CN109879982A - The preparation of fucoidan component and activity test method in a kind of sargassum fusifome - Google Patents
The preparation of fucoidan component and activity test method in a kind of sargassum fusifome Download PDFInfo
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Abstract
The invention discloses a kind of preparation of fucoidan component in sargassum fusifome and activity assays, its drip irrigation device includes that sargassum fusifome cleans drying, ethyl alcohol is added after crushing, flow back degreasing and agitation and filtration, degreasing algae powder extracts 3 times repeatedly through calcium chloride solution, and merging filtrate obtains fucoidan extracting solution；Extracting solution vacuum distillation is concentrated into the 1/5 of original volume；Dehydrated alcohol is added into concentrate and stands overnight, after centrifugal treating and washes of absolute alcohol, is dried in vacuo up to sargassum fusifome fucoidan；Sargassum fusifome fucoidan is dissolved in distilled water, it is separated using DEAE- cellulose ion-exchange column chromatography, with distillation water elution, then it is eluted respectively with the NaCl of 0.1M, 0.3M, 0.5M, 1M, products therefrom Sepharose CL-6B column chromatographic purifying, it is eluted with NaCl, eluent is analyzed using phend-sulphuric acid, collect suitable fraction preparation.The present invention has the advantages that prepare sargassum fusifome fucoidan component easy to operate.
Description
Technical field
The present invention relates to a kind of extracting methods of active material in sargassum fusifome, and more specifically it is related in a kind of sargassum fusifome
The preparation of fucoidan component and activity test method.
Background technique
Sargassum fusifome (Sargassum fusiforme or Hizikia fusiformis) belongs to Phaeophyta, Phaeophyceae, Mo Jiao
Cutleriales, Sargassaceae, Sargassum a kind of ocean cryptogam.Be known as from the eighties in last century " Chinese sargassum fusifome it
The Wenzhou City, Zhejiang Province Dongtou County in township " starts largely to propagate artificially, and main exit Japan.Sargassum fusifome substance containing various active,
It is known as the laudatory title of " longevity greens/mustard green ", as dual-purpose of drug and food seaweed value of exploiting and utilizing with higher.
Fucoidan is the important active substances in sargassum fusifome.Fucoidan is also known as fucoidin, is
A kind of water-soluble heteromeric polysaccharide.The important molecule of endothelial cell and neutrophil leucocyte identification and adherency that palatelet-selectin mediates,
It is the promising target for treating acute inflammation related disease.Fucoidan has the function of antagonism palatelet-selectin, plays anti-
Scorching effect, but which specific component of fucoidan has the function of antagonism palatelet-selectin, needs further to study.
People need to carry out separation preparation to fucoidan component during the experiment, further to the function of antagonism palatelet-selectin
Effect is evaluated.Due to sargassum fusifome is common in China coast and can large scale cultivating, more and more focus of attention sargassum fusifomes
Exploitation, and lack effective preparation process of fucoidan component in a kind of pair of sargassum fusifome at this stage.
Summary of the invention
In view of the deficienciess of the prior art, the invention reside in provide fucoidan component in a kind of sargassum fusifome
Preparation and activity test method have and prepare fucoidan component conveniently, and convenient for analyzing it to the short of money of palatelet-selectin
The function of anti-effect.
To achieve the above object, the present invention provides the following technical scheme that fucoidan group in a kind of sargassum fusifome
The preparation method divided, it is characterised in that:
Step 1: taking 500g sargassum fusifome to clean, drying to constant weight；
Step 2: Sangassivm fuciforime (Harv) Setch powder is broken to 60-120 mesh；85% ethyl alcohol of 2-4L is added into smashed sargassum fusifome, 60
DEG C reflux degreasing 2-5h, be stirred continuously therebetween, filter；
Step 3: 1M calcium chloride solution 5L will be added in degreasing algae powder, 20-40 DEG C of Extracting temperature, stir 4-8h；Filtering is received
Collect filtrate；Filter residue extracts 3 times repeatedly, merging filtrate, obtains sargassum fusifome fucoidan extracting solution；
Step 4: extracting solution being evaporated under reduced pressure at 40-80 DEG C, vacuum degree -0.05MPa and is concentrated into the 1/5 of original volume；To
Dehydrated alcohol is slowly added in concentrate, concentration of alcohol is 80% in concentrate, and 4 DEG C stand overnight, 5000-12000rpm centrifugation
Processing 30 minutes is dried in vacuo after washes of absolute alcohol to get sargassum fusifome fucoidan；
Step 5: sargassum fusifome fucoidan being dissolved in 10mL distilled water, is handed over using DEAE- cellulose ion
Change column chromatography for separation, with distillation water elution, the object marker recycled be SFF-1, then respectively with 0.1M, 0.3M, 0.5M,
Obtained label is respectively SFF-3, SFF-4, SFF-5 by the NaCL elution of 1M；
Step 6: step 5 products therefrom further being used into Sepharose CL-6B column chromatographic purifying, is washed with 0.15M NaCl
It is de-, eluent is analyzed using phend-sulphuric acid, collects suitable fraction preparation, and be respectively labeled as SFF-11, SFF-
12、SFF-21、SFF-31、SFF-32、SFF-41、SFF-42、SFF-51。
By using above-mentioned technical proposal, high-purity can effectively be extracted from sargassum fusifome by above-mentioned extracting method
Fucoidan, convenient for carrying out screening active ingredients experiment to component, the fucoidan of high-purity can make component
Separation it is more efficient, reduce impurity.
The present invention is further arranged to: when calcium chloride solution being added in step 3, while with salt acid for adjusting pH value to 3-5.
By using above-mentioned technical proposal, by the way that calcium chloride solution is added, and adjust pH value can, effective inhibition alginic acid
The dissolution of the impurity such as salt improves extraction efficiency and makes the fucoidan purity finally extracted higher.
The present invention is further arranged to: crushing sargassum fusifome described in step 2, Sangassivm fuciforime (Harv) Setch powder are broken to 80 mesh.
By using above-mentioned technical proposal, the sargassum fusifome for being crushed to 80 mesh is of moderate size, and dwells convenient for extracting sheep in step 3
Dish fucoidan.
The present invention is further arranged to: Extracting temperature is 25 degrees Celsius in step 3, stirs 6h, and Beater operator is used when stirring
Tool stirring.
By using above-mentioned technical proposal, it is stirred by stirring tool and enables to solution mixing more uniform fast
Speed improves extraction efficiency.
The present invention is further arranged to: the stirring tool is stirring rod, and the stirring rod includes rotating bar, rotating bar
Lower end is circumferentially uniformly fixed with multiple mixed poles, and the upper end of the rotating bar is fixed with hemispheric handle.
It by using above-mentioned technical proposal, drives mixed pole to rotate by rotating bar, the flow velocity of solution can be accelerated, mentioned
High mixing velocity, and to mix more uniform.
The present invention is further arranged to: the side wall of the mixed pole is equipped with mix aperture, and mix aperture is horizontally extending
And its both ends open is different at a distance from rotating bar.
By using above-mentioned technical proposal, when rotating rotating bar, mixed pole follows rotating bar to rotate, and liquid is by mixing
Kong Houhui changes its position, and the flowing of accelerating liquid improves mixing efficiency.
The present invention is further arranged to: the mixed pole is threadedly coupled with rotating bar, and the lower end of the handle is equipped with can be with
The location hole that mixed pole is threadedly coupled.
By using above-mentioned technical proposal, mixed pole can be threadedly coupled with location hole when not using stirring rod, so that
Stirring rod takes up space reduction convenient for storage.
The present invention is further arranged to: the one end of the mixed pole far from rotating bar is spherical surface.
By using above-mentioned technical proposal, when the holder for mixing boom end and solution contacts, contact area is smaller, no
Easily cause the damage of the holder of solution.
The activity test method of fucoidan component, the component including fucoidan in a kind of sargassum fusifome
SFF-11, SFF-12, SFF-21, SFF-31, SFF-32, SFF-41, SFF-42, SFF-51, it is characterised in that:
Step 1: preparing Chinese hamster ovary cell CHO and human promyelocytic leukemia HL-60, people P- is selected
Plain cDNA overall length transfection CHO cell obtains the CHO-P cell for stablizing expression people's palatelet-selectin；
Step 2: by CHO or CHO-P cell inoculation into 24 orifice plates, being incubated overnight to form cell monolayer, then by cell
It is each with the fucoidan of blocking-up type monoclonal antibody 9E1, non-blacked type monoclonal antibody AC 1.2 or 100 μ g/ml
Component 37 DEG C preincubate 1 hour, pretreated CHO-P or CHO cell monolayer is added in the HL-60 cell of calcein label
In and incubate again 1 hour, after being washed with PBS, with Tecan Infinite M200 microplate reader measure fluorescence intensity, excitation and hair
Ejected wave is long to be set separately in 485nm and 530nm, studies different component pair by comparing the percentage of different component adherent cell
The antagonism of palatelet-selectin.
By using above-mentioned technical proposal, by blocking-up type monoclonal antibody 9E1, non-blacked type monoclonal antibody and rock
Component compares in algae glycan sulfuric ester, analyzes fucoidan different component to palatelet-selectin antagonism, just
The key factor that fucoidan plays antagonism to p- selectin is analyzed in people.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of stirring rod in the present embodiment.
Appended drawing reference: 1, rotating bar；2, mixed pole；3, mix aperture；4, handle；5, location hole.
Specific embodiment
Below in conjunction with attached drawing, invention is further described in detail.
Present embodiment discloses a kind of preparation method of fucoidan component in sargassum fusifome,
Step 1: taking 500g sargassum fusifome to clean, drying to constant weight；
Step 2: Sangassivm fuciforime (Harv) Setch powder is broken to 80 mesh；85% ethyl alcohol of 2L is added into smashed sargassum fusifome, flows back at 60 DEG C
Degreasing 4h, is stirred continuously therebetween, filtering；
Step 3: 1M calcium chloride solution 5L will be added in degreasing algae powder, salt acid for adjusting pH value 4, uses by 25 DEG C of Extracting temperature
Stirring tool stirs 6h；Filtrate is collected in filtering；Filter residue extracts 3 times repeatedly, and merging filtrate obtains sargassum fusifome sulfated fucan
Ester extracting solution；
Step 4: extracting solution being evaporated under reduced pressure at 60 DEG C, vacuum degree -0.05MPa and is concentrated into the 1/5 of original volume；To concentration
Dehydrated alcohol is slowly added in liquid, concentration of alcohol is 80% in concentrate, and is stood overnight in 4 DEG C of environment, 8000rpm centrifugation
It 30 minutes, after washes of absolute alcohol, is dried in vacuo to get sargassum fusifome fucoidan；
Step 5: sargassum fusifome fucoidan being dissolved in 10mL distilled water, is handed over using DEAE- cellulose ion
Column chromatography is changed, with distillation water elution, the eluent recycled is labeled as SFF-1, then uses 0.1M, 0.3M, 0.5M, 1M respectively
NaCl elution be respectively SFF-3, SFF-4, SFF-5 by obtained label.
Step 6: step 5 products therefrom further being used into Sepharose CL-6B column chromatographic purifying, 0.15M NaCl is washed
It is de-, then eluent is analyzed using phend-sulphuric acid, collects suitable fraction preparation, and be respectively labeled as SFF-11,
SFF-12,SFF-21,SFF-31,SFF-32,SFF-41,SFF-42,SFF-51；
As shown in Figure 1, stirring tool is stirring rod, the stirring rod includes rotating bar 1, and the lower end of rotating bar 1 is spherical surface,
The lower end of rotating bar 1 is circumferentially screwed there are three mixed pole 2 and is evenly distributed, and the side wall of mixed pole 2 is equipped with mixing
Hole 3, mix aperture 3 is horizontally extending and its both ends open is different at a distance from rotating bar 1.
Mixed pole 2 far from rotating bar 1 one end be spherical surface so that stirring when mixed pole 2 end and solution holder
When contact, contact area is smaller, does not easily cause the damage of the holder of solution.Spherical hand is fixed in the upper end of rotating bar 1
Handle 4 is taken stirring rod convenient for people.The location hole 5 that can be threadedly coupled with mixed pole 2 is offered in the lower end of handle 4, works as people
Mixed pole 2 can be connect with location hole 5 without using when stirring rod, consequently facilitating people store stirring rod.
The dissolution of the impurity such as alginate can be effectively suppressed using said extracted method, and calcium chloride solution and degreasing can be made
Algae powder stirs evenly, and removes convenient for impurity, while extraction efficiency is high, purity is good, is convenient for screening active ingredients.
Embodiment two:
The analysis method of fucoidan component in a kind of sargassum fusifome, including fucoidan component SFF-
11, SFF-12, SFF-21, SFF-31, SFF-32, SFF-41, SFF-42, SFF-51, it is glutinous that this eight kinds of components carry out cell static state
Attached experiment.
Step 1: preparing Chinese hamster ovary cell CHO and human promyelocytic leukemia HL-60, people P- is selected
Plain cDNA overall length transfection CHO cell obtains the CHO-P cell for stablizing expression people's palatelet-selectin；
Step 2: by CHO or CHO-P cell inoculation into 24 orifice plates, being incubated overnight to form cell monolayer, then by cell
With blocking-up type monoclonal antibody 9E1, the fucoidan component of non-blacked type monoclonal antibody AC 1.2 or 100 μ g/ml
37 DEG C preincubate 1 hour, the HL-60 cell of calcein label is added in pretreated CHO-P or CHO cell monolayer simultaneously
It incubates again 1 hour, after being washed with PBS, measures fluorescence intensity, excitation and transmitted wave with Tecan Infinite M200 microplate reader
It is long to be set separately in 485nm and 530nm, different component is studied by comparing the percentage of different component adherent cell, and P- is selected
Select the antagonism of element.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to restrict the invention, all in design structure of the invention
Within think of, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. the preparation method of fucoidan component in a kind of sargassum fusifome, it is characterised in that:
Step 1: taking 500g sargassum fusifome to clean, drying to constant weight；
Step 2: Sangassivm fuciforime (Harv) Setch powder is broken to 60-120 mesh；2-4L85% ethyl alcohol is added into smashed sargassum fusifome, is returned at 60 DEG C
Degreasing 2-5h is flowed, is stirred continuously therebetween, is filtered；
Step 3: 1M calcium chloride solution 5L will be added in degreasing algae powder, 20-40 DEG C of Extracting temperature, stir 4-8h；Filter is collected in filtering
Liquid；Filter residue extracts 3 times repeatedly, merging filtrate, obtains sargassum fusifome fucoidan extracting solution；
Step 4: extracting solution being evaporated under reduced pressure at 40-80 DEG C, vacuum degree -0.05MPa and is concentrated into the 1/5 of original volume；To concentration
It is slowly added to dehydrated alcohol in liquid, makes concentration of alcohol 80% in concentrate, 4 DEG C stand overnight, at 5000-12000rpm centrifugation
Reason 30 minutes is dried in vacuo after washes of absolute alcohol to get sargassum fusifome fucoidan；
Step 5: sargassum fusifome fucoidan being dissolved in 10mL distilled water, DEAE- cellulose ion exchange column is utilized
Chromatography, with distillation water elution, the object marker recycled is SFF-1, then uses the NaCl of 0.1M, 0.3M, 0.5M, 1M respectively
Obtained label is respectively SFF-3, SFF-4, SFF-5 by elution；
Step 6: step 5 products therefrom is further used into Sepharose CL-6B column chromatographic purifying, is eluted with 0.15M NaCl,
Eluent is analyzed using phend-sulphuric acid, collects the preparation of suitable fraction, and be respectively labeled as SFF-11, SFF-12,
SFF-21、SFF-31、SFF-32、SFF-41、SFF-42、SFF-51。
2. the preparation method of fucoidan component in a kind of sargassum fusifome according to claim 1, it is characterised in that:
When calcium chloride solution being added in step 3, while pH value is adjusted to 3-5 with hydrochloric acid.
3. the preparation method of fucoidan component in a kind of sargassum fusifome according to claim 1, it is characterised in that:
Extracting temperature is 25 degrees Celsius in step 3, stirs 6h, is stirred when stirring using stirring tool.
4. the preparation method of fucoidan component in a kind of sargassum fusifome according to claim 3, it is characterised in that:
The stirring tool is stirring rod, and the stirring rod includes rotating bar (1), and the lower end of rotating bar (1) is circumferentially uniformly fixed with
The upper end of multiple mixed poles (2), the rotating bar (1) is fixed with hemispheric handle (4).
5. the preparation method of fucoidan component in a kind of sargassum fusifome according to claim 4, it is characterised in that:
The side wall of the mixed pole (2) is equipped with mix aperture (3), and mix aperture (3) is horizontally extending and its both ends open and rotation
The distance of bar (1) is different.
6. the preparation method of fucoidan component in a kind of sargassum fusifome according to claim 5, it is characterised in that:
The mixed pole (2) is threadedly coupled with rotating bar (1), and the lower end of the handle (4) is equipped with and can be threadedly coupled with mixed pole (2)
Location hole (5).
7. the activity test method of fucoidan component, feature in a kind of sargassum fusifome according to claim 6
Be: the one end of the mixed pole (2) far from rotating bar (1) is spherical surface.
8. in a kind of sargassum fusifome in fucoidan component analysis method, including SFF-11, SFF-12, SFF-21,
SFF-31, SFF-32, SFF-41, SFF-42, SFF-51, it is characterised in that:
Step 1: preparing Chinese hamster ovary cell CHO and human promyelocytic leukemia HL-60, by people's palatelet-selectin
CDNA overall length transfection CHO cell obtains the CHO-P cell for stablizing expression people's palatelet-selectin；
Step 2: by CHO or CHO-P cell inoculation into 24 orifice plates, cell monolayer is formed, then by cell and blocking-up type Dan Ke
The each component of fucoidan of grand antibody 9E1, non-blacked type monoclonal antibody AC1.2 or 100 μ g/mL are incubated in advance at 37 DEG C
It educates 1 hour, the HL-60 cell of calcein label is added in pretreated CHO-P or Chinese hamster ovary celI single layer and incubates 1 again and is small
When, after being washed with PBS, fluorescence intensity is measured with Tecan Infinite M200 microplate reader, excitation and launch wavelength are set separately
In 485nm and 530nm, show different component to the antagonism of palatelet-selectin by comparing the percentage of different component adherent cell
Effect.
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