CN104892784B - A kind of method of purification of fern amylose - Google Patents
A kind of method of purification of fern amylose Download PDFInfo
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Abstract
A kind of method of purification of fern amylose, the method comprises the following steps：Fern fiber crops are ground into fern fiber crops particle；Fern fiber crops particle is put into water and is extracted, in the fern amylose in the fern fiber crops particle is dissolved in water；The fern amylose aqueous solution after being extracted in step 2 is separated with fern fiber crops particle, obtains thick fern amylose solution；The thick fern amylose solution is carried out into hyperfiltration treatment by milipore filter, fern amylose purification solution is obtained；Fern amylose purification solution is mixed with absolute ethyl alcohol carries out alcohol precipitation, fern amylose is separated out from fern amylose purification solution；The fern amylose of precipitation is separated from purification mixed solution of the solution with the absolute ethyl alcohol of fern amylose described in step 5, and carries out dried process, the fern amylose after being purified, the method can be purified and obtain antioxidation activity preferably fern amylose.
Description
Technical field
The invention belongs to medicinal chemistry art, more particularly to a kind of fern amylose, a kind of specifically fern amylose
Method of purification.
Background technology
Fern fiber crops expand root tuber for rose family Potentilla Potentilla anserine L., are Tibetan medicine's common medicine, and
Name stamp agate, Zhuo Laosha once, life-prolonging time grass, panax ginseng fruit, life-prolonging time grass, lotus cauliflower, in Tibetan language claim " tall and erect Lao Sha monk ", " stamp agate ".It is a kind of
Common Tibetan medicine.Fern fiber crops have significant protect liver, anti anoxia and enhancing immunity function, are that a kind of China is distinctive, have
The plant of good medical value.Found in the research of fern fiber crops, fern amylose is rich in fern fiber crops, and fern amylose can be carried
Immune function of human body high, antifatigue, resist oxygen lack and the antidiarrheal effect such as antibacterial.Therefore, how to purify fern amylose becomes fern fiber crops
Development and application in a problem for key.In the prior art, it is general that concentration fern fiber crops are extracted using Vacuum Heat method for concentration
Polysaccharide, the method easily reduces the antioxidation activity of fern amylose, its medical value is reduced, in addition, in existing method
Only fern amylose is slightly purified, it is impure more in the thick fern amylose for purifying out, it is unfavorable for that the exploitation of fern amylose should
With.
The content of the invention
It is described the invention provides a kind of method of purification of fern amylose for above-mentioned technical problem of the prior art
This fern amylose method of purification solve it is impure more in the fern amylose that method of the prior art is extracted, and
The technical problem for easily reducing the antioxidation activity of fern amylose.
A kind of method of purification of fern amylose is the embodiment of the invention provides, the method comprises the following steps：
Step one：Fern fiber crops are ground into fern fiber crops particle；
Step 2：Fern fiber crops particle is put into water and is extracted, dissolve in the fern amylose in the fern fiber crops particle
In water, feed liquid weight ratio during extraction is 1:15-1:25, extraction time is 1.5-2.5h, and extraction temperature is 85-95 DEG C.
Step 3：The fern amylose aqueous solution after being extracted in step 2 is separated with fern fiber crops particle, obtains thick fern fiber crops
Polysaccharide solution；
Step 4：The thick fern amylose solution is carried out into ultrafiltration pretreatment, in the removing thick fern amylose solution
Solid particle.
Step 5：The thick fern amylose solution is carried out into hyperfiltration treatment by milipore filter, fern amylose purification is obtained molten
Liquid, when hyperfiltration treatment is carried out, the thick fern amylose solution sequentially passes through the milipore filter and retention that molecular cut off is 100K
Molecular weight is the milipore filter of 50K, and the fern amylose purification solution is the milipore filter and retention that the molecular cut off is 100K
Molecular weight is the solution between the milipore filter of 50K；
Step 6：Fern amylose purification solution is mixed with absolute ethyl alcohol and carries out alcohol precipitation, makes fern amylose from described
Separated out in fern amylose purification solution；
Step 7：The fern amylose that will be separated out purifies the mixed solution of solution and the absolute ethyl alcohol from the fern amylose
In separate, and carry out dried process, the fern amylose after being purified.
Further, it is 0.5 μm by aperture by the thick fern amylose solution when the ultrafiltration pretreatment is carried out
Filter membrane is removing the solid particle in the thick fern amylose solution.
Further, before hyperfiltration treatment is carried out, the milipore filter is first cleaned with sodium hydroxide solution, then entered with clear water
Row cleaning, makes the Ph values of the milipore filter reach 7.0-7.4.
Further, the feed liquid weight ratio during extraction is 1:25, extraction time is 2h, and extraction temperature is 87.5 DEG C.
Further, the fern amylose purification solution and the volume ratio of absolute ethyl alcohol are 1:3.
In sum, in the present invention, when fern amylose is purified, thick fern amylose solution is made to sequentially pass through retention molecule
Measure for the milipore filter and molecular cut off of 100K are the milipore filter of 50K, due to being by milipore filter when thick fern amylose is purified
Processed, so the method for purification can obtain more pure in the case of the inoxidizability for not reducing fern amylose
Fern amylose；After milipore filter and molecular cut off of the molecular cut off for 100K are sequentially passed through for the milipore filter of 50K, at molecular weight
Fern amylose between 50K-100K, the clearance rate of its 1,1- diphenyl -2- trinitrophenyl-hydrazine DPPH free radical can reach
74.5%, there is optimal antioxidation activity compared to the fern amylose of other molecular weight.Therefore in the present invention, can be resisted
Oxidisability preferably purify after fern amylose.Further, by the adjustment to extracting condition, thick fern amylose can be made
Yield reaches maximum, you can to obtain the fern amylose after most purifications.
Described above is only the general introduction of technical solution of the present invention, in order to better understand technological means of the invention,
And can be practiced according to the content of specification, and in order to allow the above and other objects, features and advantages of the invention can
Become apparent, below especially exemplified by preferred embodiment, and coordinate accompanying drawing, describe in detail as follows.
Brief description of the drawings
Fig. 1 be purification after fern amylose in the case of different molecular weight and different concentration, DPPH free radicals
The block diagram of clearance rate.
Specific embodiment
Further to illustrate the present invention to reach technological means and effect that predetermined goal of the invention is taken, below in conjunction with
Accompanying drawing and preferred embodiment, it is as follows that the present invention is described in detail.
The method of the purification fern amylose that the present invention is provided mainly comprises the following steps：
Step one：Fern fiber crops are ground into fern fiber crops particle；
Step 2：Fern fiber crops particle is put into water and is extracted, in the fern amylose in fern fiber crops particle is dissolved in water, extraction
When feed liquid weight ratio be 1:15-1:25, extraction time is 1.5-2.5h, and extraction temperature is 85-95 DEG C.
Step 3：The fern amylose aqueous solution after being extracted in step 2 is separated with fern fiber crops particle, obtains thick fern amylose
Solution；
Step 4：Thick fern amylose solution is carried out into ultrafiltration pretreatment, the solid particle in thick fern amylose solution is removed.
Step 5：Thick fern amylose solution is carried out into hyperfiltration treatment by milipore filter, fern amylose purification solution is obtained,
When carrying out hyperfiltration treatment, thick fern amylose solution sequentially passes through the milipore filter and molecular cut off that molecular cut off is 100K and is
The milipore filter of 50K, fern amylose purification solution is molecular cut off for the milipore filter and molecular cut off of 100K are the ultrafiltration of 50K
Solution between film；
Step 6：Fern amylose purification solution is mixed with absolute ethyl alcohol carries out alcohol precipitation, makes fern amylose from fern amylose
Separated out in purification solution；
Step 7：The fern amylose of precipitation is isolated from fern amylose purification mixed solution of the solution with absolute ethyl alcohol
Come, and carry out dried process, the fern amylose after being purified.
It is below specific embodiment：
Embodiment one
Fern fiber crops are dried treatment, and fern fiber crops are ground into fern fiber crops particle.
Fern fiber crops particle is put into water and is extracted, feed liquid mass ratio during extraction is 1:15, extraction temperature is 85 DEG C, leaching
The time is carried for 2h.It is stirred continuously in leaching process, to promote the fern amylose in fern fiber crops particle to be dissolved into water, forms fern fiber crops
The mixed solution of polysaccharide solution and fern fiber crops particle.
By the fern amylose aqueous solution after extraction and fern fiber crops particle by centrifugation, the supernatant liquor after separating is taken, obtained
To clear liquid be thick fern amylose solution.
Fraction of solid particle is also had in thick fern amylose solution now, needs to carry out when further being purified
Pretreatment removes remaining solid particle in thick fern amylose solution.In the present embodiment, thick fern amylose solution is passed through
Aperture is 0.5 μm of filter membrane to remove the solid particle in thick fern amylose solution.
Will by pre-process thick fern amylose solution hyperfiltration treatment will be carried out by milipore filter, carry out hyperfiltration treatment it
Before, milipore filter is cleaned with 0.1% sodium hydroxide solution first, then cleaned with clear water, reach the pH value of milipore filter
7.0-7.4。
Pretreated thick fern amylose solution is carried out into hyperfiltration treatment, is in the present embodiment by pretreated thick fern
It is the milipore filter of 100K, the milipore filter that molecular cut off is 50K and retention molecule that numb polysaccharide solution sequentially passes through molecular cut off
Measure the milipore filter for 5K.Fern amylose solution of the molecular weight more than 100K, molecular weight are respectively obtained for the fern fiber crops of 50K-100K are more
Sugar juice, molecular weight are the fern amylose solution of fern amylose solution and molecular weight less than 5K of 5K-50K.
Fern amylose solution after above-mentioned four kinds are purified is respectively by absolute ethyl alcohol to the fern amylose solution after purification
It is pure heavy to carry out.Its specific method is：By four kinds of solution according to the purification solution of fern amylose and the volume ratio 1 of absolute ethyl alcohol：3,
It is separately added into absolute ethyl alcohol, 15min is centrifuged under the rotating speed of 5000rpm after being sufficiently stirred for, the clear liquid that will be obtained
Go, precipitation taking-up is placed in container and is dried in 60 DEG C of -100 DEG C of thermostatic drying chambers, you can four kinds not after being purified
With the fern amylose of molecular weight.In other embodiments, the fern amylose after the purification of precipitation can also by other method from
Separated in mixed liquor.
Fern amylose after the purification best in order to obtain inoxidizability contains DPPH free radicals, it is necessary to fern amylose is used
Ethanol solution carry out the measure of inoxidizability.Accurately weigh the DPPH reagents of 7.8864mg, plus anhydrous second first when determining
Alcohol is dissolved in the volumetric flask of 100ml, then with the DPPH solution of liquid-transfering gun accurate measuring 0.1ml absolute ethyl alcohol constant volume in
In the brown volumetric flask of 100ml, the 2x10 needed for being made experiment-4The DPPH ethanol solutions of mmol/L, shake up, and are placed on lucifuge
Place preserves.Then each fern amylose after purifying is made isoconcentration for 2.5,5 and 7.5 μ g/mL solution for standby.Will be every
2x10 is added in the sample of 2ml-4[DPPH] ethanol solution 2ml of mmol/L, is shaken up, and its extinction is determined at 517nm
Degree, surveys once, per 5min until the change of absorbance is within 1%.Each sample does three Duplicate Samples, takes its average value.Root
According to following formula, show that fern amylose is in different molecular weight and various concentrations after purifying, the clearance rate of [DPPH].
The clearance rate computing formula of DPPH free radicals：
P (%)=[1- (Ai-Aj)/Ac]×100
In formula：P：Clearance rate；
Ai:The mixed solution of fern amylose solution and 2mL DPPH solution after 2mL purifications is the suction at 517nm in wavelength
Luminosity；
Aj:The mixed solution of fern amylose solution and 2mL anhydrous ethanol solvents after 2mL purifications is at wavelength is for 517nm
Absorbance；
Ac：The mixed solution of 2mL DPPH solution and 2mL anhydrous ethanol solvents is in the absorbance that wavelength is at 517nm.
Fig. 1 represent purification after fern amylose in the case of different molecular weight and different concentration, the removing of DPPH
The block diagram of rate, as shown in figure 1, when the molecular weight of fern amylose is less than 5K, its DPPH under 2.5,5 and 7.5 μ g/mL concentration
The clearance rate of free radical is respectively 70.03%, 65.27% and 70.03%；Between the molecular weight of fern amylose is in 5K-50K
When, the clearance rate of its DPPH under 2.5,5 and 7.5 μ g/mL concentration is respectively 68.07%, 70.03% and 68.07%；When fern fiber crops
When the molecular weight of polysaccharide is between 50K-100K, the clearance rate of its DPPH under 2.5,5 and 7.5 μ g/mL concentration is all
74.51%；When fern amylose molecular weight be more than 100K when, its under 2.5,5 and 7.5 μ g/mL concentration DPPH free radicals it is clear
Except rate is respectively 65.27%, 68.07% and 65.27%.Thus, it can be known that the fern amylose solution after being purified under various concentrations
Molecular weight component between 50K-100K, all with optimal DPPH free radical scavenging activities.Illustrate fern amylose in 50K-
Molecular weight component between 100K, antioxidation activity is optimal.Therefore molecular weight be its medical value of the fern amylose of 50-100K compared with
Greatly.
In order to improve the yield of fern amylose, it is necessary to be controlled to each condition when purifying fern amylose.It is appreciated that
Ground, the thick fern amylose that extraction is obtained is more, and the fern amylose obtained after purification is also more.In order to obtain optimal extraction work
Skill, to obtain most thick fern amyloses, it is necessary to calculate the yield of the thick fern amylose that different extracting conditions are obtained.
Computational methods are for feed liquid mass ratio is 1 after fern fiber crops are crushed according to extracting condition in the present embodiment:15, extraction
Temperature is 85 DEG C, and extraction time is 2h, and the extraction of fern amylose is carried out in the lab, obtains thick fern amylose solution.Then
The thick fern amylose solution that will be obtained mixes with absolute ethyl alcohol, thick fern amylose is separated out from the mixed solution of the two, passes through
Centrifugation, obtains precipitate, then precipitate is dried obtains thick fern amylose.According to below equation, thick fern fiber crops are drawn
The yield of polysaccharide：
In formula：m1- by the weighing disk and the gross mass of Thick many candies of freeze-day with constant temperature constant weight, g
m2- by freeze-day with constant temperature constant weight weighing disk quality, g
M- weigh fern fiber crops solid powder quality, g
In the present embodiment, the yield of thick fern amylose is 15.6%.Feed liquid when following each embodiments are except extraction
Mass ratio, extraction time and extraction temperature are different outer, the method for its yield for drawing thick fern amylose, the processing method of milipore filter
Difference is had no, therefore is not repeated in each of the embodiments described below.
In the present embodiment, because the method is after thick fern amylose solution is extracted out, again by thick fern amylose solution
Hyperfiltration treatment is carried out, the method for purification can obtain more pure in the case of the inoxidizability for not reducing fern amylose
Fern amylose.After milipore filter carries out hyperfiltration treatment, because molecular weight component of the fern amylose between 50K-100K, antioxygen
Change activity optimal.Therefore taking the fern amylose that molecular weight is 50-100K can obtain the fern after inoxidizability is more preferably purified again
Numb polysaccharide.
Embodiment two：
Fern fiber crops particle after grinding is extracted in being put into water, and feed liquid mass ratio during extraction is 1:15, extraction
Temperature is 95 DEG C, and extraction time is 2h.The fern amylose aqueous solution after extraction is separated with fern fiber crops particle, thick fern amylose is obtained
Solution.Under this extracting condition, the yield of thick fern amylose is 14.2%.
The method of purification obtained after thick fern amylose solution is identical with embodiment one.
Embodiment three：
Fern fiber crops particle after grinding is extracted in being put into water, and feed liquid mass ratio during extraction is 1:25, extraction
Temperature is 90 DEG C, and extraction time is 1.5h.The fern amylose aqueous solution after extraction is separated with fern fiber crops particle, thick fern fiber crops is obtained many
Sugar juice.Under this extracting condition, the yield of thick fern amylose is 15.9%.
The method of purification obtained after thick fern amylose solution is identical with embodiment one.
Example IV：
Fern fiber crops particle after grinding is extracted in being put into water, and feed liquid mass ratio during extraction is 1:20, extraction
Temperature is 95 DEG C, and extraction time is 1.5h.The fern amylose aqueous solution after extraction is separated with fern fiber crops particle, thick fern fiber crops is obtained many
Sugar juice.Under this extracting condition, the yield of thick fern amylose is 10.1%.
The method of purification obtained after thick fern amylose solution is identical with embodiment one.
Embodiment five：
Fern fiber crops particle after grinding is extracted in being put into water, and feed liquid mass ratio during extraction is 1:20, extraction
Temperature is 95 DEG C, and extraction time is 2h.The fern amylose aqueous solution after extraction is separated with fern fiber crops particle, thick fern amylose is obtained
Solution.Under this extracting condition, the yield of thick fern amylose is 12.1%.
The method of purification obtained after thick fern amylose solution is identical with embodiment one.
Embodiment six：
Fern fiber crops particle after grinding is extracted in being put into water, and feed liquid mass ratio during extraction is 1:20, extraction
Temperature is 95 DEG C, and extraction time is 2.5h.The fern amylose aqueous solution after extraction is separated with fern fiber crops particle, thick fern fiber crops is obtained many
Sugar juice.Under this extracting condition, the yield of thick fern amylose is 12.5%.
The method of purification obtained after thick fern amylose solution is identical with embodiment one.
Embodiment seven：
Fern fiber crops particle after grinding is extracted in being put into water, and feed liquid mass ratio during extraction is 1:15, extraction
Temperature is 90 DEG C, and extraction time is 2.5h.The fern amylose aqueous solution after extraction is separated with fern fiber crops particle, thick fern fiber crops is obtained many
Sugar juice.Under this extracting condition, the yield of thick fern amylose is 12.5%.
The method of purification obtained after thick fern amylose solution is identical with embodiment one.
Embodiment eight
Fern fiber crops particle after grinding is extracted in being put into water, and feed liquid mass ratio during extraction is 1:25, extraction
Temperature is 87.5 DEG C, and extraction time is 2h.The fern amylose aqueous solution after extraction is separated with fern fiber crops particle, thick fern fiber crops is obtained many
Sugar juice.Under this extracting condition, the yield of thick fern amylose is 15.88%.
The method of purification obtained after thick fern amylose solution is identical with embodiment one.
By test of many times it is known that the feed liquid mass ratio when extraction is 1:25, extraction temperature is 87.5 DEG C, extraction
When time is 2h, the yield that can obtain thick fern amylose reaches the 15.88% of maximum.
In the present invention, because the method is after thick fern amylose solution is extracted out, thick fern amylose solution is entered again
Row hyperfiltration treatment, the method for purification can obtain more pure fern in the case of the inoxidizability for not reducing fern amylose
Numb polysaccharide.After milipore filter carries out hyperfiltration treatment, because molecular weight component of the fern amylose between 50K-100K, anti-oxidant
It is active optimal.Therefore taking the fern amylose that molecular weight is 50-100K can obtain the fiber crops of the fern after inoxidizability is more preferably purified again
Polysaccharide.Further, by the adjustment to extracting condition, the yield of thick fern amylose can be made to reach maximum, you can to obtain
Fern amylose after most purifications.
The above, is only presently preferred embodiments of the present invention, and any formal limitation is not made to the present invention, though
So the present invention is disclosed above with preferred embodiment, but is not limited to the present invention, any to be familiar with this professional technology people
Member, without departing from the scope of the present invention, when making a little change or modification using the technology contents of the disclosure above
It is the Equivalent embodiments of equivalent variations, as long as being without departing from technical solution of the present invention content, according to technical spirit pair of the invention
Any simple modification, equivalent variations and modification that above example is made, still fall within the range of technical solution of the present invention.
Claims (2)
1. a kind of method of purification of fern amylose, it is characterised in that：The method comprises the following steps：
Step one：Fern fiber crops are ground into fern fiber crops particle；
Step 2：Fern fiber crops particle is put into water and is extracted, the fern amylose in the fern fiber crops particle is dissolved in water
In, feed liquid weight ratio during extraction is 1:25, extraction time is 2h, and extraction temperature is 87.5 DEG C；
Step 3：The fern amylose aqueous solution after being extracted in step 2 is separated with fern fiber crops particle, obtains thick fern amylose
Solution；
Step 4：The thick fern amylose solution is carried out into ultrafiltration pretreatment, is by aperture by the thick fern amylose solution
0.5 μm of filter membrane is removing the solid particle in the thick fern amylose solution；
Step 5：Hyperfiltration treatment will be carried out by milipore filter by the thick fern amylose solution for pre-processing, obtain fern amylose and carry
Pure solution, when hyperfiltration treatment is carried out, the thick fern amylose solution sequentially passes through the milipore filter that molecular cut off is 100K
And molecular cut off is the milipore filter of 50K, the fern amylose purification solution is the molecular cut off surpassing for 100K
Filter membrane and molecular cut off are the solution between the milipore filter of 50K；
Step 6：Fern amylose purification solution is mixed with absolute ethyl alcohol carries out alcohol precipitation, and the fern amylose purifies solution
It is 1 with the volume ratio of absolute ethyl alcohol:3, with the rotating speed centrifugation 15min of 5000rpm in centrifuge, make fern amylose from
Separated out in the fern amylose purification solution；
Step 7：Supernatant after being centrifuged described in step 6 is fallen, by the fern amylose of Precipitation from the fern amylose
Separated in purification solution and the mixed solution of the absolute ethyl alcohol, precipitation is placed in container, in 60 DEG C of -100 DEG C of perseverances
Warm drying box is dried, the fern amylose after being purified.
2. the method for purification of a kind of fern amylose according to claim 1, it is characterised in that：Before hyperfiltration treatment is carried out,
The milipore filter is first cleaned with sodium hydroxide solution, then is cleaned with clear water, the pH value of the milipore filter is reached 7.0-
7.4。
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