CN105111326B - A kind of preparation method and applications of Polysaccharide in Pleurotus eryngii component - Google Patents
A kind of preparation method and applications of Polysaccharide in Pleurotus eryngii component Download PDFInfo
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- CN105111326B CN105111326B CN201510586754.4A CN201510586754A CN105111326B CN 105111326 B CN105111326 B CN 105111326B CN 201510586754 A CN201510586754 A CN 201510586754A CN 105111326 B CN105111326 B CN 105111326B
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Abstract
The present invention relates to a kind of preparation method of Polysaccharide in Pleurotus eryngii component, this method comprises the following steps：(1) extract：Add water carry out water extraction after dry pleurotus eryngii is crushed, through being filtrated to get filtrate；The filtrate produces pleurotus eryngii water extract through being dried under reduced pressure to paste；(2) alcohol precipitation：The pleurotus eryngii water extract water adds ethanol solution alcohol precipitation after dissolving, through centrifugation, dry Polysaccharide in Pleurotus eryngii crude extract；(3) cellulose column chromatography is separated：The Polysaccharide in Pleurotus eryngii crude extract carries out gradient elution after being dissolved in water using cellulose column, collects 0.4 mol/L sodium-chloride water solution eluents；The eluent is through being dried under reduced pressure, dialysis membrane dialysis desalination, and the Polysaccharide in Pleurotus eryngii component for producing pale yellow powder shape is finally dried under reduced pressure to trapped fluid in film.Present invention process is simple, easy scale, and the Polysaccharide in Pleurotus eryngii component obtained has the effect of good inhibitor against colon carcinoma cells, can be applied to prepare drugs against colon cancer or health food.
Description
Technical field
The present invention relates to a kind of preparation method of polysaccharide component, more particularly to a kind of preparation method of Polysaccharide in Pleurotus eryngii component
And its application.
Background technology
Pleurotus eryngii（Pleurotus eryngiiQuel.）It is subordinate to Eumycota, Basidiomycetes, Agaricales, Pleurotaceae, picks up the ears
Belong to fungi, be described as " king in mushroom ", be distributed mainly on typical subtropical zone grassland-Arid desert region.Modern study shows
Polysaccharide is rich in pleurotus eryngii, polysaccharide has pharmacological effect that is antiviral, antitumor and improving immunity.
Chi Guirong reports are taken off using the extraction of chloroform-methanol mixed solvent, 95% ethanol alcohol precipitation, activated carbon decolorizing, Sevage methods
Holosaccharide in albumen, Sephadex G-50, Sephadex G-100 column chromatography for separation pleurotus eryngiis, process is cumbersome and loss amount
Greatly, and at present the preparation research for the polysaccharide component that quick obtaining inhibitor against colon carcinoma cells cell is bred has no document report from pleurotus eryngii.
The content of the invention
It is simple, easy scale Polysaccharide in Pleurotus eryngii component that the technical problems to be solved by the invention are to provide a kind of technique
Preparation method.
Another technical problem to be solved by this invention is to provide the application of the Polysaccharide in Pleurotus eryngii component.
To solve the above problems, a kind of preparation method of Polysaccharide in Pleurotus eryngii component of the present invention, comprises the following steps：
(1) extract：Dry pleurotus eryngii powder is broken to after 50 ~ 120 mesh the carry out water extraction that adds water, through being filtrated to get filtrate；The filter
Liquid produces pleurotus eryngii water extract through being dried under reduced pressure to paste；
(2) alcohol precipitation：The pleurotus eryngii water extract is dissolved with the water of 5 ~ 10 times of its quality, then adds the apricot Bao
The volume fraction of 10 ~ 20 times of mushroom water extract quality is 85% ~ 95% ethanol solution alcohol precipitation, and precipitation is collected by centrifugation, and repeats 1 ~ 3
It is secondary, merge sediment, the sediment is through dry Polysaccharide in Pleurotus eryngii crude extract；
(3) cellulose column chromatography is separated：The water that the Polysaccharide in Pleurotus eryngii crude extract adds 10 ~ 20 times of its quality is dissolved,
Then cellulose column is used respectively with water, 0.1 mol/L sodium-chloride water solutions, 0.4 mol/L sodium-chloride water solutions by 5 ~ 10 times
Column volume carry out gradient elution, collect 0.4 mol/L sodium-chloride water solution eluents；The eluent is through being dried under reduced pressure to 0.5
After the L of L ~ 1.0, dialysed the h of the h of desalination 15 ~ 24 with dialysis membrane, finally trapped fluid in film is dried under reduced pressure and produces pale yellow powder shape
Polysaccharide in Pleurotus eryngii component.
(1) middle abstract methods refer to that solid-liquid ratio is 1g to the step：10 ~ 30 mL, temperature are 80 DEG C ~ 95 DEG C, extraction time
It is 1 ~ 5 h for 1 ~ 3 time, each extraction time.
The step (1), the step (3) in be dried under reduced pressure condition each mean vacuum be 0.07 ~ 0.09 MPa, temperature
For 65 ~ 80 DEG C.
(2) middle drying condition is that temperature is 65 ~ 80 DEG C to the step.
(3) middle cellulose column is DEAE-52 to the step.
The step (3) middle dialysis membrane molecular cut off be 3000 D ~ 10000D.
Polysaccharide in Pleurotus eryngii component made from a kind of preparation method of Polysaccharide in Pleurotus eryngii component as described above is preparing resistive connection
Application in bowelcancer medicine or health food, it is characterised in that：The Polysaccharide in Pleurotus eryngii component as active principle according to a conventional method
Be made all kinds of inhibitor against colon carcinoma cells preparations with pharmaceutically acceptable any carrier, or as active principle according to a conventional method with food section
All kinds of health care based foods are made in acceptable any carrier on.
The present invention has advantages below compared with prior art：
1st, the present invention is extracted using water, can be by fat-soluble small molecule or large biological molecule and water-soluble polysaccharide in pleurotus eryngii
Separate, not only inexpensive, and easily scale is implemented.
2nd, the present invention can effectively remove sample small molecular compound using alcohol precipitation processing.
3rd, cellulose column of the present invention can be used with regeneration cycle, while cellulose column is without using organic reagent, to environment not
Pollute, while preparation method is simple, average unit cost is cheap.
4th, the Polysaccharide in Pleurotus eryngii component that is obtained of the present invention after tested, the effect with good inhibitor against colon carcinoma cells.
(1) experimental method is carried out using international MTT assay methods：First, 2 × 10 are inoculated with 96 porocyte plates4
After the colon cancer cell HCT116 of individual exponential phase, 3 multiple holes, cell attachment；Various concentrations testing sample is added, is set altogether
7 drug concentration gradients are put, are respectively：1.0 μg/mL、5.0 μg/mL、10.0 μg/mL、20 μg/mL、30 μg/mL、40
The μ g/mL and 50 μ g/mL polysaccharide component sample sets；After 72 hours, the MTT that 5 mg/mL are added in 96 orifice plate corresponding apertures is molten
The μ L of liquid 20, continue to cultivate 3 h；Supernatant in 96 orifice plates is discarded, 100 μ L DMSO dissolvings is added, 570 is detected using ELIASA
Light absorption value under nm wavelength simultaneously calculates half-inhibition concentration IC of the testing sample to Growth of Colon Cancer Cells50。
Test result indicates that, 1 ~ 3 group of polysaccharide component of embodiment has obvious inhibitory action to colon cancer cell line, according to
Secondary is 35.4 μ g/mL, 34.2 μ g/mL, 36.1 μ g/mL, and average value is 35.2 μ g/mL.
Embodiment
A kind of preparation method of Polysaccharide in Pleurotus eryngii component of embodiment 1, comprises the following steps：
(1) extract：The 1.0 Kg pleurotus eryngii powders dried are broken to after 120 mesh the carry out water extraction that adds water, through being filtrated to get filtrate；
The filtrate is that 0.07 MPa, temperature are, through being dried under reduced pressure to paste, to produce the extraction of pleurotus eryngii water under conditions of 80 DEG C in vacuum
The g of thing 402.4.
Wherein：Abstract methods refer to that solid-liquid ratio is 1g：10 mL, temperature are 95 DEG C, extraction time is 3 times, when extracting every time
Between be 1 h.
(2) alcohol precipitation：Pleurotus eryngii water extract is dissolved with the water of 5 times of its quality, then adds pleurotus eryngii water extract matter
The volume fraction of 20 times of amount is 85% ethanol solution alcohol precipitation, and precipitation is collected by centrifugation, is repeated 1 times, and merges sediment, the sediment
It is dry the g of Polysaccharide in Pleurotus eryngii crude extract 184.5 under conditions of 65 DEG C in temperature.
(3) cellulose column chromatography is separated：The water that Polysaccharide in Pleurotus eryngii crude extract adds 10 times of its quality is dissolved, and is then adopted
5 ~ 10 times are pressed with DEAE-52 cellulose columns respectively with water, 0.1 mol/L sodium-chloride water solutions, 0.4 mol/L sodium-chloride water solutions
Column volume carry out gradient elution, collect 0.4 mol/L sodium-chloride water solution eluents；The eluent is through being dried under reduced pressure to 0.5
After L, with the dialysis membrane dialysis h of desalination 24, finally to trapped fluid in film in the condition that vacuum is 0.07 MPa, temperature is 80 DEG C
Under be dried under reduced pressure the g of Polysaccharide in Pleurotus eryngii component 54.8 for producing pale yellow powder shape.
Wherein：The molecular cut off of dialysis membrane is 3000 D.
A kind of preparation method of Polysaccharide in Pleurotus eryngii component of embodiment 2, comprises the following steps：
(1) extract：The 5.0 Kg pleurotus eryngii powders dried are broken to after 50 mesh the carry out water extraction that adds water, through being filtrated to get filtrate；Should
Filtrate is that 0.09 MPa, temperature are, through being dried under reduced pressure to paste, to produce pleurotus eryngii water extract under conditions of 65 DEG C in vacuum
1845.0 g。
Wherein：Abstract methods refer to that solid-liquid ratio is 1g：30 mL, temperature are 80 DEG C, extraction time is 1 time, when extracting every time
Between be 5 h.
(2) alcohol precipitation：Pleurotus eryngii water extract is dissolved with the water of 10 times of its quality, then adds pleurotus eryngii water extract
The volume fraction that 10 times of quality is 95% ethanol solution alcohol precipitation, and precipitation is collected by centrifugation, is repeated 3 times, and merges sediment, the precipitation
Thing is dry the g of Polysaccharide in Pleurotus eryngii crude extract 864.3 under conditions of 80 DEG C in temperature.
(3) cellulose column chromatography is separated：The water that Polysaccharide in Pleurotus eryngii crude extract adds 20 times of its quality is dissolved, and is then adopted
With DEAE-52 cellulose columns respectively with water, 0.1 mol/L sodium-chloride water solutions, 0.4 mol/L sodium-chloride water solutions by 10 times
Column volume carries out gradient elution, collects 0.4 mol/L sodium-chloride water solution eluents；The eluent is through being dried under reduced pressure to 1.0 L
Afterwards, dialysed the h of desalination 15 with dialysis membrane, be finally that 0.09 MPa, temperature are under conditions of 65 DEG C in vacuum to trapped fluid in film
It is dried under reduced pressure the g of Polysaccharide in Pleurotus eryngii component 262.6 for producing pale yellow powder shape.
Wherein：The molecular cut off of dialysis membrane is 10000D.
A kind of preparation method of Polysaccharide in Pleurotus eryngii component of embodiment 3, comprises the following steps：
(1) extract：The 2.0 Kg pleurotus eryngii powders dried are broken to after 80 mesh the carry out water extraction that adds water, through being filtrated to get filtrate；Should
Filtrate is that 0.08 MPa, temperature are, through being dried under reduced pressure to paste, to produce pleurotus eryngii water extract under conditions of 75 DEG C in vacuum
796.6 g。
Wherein：Abstract methods refer to that solid-liquid ratio is 1g：20 mL, temperature are 90 DEG C, extraction time is 2 times, when extracting every time
Between be 3 h.
(2) alcohol precipitation：Pleurotus eryngii water extract is dissolved with the water of 8 times of its quality, then adds pleurotus eryngii water extract matter
The volume fraction of 15 times of amount is 90% ethanol solution alcohol precipitation, and precipitation is collected by centrifugation, is repeated 2 times, and merges sediment, the sediment
It is dry the g of Polysaccharide in Pleurotus eryngii crude extract 356.9 under conditions of 75 DEG C in temperature.
(3) cellulose column chromatography is separated：The water that Polysaccharide in Pleurotus eryngii crude extract adds 15 times of its quality is dissolved, and is then adopted
With DEAE-52 cellulose columns respectively with water, 0.1 mol/L sodium-chloride water solutions, 0.4 mol/L sodium-chloride water solutions by 8 times
Column volume carries out gradient elution, collects 0.4 mol/L sodium-chloride water solution eluents；The eluent is through being dried under reduced pressure to 0.8 L
Afterwards, dialysed the h of desalination 20 with dialysis membrane, be finally that 0.08 MPa, temperature are under conditions of 75 DEG C in vacuum to trapped fluid in film
It is dried under reduced pressure the g of Polysaccharide in Pleurotus eryngii component 105.2 for producing pale yellow powder shape.
Wherein：The molecular cut off of dialysis membrane is 8000D.
Application of the Polysaccharide in Pleurotus eryngii component in drugs against colon cancer or health food is prepared made from above-described embodiment 1 ~ 3
Refer to：The Polysaccharide in Pleurotus eryngii component is made all kinds of anti-with pharmaceutically acceptable any carrier according to a conventional method as active principle
Colon cancer preparation, or be made according to a conventional method with acceptable any carrier in Food Science as active principle all kinds of health-related
Food.
Claims (7)
1. a kind of preparation method of Polysaccharide in Pleurotus eryngii component, comprises the following steps：
(1) extract：Dry pleurotus eryngii powder is broken to after 50 ~ 120 mesh the carry out water extraction that adds water, through being filtrated to get filtrate；The filtrate passes through
It is dried under reduced pressure to paste, produces pleurotus eryngii water extract；
(2) alcohol precipitation：The pleurotus eryngii water extract is dissolved with the water of 5 ~ 10 times of its quality, then adds the pleurotus eryngii water
The volume fraction that 10 ~ 20 times of extract quality is 85% ~ 95% ethanol solution alcohol precipitation, and precipitation is collected by centrifugation, and is repeated 1 ~ 3 time, is closed
And sediment, the sediment is through dry Polysaccharide in Pleurotus eryngii crude extract；
(3) cellulose column chromatography is separated：The water that the Polysaccharide in Pleurotus eryngii crude extract adds 10 ~ 20 times of its quality is dissolved, then
Cellulose column is used respectively with water, 0.1 mol/L sodium-chloride water solutions, 0.4 mol/L sodium-chloride water solutions by 5 ~ 10 times of post
Volume carries out gradient elution, collects 0.4 mol/L sodium-chloride water solution eluents；The eluent through be dried under reduced pressure to 0.5 L ~
After 1.0 L, dialysed the h of desalination 15 h ~ 24 with dialysis membrane, finally trapped fluid in film is dried under reduced pressure and produces pale yellow powder shape
Polysaccharide in Pleurotus eryngii component.
2. a kind of preparation method of Polysaccharide in Pleurotus eryngii component as claimed in claim 1, it is characterised in that：The step (1) reclaimed water
The condition of carrying refers to that solid-liquid ratio is 1g：10 ~ 30 mL, temperature are 80 DEG C ~ 95 DEG C, extraction time is 1 ~ 3 time, each extraction time is 1
~5 h。
3. a kind of preparation method of Polysaccharide in Pleurotus eryngii component as claimed in claim 1, it is characterised in that：The step (1), institute
The condition that is dried under reduced pressure in stating step (3) each means that vacuum is 0.07 ~ 0.09 MPa, and temperature is 65 ~ 80 DEG C.
4. a kind of preparation method of Polysaccharide in Pleurotus eryngii component as claimed in claim 1, it is characterised in that：The step (2) in do
Dry condition is that temperature is 65 ~ 80 DEG C.
5. a kind of preparation method of Polysaccharide in Pleurotus eryngii component as claimed in claim 1, it is characterised in that：The step (3) middle fibre
The plain post of dimension is DEAE-52.
6. a kind of preparation method of Polysaccharide in Pleurotus eryngii component as claimed in claim 1, it is characterised in that：The step (3) in thoroughly
The molecular cut off for analysing film is 3000 D ~ 10000D.
7. made from a kind of preparation method of Polysaccharide in Pleurotus eryngii component as claimed in claim 1 prepared by Polysaccharide in Pleurotus eryngii component
Application in drugs against colon cancer or health food, it is characterised in that：The Polysaccharide in Pleurotus eryngii component as active principle routinely
Method and pharmaceutically acceptable any carrier are made all kinds of inhibitor against colon carcinoma cells preparations, or as active principle according to a conventional method with food
All kinds of health care based foods are made in acceptable any carrier in product science.
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