CN105085694A - Preparation method of urechis unicinctus viscera glycosaminoglycan and application thereof - Google Patents
Preparation method of urechis unicinctus viscera glycosaminoglycan and application thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of urechis unicinctus viscera glycosaminoglycan. The method comprises the following steps: using urechis unicinctus viscera, rubbing and homogenizing, degreasing and performing pumping filtration to obtain the urechis unicinctus viscera dry powder, then hydrolyzing the viscera dry powder by papain and trypsin, extracting a viscera glycosaminoglycan crude product from a hydrolysate, and performing dialysis and freeze drying to obtain the viscera glycosaminoglycan. The invention also relates to an application of the viscera glycosaminoglycan in preparation of food, health product and medicine for preventing and adjusting blood fat. The extraction method is simple, yield can reach 6-7%, protein content of viscera glycosaminoglycan can be greatly reduced, purity of glycosaminoglycan is increased, and the method provides a novel approach for preventing and adjusting blood fat.
Description
Technical field
The present invention relates to and extract the method for glycosaminoglycan and the application of this glycosaminoglycan from Urechis uniconctus internal organ, belong to the technological method to Urechis uniconctus comprehensive processing and utilization.
Background technology
Urechis uniconctus (Urechisunicinctus) popular name sea intestines, sea intestines, belong to Echiuroidea Echiurioidea Yi guiding principle Echiurida, without pipe Yi order Xenopneusta, thorn Yi section Urchidae, body is thick, be about 100-300mm, wide about 25-27mm, body surface is abound with the granular projection differed in size, kiss is conical, neuroseta 1 is right, thick, perianal has a circle 9-13 bar brown caudal seta, be distributed in Russia, Japan, Korea and China's coastal zone along the Huanghai Sea and the Bohai Sea, it is the benthic Common Species of northern China coastal silt bank tideland inferior segment and subtidal zone shoaling water.Urechis uniconctus is individual loose, and meat flavour is delicious, and body wall flesh rich in proteins and multiple essential amino acid are since ancient times, coastal all as famous and precious seafood in China, Japan and Korea, have higher economic worth.
Current Urechis uniconctus has realized propagating artificially, and due to traditional method only its body wall edible of people, therefore in process for processing link, the internal organ of Urechis uniconctus are dropped as waste, cause the very large waste of resource, also cause serious pollution to environment.In fact, internal organ account for 2/3 of Urechis uniconctus body quality, and research finds that in Urechis uniconctus internal organ, protein content is 18.25%, lipid content is 0.12%, total sugar content is 4.09%, containing elements such as abundant Ca, Mg, Fe, Zn, simultaneously also containing abundant EPA, DHA and DPA etc.
Along with the pay attention to day by day studied exploitation and the Carbohydrate drugs of Living marine resources, the research of marine animal active polysaccharide is also paid close attention to more and more widely, immunity is improved as glycosaminoglycan extracted from sea cucumber has, antitumor, the various active such as anticoagulation, it is active substance main in sea cucumber, the extracting method of glycosaminoglycan extracted from sea cucumber also has more research, as the Chinese invention patent " a kind of preparation method of stichopus japonicus selenka glycosaminoglycans " that application number is 201010112839.6 (application publication number CN101787084A), the patent No. is the Chinese invention patent " a kind of extracting method of holothuria leucospilota glycosaminoglycan " etc. of ZL200910305363.5 (Authorization Notice No. CN101624426B).Also containing abundant active polysaccharide material in sea cucumber internal organ, the research of discarding internal organ polysaccharide to sea cucumber not only avoid the waste of resource, also improves the additional output value of Holothurian machining.And Urechis uniconctus not only looks like nude sea cucumber, its nutritive value is also in no way inferior compared with sea cucumber, with sea cucumber internal organ polysaccharide researches for instructing, Urechis uniconctus internal organ polysaccharide is furtherd investigate, to filtering out active polysaccharide composition, for the refuse reclamation of Urechis uniconctus internal organ, the production value improving Urechis uniconctus provides important references.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method utilizing Urechis uniconctus internal organ to extract Urechis uniconctus internal organ glycosaminoglycan.
Another technical problem to be solved by this invention provides a kind of application of above-mentioned Urechis uniconctus internal organ glycosaminoglycan.
The present invention solves the technical scheme that first technical problem adopt: 1, a kind of preparation method of Urechis uniconctus internal organ glycosaminoglycan, is characterized in that comprising the following steps:
(1) pre-treatment: get fresh Urechis uniconctus internal organ, rub homogenate, add 3 ~ 4 times of vol acetone, after magnetic stirrer over night, suction filtration gets precipitation, pulverizes to obtain Urechis uniconctus internal organ dry powder after precipitation being dried;
(2) enzymolysis: by solid-liquid ratio (g/ml) 1:18 ~ 20, distilled water is added in internal organ dry powder, after 4 ~ 5h is extracted in 75 ~ 80 DEG C of water-baths, adjust ph is 6 ~ 7, adds the papoid of internal organ dry powder weight 1 ~ 1.2%, 55 ~ 65 DEG C of water-bath digestion 4 ~ 5h, adjust ph is 8.2 ~ 9.0, adds the trypsinase of internal organ dry powder weight 0.5 ~ 0.6%, 55 ~ 65 DEG C of water-bath digestion 3 ~ 4h, adjust ph is deactivation 5 ~ 15 minutes at 7.0,100 DEG C;
(3) extract: 6000rpm is centrifugal, and 8min gets supernatant liquor, and after being concentrated into 1/15 of original volume, adding ethanol to alcohol content is 80%, hold over night, and 6000rpm is centrifugal, and 8min gets precipitation, obtains internal organ glycosaminoglycan crude product;
(4) purifying: by solid-liquid ratio (mg/ml) 1:100, dialysed after distilled water by internal organ glycosaminoglycan dissolving crude product, lyophilize obtains internal organ glycosaminoglycan.
As preferably, the molecular weight that described dialysis method retains is 3.5 ~ 4.1kDa.
The application of a kind of Urechis uniconctus internal organ glycosaminoglycan as claimed in claim 1 in prevention and the food of adjusting blood lipid, healthcare products and medicine.
As preferably, the monose composition of described Urechis uniconctus internal organ glycosaminoglycan comprises glucose, glucosamine, seminose, semi-lactosi, Fucose, wood sugar, rhamnosyl, glucuronic acid and galn, with molar ratio computing, glucose: glucosamine: seminose: semi-lactosi: Fucose: wood sugar: rhamnosyl: glucuronic acid: the ratio of galn is 5.37:1.38:1:1.38:2.4:1.05:0.87:0.52:1.38, and weight-average molecular weight is 3.5 ~ 4.1kDa.
For making described Urechis uniconctus internal organ glycosaminoglycan obtain good inhibition of lipid peroxidation, the effective concentration of described Urechis uniconctus internal organ glycosaminoglycan scavenging free radicals is 2.47 ~ 10mg/ml.
Compared with prior art, the invention has the advantages that: utilize Urechis uniconctus to discard internal organ for raw material, extracted by two step enzymolysis processs and obtain the glycosaminoglycan of weight-average molecular weight between 3.5 ~ 4.1kDa, this extracting method is simple, yield can reach 6%-7%, adopt papoid to combine with trypsinase to be hydrolyzed, greatly reduce the protein content of internal organ polysaccharide, thus improve the purity of glycosaminoglycan.Simultaneously, this Urechis uniconctus internal organ glycosaminoglycan has good inhibition of lipid peroxidation in anti peroxidation of lipid model in vitro, during 10mg/ml to the clearance rate of free radical close to 90%, therefore this internal organ glycosaminoglycan can be processed into prevention and the food of adjusting blood lipid, healthcare products and medicine further, thus for prevent and adjusting blood lipid opens up new approach.
Accompanying drawing explanation
Fig. 1 is the PMP column front derivation high-efficient liquid phase chromatogram of Urechis uniconctus internal organ glycosaminoglycan in the present invention;
Fig. 2 is the PMP column front derivation high-efficient liquid phase chromatogram of 10 kinds of monose standard substance;
Fig. 3 is the High Performance Gel Permeation color atlas of Urechis uniconctus internal organ glycosaminoglycan in the present invention;
Fig. 4 is that the lipid peroxidation of Urechis uniconctus internal organ glycosaminoglycan in the present invention eliminates curve.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment 1: Urechis uniconctus internal organ glycosaminoglycan prepares one
Get fresh Urechis uniconctus, insert from its anus with scissors, cut open along belly, or cut open along back, take out all internal organ, the internal organ of taking-up are cleaned up rear mincer and pulverizes homogenate, add the acetone degreasing of 4 times of volumes, after magnetic agitation (200rpm) is spent the night, suction filtration gets precipitation, pulverizes to obtain Urechis uniconctus internal organ dry powder after precipitation being dried.In obtained internal organ dry powder, add distilled water by solid-liquid ratio (g/ml) 1:20, after 4h is extracted in 80 DEG C of water-baths, adjust ph is 7, adds the papoid of internal organ dry powder weight 1%, 60 DEG C of water-bath digestion 4h, and adjust ph is 9.0; Add the trypsinase of internal organ dry powder weight 0.5%, 60 DEG C of water-baths digestion 3h, adjust ph is deactivation 5 minutes at 7.0,100 DEG C, to make unreacted enzyme deactivation.The obtained centrifugal 8min of enzymolysis solution 6000rpm is got supernatant liquor, and by supernatant concentration to 1/15 of original volume, adding ethanol to alcohol content is 80%, hold over night, and 6000rpm is centrifugal, and 8min gets precipitation, obtains internal organ glycosaminoglycan crude product.By solid-liquid ratio (mg/ml) 1:100, internal organ glycosaminoglycan dissolving crude product is crossed after distilled water 3500 dialysis tubing dialysis, lyophilize obtains brown cotton-shaped internal organ glycosaminoglycan, and in the present embodiment, the yield of internal organ glycosaminoglycan is 6.5%.
Embodiment 2: Urechis uniconctus internal organ glycosaminoglycan prepares two
Get fresh Urechis uniconctus, insert from its anus with scissors, cut open along belly, or cut open along back, take out all internal organ, the internal organ of taking-up are cleaned up rear mincer and pulverizes homogenate, add the acetone degreasing of 3 times of volumes, after magnetic agitation (200rpm) is spent the night, suction filtration gets precipitation, pulverizes to obtain Urechis uniconctus internal organ dry powder after precipitation being dried.In obtained internal organ dry powder, add distilled water by solid-liquid ratio (g/ml) 1:18, after 5h is extracted in 75 DEG C of water-baths, adjust ph is 6, add the papoid of internal organ dry powder weight 1.4%, 65 DEG C of water-bath digestion 4h, adjust ph is 8.5, adds the trypsinase of internal organ dry powder weight 0.6%, 65 DEG C of water-bath digestion 4h, adjust ph is deactivation 15 minutes at 7.0,100 DEG C, to make unreacted enzyme deactivation.The obtained centrifugal 8min of enzymolysis solution 6000rpm is got supernatant liquor, and by supernatant concentration to 1/15 of original volume, adding ethanol to alcohol content is 80%, hold over night, and 6000rpm is centrifugal, and 8min gets precipitation, obtains internal organ glycosaminoglycan crude product.By solid-liquid ratio (mg/ml) 1:100, internal organ glycosaminoglycan dissolving crude product is crossed after distilled water 3500 dialysis tubing dialysis, lyophilize obtains brown cotton-shaped internal organ glycosaminoglycan, and in the present embodiment, the yield of internal organ glycosaminoglycan is 7%.
Embodiment 3: Urechis uniconctus internal organ glycosaminoglycan prepares three
Get fresh Urechis uniconctus, insert from its anus with scissors, cut open along belly, or cut open along back, take out all internal organ, the internal organ of taking-up are cleaned up rear mincer and pulverizes homogenate, add the acetone degreasing of 4 times of volumes, after magnetic agitation (200rpm) is spent the night, suction filtration gets precipitation, pulverizes to obtain Urechis uniconctus internal organ dry powder after precipitation being dried.In obtained internal organ dry powder, add distilled water by solid-liquid ratio (g/ml) 1:18, after 4h is extracted in 80 DEG C of water-baths, adjust ph is 6, add the papoid of internal organ dry powder weight 1.2%, 55 DEG C of water-bath digestion 5h, adjust ph is 8.2, adds the trypsinase of internal organ dry powder weight 0.6%, 55 DEG C of water-bath digestion 4h, adjust ph is deactivation 15 minutes at 7.0,100 DEG C, to make unreacted enzyme deactivation.The obtained centrifugal 8min of enzymolysis solution 6000rpm is got supernatant liquor, and by supernatant concentration to 1/15 of original volume, adding ethanol to alcohol content is 80%, hold over night, and 6000rpm is centrifugal, and 8min gets precipitation, obtains internal organ glycosaminoglycan crude product.By solid-liquid ratio (mg/ml) 1:100, internal organ glycosaminoglycan dissolving crude product is crossed after distilled water 3500 dialysis tubing dialysis, lyophilize obtains brown cotton-shaped internal organ glycosaminoglycan, and in the present embodiment, the yield of internal organ glycosaminoglycan is 6%.
Embodiment 2: Urechis uniconctus internal organ glycosaminoglycan composition confirms
2.1 monose compositions
The monose of the Urechis uniconctus internal organ polysaccharide adopting PMP pre column Derivatization to be obtained by embodiment 1 or embodiment 2 or embodiment 3 forms.
2.1.1 the all-hydrolytic of sample
Get appropriate amount of sample, use 2molL
-1tFA carries out all-hydrolytic 6h at 110 DEG C, repeatedly adds methyl alcohol rotary evaporation removing TFA to without tart flavour, washes out with appropriate distilled water.
2.1.2 derivatization conditions
Get the above-mentioned standard solution of 100 μ L to put in the EP pipe of 1.5mL, add 100 μ L0.3molL
-1naOH solution, 120 μ L0.5molL
-1pMP solution, 70 DEG C of derivative 1h, have reacted and have added 100 μ L0.3molL
-1hCl solution neutralizes, and adds 500 μ L chloroforms and repeatedly extracts centrifugal, supernatant 0.22 μm of filtering with microporous membrane, adopts efficient liquid phase chromatographic analysis.
2.1.3 chromatographic condition
Chromatographic column: AgilentXDB-C
18chromatographic column (4.6mm × 150mm, 5 μm); Moving phase: phosphate buffered saline buffer (pH6.7)/CH
3cN (82:18, V/V); Flow velocity: 1.0mLmin
-1; Column temperature: 30 DEG C; Sample size: 20 μ L; Detector: DAD (245nm).
2.1.4 simple monosaccharide composition measuring
The sample that all-hydrolytic is complete and standard substance derive under the same conditions, sample introduction 20 μ L, efficient liquid phase chromatographic analysis, record color atlas.Contrast with the appearance time of each monose standard substance, determine that simple monosaccharide forms, result as shown in Figure 1.Contrasted from Fig. 1, Fig. 2, the monose of internal organ glycosaminoglycan composition is very complicated, and not only containing neutral sugar, but also mixed polysaccharide containing acid sugar and alkalescence sugar, and the hexose also comprised in neutral sugar, five-carbon sugar and six carbon methyl are sugared.In monose composition, glucose (Glc) is main composition, secondly also containing glucosamine (GlcN), seminose (Man), semi-lactosi (Gal) and Fucose (Fuc), also containing a small amount of wood sugar (Xyl), rhamnosyl (Rha), glucuronic acid (GlcUA) and galn (GalN).Ratio is respectively Man:GlcN:Rha:GlcUA:GalN:Glc:Gal:Xyl:Fuc=1:1.38:0.87:0.53: 0.52:5.37:1.38:1.05:2.40.
The solution preparation used in the present embodiment is as follows:
Monose standard solution: mole Man, GlcN, Rha, GlcUA, GalUA, GalN, Glc, Gal, Xyl, Ara and Fucu10 kind monose standard substance being dried to constant weight such as accurately to take respectively, adds water and fully dissolves, be mixed with 1.0mgmL
-.
Phosphate buffered saline buffer: accurately take 13.6g potassium primary phosphate and 1.89g sodium hydroxide, be settled in the volumetric flask of 1L with water, after fully dissolving, crosses the filter membrane of 0.22 μm, ultrasonic degas and get final product.
Mixture of acetonitrile-phosphate buffer solution: before use by above-mentioned phosphate buffered saline buffer and the pure acetonitrile of the Import Analysis proportions according to 82:18, after degassed and get final product, now with the current.
0.3molL
-1naOH solution: precision takes 0.60g sodium hydroxide constant volume in the volumetric flask of 50mL.
0.3molL
-1hCl solution: draw the concentrated hydrochloric acid constant volume of 1.25mL in the volumetric flask of 50mL.
The preparation of PMP solution: precision takes 87.1mgPMP, by methanol constant volume in the volumetric flask of 1.0mL, matching while using.
2.2 molecular weight determination
Adopt High Performance Gel Permeation Chromatography to measure the molecular weight of Urechis uniconctus internal organ glycosaminoglycan, result as shown in Figure 3.2.2.1 in mensuration process, the chromatographic condition of High Performance Gel Permeation chromatogram is:
Chromatographic column: TSK-GELG3000SWxL gel chromatographic columns (300mm × 7.8mm); Column temperature: 35 DEG C; Moving phase: 0.1molL-
1na
2sO
4; Flow velocity: 0.5mLmin
-1; Detector: Composition distribution (RID).
2.2.2 standard curve making
With series standard dextran for molecular weight standards, get the dextran standards of 6 different molecular weights respectively, dissolve by moving phase and be mixed with 5mgmL
-1solution, respectively sample introduction 20 μ L, record color atlas, with retention time T
rfor X-coordinate, draw regression equation with molecular weight logarithm Log (Mw) for ordinate zou drawing standard curve.
2.2.3 sample molecule flow measurement
Take appropriate amount of sample, add moving phase and be dissolved into 5mgmL
-1solution, with 0.22 μm of filtering with microporous membrane, sample introduction 20 μ L, record color atlas.The retention time of sample solution is substituted into the logarithmic value that the regression equation made by standard Dextrose acid anhydride can draw molecular weight analyte, then its conversion can be drawn the molecular weight of sample.As Fig. 3 presents single and more symmetrical peak, show that the molecular weight distribution of Urechis uniconctus internal organ polysaccharide is more concentrated, and molecular mass standard curve comparison, the molecular weight calculating internal organ glycosaminoglycan mainly concentrates on about 4.1kDa.It can thus be appreciated that Urechis uniconctus internal organ glycosaminoglycan is the oligose that molecular weight distribution is comparatively single.
2.3 physical and chemical property determining
Take bovine serum albumin as standard substance, adopt Folin-phenol method to measure protein content, take potassium sulfate as standard substance, adopt bariumchloride-gelatin turbidimetry for Determination sulfate radical content, measurement result protein content is lower than 4%, and sulfate content is about 8%.2.3.1 the preparation of solution
1. 4%Na
2cO
3solution, 2. 0.2molL
-1naOH solution, 3. 1%CuSO
4solution, 4. 2% potassium sodium tartrate solution.1. will mix with 2. equal-volume before using and be made into Na
2cO
3-NaOH solution, 3. will be made into copper sulfate-potassium sodium tartrate solution with 4. mixing, then these two kinds of solution are mixed to get solution first by 50:1, solution second is doubly obtained by the front dilution one of Folin-phenol reagent.
Standard solution: accurately take the standard bovine white protein 10.0mg being dried to constant weight, puts a small amount of water dissolution in 10mL volumetric flask, adds water to scale, obtain 1.0mgmL
-1standard bovine albumin soln, get 5mL constant volume in 50mL volumetric flask, obtain 0.1mgmL
-1standard bovine albumin soln.
Test liquid: accurately take each polysaccharide sample 5.0mg being dried to constant weight, add 1mL distilled water, be mixed with 5mgmL
-1solution.
2.3.2 the mensuration of protein content
The drafting of typical curve
Draw standard bovine albumin soln 0,100,200,400,600,800,1000 μ L respectively, add water and mend to 1.0mL, add 2.5mL reagent first respectively, vortex oscillation, room temperature places 10min, then adds 0.25mL reagent second respectively, vortex oscillation again, after room temperature places 30min, ultraviolet spectrophotometer 650nm place measures light absorption value.With the albuminised total mass number of standard bovine for X-coordinate, corresponding light absorption value is ordinate zou drawing standard curve.
Sample tests
Draw test liquid respectively appropriate, same method and standard bovine white protein parallel running three times, 650nm place mensuration light absorption value, calculates the mean value of the protein content of each polysaccharide, is converted into percentage composition according to typical curve.
2.3.3 sulfate measures:
Solution preparation
0.5% gelatin solution: 1.25g gelatin is dissolved in 10mL water at 60 ~ 70 DEG C, is settled to 250mL, 4 DEG C of refrigerator hold over night.
Bariumchloride-gelatin solution: 0.5gBaCl
2be dissolved in 100mL0.5% gelatin solution, 4 DEG C of refrigerator hold over night.
K
2sO
4solution: the K claiming 105 DEG C of dry constant weights
2sO
4108.75mg, is settled to 100mL with water, obtains sulfate reference liquid (sulfate radical mass concentration) 600 μ gmL
-1.
Dilute hydrochloric acid is prepared: 1mL concentrated hydrochloric acid is diluted with water to 30mL.
The drafting of typical curve
Get respectively above-mentioned standard Kraft solution 0,0.1,0.2,0.3,0.4,0.5mL, moisturizing is to 1.0mL.Add dilute hydrochloric acid 1.0mL, then add bariumchloride-gelatin solution 0.5mL, mixing, place 20min for 25 DEG C, measure its light absorption value at 500nm place.With the total mass number of sulfate reference liquid for X-coordinate, corresponding light absorption value is ordinate zou drawing standard curve.
Sample tests
Accurately take 5mg sample and be dissolved in 4mL, 1molL
-1in HCl, tube sealing, reacts 12h in the baking oven of 105 DEG C, and sulfate radical is dissociated.Accurately pipette above-mentioned solution appropriate, by the measuring method of typical curve parallel for three times, measure its light absorption value, substitute into the mean value that equation of linear regression calculates the sulfate content of each polysaccharide sample, finally, be converted into percentage composition.)
Embodiment 3: In Vitro Anti lipid peroxidation determination of activity
Research shows that ocean glycosaminoglycan can effectively reduce the generation of lipid peroxide, is reached regulate the cardiovascular and effect delayed senility by the removing of free radical.Urechis uniconctus internal organ polysaccharide, as low-molecular-weight sulphating class glycosaminoglycan, does not also carry out deep activity research to it.For fast characterizing Urechis uniconctus internal organ glycosaminoglycan to the scavenging(action) of lipid peroxide to being applied in adjusting blood lipid and preventing cardiovascular disease, adopt In Vitro Anti Lipid Peroxidation model to evaluate its Free-radical scavenging activity.
The foundation of 3.1 In Vitro Anti Lipid Peroxidation model
Yelkin TTS is often used as cell model and carries out the research of external lipid peroxidation, and the mensuration of the oxidation-resistance in Yelkin TTS system adopts thiobarbituricacidα-(TBA) method, the C in Yelkin TTS usually
2under the katalysis of iron ion, can peroxidation be there is through concussion, can set up thus and bring out Yelkin TTS C with iron ion in the vldl contained by position and the unsaturated fatty acids in low-density lipoprotein
2the model of oxidation of the vldl on position and low-density lipoprotein, excessively unsaturated fatty acids, in order to the anti-oxidant activity of assess sample.
3.2 In Vitro Anti lipid peroxidation determinations of activity
Add 1.0mL respectively, get the polysaccharide soln of 2,4,6,8,10mg/mL, draw the suspension (V yolk: 25V0.1mol/LpH7.45PBS) of 0.4mL yolk, then add the FeSO of 0.4mL25mmol/L
47H
2o, 4mL is supplemented to PBS, vibration 15min, the TCA adding 1.0mL20% after taking-up leaves standstill, the centrifugal 10min of 3500r/min, draws 4mL supernatant liquor and adds 2mL thiobarbituricacidα-(0.8%), jump a queue and put into boiling water bath and react 15min, at spectrophotometer 532nm place's colorimetric estimation absorbance, with blank tube (6mLPBS) zero, the absorbancy not adding sample hose is A
0, the absorbancy of sample hose is A, and using Vc as positive control, sample anti-oxidant activity represents with to the inhibiting rate of lipovitellinin lipid peroxidation:
Inhibiting rate I%=(A0-A)/A0 × 100%
In formula: A
0the absorbancy of-control tube; The absorbancy of A-sample.
Detect the lipid peroxidation Scavenging activity of polysaccharide sample with TBA method, sample preparation becomes to add different concns gradient (0,2,4,6,8 and 10mg/mL), using BHT and Vc as positive control, sample anti-oxidant activity represents with to the inhibiting rate of lipovitellinin lipid peroxidation.
Obtained by comparative analysis by Fig. 4, class glycosaminoglycan in internal organ polysaccharide embodies good external inhibition of lipid peroxidation, when 10mg/mL to the clearance rate of free radical close to 90%, its median elimination concentration is 2.47mg/mL, show that Urechis uniconctus internal organ polysaccharide exists good lipid peroxide scavenging(action), infer that Urechis uniconctus internal organ class glycosaminoglycan is the activeconstituents in Urechis uniconctus internal organ.
Claims (5)
1. a preparation method for Urechis uniconctus internal organ glycosaminoglycan, is characterized in that comprising the following steps:
(1) pre-treatment: get fresh Urechis uniconctus internal organ, rub homogenate, add 3 ~ 4 times of vol acetone, after magnetic stirrer over night, suction filtration gets precipitation, pulverizes to obtain Urechis uniconctus internal organ dry powder after precipitation being dried;
(2) enzymolysis: by solid-liquid ratio (g/ml) 1:18 ~ 20, distilled water is added in internal organ dry powder, after 4 ~ 5h is extracted in 75 ~ 80 DEG C of water-baths, adjust ph is 6 ~ 7, adds the papoid of internal organ dry powder weight 1 ~ 1.4%, 55 ~ 65 DEG C of water-bath digestion 4 ~ 5h, adjust ph is 8.2 ~ 9.0, adds the trypsinase of internal organ dry powder weight 0.5 ~ 0.6%, 55 ~ 65 DEG C of water-bath digestion 3 ~ 4h, adjust ph is deactivation 5 ~ 15 minutes at 7.0,100 DEG C;
(3) extract: 6000rpm is centrifugal, and 8min gets supernatant liquor, and after being concentrated into 1/15 of original volume, adding ethanol to alcohol content is 80%, hold over night, and 6000rpm is centrifugal, and 8min gets precipitation, obtains internal organ glycosaminoglycan crude product;
(4) purifying: by solid-liquid ratio (mg/ml) 1:100, dialysed after distilled water by internal organ glycosaminoglycan dissolving crude product, lyophilize obtains internal organ glycosaminoglycan.
2. the preparation method of Urechis uniconctus internal organ glycosaminoglycan as claimed in claim 1, is characterized in that: the molecular weight that described dialysis method retains is 3.5 ~ 4.1kDa.
3. a Urechis uniconctus internal organ glycosaminoglycan as claimed in claim 1 application in the food of prevention and adjusting blood lipid, healthcare products and medicine.
4. the food of Urechis uniconctus internal organ glycosaminoglycan as claimed in claim 3 in prevention and adjusting blood lipid, healthcare products and pharmaceutical applications, it is characterized in that: the monose composition of described Urechis uniconctus internal organ glycosaminoglycan comprises glucose, glucosamine, seminose, semi-lactosi, Fucose, wood sugar, rhamnosyl, glucuronic acid and galn, with molar ratio computing, glucose: glucosamine: seminose: semi-lactosi: Fucose: wood sugar: rhamnosyl: glucuronic acid: the ratio of galn is 5.37:1.38:1:1.38:2.4:1.05:0.87:0.52:1.38, weight-average molecular weight is 3.5 ~ 4.1kDa.
5. food, healthcare products and the pharmaceutical applications of the Urechis uniconctus internal organ glycosaminoglycan as described in claim 3 or 4 in prevention and adjusting blood lipid, is characterized in that: the effective concentration of described Urechis uniconctus internal organ glycosaminoglycan scavenging free radicals is 2.47 ~ 10mg/ml.
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| CN113583084A (en) * | 2021-07-28 | 2021-11-02 | 中日友好医院(中日友好临床医学研究所) | Urechis unicinctus leach liquor protein extract and extraction method and application thereof |
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| 焦绪栋等: "单环刺螠废弃内脏中粗多糖的提取工艺优化", 《食品科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107412126A (en) * | 2017-08-06 | 2017-12-01 | 长沙小新新能源科技有限公司 | A kind of moisturiser based on marine organism extract |
| CN112516174A (en) * | 2020-12-12 | 2021-03-19 | 烟台大学 | A product containing internal organs of sea intestine and its preparation method |
| CN113583084A (en) * | 2021-07-28 | 2021-11-02 | 中日友好医院(中日友好临床医学研究所) | Urechis unicinctus leach liquor protein extract and extraction method and application thereof |
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Application publication date: 20151125 |