CN105294872A - Preparation method of carboxymethylated catathelasma ventricosum polysaccharide - Google Patents
Preparation method of carboxymethylated catathelasma ventricosum polysaccharide Download PDFInfo
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Abstract
The invention discloses a preparation method of carboxymethylated catathelasma ventricosum polysaccharide. The preparation method comprises the steps of freeze-drying catathelasma ventricosum, grinding into powder, performing water extraction and alcohol sedimentation with ethanol, and removing protein and small molecular weight impurities, so as to obtain catathelasma ventricosum polysaccharide; the preparation method of the carboxymethylated catathelasma ventricosum polysaccharide comprises the steps of dissolving 250 mg of catathelasma ventricosum polysaccharide in 10 ml of NaOH solution, fully stirring mixed liquid for 30 min, adding a mixed solution of 3 g of chloroacetic acid and 15 ml of isopropanol, stirring for 1 h, then putting in an incubator of 65 DEG C for reaction for 4 h, cooling to room temperature, then adjusting pH to be neutral with 0.5 M of HCl, dialyzing by running water overnight, wherein the molecular weight cutoff of a dialysis bag is 3000, mixing with 95% of ethanol solution in four times volume, stirring for 30 min, then putting in a refrigerator of 4 DEG C for 12 h, then centrifuging, discarding supernatant, and freeze-drying, so as to obtain the carboxymethylated catathelasma ventricosum polysaccharide.
Description
Technical field
The present invention relates to the preparing technical field of polysaccharide, be specially the preparation method of the luxuriant mushroom polysaccharide (cCVPs) of a kind of carboxymethylation shuttle handle pine.
Background technology
The metabolic disease of diabetes to be one group with hyperglycemia be feature.Hyperglycemia be then due to defect of insulin secretion or its biological action impaired, or both have concurrently and cause.Long-standing hyperglycemia during diabetes, causes various tissue, particularly eye, kidney, heart, blood vessel, neural chronic lesion, dysfunction.According to the data that the World Health Organization announced in 2014, global diabetic subject's number has reached 3.47 hundred million.
The edible fungus with pharmaceutical use is considered to one of very important medicine in Traditional Folk medicine, and application is very general.Research shows, edible fungus is to the treatment of disease mainly contained by it caused by biologically active substance.The active substance of these edible funguses can make the organ function such as liver, pancreas of diabetic subject recover normal, thus promotes the secretion of Regular Insulin and associated hormone, and the metabolic function of body is recovered.
Polysaccharide has the medical actives such as antibacterial, anti-oxidant, antitumor, anti-diabetic, the biological activity of polysaccharide will be significantly increased after polysaccharide carboxymethylation, and the water-soluble of polysaccharide and toughness can be increased, be widely used in food, reagent combination, industrial raw material.And present stage polysaccharide carboxymethylated method ubiquity: carboxymethylated polysaccharides substitution value is lower, and when increasing reagent concentration and reaction times, can have a strong impact on again the yield of carboxymethylated polysaccharides.The luxuriant mushroom polysaccharide of shuttle handle pine is not utilized to carry out carboxymethylated research at present, it should be noted that the molecular weight of the luxuriant mushroom polysaccharide of shuttle handle pine is less compared with other mushroom polysaccharide, and the size of molecular weight is often inversely proportional to substitution value and biological activity, is therefore applied to antidiabetic research with the luxuriant mushroom polysaccharide of carboxymethylated shuttle handle pine and will provides new approaches and reference for the biological activity of the chemically modified of polysaccharide and polysaccharide.
Summary of the invention
The object of the present invention is to provide a kind of carboxymethylated method of efficient edible fungi polysaccharide being representative with the luxuriant mushroom of shuttle handle pine, to solve the problem proposed in above-mentioned background technology.
For achieving the above object, the invention provides following technical scheme: the preparation method of the luxuriant mushroom polysaccharide of a kind of carboxymethylation shuttle handle pine, is characterized in that: (1) is got the luxuriant mushroom of fresh shuttle handle pine and carried out pre-treatment; (2) preparation of the luxuriant mushroom polysaccharide of shuttle handle pine, is specially: water extraction: first wash with ethanol, then centrifugal treating, the deposit sample of centrifuge tube is dry, and press solid-liquid ratio 1: 20 and mix with distilled water, then carry out ultrasonication, ul-trasonic irradiation power is 150W, often work 15min, suspend 5min, Extracting temperature 80 DEG C, extraction time 2.5h, above-mentioned water extraction step repeats extraction three times, and united extraction liquid is the luxuriant mushroom polysaccharide extraction liquid of shuttle handle pine; Alcohol settling: the luxuriant mushroom polysaccharide extraction liquid Rotary Evaporators of shuttle handle pine is concentrated, adds 95% ethanolic soln of 5 times of volumes, after stirring 0.5h, be placed in 4 DEG C of refrigerator 12h, then centrifugal treating, then abandon supernatant liquor, collecting precipitation, freeze-drying is stand-by; Remove protein: add distilled water by solid-to-liquid ratio 1: 10 and dissolve the luxuriant mushroom polysaccharide of shuttle handle pine, then add the chloroform-propyl carbinol of 1/4 times of volume, fully after vibration 30min, centrifugal treating again, obtain three layers of liquid, draw orlop polysaccharide soln, repeat this removal protein step 5 time; Remove small-molecule substance: will remove the polysaccharide extract of protein, flowing water dialysed overnight, the molecular weight cut-off of dialysis tubing is 3000, to remove small molecular weight impurity; With Rotary Evaporators, this polysaccharide extraction liquid is concentrated again, mix with 95% ethanolic soln of 4 times of volumes, after stirring 0.5h, be placed in 4 DEG C of refrigerator 12h, centrifugal treating again, centrifuge speed 5000rpm, time 5min, abandon supernatant liquor, precipitation is the luxuriant mushroom polysaccharide of shuttle handle pine, and lyophilize is for subsequent use; (3) preparation of the luxuriant mushroom polysaccharide of carboxymethylation shuttle handle pine, is specially: getting molecular weight ranges is 5.0 × 10
3-1.0 × 10
4it is in the NaOH solution of 20% that the luxuriant mushroom polysaccharide of 250mg shuttle handle pine of Da is dissolved in 10ml massfraction, fully be uniformly mixed liquid 30min, add the mixing solutions of the Virahol of 3g Mono Chloro Acetic Acid and 15ml, after stirring 1h, the thermostat container being placed in 65 DEG C reacts 4h, to be cooled to room temperature, adjust pH to neutral with the HCl of 0.5M, flowing water dialysed overnight, the molecular weight cut-off of dialysis tubing is 3000, mix with 95% ethanolic soln of 4 times of volumes, after stirring 30min, be placed in 4 DEG C of refrigerator 12h, more centrifugal, abandon supernatant liquor, namely lyophilize obtains the luxuriant mushroom polysaccharide of carboxymethylation shuttle handle pine.
Further, the pre-treatment step of the luxuriant mushroom of fresh shuttle handle pine is specially,
Clean: remove by the mud stain of plastic wipers by the luxuriant mushroom surface of fresh shuttle handle pine, unavailable water cleans, and not so the polysaccharide on surface can lose;
Stripping and slicing: by the luxuriant mushroom stripping and slicing of fresh shuttle handle pine after clean, each stripping and slicing is of moderate size and evenly;
Lyophilize: by shuttle handle pine luxuriant mushroom block put into freeze drier, obtain water content lower than 5% sample.
Further, methanol precipitation step and remove the centrifuge speed 5000rpm of the centrifugal treating in protein step, time 5min.
Compared with prior art, the invention has the beneficial effects as follows: the raw material (shuttle handle pine luxuriant mushroom) that (1) this patent is selected in Sichuan, the ground such as Yunnan and Guizhou extensively plants, it is convenient to gather, cheap, and CVPs has been proved to be has comparatively outstanding anti-diabetic activity, therefore possesses the potential quality be exploited further; (2) this patent establishes the preparation method of a kind of efficient cCVPs, wherein have rated polysaccharide molecule weight range first to the carboxymethylated impact of polysaccharide, and yield and the substitution value of final gained cCVPs reach 82.01% and 0.891 respectively.And the reaction conditions of whole preparation process is gentle, with low cost, be simple to operately easy to commercial introduction; (3) experimentation on animals proves, after carboxymethylation modification is carried out to CVPs, the biological activity of cCVPs is significantly improved, only need the cCVPs taking in 0.2g/kg/d dosage, just the level of each oxygenant in diabetic mice serum can be significantly improved, to alleviate its oxidative stress level, thus realize the treatment to diabetes.
Accompanying drawing explanation
Fig. 1 is the luxuriant mushroom polysaccharide infrared spectrogram of carboxymethylation shuttle handle pine in the present invention;
Embodiment
Below in conjunction with the accompanying drawing 1 in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Refer to Fig. 1, the invention provides the preparation method of the luxuriant mushroom polysaccharide of a kind of carboxymethylation shuttle handle pine, it is characterized in that: (1) is got the luxuriant mushroom of fresh shuttle handle pine and carried out pre-treatment; Clean: remove by the mud stain of plastic wipers by the luxuriant mushroom surface of fresh shuttle handle pine, unavailable water cleans, and not so the polysaccharide on surface can lose; Stripping and slicing: by the luxuriant mushroom stripping and slicing of fresh shuttle handle pine after clean, each stripping and slicing is of moderate size and evenly; Lyophilize: by shuttle handle pine luxuriant mushroom block put into freeze drier, obtain water content lower than 5% sample.(2) preparation of the luxuriant mushroom polysaccharide of shuttle handle pine, is specially: water extraction: first wash with ethanol, then centrifugal treating, the deposit sample of centrifuge tube is dry, and press solid-liquid ratio 1: 20 and mix with distilled water, then carry out ultrasonication, ul-trasonic irradiation power is 150W, often work 15min, suspend 5min, Extracting temperature 80 DEG C, extraction time 2.5h, above-mentioned water extraction step repeats extraction three times, and united extraction liquid is the luxuriant mushroom polysaccharide extraction liquid of shuttle handle pine; Alcohol settling: the luxuriant mushroom polysaccharide extraction liquid Rotary Evaporators of shuttle handle pine is concentrated, adds 95% ethanolic soln of 5 times of volumes, after stirring 0.5h, be placed in 4 DEG C of refrigerator 12h, then centrifugal treating, then abandon supernatant liquor, collecting precipitation, freeze-drying is stand-by; Remove protein: add distilled water by solid-to-liquid ratio 1: 10 and dissolve the luxuriant mushroom polysaccharide of shuttle handle pine, then add the chloroform-propyl carbinol of 1/4 times of volume, fully after vibration 30min, centrifugal treating again, obtain three layers of liquid, draw orlop polysaccharide soln, repeat this removal protein step 5 time; Remove small-molecule substance: will remove the polysaccharide extract of protein, flowing water dialysed overnight, the molecular weight cut-off of dialysis tubing is 3000, to remove small molecular weight impurity; With Rotary Evaporators, this polysaccharide extraction liquid is concentrated again, mix with 95% ethanolic soln of 4 times of volumes, after stirring 0.5h, be placed in 4 DEG C of refrigerator 12h, centrifugal treating again, centrifuge speed 5000rpm, time 5min, abandon supernatant liquor, precipitation is the luxuriant mushroom polysaccharide of shuttle handle pine, and lyophilize is for subsequent use; (3) preparation of the luxuriant mushroom polysaccharide of carboxymethylation shuttle handle pine, is specially: getting molecular weight ranges is 5.0 × 10
3-1.0 × 10
4it is in the NaOH solution of 20% that the luxuriant mushroom polysaccharide of 250mg shuttle handle pine of Da is dissolved in 10ml massfraction, fully be uniformly mixed liquid 30min, add the mixing solutions of the Virahol of 3g Mono Chloro Acetic Acid and 15ml, after stirring 1h, the thermostat container being placed in 65 DEG C reacts 4h, to be cooled to room temperature, adjust pH to neutral with the HCl of 0.5M, flowing water dialysed overnight, the molecular weight cut-off of dialysis tubing is 3000, mix with 95% ethanolic soln of 4 times of volumes, after stirring 30min, be placed in 4 DEG C of refrigerator 12h, more centrifugal, abandon supernatant liquor, namely lyophilize obtains the luxuriant mushroom polysaccharide of carboxymethylation shuttle handle pine.Methanol precipitation step and remove the centrifuge speed 5000rpm of the centrifugal treating in protein step, time 5min.
The defining method of the optimum molecular weight ranges 5.0 × 103-1.0 × 104Da of the luxuriant mushroom polysaccharide of above-mentioned shuttle handle pine is as follows:
(1) polysaccharide acid hydrolysis: 1. get the luxuriant mushroom polysaccharide sample of appropriate shuttle handle pine in tool plug test tube, dissolve with 0.1mol/L trifluoroacetic acid (TFA), then at 110 DEG C of Water Under solution 1h, by molecular retention specification be again 5.0 × 102 dialysis tubing this reaction solution is dialysed, to remove trifluoroacetic acid and other impurity, and collect liquid in dialysis tubing, be polysaccharide hydrolysis solution; 2., except the dialysis tubing being 1.0 × 106 except molecular retention specification is directly dialysed to polysaccharide hydrolysis, the dialysis tubing of all the other specifications all to be dialysed (dialysis tubing specification: 1.0 × 10 to the polysaccharide hydrolysis solution outside last dialysis tubing
6, 5.0 × 10
5, 3.0 × 10
5, 1.0 × 10
5, 5.0 × 10
4, 3.0 × 10
4, 1.0 × 10
4, 5.0 × 10
3, 3.0 × 10
3, 1.0 × 10
3), collect the liquid in dialysis tubing, namely obtain polysaccharide molecular weight after freeze-drying and be respectively: > 1.0 × 10
6, 5.0 × 10
5-1.0 × 10
6, 3.0 × 10
5-5.0 × 10
5, 1.0 × 10
5-3.0 × 10
5, 5.0 × 10
4-1.0 × 10
5, 3.0 × 10
4-5.0 × 10
4, 1.0 × 10
4-3.0 × 10
4, 5.0 × 10
3-1.0 × 10
4, 3.0 × 10
3-5.0 × 10
3, 1.0 × 10
3-3.0 × 10
3, the component of 10 groups of different molecular weight ranges.
(2) each component carries out substitution value and the yield that carboxymethylation compares them.
Table 1: the carboxymethylation substitution value of molecular weight on polysaccharide and the impact of yield
Each numerical value is all expressed as the mean+SD (SD) of three tests, and letter representations different in same row has significant difference (P < 0.05)
Can be obtained by table 1, the molecular weight of polysaccharide is 5.0 × 10
3-1.0 × 10
4, 3.0 × 10
3-5.0 × 10
3, 1.0 × 10
3-3.0 × 10
3three groups of substitution values be significantly higher than all the other components (p < 0.05).And molecular weight is 3.0 × 10
3-5.0 × 10
3, 1.0 × 10
3-3.0 × 10
3two groups of yield significantly lower than all the other components (p < 0.05), therefore optimum molecular weight component is 5.0 × 10
3-1.0 × 10
4the polysaccharide of molecular weight, utilizes this component to carry out Infrared spectroscopy below and experimentation on animals.
Infrared spectroscopy and bioactive mensuration are carried out to optimal component.
As seen from Figure 1 1596,1423, there is the charateristic avsorption band of-COOC-in 1327cm-1 place, wherein, 1596cm-1 place is C-O absorption peak that is symmetrical and asymmetrical stretching vibration is strong peak, 1423, strong peak in being, peak, two, 1327cm-1 place, demonstrate CVPs really by carboxymethylation, be cCVPs.
Carboxymethylated polysaccharides is to the regulating effect of diabetes oxidative stress
Laboratory animal
The healthy male ICR mouse (body weight 18 ± 2g, 5 week age) of cleaning grade is purchased from this Leco Corp. of Shanghai.Adaptability is raised in two weeks (25 DEG C of constant temperature air conditioning rooms, 12h illumination 12h is dark, has sufficient drinking-water and basal feed).
Diabetes animal model is set up
After random selecting 50 mouse fasting 12h, mouse peritoneal single injection I%STZ solution (140mg/kg), after 72 hours, tail point gets blood, blood glucose meter measuring blood, Tes-Tape measures glucose in urine, chooses the mouse of 36 blood sugar > 16.7mmol/L as diabetes model.
Experimentation on animals is divided into groups
Treat that experiment mice (is often organized 6 mouse, 30 days experimental periods) by random being assigned in following experimental group:
1. I is organized: normal mouse in contrast;
2. organize II: through STZ induction diabetic mice as negative control (gavage distilled water);
3. III is organized: (amount of gavage Glyburide is the oral Glyburide of diabetic mice of inducing through STZ: 0.02g/kg/d) as positive control;
4. IV is organized: the carboxymethylation shuttle handle luxuriant mushroom polysaccharide of pine (gavage amount is: 2g/kg/d) of normal mouse high oral dose;
5. V is organized: through the carboxymethylation shuttle handle luxuriant mushroom polysaccharide of pine (gavage amount is: 0.2g/kg/d) of the diabetic mice low oral dose of STZ induction;
6. VI is organized: through the luxuriant mushroom polysaccharide of diabetic mice oral shuttle handle pine of STZ induction.(gavage amount is: 0.2g/kg/d).
The mensuration of physical signs
1. through the raising of 30 days, all mouse fasting for trying 12 hours.The body weight of mouse and blood sugar are measured, then blood is got to eyeball of mouse, and the blood Quick spin (5000rpm, 5min, 4 DEG C) collected, to obtain serum, is preserved in-80 DEG C of refrigerators; All mouse utilize dislocation of cervical vertebra method to put to death, and cut the liver of mouse and kidney is preserved in rapidly-80 DEG C of refrigerators, to be tested.
2. the content of glucose in serum is measured.
3. the Mouse Liver of gained, nephridial tissue are placed in homogenizer homogenate (being placed on ice), centrifugal (3000rpm after acquisition homogeneous, 10min, 4 DEG C), supernatant liquid is used to the level measuring catalase (CAT), mda (MDA), superoxide-dismutase (SOD), Selenoperoxidase (GSH-Px), vitamins C and vitamin-E.
Result: by table 2 with find out diabetic mice (group II, III, V and VI) on pretreatment body weight be starkly lower than normal mouse (group I and IV), blood sugar is apparently higher than normal group (p < 0.05).Taken medicine continuously after 30 days, the blood glucose in diabetic mice level of group V and VI is significantly lower than the negative control group (p < 0.05) not taking any medicine.The luxuriant mushroom polysaccharide of shuttle handle pine after this illustrates the shuttle handle luxuriant mushroom polysaccharide of pine and carboxymethylation all has certain antidiabetic activity.Take the mouse (group VI) that mouse (group V) body weight of the luxuriant mushroom polysaccharide of the pine of the shuttle handle after carboxymethylation and blood glucose target are significantly better than having taken the luxuriant mushroom polysaccharide of shuttle handle pine.This can improve the anti-diabetic activity of polysaccharide significantly after luxuriant mushroom polysaccharide carboxymethylation of shuttle handle pine is described.Normal mouse taking dose up to after the carboxymethylation of 2g/kg/d shuttle handle pine luxuriant mushroom polysaccharide after 30 days body weight and blood glucose target there is not any exception, and after carboxymethylation shuttle handle pine luxuriant mushroom polysaccharide hypoglycemic activity only a little less than positive controls.
As can be seen from Table 3, antioxidant level in normal mouse (group I and IV) liver is apparently higher than diabetic mice (group II, III, V and VI), and the content (reaction body is oxidized the index of answering) of mda is lower than diabetic mice (p < 0.05).In the diabetic mice liver organizing V and VI after taking the medicine of 30 days continuously, antioxidant level is significantly higher than the negative control group not taking any medicine, and the content of mda is significantly lower than negative control (p < 0.05).The content taking antioxidant level in diabetic mice (group V) liver of the luxuriant mushroom polysaccharide of carboxymethylated shuttle handle pine and mda is all obviously better than taking the diabetic mice (group VI) of the luxuriant mushroom polysaccharide of shuttle handle pine, wherein organizes the Selenoperoxidase of diabetic mice liver and the level of superoxide-dismutase in V and reaches normal level.Illustrate that the luxuriant mushroom polysaccharide of shuttle handle pine after the shuttle handle luxuriant mushroom polysaccharide of pine and carboxymethylation all has certain anti-oxidant activity, carboxymethylation can improve the anti-oxidant activity of the luxuriant mushroom polysaccharide of shuttle handle pine significantly.And shuttle handle pine luxuriant mushroom polysaccharide and carboxymethylation shuttle handle pine luxuriant mushroom polysaccharide for the antioxidant of non-enzymatic level promote very limited, vitamins C and vitamin-E all do not return to normal level.When normal mouse taking dose is up to the carboxymethylation shuttle handle pine luxuriant mushroom polysaccharide after 30 days of 2g/kg/d, in body, all Antioxidant Indexes all maintain normal level, and the level of antioxidase is significantly higher than normal group, the luxuriant mushroom polysaccharide of shuttle handle pine after this illustrates carboxymethylation has extremely strong anti-oxidant activity, and is comparatively safe.
To sum up can draw, carboxymethylation can significantly improve the anti-oxidant activity of the luxuriant mushroom polysaccharide of shuttle handle pine.And the anti-diabetic characteristic that the luxuriant mushroom polysaccharide of shuttle handle pine after carboxymethylation shows, it may be the strong anti-oxidative activity due to it, reduce body oxidative stress level, thus alleviate or avoid each organ-tissue to suffer oxidativestress damage, the normal function maintaining each organ realizes.
Table 2: the body weight of the carboxymethylation shuttle handle luxuriant mushroom polysaccharide of pine on diabetic mice, the impact of blood sugar
Note: each numerical value is all expressed as mean value (the mean) ± standard deviation (SD) of three tests, and letter representations different in same row has significant difference (p < 0.05); CCVPs: the luxuriant mushroom polysaccharide of carboxymethylated shuttle handle pine; CVPs: the luxuriant mushroom polysaccharide of shuttle handle pine;
h: high dosage.
Table 3: carboxymethylation shuttle handle pine luxuriant mushroom polysaccharide on diabetic mice liver internal oxidition stress impact
Note: each numerical value is all expressed as mean value (the mean) ± standard deviation (SD) of three tests, has significant difference (P < 0.05) with letter representations different in a line; CVPs: the luxuriant mushroom polysaccharide of shuttle handle pine; CCVPs: the luxuriant mushroom polysaccharide of carboxymethylated shuttle handle pine;
h: high dosage; GSH-Px Selenoperoxidase (U/mgprotein); SOD superoxide-dismutase (U/mgprotein); CAT catalase (U/mgprotein); MDA mda (nmol/mgprotein); V
cvitamins C (μ g/mgprotein); V
evitamin-E (μ g/gtissue).
Claims (3)
1. a preparation method for the luxuriant mushroom polysaccharide of carboxymethylation shuttle handle pine, is characterized in that:
(1) get the luxuriant mushroom of fresh shuttle handle pine and carry out pre-treatment;
(2) preparation of the luxuriant mushroom polysaccharide of shuttle handle pine, is specially:
Water extraction: first wash with ethanol, then centrifugal treating, the deposit sample of centrifuge tube is dry, and press solid-liquid ratio 1: 20 and mix with distilled water, then ultrasonication is carried out, ul-trasonic irradiation power is 150W, and often work 15min, suspends 5min, Extracting temperature 80 DEG C, extraction time 2.5h, above-mentioned water extraction step repeats extraction three times, and united extraction liquid is the luxuriant mushroom polysaccharide extraction liquid of shuttle handle pine;
Alcohol settling: the luxuriant mushroom polysaccharide extraction liquid Rotary Evaporators of shuttle handle pine is concentrated, adds 95% ethanolic soln of 5 times of volumes, after stirring 0.5h, be placed in 4 DEG C of refrigerator 12h, then centrifugal treating, then abandon supernatant liquor, collecting precipitation, freeze-drying is stand-by;
Remove protein: add distilled water by solid-to-liquid ratio 1: 10 and dissolve the luxuriant mushroom polysaccharide of shuttle handle pine, then add the chloroform-propyl carbinol of 1/4 times of volume, fully after vibration 30min, centrifugal treating again, obtain three layers of liquid, draw orlop polysaccharide soln, repeat this removal protein step 5 time;
Remove small-molecule substance: will remove the polysaccharide extract of protein, flowing water dialysed overnight, the molecular weight cut-off of dialysis tubing is 3000, to remove small molecular weight impurity; With Rotary Evaporators, this polysaccharide extraction liquid is concentrated again, mix with 95% ethanolic soln of 4 times of volumes, after stirring 0.5h, be placed in 4 DEG C of refrigerator 12h, centrifugal treating again, centrifuge speed 5000rpm, time 5min, abandon supernatant liquor, precipitation is the luxuriant mushroom polysaccharide of shuttle handle pine, and lyophilize is for subsequent use;
(3) preparation of the luxuriant mushroom polysaccharide of carboxymethylation shuttle handle pine, is specially:
Getting molecular weight ranges is 5.0 × 10
3-1.0 × 10
4it is in the NaOH solution of 20% that the luxuriant mushroom polysaccharide of 250mg shuttle handle pine of Da is dissolved in 10ml massfraction, fully be uniformly mixed liquid 30min, add the mixing solutions of the Virahol of 3g Mono Chloro Acetic Acid and 15ml, after stirring 1h, the thermostat container being placed in 65 DEG C reacts 4h, to be cooled to room temperature, adjust pH to neutral with the HCl of 0.5M, flowing water dialysed overnight, the molecular weight cut-off of dialysis tubing is 3000, mix with 95% ethanolic soln of 4 times of volumes, after stirring 30min, be placed in 4 DEG C of refrigerator 12h, more centrifugal, abandon supernatant liquor, namely lyophilize obtains the luxuriant mushroom polysaccharide of carboxymethylation shuttle handle pine.
2. the preparation method of the luxuriant mushroom polysaccharide of a kind of carboxymethylation shuttle handle pine according to claim 1, is characterized in that: the pre-treatment step of the luxuriant mushroom of fresh shuttle handle pine is specially,
Clean: remove by the mud stain of plastic wipers by the luxuriant mushroom surface of fresh shuttle handle pine, unavailable water cleans, and not so the polysaccharide on surface can lose;
Stripping and slicing: by the luxuriant mushroom stripping and slicing of fresh shuttle handle pine after clean, each stripping and slicing is of moderate size and evenly;
Lyophilize: by shuttle handle pine luxuriant mushroom block put into freeze drier, obtain water content lower than 5% sample.
3. the preparation method of the luxuriant mushroom polysaccharide of a kind of carboxymethylation shuttle handle pine according to claim 1, is characterized in that: methanol precipitation step and remove the centrifuge speed 5000rpm of the centrifugal treating in protein step, time 5min.
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| CN107325199A (en) * | 2017-08-03 | 2017-11-07 | 西华师范大学 | A kind of new natural products magnificence ring bud mushroom polysaccharide CIS A and its application |
| CN115553469A (en) * | 2022-09-27 | 2023-01-03 | 六安益普罗科技有限公司 | Stomach mucosa adhesion probiotic slow-release microspheres, preparation method and food for preparing anti-helicobacter pylori or relieving gastritis |
| CN115553469B (en) * | 2022-09-27 | 2023-10-03 | 六安益普罗科技有限公司 | Stomach mucosa-adhered probiotic slow-release microsphere, preparation method and medicine for preparing helicobacter pylori resisting or stomach mucositis relieving medicine |
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