CN102153666A - Sipunculidae polysaccharide and preparation method thereof and application thereof to anti-fatigue function - Google Patents
Sipunculidae polysaccharide and preparation method thereof and application thereof to anti-fatigue function Download PDFInfo
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- CN102153666A CN102153666A CN 201110042114 CN201110042114A CN102153666A CN 102153666 A CN102153666 A CN 102153666A CN 201110042114 CN201110042114 CN 201110042114 CN 201110042114 A CN201110042114 A CN 201110042114A CN 102153666 A CN102153666 A CN 102153666A
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- Prior art keywords
- polysaccharide
- worm
- siphon
- ocean
- preparation
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Links
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- 239000005017 polysaccharide Substances 0.000 title claims abstract description 109
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 109
- 238000002360 preparation method Methods 0.000 title claims abstract description 41
- 230000002929 anti-fatigue Effects 0.000 title claims abstract description 20
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Abstract
The invention discloses a sipunculidae polysaccharide, a preparation process thereof, and application of the polysaccharide to an anti-fatigue function. The monosaccharide of the polysaccharide consists of two monosaccharides, namely arabinose (7.91 percent) and glucose (79.32 percent), and the content of polysaccharides in the polysaccharide is over 80 percent. The preparation process for the polysaccharide comprises the following steps of: sequentially adding a proper amount of clean sipunculidae raw material into alkali solution, heating and extracting, regulating the pH value, centrifuging, deproteinizing, concentrating, performing alcohol precipitation, and freeze-drying to obtain the sipunculidae polysaccharide. The polysaccharide has a remarkable anti-fatigue function. A method for extracting the sipunculidae polysaccharide is simple and practicable, low in cost, and low in energy consumption, does not pollute environment, remarkably improves the content of the polysaccharide, effectively overcomes the problems of difficulty in enrichment, difficulty in extraction and low sugar content of animal polysaccharides, ensures that the commercialization of the sipunculidae polysaccharide can be realized to a greater degree, and has good promotion and application prospect in the industry.
Description
Technical field:
The invention belongs to medical technical field, relate to a kind of marine organisms polysaccharide and its production and application, be specifically related to a kind of ocean siphon-worm polysaccharide and preparation method thereof and the application in anti-fatigue effect.
Background technology:
Fatigue is a kind of common psycho-physical phenomenon, and tired generation usually causes the reduction of work capacity and working efficiency.Especially in military operation, numerous officers and men's fatigue phenomenon ubiquity, antifatigue research has great importance for the fighting capacity that improves my army.Polysaccharide has multiple efficacies as the important active substance of a class, how effectively polysaccharide to be separated and carries out function test, has become the focus and the difficult point of domestic and international research.
The ocean siphon-worm has rich nutrient substances, has put down in writing its edible and pharmaceutical use in the multiple book on Chinese herbal medicine of China.The ocean siphon-worm is not only the seafood delights treasure of delicious flavour, also be a kind of lower fat, high protein and the high-class healthy food that is rich in zinc, iron, calcium, phosphorus, iodine several mineral materials, except that work is edible, among the people also its Dietotherapy medicament as treatment hyperactivity of deficient fire and enriching yin and nourishing kidney.Generally be used for hot eyes, flush.Summer, dog days were exposed to the sun because of burning sun, health heating, the patient of constipation, but the relief of symptoms of using.In addition, in Fujian, coastlands such as platforms, Guangdong, fine jade, fisherman's daily life of a family has dietary functions such as invigorating the spleen nourishing with the ocean Chinese caterpillar fungus feeding young children that cooks congee.
The nourishing function of ocean siphon-worm, how on the books in Ancient Times in China medicine monograph, be regarded as " extra large medicine " since ancient times, native soil, southern part of the country scenery is known in the famous herbal scholar Lee Xun Guang trip south of the Five Ridges of Tang end during five generations, is familiar with the medicinal animals and plants in the southern part of the country, show " Haiyao Bencao, Oversea Materia Medica " and put down in writing a large amount of external medicines, wherein just put down in writing " extra large silkworm excrement ", it " gives birth between the stone of mountain, the South Sea its silkworm shape; big as thumb; that sand is very white, as beautiful powdery, joint is arranged whenever " cloud." it is salty to distinguish the flavor of, and big temperature is nontoxic for the sea silkworm excrement.Main consumptive disease cold air, Chu's wind is unsuccessful.Clothes make us gloss for a long time, and qi-restoratives is thin, makes light of one's life by commiting suicide, and it is not old to prolong life ".Famous herbal scholar's LI Shi-Zhen, its equally also record the medicinal efficacy of " extra large silkworm " among the Compendium of Material Medica of showing.The traditional Chinese medical science thinks that ocean siphon-worm nature and flavor are sweet, salty, cold, and clearing lung-heat qi-restoratives, nourishing Yin and falling fire effect are arranged, and clinical practice confirms that osteopyrexia and fever, night sweat due to yin deficiency, the deficiency syndrome of the lung are coughed and disease such as breathe heavily, phlegm uncomfortable in chest is many that edible ocean siphon-worm is effective in cure.To illness such as pulmonary tuberculosis cough, neurasthenia, infantile spleen deficiencies, good result of treatment is also arranged as if stewing with ocean siphon-worm and ginger splices pot.With Huaihe River Qi Yuan meat pot ocean siphon-worm soup, not only nutritious, delicious flavour, and have the effect of enriching yin beauty treatment.Efficacious prescriptions among the people have: 1. the sea sand worm is fried in shallow oil: get sea sand worm 30g, decocting boils, 1 dose, be decocted in water for oral dose every day 2 times, have nourishing Yin and falling fire, removing heat from the lung and dissipating phlegm effect, cure mainly pulmonary tuberculosis, the cough spit, hectic fever night sweat; 2. add flavor sea sand worm soup: get sea sand worm 10g, tuber of stemona 15g, Radix Glycyrrhizae 5g; Decocting, 1 dose, be decocted in water for oral dose every day 2 times, have nourishing Yin and moistening lung, cough-relieving apophlegmatic effect, cure mainly the YIN-deficiency type pulmonary tuberculosis.
Modern medicine studies show that the ocean siphon-worm contains the various active material, can regulate the multiple function of body, contains rich in protein, trace element etc.The content of zinc reaches 2.3 * 102 μ g/g, and content of taurine is 70 times (reaching 3%) of common biology, and protein content is that arginine content is up to 6.8% more than 55%; Have significantly effect such as delay senility, anti-oxidant, cardiovascular systems is had significant protective effect.
People such as Dou Baochang find that the hydrolyzed extract of ocean siphon-worm has the effect of vasoconstriction, quickening heart rate, enhancing heartbeat output and coronary flow, and losing blood property ypotension is had remarkable boosting, and the good sedative analgesic effect is also arranged.Also there is the scholar that the composition of ocean siphon-worm is analyzed, finds that they contain 6-7 kind essential amino acid, especially with Lys, Leu, Val content is higher; Especially zinc content is very high in the trace element, can reach 2.3 * 102 μ g/g.We except the taurine in the siphon-worm of ocean, amino acid, trace element, protein etc. are studied, further investigate the polysaccharide in the siphon-worm of ocean in previous experiments especially.
The present invention relates to the preparation method of a kind of ocean siphon-worm polysaccharide, by the prepared polysaccharide of this method, content obviously increases, the reliable easily row of its preparation method, and free from environmental pollution, cost is low, is applicable to scale operation.Through pharmacodynamic experiment research, this polysaccharide has obvious anti-fatigue effect fruit.
Summary of the invention:
Be difficult to enrichment in order effectively to overcome the animal polysaccharide, be difficult to extract and low this difficult problem of sugared content, key technical problem solved by the invention is the characteristic according to the contained polysaccharide of ocean siphon-worm, adopt extraction and separation technology, develop a kind of highly purified, ocean siphon-worm extraction method of polysaccharides of being applicable to industrialized production, and disclose its application in anti-fatigue effect.
The present invention is achieved in that
Ocean of the present invention siphon-worm polysaccharide extracting process, be by alkali carry, step such as deproteinated, alcohol precipitation obtains this active ingredient.Concrete extraction process is: it is an amount of to get the ocean siphon-worm raw material that cleans up, and adds 2-8 times of 2-10% alkaline solution successively, and 30-80 ℃ is extracted 2-8h, and adjust pH is 4.0-10.0, and is centrifugal, 9000rpm, and time 30min discards precipitation, gets supernatant liquor.Adopt the trichoroacetic acid(TCA) deproteinated, to the pH value be 3.0-8.0, pour in the separating funnel after about static 50min, upper water solution is separated in static back, be concentrated to about 1/4 of original volume, ethanol with 95% and parting liquid thorough mixing stir, and make its concentration reach 20-80%, 4 ℃ of standing over night, centrifugal, lyophilize gets ocean siphon-worm polysaccharide.
Ocean of the present invention siphon-worm polysaccharide extracts optimum process: it is an amount of to get the ocean siphon-worm raw material that cleans up, and adds 6 times of 6%NaoH, and 50 ℃ are extracted 4h, and adjust pH is 7.0, and is centrifugal, 9000rpm, and time 30min discards precipitation, gets supernatant liquor.Adopt the trichoroacetic acid(TCA) deproteinated, to the pH value be 4.0, pour in the separating funnel after about static 50min, upper water solution is separated in static back, be concentrated to original volume about 1/4 till take out, ethanol with 95% and parting liquid thorough mixing stir, and its concentration is reached about 75%, 4 ℃ of static spending the night, centrifugal, lyophilize gets ocean siphon-worm polysaccharide.
The ocean siphon-worm polysaccharide that obtains with method of the present invention is pale powder, and is water-soluble, is insoluble to organic solvents such as ethanol, acetone, chloroform, and polysaccharide content is more than 80%.The monose of this ocean siphon-worm polysaccharide is formed and is made up of pectinose (7.91%) and (79.32%) 2 kind of monose of glucose; Sugared content is more than 80% in this ocean siphon-worm polysaccharide; This ocean siphon-worm polysaccharide chance Molish reagent, Fehling (Fehling) reagent, anthrone-sulphate reagent, phenolsulfuric acid reagent all show specific color reaction; The absorption of infrared spectra indicating characteristic.
Ocean siphon-worm polysaccharide process antifatigue experimental study with method of the present invention obtains has obvious anti-fatigue effect.
Advantage of the present invention:
1, this extracting method is mainly according to the characteristics of animal polysaccharide, by to pH value, Heating temperature, add the accurate selection of factors such as alkali number, alkali concn, determining alcohol and to adding the selection of reagent precedence, thereby reach to effective enrichment of polysaccharide with effectively separate.In leaching process not with an organic solvent with other specific installation, extracting method is simple, cost is low, less energy consumption, free from environmental pollution, effectively overcome the animal polysaccharide and be difficult to enrichment, be difficult to separate this difficult problem, made ocean siphon-worm polysaccharide can realize commercialization largely, good popularization and application prospect arranged industrial.
2, by extraction process relatively, this extracting method gained polysaccharide has obviously improved polysaccharide content, makes polysaccharide content surpass 80%, effectively overcomes to fall the low characteristics of animal polysaccharide content.
3, by the research of antagonism fatigue experiment, this polysaccharide sugar content height, and have the obvious anti-fatigue effect.
Description of drawings:
Fig. 1: mix monose standard substance color atlas
Fig. 2: sample chromatogram figure
Fig. 3: the Molish reagent react is differentiated figure
Fig. 4: Fehling (Fehling) reagent react is differentiated figure
Fig. 5: figure is differentiated in anthrone-sulphate reagent reaction
Fig. 6: the phenolsulfuric acid reagent react is differentiated figure
Fig. 7: infrared spectra is differentiated figure
Embodiment:
The mensuration of monosaccharide component in the siphon-worm polysaccharide of ocean:
1, sample and reagent
Ocean siphon-worm polysaccharide sample is self-control.The monose standard substance are rhamnosyl, pectinose, wood sugar, seminose, glucose, semi-lactosi, inositol, fructose, are the analytical pure biochemical reagents, all available from the poly-Science and Technology Ltd. in source, Shanghai.Trifluoroacetic acid, aceticanhydride, methyl alcohol, anhydrous sodium sulphate, chloroform are homemade analytical reagent.
2, instrument and condition
Adopt vapor-phase chromatography to measure.Detecting instrument: HP 6890 (Plus) type gas chromatograph.Detector: FID, chromatographic column: HP-INNOWAX, (30m*0.25mm*0.25 μ m).Temperature of vaporization chamber: 240 ℃, detector temperature: 260 ℃.With nitrogen as carrier gas, purity 99.999%.140 ℃ of chromatographic column initial temperature then, rise to 240 ℃ with the temperature rise rate of 10 ℃/min, keep 10min.Splitting ratio: 20/1, sample size: 1 μ l, area normalization standard measure.
3, sample preparation takes by weighing ocean siphon-worm polysaccharide 15mg,, takes out and is concentrated into driedly in 120 ℃ of hydrolysis 6h of baking oven with the 3mol/L trifluoroacetic acid, adds methyl alcohol and dries up, and is stand-by.
4, the preparation of sugared oxime acetic ester derivative
Get the dry product 5mg of ocean siphon-worm polysaccharide hydrolysis liquid, add oxammonium hydrochloride 6mg, pyridine 0.5mL places 10min in 90 ℃ of baking ovens, taking-up is put and is chilled to room temperature, adds aceticanhydride 0.3mL, places 20min again in 90 ℃ of baking ovens, be decompressed to after the taking-up dried, with chloroform dissolving, standby.
5, the preparation of monosaccharide derivatives
Precision takes by weighing standard substance rhamnosyl, pectinose, wood sugar, seminose, glucose, semi-lactosi, inositol, each 5mg of fructose respectively, add the 6mg oxammonium hydrochloride respectively, pyridine 0.5mL, place 10min in 90 ℃ of baking ovens, taking-up is put and is chilled to room temperature, adds aceticanhydride 0.3mL, in 90 ℃ of baking ovens, place 20min again, be decompressed to after the taking-up dried, with chloroform dissolving, standby.Get each 0.5ml of monosaccharide derivatives that has prepared respectively, after the mixing, make standard monosaccharide derivatives mixed solution.
6, qualitative and quantitative
Earlier with each standard monosaccharide derivatives and standard monosaccharide derivatives mixed solution sample introduction, the retention time (RT) of record chromatographic peak is also confirmed, with the sample feeding of siphon-worm polysaccharide hydrolysis thing behind derivatize for preparing, obtain the color atlas (seeing Fig. 1 and Fig. 2 respectively) of sample then.Compare with the RT of standard monosaccharide derivatives chromatographic peak and the RT of analyte derivative thing chromatographic peak, to determine which contains in the siphon-worm polysaccharide plants monose.Remake quantitative Analysis, quantivative approach is a normalization method, promptly uses percentage composition method (Area%) expression of chromatographic peak peak area, the results are shown in Table 1.
Table 1 monosaccharide component and relative percentage composition
7, conclusion
On the color atlas behind standard monose mixed solution and the sample feeding, can obtain the RT of each monose, scope is between 9～20min, and each peak resolution is good, noiseless phenomenon.According to the normalization method data processing, calculate peak area percentage (Area%), the result shows in the siphon-worm polysaccharide and mainly is made up of pectinose (7.91%) and (79.32%) 2 kind of monose of glucose, all the other compositions that have 12% approximately are the very little assorted peak of some signals, fail to carry out corresponding with selected monose standard substance, also may be other impurity, remain further to be furtherd investigate.
Embodiment 2
Ocean siphon-worm determination of polysaccharide:
1, solution preparation
1.1 the preparation precision of reference substance solution takes by weighing 105 ℃ of glucose reference substance 50mg that are dried to constant weight, puts in the 500ml volumetric flask, is dissolved in water and is diluted to scale, shakes up, promptly.
1.2 prepared ocean siphon-worm polysaccharide 20mg is got in the need testing solution preparation, puts in the 100ml volumetric flask, adds the water ultrasonic dissolution and is diluted to scale, shakes up, promptly.
2, typical curve preparation
2.1 dextrose anhydrous reference substance solution and need testing solution 0.3mL are measured in the selection of mensuration wavelength precision respectively, put in the 10mL scale test tube, thin up is to 2.0mL, add 0.2% anthrone-sulphuric acid soln colour developing, with the corresponding reagent is blank, the scan light spectrogram, glucose reference substance and trial-product all have maximum absorption at the 625nm place.
2.2 the accurate respectively reference substance solution 0.2ml that draws of the preparation of typical curve, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml, put in the 10ml tool plug test tube, add water to 2.0ml, (precision takes by weighing the 0.1g anthrone to accurate adding sulfuric acid anthrone solution, add 80% sulphuric acid soln 100ml and make dissolving, shake up) 6ml, shake up, put in the water-bath and heated 15 minutes, taking out, put into ice bath cooling 15 minutes, is blank with the reagent corresponding, according to ultraviolet visible spectrophotometry, measuring absorbancy at 625nm wavelength place, is ordinate zou with the absorbancy, and concentration is X-coordinate, getting the typical curve regression equation is: y=50.576x+0.0281, R
2=0.9967.Test-results shows that glucose quality is at 0.02～1.2mg, and is good with the linear relationship after 0.2% anthrone-sulphuric acid soln colour developing.
3, methodological study
3.1 the precision test is got with 3 parts of a need testing solution 0.3ml, by 2.2 below method colour developings, measures optical density in the 625nm place, the results are shown in Table 2, coefficient of variation (RSD) is 0.6%.
The test of table 2 precision
3.2 the replica test precision takes by weighing 3 parts in sample, each about 20mg presses the preparation of need testing solution method, colour developing, and the optical density in 625nm place each sample of mensuration the results are shown in Table 3, and coefficient of variation (RSD) is 0.6%.Show this method favorable reproducibility, reliable results.
Table 3 replica test
3.3 stability experiment
It is an amount of to get need testing solution, with 0.2% anthrone-sulphuric acid soln colour developing, does blank with reagent corresponding, put in the cuvette, on spectrophotometer, measure absorbancy, every the 10min reading in 625nm, investigate 60min altogether, result's (seeing Table 4) shows that need testing solution colour developing back is stable at 60min.
Table 4 stability test
3.4 recovery test is got totally 3 parts of the sample solution 0.3ml of known content, to wherein adding 0.2ml glucose reference substance solution, prepare need testing solution in accordance with the law and measure polysaccharide content, calculate average recovery, the average average recovery of result's (table 5) is 100.47%, and RSD is 1.03% (n=3).
Table 5 rate of recovery experiment (n=6)
4, the assay precision of sample takes by weighing each 3 parts of prepared ocean siphon-worm polysaccharide, put respectively in the 100ml volumetric flask, add water and make dissolving, shake up, precision is measured 0.4ml solution, put in the 10ml tool plug test tube, method under the sighting target directrix curve preparation from " adding water to 2.0ml ", is measured absorbancy in accordance with the law, and calculate the content of polysaccharide the need testing solution from typical curve, the results are shown in Table 6.
Table 6 assay result (n=3)
Ocean siphon-worm polysaccharide identification experiment:
1, chemistry is differentiated
1.1Molish reagent react
1.1.1 experimental principle: polysaccharide dewaters under the effect of the vitriol oil and forms furfural and derivative and that formation red-purple mixture of naphthyl alcohol effect, forms purple ring between the liquid level of the liquid glucose and the vitriol oil.
1.1.2Molish reagent preparation: get 5 gram naphthyl alcohols with 95% dissolve with ethanol to 1000mL, face and use preceding preparation, the brown bottle preservation.
1.1.3 experimental technique: get test tube, add siphon-worm polysaccharide (1% concentration) 1mL, add two Molish reagent then, shake up.The inclination test tube carefully adds about 1mL vitriol oil along tube wall, is sure not to shake, the careful colour-change that examines two-layer liquid level intersection after vertically.Water replaces sugar soln, comes again observations.
1.1.4 experimental result: see Fig. 3
1.2 Fehling (Fehling) reagent react
1.2.1 experimental principle: the fehling reagent solution of new preparation, under heating condition,, be reduced into bolarious precipitation with the aldehyde radical reaction, can be used for identifying the existence of solubility reducing sugar.When identifying the solubility reducing sugar with fehling reagent, the colour-change process of solution is: mazarine → yellow (CuOH precipitation) → brick-red (precipitation).
1.2.2 fehling reagent preparation: Fehling A: the copper sulfate that 3.5g is contained five crystal water both be dissolved in the water of 100mL nattier blue Fehling A reagent.Fehling B: the soluble tartrate of 17g five crystal water is dissolved in the 20mL hot water, adds the aqueous solution 20mL contain 5g sodium hydroxide then, be diluted to 100mL both colourless limpid Fehling B reagent.Fehling A and balanced mix during use.
1.2.3 experimental technique: add sugar to be measured (1% concentration) 1mL in vitro.Add 1mL fehling reagent (AB liquid equal-volume adds after mixing again) then.Test tube is put into the colour-change that the observation test tube occurs behind the about 2min of large beaker heating that fills 50-60 ℃ of warm water.
1.2.4 experimental result: see Fig. 4.
1.3.1 anthrone-sulphate reagent reaction
1.3.2 experimental principle: polysaccharide is met vitriol oil dehydration and is generated alditol or other derivative, can produce colour substance with the anthrone reagent condensation, and reaction back solution is blue.
1.3.2 the preparation of anthrone test solution: anthrone is dissolved in the vitriol oil, and concentration is 2mg/mL, the same day, preparation was used.
1.3.3 experimental technique:
The polysaccharide soln of preparation 0.01mg/mL is drawn 1mL, adds anthrone 4mL, mixing, and boiling water bath boils 10min, is cooled to room temperature.Water replaces sugar soln, repeats aforesaid operations, the comparative observation result.
1.3.4 experimental result: see Fig. 5
1.4 phenolsulfuric acid reagent react
1.4.1 experimental principle: polysaccharide is hydrolyzed into monose earlier under the vitriolic effect, and dehydration generates the alditol derivative rapidly, generates orange-yellow compound with phenol then.
1.4.2 phenol test solution preparation: get a certain amount of phenol, add water, prepare 5% phenol solution, face and use preceding preparation.
Working method:
1.4.3 experimental technique: add sugar soln 1mL to be measured in the test tube, add 5% phenol 1.0ml and vitriol oil 5.0ml then, shake up cooling, room temperature is placed and was observed the colour-change that occurs in the test tube in 20 minutes later on.
1.4.4 experimental result: see Fig. 6
Ocean siphon-worm polysaccharide infrared spectra identification experiment:
Get ocean siphon-worm polysaccharide, carry out infrared measurement, show that from infrared spectra Fig. 7 the siphon-worm polysaccharide has typical polysaccharide charateristic avsorption band (3 403,2 930,1 728,1 647,1 416cm
-1), wherein the absorption peak that occurs of 3 403cm mainly is that glycoside hydroxyl (associations) stretching vibration by polysaccharide causes, because after hydroxyl formed hydrogen bond association ,-O-H+ key elongated, moment of dipole increases, so 3 403cm
-1Strong and the wide peak of performance, place; 2 930cm
-1The place absorption peak corresponding with the stretching vibration of hydrocarbon key, intensity a little less than; 1 728cm
-1The absorption that the absorption peak at place causes for the C=O stretching vibration, 1 647cm
-1Near absorption peak is kharophen (C=O stretching vibration NH2COCH3), 1 416cm
-1The absorption peak at place is c-H flexural vibration peaks, and the absorption peak at 1 238cm place is that the stretching vibration of ketose class C-C key causes 1 081cm
-1Locating out the peak is the stretching vibration of C-O key in the molecule then, is the feature of carbon-oxygen bond stretching vibration.
Ocean siphon-worm polysaccharide extracting process research:
The ocean siphon-worm is a kind of marine animal, and for marine animal, polysaccharide content wherein is lower, how to take a kind of effective extracting method that animal polysaccharide is wherein carried out enrichment, has become a great problem that the animal polysaccharide extracts.Therefore present technique is mainly by examining or check various extracting method, and carries out deproteinated research, and by technology relatively, final screening draws an optimum process.Specific as follows:
(1), alkaline hydrolysis method and the comparative studies of enzymolysis process technology
1, equipment and reagent
Tissue is smashed instrument (going up Industrial Co., Ltd. of Nereid section), electric-heated thermostatic water bath (the new ripple instrument in Yuyao company), GL-21M whizzer (ShangHai City centrifugal Machine Institute) to pieces at a high speed; Sodium hydroxide (AR level, Shanghai chemical reagents corporation of Chinese Medicine group, lot number: F20090921); hydrochloric acid (AR level, Shanghai chemical reagents corporation of Chinese Medicine group, lot number: T050704); (Dehua worker company limited is won in Shanghai to 95% ethanol; lot number: F20090921), trichoroacetic acid(TCA) (import packing, lot number: LJ0910B508J); pancreatin (traditional Chinese medicines group reagent); chloroform (traditional Chinese medicines group reagent), propyl carbinol (traditional Chinese medicines group reagent), ocean siphon-worm (available from the North Sea).
2, extracting method
2.1 alkaline hydrolysis method
Get the ocean siphon-worm raw material 100g that cleans up, pulverizing, homogenate, it is long-pending to add water to hexaploid, adds 6%NaoH, and 50 ℃ are extracted 4h, and adjust pH is 8.0, and 9000rpm * 30min is centrifugal, discards precipitation, gets supernatant liquor.Ethanol with 95% and parting liquid thorough mixing stir, and its concentration is reached about 75%, and 4 ℃ of static spending the night are centrifugal, and lyophilize gets Crude polysaccharides.
2.2 enzymolysis process
Get the siphon-worm raw material 100g that cleans up, pulverizing, homogenate are pressed solid-liquid ratio and are added distilled water at 1: 6, transfer pH value to 8.0, and adding pancreatin to final concentration is 0.3%, is heated to 60 ℃, and effect 3h is heated to 100 ℃ and acts on 30min, enzymolysis reaction.Above-mentioned sample is centrifugal, 9000rpm, time 30min discards precipitation, gets supernatant liquor.Ethanol with 95% and parting liquid thorough mixing stir, and its concentration is reached about 75%, and 4 ℃ of static spending the night are centrifugal, and lyophilize gets Crude polysaccharides.
3, experimental result
Calculate result's following (seeing Table 7) by polysaccharide that above-mentioned technology is extracted being carried out assay and yield.
Table 7 Crude polysaccharides assay and yield
4, experiment conclusion
By above-mentioned two technologies are compared, the result shows that the alkaline hydrolysis method is extracted and is better than enzymolysis process.
(2) deproteinated effect examination
Because the alkaline hydrolysis method is extracted and to be better than enzymolysis process, so this experiment gos deep into the alkaline hydrolysis method, and method for removing protein is compared.
1, equipment and reagent
Tissue is smashed instrument (going up Industrial Co., Ltd. of Nereid section), electric-heated thermostatic water bath (the new ripple instrument in Yuyao company), GL-21M whizzer (ShangHai City centrifugal Machine Institute) to pieces at a high speed; Sodium hydroxide (AR level, Shanghai chemical reagents corporation of Chinese Medicine group, lot number: F20090921); hydrochloric acid (AR level, Shanghai chemical reagents corporation of Chinese Medicine group, lot number: T050704); (Dehua worker company limited is won in Shanghai to 95% ethanol; lot number: F20090921), trichoroacetic acid(TCA) (import packing, lot number: LJ0910B508J); pancreatin (traditional Chinese medicines group reagent); chloroform (traditional Chinese medicines group reagent), propyl carbinol (traditional Chinese medicines group reagent), Sipunculus nudus (available from the North Sea).
2, extracting method
2.1 technology one: alkaline hydrolysis method
Get the siphon-worm raw material 100g that cleans up, pulverizing, homogenate, it is long-pending to add water to hexaploid, adds 6%NaoH, and 50 ℃ are extracted 4h, and adjust pH is 8.0, and 9000rpm * 30min is centrifugal, discards precipitation, gets supernatant liquor.Ethanol with 95% and parting liquid thorough mixing stir, and its concentration is reached about 75%, and 4 ℃ of static spending the night are centrifugal, and lyophilize gets Crude polysaccharides.
2.2 technology two: alkaline hydrolysis enzymolysis coupling method
Get the siphon-worm raw material 100g that cleans up, pulverizing, homogenate, it is long-pending to add water to hexaploid, adds 6%NaoH, 50 ℃ are extracted 4h, adjust pH to 8.0, and adding pancreatin to final concentration is 0.3%, is heated to 60 ℃, effect 3h is heated to 100 ℃ of effect 30min, enzymolysis reaction.9000rpm * 30min is centrifugal, discards precipitation, gets supernatant liquor.Ethanol with 95% and parting liquid thorough mixing stir, and its concentration is reached about 75%, 4 ℃ of static spending the night, and 9000rpm * 300min is centrifugal, and lyophilize gets Crude polysaccharides.
2.3 technology three: alkaline hydrolysis and sevage deproteinated method
Get the siphon-worm raw material 100g that cleans up, pulverizing, homogenate, it is long-pending to add water to hexaploid, adds 6%NaoH, and 50 ℃ are extracted 4h, and adjust pH is 8.0, and 9000rpm * 30min is centrifugal, discards precipitation, gets supernatant liquor.Add sevage reagent according to 1: 1 ratio, stir, pour in the separating funnel after about static 50min, upper water solution is separated in static back, ethanol with 95% and parting liquid thorough mixing stir, and its concentration are reached about 75%, 4 ℃ of static spending the night, centrifugal, lyophilize gets Crude polysaccharides.
2.4 technology four: alkaline hydrolysis and trichoroacetic acid(TCA) deproteinated method
Get the siphon-worm raw material 100g that cleans up, pulverizing, homogenate, it is long-pending to add water to hexaploid, adds 6%NaoH, and 50 ℃ are extracted 4h, centrifugal, 9000rpm, time 30min discards precipitation, gets supernatant liquor.Adjust pH is 7.0, adds the trichoroacetic acid(TCA) deproteinated, and transferring to the pH value is 4.0, and 9000rpm * 30min is centrifugal, supernatant shifts, and ethanol with 95% and parting liquid thorough mixing stir, and its concentration is reached about 75%, 4 ℃ of standing over night, 9000rpm * 300min is centrifugal, and lyophilize gets Crude polysaccharides.
2.5 technology five: alkaline hydrolysis, enzymolysis and sevage deproteinated method
Get the siphon-worm raw material 100g that cleans up, pulverizing, homogenate add 6 times of 6%NaOH, and 50 ℃ are extracted 4h, adjust pH to 8.0, and adding pancreatin to final concentration is 0.3%, is heated to 60 ℃, effect 3h is heated to 100 ℃ of effect 30min, enzymolysis reaction.9000rpm * 30min is centrifugal, discards precipitation, gets supernatant liquor.Add sevage reagent according to 1: 1 ratio, stir, pour in the separating funnel after about static 50min, upper water solution is separated in static back, ethanol with 95% and parting liquid thorough mixing stir, and its concentration are reached about 75%, 4 ℃ of static spending the night, centrifugal, lyophilize gets Crude polysaccharides.
2.6 technology six: alkaline hydrolysis, enzymolysis and trichoroacetic acid(TCA) deproteinated method
Get the siphon-worm raw material 100g that cleans up, pulverizing, homogenate add water to 6 times of volumes, add 6%NaoH, 50 ℃ are extracted 4h, and adjust pH transfers to 8.0, and adding pancreatin to final concentration is 0.3%, is heated to 60 ℃, effect 3h is heated to 100 ℃ of effect 30min, enzymolysis reaction.9000rpm * 30min is centrifugal, discards precipitation, gets supernatant liquor.Adjust pH to 7.0 adds the trichoroacetic acid(TCA) deproteinated, to the pH value be 4.0,9000rpm * 30min is centrifugal, supernatant shifts, and ethanol with 95% and parting liquid thorough mixing stir, and its concentration is reached about 75%, 4 ℃ of static spending the night, 9000rpm * 300min is centrifugal, and lyophilize gets Crude polysaccharides.
3, experimental result
Calculate result's following (seeing Table 8) by polysaccharide that above-mentioned technology is extracted being carried out assay and yield.
Table 8 Crude polysaccharides assay and yield
4, experiment conclusion
By above-mentioned technology is compared, the result shows that technology four prepared determination of polysaccharide results are best, and yield is higher.
Ocean siphon-worm polysaccharide antifatigue experimental study:
Fatigue refers generally to the organism physiology process can not continue its function on specified level, maybe can not keep the decline that predetermined exercise intensity causes the body ﹠ mind state, thereby causes the decline of work capacity and working efficiency.The consumption of energy substance, the accumulation of metabolic substd are to produce tired major reason.Especially in military operation, numerous officers and men's fatigue phenomenon ubiquity, antifatigue research has great importance for the fighting capacity that improves my army.The raising of exercise tolerance is that anti-fatigue ability is strengthened the strongest macro manifestations, and the length of swimming time can be reacted the degree of animal movement fatigue.The swimming with a load attached to the body time length is adopted in this experiment, the antifatigue effect of research ocean siphon-worm polysaccharide.
(1) antifatigue experiment one (swimming with a load attached to the body experiment)
1, experiment equipment
Electronic balance (model TB-214, Beijing Sai Duolisi instrument system company limited); Table balance (Shanghai medicine equipment eight factories); American ginseng capsule (Xiamen City Fu Ningchun protective foods company limited, authentication code: the strong word G20030007 of state's food); Ocean siphon-worm polysaccharide (laboratory self-control).
2, laboratory animal and grouping
60 of BalB/c male mices, age in 7-8 week, 20 ± 1g, (available from west, Shanghai pul-Bi Kai laboratory animal company limited, production licence: SCXK (Shanghai) 2007-0003 occupancy permit: SYXK (Shanghai) 2007-0003).Be divided into 5 groups at random, 12 every group, be respectively totally 5 groups of blank groups, positive controls (0.5g/kg), the basic, normal, high dosage group of siphon-worm polysaccharide (being 0.2g/kg, 0.4g/kg, 0.6g/kg), and carry out mark with picric acid.
3, experimental technique
Give all mouse stomach administrations, successive administration 10d, mouse is weighed after the last administration.Behind the 30min (irritate stomach before not on an empty stomach), make the bear a heavy burden sheet lead of 10% body weight of afterbody, 4 stainless steel vats, diameter 80cm makes depth of water 45cm, constantly adds hot water and makes water temperature remain on 25 ± 1 ℃, opens air-conditioning simultaneously, and keeping room temperature is 25 ℃.The time of record mouse swimming, promptly mouse begins all not have to head time till 8s can not emerge in the entry from entry.
4, medicine preparation
Low dose group preparation: take by weighing the siphon-worm polysaccharide powder that 0.72g extracts in earlier stage and be dissolved in the 36ml distilled water, abundant mixing, the equal 3ml of every test tube, totally 12 pipes are standby.
In dosage assembly system: take by weighing the siphon-worm polysaccharide powder that 1.44g extracts in earlier stage and be dissolved in the 36ml distilled water, abundant mixing, the equal 3ml of every test tube, totally 12 pipes are standby.
High dose group preparation: take by weighing the siphon-worm polysaccharide powder that 2.16g extracts in earlier stage and be dissolved in the 36ml distilled water, abundant mixing, the equal 3ml of every test tube, totally 12 pipes are standby.
The preparation of American ginseng capsule solution: with the American ginseng capsule strip off, the inner powder 0.9g of weighing is dissolved in it in 36ml distilled water, and the mixing that fully vibrates shakes up the back branch and installs in 12 test tubes, and every pipe 3ml is standby.
5, experimental result
Record mouse swimming time, promptly mouse begins all not have to head time till 8s can not emerge in the entry from entry, through as follows to its statistics, sees Table 9
Table 9 mouse swimming test statistics (unit: second)
Compare #P＜0.05 with the blank group, ##P＜0.01
Compare with positive controls: * P＜0.05
6, experiment conclusion
6.1 positive controls, the basic, normal, high dosage group of siphon-worm polysaccharide all have significant differences (p＜0.01) with blank group.
6.2 siphon-worm polysaccharide low dose group is compared with positive controls, has significant difference (p＜0.05).
6.3 siphon-worm polysaccharide low dose group is better than the middle and high dosage group of siphon-worm polysaccharide to the antifatigue effect of mouse.
7, precaution
7.1 when the mouse swimming with a load attached to the body was tested, water temperature should be strict controlled in 25 ± 1 ℃, water temperature can produce considerable influence to swimming time.
Calculate by 10% body weight 7.2 bear a heavy burden, and do not adopt most of documents described 5%, the heavier swimming time of can more obvious difference respectively organizing mouse bears a heavy burden.
7.3 should select thinner galvanized wire when selecting galvanized wire, elder generation hammers galvanized wire into shape with hammer flat, and then ties up equably at mouse tail, so both has been difficult for landing, can not damage mouse tail again.
What 7.4 this positive drug adopted is the American ginseng capsule preparation, because insoluble effective constituent is arranged, so when reagent preparation, should fully grind porphyrize.When irritating stomach, fully mixing stops up in order not cause syringe needle, can only extract 0.2ml, on a small quantity repeatedly at every turn.
(2) antifatigue experiment two (biochemical indicator mensuration)
1, experiment equipment
American ginseng capsule (Xiamen City Fu Ningchun protective foods company limited, authentication code: the strong word G20030007 of state's food); Siphon-worm polysaccharide (laboratory self-control); Electronic balance (model TB-214, Beijing Sai Duolisi instrument system company limited); Table balance (Shanghai medicine equipment eight factories).
2, laboratory animal and grouping
65 of BalB/c male mices, age in 7-8 week, 20 ± lg, (available from west, Shanghai pul-Bi Kai laboratory animal company limited, production licence: SCXK (Shanghai) 2007-0003 occupancy permit: SYXK (Shanghai) 2007-0003).Be divided into 5 groups at random, 13 every group, be respectively totally 5 groups of blank groups (distilled water group), positive controls (Radix Panacis Quinquefolii 0.5g/kg), the basic, normal, high dosage group of siphon-worm polysaccharide (being 0.2g/kg, 0.4g/kg, 0.6g/kg), and carry out mark with picric acid.
3, medicine preparation
Low dose group preparation: take by weighing the siphon-worm polysaccharide powder that 0.72g extracts in earlier stage and be dissolved in the 36ml distilled water, abundant mixing, the equal 3ml of every test tube, totally 12 pipes are standby.
In dosage assembly system: take by weighing the siphon-worm polysaccharide powder that 1.44g extracts in earlier stage and be dissolved in the 36ml distilled water, abundant mixing, the equal 3ml of every test tube, totally 12 pipes are standby.
High dose group preparation: take by weighing the siphon-worm polysaccharide powder that 2.88g extracts in earlier stage and be dissolved in the 36ml distilled water, abundant mixing, the equal 3ml of every test tube, totally 12 pipes are standby.
The preparation of Radix Panacis Quinquefolii solution: with the American ginseng capsule strip off, the inner powder 0.9g of weighing is dissolved in it in 36ml distilled water, and the mixing that fully vibrates shakes up the back branch and installs in 12 test tubes, and every pipe 3ml is standby.
4, experimental technique
Give all mouse stomach administrations, successive administration 12d, mouse is 30min after the last administration, makes not swimming with a load attached to the body.At 4 stainless steel vats, diameter 80cm makes depth of water 45cm, constantly adds hot water and makes water temperature remain on 25 ± 1 ℃, opens air-conditioning simultaneously, and keeping room temperature is 25 ℃.Take out mouse in swimming behind the 38min, wipe with thieving paper immediately, and dry up with hair dryer, put in people's bucket rest 20min posterior orbit and get blood, the gained blood sample carries out the mensuration of serum urea nitrogen and serum lactic dehydrogenase.After getting blood and finishing, the dislocation of animal cervical vertebra is put to death, get hindlimb muscle and liver immediately, behind normal saline flushing, blot, take by weighing muscle and liver, put into the physiological saline of precooling respectively and remove its blood, and blot, weigh with thieving paper with filter paper, stand-by.
5, experimental result
5.1 it is centrifugal that the serum urea nitrogen analysis carries out above-mentioned blood sample, separation of serum.Precision is got serum 20ul, and strictness is operated according to serum urea nitrogen test kit specification sheets, measures in the 30min under 620nm and respectively manages the mice serum absorbancy, is scaled the serum urea nitrogen content, the results are shown in Table 10.
The assay statistics of table 10 mice serum blood urea nitrogen (unit: second)
Compare #P＜0.05 with the blank group, ##P＜0.01
Compare with positive controls: * P＜0.05, * * P＜0.01
5.2 it is centrifugal that the assay of serum lactic dehydrogenase carries out above-mentioned blood sample, separation of serum.Precision is got serum 20ul, and strictness is operated according to lactic dehydrogenase enzyme reagent kit specification sheets, measures in the 30min under 490nm and respectively manages the mice serum absorbancy, is scaled the content of serum lactic dehydrogenase, the results are shown in Table 11.
The assay statistics of table 11 mouse serum lactic dehydrogenase (unit: second)
Compare #P＜0.05 with the blank group, ##P＜0.01
Compare with positive controls: * P＜0.05, * * P＜0.01
6, experiment conclusion
6.1 from the content statistics of table 2 serum urea nitrogen as can be known, with the blank group relatively, low dosage and middle dosage group obviously are better than blank group, have highly significant meaning (P＜0.01); Compare with positive group, low dosage and middle dosage group obviously are better than positive group, also have highly significant meaning (P＜0.01).From table 3 serum lactic dehydrogenase content statistics as can be known, low dosage obviously is better than blank group, has highly significant meaning (P＜0.01); Compare with positive group, low dosage is better than positive group, has significance meaning (P＜0.05).
Claims (9)
1. ocean siphon-worm polysaccharide with anti-fatigue effect is characterized in that this polysaccharide is is raw material with the ocean siphon-worm, cleans up, pulverizing, homogenate, adds alkaline solution according to this, and heating is extracted, and adjust pH is centrifugal then, discards precipitation, supernatant liquor.Through deproteinated, adjust pH leaves standstill, and separates upper water solution, concentrate, and alcohol precipitation, standing over night, centrifugal, lyophilize, promptly.
2. ocean as claimed in claim 1 siphon-worm polysaccharide is characterized in that its monose is formed and is made up of pectinose (7.91%) and (79.32%) 2 kind of monose of glucose.
3. ocean as claimed in claim 1 siphon-worm polysaccharide wherein polysaccharide content is more than 80%.
4. the preparation method of ocean as claimed in claim 1 siphon-worm polysaccharide is characterized in that this polysaccharide obtains by following method: it is an amount of to get the ocean siphon-worm raw material that cleans up, and adds alkaline solution successively, and heating is extracted, adjust pH, centrifugal, 9000rpm, time 30min discards precipitation, gets supernatant liquor.Adopt the trichoroacetic acid(TCA) deproteinated, pour in the separating funnel after leaving standstill about 50min, leave standstill the back and separate upper water solution, be concentrated to about 1/4 of original volume, ethanol with 95% precipitates, and thorough mixing stirs, 4 ℃ of static spending the night, centrifugal, lyophilize gets the siphon-worm polysaccharide.
5. preparation method as claimed in claim 4 is characterized in that: the alkaline solution of adding is a kind of in sodium hydroxide, potassium hydroxide, calcium hydroxide, the sodium bicarbonate, and the alkaline concentration that is added is 2-10%, and the 2-8 that adds volume and be primary liquid doubly.
6. preparation method as claimed in claim 4 is characterized in that: it is 30-80 ℃ that temperature is extracted in heating, extracts 2-8h, and adjust pH is 4.0-10.0.
7. preparation method as claimed in claim 4 is characterized in that: adopt the trichoroacetic acid(TCA) deproteinated, to the pH value be 3.0-8.0.
8. preparation method as claimed in claim 4 is characterized in that: the ultimate density of alcohol precipitation is 20-80%.
9. polysaccharide as claimed in claim 1 is characterized in that having obvious anti-fatigue effect.
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| CN103613678A (en) * | 2013-11-19 | 2014-03-05 | 广西壮族自治区海洋研究所 | Sipunculus nudus polysaccharide sulfation modification method |
| CN105918461A (en) * | 2016-04-08 | 2016-09-07 | 宁波大学 | Low-calorific-value yogurt containing phascolosoma esculenta polysaccharide and preparation method thereof |
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| CN1430975A (en) * | 2003-01-23 | 2003-07-23 | 上海澳博海洋生物技术开发有限公司 | Extractive of ocean star worm, its preparing method and application |
| CN1748714A (en) * | 2004-09-14 | 2006-03-22 | 李妍妍 | Method for preparing biological active substance of siphon-worm and its products |
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| CN1430975A (en) * | 2003-01-23 | 2003-07-23 | 上海澳博海洋生物技术开发有限公司 | Extractive of ocean star worm, its preparing method and application |
| CN1748714A (en) * | 2004-09-14 | 2006-03-22 | 李妍妍 | Method for preparing biological active substance of siphon-worm and its products |
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| 《Carbohydrate Polymers》 20080110 Liang RenJie Orthogonal test design for optimization of the extraction of polysaccharides from Phascolosoma esulenta and evaluation of its immunity activity 第558-563页 1-3,9 第73卷, 第4期 * |
| 《中华航海医学与高气压医学杂志》 20030630 沈先荣等 海洋星虫提取物的抗疲劳作用研究 第112-114页 1-9 第10卷, 第2期 * |
| 《中国热带医学》 20071231 夏乾峰等 方格星虫多糖抗菌活性的初步研究 第2192-2193页 1-9 第7卷, 第12期 * |
| 《食品与生物技术学报》 20060731 张桂和等 方格星虫多糖分离纯化及性质鉴定 第63-66页 1-9 第25卷, 第4期 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103613678A (en) * | 2013-11-19 | 2014-03-05 | 广西壮族自治区海洋研究所 | Sipunculus nudus polysaccharide sulfation modification method |
| CN103613678B (en) * | 2013-11-19 | 2015-12-02 | 广西壮族自治区海洋研究所 | A kind of Sipunculus nudus polysaccharide sulfation modification method |
| CN105918461A (en) * | 2016-04-08 | 2016-09-07 | 宁波大学 | Low-calorific-value yogurt containing phascolosoma esculenta polysaccharide and preparation method thereof |
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