CN109142131A - The extracting method of trollflower polysaccharide and its detection method of external activity - Google Patents
The extracting method of trollflower polysaccharide and its detection method of external activity Download PDFInfo
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Abstract
The invention discloses a kind of extracting method of trollflower polysaccharide and its detection methods of external activity, polysaccharide are extracted using the method for water body alcohol precipitation, with Sevag method isolating protein；Using hydrometer method, polysaccharide moisturizing and hygroscopicity are studied by doing comparison with traditional moisturizer；L-AA and polysaccharide are compared, by measurement sample to the clearance rate of total reducing power, DPPH and ABTS free radical, to study the in vitro anti-oxidation of trollflower polysaccharide.
Description
Technical field
The present invention relates to Chinese herbal medicine detection fields, and in particular to a kind of extracting method and its external activity of trollflower polysaccharide
Detection method.
Background technique
Trollflower is the dry flower and petal of ranunculaceae plant trollflower, is distributed widely in northwest, southwest, northeast ground
Area.Supplementary Amplifications of the Compendium of Materia Medica calls its " bitter, cold in nature, nontoxic ", can treat aphtha, throat swelling and pain, superficial heat pingival atrophy, ear pain, mesh
Bitterly, and have " improving eyesight, Xie Lan barrier " and other effects.The a variety of active ingredients such as polysaccharide, flavones, alkaloid are mainly contained in trollflower, are had
There is the clearing heat and detoxicating refreshing oneself, promoting digestion that nourishes the liver to improve visual acuity, has to inflammations such as stomatitis pharyngitis tonsillitis acute myringitis
Obvious curative effects.
Summary of the invention
The purpose of the present invention is to provide a kind of extracting method of trollflower polysaccharide and its detection methods of external activity.
To achieve the above object, the technical scheme adopted by the invention is as follows:
The extracting method of trollflower polysaccharide, includes the following steps:
Suitable quantity of water is added into trollflower petal or the powder of scape in the ratio of solid-liquid ratio 1: 60, under conditions of 80 DEG C
Heating water bath 3h, is stirred every 30min with glass bar primary, is extracted three times repeatedly, is filtered to obtain clear liquid, merge supernatant to get；
The activity of above-mentioned trollflower polysaccharide detects by the following method:
A, polysaccharide hygroscopicity detects:
Trollflower polysaccharide sample is contained in aluminium box and after drying to constant weight in 50 DEG C of baking oven, is put in and fills saturation sulfuric acid
In the drier of ammonium salt solution and saturated sodium carbonate solution, that is, being placed in relative humidity is 81% and 43% (RH81% and RH43%)
Drier in, and entire drier is placed in constant incubator, 25 DEG C of constant temperature, respectively at place 4h, 8h, 12h, 20h,
Quality is accurately weighed with assay balance after 44h, is calculate by the following formula the hydroscopicity of polysaccharide: hydroscopicity (%)=(sample moisture
Incrementss/sample proper mass) × 100%；
B, polysaccharide moisture retention detects
In the discoloration silica gel cleaned and dry drier bottom addition is dry, 20ml is added in every part of trollflower polysaccharide sample
Distilled water, and quality is accurately weighed again, it is placed in drier, and entire drier is placed in 25 DEG C of constant incubators
In, quality is accurately weighed with assay balance after placing 4h, 8h, 12h, 20h, 44h, is calculate by the following formula the moisturizing of polysaccharide
Rate: moisturizing rate (%)=(reduction amount of sample moisture/sample proper mass) × 100%；
C, the anti-oxidant detection of polysaccharide
(1) measurement of total reducing power
1ml different quality concentration polysaccharide solution is taken, 2.5ml is added, 3ml mass is added in the phosphate buffer of pH6.6 later
The potassium ferricyanide aqueous solution of score 1% keeps the temperature 10min in 50 DEG C of water-baths after mixing well；It cools down rapidly, adds after taking-up
The trichloroacetic acid solution of 2.5ml mass fraction 10%, oscillation shake up, and in 3000r/min, are centrifuged 10min；Pipette supernatant
2.5ml adds the ferric chloride aqueous solutions of 2.5ml distilled water and 0.5ml mass fraction 0.1% in test tube, is protected from light at room temperature anti-
20min is answered, light absorption value is measured at 700nm；
(2) measurement of DPPH Scavenging activity
The polysaccharide solution of 1ml different quality concentration is taken, 3ml is added, the DPPH free radical ethanol solution of 0.2mmol/L mixes
4.0ml ethanol solution is added after even, after being protected from light 30min at room temperature, absorbance is measured at 517nm, as follows
Sample is calculated to the clearance rate of DPPH:
In formula: AcFor the light absorption value of blank sample, AsFor the light absorption value after various concentration sample is added.
(3) measurement of ABTS free radical scavenging ability
The ABTS solution 10ml of 7mmol/L is mixed with the potassium persulfate solution 10ml of final concentration of 2.45mmol/L, in
It places 14-16 hours under the conditions of room temperature, darkness, is diluted to using the preceding phosphate buffer with the pH=7.4 of 0.2mol/L
Absorbance be 0.72~1.2 to get arrive ABTS free radical working solution；
Take each 200 μ L of the polysaccharide solution of different quality concentration, ABTS free radical working solution 4ml be added, react after 6min
Absorbance is surveyed at wavelength 734nm, calculates sample as follows to the clearance rate of ABTS free radical.
In formula: AcFor the light absorption value of blank sample, AsFor the light absorption value after various concentration sample is added.
The invention has the following advantages:
Polysaccharide is extracted using the method for water body alcohol precipitation, with Sevag method isolating protein；Using hydrometer method, by being protected with tradition
Humectant does comparison to study polysaccharide moisturizing and hygroscopicity；L-AA and polysaccharide are compared, gone back by measurement sample to total
The clearance rate of former power, DPPH and ABTS free radical, to study the in vitro anti-oxidation of trollflower polysaccharide.
Detailed description of the invention
Fig. 1 is glucose standard curve.
Fig. 2 is protein standard curve.
Fig. 3 is rear absorbance of the petal polysaccharide at 595nm before purification.
Fig. 4 is rear scape polysaccharide absorbance at 595nm before purification.
Fig. 5 is the hydroscopicity of RH81% polysaccharide and reference substance.
Fig. 6 is the hydroscopicity of RH43% polysaccharide and reference substance.
Fig. 7 is petal polysaccharide moisturizing rate.
Fig. 8 is flower (not) polysaccharide moisturizing rate.
Fig. 9 is scape polysaccharide moisturizing rate.
Figure 10 is stem (not) polysaccharide moisturizing rate.
Figure 11 is total reducing power.
Figure 12 is DPPH free radical scavenging activity.
Figure 13 is ABTS free radical scavenging activity.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
Embodiment
Make glucose standard curve
Precision weighs dry (105 DEG C) to the glucose 50mg of constant weight, is settled to 100ml, obtains Standard glucose solution.It takes
Standard glucose solution 0.0ml, 4.0ml, 8.0ml, 12.0ml, 16.0ml, 20.0ml are settled to 100ml respectively and obtain difference
Concentration dilution liquid.The accurate above-mentioned dilution 0.5ml of absorption is placed in the test tube of 10.0ml respectively, and 5% phenol 1.0ml is added,
It is rapidly added the concentrated sulfuric acid of 5.0ml, is shaken up, 30min is reacted in 40 DEG C of water-baths, then sets cold bath 10min.In 490nm wavelength
Under, absorbance, which is measured, using glucose content as abscissa using absorbance value as ordinate, obtains regression equation: y=5.7729x+
0.0005.R2=0.9994；As shown in Figure 1, equation of linear regression related coefficient square be 0.9994, have good correlation
Property, glucose solution is 0~0.12mg/ml to effective use scope of the Trendline.
Make protein standard curve
Standard protein curve is made using Coomassie brilliant blue and bovine serum albumin.Coomassie brilliant G-250 10mg is taken to be dissolved in
In 5mL95% ethyl alcohol, 85% phosphoric acid of 10mL is added, is diluted to 100mL with distilled water.Meanwhile making the cow's serum of 0.1mg/ml
Protein solution, takes 1ml bovine serum albumin, and gradient is set as 0,0.02,0.04,0.06,0.08,0.10mg/mL.5m is separately added into examine
Mas bright blue solution measures absorbance at 595nm.Obtain calcium: y=7.2886x+0.0106.R2=0.9991；
As shown in Figure 2, equation of linear regression related coefficient square be 0.9991, have good correlation, protein solution is to this
Effective use scope of Trendline is 0~0.12mg/ml.
The extraction of polysaccharide
The method of this experimental selection water extract-alcohol precipitation extracts the polysaccharide in trollflower.Its principle is big using polysaccharide as polarity
This property of molecule makes polysaccharide be dissolved in the water, concentrated extracting solution with water as solvent by heating.Polysaccharide is recycled not dissolve in
The property of ethyl alcohol is precipitated out polysaccharide from precipitating.It is specific: 1800g water (material being added into 30g petal or the powder of scape
Liquor ratio 1: 60), heating water bath 3h under conditions of 80 DEG C is stirred once every 30min glass bar, is extracted three times, is taken out repeatedly
Clear liquid is filtered to obtain, merges supernatant and is placed in the refrigeration of 2500ml white plastic bucket, waits subsequent de- albumen processing.
Polysaccharide takes off albumen
This experiment takes off albumen using Sevag method, prepares mix reagent: chloroform: n-butanol 4: 1 (volume ratio).It will
Sample mixes at 4: 1 by volume with mix reagent, is placed in constant-temperature table 150rpm, vibrates 30min, stands and divides in separatory funnel
Layer and liquid separation.
The thick yield of polysaccharide, deproteinizing rate are calculated according to following formula
Protein removal rate formula:
In formula: MBeforeFor protein quality before resin treatment, μ g；MAfterwardsFor protein quality after resin treatment, μ g.
The moisturizing moisture absorption of polysaccharide
Sucting wet experiment
The aluminium box for holding sample is cleaned and drying to constant weight in 50 DEG C of baking oven, and by sample and control sample: hyaluronic acid
Sodium, chitosan, sodium alginate, PEG-6000, equally in baking oven 50 DEG C drying to constant weight.It dries to constant weight to sample and aluminium box,
Accurately weigh above-mentioned sample and control sample, and make a record, the aluminium box for filling sample is put in fill saturated ammonium sulfate solution and
In the drier of saturated sodium carbonate solution, that is, it is placed in the drier that relative humidity is 81% and 43% (RH81% and RH43%)
In, and entire drier is placed in constant incubator, 25 DEG C of constant temperature, it is used after placing 4h, 8h, 12h, 20h, 44h
Assay balance accurately weighs quality.
Hydroscopicity (%)=(incrementss of sample moisture/sample proper mass) × 100%
Moisturizing experiment
In the discoloration silica gel cleaned and dry drier bottom addition is dry, 20ml distilled water is added in every part of sample, and
Quality is accurately weighed again, is placed in drier, and entire drier is placed in 25 DEG C of constant incubators, respectively at putting
It sets and accurately weighs quality with assay balance after 4h, 8h, 12h, 20h, 44h.
Moisturizing rate (%)=(reduction amount of sample moisture/sample proper mass) × 100%
Polysaccharide it is anti-oxidant
The measurement of total reducing power
1ml different quality concentration polysaccharide solution is taken, 2.5ml is added, 3ml mass is added in the phosphate buffer of pH6.6 later
The potassium ferricyanide aqueous solution of score 1% keeps the temperature 10min in 50 DEG C of water-baths after mixing well；It cools down rapidly, adds after taking-up
The trichloroacetic acid solution of 2.5ml mass fraction 10%, oscillation shake up, and in 3000r/min, are centrifuged 10min.Pipette supernatant
2.5ml adds the ferric chloride aqueous solutions of 2.5ml distilled water and 0.5ml mass fraction 0.1% in test tube, is protected from light at room temperature anti-
20min is answered, light absorption value is measured at 700nm.(light absorption value is bigger, and reducing power is stronger) is opposed with the Vc of identical mass concentration gradient
According to detecting according to the method described above.
The measurement of DPPH Scavenging activity
The polysaccharide solution of 1ml different quality concentration is taken, 3ml is added, the DPPH free radical ethanol solution of 0.2mmol/L mixes
4.0ml ethanol solution is added after even, after being protected from light 30min at room temperature, absorbance is measured at 517nm, blank is to distill
Water replaces polysaccharide solution as control.It is compared with the Vc of identical mass concentration gradient, is detected according to the method described above.By following public affairs
Formula calculates sample to the clearance rate of DPPH.
In formula: AcFor the light absorption value of blank sample, AsFor the light absorption value after various concentration sample is added.
The measurement of ABTS free radical scavenging ability
The ABTS solution 10ml of 7mmol/L is mixed with the potassium persulfate solution 10ml of final concentration of 2.45mmol/L, in
It is placed 14-16 hours under the conditions of room temperature, darkness, it is using the preceding phosphate buffer (PBS) (pH=7.4) with 0.2mol/L that its is dilute
Releasing to absorbance is 0.72~1.2 to get to ABTS free radical working solution, ready-to-use.
Take each 200 μ L of the polysaccharide solution of different quality concentration, ABTS free radical working solution 4ml be added, react after 6min
Absorbance is surveyed at wavelength 734nm, is replaced sample to do blank test with distilled water, is compared with the Vc of identical mass concentration gradient,
It detects according to the method described above.Sample is calculated as follows to the clearance rate of ABTS free radical.
In formula: AcFor the light absorption value of blank sample, AsFor the light absorption value after various concentration sample is added.
Results and discussion
Polysaccharide deproteinizing rate
Petal polysaccharide and scape polysaccharide take off 2 albumen respectively, petal and scape polysaccharide deproteinizing rate be respectively 35.96% with
39.44%.As a result as shown in Figure 3, Figure 4.
The moisturizing hygroscopicity of polysaccharide
Polysaccharide hygroscopicity
It has been investigated under the conditions of constant temperature (25 DEG C) relative humidity 81% (RH81%) and has extracted polysaccharide in white flower valve and scape
Hygroscopicity, and compared with not Deproteinated petal and scape polysaccharide and several traditional moisturizer, the hydroscopicity of each sample
Increase with standing time and increase, as shown in Figure 5.The rapid development in 20h, glycerol and sodium alginate hydroscopicity continue after 20h
Increase, remaining sample hydroscopicity tends to mitigate.After placing 4h, hydroscopicity sequence are as follows: glycerol > PEG-400 > sodium alginate > is saturating
Bright matter acid sodium > stem takes off the non-> of > flower and spends the non-> chitosan > PEG-6000 of de- > stem；After 8h, sequence are as follows: glycerol > PEG-400
The non-> of > Sodium Hyaluronate > flower spends de- > sodium alginate > stem to take off the non-> chitosan > PEG-6000 of > stem；After 12h, sequence
Are as follows: the non-> sodium alginate > Sodium Hyaluronate > of glycerol > PEG-400 > flower spends de- > stem to take off the poly- > sugar > of the non-> shell of > stem
PEG-6000；After 20h, sequence are as follows: the non-> Sodium Hyaluronate > stem of glycerol > PEG-400 > sodium alginate > flower takes off the non-> of > stem
Spend de- > chitosan > PEG-6000；After 44h, sequence are as follows: the non-> of the non-> flower of glycerol > alginic acid > sodium > PEG-400 > stem is saturating
Bright matter acid sodium > stem takes off > and spends de- > chitosan > PEG-6000, and not after 20h, hydroscopicity rises rapidly stem, moisture absorption after 44h
Rate: the non-> stem of stem non-> flower take off > spend it is de-, stem not with flower not higher than Sodium Hyaluronate, stem it is de- with spend it is de- higher than chitosan and
PEG-6000.In 12h, the non-hydroscopicity of flower exceeds sodium alginate.By data and chart it is found that in RH81%, lily feet is extracted from
Flower petal and the polysaccharide of scape have preferable hygroscopicity, and former polysaccharide has the moisture absorption for slightly showing color compared with the polysaccharide of deproteination
Property.There is a possibility that the above results in following discussion, and the moisture absorption gender gap from figure between four groups of polysaccharide is not special
Greatly, possible cause: the hygroscopic influence of 1. floating preteins confrontation polysaccharide does not serve conclusive；2. free protein removes
Number only 2 times, there are still relatively large number of protein in polysaccharide, cause the polysaccharide of de- albumen and non-deproteinization in hygroscopicity
Difference is simultaneously little.
It has been investigated under the conditions of constant temperature (25 DEG C) relative humidity 43% (RH43%) and has extracted from polysaccharide in petal and scape
Hygroscopicity, and compared with not Deproteinated petal and scape polysaccharide and several traditional moisturizer, the hydroscopicity of each sample
Increase with standing time and increase, as shown in Figure 6.The rapid development in 20h, glycerol, sodium alginate and PEG-400 inhale after 20h
Wet rate continues to increase, remaining sample hydroscopicity tends to mitigate.After placing 44h, hydroscopicity sequence are as follows: glycerol > sodium alginate >
The non-> stem of PEG-400 > Sodium Hyaluronate > flower takes off > and spends the non-> chitosan > PEG-6000 of de- > stem；After 20h, sequence are as follows: sweet
The non-> stem of oily > PEG-400 > Sodium Hyaluronate > sodium alginate > flower takes off > and spends the non-> chitosan > PEG-6000 of de- > stem；
After 12h, sequence are as follows: the non-> sodium alginate > of glycerol > PEG-400 > Sodium Hyaluronate > flower spends de- > stem to take off the non-> shell of > stem
Glycan > PEG-6000；After 8h: the non-> of glycerol > Sodium Hyaluronate > PEG-400 > flower spends de- > stem to take off > sodium alginate > stem
Non- > chitosan > PEG-6000；After 4h: the non-> of glycerol > Sodium Hyaluronate > PEG-400 > chitosan > flower spends de- > stem de-
The non-> PEG-6000 of > sodium alginate > stem.In RH43%, 44h, polysaccharide hydroscopicity sequence are placed are as follows: the de- > of the non-> stem of flower spends de-
Not, respectively flower is not 41.47%, stem is de- 38.03%, spends de- 36.45%, stem not 35.40% for > stem.When placing 8h, polysaccharide
Hydroscopicity is higher than sodium alginate, chitosan.Following discussion is likely to occur the possible cause of the above results, is observed by chart and data
It was found that its otherness is not particularly evident although height can be discharged in hygroscopicity of four groups of polysaccharide under the conditions of RH43%.It is former
Because possible as follows: the hygroscopic influence of 1. floating preteins confrontation polysaccharide does not serve conclusive；2. free protein removes
Number only 2 times, there are still relatively large number of protein in polysaccharide, cause the polysaccharide of de- albumen and non-deproteinization in hygroscopicity
Difference is simultaneously little.Under conditions of 3.RH43% compared with RH81% under the conditions of, relatively dry, therefore hygroscopic while can investigate investigating
Moisture retention, therefore result will receive certain influence.
Polysaccharide moisture retention
Investigation extracts from more in petal and scape under constant temperature (25 DEG C), the drying condition created by 500g discoloration silica gel
The moisture retention of sugar, and compared with not Deproteinated petal and scape polysaccharide and several traditional moisturizer, each sample moisturizing
Rate reduces at any time.Wherein PEG-6000 moisture retention is most strong, and moisturizing rate is still up to 93.59% after 44h.
De- albumen treated petal polysaccharide moisture retention result are as follows: after placing 4h, moisturizing rate sequence are as follows: PEG-400 > shell
Glycan > Sodium Hyaluronate > PEG-6000 > sodium alginate > spends > glycerol；After 8h, sequence are as follows: PEG-6000 > sodium alginate
> Sodium Hyaluronate > spends > chitosan > glycerol > PEG-400；After 12h, sequence are as follows: PEG-6000 > sodium alginate > is transparent
Matter acid sodium > spends > glycerol > chitosan > PEG-400；After 20h: PEG-6000 > sodium alginate > flower > glycerol > hyalomitome
Sour sodium > PEG-400 > chitosan；After 44h: PEG-6000 > flower > Sodium Hyaluronate > sodium alginate > glycerol > PEG-400
> chitosan.After 44h, be up to 93.29% by Thick many candies its moisturizing rate that petal extracts, be higher than Sodium Hyaluronate, sodium alginate,
Glycerol, PEG-400 and chitosan.As a result as shown in Figure 7.
De- albumen treated petal polysaccharide moisture retention result are as follows: after 4h: PEG-400 > chitosan > Sodium Hyaluronate
The non-> glycerol of > PEG-6000 > sodium alginate > flower；After 8h: the non-> of PEG-6000 > sodium alginate > Sodium Hyaluronate > flower
Chitosan > glycerol > PEG-400；After 12h: the non-> sodium alginate > Sodium Hyaluronate > glycerol > shell of PEG-6000 > flower is poly-
Sugared > PEG-400；20h moisturizing rate sequence: the non-> sodium alginate > glycerol > Sodium Hyaluronate > PEG-400 of PEG-6000 > flower
> chitosan；After placing 44h, moisturizing rate sequence are as follows: the non-> Sodium Hyaluronate > sodium alginate > glycerol > of PEG-6000 > flower
PEG-400 > chitosan；After 12h, the moisturizing rate of flower not is higher than sodium alginate, Sodium Hyaluronate, glycerol, chitosan and PEG-
400.As a result as shown in Figure 8.
De- albumen treated scape polysaccharide moisture retention result are as follows: after 4h, moisturizing rate sequence: PEG-400 > chitosan >
Sodium Hyaluronate > PEG-6000 > sodium alginate > stem > glycerol；After 8h, sequence are as follows: PEG-6000 > sodium alginate > is transparent
Matter acid sodium > chitosan > stem > glycerol > PEG-400；After 12h: PEG-6000 > sodium alginate > Sodium Hyaluronate > glycerol
The poly- > sugar > PEG-400 of > stem > shell；After 20h: PEG-6000 > sodium alginate > glycerol > stem > Sodium Hyaluronate > PEG-
400 > chitosans；After 44h: PEG-6000 > Sodium Hyaluronate > stem > sodium alginate > glycerol > PEG-400 > chitosan.
The polysaccharide of scape is extracted from 20h, moisturizing rate is higher than Sodium Hyaluronate, PEG-400 and chitosan；When placing 44h,
Its moisturizing rate is 93.41%, is higher than sodium alginate, glycerol, PEG-400 and chitosan.As a result as shown in Figure 9.
De- albumen treated scape polysaccharide moisture retention result are as follows: sequence after 4h are as follows: PEG-400 > chitosan > is transparent
The non-> glycerol of matter acid sodium > PEG-6000 > sodium alginate > stem；Sequence after 8h are as follows: PEG-6000 > sodium alginate > hyalomitome
The sour non-> glycerol > PEG-400 of sodium > chitosan > stem；Sequence after 12h are as follows: the non-> of PEG-6000 > sodium alginate > stem is transparent
Matter acid sodium > glycerol > chitosan > PEG-400；After 20h, moisturizing rate sequence are as follows: the non-> sodium alginate > of PEG-6000 > stem is sweet
Oily > Sodium Hyaluronate > PEG-400 > chitosan；After 44h, moisturizing rate sequence are as follows: PEG-6000 > Sodium Hyaluronate > stem is not
> sodium alginate > glycerol > PEG-400 > chitosan；In 12h, the non-moisturizing rate of stem is lower than sodium alginate and PEG-6000,
In 20h, the non-moisturizing rate highest of stem, is only below PEG-6000, and when placing 44h, the non-polysaccharide moisturizing rate of stem is lower than Sodium Hyaluronate
With PEG-6000.The results are shown in Figure 10.
To sum up data and chart discovery, the polysaccharide for extracting from scape and petal all have good moisture retention, and in petal
Polysaccharide slightly higher moisture retention is shown compared with the polysaccharide in scape, and not Deproteinated scape and petal polysaccharide also show that quite
Moisture retention, first analysis there is a possibility that the above results: 1. free proteins do not rise conclusive on polysaccharide moisture retention
Effect；2. protein content still has biggish ratio in gained polysaccharide solution since polysaccharide solution has only taken off protein twice
Example, therefore its difference in the research of moisture retention is not apparent；It is different that 3 samples dissolve insufficient and each sample dissolution degree；
4. drier space is relatively large；5 test periods, which are not enough, causes data differences unobvious.
The inoxidizability of polysaccharide
The measurement of total reducing power
As shown in Figure 11, with the increase of polysaccharide concentration, total reducing power is continuously improved.In concentration 0.1mg/ml,
Total reducing power sequence: the non-> stem of stem takes off > and spends the non-> Vc of de- > flower (10 times of dilution)；In 0.3mg/ml, total reducing power: stem is not
It is de- that > stem takes off the non-> stem of > Vc > flower；When 0.5mg/ml, sequence are as follows: the non-> Vc > of stem spends the non-> stem of de- > flower de-；0.7mg/ml
When, sequence are as follows: the non-> Vc > of stem spends the non-> stem of de- > flower de-；When 0.8mg/ml, sequence are as follows: the non-> Vc > of stem spends the non-> of de- > flower
Stem is de-；When 0.9mg/ml, sequence are as follows: the non-> Vc > of stem spends the non-> stem of de- > flower de-.After really diluting 10 times due to Vc concentration
, therefore total reducing power of polysaccharide is lower than Vc.Cross plot and number are it has been found that four groups of polysaccharide all have preferable reducing power, and stem is not
Highest, absorbance reach 1.318 in lmg/ml concentration；Followed by spend de-, absorbance 0.826；The non-absorbance highest of flower
It is 0.778；Stem desorption luminosity is up to 0.725.
The measurement of DPPH free radical scavenging activity
As shown in figure 12, the inoxidizability of Vc is very high, and DPPH free radical scavenging activity is average 90% or more.DPPH free radical
Clearance rate is increased with the raising of polysaccharide concentration.When concentration is 0.1mg/ml, free radical scavenging activity sequence are as follows: Vc > stem is not
> spends the non-> stem of de- > flower de-；When concentration is 0.2mg/ml, free radical scavenging activity sequence are as follows: the non-> of Vc > stem spends de- > flower not
> stem is de-；When concentration is 0.4mg/ml, free radical scavenging activity sequence are as follows: Vc > spends the non-non- > stem of > stem of de- > flower de-；Dense
When degree is 0.6mg/ml, sequence are as follows: Vc > spends the non-> stem of the non-> flower of de- > stem de-；When concentration is 0.8mg/ml, free radical is clear
Except rate sequence are as follows: the non-non- > of > stem of Vc > flower spends de- > stem de-；When concentration is 1.0mg/ml, free radical scavenging activity sequence are as follows:
The non-non- > of > stem of Vc > flower spends de- > stem de-.In summary, the polysaccharide for extracting from petal and scape all has preferable DPPH certainly
The ability removed by base.When concentration reaches lmg/ml, from data and in icon it has been observed that the DPPH of each group polysaccharide is free
Base clearance rate difference is smaller, and not Deproteinated polysaccharide has slightly higher clearance rate compared with the polysaccharide of 2 albumen of removing.Analysis may
The reason of the above results occur: 1. free proteins do not serve conclusive on polysaccharide removing DPPH free radical；2. by
In deproteination matter only 2 times, in Thick many candies still contain relatively large number of protein, therefore the polysaccharide of non-deproteinization and removing 2 times
The polysaccharide of protein difference and little on free radical scavenging activity.3. being only that part removes floating preteins due to taking off albumen step
Matter, glycoprotein do not remove, so glycoprotein might have facilitation in its oxidation resistance；4. color in polysaccharide solution
Element, bioflavonoid, polyphenols can have a certain impact to polysaccharide anti-oxidative.
The measurement of ABTS free radical scavenging activity
As shown in figure 13, the polysaccharide for extracting from trollflower petal and scape all has good ABTS radical scavenging activity
Power.When concentration is 0.1mg/ml, ABTS free radical scavenging activity sequence are as follows: the de- > of the non-non- > stem of > stem of Vc > flower spends de-；
When 0.2mg/ml, free radical scavenging activity sequence are as follows: the non-> of Vc > stem spends the non-> stem of de- > flower de-；When 0.4mg/ml, free radical is clear
Except rate sequence are as follows: the non-> of the non-> flower of Vc > stem spends de- > stem de-；When 0.6mg/ml: the de- > of the non-> stem of the non-> flower of Vc > stem spends de-；
When 0.8mg/ml: the de- > of the non-> stem of the non-> flower of Vc > stem spends de-；When 1.0mg/ml: the non-> of the non-> flower of Vc > stem spends de- > stem de-.
When polysaccharide concentration reaches highest, ABTS free radical scavenging activity is suitable with the clearance rate of Vc, is respectively as follows: the non-clearance rate of flower and is
95.09%；The non-clearance rate of stem is 95.25%；Spending de- clearance rate is 93.56%；It is 90.69% that stem, which takes off clearance rate,；Vc clearance rate
It is 96.17%, it is seen that the polysaccharide for extracting from petal and scape has good ABTS free radical scavenging ability, and not Deproteinated
Polysaccharide Scavenging activity will be slightly better than deproteination matter, but difference is not obvious.There may be the possibility of the above results originals for analysis
Cause: 1. free proteins do not serve conclusive on polysaccharide removing ABTS free radical；2. due to only removing 2 albumen
Matter, still remaining relatively large number of protein in polysaccharide, therefore cause de- albumen and not Deproteinated polysaccharide in ABTS radicals scavenging
Upper difference is not obvious.3. being only that part removes free protein, glycoprotein does not remove, so sugared due to taking off albumen step
Albumen might have facilitation in its oxidation resistance；4. pigment, bioflavonoid, polyphenols meeting in polysaccharide solution
It has a certain impact to polysaccharide anti-oxidative.
In conclusion petal and scape deproteinizing rate are respectively 35.96% and 39.44%；At 25 DEG C, RH81% condition
Under, polysaccharide in 20h polysaccharide hygroscopicity be better than Sodium Hyaluronate, be lower than glycerol, sodium alginate, PEG-400, be higher than chitosan,
PEG-6000；At 25 DEG C, under the conditions of RH43%, polysaccharide polysaccharide hygroscopicity in 44h is lower than glycerol, sea close to Sodium Hyaluronate
Mosanom, PEG-400 are higher than chitosan, PEG-6000；At 25 DEG C, dry discoloration silica gel condition, when 44h, petal polysaccharide moisturizing
Property be lower than PEG-6000, better than other traditional moisturizer；When 44h, scape polysaccharide moisture retention is lower than PEG-6000, hyaluronic acid
Sodium；Scape and petal polysaccharide have good total reducing power, petal and scape polysaccharide to the clearance rate best result of DPPH free radical
Not Wei 82.62% and 82.19%, the clearance rate highest difference of petal (do not take off albumen) and scape (not taking off albumen) DPPH free radical
For 79.6% and 80.81%；Petal and scape polysaccharide are respectively 93.56% He to the clearance rate highest of ABTS free radical
90.68%；Petal (not taking off albumen) and scape (not taking off albumen) are respectively 95.09% He to the clearance rate highest of ABTS free radical
95.25%.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make a variety of changes or modify within the scope of the claims, this not shadow
Ring substantive content of the invention.In the absence of conflict, the feature in embodiments herein and embodiment can any phase
Mutually combination.
Claims (2)
1. the extracting method of trollflower polysaccharide, characterized by the following steps:
Suitable quantity of water is added into trollflower petal or the powder of scape in the ratio of solid-liquid ratio 1: 60, water-bath under conditions of 80 DEG C
Heat 3h, stirred every 30min with glass bar primary, extract repeatedly three times, filter to obtain clear liquid, merge supernatant to get.
2. the external activity detection method of trollflower polysaccharide, it is characterised in that:
A, polysaccharide hygroscopicity detects:
Trollflower polysaccharide sample is contained in aluminium box and after drying to constant weight in 50 DEG C of baking oven, is put in that fill saturated ammonium sulfate molten
In the drier of liquid and saturated sodium carbonate solution, that is, it is placed in the drier that relative humidity is 81% and 43%, and will be entire
Drier is placed in constant incubator, 25 DEG C of constant temperature, accurate with assay balance after placing 4h, 8h, 12h, 20h, 44h
Quality is weighed, is calculate by the following formula the hydroscopicity of polysaccharide: hydroscopicity (%)=(incrementss of sample moisture/sample proper mass) ×
100%；
B, polysaccharide moisture retention detects
In the discoloration silica gel cleaned and dry drier bottom addition is dry, 20ml distillation is added in every part of trollflower polysaccharide sample
Water, and quality is accurately weighed again, it is placed in drier, and entire drier is placed in 25 DEG C of constant incubators, point
Quality is not weighed accurately with assay balance after placing 4h, 8h, 12h, 20h, 44h, is calculate by the following formula the moisturizing rate of polysaccharide: being protected
Wet rate (%)=(reduction amount of sample moisture/sample proper mass) × 100%；
C, the anti-oxidant detection of polysaccharide
(1) measurement of total reducing power
1ml different quality concentration polysaccharide solution is taken, 2.5ml is added, 3ml mass fraction is added in the phosphate buffer of pH6.6 later
1% potassium ferricyanide aqueous solution keeps the temperature 10min in 50 DEG C of water-baths after mixing well；It cools down rapidly, adds after taking-up
The trichloroacetic acid solution of 2.5ml mass fraction 10%, oscillation shake up, and in 3000r/min, are centrifuged 10min；Pipette supernatant
2.5ml adds the ferric chloride aqueous solutions of 2.5ml distilled water and 0.5ml mass fraction 0.1% in test tube, is protected from light at room temperature anti-
20min is answered, light absorption value is measured at 700nm；
(2) measurement of DPPH Scavenging activity
The polysaccharide solution of 1ml different quality concentration is taken, 3ml, the DPPH free radical ethanol solution of 0.2mmol/L, after mixing is added
4.0ml ethanol solution is added, after being protected from light 30min at room temperature, absorbance is measured at 517nm, is calculated as follows
Clearance rate of the sample to DPPH:
In formula: AcFor the light absorption value of blank sample, AsFor the light absorption value after various concentration sample is added.
(3) measurement of ABTS free radical scavenging ability
The ABTS solution 10ml of 7mmol/L is mixed with the potassium persulfate solution 10ml of final concentration of 2.45mmol/L, in room temperature,
It is placed 14-16 hours under the conditions of darkness, is diluted to absorbance using the preceding phosphate buffer with the pH=7.4 of 0.2mol/L
For 0.72~1.2 to get arrive ABTS free radical working solution；
It takes each 200 μ L of the polysaccharide solution of different quality concentration, ABTS free radical working solution 4ml is added, react after 6min in wavelength
Absorbance is surveyed at 734nm, calculates sample as follows to the clearance rate of ABTS free radical.
In formula: AcFor the light absorption value of blank sample, AsFor the light absorption value after various concentration sample is added.
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