CN110251502A - A kind of application of fucoxanthin in anti-first subtracts - Google Patents
A kind of application of fucoxanthin in anti-first subtracts Download PDFInfo
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Abstract
The invention belongs to marine resources natural products technology field of processing and application, and in particular to a kind of anti-first caused by cadmium poisoning of the fucoxanthin from marine organisms subtract in application.From extracted in marine algae obtain fucoxanthin it is anti-to cadmium poisoning caused by first subtract in application.It is extracted as adding raw materials into water, then adds enzyme preparation again and digested, organic solvent is added after enzymatic hydrolysis and extracts fucoxanthin.Fucoxanthin sterling is obtained by separating and purifying technology again after extracting fucoxanthin.The fucoxanthin that anti-first of the present invention subtracts has the characteristics that significant in efficacy, safe ready, has no toxic side effect.
Description
Technical field
The invention belongs to marine resources natural products technology field of processing and application, and in particular to one kind is originated from marine organisms
Fucoxanthin anti-first caused by cadmium poisoning subtract in application.
Background technique
Fucoxanthin, also known as fucoxanthine or pheophytin are the containing oxygen derivatives of carrotene, belong in carotenoid
Lutein class, be a kind of fat-soluble pigment, be deflection polarity carotenoid.In nature, fucoxanthin is distributed widely in algae
In class and invertebral zooblast, the algae of remaining each guiding principle and whip is determined other than true eyespot algae guiding principle and Xanthophyceae in different whip algae door
For algae in algae door using fucoxanthin as major carotenoids, the algae content of especially Phaeophyceae and Bacillariophyceae is more rich
Richness, the amount of fucoxanthin accounts for 10% or more of carotenoid total output in nature in brown alga, and diatom is this kind of slender
Golden brown is presented because it contains a large amount of fucoxanthin in born of the same parents' microalgae.
In recent years, for the source of fucoxanthin, extraction and isolation and purification method, biosynthesis pathway, physiological activity and
The research that its metabolic way etc. in vivo is carried out is always Hot Contents, is on the one hand because marine resources are rich
Richness, fucoxanthin are from a wealth of sources;On the other hand, fucoxanthin unique structure shows to have in terms of a variety of pharmacological activity and answer
With the potential value advantage of prospect, wherein anti-inflammatory, antitumor, anti-fat, anti-oxidant, anti-acne, anti-diabetic, anti-malarial and anti-blood
The physiological activity such as rouge are partially confirmed that other potential physiological activity are carried out positive research and exploration by scientist
In.
Cadmium is not human essential elements, and is a kind of environmental contaminants, and under normal circumstances, excessively taking in cadmium can lead to
Human hair gives birth to cadmium poisoning.Cadmium is classified as the food contaminant of primary study by the World Health Organization;International cancer research institution (IARC)
Cadmium is classified as human carcinogen, can the mankind be caused with serious Health cost;U.S.'s poisonous substance and disease registration administration (ATSDR) will
Cadmium is classified as the 7th substance being detrimental to health;China is also that cadmium is classified as to the key monitoring index for implementing prediction emission
One of.Cadmium ion can directly contribute thyroid microcancer, can also be by causing other lesions to subtract to cause first indirectly.
Data show that China has more than 200,000,000 first shapes at present in international thyroid gland Knowledge Promotion week news briefing in 2013
Gland disease patient.Most patients need long-term or lifelong medication, but the drug of various treatment thyroid diseases has its pair
It acts on, and has certain indication, therefore seek one kind thyroid disease therapeutic agent without side-effects just to seem especially heavy
It wants.
Summary of the invention
The purpose of the present invention is to provide a kind of fucoxanthin from marine organisms anti-first caused by cadmium poisoning to subtract
In application.
The technical solution adopted by the present invention to achieve the above object:
A kind of application of fucoxanthin in anti-first subtracts extracts from marine algae and obtains fucoxanthin anti-to cadmium poisoning
Caused first subtract in application.
The fucoxanthin is by raw material of marine algae through enzymatic hydrolysis, organic solvent extracting and developing purifying gained.
Further:
1) 0-30 times of volume of its quality (w/v) distilled water is added raw materials into, is configured to dirty solution, then be added into dirty solution
The 0.1-5wt% enzyme of its volume mass, at 25-45 DEG C of temperature, shaking table revolution 80-150r/min, oscillation enzymatic hydrolysis 2-10 hours, i.e.,
Obtain marine algae enzymolysis liquid;
The raw material reaches height for fresh sample water content and can directly be digested without additional addition moisture.
2) in algae enzymolysis liquid fucoxanthin extraction: by above-mentioned acquisition marine algae enzymolysis liquid with 3000-5000r/min
Speed, be centrifuged 5-20 minute, abandoning supernatant, sediment is extracted through 10-30 times of its quality of organic solvent A, and after pass through
It crosses suction filtration and obtains thick fucoxanthin extracting solution;
3) fucoxanthin isolates and purifies in crude extract: crude extract being become solid by reduced pressure, is added its quality
The organic solvent B (w/v) of 1-2 times of volume dissolves;Silicagel column carries out initial gross separation purifying on dissolved sample, collects Chinese red
Efflux, then be concentrated under reduced pressure, it is isolated and purified after concentration using preparative efficient liquid phase, collects fucoxanthin sterling
The liquid of appearance time outflow, the fucoxanthin sterling for being 95% or more through being concentrated under reduced pressure to give purity.
The enzyme be cellulase and or pectase;Wherein, when two kinds of enzymes add simultaneously, two kinds of enzymes are 1- in mass ratio
5:1-5.
The raw material is marine algae or its processing waste material;Wherein, raw material can crush or entirety directly uses;The ocean
Algae be the mixing of one or more of fresh or dry kelp, sargassum, sargassum fusifome, Phaeodactylum tricornutum.
Step 2) the precipitating is impregnated through organic solvent A, carries out extraction at least once then to get fucoxanthin
Crude extract;Wherein, it is extracted as impregnating sediment through organic solvent A every time, in 25-50 DEG C after immersion, in revolution 100-150r/
It is extracted 2-5 hours under min oscillating condition, realizes primary extract;It is filtered after extracting every time, filters remaining solid residue again
Secondary to merge the smoke filtrate extracted every time through organic solvent A soak extraction, total extraction time is 2-10 hours;The organic solvent A
For ethyl alcohol or methanol.It is only single-trial extraction during said extracted, then extraction time is 2-10 hours;It is extracted if multiple, often
Secondary extraction time 2-5 hours, total extraction time 2-10 hours.
Step 3) the silicagel column purification condition: eluant, eluent is acetone, n-hexane, ether, petroleum ether, ethyl acetate
In any two kinds by 1-10:1-10 volume ratios mix, collect Chinese red efflux;Preparative efficient liquid phase isolates and purifies item
Part: eluant, eluent is methanol, any two kinds volume ratios by 10-90:10-90 mix in acetonitrile, ethyl alcohol, collects fucoxanthin mark
The eluent flowed out in quasi- product appearance time section;The organic solvent B is anhydrous methanol, dehydrated alcohol or methylene chloride.
A kind of anti-preparation subtracted to first caused by cadmium poisoning, preparation obtain rock algae Huang containing described extract from marine algae
Element.
Cadmium poisoning first reduces mouse model, and feeding subtracts model mice concentration with the cadmium poisoning first of caddy modeling as 0.025%-
0.1% fucoxanthin isolated and purified 5-20 days, passes through follicular epithelial cell morphosis and T4, T3, oxidative stress
Horizontal variation shows that the first caused by the fucoxanthin of separating-purifying in seaweed has the poisoning of apparent Cadmium resistance subtracts effect.
It is deactivating that detection isolates and purifies first caused by fucoxanthin Cadmium resistance is poisoned:
1) cadmium poisoning first is established with caddy and reduces mouse model.
2) feeding model mice concentration is the fucoxanthin 5-20 that 0.1-2.0g/L separates that 0.025%-0.1% is isolated and purified
It.
3) pass through Mouse thyroid follicular epithelial cells morphosis and the variation table of blood cadmium content, oxidative stress
The bright first caused by the fucoxanthin of separating-purifying in seaweed has the poisoning of apparent Cadmium resistance subtracts effect.
Advantage for present invention
1. the fucoxanthin that the present invention obtains is to isolate and purify gained from marine algae, it is natural active product, has
Nontoxicity, the high distinguishing feature of activity
2. can be achieved from many aspects by the visible gained fucoxanthin of the present invention to thyroid protective effect:
(1) fucoxanthin has significant protective effect to Thyroid follicular epithelial cell;(2) fucoxanthin has apparent protection to make in liver
With, and liver is the important place of thyroid hormone metabolism, protection liver maintains the normal performance of thyroid hormone function.
(3) fucoxanthin can promote the metabolism of cadmium in body.
Detailed description of the invention
Fig. 1 is micro-structure diagram of the thyroid follicle provided in an embodiment of the present invention under 20 times of object lens of microscope, wherein
(a) thyroid gland microstructure is normally organized;(b) negative control group thyroid gland microstructure;(c) the micro- knot of positive controls thyroid gland
Structure;(d) separating-purifying fucoxanthin group thyroid gland microstructure.
Fig. 2 is that gained fucoxanthin high-efficient liquid phase chromatogram is isolated and purified in seaweed provided in an embodiment of the present invention.
Fig. 3 is micro-structure diagram of the liver provided in an embodiment of the present invention under 40 times of object lens of microscope, wherein (a) is normal
Group liver microstructure;(b) negative control group liver microstructure;(c) separating-purifying fucoxanthin group liver micro-structure diagram.
Specific embodiment
The invention will be further described below, and protection scope of the present invention is not only limited to following embodiment.
Embodiment 1
1. 5 times of volume (w/v) distilled water of its quality are added after new fresh sargassum is crushed with pulverizer, it is configured to muddiness
Liquid, then the 0.5wt% cellulase (v/w) of its volume is added into dirty solution, it is placed in 40 DEG C of enzymatic hydrolysis 4 in constant-temperature shaking incubator
Hour, shaking table revolution 120r/min obtains sargassum enzymolysis liquid;
2. the extraction of fucoxanthin in sargassum enzymolysis liquid: by above-mentioned acquisition sargassum enzymolysis liquid with the speed of 3000r/min
Degree is centrifuged 10 minutes, abandons supernatant, the dehydrated alcohol (w/v) that 20 times of its quality is added into sediment is impregnated, in 40 DEG C of perseverances
Pigment is extracted in warm shaking table, shaking table revolution 120r/min is extracted 4 hours, is then protected from light suction filtration and obtains fucoxanthin crude extract;
3. fucoxanthin isolates and purifies in crude extract: crude extract being become solid by reduced pressure, is added its quality 2
Methylene chloride (w/v) dissolution of times volume.Silicagel column carries out initial gross separation purifying on dissolved sample, and eluant, eluent is petroleum
Ether: ethyl acetate=7:3 (v/v) is collected Chinese red efflux, is concentrated under reduced pressure, using the efficient liquid of preparative after concentration
Mutually isolated and purified, condition: acetonitrile: water=90:10 (v/v), flow velocity 10ml/min, sample volume 1ml, Detection wavelength are
2489 ultraviolet/visible light detector of 448nm, Waters, chromatographic column are Sunfire Prep C18 (19X150mm, 10 μm);It receives
Collect fucoxanthin sterling appearance time outflow liquid, through be concentrated under reduced pressure to give purity 96% fucoxanthin sterling (referring to
Fig. 2).
Embodiment 2
1. 1 times of volume (w/v) distilled water of its quality is added in fresh Phaeodactylum tricornutum, it is configured to dirty solution, then to muddiness
The 0.5wt% cellulase (v/w) of its volume and the 0.5wt% pectase of its volume are added in liquid, is placed in constant-temperature shaking culture
It is digested 6 hours for 30 DEG C in case, shaking table revolution 150r/min obtains Phaeodactylum tricornutum enzymolysis liquid;
2. the extraction of fucoxanthin in Phaeodactylum tricornutum enzymolysis liquid: by above-mentioned acquisition Phaeodactylum tricornutum enzymolysis liquid with 3000r/
The speed of min is centrifuged 10 minutes, abandons supernatant, and the anhydrous methanol (w/v) that 10 times of its quality is added into sediment is impregnated,
Pigment is extracted in 40 DEG C of constant-temperature tables, shaking table revolution 150r/min extracts 3 hours, is then protected from light suction filtration, filtered solid
The anhydrous methanol (w/v) that residue adds 10 times of its quality is impregnated, and pigment, shaking table revolution are extracted in 40 DEG C of constant-temperature tables
150r/min is extracted 3 hours, is then protected from light suction filtration, merges suction filtration liquid twice, obtain fucoxanthin crude extract;
3. fucoxanthin isolates and purifies in crude extract: crude extract being become solid by reduced pressure, is added its quality 2
Methylene chloride (w/v) dissolution of times volume.Silicagel column carries out initial gross separation purifying on dissolved sample, and eluant, eluent is acetone:
N-hexane=1:3 (v/v) is collected Chinese red efflux, is concentrated under reduced pressure, and carries out after concentration using preparative efficient liquid phase
It isolates and purifies, condition: acetonitrile: water=90:10 (v/v), flow velocity 10ml/min, sample volume 1ml, Detection wavelength 448nm,
2489 ultraviolet/visible light detector of Waters, chromatographic column are Sunfire Prep C18 (19X150mm, 10 μm);Collect rock algae
The liquid of flavine pure product appearance time outflow, is concentrated under reduced pressure to give fucoxanthin sterling of the purity 96.5%.
Embodiment 3
1. dry sea mustard powder is broken into 1mm220 times of volume (w/v) distilled water of its quality are added thereto, match for size particles
Dirty solution is made, then the 0.5wt% cellulase (v/w) of its volume and the 0.5wt% pectin of its volume is added into dirty solution
Enzyme is placed in constant-temperature shaking incubator and digests 6 hours for 30 DEG C, and shaking table revolution 150r/min obtains thallus laminariae enzymolysis liquid;
2. the extraction of fucoxanthin in thallus laminariae enzymolysis liquid: by above-mentioned acquisition thallus laminariae enzymolysis liquid with the speed of 4000r/min
Degree is centrifuged 15 minutes, abandons supernatant, and the anhydrous methanol (w/v) that 20 times of its quality is added into sediment dissolved, lysate in
30 DEG C, in constant-temperature table revolution 150r/min, extraction time 6 hours, be then protected from light lower suction filtration and obtain fucoxanthin crude extract;
3. fucoxanthin isolates and purifies in crude extract: crude extract being become solid by reduced pressure, is added its quality 2
Methylene chloride (w/v) dissolution of times volume.Silicagel column carries out initial gross separation purifying on dissolved sample, and eluant, eluent is acetone:
N-hexane=1:3 (v/v) is collected Chinese red efflux, is concentrated under reduced pressure, and carries out after concentration using preparative efficient liquid phase
It isolates and purifies, condition: acetonitrile: water=90:10 (v/v), flow velocity 10ml/min, sample volume 1ml, Detection wavelength 448nm,
2489 ultraviolet/visible light detector of Waters, chromatographic column are Sunfire Prep C18 (19X150mm, 10 μm);Collect rock algae
The liquid of flavine pure product appearance time outflow, is concentrated under reduced pressure to give fucoxanthin sterling of the purity 96.8%.
Application examples
Subtract drug using the above-mentioned fucoxanthin isolated and purified from marine algae as first caused by Cadmium resistance is poisoned is prepared
First caused by being poisoned using, Cadmium resistance subtract effect test and its effect it is as follows:
It isolates and purifies first caused by the Cadmium resistance poisoning of fucoxanthin and subtracts test:
Experimental animal: 120 male Kunming strain mice 18-22g/ are only.
Animal model: 100 mouse feeding 0.003g/L caddy 30 days, creation cadmium poisoning first subtract model mice.
Drug is prepared: being isolated and purified fucoxanthin with the various embodiments described above and is dissolved through a small amount of ethyl alcohol, then plus pure water difference
It is configured to the suspension that fucoxanthin content is 0.025%, 0.05%, 0.1%.
Experimental group: experimental group is respectively normal group and animal model group;Animal model group is that cadmium poisoning first subtracts model
Mouse, and be randomly divided into 5 groups, every group of 20 mouse, respectively negative control group, positive controls and fucoxanthin are low
Dosage, middle dosage and high dose group.
20 normal groups (the daily feeding pure water of healthy mice, water source supply are sufficient);
20 negative control groups (first reduces the daily feeding pure water of mouse, and water source supply is sufficient);
(first reduces the daily feeding thyroid tablet of mouse to 20 positive controls, and taking dose is supplied referring to specification, water source is taken
To abundance);
20 low dosages isolate and purify fucoxanthin group, and (first reduces the suspension that the daily feeding concentration of mouse is 0.025%
20ml, water source supply are sufficient);
20 middle dosages isolate and purify fucoxanthin group, and (first reduces the suspension that the daily feeding concentration of mouse is 0.05%
20ml, water source supply are sufficient);
20 high doses isolate and purify fucoxanthin group (first reduce the daily feeding concentration of mouse be 0.1% suspension 20ml,
Water source supply is sufficient)
Experimental method:
Cadmium poisoning first subtracts mouse feeding pure water, thyroid tablet, containing the various concentration suspension for isolating and purifying fucoxanthin
Heart extracting blood after 14 days takes parathyroid tissue to carry out hormone-content measurement and thyroid follicle structure observation.Hormone-content measurement
Utilize enzyme-linked immunoassay kit;Thyroid follicle structure observation is dyed using conventional section HE, microscopy under microscope (referring to
Table 1-3, Fig. 1 and Fig. 3).
Testing index: thyronine T4 is horizontal, and triiodo thryonine T3 is horizontal, the micro- knot of follicular epithelial cell
Structure variation.
1 each group mouse T4 of table is horizontal
Feeding isolates and purifies fucoxanthin group first reduction mouse T4 level and is significantly restored, poor without conspicuousness with normal group
It is different, it is significantly higher than negative control group, and there was no significant difference with positive controls, it was demonstrated that the fucoxanthin of separating-purifying in seaweed
First caused by cadmium poisoning is subtracted and is improved significantly.
From Fig. 1 thyroid morphology structure: compared with normal group, negative control group Mouse thyroid follicular epithelial cells
There is obvious hyperplasia, thickening phenomenon;Folliculus morphosis is imperfect, concave or convex;Intracavitary colloid vacuolation is obvious.Feeding is not
The fucoxanthin group first that isolates and purifies with dosage reduces rat thyroid follicular epithelial cells hyperplasia, thickening phenomenon and is obviously changed
It is kind;Follicular structure tends to completely without deformation, is in regular circle or ellipse;Colloid in follicular cavity is evenly distributed, without obvious
Colloid vacuole.There is epithelial cell thickening phenomenon in positive controls, a small number of colloid vacuoles occur, it was demonstrated that thyroid tablet is to first shape
The protective effect of gland follicle epithelial cell is significant not as good as the fucoxanthin of separating-purifying.The fucoxanthin of separating-purifying can in seaweed
To protect the structural integrity of follicular epithelial cell, guarantee the normal performance of thyroid function.
2 each group mouse T3 of table is horizontal
In feeding, low dosage separating-purifying fucoxanthin group cadmium poisoning first reduce mouse T3 level and significantly restored, and just
Often there was no significant difference for group, and is significantly higher than negative control group, and is higher than positive controls.Prove the rock isolated and purified in seaweed
Phycoxanthine restores have apparent facilitation to the horizontal of T3.
Cd is horizontal in 3 each group mouse blood of table
The middle and high dosage separating-purifying fucoxanthin group cadmium poisoning first of feeding subtracts cadmium level in mouse blood and is significantly dropped
It is low, and substantially less than negative control group and positive controls, it was demonstrated that the fucoxanthin isolated and purified in seaweed is to cadmium in body
Metabolism has apparent facilitation.
Fucoxanthin has significant protective effect to liver as seen from Figure 3, and liver is the weight of thyroid hormone metabolism
Place is wanted, protection liver maintains the normal performance of thyroid hormone function.Liver cell Morphology result such as Fig. 3 institute
Show: normal group liver cell arrangement is close, and nucleus is located at cell center, cell membrane distinct;Negative control group liver cell is close
Degree becomes smaller, and arrangement is loose, and cell membrane boundary is unintelligible;The liver of positive controls and feeding high dose separating-purifying fucoxanthin group
Dirty damage phenomenon has clear improvement, and liver cell density is higher than negative control group, and cell membrane distinct, nucleus are located at cell
Center.Therefore high dose fucoxanthin (0.1%) improves significantly to the hepar damnification tool that caddy induces, and keeps liver thin
Born of the same parents' institutional framework tends to normalization.
The fucoxanthin of various concentration, which subtracts first caused by cadmium poisoning, improvement result, and improvement and rock algae are yellow
The concentration of element has no apparent dose-effect relationship, but high dose fucoxanthin (0.1% or more) might have and preferably change
Kind effect.
Claims (7)
1. a kind of application of fucoxanthin in anti-first subtracts, it is characterised in that: extraction obtains fucoxanthin and exists from marine algae
It is anti-to first caused by cadmium poisoning subtract in application.
2. by application of the fucoxanthin described in claim 1 in anti-first subtracts, it is characterised in that: the fucoxanthin is with ocean
Algae is raw material through enzymatic hydrolysis, organic solvent extracting and developing purifying gained.
3. by application of the fucoxanthin as claimed in claim 1 or 2 in anti-first subtracts, it is characterised in that:
1) 0-30 times of volume of its quality (w/v) distilled water is added raw materials into, is configured to dirty solution, then its body is added into dirty solution
The 0.1-5wt% enzyme of product quality, at 25-45 DEG C of temperature, shaking table revolution 80-150r/min, oscillation digests 2-10 hours to get arriving
Marine algae enzymolysis liquid;
2) in algae enzymolysis liquid fucoxanthin extraction: by above-mentioned acquisition marine algae enzymolysis liquid with the speed of 3000-5000r/min
Degree is centrifuged 5-20 minutes, abandons supernatant, sediment is extracted through 10-30 times of its quality of organic solvent A, then by taking out
Filter obtains thick fucoxanthin extracting solution;
3) fucoxanthin isolates and purifies in crude extract: crude extract being become solid by reduced pressure, is added 1-2 times of its quality
The organic solvent B (w/v) of volume dissolves;Silicagel column carries out initial gross separation purifying on dissolved sample, collects Chinese red outflow
Liquid, then be concentrated under reduced pressure, it is isolated and purified after concentration using preparative efficient liquid phase, collects fucoxanthin sterling appearance
The liquid of time outflow, the fucoxanthin sterling for being 95% or more through being concentrated under reduced pressure to give purity.
4. by application of the fucoxanthin described in claim 3 in anti-first subtracts, it is characterised in that: the enzyme be cellulase and
Or pectase;Wherein, when two kinds of enzymes add simultaneously, two kinds of enzymes are 1-5:1-5 in mass ratio.
5. by application of the fucoxanthin described in claim 3 in anti-first subtracts, it is characterised in that: step 2) the precipitating warp
Organic solvent A is impregnated, and carries out extraction at least once then to get fucoxanthin crude extract;Wherein, it is extracted as to precipitate every time
Object is impregnated through organic solvent A, in 25-50 DEG C after immersion, is extracted 2-5 hours under revolution 100-150r/min oscillating condition, real
It is now primary to extract;It is filtered after extracting every time, filters remaining solid residue and dissolve extraction through organic solvent A again, merge several
Secondary to extract resulting smoke filtrate, total extraction time is 2-10 hours;The organic solvent A is ethyl alcohol or methanol.
6. by application of the fucoxanthin described in claim 3 in the caused first of Cadmium resistance poisoning subtracts, it is characterised in that: the step 3)
Silicagel column purification condition: eluant, eluent be acetone, n-hexane, ether, petroleum ether, in ethyl acetate any two kinds press 1-10:
The volume ratio of 1-10 mixes, and collects Chinese red efflux;Preparative efficient liquid phase purification condition: eluant, eluent be methanol,
Any two kinds volume ratios by 10-90:10-90 mix in acetonitrile, ethyl alcohol, collect in fucoxanthin standard items appearance time section
The eluent of outflow;The organic solvent B is anhydrous methanol, dehydrated alcohol or methylene chloride.
7. a kind of anti-preparation subtracted to first caused by cadmium poisoning, it is characterised in that: preparation is containing described in claim 1 from ocean algae
It is extracted in class and obtains fucoxanthin.
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