CN104774276B - A kind of yew amylose and its preparation method and application - Google Patents
A kind of yew amylose and its preparation method and application Download PDFInfo
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- CN104774276B CN104774276B CN201510119095.3A CN201510119095A CN104774276B CN 104774276 B CN104774276 B CN 104774276B CN 201510119095 A CN201510119095 A CN 201510119095A CN 104774276 B CN104774276 B CN 104774276B
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- yew
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- yew amylose
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- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a kind of yew amylose and its preparation method and application.The preparation method include isolate and purify, isolate and purify including：Using NaCl solution as eluant, eluent, Thick many candies are separated using DEAE sepharose fast flow resin columns, the target fraction that concentration elutes for 0.3M NaCl solution is obtained；Using deionized water as eluant, eluent, target fraction is separated using the gel columns of Sephdex G 10, yew amylose is obtained when retention time is 30min.The weight average molecular weight of the yew amylose is 73527Da, by glucose, xylose, mannose, arabinose, rhamnose and galactolipin with mol ratio 1：0.38：0.31：3.11：1.34：1.92 composition；Main chain has significant apoptosis-induced effect to stomach cancer cell SGC 7901 using (1 → 3) arabinose and (1 → 4) galactolipin as skeleton.
Description
Technical field
Technology is effectively utilized the present invention relates to a kind of taxus resource, and in particular to a kind of yew amylose and its preparation side
Method and application.
Background technology
Powder basic moiety is just extracted from european yew (Taxus baccata) leaf early in Lucas in 1856
Taxine, but progress afterwards is very slow, until 1960s spectral technique develops rapidly the research for just having deep people.
The scientist of 1980s US and European discloses the anticancer therapeutic of taxol in succession, and the whole world has started research red bean
The climax of China fir.It is due to that content of taxol is very low in Chinese yew although taxol has good anticancer effect, and many distributions
In bark, part producing business largely cuts down Chinese yew in order to improve Taxol Yield, makes taxus resource by serious
Destruction.All gone out of use substantially in addition, being extracted the Chinese yew after taxol, cause the serious wasting of resources and environmental pollution.
Fall sharply to solve taxus resource, the wasting of resources and the problems such as environmental pollution, taxus resource utilization with
In exploitation, the new technology of research and development planting yew is not only wanted, it is often more important that will extract after taxanes substance
Chinese yew residue is recycled, and furthers investigate the exploitation of wherein new active constituent.At present in Chinese yew small molecule active
In the exploitation of material, many progress have all been obtained both at home and abroad, in addition to taxol has anti-tumor biological, in Chinese yew also
Contain 20 various taxanes class diterpene compounds and other water-soluble bioactive substances.In recent years, to taxus resource
Using the research also no longer rested on to small-molecule active substance, increasing research shows in Chinese yew containing substantial amounts of
Bioactive substance --- polysaccharide, and these polysaccharide have the immune isoreactivity of antitumor, regulation.
Yew amylose is the material with bioactivity separated from Chinese yew, with strengthen immunity, is prevented
Only myocardial infarction and a variety of functions such as antitumor, have broad application prospects.Former phenanthrene etc. have studied yew amylose with mtt assay
TMP90W is to the human breast cancer cell line Bcap-37 of in vitro culture and the growth inhibition effect of lung cell A549.TMP90W is to MCF-7
There is different degrees of inhibitory action with A549, and with concentration dependent.Although its inhibitory action is suitable compared with positive control medicine
Platinum is smaller, but TMP90W small toxicity, so antitumor drug candidate can be used as.Yin etc. successively isolates many from Chinese yew
Sugared TMP70W and TMP70S-1, and suppression of two kinds of yew amyloses to the tumour cell of in vitro culture have detected using mtt assay
Effect.As a result show that TMP70W is respectively provided with inhibitory action to K562 cells, MCF-7 cells；TMP70S-1 is to human cervical carcinoma cell
Hela and human fibrosarcoma cell HT1080 are also inhibited, and with concentration dependent.The research such as Kong Fanzhi finds red
Beans China fir polysaccharide, which has, to be alleviated and stable late period lung, stomach, mammary gland, prostate, the curative effect of colorectal cancer, can protect the T cell of human body
The immunologic function of cancer patient is set to be maintained at relatively normal state.Yew amylose toxic side effect is small, and pharmacological action is strong, can subtract
Few chemotherapeutics consumption, reduces toxic and side, is the good ancillary drug of cancer patient's clinical chemotherapy, possesses good
Good prospect in medicine.
In view of the significant bioactivity of yew amylose, deeply develops new yew amylose and just shows for disease treatment
Obtain very necessary.
The content of the invention
The invention provides a kind of yew amylose, the yew amylose has significant apoptosis-induced to gastric carcinoma cells
Effect.
A kind of yew amylose, its preparation method includes degreasing, extraction, alcohol precipitation and isolated and purified, the separation successively
Purifying includes：
(1) using NaCl solution as eluant, eluent, alcohol precipitation is obtained using DEAE sepharose fast flow resin columns
Thick many candies are separated, and obtain the target fraction that concentration elutes for 0.3M NaCl solution；
(2) using deionized water as eluant, eluent, target fraction is separated using Sephdex G-10 gel columns, retained
Time obtains the yew amylose when being 30min.
Specifically, the preparation method of the yew amylose includes：
1st, degreasing：Branches and leaves of yew chip is taken, is placed in apparatus,Soxhlet's and uses petroleum ether refluxing extraction, to be after extracting
Oil stain is not left after petroleum ether volatilization for degreasing terminal, the branches and leaves of yew chip after degreasing is placed in ventilation at room temperature and dried.
2nd, extract：
1. Microwave Extraction is first carried out to branches and leaves of yew chip with water, then is placed in extraction in 95 DEG C of water-baths, water is obtained respectively
Extract solution and residue；
Preferably, the Microwave Extraction is：40~50s is extracted under 900~1400W power, extraction time is extremely
It is few three times, 4~5min is spaced between adjacent extraction twice；Microwave Extraction is conducive to promoting the water-soluble thing in branches and leaves of yew
Matter is more easy to discharge, and improves polysaccharide extract rate；Interval repeatedly extract and can avoided because of an extraction time oversize temperature too
High and composition inside destroying；
2. alkali carries are carried out to the residue, extracted supernatant and the water after filtering to take supernatant, regulation supernatant pH to 7.0
Liquid is mixed, and concentration obtains concentrate；
Preferably, using concentration to carry out alkali carries, the solid-liquid of the residue and alkali lye for 1.0~1.2mol/L alkali lye
Than for 1g:18~20L, 110~120min of alkali carries at 70~80 DEG C；
More preferably：Use concentration for 1.0mol/L alkali lye progress alkali carries, the solid-to-liquid ratio of residue and alkali lye is 1g:
20L, the alkali carries 120min at 75 DEG C.
3rd, alcohol precipitation：Alcohol precipitation is stayed overnight to the concentrate in the case where temperature is less than 4 DEG C for 75~80% ethanol with volume fraction,
Centrifuge and clean sediment, obtain Thick many candies.
4th, isolate and purify：
(1) using NaCl solution as eluant, eluent, alcohol precipitation is obtained using DEAE sepharose fast flow resin columns
Thick many candies are separated, and obtain the target fraction that concentration elutes for 0.3M NaCl solution；
The gradient of NaCl solution is followed successively by：0.1M, 0.3M, 0.5M, 0.7M, elution speed are 11.5mL/min, are washed
Lift-off product is 1L；
(2) using deionized water as eluant, eluent, (deionization is separated to target fraction using Sephdex G-10 gel columns
Water flow velocity is 0.6mL/min), obtain the yew amylose when retention time is 30min.
The yew amylose of acquisition is carried out gel permeation chromatography (Gel Permeation Chromatograp, GPC) and
Ultraviolet spectra (Ultraviolet Spectrophotometry, UV) is analyzed, it is known that its weight average molecular weight is 73527Da, and
Absworption peak is single symmetrical peak.
Gas chromatographic analysis is carried out to the yew amylose of acquisition, it is known that the yew amylose is by glucose, xylose, sweet dew
Sugar, arabinose, rhamnose and galactolipin are with mol ratio 1：0.38：0.31：3.11：1.34：1.92 composition.
Infrared spectrum analysis is carried out to the yew amylose of acquisition, finds that there is following absorb in its infrared absorption pattern
Peak：3413cm-1, 2923cm-1, 2846cm-1, 1735cm-1, 1614cm-1, 1415cm-1, 1328cm-1, 1242cm-1, 1139cm-1, 1090cm-1, 1072cm-1, 1024cm-1, 957cm-1, 893cm-1, 838cm-1, 771cm-1, show in the yew amylose
Glycosidic bond is mainly α-type.
Nuclear magnetic resonance spectroscopy is carried out to the yew amylose of acquisition, it is found that the monose anomer for constituting the yew amylose is equal
For α configurations；Also contain a small amount of acetyl group in polysaccharide structures.
Periodate oxidation and Smith degradation analysis are carried out to the yew amylose of acquisition, find to contain in the yew amylose
Have 1 →, 1 → 3,1 → 2,1 → 6 or 1 → 2,6 glycosidic bond；Methylation analysis is carried out to the yew amylose of acquisition, finding should
The main chain of yew amylose using (1 → 3)-arabinose and (1 → 4)-galactolipin as basic framework, branch mainly contain (1 → 3,
6)-arabinose and (1 → 3,6)-mannose, non reducing end mainly contain arabinose, glucose and galactolipin.
Present invention also offers application of the yew amylose in anti-gastric cancer medicament is prepared.Preferably, described anti-
Gastric cancer medicament is used for the growing multiplication for suppressing SGC-7901 cells.Also, it is to SGC- when the yew amylose is combined with 5-FU
The apoptosis-induced effect of 7901 cells can be significantly improved, i.e., both have synergy.
Compared with prior art, beneficial effects of the present invention are：
(1) present invention first carries out Microwave Extraction to branches and leaves of yew in extraction process and is carrying out 95 DEG C of hot water extractions, micro-
Ripple, which is extracted, is conducive to promoting the water-soluble substances in branches and leaves of yew to be more easy to discharge, and improves polysaccharide extract rate；
(2) present invention during isolating and purifying using successively using DEAE sepharose fast flow resin columns and
Sephdex G-10 gel columns are separated to Thick many candies, final to obtain the yew amylose that weight average molecular weight is 73527Da, should
Yew amylose has significant apoptosis-induced effect to SGC-7901 cells.
Brief description of the drawings
Fig. 1 is elution curves of the yew amylose CPTC-2 in Sephdex G-10 gel columns；
Fig. 2 is yew amylose CPTC-2 ultraviolet all band scanning spectra；
Fig. 3 is yew amylose CPTC-2 gpc analysis collection of illustrative plates；
Wherein, Minutes represents (residence time) minute, and MV represents voltage millivolt, and Mw represents weight average molecular weight；
Fig. 4 is the GC analysis collection of illustrative plates that standard mixes monose and yew amylose CPTC-2 hydrolysis monose；
Wherein, min represents (retention time) minute, and The peak height (nC) represent that (nC represents each peak group to peak height
The response divided)；A curve tables indicating quasi- mixing monose, B curves represent that yew amylose CPTC-2 hydrolyzes monose；
Fig. 5 is yew amylose CPTC-2 infrared spectrogram；
Wherein, Wavenumber cm-1Represent wave number (cm-1), Transmittance [%] represents light transmittance (%)；
Fig. 6 is yew amylose CPTC-2's1H spectrograms；
Fig. 7 is yew amylose CPTC-2's13C spectrograms；
Fig. 8 A are the flow cytometric analysis results figure of normal SGC-7901 cells in embodiment 2；
Wherein, Count% represents each cell cycle phase percentage of cells, and Population represents cell population, Cells
Total cell is represented, Apo represents apoptotic cell, and G0/1 represents that the cell cycle is in the cell of G0/1 phases, and S represents that the cell cycle is in
The cell of S phases, G2/M represents that the cell cycle is in the cell of G2/M phases；%Parent represents percentage of cells, and Mean represents flat
Equal fluorescence intensity, %CV represents the fluorescence intensity coefficient of variation (%), and PI-A represents fluorescence intensity；Similarly hereinafter；
Fig. 8 B be embodiment 2 in handled through CPTC-2 after SGC-7901 cells flow cytometric analysis results figure；
Fig. 9 A are the flow cytometric analysis results figure of normal SGC-7901 cells in embodiment 3；
Fig. 9 B be embodiment 3 in handled through 5-FU after SGC-7901 cells flow cytometric analysis results figure；
Fig. 9 C be embodiment 3 in handled through CPTC-2 after SGC-7901 cells flow cytometric analysis results figure；
Fig. 9 D be embodiment 3 in after CPTC-2 and 5-FU Combined Treatments SGC-7901 cells flow cytometric analysis results
Figure.
Embodiment
The present invention is described in further detail with reference to the accompanying drawings and detailed description.
The extraction and identification of the yew amylose of embodiment 1
1st, the extraction of yew amylose
A kind of extracting method of yew amylose of the present embodiment, comprises the following steps：
(1) degreasing：
Dry branches and leaves of yew chip 10.0g is taken, it is 60~90 DEG C of oil to be placed in 250mL apparatus,Soxhlet'ses with boiling point
Ether refluxing extraction, bath temperature is 80 DEG C；Using after extracting be petroleum ether volatilization after do not leave oil stain as degreasing terminal, after degreasing
Branches and leaves of yew chip be placed at room temperature ventilation dry, accumulation save backup together.
(2) extract：
1. Microwave Extraction is first carried out with water to branches and leaves of yew chip, and (power 900W, extraction time 45s, extraction time is
Three time, 5min is spaced between adjacent extraction twice or is extracted next time after after system cooling), then be placed in 95 DEG C of water-baths
Extract repeatedly, untill the solution that extraction is obtained determines through Phenol-sulphate acid method and DNS methods and is substantially free of polysaccharide；Water is obtained respectively
Extract solution and residue；
2. concentration is used to carry out alkali carries to the residue after washing for 1.0mol/L NaOH solution, residue is molten with NaOH
The solid-to-liquid ratio of liquid is 1g:20L, 120min is extracted at 75 DEG C；Supernatant is filtered to take after the completion of extraction, after HCl supernatants pH to 7.0
By supernatant and step 1. in aqueous extract mix, Rotary Evaporators concentration obtains concentrate；
(3) alcohol precipitation：
Alcohol precipitation, 5000r/min centrifugations are stayed overnight to concentrate at 4 DEG C for 75% ethanol with volume fraction, and is used successively
Ethanol, acetone, ether clean sediment each three times；Under the conditions of gravity-flow ventilation, acetone and the ether volatilization allowed in sediment；It is molten
It is after agent volatilization completely, sediment is soluble in water, dialysed, decolourized after filtering, desalination, it is lyophilized after floccule be slightly many
Sugared CPTC.The recovery rate of Thick many candies is 3.45%, and polyoses content is 5.20%.
(4) isolate and purify：
1. lyophilized rear Thick many candies CPTC is taken, Tris-HCl buffer solutions are dissolved in, upper DEAE sepharose fast flow are cloudy
Ion exchange column, is first washed off unadsorbed material with level pad, and the gradient NaCl then prepared with level pad is molten
(gradient of NaCl solution is followed successively by liquid：0.1M, 0.3M, 0.5M, 0.7M, elution volume are 1L) with 11.5mL/min's
Flow velocity is eluted, and collects eluent with automatic fraction collector, and the polyoses content in each eluent is determined with Phenol-sulphate acid method.Will
Each component is collected, and rotated evaporation is lyophilized.
Through DEAE sepharose fast flow anion-exchange columns be separated to three kinds of polysaccharide CPTC-1a, CPTC-1b and
The NaCl solution that CPTC-2, wherein CPTC-1a, CPTC-1b are 0.1M is eluted, and CPTC-2 be 0.3M NaCl it is molten
What liquid was eluted.
2. polysaccharide component CPTC-2 is taken to be dissolved in Tris-HCl buffer solutions, upper Sephdex G-10 gel columns use deionization
Water is eluted with 0.6mL/min flow velocity, with the electrical conductivity of conductivity meter measure eluent to characterize the content of NaCl in eluent,
Refined polysaccharide CPTC-2 (elution time or retention time are 30min) is obtained, elution curve is as shown in Figure 1.
2nd, the identification of yew amylose
(1) purity testing
By the refined polysaccharide CPTC-2 polysaccharide solutions soluble in water for being configured to concentration for 1mg/mL, using deionized water as blank
Control, carries out ultraviolet spectra all band scanning in the range of 200-400nm, and scanning result is shown in Fig. 2.Fig. 2 result shows to refine
Albumen and nucleic acid are free of in polysaccharide CPTC-2, is homogeneous components.
(2) molecular weight determination
Polysaccharide is closely related by the elution volume and molecular weight of Sephdex G-10 gel columns, in certain molecular weight model
In enclosing, residence time (RT) and the logarithm (lg of polysaccharide molecular weight) linear.Using this feature, first with different points
The dextran standards (1000,5000,12000,50000,150000,270000,410000,670000) of son amount are standard sample
Product, draw standard curve, operating condition：Waters highly effective liquid phase chromatographic systems (515Waters Pump, Ultrahydrogel
TM250 and the columnss in series of Ultrahydrogel TM 2000, Waters2410 differential refraction detectors), mobile phase is NaNO3It is molten
Liquid, flow velocity is 0.8mL/min, 30 DEG C of column temperature；Then the residence time (RT) according to polysaccharide sample under identical chromatographic conditions, lead to
Cross standard curve and obtain refined polysaccharide CPTC-2 weight average molecular weight for 73527Da.
Gpc analysis is further carried out to refined polysaccharide CPTC-2, to characterize the uniformity of polysaccharide；Gpc analysis collection of illustrative plates such as Fig. 3
It is shown.As shown in figure 3, refined polysaccharide CPTC-2 absworption peak is single symmetrical peak, show that its uniformity is higher.
(3) measure of monose composition
5mg glucose, xylose, arabinose, mannose, rhamnose, galactolipin are respectively weighed, 0.1% hydroxide is added
Sodium solution 3ml, appropriate sodium borohydride, sealing room temperature reduction 12h, or shaking；Acetic acid is added dropwise and decomposes excess sodium borohydrate extremely
Bubble-free, adds 2ml methanol (repeatedly 4 times) and removes unnecessary borate, decompression is spin-dried for, and residue is heated in 105 DEG C of baking ovens
15min to remove moisture completely；By 1:1 (V/V) ratio adds 2ml acetic anhydrides and pyridine, 100 DEG C of heating water bath 1h, reaction solution
It is concentrated under reduced pressure, is extracted with 1ml dichloromethane, takes extract to carry out GC analyses；Chromatographic condition：Agilent6829N gas chromatographs
(FID), SE-54 capillary columns 30m × 0.32mm (Agilent companies), 250 DEG C of temperature of vaporization chamber, 250 DEG C of detector temperature,
Temperature programming：200 DEG C (1min) is warming up to 220 DEG C (3min) with 0.5 DEG C/min.
10mg refined polysaccharide CPTC-2 are taken, are put into l0ml ampoule bottles, 2mol/L trifluoracetic acids (TFA) 4ml is added, is filled with
N2, tube sealing hydrolyzes 6h at 100 DEG C；Solution decompression after complete hydrolysis is evaporated (<40 DEG C), then add methanol and be evaporated, repeat
This operation 5 times, to be freezed after removing TAF, plus distilled water dissolving completely；Take it is lyophilized after Polysaccharides 5mg, do with it is above-mentioned identical
Derivation process, then GC analyze, ibid, analysis result is shown in Fig. 4 to analysis condition.
Fig. 4 result shows that it is glucose, xylose, mannose, arabinose, rhamnose, half to constitute CPTC-2 monose
Lactose (the wherein content highest of arabinose), their mol ratio is：1:0.38:0.31:3.11:1.34:1.92.
(4) infrared spectrum is detected
2mg refined polysaccharide CPTC-2 is weighed, finely ground rear tabletting is mixed with KBr, it is directly infrared in the types of Nicolet 5700
Determined under spectrometer (ThermoFisher Co.), spectroscopic assay scope 4000-400cm-1, DTCS, KBr detectors and MCT are examined
Device is surveyed, resolution ratio is better than 0.09cm-1, testing result is shown in Fig. 5.
In Fig. 5, in 3413cm-1Place's peak value is that the stretching vibration of the O-H on hydroxyl in polysaccharide causes, and peak shape is very wide, it is seen that
Hydroxyl associates intermolecular, is not free hydroxyl；In 2923cm-1It is-the CH in polysaccharide to locate peak value2, C- in-CH groups
H antisymmetric stretching vibration causes；2846cm-1It is-the CH in polysaccharide to locate peak value2, in-CH groups C-H symmetrical stretching vibration
Cause；In 1735cm-1、1614cm-1Place's absworption peak is that the stretching vibration of carbonyl or the-C=O on acetyl group in polysaccharide cause；
1415cm-1、1328cm-1、1242cm-1The absworption peak at place is the flexural vibrations of C-O stretching vibrations, C-H or O-H in polysaccharide
Cause；In 1139cm-1、1090cm-1、1072cm-1、1024cm-1The absworption peak at place is by the C-O-C in polysaccharide, C-O-H groups
In C-O, C-C stretching vibration cause；In 957cm-1The absworption peak at place causes for the vibration of D- glucopyranose rings；
893cm-1The absworption peak at place becomes the characteristic peak of angular oscillation, i.e. β-type glycosidic bond characteristic peak for the C-H of pyranoid ring β-type；In 838cm-1The absworption peak at place is the characteristic peak that α-type C-H becomes angular oscillation, i.e. α-type glycosidic bond characteristic peak；In 771cm-1The absworption peak at place is
α-pyranoid ring symmetrical stretching vibration.
(5) nuclear magnetic resonance spectroscopy
Take polysaccharide sample 30mg to load nuclear magnetic tube, be dissolved in 0.5ml heavy water, carried out on NMR1H-NMR,13C-
The spectrum analyses such as NMR.With D2O makees solvent, and TMS makees the measure of progress nmr spectrum under the conditions of internal standard, 600HZ.Analysis knot
Fruit sees Fig. 6, Fig. 7, table 1.
The CPTC-2 of table 1 1H spectrum chemical shifts
From table 1, the sugar that the proton peak at δ 5.22 shows to constitute CPTC-2 is mainly a kind of configuration, i.e. α-pyrans
Type sugar.In addition, by13Peak of the C spectrograms at δ 79.1 and δ 79.3 is it is also seen that composition CPTC-2 monose anomer is configured as α
Configuration.
(6) periodate oxidation and Smith degradation analysis
1. 15mmol/L NaIO are taken450ml(320.8mg NaIO4, it is settled to 100ml), lucifuge is wrapped with tinfoil, is taken
0.1ml is diluted to measure light absorption value at 25ml, 223nm and can used (now with the current) more than 0.6 side；
2. CPTC-2 sugar-likes 5mg (recording exact mass) is accurately weighed, is dissolved in a small amount of water in 25ml volumetric flasks, so
15mmol/L NaIO are added afterwards4Constant volume；Wrap lucifuge with tinfoil, be placed on low temperature dark place reaction, interval time (0h, 6h,
12h, 24h, 36h, 48h, 60h ...) sampling 0.1ml, with 250 times of distilled water diluting, blank control is made with distilled water,
Densitometric value at 223nm wavelength, untill OD value is constant, record reaction end time (60h)；
3. simultaneously by remaining 15mmol/L NaIO4It is same with response sample to place as control, taken at the end of question response
Go out 0.1ml, after 250 times of dilution, (being shown in Table 2) is diluted by different proportion with distilled water, per three repetitions of concentration, is determined at 223nm
OD value, using OD values as ordinate, NaIO4Concentration is abscissa, makes standard curve；
Table 2
4. by looking into standard curve, the consumption of periodic acid is calculated.
Periodic acid consumption (mmol)=(periodic acid concentration after periodic acid concentration-reaction before reaction) × reaction volume；
5. the oxidation solution in taking 1ml above-mentioned steps 2., plus 1 and drips phenolphthalein indicator, is dripped with 0.005mol/L NaOH solutions
It is fixed, calculate formic acid growing amount；
Formic acid growing amount (mmol)=(volume of NaOH actual concentrations × titration) × reaction volume；
6. Smith degrades：Plus ethylene glycol terminates Malaprade reaction, flowing water and distilled water are respectively dialysed 24h；It is concentrated into
5ml, adds KBH435mg reduction is stayed overnight；It is 6-7 that pH is neutralized to 50% acetic acid, and flowing water and distilled water are respectively dialysed 24h, and concentration is frozen
It is dry.Take it is lyophilized after polysaccharide polyol, add 2mol/L TFA solution 3ml, 100 DEG C of hydrolysis 6h, hydrolyzate through be concentrated under reduced pressure into it is dry with
TFA is removed, GC analyses are then done.
The result of calculation of step 4. and 5. shows that every mole of saccharide residue consumes periodic acid 1.052mol, every mole of saccharide residue
The amount for generating formic acid is 0.479mol.
Periodate oxidation has formic acid generation when completing, and illustrates there is non reducing end or 1 → 6 glycosidic bond；Periodic acid disappears
Consumption is far longer than two times of formic acid growing amount, illustrates there is type of the substantial amounts of consumption periodic acid without generating formic acid, can
There can be 1 → 2,6,1 → 4,1 → 4,6 of bonding；Each saccharide residue consumption periodic acid 1.052mmol, generates 0.479mmol formic acid,
Illustrate to have 62.5% saccharide residue to be oxidized, also 37.5% saccharide residue is not oxidized, not oxidized saccharide residue may
Mainly to exist with 1 → 3 glycosidic bond.
From GC analysis results understand CPTC-2 degraded through Smith after product in containing glycerine, erythrite, arabinose and
Glucose.There is glycerine in product, illustrate in CPTC-2 containing 1 →, 1 → 2,1 → 6 or 1 → 2,6 grade glycosidic bonds；Have in product
Erythrite, illustrates there is 1 → 4 glycosidic bond；Contain arabinose and glucose in product, illustrate arabinose in CPTC-2 with
Glucose contains not by the glycosidic bond of periodate oxidation, i.e. 1 → 3 glycosidic bond.
(7) methylation analysis
1. methylate
CPTC-2 polysaccharide sample 10mg, put in 50mL round-bottomed flasks, are put into magnetic stir bar, then in being placed with five oxidations two
Dried 1 day in the vacuum desiccator of phosphorus；Plus 5ml dimethyl sulfoxides, stirring and dissolving 10min, plus 100mg sodium hydroxide powders, continue
Stirring reaction 20min；Iodomethane 1ml (20min is added) is added dropwise, stirring reaction 2h is then proceeded to；Plus 5ml water stirring reactions
10min, destroys excess iodomethane；Reaction solution is transferred to bag filter (molecular cut off 1000Da), and flowing water is dialysed 2 days；Solution turns
Enter round-bottomed flask, be freeze-dried；The above-mentioned methylation procedure of residue repetition 2~3 times after drying.
2. derivatization
2~3mg of methylate, plus 2mol/L tri- Buddhist acetic acid 3ml, 120 DEG C of hydrolysis 2h are taken, takes out, lets cool to room temperature,
Hydrolyzate is transferred to 25ml pear-shaped flasks, 60 DEG C of water-bath evaporated under reduced pressure, then Jia 1~2ml methanol is evaporated 3 times, plus 50mg NaBH4
With 2ml water, mix, more than room temperature reductive hydrolysis 4h, the excessive NaBH of glacial acetic acid destruction is added dropwise4, to pH value of solution 4~5,
60 DEG C of water-baths decompression is steamed to thick, plus methanol：Glacial acetic acid (5:1) 1~2ml of solution is evaporated several times, is finally evaporated to powdered,
Put in 105 DEG C of baking ovens and dry 10min, then add 2~3ml acetic anhydrides, 105 DEG C of acetylation 1h are taken out, and 2ml water is added while hot,
Shake up, room temperature place 30min, or shake, 50 DEG C of water-bath evaporated under reduced pressure, plus 5ml water be suspended, then plus 5mL chloroforms extraction, chlorine
Imitative layer is washed with water 3 times, standby then with anhydrous sodium sulfate drying.
3. GC-MS is tested and analyzed
Chromatographic condition：Chromatographic column：HP-5ms, 60m × 0.25mm × 0.25 μm, Agilent；
Carrier gas：He；
Flow rate of carrier gas：1mL/min；
Injection port：250℃；
Column oven temperature programming：120 DEG C of initial temperature, 3 DEG C/min is warming up to 190 DEG C, and 2 DEG C/min is warming up to 250 DEG C, guarantor
Warm 5min；
Sampling volume：1μL；
Mass Spectrometry Conditions：EI sources, 70eV, 230 DEG C, 50~500m/e of scanning range, 150 DEG C of quadrupole rod temperature.
Methylating for CPTC-2 the results are shown in Table 3.
The CPTC-2 of table 3 result that methylates
The result of table 3 shows：After the CPTC-2 of exhaustive methylation is further analyzed through GC-MS, 9 kinds of residues are generated, point
Wei 2,3,4,6-Me4-Ara, 2,4,6-Me3-Ara, 2,3,4,6-Me4-Glc, 2,3,4,6-Me4-Gal, 2,4-Me2-
Ara, 2,3,6-Me3-Gal, 2,4,6-Me3-Glc, 2,3,4-Me3-Rha and 2,4-Me2-Man, mol ratio is respectively 0.66:
1.81:0.22:0.26:0.09:1.00:0.06:0.20:0.18.It follows that CPTC-2 is mainly with 1 → 3- arabinoses
It is skeleton with 1 → 4- galactolipins, branch is mainly made up of 1 → 3,6- arabinoses and 1 → 3,6- mannose, irreducibility end
End is mainly made up of arabinose, glucose and galactolipin.
The CPTC-2 anti tumor activity in vitro of embodiment 2 is tested
1st, MTS methods are detected
The SGC-7901 cells of exponential phase of growth are in 0.25% Trypsin Induced, cell suspension is made, is inoculated in
On 96 well culture plates, per the μ L of hole 100,5%CO is put2, cultivate 24h in 37 DEG C of incubator；For CPTC-2 experimental groups, then to 96
The yew amylose configured with the RPMI-1640 basic culture solutions without serum is added in well culture plate, per the μ L of hole 100, is made many
Sugared final concentration is respectively 0,12.5,25,50,100,150,200,300 μ g/ml, while setting blank control, negative control and Huang
Astragalus polysaccharides (50 μ g/ml) positive control, every group of 3 repetitions；Continue to cultivate after 48h, the MTS (5mg/ that PBS is prepared are added per hole
ML) 20 μ L, continue to cultivate 2h, OD values are directly surveyed at 490nm wavelength with ELIASA, press formula and calculate inhibiting rate (RI)：
Inhibiting rate (RI)=(negative control group OD values-experimental group OD values)/negative control group OD value × 100%.Calculate knot
Fruit is shown in Table 4.
Influences of the CPTC-2 of table 4 to SGC-7901gastriccarcinomacellline activity
From table 4, it can be seen that with the increase of concentration, proliferation inhibition rates of the CPTC-2 to SGC-7901gastriccarcinomacellline
The trend increased is presented, when concentration is 12.5 μ g/mL, inhibiting rate, than relatively low, is 8.28%；When concentration increases to 300 μ g/mL
When, inhibiting rate has been also added to 36.53%, and compared with control group, with significant difference (P ＜ 0.01), illustrates CPTC-
The propagation of 2 pairs of SGC-7901gastriccarcinomacelllines significantly inhibits effect, and with concentration dependent, calculates its IC50It is worth and is
758.75μg/mL。
2nd, flow cytometry
SGC-7901 cells are inoculated in 6 porocyte culture plates, inoculum density is 1 × 105Added after/hole, culture 24h
Medicine is tested, wherein setting blank control group, CPTC-2 groups (350 μ g/mL), continues to cultivate 48h, Trypsin Induced, 1000r/
Min low-speed centrifugals 5min, 4 DEG C of PBS are washed 2 times, and collected after centrifugation cell mass is fixed with 70% ethanol of 4 DEG C of precoolings again, juxtaposition
More than the 12h in -20% refrigerator.Fixed to finish, low-speed centrifugal, cell mass is washed with 4 DEG C of PBS and centrifuged again, then adds PI mixing
Dye liquor (45mg/L PI, 10mg/L RNaseA, 0.1%TritonX-100), lucifuge dyeing l h, flow cytometer collection,
ModFit LT2.0EP software analysis.Analysis result is as shown in figs. 8 a and 8b.
From Fig. 8 A and 8B as can be seen that occurred in that in blank control group and CPTC-2 groups apoptosis cell (Apo,
Apoptotic cell), wherein the apoptosis rate of control group is that the apoptosis rate of 2.9%, CPTC-2 groups is that 4.4%, CPTC-2 groups are bright
It is aobvious to be higher than control group, illustrate that CPTC-2 can be by inducing SGC-7901 Apoptosis to play antitumor action.
The CPTC-2 of embodiment 3 combines 5-FU antitumor synergistic effect
1st, MTS methods are detected
Experiment set control group, 5-FU groups (50 μ g/mL), the high, medium and low dosage groups of CPTC-2 (300,150,50 μ g/mL) with
And high, medium and low (300,150,50 μ g/mL)+5-FU (the 50 μ g/mL) groups of CPTC-2, every group of 3 repetitions.
The SGC-7901 cells of exponential phase of growth are in 0.25% Trypsin Induced, cell suspension is made, is inoculated in
On 96 well culture plates, per the μ L of hole 100,5%CO is put2, cultivate 24h in 37 DEG C of incubator, add each group working solution, continue to cultivate
After 48h, the μ L of MTS (5mg/mL) 20 that PBS is prepared are added per hole, continues to cultivate 2h, is directly surveyed with ELIASA at 490nm wavelength
OD values, press formula and calculate inhibiting rate (RI)：
Inhibiting rate (RI)=(control group OD values-experimental group OD values)/control group OD value × 100%；Result of calculation is shown in Table 5.
The CPTC-2 of table 5 and influence of the 5-FU synergy to SGC-7901 cytoactives
SGC-7901 cells shows are normal proliferative state in control group as can be seen from Table 5, after independent 5-FU processing, carefully
Born of the same parents' number significantly reduces (P compared with control group<0.01), cell propagation is suppressed, while it is thin to SGC-7901 to measure independent 5-FU
The inhibiting rate of born of the same parents is 54.21%；Effect of the influence significantly lower than 5-FU that independent CPTC-2 breeds to SGC-7901 cells, suppresses
Rate increases with the increase of CPTC-2 concentration, and inhibiting rate when 300 μ g/mL is 44.21%；CPTC-2 combines 5-FU
Cell proliferating number mesh after effect is significantly reduced, and inhibiting rate reaches 61.93%, hence it is evident that higher than the effect effect that 5-FU is used alone
Really.Illustrate that CPTC-2 can significantly improve inhibited proliferations of the 5-FU to SGC-7901 cells.
2nd, flow cytometry
Experiment sets control group, 5-FU groups (50 μ g/mL), CPTC-2 groups (300 μ g/mL), CPTC-2 (300 μ g/mL) joints
5-FU (50 μ g/mL) group, every group of 3 repetitions.
SGC-7901 cells are inoculated in 6 porocyte culture plates, inoculum density is 1 × 105Added after/hole, culture 24h
Test medicine, continuation culture 48h, Trypsin Induced, 1000r/min low-speed centrifugals 5min, 4 DEG C of PBS are washed 2 times, after centrifugation
Collect cell mass to be fixed with 70% ethanol of 4 DEG C of precoolings again, be placed in more than 12h in -20 DEG C of refrigerators.It is fixed to finish, low-speed centrifugal,
Cell mass is washed with 4 DEG C of PBS and centrifuged again, then adds PI mixing dye liquor (45mg/L PI, 10mg/L RNaseA, 0.1%
TritonX-100), lucifuge dyeing lh.Flow cytometer is gathered, ModFit LT2.0EP software analysis.Analysis result such as Fig. 9 A,
Shown in 9B, 9C and 9D.
CPTC-2 groups are can be seen that compared with control group from Fig. 9 A, 9B, 9C and 9D, apoptosis rate is 3.2%, slightly higher than
Control group, the cell reduction of this group of S phase, cell-cycle arrest is in the G0/1 phases；5-FU groups are compared with control group, and apoptosis rate is
8.1%, relative to single CPTC-2 groups, apoptosis rate substantially increases, and 5-FU group G0/1 phase cells substantially increase, S phases and G2/M
Phase cell is substantially reduced, and the cell cycle is also to be blocked in the G0/1 phases；CPTC-2 combines the 5-FU group S phases and G2/M phase cells substantially drop
Low, apoptosis rate is 14.5%, and apoptosis phase cell significantly increases compared with control group and single CPTC-2 groups and single 5-FU groups
Plus.It can thus be appreciated that yew amylose can significantly improve inhibited proliferations of the 5-FU to SGC-7901 cells.
Claims (6)
1. a kind of application of yew amylose in anti-gastric cancer medicament is prepared, it is characterised in that the yew amylose, it is prepared
Method includes degreasing, extraction, alcohol precipitation and isolated and purified successively, it is described isolate and purify including：
(1) using NaCl solution as eluant, eluent, using DEAE sepharose fast flow resin columns alcohol precipitation is obtained it is thick many
Sugar is separated, and obtains the target fraction that concentration elutes for 0.3M NaCl solution；
(2) using deionized water as eluant, eluent, target fraction is separated using Sephdex G-10 gel columns, in retention time
The yew amylose is obtained during for 30min；
In step (1), the gradient of NaCl solution is followed successively by：0.1M, 0.3M, 0.5M, 0.7M, elution volume are 1L；
In step (2), deionized water flow velocity is 0.6mL/min.
2. application as claimed in claim 1, it is characterised in that the anti-gastric cancer medicament is used for the life for suppressing SGC-7901 cells
Long propagation.
3. application as claimed in claim 1 or 2, it is characterised in that the extraction includes：
1. Microwave Extraction is first carried out to branches and leaves of yew chip with water, then is placed in extraction in 95 DEG C of water-baths, water is obtained respectively and is extracted
Liquid and residue；
2. alkali carries are carried out to the residue, mixed supernatant with the aqueous extract after filtering to take supernatant, regulation supernatant pH to 7.0
Close, concentration obtains concentrate.
4. application as claimed in claim 3, it is characterised in that step 1. in, the Microwave Extraction is：900~1400W's
40~50s is extracted under power, extraction time is at least three times, 4~5min is spaced between adjacent extraction twice.
5. application as claimed in claim 3, it is characterised in that step 2. in, use concentration for 1.0~1.2mol/L alkali lye
Alkali carries are carried out, the solid-to-liquid ratio of the residue and alkali lye is 1g:18~20L, 110~120min of alkali carries at 70~80 DEG C.
6. application as claimed in claim 1 or 2, it is characterised in that the alcohol precipitation is：With the second that volume fraction is 75~80%
Alcohol stays overnight alcohol precipitation in the case where temperature is less than 4 DEG C to concentrate.
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