CN102961395B - Sargassum henslowianum polysaccharide or sargassum henslowianum polysaccharide containing extract and applications thereof - Google Patents
Sargassum henslowianum polysaccharide or sargassum henslowianum polysaccharide containing extract and applications thereof Download PDFInfo
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Abstract
The invention discloses a sargassum henslowianum polysaccharide or sargassum henslowianum polysaccharide containing extract and applications thereof. The sargassum henslowianum polysaccharide or sargassum henslowianum polysaccharide containing extract can be used as an antioxidant or used for preparing an antioxidant. The molecular weight of the sargassum henslowianum polysaccharide is distributed between 1.5 million Daltons and 9 million Daltons. The antioxidant activity of the sargassum henslowianum polysaccharide disclosed by the invention is significant; and when the sargassum henslowianum polysaccharide is low in concentration, an extremely high clearance rate can be achieved, and a certain dose-effect relationship is formed between the clearance rate and the concentration of the sargassum henslowianum polysaccharide. When the concentration of the sargassum henslowianum polysaccharide is 0.07 mg/mL, the clearance rate of the sargassum henslowianum polysaccharide to hydroxyl radicals can reach more than 65%; and when the concentration of the sargassum henslowianum polysaccharide is 0.07 mg/mL, the clearance rate of the sargassum henslowianum polysaccharide to superoxide anion free radicals can reach more than 40%.
Description
Technical field
The present invention relates to functional health-care food or biomedicine field, be specifically related to a kind of Heng Shi sargassan or the extract that contains Heng Shi sargassan and application thereof.
Background technology
Modern disease pharmacological research shows, free radical is relevant with numerous disease, as atherosclerosis, hepatopathy, diabetes, organism aging process, cancer etc.Antioxidant can not only be removed free radical, and can suppress the generation of free radical.At present due to some synthetized oxidation preventive agents, as dibenzylatiooluene (BHT), Butylated hydroxyanisole (BHA) are found to have the toxic and side effects such as carcinogenic in various degree, thereby stop using at u.s.a. and japan.Along with the enhancing of people's environmental protection consumption consciousness, natural plants oxidant becomes the focus of research day by day, therefore recent years, the antioxidation of marine bioactivity polysaccharide is in widespread attention, research shows that marine bioactivity polysaccharide has compared with high anti-oxidation ability, and sargassum thunbergii polysaccharide is Scavenger of ROS free radical effectively.Sea grass polysaccharide energy superoxide dismutase (SOD) vigor reduces the content of lipofuscin (LPO) in liver, spleen.Therefore marine bioactivity polysaccharide is widely used in the industry such as food, medicines and health protection.
Along with modern analysis and isolation technics are applied to the exploitation of living marine resources, Sargassum is considered to contain one of the abundantest source of novel active secondary metabolite.Research shows, Sargassum polysaccharides can promote proliferation of mouse T lymphocytes reaction, cell is had to the multiple efficacies such as immunoregulation effect, antioxidation, antitumor action, antimutagenic effect, Cell differentiation inducing activity, antiviral.Along with extensive use and people's the going deep into the important living matter understanding of this class of Sargassum polysaccharides of Sargassum, to Sargassum polysaccharides, bioactive research more and more comes into one's own, meanwhile, Sargassum polysaccharides is having huge market prospect aspect functional food, new drug development, natural food anticorrosive additive and cosmetic material.
Heng Shi Alga Sgrgassi Enerves (C.Agardh) is subordinate to Phaeophyta, Fucales, and Sargassaceae, Sargassum, frond pitchy, more coarse, high about 1m, holdfast plate-like, diameter 1cm-1.5cm, grows one or two trunk on it, and trunk is shorter, cylindric.Heng Shi Alga Sgrgassi Enerves is grown on low tide band rock, and in Hong Kong, field, Pu peace Tan in Shantou, Shang Chuandao, Naozhou Island of Zhanjiang, Fujian Province etc. all has distribution, resource is quite abundant, easily obtains.Alga Sgrgassi Enerves is among the people can not directly be eaten, and is non-edible seaweed, only has fraction to be used as the raw material of feedstuff, algin, beverage and medical industry, and most of Alga Sgrgassi Enerves is not also comprehensively developed.For further exploitation marine resources, expand the range of application of this large-scale economic algae of Alga Sgrgassi Enerves and improve its exploitation and be worth, be necessary it to carry out evaluated biological activity, to provide certain scientific theory reference for the comprehensive utilization of Alga Sgrgassi Enerves.
Radial flow chromatogram (Radial Flow Chromatography, RFC) as a kind of novel chromatographic isolation analytical technology, can be used for the separation and purification of polysaccharide and protein, radial chromatographic column is made up of two porous permeable plexiglas concentric drums and marginal chromatography separation media, its unique Radial Flow design, mobile phase is carried sample and is radially moved, the fluid that is different from conventional axial chromatographic column passes through, thereby it is high to have overcome traditional colour spectral technology operating pressure, flow velocity is low, in the time of extensive processing, be difficult for the shortcomings such as amplification, make it have unrivaled advantage.Application number is the radial flow chromatogram separation method that discloses a kind of shiitake mushroom hypha polysaccharide in the Chinese invention patent application of CN201010268285.9, and whole radial chromatography purification has only been used 52min, and polysaccharide recovery reaches 87.1%.Application number is to disclose method of protein in a kind of radial flow chromatogram to remove crude polysaccharides in the Chinese invention patent application of CN2001063568.X, utilize radial flow chromatographic column to carry out purification to ganoderan, polysaccharide recovery and albumen removal efficiency can reach respectively 81.4%, 99.7%.
Application number is to disclose a kind of algae polysaccharide and preparation method thereof and the application in Medicated cigarette in the Chinese invention patent application of CN 201110333024.5, the preparation method of algae polysaccharide comprises: dry after marine algae is cleaned, after pulverizing, in water, extract, obtain extracting solution, wherein, the weight ratio of marine algae and water is 1:50-60; After extracting solution is cooling, obtain filtrate through centrifugal and vacuum filtration; By filtrate precipitate with ethanol; Collecting precipitation; After lyophilization, obtain polysaccharides of marine algae; Or; Filtrate is obtained to polysaccharides of marine algae with radial flow chromatogram method separating-purifying postlyophilization; Described marine algae is many ribs algae, Heng Shi Alga Sgrgassi Enerves or Sargassum phyllocystum Tseng et Lu,Sargassum horneri (Turn.) C. Ag. (Fucus horneri (Turn.)C.Ag.,Spongocarpus horneri Kutz.); This algae polysaccharide is applied in Medicated cigarette, and the flue gas fragrance matter of Medicated cigarette and perfume quantity increase, and the form of flue gas is good, and the dry sensation of flue gas reduces, and mellow and full sense, raw Feng sense and time sweet sense strengthen, and cigarette perfume (or spice) is characteristic.But its for algae polysaccharide other biological activity and application do not make a search.
Summary of the invention
The object of the present invention is to provide a kind of have the Heng Shi sargassan of antioxidant activity or the extract that contains Heng Shi sargassan.
The extract that the present invention also provides a kind of Heng Shi sargassan or contained Heng Shi sargassan as or prepare the application in antioxidant.
An application for Heng Shi sargassan or the extract that contains Heng Shi sargassan, described is applied as: Heng Shi sargassan or the extract that contains Heng Shi sargassan as or prepare the application in antioxidant.
In the present invention, be Heng Shi sargassan owing to playing the active substance of antioxygenic property, therefore Heng Shi sargassan or the extract that contains Heng Shi sargassan all have antioxygenic property.
The molecular weight distribution of described Heng Shi sargassan is 1,500,000-9,000,000 dalton (Da).Specifically can select molecular weight distribution at 1,500,000-7,000,000 daltonian Heng Shi sargassans, molecular weight distribution in 1,600,000-9,000,000 daltonian Heng Shi sargassans or molecular weight distribution at 1,500,000-2,000,000 daltonian Heng Shi sargassans.Comprehensively hydroxyl radical free radical is had to the significant antioxygenic properties such as active, scavenging action to ultra-oxygen anion free radical of removing, molecular weight distribution is best at the antioxygenic property of 1,600,000-9,000,000 daltonian Heng Shi sargassans.Measure the IC of Heng Shi sargassan through the algoscopy of removing ultra-oxygen anion free radical ability
50value can reach 0.0424mg/mL-0.0886mg/mL; Measure the IC of Heng Shi sargassan through the algoscopy of removing hydroxyl radical free radical ability
50value can reach 0.0075mg/mL-0.0180mg/mL.
The described extract that contains Heng Shi sargassan can be the extract of arbitrary form, can be both the extracting solution that contains Heng Shi sargassan, can be also the crude extract that contains Heng Shi sargassan or purified etc.In the described extract that contains Heng Shi sargassan, the quality percentage composition of Heng Shi sargassan generally can reach 19%-28%, as can be 19.47%-27.52%.In the described extract that contains Heng Shi sargassan, can also contain the materials such as protein, the quality percentage composition of protein is generally 0.1%-1.5%, as can be 0.1%-0.53%.
The preparation method of described Heng Shi sargassan or the extract that contains Heng Shi sargassan is simpler, generally adopts water extraction preparation, comprising:
(1) taking Heng Shi Alga Sgrgassi Enerves as raw material, extract taking water as extractant, obtain Heng Shi sargassan extracting solution;
(2) after Heng Shi sargassan extracting solution is concentrated and precipitate with ethanol, be precipitated i.e. Heng Shi Alga Sgrgassi Enerves crude polysaccharides;
(3) by the extract that obtains Heng Shi sargassan or contain Heng Shi sargassan after Heng Shi Alga Sgrgassi Enerves crude polysaccharides or Heng Shi sargassan extracting solution purification.
The described extract that contains Heng Shi sargassan can be selected one or more in the material that contains Heng Shi sargassan that described Heng Shi sargassan extracting solution, Heng Shi Alga Sgrgassi Enerves crude polysaccharides etc. exist with any form in preparation process.
In step (1), the condition optimization of described extraction is: ultrasound wave assisted extraction.The optimum condition of described ultrasound wave assisted extraction is: ultrasonic power 480w, dutycycle 1:1, extraction time 30min.
The described preferred Heng Shi Alga Sgrgassi Enerves of Heng Shi Alga Sgrgassi Enerves powder, therefore Heng Shi Alga Sgrgassi Enerves preferably carried out pulverization process before extracting, and preferably 40 DEG C of-50 DEG C of crushed after being dried, crossed 80 order-100 mesh sieves, obtained 80 order-100 order Heng Shi Alga Sgrgassi Enerves powder.
The grams of described Heng Shi Alga Sgrgassi Enerves is preferably 1:50-70, more preferably 1:60 with the ratio of the milliliter number of water.
Best cooling standing 1h-1.5h after described extraction completes, centrifugal, get supernatant, supernatant is Heng Shi sargassan extracting solution.
In step (2), described concentrated comprising: Heng Shi sargassan extracting solution rotary evaporation is concentrated into the 1/10-1/8 that volume is original volume.
Described precipitate with ethanol comprises: in the Heng Shi sargassan extracting solution after concentrated, add alcoholic solution make solution in the final volume percentage concentration of alcohol be 30%-80%, hold over night, centrifugal, be precipitated.Described precipitate with ethanol is preferably substep precipitate with ethanol, specifically comprise: in the Heng Shi sargassan extracting solution after concentrated substep add alcoholic solution make solution in the final volume percentage concentration of alcohol raise successively from 30%-80%, every step all needs hold over night, centrifugal, obtain respectively the precipitation that different alcohol precipitation concentrations are corresponding; More preferably: in the Heng Shi sargassan extracting solution after concentrated, substep adds alcoholic solution, the final volume percentage concentration that makes successively alcohol in solution is 30%, 60%, 80%, every step all needs hold over night, centrifugal, obtain respectively the precipitation that different alcohol precipitation concentrations are corresponding, the molecular weight distribution of the Heng Shi sargassan in described precipitation respectively is 1,500,000-7,000,000 Da, 160-900 ten thousand Da, 150-200 ten thousand Da.
It is the alcoholic solution of 95%-100% that described precipitate with ethanol preferably adopts concentration expressed in percentage by volume.
In step (3), described purification preferably adopts radial flow chromatogram to carry out purification.The step that radial flow chromatogram carries out purification comprises: Heng Shi Alga Sgrgassi Enerves crude polysaccharides is dissolved in the water and is made into the sample that concentration is 10mg-20mg/mL, or directly use Heng Shi sargassan extracting solution, centrifugal, remove portion impurity, remove finely ground particle substance with microporous filter membrane, cross weakly-basic anion radial column, carry out eluting with pure water, collect eluent, until sugar-free detects in effluent, eluent rotary evaporation is concentrated, the extract that lyophilization obtains the Heng Shi sargassan after purification or contains Heng Shi sargassan.The extract that the final product of described preparation method can be divided into Heng Shi sargassan or contain Heng Shi sargassan depending on degree of purification difference, Heng Shi sargassan is polysaccharide free from foreign meter in theory.
When described radial flow chromatogram carries out purification, applied sample amount is preferably 50mL, and loading flow velocity is preferably 5mL/min, and elution flow rate is preferably 40mL/min.
Described radial flow chromatogram can adopt SUPE RFL0-250A103S weakly-basic anion radial column.
In the each step of the present invention, described centrifugal condition optimization is rotating speed 10000rpm(rev/min), 4 DEG C of temperature, frozen centrifugation 10min.
In the each step of the present invention, the condition optimization of described rotary evaporation is that rotating speed 20-40rpm, temperature 40-50 DEG C and vacuum are 0.1MPa.
In the each step of the present invention, described precipitation all can be carried out lyophilization, and described cryodesiccated condition optimization is vacuum lyophilization 48h under-40 DEG C, 0.10mbar.
Antioxidation experiment: the extract that contains Heng Shi sargassan and Heng Shi sargassan that extraction is obtained carry out antioxidation in vitro test, result shows, extracts the extract that contains Heng Shi sargassan and the Heng Shi sargassan that obtain and has very high removing ultra-oxygen anion free radical, the activity of hydroxyl radical free radical.
Computing formula used in the present invention is as follows:
Heng Shi Alga Sgrgassi Enerves crude polysaccharides yield=Heng Shi Alga Sgrgassi Enerves crude polysaccharides quality ÷ Heng Shi Alga Sgrgassi Enerves raw materials quality × 100%;
Total yield=respectively walk yield sum;
Quality × 100% of polysaccharide in the front product of the quality ÷ purification of polysaccharide in polysaccharide recovery=purification afterproduct;
Quality × 100% of protein in product before removal of protein rate=(before purification in product in quality-purification afterproduct of protein the quality of protein) ÷ purification.
The beneficial effect of advantage of the present invention and generation:
1, Heng Shi sargassan of the present invention or the extract that contains Heng Shi sargassan all can be used as the effective Natural antioxidant, also can be used for preparing antioxidant, and human body is had no side effect.Because Heng Shi Alga Sgrgassi Enerves is a kind of kelp distributed more widely, wide material sources, can reduce the cost of antioxidant.
2, the present invention extracts antioxidant activity polysaccharide initiatively from Heng Shi Alga Sgrgassi Enerves, start the new way that Heng Shi Alga Sgrgassi Enerves utilizes, solve Heng Shi Alga Sgrgassi Enerves and utilized inadequate problem, improve the utilization rate of Heng Shi Alga Sgrgassi Enerves, increase economic benefit, for the exploitation of Heng Shi Alga Sgrgassi Enerves are laid a good foundation.
3, the antioxidant activity of Heng Shi sargassan of the present invention is remarkable, just can reach very high clearance rate when Heng Shi sargassan low concentration, and clearance rate and Heng Shi sargassan concentration are certain dose-effect relationship.In the time that Heng Shi sargassan concentration is 0.07mg/mL, Heng Shi sargassan can reach more than 65% the clearance rate of hydroxyl radical free radical; In the time that Heng Shi sargassan concentration is 0.07mg/mL, Heng Shi sargassan can reach more than 40% the clearance rate of ultra-oxygen anion free radical.
4, in optimum condition of the present invention, adopt ultrasonic auxiliary extraction method to extract Heng Shi sargassan, extraction time only needs 30min, compared with traditional hot water extraction (2-3h), can shorten time 4-6 doubly, significantly increase extraction efficiency, reduce preparation cost.
5, the present invention utilizes radial flow chromatogram to take preliminary purification sargassan, and polysaccharide recovery can reach 92.06%-97.79%, albumen removal efficiency reach 55.08%-92.86%, the purity that obtains polysaccharide significantly increases.
6, the radial flow chromatogram that the present invention adopts is compared with traditional axial flow chromatograph, have that loading flow velocity is fast, operating pressure is low (being no more than 0.1MPa), the advantages such as eluant (pure water) consumption is few and pollution-free, linear amplification is easy, quantity of sample handling is large, thereby significantly reduce production costs, improve the quality of products.
7, the present invention is not affecting under the prerequisite of separation efficiency, the chromatographic isolation operating time short (generally only needing 40min-60min), be conducive to improve the biological activity of Heng Shi sargassan, increase the response rate and polyoses content, meet the requirement of functional health-care food or biological medicine industry.
Brief description of the drawings
Fig. 1 is the variation of Heng Shi sargassan content before and after radial flow;
Fig. 2 is the variation of protein content before and after radial flow;
Fig. 3 is the scavenging action of Heng Shi sargassan to hydroxyl radical free radical;
Fig. 4 is the scavenging action of Heng Shi sargassan to ultra-oxygen anion free radical.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is conducted further description, but protection scope of the present invention is not limited in this:
Dutycycle refers to that square wave in one-period is the ratio of time and the square wave of high value time that is low value.
The method of testing that polysaccharide molecular weight distributes comprises: high performance liquid chromatography, make standard substance with the glucosan of different molecular weight, measure the relation between its appearance time and molecular weight, drawing standard curve, show that according to the appearance time of polysaccharide sample polysaccharide molecular weight distributes.
The mixture that Heng Shi sargassan of the present invention is multiple polysaccharide.
Embodiment 1:
(1) acquisition of Heng Shi Alga Sgrgassi Enerves extract
Get Heng Shi Alga Sgrgassi Enerves appropriate, carry out rinsing with clear water, naturally dry in the shade, be placed in 45 DEG C of oven dry in baking oven, pulverize with pulverizer, cross 90 mesh sieves.Accurately take 90 order Heng Shi Alga Sgrgassi Enerves powder 30g, be placed in the beaker of 2L, add the distilled water of 1800mL, pH7.0, carry out ultrasound wave assisted extraction 30min with ultrasonic power 480w, dutycycle 1:1.Cooling standing 1h after ultrasonic completing, then taking rotating speed as 10000rpm, 4 DEG C of frozen centrifugation 10min of temperature, obtain supernatant of 1600mL and precipitation.
(2) acquisition of different molecular weight component
Get supernatant of above-mentioned gained 1600mL, rotary evaporation be concentrated into volume be 200mL(be supernatant volume 1/8), add concentration expressed in percentage by volume be 95% ethanol water to make the final volume percentage concentration of ethanol in sample solution be 30%, hold over night.Sample after hold over night is at 1000rpm, and 4 DEG C of centrifugal 10min, separate and obtain secondary supernatant and precipitation, and gained precipitation is the Heng Shi Alga Sgrgassi Enerves crude polysaccharides that under 30% concentration of alcohol, precipitate with ethanol gets off, and precipitation is carried out lyophilization, is denoted as: Heng Shi Alga Sgrgassi Enerves crude polysaccharides F
1, for subsequent use.In secondary supernatant, progressively add concentration expressed in percentage by volume and be 95% ethanol water, make ethanol final concentration reach successively 60%(concentration expressed in percentage by volume), after hold over night at 1000rpm, 4 DEG C of centrifugal 10min, separation obtains three supernatant and precipitation, gained precipitation is the Heng Shi Alga Sgrgassi Enerves crude polysaccharides under 60% alcohol precipitation concentration, and precipitation is carried out lyophilization, is denoted as: Heng Shi Alga Sgrgassi Enerves crude polysaccharides F
2, for subsequent use.In three supernatant, continuing to add concentration expressed in percentage by volume is 95% ethanol water, make ethanol final concentration reach 80%(concentration expressed in percentage by volume), after hold over night at 1000rpm, 4 DEG C of centrifugal 10min, separation obtains four supernatant and precipitation, and gained precipitation is the Heng Shi Alga Sgrgassi Enerves crude polysaccharides under 80% alcohol precipitation concentration, and precipitation is carried out lyophilization, be denoted as: Heng Shi Alga Sgrgassi Enerves crude polysaccharides F3, for subsequent use.Three kinds of Heng Shi Alga Sgrgassi Enerves crude polysaccharides are taupe flocculence solid, and yield is respectively 1.2667%, 1.2477%, 1.7420%, and total yield is 4.2564%.
Above-mentioned cryodesiccated condition is vacuum lyophilization 48h under-40 DEG C, 0.10mbar.
Heng Shi Alga Sgrgassi Enerves crude polysaccharides F
1the quality percentage composition of middle Heng Shi sargassan is 21%, and the quality percentage composition of protein is that the molecular weight distribution of 1.18%, Heng Shi sargassan is 1,500,000-7,000,000 dalton.
Heng Shi Alga Sgrgassi Enerves crude polysaccharides F
2the quality percentage composition of middle Heng Shi sargassan is 27%, and the quality percentage composition of protein is that the molecular weight distribution of 1.4%, Heng Shi sargassan is 1,600,000-9,000,000 dalton.
Heng Shi Alga Sgrgassi Enerves crude polysaccharides F
3the quality percentage composition of middle Heng Shi sargassan is 28%, and the quality percentage composition of protein is that the molecular weight distribution of 1.42%, Heng Shi sargassan is 1,500,000-2,000,000 dalton.
(3) radial flow chromatogram purification
Separating medium pretreatment: getting that NaOH aqueous solution soaking 3h → pure water that appropriate A103S weak base anion exchanger (macropore weak base phenylethylene resin series) filler → mass percentage concentration is 2% washs to neutrality → mass percentage concentration is that 3%HCl aqueous solution soaking 3h → pure water washs to the NaOH aqueous solution soaking 3h → pure water washing that is 2% of neutrality → mass percentage concentration to neutral.
Dress post: will, after pretreated ion-exchanger is degassed, manually fill in radial flow chromatographic column; After dress post, with the first back flush pillar of 5 times of volume pure water, then with 5 times of volume pure water forwards flushing pillars, so that pillar balance;
Loading, eluting: get the Heng Shi Alga Sgrgassi Enerves crude polysaccharides F after 1.000g lyophilizing
1be dissolved in 50mL distilled water, being made into concentration is 20mg/mL solution, 1000rpm, 4 DEG C of centrifugal 10min, get supernatant and cross 0.45 μ m microporous filter membrane, by constant flow pump loading under the flow velocity of 5mL/min, then carry out eluting with distilled water with 40mL/min, detect without polysaccharide to phenolsulfuric acid method, automatically fraction collector is collected eluent, measuring space polyoses content simultaneously, whole elution process 40min consuming time.
Collect eluent: all eluents are collected, and merging mixes, measure polysaccharide and protein content wherein by phenolsulfuric acid method and Folin-phenol method respectively, calculate polysaccharide recovery and removal of protein rate;
Filler regeneration: ion-exchanger used is reusable after regeneration, utilizes 2mol/L NaCl aqueous solution eluting, to eluent water white transparency, and detects without albumen substantially by Folin-phenol method, then is eluted to without NaCl with distilled water;
Concentrated: eluent adopts vacuum concentration, reduce the loss in concentration process, its condition is that rotating speed 20rpm, temperature (45 ± 5) DEG C, vacuum are 0.1MPa.
Dry: after eluent is concentrated under-40 DEG C, 0.10mbar vacuum lyophilization 48h, obtain the product after purification: Heng Shi sargassan extract F
1.
Heng Shi sargassan extract F
1the quality percentage composition of middle Heng Shi sargassan is 19.47%, and the quality percentage composition of protein is that the molecular weight distribution of 0.53%, Heng Shi sargassan is 1,500,000-7,000,000 dalton.Heng Shi sargassan extract F before and after radial flow chromatogram as seen from Figure 1
1polyoses content change not quite, the loss of polysaccharide is little, polysaccharide recovery is up to 92.06%, the variation of protein before and after radial flow chromatogram is less as shown in Figure 2, due to F
1in sugar mainly formed by glycoprotein, so albumen removal efficiency is not high, reach 55.08%.Because radial flow chromatogram purification required time is shorter, whole elution process only needs 40min, and polysaccharide natural anti-oxidation activity is not damaged.
Embodiment 2:
Other operate as embodiment 1, and difference is:
Loading, eluting: get the Heng Shi Alga Sgrgassi Enerves crude polysaccharides F after 800mg lyophilizing
2be dissolved in 40mL distilled water, being made into concentration is 20mg/mL solution, 1000rpm, 4 DEG C of centrifugal 10min, get supernatant and cross 0.45 μ m microporous filter membrane, by constant flow pump loading under the flow velocity of 5mL/min, then carry out eluting with distilled water with 40mL/min, detect without polysaccharide to phenolsulfuric acid method, automatically fraction collector is collected eluent, measuring space polyoses content simultaneously, whole elution process 42min consuming time.
The polysaccharide recovery of radial flow chromatogram front and back Heng Shi sargassan extract F2 is up to 97.79%, if Fig. 2 albumen removal efficiency is up to 92.86% as seen from Figure 1.Whole elution process only needs 42min.
Heng Shi sargassan extract F
2the quality percentage composition of middle Heng Shi sargassan is 26%, and the quality percentage composition of protein is that the molecular weight distribution of 0.1%, Heng Shi sargassan is 1,600,000-9,000,000 dalton.
Embodiment 3:
Other operate as embodiment 1, and difference is:
Loading, eluting: get the Heng Shi Alga Sgrgassi Enerves crude polysaccharides F after 500mg lyophilizing
3be dissolved in 50mL distilled water, being made into concentration is 10mg/mL solution, 1000rpm, 4 DEG C of centrifugal 10min, get supernatant and cross 0.45 μ m microporous filter membrane, by constant flow pump loading under the flow velocity of 5mL/min, then carry out eluting with distilled water with 40mL/min, detect without polysaccharide to phenolsulfuric acid method, automatically fraction collector is collected eluent, measuring space polyoses content simultaneously, whole elution process 40min consuming time.
Heng Shi sargassan extract F before and after radial flow chromatogram as seen from Figure 1
3polysaccharide recovery up to 96.39%, if Fig. 2 albumen removal efficiency is up to 92.96%.Whole elution process only needs 40min.
Heng Shi sargassan extract F
3the quality percentage composition of middle Heng Shi sargassan is 27.52%, and the quality percentage composition of protein is that the molecular weight distribution of 0.1%, Heng Shi sargassan is 1,500,000-2,000,000 dalton.
Below in conjunction with specific experiment data, the antioxidant activity of Heng Shi sargassan of the present invention is described further:
(1) scavenging action of Heng Shi sargassan to hydroxyl radical free radical
The system of utilizing Fenton reaction to prepare hydroxy radical (OH) is the classical radical former of the one system being at home and abroad widely used, its ultimate principle is that hydrogen peroxide is followed ferrous ion reaction, generate water and oxygen, in course of reaction, produce hydroxyl radical free radical (OH) simultaneously, and meanwhile, salicylic acid possesses again specific recognition ability to hydroxyl radical free radical (OH).What in system, add that salicylic acid can be sharp captures hydroxyl radical free radical (OH), generates purple product, and this product possesses strong absworption peak at 510nm place.If but add the material with scavenging action, can compete with salicylic acid, thus coloured product growing amount is reduced, and absorbance reduces.As shown in Figure 3, result shows, the Heng Shi sargassan extract F of three kinds of different molecular weight ranges after the purification obtaining in embodiment 1,2,3
1, Heng Shi sargassan extract F
2, Heng Shi sargassan extract F
3all there is the effect of very strong removing hydroxyl radical free radical, when low concentration (this concentration has been converted into Heng Shi sargassan concentration), just can reach very high clearance rate, and clearance rate and polysaccharide concentration are certain dose-effect relationship, when in system, polysaccharide concentration is 0.07mg/mL, the clearance rate of three kinds of polysaccharide all can reach more than 65%, wherein Heng Shi sargassan extract F
2removing ability to hydroxyl radical free radical is the strongest, reaches the IC of 71.84%, three kind of polysaccharide in the time of 0.07mg/mL
50value is respectively 0.0084mg/mL, 0.0075mg/mL, 0.0180mg/mL, and removing ability is F
2>F
1>F
3, above data show that Heng Shi sargassan has significant removing active to hydroxyl radical free radical, wherein molecular weight distribution is best in the removing activity of 1,600,000-9,000,000 daltonian Heng Shi sargassans.
(2) scavenging action of Heng Shi sargassan to ultra-oxygen anion free radical
Pyrogallol is rapid autoxidation under alkali condition, produces superoxide anion (O2) in autoxidation process, and superoxide anion (O2) accelerates pyrogallol autoxidation speed, generates coloured intermediate product simultaneously, and coloured product has strong light absorption at 325nm.This system is usually used in measuring superoxide dismutase (SOD) or the inhibitory action of quasi-SOD active substance to O2.Consider from kinetics angle, if active substance has scavenging action to O2, can slow down the carrying out of this reaction.Because autoxidation rate dependent is in the concentration of superoxide anion (O2), remove superoxide anion (O2), suppress automatic oxidation reaction, stop the accumulation of intermediate product, thereby evaluate the ability of extracting solution removing superoxide anion (O2).As shown in Figure 4, result shows, the Heng Shi sargassan extract F of three kinds of different molecular weight ranges after the purification obtaining in embodiment 1,2,3
1, Heng Shi sargassan extract F
2, Heng Shi sargassan extract F
3all there is the effect of very strong removing ultra-oxygen anion free radical, when low concentration (this concentration has been converted into Heng Shi sargassan concentration), just can reach very high clearance rate, and clearance rate and polysaccharide concentration are certain dose-effect relationship, Heng Shi sargassan extract F
2removing ability to ultra-oxygen anion free radical is the strongest, reaches the IC of 74.34%, three kind of polysaccharide in the time of 0.07mg/mL
50value is respectively 0.0886mg/mL, 0.0424mg/mL, 0.0605mg/mL, and removing ability is F
2>F
3>F
1, above data show that Heng Shi sargassan has significant removing active to ultra-oxygen anion free radical, wherein molecular weight distribution is best in the removing ability of 1,600,000-9,000,000 daltonian Heng Shi sargassans.
Above-mentioned experimental result shows, Heng Shi sargassan of the present invention or the extract containing Heng Shi sargassan have antioxygenic property, can as or prepare antioxidant.
In the present invention, because the active substance that plays antioxygenic property is Heng Shi sargassan, therefore Heng Shi sargassan or the extract that contains Heng Shi sargassan all have antioxygenic property, in the power of its antioxygenic property and system, the concentration of Heng Shi sargassan is certain dose-effect relationship, in general system, the concentration of Heng Shi sargassan is higher, antioxygenic property is stronger, and when excessive concentration, antioxygenic property tends to be steady.
In preparation method of the present invention, the moderate change of parameter does not affect the preparation of Heng Shi sargassan, and therefore in preparation method of the present invention, the combination of arbitrary parameter all can realize the preparation of Heng Shi sargassan.Do not repeat them here.
Claims (5)
1. the application of a Heng Shi sargassan or the extract that contains Heng Shi sargassan, it is characterized in that, described Heng Shi sargassan or the extract that contains Heng Shi sargassan are in the application of preparing in antioxidant, and the molecular weight distribution of described Heng Shi sargassan is 1,500,000-9,000,000 dalton.
2. the application of Heng Shi sargassan according to claim 1 or the extract that contains Heng Shi sargassan, is characterized in that, the molecular weight distribution of described Heng Shi sargassan is 1,500,000-7,000,000 dalton.
3. the application of Heng Shi sargassan according to claim 1 or the extract that contains Heng Shi sargassan, is characterized in that, the molecular weight distribution of described Heng Shi sargassan is 1,600,000-9,000,000 dalton.
4. the application of Heng Shi sargassan according to claim 1 or the extract that contains Heng Shi sargassan, is characterized in that, the molecular weight distribution of described Heng Shi sargassan is 1,500,000-2,000,000 dalton.
5. according to the application of the Heng Shi sargassan described in claim 1-4 any one or the extract that contains Heng Shi sargassan, it is characterized in that, in the described extract that contains Heng Shi sargassan, the quality percentage composition of Heng Shi sargassan is 19%-28%.
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| 吉宏武，等.湛江海域14 种主要海藻SOD含量与活力测定.《食品研究与开发》.2006,第27卷(第9期),102-106. * |
| 谌素华,等.亨氏马尾藻化学成分分析及其营养学评价.《食品研究与开发》.2010,第31卷(第5期),154-155. * |
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