CN107880147A - Chinese chestnut active anticancer polysaccharide and its isolation and purification method - Google Patents
Chinese chestnut active anticancer polysaccharide and its isolation and purification method Download PDFInfo
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- 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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
The invention provides a kind of Chinese chestnut active anticancer polysaccharide and its isolation and purification method, it is related to natural medicine field.The preparation method includes：Chinese chestnut Thick many candies are obtained using solvent extraction method, Chinese chestnut Thick many candies solution is transferred in bag filter and dialysed, obtains chestnut polysaccharide dialysate；And chestnut polysaccharide dialysate is separated with column chromatography, using deionized water as eluant, eluent, eluant, eluent flow velocity is 25~35mL/h.This method, by the way of dialysis and column chromatography are combined, the small-molecule substance in Chinese chestnut Thick many candies is removed, chestnut polysaccharide content is high in resulting product, and active anticancer is notable.
Description
Technical field
The present invention relates to natural medicine field, in particular to a kind of Chinese chestnut active anticancer polysaccharide and its isolates and purifies
Method.
Background technology
Chinese chestnut is a kind of with higher edible and medical value nut based food.Chinese chestnut is conventional medium-height grass among the people
Medicine, it has Kidney-invigorating and tendon-reinforcing, nourishing stomach and spleen, promoting blood circulation and hemostasis and other effects, and it is possible to treat, waist pin is weak, injured swells and ache, anti-
The common symptons such as stomach, diarrhea, cold carbuncle.In addition, the research of recent years shows, Chinese chestnut also has different physiological roles, such as：Raising is exempted from
Epidemic disease power, anticoagulation, antifatigue, anti-aging, hypotensive and hypoglycemic etc..
Modern pharmacological research shows that chestnut polysaccharide has certain reducing power, to lipid oxidation, Fenton reaction productions
Raw hydroxy radical and NO free radicals is respectively provided with certain elimination or inhibitory action, and polysaccharide concentration is higher, its anti-oxidant work
Property is stronger.Chestnut polysaccharide has certain ability for removing hydroxyl radical free radical, ultra-oxygen anion free radical and hydrogen peroxide.Mesh
Before, using the chestnut polysaccharide obtained by common extracting method, purity is relatively low, and effect is not good enough, is unfavorable for being developed further into product.
The content of the invention
The first object of the present invention is to provide a kind of isolation and purification method of Chinese chestnut active anticancer polysaccharide, using dialysis
The mode being combined with column chromatography, the small-molecule substance in Chinese chestnut Thick many candies is removed, chestnut polysaccharide in resulting product
Content is high.
The second object of the present invention is to provide a kind of Chinese chestnut active anticancer polysaccharide prepared by the above method, this Chinese chestnut
The content of active component is high in polysaccharide, and antitumaous effect is notable.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of isolation and purification method of Chinese chestnut active anticancer polysaccharide, it includes：
Chinese chestnut Thick many candies are obtained with solvent extraction method, Chinese chestnut Thick many candies solution is transferred in bag filter and dialysed, is obtained
Chestnut polysaccharide dialysate；And separate the chestnut polysaccharide dialysate, using deionized water as eluant, eluent, eluant, eluent with column chromatography
Flow velocity is 25~35mL/h.
A kind of Chinese chestnut active anticancer polysaccharide as obtained by above-mentioned isolation and purification method.
Compared with prior art, beneficial effects of the present invention for example including：
Chestnut polysaccharide preparation method provided by the invention, by dialysis, remove the small molecule thing that molecular weight is less than 7000
Matter so that the purity of chestnut polysaccharide improves；In conjunction with column chromatography, the non-polysaccharide material in chestnut polysaccharide dialysate is further removed
Matter so that the purity of chestnut polysaccharide is further improved in last products therefrom.Inventor, which studies, to be found, chestnut polysaccharide pair
The Cancerization cell of kinds cancer shows stronger inhibitory activity, can effectively suppress the propagation of its related cancerous tumor cell.
Therefore, the chestnut polysaccharide obtained by the present invention, antitumaous effect is notable, can be controlled as the anti-cancer active matter of wide spectrum for preparing
Treat the medicine of cancer.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 is Sevage methods number of processes and polysaccharide loss rate, protein removal rate relation in experimental example 2；
Fig. 2 is activated carbon in experimental example 2, hydrogen peroxide, the polysaccharide loss rate of tri- kinds of discoloration methods of macroreticular resin AB-8 and de-
The comparison of color rate；
Fig. 3 is chestnut polysaccharide DEAE-52 cellulose chromatography elution curves in experimental example 2.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products that can be obtained by commercially available purchase.
Present embodiment provides a kind of preparation method for separating and purifying of chestnut polysaccharide, and it includes：
Step S1:Chinese chestnut Thick many candies are prepared using solvent extraction method.
It is more preferable, the Chinese chestnut powder for crossing 40~60 mesh sieves is mixed with Extraction solvent, carried out using solvent extraction method
Extraction, the extract solution of gained is concentrated, precipitated, and Chinese chestnut Thick many candies are made.
Wherein, solvent extraction method includes：Soxhlet extraction, hot water extraction method, extraction, pressurized liquid extraction method,
Enzyme assisted extraction method, microwave loss mechanisms, ultrasonic extraction, supercritical extraction etc..Carried by said extracted method from Chinese chestnut
Obtain Chinese chestnut Thick many candies solution.
Further, when solvent extraction method is pressurized liquid extraction method, it includes：Chinese chestnut powder is mixed with diatomite
Load extraction container, by extractant of deionized water in temperature be 50~80 DEG C, pressure be under 3~9MPa extraction 4~
12min, circulate 2~4 times.
Further, when solvent extraction method is ultrasonic extraction, it includes：Chinese chestnut powder is mixed with water, in work(
Rate is 500~1000W, temperature is 0.5~1h of ultrasound at 20~50 DEG C, is circulated 1~3 time.
Further, solvent extraction method is microwave loss mechanisms, and it includes：The Chinese chestnut powder is mixed with water, in microwave
Extracted in container, extract 500~1000W of power, centrifuged after extracting 0.5~1min.
Further, solvent extraction method is extraction, and it includes：The Chinese chestnut powder is mixed with Extraction solvent, in 40
Extract at~80 DEG C, centrifuged after extracting 0.5~2h.Wherein, Extraction solvent is water or sour water, and more preferably, sour water is salt
Aqueous acid, further, the concentration of aqueous hydrochloric acid solution is 0.5-1mol/L.
Step S2：The Chinese chestnut Thick many candies solution obtained by solvent extraction method is transferred in bag filter and dialysed, is obtained
Chestnut polysaccharide dialysate.
Further, when carrying out dialysis treatment to Chinese chestnut Thick many candies solution, change water every 3~5h and (or changed every 4h
Water), dialyse 1.5~3 days, either 1.8~2.5 days or for 2 days.
Further, before dialysis treatment is carried out to Chinese chestnut Thick many candies solution, in addition to Chinese chestnut Thick many candies solution is entered
Row decolorization.Wherein, decolorization includes：Activated carbon decolorizing method, macroreticular resin decoloring method, hydrogen peroxide for decoloration method.
Wherein, activated carbon decolorizing method includes：Chinese chestnut Thick many candies solution is mixed with activated carbon, taken off at 30~50 DEG C
Color, time are 25~35min, and the pH value of the Chinese chestnut Thick many candies solution after regulation decolouring is 6.5~7.5.Preferably, bleaching temperature
For 30~50 DEG C, it is either 35~45 DEG C or is 38~42 DEG C, or for 40 DEG C.Bleaching time is 25~35min, or
For 28~32min, or it is 30min.The pH value of Chinese chestnut Thick many candies solution after regulation decolouring is 6.5~7.5, or is 6.8
~7.2, or be 7.0.
Further, macroreticular resin decoloring method includes：It is slightly more that post separation Chinese chestnut is filled using macroreticular resin AB-8 after pretreatment
Sugar juice, using deionized water as eluant, eluent, eluant, eluent flow velocity is that 250~350mL/h (or is 270~330mL/h, Huo Zhewei
290~310mL/h, or be 300mL/h), Fractional Collections, every 25~35min (or be 28~32min, Huo Zhewei
30min) collect one bottle.
Further, before dialysis treatment is carried out to Chinese chestnut Thick many candies solution, in addition to Chinese chestnut Thick many candies solution is entered
The de- albumen processing of row.More specifically, taking off albumen processing includes：Isometric Sevage reagents are added in Chinese chestnut crude extract
(chloroform/n-butanol=3~6:1, V/V), mixture acutely shakes 50~70min, removes precipitating proteins, repeat more than
Step 3~7 time, obtain Chinese chestnut Thick many candies and take off protein liquid.
Step S2:Chestnut polysaccharide dialysate is separated with column chromatography, using deionized water as eluant, eluent, eluant, eluent flow velocity is 25
~35mL/h, is either 27~33mL/h or is 29~31mL/h, or is 30mL/h.
Further, column chromatography includes cellulose chromatography method or gel filtration chromatography method.
Further, the cellulose used in cellulose chromatography method is DEAE- cellulose.Enter with DEAE- celluloses
Before luggage post, in addition to the activated processing of DEAE celluloses after, add 3~5 times of volumes deionized water, soak 12~48h
Afterwards, immersion is washed respectively with acid, alkali, water, be washed till neutrality, fill post.
Further, in column chromatography, be washed with deionized water it is de- during, Fractional Collections, every 8~12mL (or
For 9~11mL, or it is 10mL) pipe is collected, processing is merged using Phenol sulfuric acid procedure detection polyoses content.
The chestnut polysaccharide obtained by the above method, its weight average molecular weight are 5 × 102~1 × 107Da.Chinese chestnut has multiple
Kind, such as：Yanshan Mountain Kui Li, Yanshan Mountain brachyplast, abide by beautiful, the Yanshan Mountain is early rich, big plate is red, swallow king, Qianxi are early red, late red, the purple amber in Qianxi,
Great Feng, distant chestnut ten, oily hazel and dirty oil hazel etc..The present invention have studied from respectively not using the Chinese chestnut of above-mentioned kind as research object
With the active anticancer of obtained chestnut polysaccharide in the Chinese chestnut of germline.
Kui Li research find, from the Kui Li of the Yanshan Mountain extraction made from chestnut polysaccharide, its have to liver cancer, stomach cancer and lung cancer compared with
Good active anticancer, is in particular in, HepG2 and BEL-7402 cells to liver cancer and to the BGC-823 cells of stomach cancer
There is stronger inhibitory action.The chestnut polysaccharide made from extraction from the brachyplast of the Yanshan Mountain, it has preferable active anticancer to liver cancer, tool
Body surface is present, and it can suppress HepG2 hepatoma cell proliferations.From abiding by beautiful Chinese chestnut chestnut polysaccharide made from extraction, its to liver cancer,
Lung cancer is respectively provided with preferable active anticancer, is in particular in, it can suppress HepG2 liver cancer cells, A-549 lung carcinoma cells increase
Grow.
Thus, from Yanshan Mountain Kui Li, Yanshan Mountain brachyplast and the chestnut polysaccharide extracted in beautiful Chinese chestnut is abided by, the anticancer with more wide spectrum
Activity is especially the most notable to the active anticancer of liver cancer.
The feature and performance of the present invention are described in further detail with reference to embodiments：
Embodiment 1
The present embodiment provides a kind of chestnut polysaccharide, and its preparation method includes：
A. abstraction pool is loaded after being sufficiently mixed rice-chestnut powder and diatomite, using deionized water as extractant, extraction temperature
70 DEG C, extraction time 8min, extracting pressure 6MPa, circulate 2 times.Extract solution after the completion of extraction to chestnut polysaccharide centrifuged,
Chinese chestnut Thick many candies are obtained after precipitation.
B. Chinese chestnut Thick many candies solution is transferred in bag filter, dialysed with deionized water, water is changed once every 3h, dialysed
1.5 days, to remove the small molecule of residual and ion, obtain chestnut polysaccharide dialysate.
C. by Chinese chestnut dialyzate Sephadex G-100 gel filtration chromatographies, using water as eluant, eluent, the elution speed is controlled to be
25ml/h, fraction collection, often pipe 8mL, pipe take a small amount of eluent, with Phenol sulfuric acid procedure detect polyoses content, until sugar-free examine
Untill going out.The eluent containing polysaccharide component is collected, obtains chestnut polysaccharide.
Embodiment 2
The present embodiment provides a kind of chestnut polysaccharide, and its preparation method for separating and purifying includes：
A. Chinese chestnut powder and deionized water are mixed with liquid ratio 40ml/g ratio, carried with ultrasonic wave biological abstraction instrument
Taking, ultrasonic power 870W, 30 DEG C, ultrasonic time 50min of extraction temperature, centrifugation merges supernatant, extract solution is concentrated,
Chinese chestnut Thick many candies are obtained after precipitation；
B. Chinese chestnut Thick many candies solution is transferred in bag filter, dialysed with deionized water, water is changed once every 4h, dialysis 2
My god, to remove the small molecule of residual and ion, obtain chestnut polysaccharide dialysate.
C. by Chinese chestnut dialyzate DEAE-52 cellulose chromatographies, using water as eluant, eluent, it is 30mL/ to control elution speed
H, fraction collection, often pipe 10mL, pipe take a small amount of eluent, with Phenol sulfuric acid procedure detect polyoses content, until sugar-free detection be
Only.The eluent containing polysaccharide component is collected, obtains chestnut polysaccharide.
Embodiment 3
The present embodiment provides a kind of chestnut polysaccharide, and its preparation method for separating and purifying includes：
A. rice-chestnut powder and deionized water are mixed with liquid ratio 30ml/g ratio, in being extracted in micro-wave oven, extracts power
800W, extraction time 50s, centrifugation merge supernatant, extract solution is concentrated, precipitate after obtain Chinese chestnut Thick many candies.
B. by Chinese chestnut Thick many candies solution and Sevage reagents (chloroform/n-butanol=4:1, V/V) equal proportion mixes.It is mixed
Compound acutely shakes 30min, removes precipitating proteins, repeats above step 7 times, obtains chestnut polysaccharide and takes off protein liquid.
C. Chinese chestnut Thick many candies solution is mixed with activated carbon, decolourized at 40 DEG C, time 30min, regulation is decolourized
The pH value of Chinese chestnut Thick many candies solution afterwards is 7.0, obtains Chinese chestnut Thick many candies destainer.
D. Chinese chestnut Thick many candies destainer is transferred in bag filter, dialysed with deionized water, water is changed once every 5h, dialysed
3 days, to remove the small molecule of residual and ion, obtain chestnut polysaccharide dialysate.
E. by Chinese chestnut dialyzate DEAE-52 cellulose chromatographies, using water as eluant, eluent, it is 35mL/ to control elution speed
H, fraction collection, often pipe 12mL, pipe take a small amount of eluent, with Phenol sulfuric acid procedure detect polyoses content, until sugar-free detection be
Only.The eluent containing polysaccharide component is collected, obtains chestnut polysaccharide.
Embodiment 4
The present embodiment provides a kind of chestnut polysaccharide, and its preparation method for separating and purifying includes：
A. rice-chestnut powder and 0.72mol/L hydrochloric acid are mixed with liquid ratio 20ml/g ratio, extracts, carry in water-bath
Take temperature 70 C, extraction time 40min, centrifugation merges supernatant, extract solution is concentrated, precipitate after to obtain Chinese chestnut slightly more
Sugar.
B. by Chinese chestnut Thick many candies solution and Sevage reagents (chloroform/n-butanol=6:1, V/V) equal proportion mixes.It is mixed
Compound acutely shakes 50min, removes precipitating proteins, repeats above step 3 times, obtains chestnut polysaccharide and takes off protein liquid.
C. the chestnut polysaccharide is taken off into protein liquid macroreticular resin AB-8 column chromatographies to decolourize, using water as eluant, eluent, control is washed
De- speed is 350ml/h, fraction collection, collects a pipe every 25min, polyoses content is detected with Phenol sulfuric acid procedure, until sugar-free
Untill detection.The eluent containing polysaccharide component is collected, obtains chestnut polysaccharide destainer.
D. Chinese chestnut Thick many candies destainer is transferred in bag filter, dialysed with deionized water, water is changed once every 4h, dialysed
2 days, to remove the small molecule of residual and ion, obtain chestnut polysaccharide dialysate.
E. by the Chinese chestnut dialyzate DEAE-52 cellulose chromatographies, using water as eluant, eluent, the elution speed is controlled to be
30mL/h, fraction collection, often pipe 10mL, pipe take a small amount of eluent, with Phenol sulfuric acid procedure detect polyoses content, until sugar-free examine
Untill going out.The eluent containing polysaccharide component is collected, obtains chestnut polysaccharide.
Embodiment 5
The present embodiment provides a kind of chestnut polysaccharide, and its preparation method for separating and purifying includes：
A. rice-chestnut powder and deionized water are mixed with liquid ratio 35ml/g ratio, extracted in water-bath, Extracting temperature
65 DEG C, extraction time 1h, centrifugation merge supernatant, extract solution is concentrated, precipitate after obtain Chinese chestnut Thick many candies.
B. by Chinese chestnut Thick many candies solution and Sevage reagents (chloroform/n-butanol=3:1, V/V) equal proportion mixes.It is mixed
Compound acutely shakes 70min, removes precipitating proteins, repeats above step 3 times, obtains chestnut polysaccharide and takes off protein liquid.
C. chestnut polysaccharide is taken off into protein liquid macroreticular resin AB-8 column chromatographies to decolourize, using water as eluant, eluent, control elution speed
Spend and managed for 300mL/h, fraction collection every 30min collections one, polyoses content is detected with Phenol sulfuric acid procedure, until sugar-free detects
Untill.The eluent containing polysaccharide component is collected, obtains chestnut polysaccharide destainer.
D. Chinese chestnut Thick many candies destainer is transferred in bag filter, dialysed with deionized water, water is changed once every 4h, dialysed
2 days, to remove the small molecule of residual and ion, obtain chestnut polysaccharide dialysate.
E. by the Chinese chestnut dialyzate DEAE-52 cellulose chromatographies, using water as eluant, eluent, the elution speed is controlled to be
30mL/h, fraction collection, often pipe 10mL, pipe take a small amount of eluent, with Phenol sulfuric acid procedure detect polyoses content, until sugar-free examine
Untill going out.The eluent containing polysaccharide component is collected, obtains chestnut polysaccharide.
Experimental example 1
The comparison of Different Extraction Method：
The extracting method of the Chinese chestnut Thick many candies provided using a steps in embodiment 1~5 (is followed successively by：Accelerate solvent extraction
Method, ultrasonic assistant extraction method, microwave loss mechanisms, sour formulation, water extraction) prepare Chinese chestnut Thick many candies, and in every kind of method
Extraction time, solvent load and extraction yield investigated, as a result as shown in table 1：
The comparison of the Different Extraction Method of the Chinese chestnut Thick many candies of table 1.
From the Different Extraction Method of table 1 relatively：Time used in pressurized liquid extraction method is short, and circulation time of 2 times is only
16min, other 4 kinds of assisted extraction methods required times are longer than pressurized liquid extraction method, and to reach pressurized liquid extraction method
Similar extraction effect needs 12h or so；In addition, pressurized liquid extraction method solvent usage amount is few, and extract small volume, polysaccharide concentration
Height, vacuum concentration step is eliminated, improve extraction efficiency, the extraction of polysaccharide extract rate specific heat water, Microwave Extraction, acid extraction difference
Improve 3.20,13.86,6.50 percentage points.Pressurized liquid extraction method is compared with ultrasonic wave extraction, although recovery rate reduces
2.37 percentage points, but within the equal time (16min), the polysaccharide extract rate of ultrasonic assistant extraction is only 8.72%,
Well below pressurized liquid extraction method.
Experimental example 2
The different purification process of chestnut polysaccharide compare：
1.Sevage methods remove removing protein：
Using step b in embodiment 3, i.e., isometric Sevage reagents (chloromethanes of V tri- are added in Chinese chestnut Thick many candies solution
Alkane：V n-butanol=4:1), mixture acutely shakes 30min, removes precipitating proteins, takes a small amount of supernatant to be measured.More than repeating
Step 7 time, obtain chestnut polysaccharide and take off protein liquid.
Using Coomassie Brilliant Blue, protein standard curve, Y=7.7895x+0.017, its coefficient of determination R are built2For
0.9987.Through Sevage method techniques deproteinized operation totally 7 times, polysaccharide and protein content in gained supernatant every time, meter are determined
Calculate polysaccharide loss rate and protein removal rate.
As a result it as shown in figure 1, after 7 Sevage methods go isolating protein technique, can reach the clearance of protein
80%, deproteinized effect is preferably and polysaccharide loss rate is low.
2. decolourize：
Using different methods, to the above-mentioned de- protein liquid progress of chestnut polysaccharide that gained after removing protein is removed using Sevage methods
Decolourize：
(1) discoloration method：
A. activated carbon decolorizing method：40 DEG C of temperature, activated carbon addition are 2%, decolouring 30min, are adjusted to pH 7 and calculate polysaccharide
Percent of decolourization and loss late.
B. hydrogen peroxide decoloring method：40 DEG C of temperature, add 20%H2O2, decolouring 3h, pH 7 is adjusted to, calculates polysaccharide percent of decolourization
And loss late.
C. macroreticular resin AB-8 column chromatographies decoloring method：Using deionized water as eluant, eluent, eluant, eluent flow velocity is 300mL/h, point
Section is collected, and one bottle is collected every 30min.Polyoses content is detected with Phenol sulfuric acid procedure, light absorption value is determined at 490nm, will be contained
The eluent of polysaccharide, which is collected, to be merged, and concentration is settled to 100ml, calculates polysaccharide percent of decolourization and loss late.
(2) decolouring result：
Because polysaccharide component is complex in extract solution, it is difficult to the species of pigment is determined, and the decolouring to natural products is temporary
Method without standard, the Chinese chestnut Thick many candies destainer 1ml of gained after above-mentioned three kinds of methods decolouring is taken, is diluted to 50ml, use is ultraviolet
Its light absorption value of spectrophotometry, the obtained the maximum absorption for obtaining chestnut polysaccharide coloring matter is 400nm.Polysaccharide solution is determined to decolourize
Front and rear light absorption value, reference is made with distilled water, calculates percent of decolourization formula:
Absorbance × 100% before percent of decolourization=(absorbance after absorbance-decolouring before decolouring)/decolouring
As a result as shown in Fig. 2 experiment compare activated carbon decolorizing, hydrogen peroxide for decoloration, macroreticular resin AB-8 decolourize result can
Know, polysaccharide loss rate：Macroreticular resin AB-8 decolouring ＜ activated carbon decolorizing ＜ hydrogen peroxide for decoloration, percent of decolourization：Macroreticular resin AB-8 takes off
Color ＞ activated carbon decolorizing ＞ hydrogen peroxide for decoloration, it can be seen that：Macroreticular resin AB-8 decolorizing effects are better than other two methods.Cause
This, the removing of pigment is advisable with macroreticular resin AB-8 chromatographic decolorizations, and after the method is decolourized, polysaccharide loss rate is 28.44, percent of decolourization
For 86.36%.
3. purifying：
Obtained Chinese chestnut Thick many candies are extracted with pressurized liquid extraction method, are operated through removing protein, macroreticular resin decolourizes, dialysis
Afterwards, DEAE-52 celluloses method, Sephadex G-100 gel filtration chromatographies are further purified.By gained washing containing polysaccharide
De- liquid, which is collected, to be merged, and after concentration, produces holosaccharide liquid, holosaccharide liquid obtains Chinese chestnut holosaccharide through ethanol precipitation after freeze-drying.
Isolated and purified through DEAE-52 cellulose chromatographies and Sephadex G-100 gel filtration chromatographies, with phenol-sulphur
Acid system detects polyoses content, and an only main peak, and peak shape almost symmetry, it is one-component to show the chestnut polysaccharide, as a result such as
Shown in Fig. 3.
Experimental example 3
This experimental example is detected using mtt assay to the active anticancer of gained chestnut polysaccharide in embodiment 2：
First, detection method：
By cancer cell inoculation in 96 holes, per hole 100mL (containing 1000 cancer cells), be placed in saturated humidity, 37 DEG C and 5%
CO2Dosing after culture 24h in incubator, given the test agent sets 5 concentration (being respectively 1000,100,10,1,0.1 μ g/mL), per concentration
3 parallel holes, put culture 4 days in incubator.Discard nutrient solution, MTT solution is added per hole (0.4mg/mL, RPMI1640 are prepared)
100ml, 37 DEG C of incubation 4h.Supernatant is abandoned, DMSO 150mL are added per hole, dissolves Fomazan particles, after gentle agitation, uses enzyme
Mark instrument determines OD values under Detection wavelength 540nm, reference wavelength 405nm.(positive control drug is taxol).
As a result calculate：Dose-effect curve is can obtain with the various concentrations of medicine and the inhibiting rate mapping to cell, therefrom
Obtain half-inhibition concentration (IC50)。
2nd, result of the test：
With human colon cancer cell HCT-116, hepatocellular carcinoma H22, stomach cancer cell BGC-823, lung carcinoma cell NCI-
H1650, ovarian cancer cell A2780, liver cancer cells BEL-7402, lung carcinoma cell A-549 are experiment cell, and Chinese chestnut is extracted in test
Polysaccharide is to the inhibitory action of a variety of cancer cells, as a result as shown in table 2.
Table 2. extracts histamine result of the chestnut polysaccharide to a variety of cancer cells
Table 2 shows that Yanshan Mountain Kui Li chestnut polysaccharides have very strong suppression BEL-7402 human hepatoma cell proliferations activity, and living
Property is better than positive drug taxol.In addition, Yanshan Mountain Kui Li chestnut polysaccharides to HepG2 human liver cancer cells, NCI-H1650 lung carcinoma cells and
BGC-823 stomach cancer cells also have stronger inhibitory activity.Abiding by beautiful and Yanshan Mountain brachyplast chestnut polysaccharide has very strong suppression HepG2 people
Hepatoma cell proliferation activity, activity is suitable with taxol, and abides by beautiful chestnut polysaccharide to human colon cancer cell HCT-116 and lung cancer
Cell A-549 also has certain inhibitory action.Big peak chestnut polysaccharide is to human colon cancer cell HCT-116 and ovarian cancer cell
A2780 has stronger inhibitory action, and dirty oil hazel chestnut polysaccharide then there is stronger suppression to make ovarian cancer cell A2780
With.
Although illustrate and describing the present invention with specific embodiment, but will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of isolation and purification method of Chinese chestnut active anticancer polysaccharide, it is characterised in that it includes：
Chinese chestnut Thick many candies are obtained with solvent extraction method, Chinese chestnut Thick many candies solution is transferred in bag filter and dialysed, obtains plate
Chestnut polysaccharide dialysate；And separate the chestnut polysaccharide dialysate, using deionized water as eluant, eluent, eluent stream with column chromatography
Speed is 25~35mL/h.
2. isolation and purification method according to claim 1, it is characterised in that the solvent extraction method extracts including pressurized solvent
Follow the example of, the pressurized liquid extraction method includes：The Chinese chestnut powder is mixed with diatomite and loads extraction container, with deionized water
For extractant in temperature be 50~80 DEG C, pressure be under 3~9MPa extraction 4~12min, circulate 2~4 times.
3. isolation and purification method according to claim 1, it is characterised in that the column chromatography includes cellulose chromatography
Method or gel filtration chromatography method.
4. isolation and purification method according to claim 3, it is characterised in that the fibre used in the cellulose chromatography method
It is DEAE- celluloses to tie up element.
5. isolation and purification method according to claim 1, it is characterised in that in the column chromatography, with deionization
During water elution, every 8~12mL collects a pipe, and processing is merged using Phenol sulfuric acid procedure detection polyoses content.
6. isolation and purification method according to claim 1, it is characterised in that carried out thoroughly to the Chinese chestnut Thick many candies solution
Before analysis processing, in addition to decolorization is carried out to the Chinese chestnut Thick many candies solution.
7. isolation and purification method according to claim 6, it is characterised in that the decolorization includes：Activated carbon decolorizing
Method, macroreticular resin decoloring method, hydrogen peroxide for decoloration method.
8. isolation and purification method according to claim 7, it is characterised in that the macroreticular resin decoloring method includes：Using
Macroreticular resin AB-8 fills Chinese chestnut Thick many candies solution described in post separation, using deionized water as eluant, eluent, eluant, eluent flow velocity after pretreatment
For 250~350mL/h, Fractional Collections, one bottle is collected every 25~35min.
9. isolation and purification method according to claim 1, it is characterised in that carried out thoroughly to the Chinese chestnut Thick many candies solution
Before analysis processing, in addition to de- albumen processing is carried out to the Chinese chestnut Thick many candies solution using Sevage methods.
A kind of 10. Chinese chestnut active anticancer polysaccharide as obtained by isolation and purification method according to any one of claims 1 to 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711426638.1A CN107880147A (en) | 2017-12-26 | 2017-12-26 | Chinese chestnut active anticancer polysaccharide and its isolation and purification method |
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| CN109287871A (en) * | 2018-09-18 | 2019-02-01 | 青岛农业大学 | A method of animal semen quality is improved using chestnut polysaccharide |
| CN112973641A (en) * | 2021-02-01 | 2021-06-18 | 仲恺农业工程学院 | Chestnut shell treatment process |
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| CN109287871A (en) * | 2018-09-18 | 2019-02-01 | 青岛农业大学 | A method of animal semen quality is improved using chestnut polysaccharide |
| CN109287871B (en) * | 2018-09-18 | 2021-12-28 | 青岛农业大学 | Method for improving animal semen quality by using Chinese chestnut polysaccharide |
| CN112973641A (en) * | 2021-02-01 | 2021-06-18 | 仲恺农业工程学院 | Chestnut shell treatment process |
| CN112973641B (en) * | 2021-02-01 | 2023-06-23 | 仲恺农业工程学院 | Chestnut shell treatment process |
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