CN109400749A - A kind of carboxymethylpachymaran CMP44 and the preparation method and application thereof - Google Patents

A kind of carboxymethylpachymaran CMP44 and the preparation method and application thereof Download PDF

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CN109400749A
CN109400749A CN201811015738.XA CN201811015738A CN109400749A CN 109400749 A CN109400749 A CN 109400749A CN 201811015738 A CN201811015738 A CN 201811015738A CN 109400749 A CN109400749 A CN 109400749A
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cmp44
carboxymethylpachymaran
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张学武
刘晓菲
徐晓飞
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South China University of Technology SCUT
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Abstract

The invention discloses a kind of carboxymethylpachymaran CMP44 and the preparation method and application thereof.The preparation method is combined using DEAE-52 cellulose chromatographic column, Sephadex-G200+Sephadex-G150 chromatographic column, is isolated and purified to existing carboxymethyl Poria cocos Thick many candies, is obtained CMP44 using 0.2mol/L NaCl solution as eluent.Carboxymethylpachymaran CMP44 produced by the present invention has anti-oxidant, antitumor, anti-inflammatory and immunoregulatory activity, can be applied to functional food and field of biological pharmacy.

Description

A kind of carboxymethylpachymaran CMP44 and the preparation method and application thereof
Technical field
The invention belongs to functional food and field of biological pharmacy, and in particular to a kind of carboxymethylpachymaran CMP44 and its Preparation method and application.
Background technique
Poria cocos is the dry sclerotia of polyporaceae fungus Poria cocos (Schw.) Wolf, is recorded in " middle traditional Chinese medicines Allusion quotation ", it ranks among Chinese medicine " four monarch's eight delicacies ", also known as beautiful spirit, Fu spirit, Song Ling, Fu Tu etc. have very high tonic and medicinal valence Value has clearing damp diuresis, strengthening the spleen and stomach, calming heart and tranquilizing mind and other effects, is a kind of traditional Chinese medicine of dual-purpose of drug and food.Pachymaran For the principle active component of Poria cocos, 80% of poria cocos sclerotium or more is accounted for.Water solubility can be obtained through carboxymethyl-modification in pachymaran Carboxymethylpachymaran has the effects that antitumor, anti-inflammatory, Immune-enhancing effect.Currently, for pachymaran carboxymethyl-modification, Purify and evaluate its bioactivity, and analyze the systematic researches such as its structure-activity relationship work it is still at an early stage, especially The research of the anti-inflammatory effect of purifying carboxymethylpachymaran is rarely reported.
Summary of the invention
The present invention utilizes ion exchange column and sephadex chromatography column using carboxymethyl Poria cocos Thick many candies as raw material Polysaccharide is isolated and purified, and Structural Identification has been carried out to purified components CMP44, demonstrates its antioxidation activity in vitro, antitumor work Property and extracorporeal anti-inflammatory activity.The object of the present invention is to provide one kind to have anti-oxidant, antitumor, anti-inflammatory and immunological regulation living The carboxymethylpachymaran CMP44 and the preparation method and application thereof of property.
The carboxymethylpachymaran that the present invention uses can with have disclosed Chinese patent application (application number: 201510905843.0) any preparation method recorded is made, such as may comprise steps of: (1) prepare Poria cocos lye: Poria cocos is crushed, is soaked in water, sodium hydroxide or potassium hydroxide is then added, Poria cocos lye is obtained after Poria cocos has been dissolved；(2) Decoloration, substitution reaction: hydrogen peroxide is slowly added in Poria cocos lye, is then added into the Poria cocos lye after the completion of decoloration solid Body shape monoxone carries out carboxymethyl substitution reaction, obtains carboxymethylpachymaran lye；(3) it neutralizes, alcohol precipitation: by ethyl alcohol and ice vinegar Acid-mixed is closed, and obtains acetate ethanol solution, the carboxymethylpachymaran lye that step (2) obtains is mixed with acetate ethanol solution, Precipitating is collected by filtration in carboxymethylpachymaran Precipitation, and by precipitating ethanol washing, to be dried to obtain carboxymethyl Poria cocos more Sugar.If there is the parameter of not special detailed description, it is referred to this application document.
The purpose of the present invention is realized at least through one of following technical solution.
A kind of preparation method of carboxymethylpachymaran CMP44, the polysaccharide be using DEAE-52 cellulose chromatographic column, The combination of Sephadex-G200+Sephadex-G150 chromatographic column, isolates and purifies existing carboxymethyl Poria cocos Thick many candies, with 0.2mol/L NaCl solution is that eluent obtains carboxymethylpachymaran CMP44.
Further, the preparation method of the carboxymethylpachymaran CMP44 includes: that carboxymethyl Poria cocos Thick many candies are dissolved in Upper DEAE-52 cellulose ion-exchange chromatography column purification, is eluted, phend-sulphuric acid is tracked with NaCl solution in ultrapure water Eluting peak is collected in detection, and vacuum concentration is freeze-dried after dialysis, and carboxymethylpachymaran CMP3 is made.CMP3 is dissolved in again Upper Saphadex-G200 and Saphadex-G150 chromatographic column is further purified in ultrapure water, NaCl solution elution, and vacuum is dense Contracting, is freeze-dried after dialysis, and carboxymethylpachymaran CMP44 is made.
Further, the existing carboxymethyl Poria cocos Thick many candies are as disclosed in 105348407 A of Chinese patent literature CN The preparation process of carboxymethylpachymaran is made.
Further, the monosaccharide group of the polysaccharide CMP44 becomes D-Glucose, and molecular weight is 20.96 × 104Da, structure For (1 → 3)-callose, contain (1 → 6) and (1 → 2) glycosidic bond, there is triple-helix structure.
The carboxymethylpachymaran CMP44 as made from a kind of preparation method of carboxymethylpachymaran CMP44.Institute Polysaccharide CMP44 is stated with antioxidation activity in vitro, CMP44 is respectively 4.85mg/ to the EC50 value of DPPH, ABTS+, OH mL、4.83mg/mL、2.94mg/mL。
Further, the polysaccharide CMP44 in 31.25-1000 μ g/mL concentration range to colon cancer cell HT-29, HepG-2 cell, breast cancer cell MCF-7, stomach cancer cell SGC-7901 and lung cell A549 cell Proliferation embody Inhibiting effect out, corresponding IC50 value are respectively 140.5,264.3,256.4,102.5,313.2 μ g/mL.
Further, the mouse monokaryon macrophage that the polysaccharide CMP44 can inhibit lipopolysaccharides (LPS) to induce RAW264.7 discharges nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF- α) and Interleukin -1β (IL- 1 β), in 31.25-1000 μ g/mL concentration range, to the inhibiting rate of NO, IL-6 of LPS induction, TNF-α and IL-1 β generation Difference 46.87%, 44.33%, 79.48% and 34.72% has anti-inflammatory activity.
Further, RAW264.7 cell can be activated to secrete NO, IL-6, TNF-α when the polysaccharide CMP44 is induced without LPS It and in IL-1 β, 31.25-1000 μ g/mL concentration range is 3.17 times of control group, 13.37 respectively to its secretory volume maximum value Again, 2.34 times and 8.50 times, there is immunoregulation effect.
The polysaccharide CMP44 provided by the invention can be applied in preparing functional food and bio-pharmaceuticals, polysaccharide CMP44 With anti-oxidant, antitumor, anti-inflammatory and immunoregulatory activity.
Compared with prior art, the invention has the advantages that and technical effect:
The present invention is in addition to using DEAE-52 cellulose chromatographic column, it is often more important that has used Sephadex-G200+ Two kinds of chromatographic column combinations of Sephadex-G150, isolate and purify carboxymethyl Poria cocos Thick many candies, molten with 0.2mol/L NaCl Liquid is that eluent obtains CMP44.Experiment show CMP44 produced by the present invention be it is a kind of have it is anti-oxidant, antitumor, anti-inflammatory and The polysaccharide of immunoregulatory activity.
Detailed description of the invention
Fig. 1 is DEAE-52 cellulose chromatographic column elution curve (abscissa is pipe number, and ordinate is absorbance value).
Fig. 2 is Sephadex-G200 and Sephadex-G150 sephadex chromatography column elution curve.
The HPGPC chromatogram that Fig. 3 is CMP44 (abscissa is time min, and ordinate is voltage mv).
Fig. 4 is CMP44 infrared spectrogram (abscissa is wave number, and ordinate is transmitance).
Fig. 5 is that the GC of hybrid standard product (A) schemes.
Fig. 6 a, Fig. 6 b are respectively hybrid standard product (A) and the Smith catabolite GC figure of CMP44 (B).
Fig. 7 a, Fig. 7 b are respectively 1H (A) and 13C (B) the NMR figure of CMP44 (abscissa is chemical shift).
Fig. 8 is that the maximum absorption wavelength of CMP44 and Congo red mixed liquor changes comparison diagram (abscissa under different alkali concentrations For the concentration of NaOH, ordinate is maximum absorption wavelength).
Fig. 9 a, Fig. 9 b are that (abscissa is dense to influence (x ± s, the n=5) comparison diagram of CMP44 to RAW264.7 cell Proliferation Degree, ordinate are the relative activity of RAW264.7 cell).
Figure 10 a, Figure 10 b are influence (x ± s, n=5) comparison diagram (abscissa that CMP44 discharges NO to RAW264.7 cell For concentration, ordinate is the concentration of NO).
Specific embodiment
Below in conjunction with specific example, the invention will be further described, but implementation and protection scope of the invention is not limited to This.If being that those skilled in the art can refer to now it is noted that having the process or parameter of not special detailed description below Have technology understand or realize.
The purifying of carboxymethylpachymaran
Carboxymethyl Poria cocos Thick many candies 100-300mg is dissolved in 1-10mL ultrapure water upper DEAE-52 cellulose ion switching layer Analyse column purification, with 0.2mol/L NaCl solution elute, flow velocity 0.45mL/min, 12min/ pipe collect, phend-sulphuric acid into Eluting peak is collected in line trace detection, and vacuum concentration is freeze-dried after dialysis, and carboxymethylpachymaran CMP3 is made.Again will CMP350-150mg is dissolved in 1-5mL ultrapure water upper Saphadex-G200 and Saphadex-G150 chromatographic column and is further purified, The elution of 0.2mol/L NaCl solution, flow velocity 0.3mL/min, 10min/ pipe are collected, and vacuum concentration is freeze-dried after dialysis, Carboxymethylpachymaran CMP44 is made.
Carboxymethylpachymaran Structural Identification
Purity testing:CMP44 sample 10mg is dissolved in 100mL distilled water, takes 1.0mL prepare liquid in 25 mL colorimetric cylinders, mends Add distilled water to 2.00mL, adds 5% (mass concentration) phenol solution 1mL, mixing is rapidly added the dense sulphur of 5.00mL after shaking up Acid, boiling water bath 30min stand cooling, and using the pipe that glucose standards solution is not added as blank, absorbance is surveyed at 490nm wavelength Value.Prepare liquid does five in parallel.Polyoses content in sample is calculated according to glucose standard curve.
Molecular weight determination:GPC chromatographic condition: gel permeation chromatograph (Water Breeze GPC), chromatographic column TOSOH The TSK-GEL G-5000PWXL column (7.8mm × 300mm) and TSK-GEL G-3000PWXL column of company (7.8mm × 300mm) series connection；35 DEG C of column temperature；Mobile phase 0.02mol/L KH2PO4 solution, pH 6.0；Flow velocity 0.6mL/min. 2414 type differential refraction detector of detector.
GPC calibration curve equation: dextran standard series is made into the standard of 1.0mg/mL concentration with mobile phase respectively Solution, 20 μ L of sample volume acquire GPC chromatogram.
Ultraviolet spectral analysisCMP44 sample 1mg is weighed, is dissolved with distilled water, the polysaccharide that concentration is 1mg/mL is configured to Solution is control with distilled water, is scanned analysis in 200~400nm wave-length coverage.
Infrared spectrum analysisCMP44 sample 2mg is weighed, addition has dried KBr powder in right amount, ground uniform tabletting, FT- IR 400~4000cm-1 of instrument is scanned analysis in section.
Monosaccharide composition analysisGC chromatographic condition: chromatograph (Aglient 6890N gas chromatograph)；Chromatographic column DB- 1701 capillary columns (0.25 μm of the μ m of 30.0m × 320) are washed needle 3 times with pyridine before sample introduction, and acetone is washed needle 3 times；Injection port temperature 250 DEG C of degree, split ratio 20:1；Carrier gas is nitrogen, flow velocity 40mL/min；Hydrogen flow rate is 25mL/min, and air velocity is 450mL/min.Using temperature programming: 180 DEG C of initial temperature, keeping constant temperature 2min, be then warming up to 220 with 2 DEG C/min speed DEG C, it keeps constant temperature 1min, then 7 DEG C/min to be warming up to 250 DEG C, keeps constant temperature 1min；Detector is fid detector, and temperature is 250℃；1.0 μ L of sample volume.
Sample hydrolysis: CMP44 sample 10mg is weighed in ampoule bottle, and trifluoroacetic acid (TFA) solution of 2mol/L is added 4mL, alcohol blast burner sealing, which is placed at 110 DEG C, hydrolyzes 8h.Be added proper amount of methanol after hydrolyzate is cooled to room temperature, 45 DEG C with Lower reduced pressure is evaporated, and repeats 3 to 5 times to completely remove TFA.
Monosaccharide derivatization: 0.5mL pyridine and 10mg hydroxylamine hydrochloride are added into hydrolysate, heats and shakes in 90 DEG C of water-baths Reaction 30min is swung, taking-up is cooled to room temperature, and adds 0.5mL acetic anhydride, and the reaction was continued in 90 DEG C of water-baths, and 30min carries out acetyl Change；Monosaccharide standard is performed the derivatization using same operation as control.By above-mentioned monosaccharide gas phase derivative through 0.45 μm of micropore According to GC conditions, upper machine analysis after membrane filtration.
Periodate oxidation and Smith degradation analysisPeriodate oxidation: CMP44 sample 20mg is weighed, 15mmol/ is dissolved in The NaIO4 solution of L.Solution is placed in dark at room temperature reaction, interval 6h sampling, with ultraviolet specrophotometer at 223nm after mixing Absorbance value is measured, until 1.5 mL ethylene glycol are added when its is constant into solution to terminate reaction.It is bent according to sodium metaperiodate standard Line computation goes out periodic acid consumption.And 20min will be placed containing ethylene glycol solution, it takes 2mL solution in conical flask, adds phenolphthalein Indicator, NaOH standard solution are titrated, and formic acid production quantity is calculated.Remainder is used for Smith degradation reaction.
Smith degradation: ethylene glycol treated solution is dialysed with bag filter, and flowing water, purified water are respectively for 24 hours.Add after concentration Enter sodium borohydride reduction, overnight (as 24 hours).Then 50% acetic acid tune pH value neutralizes remaining sodium borohydride, flowing water to 6~7 And purified water is respectively dialysed for 24 hours.It is concentrated to dryness, 2mol/L TFA is added, 110 DEG C of tube sealings hydrolyze 8h, machine on GC is carried out after acetylation Analysis, chromatographic condition is the same as above-mentioned GC chromatographic condition.Glucose, glycerine and erythrite perform the derivatization work using same operation For control.
Nuclear magnetic resonance spectroscopy: CMP44 sample 15mg is dissolved in 0.5mL heavy water, is transferred in nuclear magnetic tube after dissolution, in Hydrogen spectrum (1H-NMR), carbon spectrum (13C-NMR), hydrocarbon relationship spectrum (HSQC, HMBC) and hydrogen hydrogen are carried out in 600MHz Nuclear Magnetic Resonance Relationship composes (H-H COSY) analysis, is analyzed using MestReNova-11.0.2 software map.
Triple-helix structure analysis: CMP44 sample 2mg is weighed, distilled water is added and 100 μm of Congo red reagents of ol/L are each 2mL, then be gradually added into 4mol/L NaOH solution, makes NaOH concentration in solution be gradually increased to 0.5mol/L, then 400~ It is scanned in 600nm wave-length coverage, measures maximum absorption wavelength under different NaOH concentration.The Congo of polysaccharide sample is not added Red solution is control.NaOH concentration is abscissa, and a length of ordinate of maximum absorption wave draws curve.
Antioxidation activity in vitro experiment
Sample solution is prepared:It is dense that carboxymethylpachymaran CMP44 and ascorbic acid Vc are made into quality with distilled water respectively Degree is the mother liquor of 10mg/mL and 0.5mg/mL, and it is stand-by to be diluted to various concentration gradient solution.
Reducing power measurement: various concentration testing sample solution 1mL is taken, the 0.2mol/L phosphate-buffered of pH 6.6 is added Liquid 2.5mL, 1% potassium ferricyanide solution 2.5mL are uniformly mixed, and after 50 DEG C of water-bath 20min, taking-up is cooling rapidly, then plus Enter 10% solution of trichloroacetic acid 2.5mL, be uniformly mixed, 3000r/min is centrifugated 10min, takes supernatant 2.5mL in test tube In, add distilled water 2.5mL, 0.1% liquor ferri trichloridi 0.5mL, react at room temperature 5min, returned to zero with distilled water, is surveyed in 700nm Fixed its light absorption value A, parallel laboratory test is three times.Using Vc as positive control.
The measurement of DPPH free radical (DPPH) scavenging capacityVarious concentration testing sample solution 1mL is taken, 60 μm of ol/ are added DPPH solution (with the preparation of 50% ethanol solution) 3mL of L, is uniformly mixed, standing reaction 30min is protected from light at room temperature, in 517nm Absorbance value A is measured at wavelength1, parallel laboratory test is three times.Using Vc as positive control.
DPPH free radical scavenging activity (%)=[1- (A1- A2)/A0] × 100%
In formula, A0- distilled water replaces the absorbance value of sample solution；A1The absorbance value of-sample solution； A2- ethyl alcohol Solution replaces the absorbance value of DPPH solution.
ABTS free radical (ABTS+) scavenging capacity measurementABTS working solution: 7mmoL/L ABTS solution and 2.45 mmoL/ It is spare that L K2S2O8 solution (final concentration) is protected from light 12~16h at room temperature.PBS (0.01mol/L, pH7.4) is used using preceding Being diluted to absorbance value under 734nm wavelength is 0.70 ± 0.02.ABTSRadicals scavenging measurement: 0.2mL various concentration it is to be measured Sample solution is mixed with 3.8mL ABTS working solution, is protected from light 6min, absorbance value A1 is measured at 734nm wavelength, in parallel Experiment is three times.Using Vc as positive control.ABTS free radical scavenging activity is calculated by formula (3-2):
ABTS free radical scavenging activity (%)=[1- (A1- A2)/A0] × 100%
In formula, A0- plus sample solution absorbance value；A1The absorbance value of-addition sample solution；A2- with ultrapure Water replaces the absorbance value of ABTS working solution.
The measurement of hydroxyl radical free radical (OH) scavenging capacity9mmoL/L FeSO is sequentially added in reaction system4Solution 2mL and 9mmoL/L salicylic acid solution 2mL adds various concentration testing sample solution 2mL, is eventually adding 8.8mmoL/L hydrogen peroxide The entire reaction of 2mL starting, 37 DEG C of constant temperature water baths react 30min.Using distilled water as reference, each reaction is measured at 510nm wavelength Liquid absorbance value A1, parallel laboratory test is three times.Using Vc as positive control.
Hydroxyl radical free radical clearance rate (%)=[1- (A1- A2)/A0] × 100%
In formula, A0- distilled water replaces the absorbance value of sample solution；A1The absorbance value of-sample solution； A2- distillation Water replaces salicylic background absorbance value.
Ultra-oxygen anion free radical (O2 -) scavenging capacity measurementTake various concentration testing sample solution 1mL, pH 8.2Tris-HCl buffer 4.5mL and distilled water 3.2mL, in 37 DEG C of water-bath 10min, addition is preheated thereafter 25mmol/L pyrogallol solution 0.4mL, oscillation mix, are immediately placed in cuvette and measure its absorbance value at 320nm wavelength A1, every 30s measurement 1 time, total 4min, reaction is finally terminated with the dense HCl solution of 0.5mL, parallel laboratory test is three times.It is the positive with Vc Control.
Ultra-oxygen anion free radical clearance rate (%)=(1- Δ A1/ΔA0) × 100%
In formula, Δ A0- plus sample solution autoxidation rate；ΔA1The autoxidation rate of-addition sample solution.
Anti tumor activity in vitro experiment
Sample solution is preparedCarboxymethylpachymaran CMP44 is respectively with DMEM high glycosyl basal culture medium and McCoy ' s 5A Basal medium is configured to 1000 μ g/mL mother liquors, with 0.22 μm of membrane filtration degerming under gnotobasis, and successively twice of dilution It is stand-by at various concentration gradient solution.
Cell cultureWith the sugared complete medium of DMEM high (percent by volume is pressed, 10% fetal calf serum, 1% penicillin-are contained Streptomysin) culture HepG-2, MCF-7, SGC-7901, A549 cell；McCoy ' s 5A complete medium (contains 10% tire ox blood Clearly, 1% Pen .- Strep) culture HT-29 cell.Logarithmic growth phase cell is accordingly tested.
Cell proliferating determiningUsing MTT colorimetrically analysing CMP44 to the growth inhibition effect of five kinds of cells.Take logarithm raw Long-term 100 μ L of cell is seeded in 96 orifice plates, is placed in incubator and is cultivated 48h.Being separately added into concentration is 31.25-1000 μ The CMP44 sample and positive drug 5 FU 5 fluorouracil (5-Fu) of g/mL, every 200 μ L of hole, setting blank cultures are control, often A sample is all provided with 5 multiple holes, is placed in incubator and continues to cultivate 48h.PBS board-washing, be added 5mg/mL 20 μ L of MTT solution and 180 μ L of corresponding basic culture solution, cultivates 4h in incubator.The culture solution containing MTT is sucked, 150 μ L of DMSO vibration is added 15min is swung, absorbance value is measured at 490nm.
Cell proliferation inhibition rate (%)=(1-ODAdministration/ODControl) × 100%.
Extracorporeal anti-inflammatory activity experiment
Sample solution is preparedCarboxymethylpachymaran CMP44 is configured to the more of 1000 μ g/mL with RPMI-1640 culture medium It is stand-by to be successively diluted to various concentration gradient solution twice with 0.22 μm of membrane filtration degerming under gnotobasis for sugared mother liquor.
Cell cultureWith the RPMI-1640 complete medium culture mouse containing 8% fetal calf serum (56 DEG C of inactivation 30min) Macrophage RAW264.7.Logarithmic growth phase cell is accordingly tested.
Cytotoxicity assay, measurement strong and weak to the toxicity of RAW264.7 cell is handled using MTT colorimetric determination CMP44 Operate same 1.2.4.3.
Experimental groupLogarithmic growth phase RAW264.7 cell, blood cell plate count adjust suspension density about 1 × 105 A/mL, 100 μ L/ pore volumes are inoculated in 96 porocyte culture plates after piping and druming uniformly repeatedly, are placed in 37 DEG C, containing 5%CO2Incubator After interior culture for 24 hours, culture solution is sucked, is administered processing: Control group, 100 μ of cell culture medium according to following experimental group L；LPS group: the 100 μ L of cell culture medium of the final concentration of 1 μ g/mL of LPS；LPS+ polysaccharide sample group: the final concentration of 1 μ g/ containing LPS The 100 μ L of cell culture medium of mL and 31.25-1000 μ g/mL concentration polysaccharide sample；Polysaccharide sample group: 31.25-1000 μ g/mL The 100 μ L of cell culture medium of concentration polysaccharide sample.Every group of sample sets parallel 5 multiple holes.Sealing plate is placed in incubator and is incubated for For 24 hours, supernatant is collected.
The measurement of NO burst sizeRAW264.7 cell NO burst size of the carboxymethylpachymaran CMP44 to LPS stimulation induction Using Griess reagent method[13]Measurement.It takes the 100 μ L of supernatant collected in 96 orifice plates, isometric Griess reagent is added (0.1%1- naphthalene ethylene diamine and the 1% p-aminobenzene sulfonic acid mixed in equal amounts for being dissolved in 5% phosphoric acid), at room temperature oscillating reactions Absorbance value is measured after 10min at 540nm wavelength, according to NO2 -Standard curve (NaNO20~200 μm of ol/L gradient of solution is dense Degree is reacted with Griess reagent, NO2 -Concentration be abscissa, 540nm locate absorbance value be ordinate, drafting curve), calculate The inhibiting rate that the burst size and polysaccharide sample of NO discharges NO in cell supernatant.
Cytokine release measures fixedCarboxymethylpachymaran CMP44 is white to the RAW264.7 cell of LPS stimulation induction Interleukin -6 (IL-6), tumor necrosis factor-alpha (TNF-α) and Interleukin -1β (IL-1 β) burst size use Mouse TNF-α Elisa kit, Mouse IL-6Elisa kit and Mouse IL-1 β Elisa kit measurement, are operated to specifications.
Application Example 1
It is pure that carboxymethyl Poria cocos Thick many candies 180mg is dissolved in 5mL ultrapure water upper DEAE-52 cellulose ion-exchange chromatography column Change (Fig. 1), with 0.2mol/L NaCl solution elute, flow velocity 0.45mL/min, 12min/ pipe collect, phend-sulphuric acid into Eluting peak is collected in line trace detection, and vacuum concentration is freeze-dried after dialysis, and carboxymethylpachymaran CMP3 is made.Again will CMP3 80mg is dissolved in 2mL ultrapure water upper Saphadex-G200 and Saphadex-G150 chromatographic column and (Fig. 2) is further purified, The elution of 0.2mol/L NaCl solution, flow velocity 0.3mL/min, 10min/ pipe are collected, and vacuum concentration is freeze-dried after dialysis, Carboxymethylpachymaran CMP44 is made.Structural Identification the result shows that (Fig. 3-8), CMP44 be a kind of molecular weight be 20.96 × 104(1 → 3)-callose of Da, containing a small amount of (1 → 6) and (1 → 2) glycosidic bond, monosaccharide group becomes D-Glucose, has Triple-helix structure.
Application Example 2
It chooses CMP44 sample (0.2-10mg/mL) and positive control Vc solution (0.005-0.5mg/mL) is resisted in vitro Oxidation experiment.The result shows that CMP44 has reducing power, and to DPPH free radical (DPPH), ABTS free radical (ABTS +), hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2-) show scavenging capacity, be in dose dependent, In be respectively 4.85mg/mL, 4.83mg/mL, 2.94mg/mL to the EC50 value of DPPH, ABTS+, OH.
Application Example 3
Choose the concentration gradient CMP44 sample and positive drug 5 FU 5 fluorouracil (5- that concentration is 31.25-1000 μ g/mL Fu anticancer experiment in vitro) is carried out.The result shows that (table 1-2), CMP44 is in 31.25-1000 μ g/mL concentration range to colon Cancer cell HT-29, HepG-2 cell, breast cancer cell MCF-7, stomach cancer cell SGC-7901 and lung cell A549 Cell Proliferation embodies inhibiting effect, and IC50 value is respectively 140.5,264.3,256.4,102.5,313.2 μ g/mL.
1 CMP44 of table to the inhibitory effects of five kinds of cancer cells (N=5) (%)
25 FU 5 fluorouracil of table (positive control) to the inhibitory effects of five kinds of cancer cells (N=5) (%)
A-f represents the significant difference p between various concentration in table 1 and table 2, according to the descending row of alphabetic order It arranges, is p < 0.05, significant difference between adjacent letters.
Application Example 4
It chooses 31.25-1000 μ g/mL concentration gradient CMP44 sample and carries out extracorporeal anti-inflammatory experiment.The result shows that (see Fig. 9- 10, table 3), CMP44 can significantly inhibit the mouse monokaryon macrophage RAW264.7 release nitric oxide of lipopolysaccharides (LPS) induction (NO), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and Interleukin -1β (IL-1 β), 31.25~1000 μ g/mL In concentration range, 46.87% is respectively reached to NO, IL-6, TNF-α and the IL-1 β of LPS the induction inhibiting rate generated, 44.33%, 79.48% and 34.72%, there is anti-inflammatory activity；CMP44 can activate RAW264.7 cell point when no LPS is induced Secrete in NO, IL-6, TNF-α and IL-1 β, 31.25-1000 μ g/mL concentration range is respectively to its secretory volume maximum value 3.17 times, 13.37 times, 2.34 times and 8.50 times of Control group have immunoregulation effect.
3 CMP44 of table to RAW264.7 cell secretion of cytokines influence (N=5)
In table 3, compared with Control group, #p < 0.05, ##p < 0.01；Compared with LPS group, * p < 0.05, * * P < 0.01.

Claims (9)

1. a kind of preparation method of carboxymethylpachymaran CMP44, it is characterized in that the polysaccharide is to utilize DEAE-52 cellulose layer Column, the combination of Sephadex-G200+Sephadex-G150 chromatographic column are analysed, existing carboxymethyl Poria cocos Thick many candies separate pure Change, obtains carboxymethylpachymaran CMP44 by eluent of 0.2mol/L NaCl solution.
2. a kind of preparation method of carboxymethylpachymaran CMP44 as described in claim 1, it is characterised in that the existing carboxylic The preparation process of methyl Poria cocos Thick many candies carboxymethylpachymaran as disclosed in 105348407 A of Chinese patent literature CN is made.
3. a kind of preparation method of carboxymethylpachymaran CMP44 as described in claim 1, it is characterised in that the polysaccharide The monosaccharide group of CMP44 becomes D-Glucose, and molecular weight is 20.96 × 104Da, structure are (1 → 3)-callose, containing (1 → 6) with (1 → 2) glycosidic bond, there is triple-helix structure.
4. the carboxymethyl Fu as made from a kind of any one of claims 1 to 3 preparation method of carboxymethylpachymaran CMP44 Siberian cocklebur polysaccharide CMP44.
5. a kind of carboxymethylpachymaran CMP44 as claimed in claim 4, it is characterised in that the polysaccharide CMP44 has body Outer antioxidant activity, CMP44 to the EC50 value of DPPH, ABTS+, OH be respectively 4.85mg/mL, 4.83mg/mL, 2.94mg/mL。
6. a kind of carboxymethylpachymaran CMP44 as described in claim 1, it is characterized in that the polysaccharide CMP44 is in 31.25- To colon cancer cell HT-29, HepG-2 cell, breast cancer cell MCF-7, stomach cancer cell in 1000 μ g/mL concentration ranges SGC-7901 and lung cell A549 cell Proliferation embody inhibiting effect, corresponding IC50 value is respectively 140.5,264.3, 256.4、102.5、313.2μg/mL。
7. a kind of carboxymethylpachymaran CMP44 as described in claim 1, it is characterized in that the polysaccharide CMP44 can inhibit rouge Mouse monokaryon macrophage RAW264.7 release nitric oxide (NO) of polysaccharide (LPS) induction, interleukin-6 (IL-6), tumour are bad Necrosis factor-α (TNF-α) and Interleukin -1β (IL-1 β), in 31.25-1000 μ g/mL concentration range, to LPS induction NO, The inhibiting rate difference 46.87%, 44.33%, 79.48% and 34.72% that IL-6, TNF-α and IL-1 β are generated, has anti-inflammatory work Property.
8. a kind of carboxymethylpachymaran CMP44 as described in claim 1, it is characterized in that the polysaccharide CMP44 is induced without LPS When can activate RAW264.7 cell secretion NO, IL-6, TNF-α and IL-1 β.
9. any one of claim 4~8 a kind of application of carboxymethylpachymaran CMP44, it is characterized in that the polysaccharide CMP44 is preparing the application in functional food and bio-pharmaceuticals, and polysaccharide CMP44 is with anti-oxidant, antitumor, anti-inflammatory and immune tune Section activity.
CN201811015738.XA 2018-08-31 2018-08-31 A kind of carboxymethylpachymaran CMP44 and the preparation method and application thereof Pending CN109400749A (en)

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