Volume）Ethanol precipitation position TCP-60, is dissolved in 150ml distilled water
In, filtering is divided into trametes robinioplila low molecule through being concentrated under reduced pressure into outside part and bag outer portion in volume 50ml, dialysis collecting bag, bag
Measure component -2（TO-2）, bag is interior partly to be separated by DEAE-52 ion exchange column chromatographies, and distilled water and 1.0Mol/ is respectively adopted
LNaCl solution is eluted, and 3.0L is collected at each position, and water elution part is directly thickened to do, and sodium chloride elution fraction is in concentration
Dialysis desalination is carried out afterwards, and dialysis procedure is that flowing water is dialysed three days, and distilled water is dialysed two days.Then each section is respectively adopted
Sepharose CL-6B agarose Gel columns are isolated and purified, untill high performance liquid chromatography is detected as sterling, are passed through
Above-mentioned pillar layer separation process, is obtained 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 GL-PP TP-3 of homogeneous components（12.1g）.
Weigh appropriate trametes robiniophila 80%（Volume）Ethanol precipitation position TCP-80, is dissolved in 150ml distilled water
In, filtering is concentrated under reduced pressure into outside part and bag outer portion in volume 50ml, dialysis collecting bag, bag and is divided into trametes robinioplila low molecule amount
Component -3（TO-3）, bag is interior partly to be 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, and sodium chloride elution fraction enters after concentration
Row dialysis desalination, dialysis procedure is that flowing water is dialysed three days, and distilled water is dialysed two days.Then each section is respectively adopted Sepharose
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 GL-PP 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. 2).
Embodiment 2：Huai Er albumen（TP-4）The research of chemical constitution
The present embodiment have studied the Huai Er albumen TP-4 of the preparation of embodiment 1 chemical constitution.
First, Huai Er albumen（TP-4）Chemical constitution research method
1st, purity and the measure of molecular weight
Ultra high efficiency liquid phase-gel chromatography-EISD（UPLC-GPC-ELSD）Instrument configuration and chromatostrip
Part：It is prepared by U.S. Waters UPLC, TSK-3000GPC chromatographic columns, automatic sampler, Millipore ultra-pure waters ion-exchanger
High purity water（0.45 μm of cellulose acetate membrane filtration）；Flow velocity 0.3ml/min.
The preparation of standard curve：Appropriate dextran standard items are weighed respectively, are added deionized water, are configured to dense
0.5mg/ml reference substance solution is spent, UPLC detections is carried out one by one afterwards, as a result sees Fig. 3.
It is prepared by sample solution：GL-PP TP-4 prepared by a certain amount of embodiment 1 is weighed respectively, adds appropriate deionization
Water, is configured to the solution that concentration is 1mg/ml, Millipore0.22 μm of water system membrane filtration, sample detection.
2nd, monosaccharide composition analysis
Complete sour water solution：The GL-PP TP-4 (10mg) that precision weighs the preparation of embodiment 1 is put into heavy wall pressure bottle, plus
Enter 4ml 2Mol/L trifluoroacetic acids（TFA）, inflated with nitrogen, after tube sealing, 100 DEG C hydrolyze 2 hours, evaporated under reduced pressure.
The ion chromatography of sample
Instrument configuration and chromatographic condition：Dionex ICS3000 type chromatography of ions, CarboPac PA20 analytical columns, 150 ×
3mm, S/N002823, CarboPac PA20 guard columns, 50*3mm, S/N002652, leacheate composition and flow velocity 1-25min, 1m
Mol/L KOH；25.1-32min, 30mMol/L KOH,；32.1-35min, 1m Mol/L KOH；0.45mL/min, the μ of sample introduction 10
L。
The preparation of reference substance solution：Take arabinose, galactolipin, glucose, xylose, mannose reference substance appropriate, spend
Ionized water is dissolved into the respectively reference substance solution containing 10.0mg/L, shakes up, produces.
It is prepared by standard curve：Precision draws reference substance mixing stock solution in right amount, is diluted to respectively with deionized water
After 0.5mg/L, 1mg/L, 5mg/L, 10mg/L, 15mg/L standard liquid, 0.45 μm of filtering with microporous membrane, ion is carried out successively
Chromatographic determination, chromatography of ions condition is：Dionex ICS3000 type chromatography of ions, CarboPac PA20 analytical columns, 150 ×
3mm, S/N002823, CarboPacPA20 guard column, 50*3mm, S/N002652, leacheate composition and flow velocity 1-25min, 1m
Mol/LKOH；25.1-32min, 30m Mol/L KOH,；32.1-35min, 1m Mol/L KOH；0.45mL/min, the μ of sample introduction 10
L.Using integrating peak areas value as ordinate（Y）, using each standard concentration as abscissa（X）, draw each reference substance standard curve simultaneously
Regression equation is calculated, 1 and Fig. 4 is the results are shown in Table.
The linear relationship of table 1 investigates result
It is prepared by need testing solution：The complete acid hydrolysis products of GL-PP TP-4 prepared by embodiment 1 redissolve to be gone in 50ml
Ionized water, ultrasound makes dissolving in 10 minutes, takes solution appropriate, crosses 0.22 μm of aperture water system filter membrane and the SPEs of DIONEX RP II
Pillar.
It is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, ion chromatograph is injected, is produced.
3rd, methylation analysis
GL-PP exhaustive methylation：The GL-PP TP-4 of the appropriate preparation of embodiment 1 is weighed in reaction bulb, is put into true
5h is dried in empty drying box（50℃）, the DMSO2ml treated through over-molecular sieve is added, sonic oscillation 5 minutes treats that sample is complete
After dissolving, addition grinds to form powdery NaOH20mg, while catching up with air in most bottle with nitrogen, ultrasound 10 minutes, stand 90 at room temperature
Minute, after the DMSO frosts completely in question response bottle, 0.1ml iodomethane is added dropwise（This process takes around 15~20 points
Clock）, simultaneous reactions thing can slowly thaw, and gradually clarify, until as glassy yellow.Then ultrasound 10 minutes, stand 30 minutes.
Reduced pressure at room temperature distills, and removes most excessive iodomethane, is dialysed one day with water, decompression steaming to 2ml or so.Then at drying after freeze-drying
Dry 5 hours, repeated after aforesaid operations 2 times in device, take a small amount of sample to carry out infrared detection, if infrared spectrum Central Plains
3300cm-1The strong and wide hydroxyl peak in place disappears, and 2900cm-1Place methyl peak is significantly increased, and shows the permethylated reaction of sample
Complete.
It is prepared by partial methylation ALDI alcohol acetonyl ester：The sample of exhaustive methylation is dissolved in 3mL90%（Volume）'s
In formic acid solution, tube sealing, depolymerization 6h at 100 DEG C is added to be concentrated under reduced pressure at 2~3mL methanol, 40 DEG C into reaction bulb and is evaporated, weight
Three times are operated more than multiple to eliminate excessive formic acid, 2Mol/L TFA solution 4mL are then added into the sample after depolymerization, after sealing
In hydrolyzing the solution in 2h, reaction bulb at 110 DEG C in evaporated under reduced pressure at 40 DEG C, 2~3mL methanol is added, is evaporated, be more than repetition
Operate repeatedly to eliminate excessive TFA.After sample after hydrolysis is dissolved with 3~4mL distilled water, about 20mg NaBH are added4In room
The lower reduction 3h of temperature, then adjusts pH value to 5 or so with glacial acetic acid, adds after 1~2mL methanol and a drop glacial acetic acid, then evaporated under reduced pressure,
Aforesaid operations are repeated repeatedly to eliminate excessive acetic acid.Sample through above-mentioned processing is placed in P2O5It is dried under reduced pressure in vacuum desiccator
One day, after 110 DEG C of dry 10~15min, 3mL aceticanhydrides are added, 110 DEG C of reaction 1h add 2mL toluene into reaction solution,
Decompression boils off unreacted aceticanhydride at 40 DEG C after vibration, so repeatedly to eliminate aceticanhydride.Then by the sample after acetylation
It is dissolved in chloroform, adds isometric distillation water washing chloroform layer 3 times, eliminate water layer, chloroform layer adds anhydrous sodium sulfate and done
Dry 10min, filtering, chloroformic solution reduced pressure at room temperature is concentrated into after 0.1mL or so, carries out Gc-mss（GC-MS）.Makings
Condition be：50 DEG C of initial temperature, heating schedule is 40 DEG C/min, to 215 DEG C, holding 40min, 250 DEG C of detector temperature,
DB-5 Capillary GC-MS chromatogram post detections.
4th, amino acid analysis：
The GL-PP TP-4 for weighing the appropriate preparation of embodiment 1 is put into test tube, adds 4ml 6Mol/L HCl 100
Hydrolyzed 6 hours at DEG C, boil off HCl, centrifuged, filtering, filtrate is analyzed with amino-acid analyzer.
5th, IR spectrum analyses：
The GL-PP TP-4 of the preparation of 1mg embodiments 1 is weighed, is dried in vacuum overnight, secondary daily pellet technique is carried out
IR is detected.
2nd, Huai Er albumen（TP-4）Chemical constitution result of study
The Huai Er albumen prepared according to the research method of Part I to embodiment 1（TP-4）Chemical constitution carry out
Research, result of study is as follows：
1st, purity and molecular weight
TP-4 is dark brown powder shape material, soluble in water, DMSO, and methanol, ethanol insoluble in high concentration etc. are organic molten
Agent, the UPLC-GPC-ESLD collection of illustrative plates of the GL-PP（See Fig. 5）A symmetrical narrow chromatographic peak is presented, it is one to point out it
Pure GL-PP material, is contrasted with molecular weight standards, and the molecular weight of the GL-PP is 1.41 × 106Da（See Fig. 3）,
Lowry reactions are positive, and have weak absorption at UV scanning 280nm, show that the material contains albumen.
2nd, monosaccharide composition analysis
After the complete sour water solutions of TP-4, a hydrolysate part has directly carried out ion chromatography, with standard monose pair
According to analysis result shows that the polysaccharide is made up of arabinose, galactolipin, glucose, xylose and mannose, does not have in detection process
It is found the chromatographic peak related to uronic acid and nitrogen acetylamino sugars to show, it is a neutral polysaccharide to point out it（Table 2, is shown in figure
6）.
The Huai Er albumen TP-4 monose of table 2 is constituted and ratio（Mass ratio）Ion chromatography result
By monosaccharide composition analysis as can be seen that Huai Er albumen TP-4 contains 5 kinds of monose, wherein with arabinose,
The content highest of xylose, is secondly glucose, galactolipin and mannose content are less.
3rd, methylation analysis
In order to confirm the saccharide residue connected mode in TP-4, TP-4 is methylated using Needs methods, in three first
After base, by its with 90% formic acid depolymerization, with the complete sour water solutions of 2Mol/LTFA, NaBH4It is prepared by reduction and aceticanhydride acetylation
Into ALDI alcohol acetic ester derivative, GC-MS analyses are carried out, 3 are the results are shown in Table.
Table 3TP-4 methylation analysis results
Methylation analysis shows that the structure of Huai Er -4 is extremely complex.Saccharide residue containing 8 kinds of different connections, wherein,
Arabinose is with 1,1,2 and 1, and 5 hydroxyls are interconnected with other saccharide residues formation glycosidic bond, and xylose is with 1,1,3
Hydroxyl is interconnected with other saccharide residues formation glycosidic bond, and glucose is mutual with Isosorbide-5-Nitrae-position hydroxyl and other saccharide residues formation glycosidic bond
Be connected, galactolipin with 1,6 hydroxyl and other saccharide residues formation glycosidic bond interconnect, mannose with 1,3,6 hydroxyl with
Other saccharide residues formation glycosidic bond is interconnected.
In order to further confirm that TP-4 chain structures, Partial acid hydrolysis has been carried out to it（0.3Mol/LTFA, 8 hours）, by water
Solution product is dialysed, and after freeze-drying, is obtained TP-4 catabolite, is named as TP-4-in, the same with TP-4,
Monosaccharide composition analysis and methylation analysis are carried out to TP-4-in, as a result such as table 4.
Table 4TP-4-in methylation analysis results
TP-4-in arabinose and being decreased obviously for xylose signal compared with TP-4 point out both sugar to be distributed in
TP-4 side chain, and the relative scale of Isosorbide-5-Nitrae connection glucose residue and other saccharide residues rises, and points out Isosorbide-5-Nitrae connection glucose residual
Base is constitutes the saccharide residue of TP-4 main chains, and 1,3,6 connection mannose residue almost disappears, the appearance of 1,6 connection mannose residues
Also 1,6 connection mannose residues are pointed out to constitute the saccharide residue of TP-4 main chains, TP-4 main chain connects mannose residue 1,3,6
O-3 positions formed branch.Note：Substantial amounts of non-reducing end saccharide residue comes off relevant with methylation analysis reaction process saccharide residue.
Comprehensive analysis TP-4 and TP-4-in methylation analysis results are can be found that, it may be determined that Huai Er albumen TP-
4 be the extremely complex GL-PP of a chemical constitution, and it is mainly by arabinose, xylose, galactolipin, glucose, mannose
The main mode with 1,1,2-, 1,5- connection of composition, wherein arabinose is present, and xylose is mainly deposited in the mode of 1,1,3- connection
, galactolipin exists in the mode of 1,6- connections, and glucose exists in the way of Isosorbide-5-Nitrae-connection, mannose with 1,3,6- connection
Mode is present.
4th, amino acid analysis：
In order to analyze the composition of the amino acid in Huai Er albumen TP-4 and ratio, using hydrochloric acid water solution combination, Hitachi is automatic
Amino-acid analyzer, amino acid composition and proportion grading have been carried out to it, 5 are the results are shown in Table.
The Huai Er albumen TP-4 amino acid composition analysis results of table 5
5th, IR spectrum analyses：
TP-4 infrared spectrum is in 1735cm-1Without absorption, TP-4 is pointed out to be free of uronic acid.
The trametes robiniophila of embodiment 3 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 molecule amount group
Divide TO-1, TO-2, TO-3, TO-4；The thick total floating preteins part TFP of trametes robinioplila）And negative control medicine glucan（Dextran）With
Mouse boosting cell is co-cultured, 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 see, 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 can not 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, is incubated altogether after 48h with Huai Er,3H-TdR results show
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, after 24 hours, flow cytometer detection T, B cell ratio
Example.As a result Fig. 9 is seen.
Conclusion：Huai Er TP-1, TP-2, TP-3, TP-4 are stimulated after mouse boosting cell or human peripheral PBMC, with compareing
Medicine glucan（Dextran）Compare, B cell ratio is substantially raised, and T cell ratio is without significant change.
4）Huai Er is stimulated after mouse boosting cell, T, CD69 expression 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）Co-cultured with mouse boosting cell, after 6 hours, flow cytometer detection T, B cell surface C D69
Expression.As a result Figure 10 is seen.
Conclusion：Huai Er is stimulated after mouse boosting cell 6h, and B cell starts high expression CD69, and T cell is without obvious change
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,
The thick total floating preteins part TFP of TP-4, lower-molecular-weight component TO-1, TO-2, TO-3, TO-4, trametes robinioplila is with compareing polysaccharide glucan
（Dextran）, lipopolysaccharides（LPS）Co-culture, after 48 hours, use Griess reagent box（Griess Reagent kit）Detection is thin
NO content, is as a result shown in Figure 11 in born of the same parents' culture supernatant.
Conclusion：NO testing results show, Huai Er being capable of Stimulated Macrophages secretion NO, and secretory volume and polysaccharide
Concentration is in dosage correlation.NO secretion is then without significant changes after lower-molecular-weight component is stimulated.
Inhibitory action of the Huai Er albumen of embodiment 4 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）.
Testing sample：TP-4：Huai Er protein-4, is made by embodiment 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, 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 with PBS one time, be put in super-clean bench standby.
（3）Prepare 20%FBS, plus dual anti-M199 nutrient solutions.
（4）The vascular endothelial cell for taking the human tumor of recovery to originate, suctions out nutrient solution, is washed once, used after centrifugation with PBS
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）After 12 hours, prepare and contain 1%-5%FBS, 10-100ng/ml bFGF M199 culture mediums.
（7）96 orifice plates are taken out, culture medium is suctioned out with the volley of rifle fire, M199 and medicine containing 1%-5%FBS and bFGF are added, often
100 microlitres of hole, if control group and zeroing group.And add testing sample, respectively TCP, TP-1, TP-2, TP-3 and TP-4.It is each
Sample sets 4 concentration, respectively 10 μ g/L, 100 μ g/L, 500 μ g/L and 1000 μ g/L.Then connect under 37 °C, 5% CO2
It is continuous to be incubated.
（8）After 72 hours, culture medium, plus MTT (concentration 0.5mg/ml, filtration sterilization) are sucked, is incubated 4-6 hours.
（9）MTT is sucked, plus 100 microlitres of DMSO, 570nm survey OD.
2nd, experiment conclusion：
Huai Er protein-4（TP-4）There is significant inhibitory action to the propagation of tumor vascular endothelial cell.As a result see
Figure 12.Each administration group compared with control group relatively after, and Analysis of variance and afterwards T examine.* * P ＜ 0.001, represent two groups of ratios
Relatively there is pole significant difference.
Claims (12)
1. a kind of Huai Er albumen, the monose composition of the GL-PP is arabinose, galactolipin, glucose, xylose and sweet
Dew sugar, its mass ratio is 8.9：1.6：3.4：7.4：1.3；The preparation method of the GL-PP comprises the following steps：
(1) mass concentration is removed into floating preteins using Sevage methods for the 2%-20% extractive of locust tree microellobosporia aqueous solution, received
Collect carbohydrate fraction；
(2) carbohydrate fraction for obtaining step (1) is added in absolute ethyl alcohol, and it is 75%-80%'s to form it into alcohol volumetric concentration
Sugar juice, centrifugation, obtains sediment；
(3) sediment for obtaining step (2) is dissolved in water, filtering, part in concentration, dialysis collecting bag；
(4) part is separated in the bag being collected into using ion exchange column chromatography to step (3), wherein the eluent used is
The 0.85mol/L-1.5mol/L NaCl aqueous solution；
(5) product afforded to step (4) NaCl aqueous solution carries out dialysis desalination；And
(6) product after step (5) dialysis desalination is isolated and purified using Sepharose CL-6B agarose Gel columns.
2. GL-PP according to claim 1, it is characterised in that the weight average molecular weight of the GL-PP is 7.0 ×
105-2.0×106Da。
3. GL-PP according to claim 2, it is characterised in that the weight average molecular weight of the GL-PP is 1.41 ×
106Da。
4. GL-PP 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. GL-PP according to claim 1, it is characterised in that in step (2), the alcohol volume of the sugar juice is dense
Spend for 80%.
6. GL-PP according to claim 1, it is characterised in that in step (4), the eluent is 1.0mol/L
The NaCl aqueous solution.
7. a kind of preparation method of the GL-PP any one of claim 1 to 6, the preparation method includes following step
Suddenly：
(1) mass concentration is removed into floating preteins using Sevage methods for the 2%-20% extractive of locust tree microellobosporia aqueous solution, received
Collect carbohydrate fraction；
(2) carbohydrate fraction for obtaining step (1) is added in absolute ethyl alcohol, and it is 75%-80%'s to form it into alcohol volumetric concentration
Solution, centrifugation, obtains sediment；
(3) sediment for obtaining step (2) is dissolved in water, filtering, part in concentration, dialysis collecting bag；
(4) part is separated in the bag being collected into using ion exchange column chromatography to step (3), wherein the eluent used is
The 0.85mol/L-1.5mol/L NaCl aqueous solution；
(5) product afforded to step (4) NaCl aqueous solution carries out dialysis desalination；And
(6) product after step (5) dialysis desalination is isolated and purified using Sepharose CL-6B agarose Gel columns.
8. preparation method according to claim 7, it is characterised in that in step (1), the extractive of locust tree microellobosporia water
The mass concentration of solution is 8%.
9. preparation method according to claim 7, it is characterised in that in step (2), the alcohol volume of the sugar juice is dense
Spend for 80%.
10. preparation method according to claim 7, it is characterised in that in step (4), the eluent is 1.0mol/
The L NaCl aqueous solution.
11. preparation method according to claim 7, it is characterised in that the preparation method comprises the following steps：
(1) accurate to weigh extractive of locust tree microellobosporia trametes robiniophila 300g, the appropriate distilled water of addition is diluted to mass concentration and is
8% weak solution, removes floating preteins using Sevage methods afterwards, repeats 5 times, collects carbohydrate fraction；
(2) absolute ethyl alcohol is slowly added into the locust tree microellobosporia carbohydrate fraction obtained to step (1) becomes alcohol volumetric concentration and be
80% sugar juice, after 4 DEG C stand 24 hours, with 3000 revs/min of centrifugation 10 minutes, precipitation obtains sediment；
(3) sediment that appropriate step (2) is obtained is weighed, is dissolved in 150ml distilled water, filters, is concentrated under reduced pressure into body
Product 50ml；Part in dialysis collecting bag, and
(4) part is separated in the bag being collected into using DEAE-52 ion exchange column chromatographies to step (3), using 1.0mol/
L NaCl solutions are eluted, and elution fraction carries out dialysis desalination after concentration, and dialysis procedure is that flowing water is dialysed three days, 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.
12. purposes of the GL-PP in the medicine for preparing treatment tumour any one of claim 1 to 6.
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