CN105713104B - A kind of synthesis and its application of selenizing Fenugreek Polysaccharides - Google Patents
A kind of synthesis and its application of selenizing Fenugreek Polysaccharides Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
Abstract
The invention discloses a kind of method for synthesizing selenizing Fenugreek Polysaccharides, including：The preparation of Fenugreek Polysaccharides formamide solution；The preparation of ionic liquid；Activation of the ionic liquid to selenous acid；The synthesis of selenizing Fenugreek Polysaccharides.The present invention is using ionic liquid activation selenous acid, prepare high activity selenizing reagent, the selenizing Fenugreek Polysaccharides with bioactivity have been efficiently synthesized in a mild condition, Se content is up to 27,554 29773 μ g/g, agents useful for same environmental protection, prepare simply, without using poisonous intermediate containing selenium, no discharging of waste liquid, the time required to effectively shortening reaction, application of the Fenugreek Polysaccharides as natural active matter has been expanded.Activity test shows that the selenizing Fenugreek Polysaccharides that the present invention synthesizes have significant In-vitro Inhibitory Effect to HepG2 liver cancer cells.
Description
Technical field
The invention belongs to boiomacromolecule and chemosynthesis technical field, is related to a kind of synthesis side of selenizing Fenugreek Polysaccharides
Method；The present invention also relates to the medical usage of selenizing Fenugreek Polysaccharides --- the inhibitor application as HepG2 liver cancer cells
In preparing in cancer therapy drug.
Background technology
Selenium（Se）The essential trace element of human body, the raising of life immunity function, anticancer, it is anti-oxidant, prevent
All many-sides such as trophism hepatonecrosis play an important role.In biological metabolism, selenium is also glutathione peroxidase
Active component, therefore, effect of the selenium to human body are particularly important.But selenium element is in earth skewness, in China about
3/5ths regional selenium deficiency, and Se content is all extremely low, the effect typically up to less than Selenium Supplement in ordinary food.In nature
Boundary, the existence form of selenium mainly include two kinds of inorganic selenium and Organic Selenium.Compared with inorganic selenium, Organic Selenium be it is safer, active more
High material containing selenium, it is more notable than inorganic selenium on challenge.The main source of Organic Selenium is natural selenium-rich biology
With it is artificial synthesized, it is numerous be rich in Organic Selenium material in, selenium polysaccharide receives very big concern.Selenium polysaccharide is as a kind of organic selenizing
Compound, possess the activity of selenium and both polysaccharide.The bioactivity of selenium polysaccharide is generally higher than polysaccharide and selenium, and toxicity is smaller, and selenizing
Polysaccharide afterwards is easier to be absorbed and utilized by organism.
The preparation method of selenium polysaccharide includes natural plant polyose containing selenium, and microorganism enrichment culture metabolism selenium polysaccharide closes with artificial
Into selenium polysaccharide.Artificial synthesized selenium polysaccharide be more convenient and controllable method, at present domestic and foreign literature and patent mainly use with
Lower three kinds of methods prepare selenium polysaccharide：Selenizing modification is carried out to polysaccharide using monomer selenium, selenous acid or sodium selenite under temperate condition；
Active using chemical property, the intermediate with acyl chlorides structure is modified as selenizing reagent；Functional gene containing selenium is connect
Branch is on polysaccharide molecule.Liang Shuxuan etc. is reacted using the lower sodium selenate of glacial acetic acid catalysis with LBP-X, gained selenizing LBP-X
Se content is in 606-3762 μ g/g（Food research and development, 2011,3,160-164）；Li Zhizhou etc. utilizes chemical synthesis,
Polyporus selenium polysaccharide is prepared as raw material using grifola polysaccharide and sodium selenite, using continuous or ultrasonic assistant chemical synthesis process（Food
Product and machinery, 2013,1,31-35, do not report Se content）.Patent CN 1560088A have disclosed selenizing Glucomannan a kind of
Preparation method, by selenium simple substance oxidant effect under be oxidized to Se6+, in Se6+Ethanol and salt are added in the aqueous solution of ion
Acid, selenylation reaction liquid is obtained, selenylation reaction liquid is reacted with Glucomannan, selenizing Glucomannan, gained selenizing Portugal is made
Sweet glycan Se content is 125.4-197.3 μ g/g, and selenium is Se in derivative than relatively low6+；The disclosures of patent ZL 88103347
A kind of preparation method of selenide of carragheen, using selenium powder as raw material, is dissolved with nitric acid and is prepared into selenium liquid, adds Kappa- Irish moss
Sol solution carries out selenylation reaction, but this method products therefrom Se content is relatively low（2500-15000 ppm）, and accessory substance hydrogen selenide
It is poisonous.Patent CN102653566A discloses a kind of method that organic method prepares astragalus polysaccharide selenide, and this method is by astragalus polyose
Crude product synthesizes sterling astragalus polyose under enzyme degradation, then is reacted with chlorination seleniyl, obtains the astragalus polyose selenium content of selenizing
For 14305-24864 μ g/g, but this method selenizing time is grown（12-48h）, reactant chlorination seleniyl is not easy to prepare, toxicity
Greatly, large-scale industrial production is not suitable for.Patent ZL 200910162003.4 discloses a kind of organic method and prepares selenizing sand sagebrush (Artemisia filifolia)
The method of polysaccharide or fern amylose, by fern amylose or artemisia desertorum polysaccharide and seleniyl chlorine react selenizing artemisia desertorum polysaccharide or fern fiber crops
Polysaccharide, Se content 12000-26000 μ g/g.But the seleniyl chlorine synthesis that this method uses is difficult and seleniyl chlorine has toxicity,
In course of reaction, in atmosphere, seleniyl chlorine can be oxidized, and may be contained in obtained seleniyl chlorine for exposure when adding seleniyl chlorine
There are the impurity such as selenium acyl chlorides.
The polysaccharide selenizing modification technique generally existing intermediate containing selenium mentioned in above patent and document prepares complicated and poison
Property it is big, the reaction time is long, selenizing polysaccharide Se content and yield are low, side reaction is more, biological effect is not clear the shortcomings of.Ionic liquid
With its heat endurance is good, efficiency high, environment-friendly and recyclable recycling the advantages that and be widely used in all kinds of organic syntheses
In reaction.By consulting domestic and foreign literature and patent, do not report that ionic liquid is as catalyst using Fenugreek Polysaccharides as raw material
Synthesize method and the application of high Se content selenizing Fenugreek Polysaccharides.
The content of the invention
It is an object of the invention to provide a kind of synthetic method of selenizing Fenugreek Polysaccharides.
Another object of the present invention is to provide a kind of inhibitor use of selenizing Fenugreek Polysaccharides as HepG2 liver cancer cells
In preparing cancer therapy drug.
First, the synthesis of selenizing Fenugreek Polysaccharides
1. the preparation of Fenugreek Polysaccharides formamide solution：
Fenugreek Polysaccharides are stirred into 2 ~ 5 h at 60 DEG C ~ 80 DEG C under 300 ~ 500 rpm rotating speed be dissolved in formamide,
Obtain the formamide solution of Fenugreek Polysaccharides.
The weight average molecular weight of above-mentioned Fenugreek Polysaccharides is 2.303 × 106 Da, total sugar content 87.2%.Fenugreek Polysaccharides with
The mass volume ratio of formamide is 2.5 ~ 4 mg/mL.
2. the preparation of ionic liquid
The concentrated sulfuric acid is added drop-wise in 1-METHYLPYRROLIDONE at -10 DEG C ~ 0 DEG C with 50 ~ 80 drops/min speed, 300 ~
The h of 800 rpm stirring reactions 10 ~ 18, washed 2 ~ 3 times with ethyl acetate at room temperature after completion of the reaction, collect lower floor's thick liquid,
It is dried in vacuo to obtain ionic liquid.
Wherein：The volume ratio of 1-METHYLPYRROLIDONE and the concentrated sulfuric acid is 2:1~2.5:1, the body of ethyl acetate and ionic liquid
Product is than being 0.5:1~1:1, vacuum drying condition is the MPa of vacuum 0.06 ~ 0.08,50 ~ 70 DEG C of drying temperature, the h of time 6 ~ 12.
3. activation of the ionic liquid to selenous acid
In 60 ~ 80 DEG C and N2Under conditions of protection, selenous acid is added into 1 ~ 3 h of reaction in above-mentioned ionic liquid to activate
Selenous acid afterwards.
The mass volume ratio of above-mentioned selenous acid and ionic liquid is 0.125 ~ 0.2 g/mL.
4. the synthesis of selenizing Fenugreek Polysaccharides
The formamide solution of Fenugreek Polysaccharides is added drop-wise into ionic liquid with 30 ~ 60 drops/min speed with dropping funel to live
In the selenous acid changed, in 60 ~ 80 DEG C and N22 ~ 4 h are reacted under conditions of protection, are completely cutting off the bar of air after question response
Add acetone under part to wash 10 ~ 15 times, sediment is fitted into flowing water in the Da of molecular cut off 8000 ~ 14000 bag filter dialyses
24 ~ 48 h, deionized water 12 ~ 24 h of dialysis, dialysis finishes thing in back pkt. is concentrated under reduced pressure into original volume 1/20 ~ 1/30, dense
Absolute ethyl alcohol is added in contracting liquid（Ethanol content of the total volume 70 ~ 80%）Precipitation, is collected by centrifugation lower sediment thing, is freeze-dried,
Obtain selenizing Fenugreek Polysaccharides.
Wherein：The volume ratio of the formamide solution of Fenugreek Polysaccharides and the selenous acid of activation is 10 ~ 25, and stirring condition is
The volume ratio of 300 ~ 800 rpm, acetone and reaction solution is 2 ~ 3（v/v）, the temperature that is concentrated under reduced pressure be 55 ~ 65 DEG C, vacuum 0.06 ~
0.08 MPa, centrifugal rotational speed are 4000 ~ 5000 rpm/min, the min of centrifugation time 10 ~ 15, and freeze-drying condition is temperature -60
~ -50 DEG C, the Pa of vacuum 5 ~ 20, the h of drying time 24 ~ 48.
2nd, selenizing Fenugreek Polysaccharides are in vitro to the inhibitory action of HepG2 liver cancer cells
Take the logarithm the HepG-2 cells in growth period, with 0.25% Trypsin Induced, and with containing 15% NBCS
Concentration 3 × 10 is made in DMEM culture mediums5Individual/mL single cell suspension, constantly shaking cell suspension makes cell number uniform, with 0.2
Ml/ holes are inoculated in 96 well culture plates（Edges of boards edge seals sterilized water, and blank group adds the not celliferous culture medium in 3 holes）, put CO2Incubator
Inherent 37 DEG C, saturated humidity, 5%CO2Under the conditions of cultivate.
Cell attachment after 24 h is cultivated, changes pastille culture medium, adds 190 μ L culture mediums and 10 μ L sample liquid per hole（Blank
Group and control group add 10 μ l PBS liquid）, the μ g/mL of medicine final concentration of 20,50,100,200,500,1000,5000, per concentration
6 holes are repeated, and set the control group for being not added with sample and blank zeroing group.Put CO2In incubator, 37 DEG C, saturated humidity, 5%CO2Bar
Cultivated under part.
After the h of sample treatment 48, MTT solution 20 μ L are added per hole, continue to cultivate 4 h at 37 DEG C.Carefully discard in hole
Clear liquid, and 150 μ L DMSO are added, 10 min are vibrated, are completely dissolved purple crystal.Extinction is surveyed at 490 nm with ELIASA
Value, control group are returned to zero with blank group, and calculate inhibiting rate by following equation, and calculate half according to the inhibiting rate of various concentrations sample
Number inhibition concentration（IC50）：
Inhibiting rate（%）=（1- experimental groups mean OD value/control group mean OD value）×100%
To the IC of HepG2 liver cancer cells50For 527.4 ~ 1230.8 μ g/mL, there is significant external suppression HepG2 cells
Effect, can be as the inhibitor of HepG2 liver cancer cells, for developing cancer therapy drug.
Beneficial effects of the present invention：
1st, the present invention synthesizes selenizing Fenugreek Polysaccharides using Fenugreek Polysaccharides as raw material using ionic liquid-catalyzed selenous acid,
Ionic liquid is nontoxic, environmentally friendly, preparation is simple, without using poisonous intermediate containing selenium, no discharging of waste liquid, effectively shortens reaction
Required time, the selenizing polysaccharide of high Se content is obtained under mild conditions.
2nd, it is prepared for, with the significant external selenizing Fenugreek Polysaccharides for suppressing HepG2 cytoactives, having expanded faenum graecum
Application of the polysaccharide as natural active matter；
3rd, the Se content of selenizing Fenugreek Polysaccharides prepared by the present invention can be adjusted by reaction condition and optimized, to meet not
Same domain requirement；
4th, the present invention does not need special installation, and simple controllable, cost is low, is adapted to popularization and application.
Brief description of the drawings
The infrared spectrogram of Fig. 1 Fenugreek Polysaccharides and its selenizing Fenugreek Polysaccharides.
The x-ray photoelectron power spectrum of Fig. 2 Fenugreek Polysaccharides and selenizing Fenugreek Polysaccharides（XPS）Figure.
Embodiment
The synthetic method of selenizing Fenugreek Polysaccharides of the present invention is further described below by specific embodiment.
Embodiment 1
（1）The preparation of Fenugreek Polysaccharides formamide solution：200 mg Fenugreek Polysaccharides are turned at 70 DEG C with 500 rpm
2 h of the lower stirring of speed are dissolved in 60 mL formamides, obtain the formamide solution of Fenugreek Polysaccharides.
（2）The preparation of ionic liquid：The 5 mL concentrated sulfuric acids are added drop-wise to 10 mL's at -10 DEG C with 60 drops/min speed
In 1-METHYLPYRROLIDONE, the h of 800 rpm stirring reactions 12, washed 2 times with 15 mL ethyl acetate at room temperature after completion of the reaction,
Lower floor's thick liquid is collected, being dried in vacuo 6 h at the MPa of vacuum 0.07,60 DEG C obtains ionic liquid.
（3）Activation of the ionic liquid to selenous acid：In 80 DEG C and N2Under conditions of protection, 0.6 g selenous acid is added to 4
Selenous acid after 3 h are reacted in mL ionic liquids must activating.
（4）The synthesis of selenizing Fenugreek Polysaccharides：By the formamide solution dropping funel of 60 mL Fenugreek Polysaccharides with 40
Drop/min speed is added drop-wise in the selenous acid that 4 mL ionic liquids have activated, in 70 DEG C and N24 h are reacted under conditions of protection,
128 mL acetone are added after question response under conditions of air is completely cut off, are washed 10 times at room temperature, sediment is loaded and retained
24 h of flowing water dialysis in the Da of molecular weight 8000 ~ 14000 bag filter, deionized water 18 h of dialysis, dialysis finish thing in back pkt. and existed
0.08 MPa, the 1/30 of original volume is concentrated under reduced pressure at 60 DEG C, absolute ethyl alcohol is added in concentrate（Ethanol content accounts for cumulative volume
70%）Precipitate, 10 min are centrifuged under 5000 rpm and collect lower sediment thing, 36 h are freeze-dried at 5 Pa, -55 DEG C, obtain selenium
Change Fenugreek Polysaccharides.
The selenizing Fenugreek Polysaccharides are kermesinus amorphous powder, and total sugar content 80.3%, solubility is in water
18.5mg/mL（25℃）, Se content is 28128 μ g/g, and molecular weight is 2.376 × 106Da, to the body of HepG2 liver cancer cells
Outer IC50It is worth for 591.1 μ g/mL.
Below by Determination of Selenium, infrared spectrogram, X-ray photoelectron spectroscopic analysis（XPS）, molecular weight determination to this
Structure, the activity for the selenizing Fenugreek Polysaccharides that embodiment 1 synthesizes are analyzed, and are explained：
1. the measure of Se content：Determination of Selenium is carried out to sample using fluorimetry.
Dry testing sample is weighed, adds nitration mixture（HNO3:H2SO4:HClO4=1:1:4）, at 135 DEG C after left undisturbed overnight
Middle resolution 1~2 hour, by digestion solution distilled water diluting constant volume, take 10mL add 0.2 mol/L EDTA-2Na solution and
0.5 mol/L each 2~4mL of hydroxylamine hydrochloride, 5~10min is stood after shaken well, and dark place adds 0.1% 2,3- diaminonaphthalenes
2~4mL of solution, 4~6min of boiling water bath, after taking-up is cooled to room temperature, the mL of hexamethylene 5 is added, abundant oscillation extraction, takes hexamethylene
Alkane layer determines fluorescent value in fluorescent sub-photometer（The nm of excitation wavelength 376, the nm of launch wavelength 520）, with working curve method meter
Calculate Se content.
2. infrared spectrum map analysis
Fig. 1 is the infrared spectrogram of Fenugreek Polysaccharides and the selenizing Fenugreek Polysaccharides of the invention prepared.Faenum graecum is more in figure
The infrared spectrum of sugar shows that it has the characteristic peak of polysaccharide：3400 cm-1The extremely wide strong absworption peak in left and right is the O-H of polysaccharide hydroxyl
Stretching vibration, 2922.22 cm-1Locate the stretching vibration for c h bond.Contrasted, can seen by the infrared spectrum with Fenugreek Polysaccharides
Go out selenizing Fenugreek Polysaccharides in addition to polysaccharide parent characteristic absorption peak, in 1149 cm-1Place and 669 cm-1There is new absorption in place
The stretching vibration at peak, respectively Se=O and C-O-Se flexural vibrations, illustrate the part-OH in Fenugreek Polysaccharides structure by sub- selenium
Acidic group substitutes, by selenizing.
3. XPS analysis
Fig. 2 is the XPS spectrum of Fenugreek Polysaccharides and selenizing Fenugreek Polysaccharides.It is not any in the Se 3d spectrums of Fenugreek Polysaccharides
Peak, and there are very strong Se 3d in 58.1 eV and combine energy peak in selenizing Fenugreek Polysaccharides, Se valence state is Se4+, illustrate cucurbit
By selenizing, the selenium in selenizing Fenugreek Polysaccharides occurs Abbado sugar in the form of monohydrate selenium dioxide ester.
4. molecular weight determination
Using gel chromatography-dynamic static light scattering combination measure, the weight of the selenizing Fenugreek Polysaccharides prepared in the present invention is equal
Molecular weight is 1.533 × 106 Da ~2.616×106 Da。
Embodiment 2
（1）The preparation of Fenugreek Polysaccharides formamide solution：250 mg Fenugreek Polysaccharides are turned at 80 DEG C with 300 rpm
4 h of the lower stirring of speed are dissolved in 100 mL formamides, obtain the formamide solution of Fenugreek Polysaccharides.
（2）The preparation of ionic liquid：The 4 mL concentrated sulfuric acids are added drop-wise to 10 mL N- at 0 DEG C with 50 drops/min speed
In methyl pyrrolidone, the h of 600 rpm stirring reactions 18, washed 3 times, collected with 7 mL ethyl acetate at room temperature after completion of the reaction
Lower floor's thick liquid, at the MPa of vacuum 0.06,70 DEG C being dried in vacuo 8 h obtains ionic liquid.
（3）Activation of the ionic liquid to selenous acid：In 60 DEG C and N2Under conditions of protection, 0.8 g selenous acid is added to 4
Selenous acid after 2 h are reacted in mL ionic liquids must activating.
（4）The synthesis of selenizing Fenugreek Polysaccharides：By the formamide solution dropping funel of 100 mL Fenugreek Polysaccharides with 60
Drop/min speed is added drop-wise in the selenous acid that 4 mL ionic liquids have activated, in 60 DEG C and N22 h are reacted under conditions of protection,
312 mL acetone are added after question response under conditions of air is completely cut off, are washed 12 times at room temperature, sediment is loaded and retained
48 h of flowing water dialysis in the Da of molecular weight 8000 ~ 14000 bag filter, deionized water 12 h of dialysis, dialysis finish thing in back pkt. and existed
0.06 MPa, the 1/25 of original volume is concentrated under reduced pressure at 65 DEG C, absolute ethyl alcohol is added in concentrate（Ethanol content accounts for cumulative volume
80%）Precipitate, 12 min are centrifuged under 4000 rpm and collect lower sediment thing, 48 h are freeze-dried at 20 Pa, -60 DEG C, are obtained
Selenizing Fenugreek Polysaccharides.
The selenizing Fenugreek Polysaccharides are kermesinus amorphous powder, and total sugar content 84.9%, solubility is 23.6 in water
mg/mL（25℃）, Se content is 27554 μ g/g, and molecular weight is 1.533 × 106Da, to the external IC of HepG2 liver cancer cells50
It is worth for 527.4 μ g/mL.
Embodiment 3
（1）The preparation of Fenugreek Polysaccharides formamide solution：160 mg Fenugreek Polysaccharides are turned at 60 DEG C with 400 rpm
5 h of the lower stirring of speed are dissolved in 40 mL formamides, obtain the formamide solution of Fenugreek Polysaccharides.
（2）The preparation of ionic liquid：The 5 mL concentrated sulfuric acids are added drop-wise to 10 mL N- at -5 DEG C with 80 drops/min speed
In methyl pyrrolidone, the h of 300 rpm stirring reactions 10, washed 3 times, received with 15 mL ethyl acetate at room temperature after completion of the reaction
Collect lower floor's thick liquid, being dried in vacuo 12 h at the MPa of vacuum 0.08,50 DEG C obtains ionic liquid.
（3）Activation of the ionic liquid to selenous acid：In 70 DEG C and N2Under conditions of protection, 0.6 g selenous acid is added to 4
Selenous acid after 1 h is reacted in mL ionic liquids must activating.
（4）The synthesis of selenizing Fenugreek Polysaccharides：By the formamide solution dropping funel of 40 mL Fenugreek Polysaccharides with 30
Drop/min speed is added drop-wise in the selenous acid that 4 mL ionic liquids have activated, in 80 DEG C and N23 h are reacted under conditions of protection,
110 mL acetone are added after question response under conditions of air is completely cut off, are washed 15 times at room temperature, sediment is loaded and retained
36 h of flowing water dialysis in the Da of molecular weight 8000 ~ 14000 bag filter, deionized water 24 h of dialysis, dialysis finish thing in back pkt. and existed
0.07 MPa, the 1/20 of original volume is concentrated under reduced pressure at 55 DEG C, absolute ethyl alcohol is added in concentrate（Ethanol content accounts for cumulative volume
75%）Precipitate, 15 min are centrifuged under 4500 rpm and collect lower sediment thing, 24 h are freeze-dried at 15 Pa, -50 DEG C, are obtained
Selenizing Fenugreek Polysaccharides.
The selenizing Fenugreek Polysaccharides are kermesinus amorphous powder, and total sugar content 78.1%, solubility is 20.2 in water
mg/mL（25℃）, Se content is 29773 μ g/g, and molecular weight is 2.616 × 106Da, to the external IC of HepG2 liver cancer cells50
It is worth for 630.6 μ g/mL.
Comparative example 1
（1）The preparation of Fenugreek Polysaccharides formamide solution：250 mg Fenugreek Polysaccharides are turned at 80 DEG C with 300 rpm
4 h of the lower stirring of speed are dissolved in 100 mL formamides, obtain the formamide solution of Fenugreek Polysaccharides.
（2）The synthesis of selenizing Fenugreek Polysaccharides：0.8 g selenous acid is added to the formamide solution of above-mentioned Fenugreek Polysaccharides
In, in 60 DEG C and N22 h are reacted under conditions of protection, 312 mL third are added under conditions of air is completely cut off after question response
Ketone, wash 12 times at room temperature, sediment be fitted into 48 h of flowing water dialysis in the Da of molecular cut off 8000 ~ 14000 bag filter,
Deionized water 12 h of dialysis, dialysis finishes thing in back pkt. and is concentrated under reduced pressure into the 1/25 of original volume at 0.06 MPa, 65 DEG C, dense
Absolute ethyl alcohol is added in contracting liquid（Ethanol content of the total volume 80%）Precipitate, 12 min collections lower floor is centrifuged under 4000 rpm and is sunk
Starch, 48 h are freeze-dried at 20 Pa, -60 DEG C, obtain selenizing Fenugreek Polysaccharides.
The selenizing Fenugreek Polysaccharides are pale red amorphous powder, and total sugar content 82.8%, solubility is 16.6 in water
mg/mL（25℃）, Se content is 4060 μ g/g, and molecular weight is 1.236 × 106Da, to the external IC of HepG2 liver cancer cells50
It is worth for 1230.8 μ g/mL.
Each parameter of the comparative example is corresponding with embodiment 2, and the difference of the two is：Embodiment 2 is sub- using ionic liquid activation
Formamide solution after selenic acid with Fenugreek Polysaccharides is reacted.
Table 1 is that 1-3 of the embodiment of the present invention and comparative example prepare selenizing Fenugreek Polysaccharides reaction temperature, reaction time and selenium and contained
The list of amount：
As can be seen from Table 1：Reaction temperature that Se content and selenous acid are added in the formamide solution of Fenugreek Polysaccharides and
Reaction time is relevant.
Claims (8)
1. a kind of synthetic method of selenizing Fenugreek Polysaccharides, is comprised the following steps that：
（1）The preparation of Fenugreek Polysaccharides formamide solution：By Fenugreek Polysaccharides at 60 DEG C ~ 80 DEG C, 300 ~ 500 rpm stirrings 2 ~
5 h are dissolved in formamide, obtain the formamide solution of Fenugreek Polysaccharides；
（2）The preparation of ionic liquid：The concentrated sulfuric acid is added drop-wise to N- methyl pyrroles at -10 DEG C ~ 0 DEG C with 50 ~ 80 drops/min speed
In pyrrolidone, the h of 300 ~ 800 rpm stirring reactions 10 ~ 18, washed 2 ~ 3 times, collected with ethyl acetate at room temperature after completion of the reaction
Lower floor's thick liquid, 6 ~ 12 h of vacuum drying obtain ionic liquid at 0.06 ~ 0.08 MPa, 50 ~ 70 DEG C；
（3）Activation of the ionic liquid to selenous acid：In 60 ~ 80 DEG C and N2Under conditions of protection, selenous acid is added to above-mentioned steps
（2）Selenous acid after 1 ~ 3 h of reaction must be activated in ionic liquid；
（4）The synthesis of selenizing Fenugreek Polysaccharides：By step（1）In the obtained formamide solution dropping funel of Fenugreek Polysaccharides
Step is added drop-wise to 30 ~ 60 drops/min speed（3）In the selenous acid that intermediate ion liquid has activated, in 60 ~ 80 DEG C and N2Protection
Under conditions of, 2 ~ 4 h are reacted under 300 ~ 800 rpm, after question response, under conditions of air completely cut off adding acetone washs 10
~ 15 times, it is saturating that sediment is fitted into 24 ~ 48 h of flowing water dialysis, deionized water in the Da of molecular cut off 8000 ~ 14000 bag filter
Analyse 12 ~ 24 h, dialysis finish in back pkt. thing under the MPa of vacuum 0.06 ~ 0.08,55 ~ 65 DEG C be concentrated under reduced pressure into the 1/ of original volume
20 ~ 1/30, absolute ethyl alcohol precipitation is added in concentrate, 4000 ~ 5000 rpm centrifuge 10 ~ 15 min and collect lower sediment thing,
24 ~ 48 h are freeze-dried at 5 ~ 20 Pa, -60 ~ -50 DEG C, obtain selenizing Fenugreek Polysaccharides.
2. the synthetic method of selenizing Fenugreek Polysaccharides as claimed in claim 1, it is characterised in that：Step（1）In, Fenugreek Polysaccharides
Weight average molecular weight be 2.303 × 106 Da, total sugar content 87.2%.
A kind of 3. synthetic method of selenizing Fenugreek Polysaccharides as claimed in claim 1, it is characterised in that：Step（1）In, faenum graecum
The mass volume ratio of polysaccharide and formamide is 2.5 ~ 4 mg/mL.
A kind of 4. synthetic method of selenizing Fenugreek Polysaccharides as claimed in claim 1, it is characterised in that：Step（2）In, N- methyl
The volume ratio of pyrrolidones and the concentrated sulfuric acid is 2:1~2.5:1.
A kind of 5. synthetic method of selenizing Fenugreek Polysaccharides as claimed in claim 1, it is characterised in that：Step（3）In, selenous acid
Mass volume ratio with ionic liquid is 0.125 ~ 0.2 g/mL.
A kind of 6. synthetic method of selenizing Fenugreek Polysaccharides as claimed in claim 1, it is characterised in that：Step（4）In, faenum graecum
The volume ratio of the formamide solution of polysaccharide and the selenous acid of activation is 10 ~ 25.
A kind of 7. synthetic method of selenizing Fenugreek Polysaccharides as claimed in claim 1, it is characterised in that：Step（4）In, acetone and
The volume ratio of reaction solution is 2 ~ 3.
8. described in claim 1 a kind of selenizing Fenugreek Polysaccharides synthetic method prepare selenizing Fenugreek Polysaccharides as
The inhibitor of HepG2 liver cancer cells is applied in cancer therapy drug.
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