CN106478831A - A kind of Paeonia suffruticosa polysaccharide and its preparation method and application - Google Patents
A kind of Paeonia suffruticosa polysaccharide and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of Paeonia suffruticosa polysaccharide and its preparation method and application, belong to natural product application.This preparation method comprises the following steps：Pulverize Paeonia suffruticosa raw material and be 400 600 μm to its particle diameter；The supernatant carrying out extracting separation and collect after separating to the Paeonia suffruticosa raw material after pulverizing obtains crude extract, and extraction separates and carries out in the following manner：By solid-liquid ratio 5 20g：Extractant is mixed by 80 400mL with the Paeonia suffruticosa raw material after pulverizing, extracts 30 100min, supernatant is collected by filtration；Crude extract is mixed with protein denaturant, stands 12 48h under conditions of 28 DEG C, centrifugation, collect the supernatant after centrifugation and obtain crude polysaccharides solution；Add ethanol solution to obtain solution to be concentrated in crude polysaccharides solution, after concentration, obtain Paeonia suffruticosa polysaccharide.The method is simple, easy to operate.There is through the Paeonia suffruticosa polysaccharide that the method is prepared the effect of stronger removing free radical, can be applicable in free radical scavenger.
Description
Technical field
The present invention relates to natural product application, and particularly to a kind of Paeonia suffruticosa polysaccharide and its preparation method and application.
Background technology
Free radical, is chemically also referred to as " free radical ", the molecule referring to compound under the external conditions such as photo-thermal, covalent bond
Atom or the group with unpaired electron homolysis occurring and being formed.Radical reaction is anti-in burning, aerochemistry, polymerization
, critically important role should be played the part of in plasma chemistry, biochemistry and other various chemistry subjects.Sunlight in external environment
Radiation, air pollution, smoking, pesticide etc. all can make human body produce more reactive oxygen free radical, make nucleic acid mutation, this is that the mankind decline
Old and ill root.
Activity in vivo oxygen-derived free radicals have certain function, such as immunity and signal transduction process.But excessive active oxygen is certainly
Destruction just being had by base, leading to human normal cell and the damage of tissue, thus causing multiple diseases.As heart disease, old
Dementia disease, parkinson disease and tumor.Numerous medical researches and clinical trial prove：Human body cell electronics is ten thousand diseases by plunder
Source, free radical ROS is a kind of material (unsaturated electron species) lacking electronics, fights for electronics everywhere after entering human body, if
Seize the electronics of cell protein molecule, make protein connect side chain occur alkylation, formed distortion molecule and carcinogenic.This distortion
Molecule, due to oneself lacking electronics, goes to capture the electronics of neighboring molecule again, so that neighboring molecule is also distorted and carcinogenic again.This
Sample, vicious cycle will form the protein molecular of a large amount of distortion, and gene mutation forms a large amount of cancerous cell, cancer finally.And
When free radical or distortion molecule grabbed gene electronics when, people will directly cancer.After human body obtains anion, due to negative
Ion is negatively charged unnecessary electronics, it is possible to provide a large amount of electronics, and blocks vicious cycle, and cancerous cell is prevented from or is suppressed.
Drug variety currently used for removing free radical is less and some of which also has side effect, thus finds high from natural plants
Effect, the free radical inhibitors of Small side effects are the focus of research both at home and abroad at present.
Paeonia suffruticosa (Paeonia suffruticosa Andr.), also known as Mus aunt, wooden Radix Paeoniae, Luoyang flower and wealth and rank flower, belong to
Undershrub Paeoniaceae plant, is mainly grown in the areas such as China Yangtze river basin and the Huanghe valley, and it is rich in multiple nutritional components certainly
All it has been used as medicine with its root bark since Gu, name says " Cortex Moutan ".《Xinhua's book on Chinese herbal medicine outline》Described in Paeonia suffruticosa cold nature, bitter is pungent, nontoxic, enter
The heart, liver and kidney channel.There is the function of clearing away liver-fire, promoting blood circulation to remove blood stasis.Recent years, research finds that Paeonia suffruticosa can be used for treating macule haematemesis, swelling
Swollen sore, the deficiency of YIN send out the disease such as gesture and lossless hectic fever due to YIN-deficiency.Chinese scholars take to the chemical composition of Paeonia suffruticosa and its applied research at present
Obtained certain progress, but the system that also needs to furtherd investigate structure and the activity of polysaccharide, flavonoid and polyphenols.
Content of the invention
It is an object of the invention to provide a kind of preparation method of Paeonia suffruticosa polysaccharide, this preparation method simple to operate it is easy to real
Existing, prepared polysaccharide material content is more and scavenging action of to free radical is stronger.
Another object of the present invention is to providing a kind of Paeonia suffruticosa polysaccharide being prepared by above-mentioned preparation method, this polysaccharide has
There is good free radical scavenging effect.Its raw material is natural product, and toxic and side effects are little, can be widely applied to various free radical scavengings
In agent.
The third object of the present invention is to provide a kind of application of Paeonia suffruticosa polysaccharide, will Paeonia suffruticosa polysaccharide answer as active component
For in free radical scavenger.
The present invention solves its technical problem and employs the following technical solutions to realize：
The present invention proposes a kind of preparation method of Paeonia suffruticosa polysaccharide, and it comprises the following steps：
Pulverize Paeonia suffruticosa raw material and be 400-600 μm to its particle diameter, Paeonia suffruticosa raw material is selected from Paeonia suffruticosa blade or Paeonia suffruticosa kind skin；
Then the supernatant carrying out extracting separation and collect after separating to the Paeonia suffruticosa raw material after pulverizing obtains crude extract, extraction point
From carrying out in the following manner：By solid-liquid ratio 5-20g：Extractant is mixed by 80-400mL with the Paeonia suffruticosa raw material after pulverizing, extracts 30-
100min, is collected by filtration supernatant；Extractant is hydrophilic organic solvent；
Then crude extract is mixed with protein denaturant, under conditions of 2-8 DEG C stand 12-48h, centrifugation, collect from
Supernatant after the heart obtains crude polysaccharides solution；
Then add ethanol solution to obtain solution to be concentrated in crude polysaccharides solution, after concentration, obtain Paeonia suffruticosa polysaccharide.
The present invention also proposes a kind of polysaccharide material being prepared gained by above-mentioned preparation method.
The invention allows for a kind of application in preparing free radical scavenger for Paeonia suffruticosa polysaccharide.
A kind of beneficial effect of Paeonia suffruticosa polysaccharide of the embodiment of the present invention and its preparation method and application is：By entering to Paeonia suffruticosa
Row is pulverized, and increased the contact area between raw material and extractant, improves leaching velocity.With hydrophilic organic solvent for extracting
Agent, both can guarantee that leaching velocity was fast, extractum purity is high, impurity is few, and can also reduces cost.5-20g：80-400mL's
Solid-liquid ratio can make the active component extraction effect in Paeonia suffruticosa preferable；Extraction time elects 30-100min as, it is to avoid because of extraction time mistake
Short cause active substance dissolution rate low, extraction time is long and the impurity such as oils and fatss in plant cell, tannin can be made excessive
Dissolution, increases the defects such as the difficulty isolating and purifying.Crude extract is mixed the protein that can make in crude extract with protein denaturant
Degeneration, under the conditions of 2-8 DEG C, standing can make the abundant degeneration of protein precipitate, and makes the separating effect of Paeonia suffruticosa polysaccharide and extraction rate reached to
Good.The method is simple to operate, it is easy to accomplish, the Paeonia suffruticosa polysaccharide prepared has good free radical scavenging effect.Due to this
Bright raw material is natural product, and toxic and side effects are little, thus the polysaccharide material prepared by it to can be widely applied to various free radicals clear
Except in agent.
Specific embodiment
Purpose, technical scheme and advantage for making the embodiment of the present invention are clearer, below will be in the embodiment of the present invention
Technical scheme be clearly and completely described.In embodiment, unreceipted actual conditions person, builds according to normal condition or manufacturer
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can be by the commercially available conventional product bought and obtain
Product.
Below Paeonia suffruticosa polysaccharide of the embodiment of the present invention and its preparation method and application is specifically described.
The preparation method of Paeonia suffruticosa polysaccharide provided in an embodiment of the present invention, with the distinctive Paeonia suffruticosa of China as raw material, its petal is big
And fragrant, bright-colored, the reputation having " aromatic " claims, and represents luxurious and dignified.Specifically, the Paeonia suffruticosa in the embodiment of the present invention
" Paeonia ostii " blade in Tongling Feng huangshan Mountain for example can be chosen or to plant one of skin be raw material, also can simultaneously will blade and to plant skin common
As raw material.In preparation process, first raw material can be pulverized, to increase the contact area between raw material and extractant, improve
Leaching velocity.Wherein, blade and plant skin grinding particle size for example can for 400-600 μm it is preferred that blade and plant skin pulverizing
Granularity for example can also be 420-580 μm, in this particle size range the blade of Paeonia suffruticosa or plant skin in effective ingredient dissolution rate larger.
Raw material preferably picks up from the fresh Paeonia suffruticosa of full-bloom stage, the active constituent content highest in the Paeonia suffruticosa in this period, is conducive to improving subsequently
The content of the effective ingredient in extract.
As preferred, before pulverising step, Paeonia suffruticosa can also be carried out with the operations such as remove impurity, cleaning and drying.Wherein
Remove impurity and cleaning can avoid the interference to preparation effect for other impurity, drying can the cell membrane of partial destruction raw material and cell wall,
Beneficial to extraction.Wherein, baking temperature for example can be chosen as 20-45 DEG C, preferably 35-37 DEG C.The preferred temperature range of here
Interior, the active component in raw material can farthest retain not by high temperature.
Preferably, the embodiment of the present invention adopts extraction, using hydrophilic organic solvent as extractant to former after pulverizing
Material is extracted.Using rule of similarity, this hydrophilic organic solvent is preferably for example the aqueous solution of ethanol, both can guarantee that
Leaching velocity is fast, the purity of extractum is high, impurity is few, and low cost.When from dehydrated alcohol-aqueous solution as extractant
When, the volume fraction of dehydrated alcohol can be for example 70%-90%, preferably 80%, ethanol solution under this volume fraction
Polarity moderate so that the most effective ingredient in Paeonia suffruticosa are soluble in this solution, extract that very fast and impurity is less.This
Outward, also can be selected for methanol-water solution etc..
Paeonia suffruticosa raw material after extractant and pulverizing is, for example, 5-20g with solid-liquid ratio：80-400mL mixes, and obtains waiting to carry
Thing.As preferred, solid-liquid ratio can be for example 10g：100mL, under this ratio, the active component extraction effect in Paeonia suffruticosa is
Good.
Further, will treat that extract carries out extracting for the first time.As preferred, in the embodiment of the present invention first time extraction
Using water-bath extraction, the temperature of extraction for example can be 60-85 DEG C, and the time of extraction is preferably 30-100min, obtains the first extraction
Thing.Active substance dissolution rate can be caused low because extraction time is too short or Extracting temperature is too low, extraction time is long or Extracting temperature
Too high and the excessive dissolutions of impurity such as oils and fatss in plant cell, tannin can be made, when increasing the difficulty isolating and purifying, therefore extracting
Between be preferably 40min, Extracting temperature be preferably 80 DEG C.
Additionally, extracting mode can also adopt microwave extraction, ultrasonic extraction, supercritical extraction etc..
First extract is carried out filtering for the first time, for example, can be centrifugal filtration, the supernatant after collecting separately, obtain the
One separator.The rotating speed of the centrifugation apparatus used by this centrifugal filtration for example may be configured as 3000-5000r/min, centrifugal filtration
Time can be for example 10-35min.As preferred, the first extract of the embodiment of the present invention is under conditions of 4000r/min
Centrifugation 20min, under the conditions of this, centrifugal effect is optimal.
In order to reduce the loss rate of polysaccharide in raw material, after also can filtering first time, remaining precipitation is carried for the second time
Take, such as after can filtering first time, the precipitation of gained presses solid-liquid ratio 5-20g again with said extracted agent：80-400mL mixes,
Under the conditions of 60-85 DEG C, second water-bath extraction 30-100min, obtains the second extract the centrifugation in 3000-5000r/min
Under the conditions of second filter 10-35min, collect supernatant and obtain the second separator.Wherein, second filter it is also preferred that in 4000r/
It is centrifuged 20min under conditions of min.Additionally, according to practical operation situation, also can proceed many on the basis of extracting at second
Secondary extraction separates.
The supernatant collecting gained after separating, as crude extract, this crude extract is mixed with protein denaturant, wherein, egg
White matter denaturant for example can be selected for solution of trichloroacetic acid, and crude extract and solution of trichloroacetic acid by the volume ratio that feeds intake can be for example
1mL:6-20mL mixes, preferably 1mL:12mL.Wherein, the mass percent of the trichloroacetic acid in solution of trichloroacetic acid for example may be used
Think 6-15%, preferably 10%, should under the conditions of trichloroacetic acid be more easy to make protein denaturation and form insoluble substance.Additionally,
Protein denaturant in the embodiment of the present invention is also with from materials such as carbamide, guanidine hydrochlorides.
Mixed crude extract and protein denaturant are stood 12-48h under conditions of 2-8 DEG C, is preferable over 4 DEG C of ice
It is centrifuged to remove protein portion after cold preservation standing 24h in case, collect supernatant and obtain crude polysaccharides solution.Wherein, the present invention is implemented
The centrifugation time that example is adopted can be for example 15-30min, and centrifugal rotational speed can be for example 3000-5000r/min.Preferably,
This centrifugation time is 10min and centrifugal rotational speed is 4000r/min, and protein component can be made under this centrifugal condition to precipitate completely.
Remove the protein precipitation after centrifugation, and add dehydrated alcohol aqueous solution to obtain solution to be concentrated in crude polysaccharides solution,
Preferably, the volume fraction of contained ethanol solution in this solution to be concentrated is more than or equal to 80%, is beneficial to polysaccharide material
Dissolving and concentration.
Preferably, for example above-mentioned solution to be concentrated can be concentrated into its moisture content to be less than or equal to 10%, obtain Paeonia suffruticosa many
Sugar.As preferred, for example solution to be concentrated can be turned for 30-50 in 45-55 DEG C of water-bath, rotating speed in the embodiment of the present invention/
Min and vacuum are rotary evaporation under conditions of 0.07-0.09MPa, remove the ethanol in organic faciess, obtain extractum shape
Paeonia suffruticosa polysaccharide, the concentrating under reduced pressure carrying out under the conditions of being somebody's turn to do, be conducive to avoiding the structure of active component in Paeonia suffruticosa to be subject in recycling design
To destruction.
With reference to embodiments the feature and performance of the present invention is described in further detail.
Embodiment 1
By the blade of fresh Paeonia ostii with plant skin and be crushed to granularity respectively and be 400 μm and 420 μm, with methanol-water solution for carrying
Take agent, the wherein percentage by volume of methanol is 80%.By solid-liquid ratio 5g：80mL by extractant with pulverize after blade and kind skin mix
Close, obtain treating extract.Under conditions of 60 DEG C, water-bath extraction 100min, obtains the first extract.By this first extract in
The pelleted by centrifugation 35min of 3000r/min, collects the supernatant after centrifugation and obtains the first separator.By the first separator and quality hundred
Fraction be 6% solution of trichloroacetic acid with the volume ratio that feeds intake as 1mL:6mL mixes, after cold preservation standing 48h in 2 DEG C of the refrigerator,
It is centrifuged 30min under the centrifugal condition of 3000r/min, removes protein portion, the supernatant after collection this time centrifugation obtains slightly many
Sugar juice.Dehydrated alcohol is added to obtain solution to be concentrated and make the volume of the ethanol solution in solution to be concentrated in crude polysaccharides solution
Fraction is 80%, by the vacuum degree condition backspin of water-bath, the rotating speed of 50 turns/min and 0.07MPa in 45 DEG C for this solution to be concentrated
Turn and be evaporated to its moisture content for 10%, obtain Paeonia suffruticosa polysaccharide.Male containing 0.085ug in 1ul crude polysaccharides solution in the present embodiment
Red polysaccharide.
Embodiment 2
The blade of fresh Paeonia ostii and kind skin are carried out remove impurity, cleaning, drying and be crushed to respectively granularity at 20 DEG C is 600
μm and 580 μm, with dehydrated alcohol-aqueous solution as extractant, wherein dehydrated alcohol percentage by volume be 70%.By solid-liquid ratio
5g：Extractant is mixed by 400mL with the blade after pulverizing, obtains treating extract.Under conditions of 85 DEG C, water-bath extraction 30min, obtains
To the first extract.By this first extract in the pelleted by centrifugation 10min of 5000r/min, collect supernatant and obtain the first separator.
Remaining precipitation after centrifugation is pressed solid-liquid ratio 5g with dehydrated alcohol-aqueous solution：400mL mixes, and second under conditions of 85 DEG C
Secondary water-bath extracts 30min, obtains the second extract.By this second extract in the pelleted by centrifugation 10min of 5000r/min, collect
Supernatant obtains the second separator.First separator and the second separator are merged, and with the volume ratio that feeds intake as 1mL:20mL and matter
Amount percent is 15% solution of trichloroacetic acid mixing, after cold preservation standing 12h in 8 DEG C of the refrigerator, in the centrifugation of 5000r/min
Under the conditions of be centrifuged 15min, remove protein portion, collect the supernatant after being this time centrifuged and obtain crude polysaccharides solution.Molten to crude polysaccharides
In liquid add dehydrated alcohol obtain solution to be concentrated and make the ethanol solution in solution to be concentrated volume fraction be 90%, this is treated
Concentrated solution in 55 DEG C water-bath, under the rotating speed of 30 turns/min and the vacuum degree condition of 0.08MPa rotation be evaporated to its moisture content
For 8%, obtain Paeonia suffruticosa polysaccharide.Paeonia suffruticosa polysaccharide containing 0.056ug in 1ul crude polysaccharides solution in the present embodiment.
Embodiment 3
By the blade of fresh Paeonia ostii and plant skin carry out remove impurity, cleaning, dry at 45 DEG C and be crushed to granularity be 420 μm and
600 μm, with dehydrated alcohol-aqueous solution as extractant, the percentage by volume of wherein dehydrated alcohol is 90%.By solid-liquid ratio 20g：
Extractant is mixed by 80mL with planting skin after pulverizing, obtains treating extract.Under conditions of 72 DEG C, water-bath extraction 65min, obtains first
Extract.By this first extract in the pelleted by centrifugation 22.5min of 4000r/min, collect supernatant and obtain the first separator.Will be from
After the heart, remaining precipitation and dehydrated alcohol-aqueous solution press solid-liquid ratio 20g：80mL mixes, and second water under conditions of 72 DEG C
Bath extraction 65min, obtains the second extract.By this second extract in the pelleted by centrifugation 22.5min of 4000r/min, in collection
Clear liquid obtains the second separator.First separator and the second separator are merged, and with the volume ratio that feeds intake as 1mL:13mL and quality
Percent is 10.5% solution of trichloroacetic acid mixing, after cold preservation standing 30h in 5 DEG C of the refrigerator, in the centrifugation of 4000r/min
Under the conditions of be centrifuged 22.5min, remove protein portion, collect the supernatant after being this time centrifuged and obtain crude polysaccharides solution.To crude polysaccharides
In solution add dehydrated alcohol obtain solution to be concentrated and make the ethanol solution in solution to be concentrated volume fraction be 85%, should
Solution to be concentrated in 50 DEG C water-bath, under the rotating speed of 40 turns/min and the vacuum degree condition of 0.09MPa rotation to be evaporated to it aqueous
Rate is 6%, obtains Paeonia suffruticosa polysaccharide.Paeonia suffruticosa polysaccharide containing 0.025ug in 1ul crude polysaccharides solution in the present embodiment.
Embodiment 4
By the blade of fresh Paeonia ostii and plant skin carry out remove impurity, cleaning, dry at 37 DEG C and be crushed to granularity be 580 μm and
400 μm, with dehydrated alcohol-aqueous solution as extractant, the percentage by volume of wherein dehydrated alcohol is 80%.By solid-liquid ratio 20g：
Extractant is mixed by 400mL with the blade after pulverizing and kind skin, obtains treating extract.Water-bath extraction 40min under conditions of 80 DEG C,
Obtain the first extract.By this first extract in the pelleted by centrifugation 20min of 4000r/min, collect supernatant and obtain the first separation
Thing.Remaining precipitation after centrifugation is pressed solid-liquid ratio 20g with dehydrated alcohol-aqueous solution：400mL mixes, and under conditions of 80 DEG C
Second water-bath extracts 40min, obtains the second extract.By this second extract in the pelleted by centrifugation 20min of 4000r/min,
Collect supernatant and obtain the second separator.First separator and the second separator are merged, and with the volume ratio that feeds intake as 1mL:12mL
Mix with the solution of trichloroacetic acid that mass percent is 10%, after cold preservation standing 24h in 4 DEG C of the refrigerator, in 4000r/min's
It is centrifuged 10min under centrifugal condition, removes protein portion, collect the supernatant after being this time centrifuged and obtain crude polysaccharides solution.To slightly many
In sugar juice add dehydrated alcohol obtain solution to be concentrated and make the ethanol solution in solution to be concentrated volume fraction be 95%, will
This solution to be concentrated in 55 DEG C water-bath, under the rotating speed of 40 turns/min and the vacuum degree condition of 0.09MPa rotation be evaporated to it and contain
Water rate is 4%, obtains Paeonia suffruticosa polysaccharide.Paeonia suffruticosa polysaccharide containing 0.078ug in 1ul crude polysaccharides solution in the present embodiment.
Embodiment 5
Fresh Paeonia ostii is carried out remove impurity, cleaning, drying at 35 DEG C and its blade and kind skin are all crushed to granularity is 500
μm, with dehydrated alcohol-aqueous solution as extractant, the percentage by volume of wherein dehydrated alcohol is 80%.By solid-liquid ratio 10g：100mL
Extractant is mixed with the blade after pulverizing and kind skin, obtains treating extract.Under conditions of 80 DEG C, water-bath extraction 40min, obtains
First extract.By this first extract in the pelleted by centrifugation 20min of 4000r/min, collect supernatant and obtain the first separator.Will
After centrifugation, remaining precipitation and dehydrated alcohol-aqueous solution press solid-liquid ratio 10g：100mL mixes, and second under conditions of 80 DEG C
Water-bath extracts 40min, obtains the second extract.By this second extract in the pelleted by centrifugation 20min of 4000r/min, in collection
Clear liquid obtains the second separator.First separator and the second separator are merged, and with the volume ratio that feeds intake as 1mL:12mL and quality
Percent is 10% solution of trichloroacetic acid mixing, after cold preservation standing 24h in 4 DEG C of the refrigerator, in the centrifugation bar of 4000r/min
It is centrifuged 10min under part, removes protein portion, collect the supernatant after being this time centrifuged and obtain crude polysaccharides solution.To crude polysaccharides solution
Middle add dehydrated alcohol obtain solution to be concentrated and make the ethanol solution in solution to be concentrated volume fraction be 90%, by this treat dense
Contracting liquid in 50 DEG C water-bath, under the rotating speed of 40 turns/min and the vacuum degree condition of 0.09MPa rotation be evaporated to its moisture content and be
2%, obtain Paeonia suffruticosa polysaccharide.Paeonia suffruticosa polysaccharide containing 0.091ug in 1ul crude polysaccharides solution in the present embodiment.
Embodiment 6
Fresh Paeonia ostii is carried out remove impurity, cleaning, dries at 32.5 DEG C and by its blade and plant skin and be all crushed to granularity and be
500 μm, with methanol-water solution as extractant, the percentage by volume of wherein methanol is 80%.By solid-liquid ratio 10g：100mL will carry
Blade after taking agent and pulverizing and kind skin mix, and obtain treating extract.Under conditions of 80 DEG C, water-bath extraction 40min, obtains first
Extract.By this first extract in the pelleted by centrifugation 20min of 4000r/min, collect supernatant and obtain the first separator.To be centrifuged
Remaining precipitation and methanol-water solution press solid-liquid ratio 10g afterwards：100mL mixes, and second water-bath extraction under conditions of 80 DEG C
40min, obtains the second extract.By this second extract in the pelleted by centrifugation 20min of 4000r/min, collect supernatant and obtain the
Two separators.First separator and the second separator are merged, and with the volume ratio that feeds intake as 1mL:12mL is mixed with urea liquid,
After cold preservation standing 24h in 4 DEG C of the refrigerator, it is centrifuged 10min under the centrifugal condition of 4000r/min, removes protein portion, receive
Supernatant after collection is this time centrifuged obtains crude polysaccharides solution.Add in crude polysaccharides solution dehydrated alcohol obtain solution to be concentrated making treat dense
The volume fraction of the ethanol solution in contracting liquid is 90%, by the rotating speed of the water-bath in 50 DEG C for this solution to be concentrated, 40 turns/min
And under the vacuum degree condition of 0.09MPa, rotation is evaporated to its moisture content for 9%, obtains Paeonia suffruticosa polysaccharide.In the present embodiment, 1ul is thick
Paeonia suffruticosa polysaccharide containing 0.083ug in polysaccharide solution.
Embodiment 7
Fresh Paeonia suffruticosa is carried out remove impurity, cleaning, dries at 36 DEG C and its blade is crushed to granularity for 500 μm, with first
Alcohol-water solution is extractant, and wherein the percentage by volume of methanol is 80%.By solid-liquid ratio 10g：100mL is by extractant and pulverizing
Blade mixing afterwards, obtains treating extract.Under conditions of 80 DEG C, water-bath extraction 40min, obtains the first extract.This first is carried
Take thing in the pelleted by centrifugation 20min of 4000r/min, collect supernatant and obtain the first separator.By remaining precipitation and first after centrifugation
Alcohol-water solution presses solid-liquid ratio 5g：80mL mixes, and second water-bath extraction 100min under conditions of 60 DEG C, obtains second and carries
Take thing.By this second extract in the pelleted by centrifugation 35min of 3000r/min, collect the supernatant after being this time centrifuged and obtain second point
From thing.First separator and the second separator are merged, and with the volume ratio that feeds intake as 1mL:12mL is mixed with guanidine hydrochloride solution, in
After cold preservation standing 24h in 4 DEG C of refrigerator, it is centrifuged 10min under the centrifugal condition of 4000r/min, removes protein portion, collect
Supernatant obtains crude polysaccharides solution.Dehydrated alcohol is added to obtain solution to be concentrated and make the anhydrous second in solution to be concentrated in crude polysaccharides solution
The volume fraction of alcoholic solution is 90%, by this solution to be concentrated in 50 DEG C water-bath, the rotating speed of 40 turns/min and 0.09MPa true
Under the conditions of reciprocal of duty cycle, rotary evaporation is 7% to its moisture content, obtains Paeonia suffruticosa polysaccharide.Contain in 1ul crude polysaccharides solution in the present embodiment
The Paeonia suffruticosa polysaccharide of 0.053ug.
Embodiment 8
Fresh Paeonia suffruticosa is carried out remove impurity, cleaning, dries at 36 DEG C and its kind of corium farinosum is broken to granularity for 500 μm, adopt
Microwave extraction obtains the first extract.By this first extract in the pelleted by centrifugation 20min of 4000r/min, collect supernatant and obtain
First separator.Obtain the second extract by after remaining precipitation after centrifugation again microwave extraction.By this second extract in
The pelleted by centrifugation 20min of 4000r/min, collects supernatant and obtains the second separator.First separator and the second separator are merged,
And with the volume ratio that feeds intake as 1mL:12mL is mixed with guanidine hydrochloride solution, after cold preservation standing 24h in 4 DEG C of the refrigerator, in 4000r/
It is centrifuged 10min under the centrifugal condition of min, removes protein portion, collect the supernatant after being this time centrifuged and obtain crude polysaccharides solution.To
Dehydrated alcohol is added to obtain solution to be concentrated and make the volume fraction of the ethanol solution in solution to be concentrated be in crude polysaccharides solution
90%, by this solution to be concentrated in 50 DEG C water-bath, rotary evaporation under the rotating speed of 40 turns/min and the vacuum degree condition of 0.09MPa
It is 5% to its moisture content, obtain Paeonia suffruticosa polysaccharide.Paeonia suffruticosa polysaccharide containing 0.033ug in 1ul crude polysaccharides solution in the present embodiment.
Embodiment 9
Fresh Paeonia suffruticosa is carried out remove impurity, cleaning, drying at 36 DEG C and its blade and kind skin are all crushed to granularity is 500
μm, the first extract is obtained using ultrasonic extraction.By this first extract in the pelleted by centrifugation 20min of 4000r/min, collect
Supernatant obtains the first separator.Obtain the second extract by after remaining precipitation after centrifugation again ultrasonic extraction.By this second
Extract, in the pelleted by centrifugation 20min of 4000r/min, is collected supernatant and is obtained the second separator.By the first separator and second point
Merge from thing, and with the volume ratio that feeds intake as 1mL:12mL is mixed with guanidine hydrochloride solution, after cold preservation standing 24h in 4 DEG C of the refrigerator,
It is centrifuged 10min under the centrifugal condition of 4000r/min, removes protein portion, the supernatant after collection this time centrifugation obtains slightly many
Sugar juice.Dehydrated alcohol is added to obtain solution to be concentrated and make the volume of the ethanol solution in solution to be concentrated in crude polysaccharides solution
Fraction is 90%, by the vacuum degree condition backspin of water-bath, the rotating speed of 40 turns/min and 0.09MPa in 50 DEG C for this solution to be concentrated
Turn and be evaporated to its moisture content for 3%, obtain Paeonia suffruticosa polysaccharide.Paeonia suffruticosa containing 0.077ug in 1ul crude polysaccharides solution in the present embodiment
Polysaccharide.
Embodiment 10
Fresh Paeonia suffruticosa is carried out remove impurity, cleaning, drying at 36 DEG C and its blade and kind skin are all crushed to granularity is 500
μm, the first extract is obtained using supercritical extraction.By this first extract in the pelleted by centrifugation 20min of 4000r/min,
Collect supernatant and obtain the first separator.Obtain the second extract by after remaining precipitation after centrifugation again supercritical extraction.
By this second extract in the pelleted by centrifugation 20min of 4000r/min, collect supernatant and obtain the second separator.By the second extract
After centrifugation, remaining precipitation obtains the 3rd extract after carrying out third time supercritical extraction.By the 3rd extract in
The pelleted by centrifugation 20min of 4000r/min, collects supernatant and obtains the 3rd separator, by the first separator, the second separator and the 3rd
Separator merges, and with the volume ratio that feeds intake as 1mL:12mL is mixed with guanidine hydrochloride solution, cold preservation standing 24h in 4 DEG C of refrigerator
Afterwards, it is centrifuged 10min under the centrifugal condition of 4000r/min, removes protein portion, collect the supernatant after being this time centrifuged and obtain slightly
Polysaccharide solution.Dehydrated alcohol is added to obtain solution to be concentrated and make the body of the ethanol solution in solution to be concentrated in crude polysaccharides solution
Fraction is 90%, by this solution to be concentrated in 50 DEG C water-bath, under the rotating speed of 40 turns/min and the vacuum degree condition of 0.09MPa
Rotary evaporation is 1% to its moisture content, obtains Paeonia suffruticosa polysaccharide.Male containing 0.081ug in 1ul crude polysaccharides solution in the present embodiment
Red polysaccharide.
Test example 1
Using following free radical scavenging Experiment on Function method, the Paeonia suffruticosa polysaccharide material of gained in embodiment is purged
Rate is tested.
Method of testing：Repeat above example 1-10 be obtained enough Paeonia suffruticosa blade polysaccharide, Paeonia suffruticosa kind skin polysaccharide with
And Paeonia suffruticosa blade and kind skin mixing polysaccharide, as test group, with commercially available polysaccharide as a control group, are each configured to variable concentrations
Polysaccharide solution as prepare liquid, its concentration be followed successively by 0.025ug/ul, 0.05ug/ul, 0.075ug/ul, 0.1ug/ul and
0.125ug/ul.
The reaction solution that mensure determinand acts on to free radical scavenging includes：Prepare liquid, 0.2M and pH be 7.4 phosphoric acid delay
Rush liquid, 7.5mmol/LFeSO4, ultra-pure water, 5.0mmol/L orthophenanthroline solution and 0.1% hydrogenperoxide steam generator.Reaction needs altogether
Blank tube, do not damage pipe, damage pipe, sample blank pipe and sample-adding pipe amount to 5 reaction systems.Wherein, add 2mL in blank tube
Phosphate buffer and 8mL ultra-pure water that concentration is 7.4 for 0.2mol/L and pH；Do not damage pipe to add 2mL concentration is 0.2mol/L
And pH is 7.4 phosphate buffer, the FeSO of the 7.5mmol/L of 2mL4, the ultra-pure water of 5.4mL and 1.6mL orthophenanthroline molten
Liquid；Damage and in pipe, add phosphate buffer, the FeSO of the 7.5mmol/L of 1mL that 2mL concentration is 7.4 for 0.2mol/L and pH4、
The ultra-pure water of 4.4mL, the orthophenanthroline solution of 1.6mL and the hydrogenperoxide steam generator of 1mL；2mL concentration is added in sample blank pipe
The polysaccharide solution to be measured of the phosphate buffer, 7mL ultra-pure water and each concentration of 1mL that are 7.4 for 0.2mol/L and pH；Sample-adding Guan Zhongjia
Enter phosphate buffer, the 7.5mmol/LFeSO of 1mL that 2mL concentration is 7.4 for 0.2mol/L and pH4, 3.4mL ultra-pure water,
The polysaccharide solution to be measured of 1.6mL orthophenanthroline solution, 1mL hydrogenperoxide steam generator and each concentration of 1mL.Each after addition reagent
1h placed in 40 DEG C of water bath with thermostatic control by test tube, respectively using sample blank pipe and blank tube as control tube zero setting, in 536nm ripple
Strong point is measured and is damaged pipe, do not damage pipe and variable concentrations Paeonia suffruticosa polysaccharide determinand and the extinction number of degrees of commercially available polysaccharide sample-adding pipe
Value, calculates the Paeonia suffruticosa polysaccharide and commercially available polysaccharide clearance rate to hydroxyl radical free radical.Computing formula is as follows：
Hydroxyl radical free radical clearance rate (%)=[(A3-A2)/(A1-A2)] × 100%.
Wherein：A1、A2It is respectively the absorbance not damaging and damaging pipe；A3For variable concentrations Paeonia suffruticosa polysaccharide and commercially available
Polysaccharide is loaded the absorbance of pipe.
Test result is as follows：In embodiment 1-10, the Paeonia suffruticosa polysaccharide prepared by embodiment 5 acts on to free radical scavenging
The strongest, its reason be this embodiment with Paeonia suffruticosa kind skin and blade collectively as raw material, the technique employed in its preparation process
Condition is optimal value.Additionally, concentration be respectively 0.025ug/ul, 0.05ug/ul, 0.075ug/ul, 0.1ug/ul and
During 0.125ug/ul, Paeonia suffruticosa blade polysaccharide, Paeonia suffruticosa kind skin polysaccharide and Paeonia suffruticosa blade and plant skin mixing polysaccharide corresponding hydroxyl from
As shown in table 1 by base clearance rate：
The hydroxyl radical free radical clearance rate of Paeonia suffruticosa polysaccharide under table 1 variable concentrations
As seen from Table 1, under the conditions of same concentration, Paeonia suffruticosa kind skin polysaccharide, Paeonia suffruticosa blade polysaccharide and Paeonia suffruticosa blade with
Plant skin mixing polysaccharide the scavenging action of hydroxyl radical free radical is strengthened successively；The ability of this three kinds of polysaccharide scavenging hydroxyl is all with many
The increase of sugared concentration and strengthen, and be in significant positive correlation.In addition, Paeonia suffruticosa blade polysaccharide can also be drawn from test result
And Paeonia suffruticosa blade is all remarkably higher than the free radical scavenging effect of commercially available polysaccharide with kind skin mixing polysaccharide, therefore embodiment of the present invention institute
The Paeonia suffruticosa polysaccharide prepared has stronger free radical scavenging effect.
Therefore, Paeonia suffruticosa polysaccharide can be applied to prepare free radical clear by the scavenging action to free radical using Paeonia suffruticosa polysaccharide
Except agent.And preferably, Paeonia suffruticosa polysaccharide can be prepared using above-mentioned Paeonia suffruticosa polyoses producing method.Additionally, this polysaccharide
Material is derived from natural plants, and toxic and side effects are little, also can use it for for example removing the health care of radical type in various health product
In food.
In sum, the embodiment of the present invention, by pulverizing to Paeonia suffruticosa, increased the contact between raw material and extractant
Area, improves leaching velocity.With hydrophilic organic solvent as extractant, both can guarantee that leaching velocity is fast, extractum purity is high,
Impurity is few, and can also reduces cost.With 5-20g：80-400mL can make the active component in Paeonia suffruticosa extract effect as solid-liquid ratio
Fruit is preferably；It is used in the mode that 60-85 DEG C of water-bath extracts 30-100min, can avoid causing active substance because extraction time is too short
Dissolution rate is low, and extraction time is long and can make the excessive dissolutions of impurity such as oils and fatss in plant cell, tannin, increase separate pure
The defects such as the difficulty changed.Crude extract is mixed the protein that can make in crude extract with the solution of trichloroacetic acid in protein denaturant
Degeneration, under the conditions of 2-8 DEG C, standing can make the abundant degeneration of protein precipitate, and makes the separating effect of Paeonia suffruticosa polysaccharide and extraction rate reached to
Good.The method is simple to operate, it is easy to accomplish, the Paeonia suffruticosa polysaccharide prepared has good free radical scavenging effect.Due to this
Bright raw material is natural product, and toxic and side effects are little, thus the polysaccharide material prepared by it to can be widely applied to various free radicals clear
Except in agent.
Embodiments described above is a part of embodiment of the present invention, rather than whole embodiments.The reality of the present invention
The detailed description applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected enforcement of the present invention
Example.Based on the embodiment in the present invention, those of ordinary skill in the art are obtained under the premise of not making creative work
Every other embodiment, broadly falls into the scope of protection of the invention.
Claims (10)
1. a kind of preparation method of Paeonia suffruticosa polysaccharide is it is characterised in that it comprises the following steps：
Pulverize Paeonia suffruticosa raw material and be 400-600 μm to its particle diameter, described Paeonia suffruticosa raw material is selected from Paeonia suffruticosa blade or Paeonia suffruticosa kind skin；
The supernatant carrying out extracting separation and collect after separating to the described Paeonia suffruticosa raw material after pulverizing obtains crude extract, extracts to separate and presses
In the following manner is carried out：By solid-liquid ratio 5-20g：Extractant is mixed by 80-400mL with the described Paeonia suffruticosa raw material after pulverizing, extracts 30-
100min, is collected by filtration supernatant, and described extractant is hydrophilic organic solvent；
Described crude extract is mixed with protein denaturant, stands 12-48h under conditions of 2-8 DEG C, centrifugation, after collecting centrifugation
Supernatant obtain crude polysaccharides solution；
Add ethanol solution to obtain solution to be concentrated in described crude polysaccharides solution, after concentration, obtain Paeonia suffruticosa polysaccharide.
2. preparation method according to claim 1 is it is characterised in that described Paeonia suffruticosa raw material is selected from Paeonia suffruticosa kind skin and will be described
Paeonia suffruticosa raw material pulverizing granularity is to 420-580 μm.
3. preparation method according to claim 1 is it is characterised in that described extractant is 70%- for percentage by volume
The aqueous solution of 90% ethanol.
4. preparation method according to claim 1 is it is characterised in that extracted to the described Paeonia suffruticosa raw material after pulverizing
Mode extracts for water-bath, and the bath temperature of water-bath extraction is 60-85 DEG C.
5. preparation method according to claim 1 is it is characterised in that described protein denaturant for mass percent is
The solution of trichloroacetic acid of 6%-15%.
6. preparation method according to claim 5 is it is characterised in that the throwing of described crude extract and described solution of trichloroacetic acid
Material volume ratio is 1:6-20.
7. preparation method according to claim 1 is it is characterised in that described crude extract is mixed with described protein denaturant
The time being centrifuged after standing is 15-30min, and rotating speed is 3000-5000r/min.
8. a kind of Paeonia suffruticosa polysaccharide is it is characterised in that the preparation method system of its Paeonia suffruticosa polysaccharide described in any one of claim 1-8
Standby and obtain.
9. application in preparing free radical scavenger for the Paeonia suffruticosa polysaccharide.
10. Paeonia suffruticosa polysaccharide removes the application in the health food of radical type in preparation.
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