CN105566509A - Suillus granulatus fruiting body polysaccharide with antioxidant activity in vivo and preparation method thereof - Google Patents
Suillus granulatus fruiting body polysaccharide with antioxidant activity in vivo and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the developing field of edible mushroom resources, and in particular relates to a suillus granulatusfruiting body polysaccharide with antioxidant activity in vivo and a preparation method of the suillus granulatusfruiting body polysaccharide. The preparation method comprises the following steps of (1) cleaning, airing and smashing fruiting bodies; (2) boiling and extracting by using deionized water, and combining extraction solutions; (3) concentrating the extraction solution and adding ethanol, collecting sediments, and drying the sediments to obtain a crude polysaccharide; (4) adding an Sevag reagent into the crude polysaccharide, removing a protein layer and a chloroform layer, repeating the operations for a water layer to obtain polysaccharide self-glucosylating protein (SGP); (5) performing elution fractionation for the polysaccharide SGP, collecting the eluant, dialyzing the eluant to remove salt, freezing and drying to obtain polysaccharide SGP II; (6) performing elution fractionation for the polysaccharide SGP II, collecting the a component, dialyzing the component to remove salt, freezing and drying to obtain polysaccharide SGP II-b. The preparation method disclosed by the invention is simple in operation, and the target product has remarkable antioxidant activity in vivo, can reduce free radicals in vivo, and has the anti-aging function.
Description
Technical field
The invention belongs to domestic fungus resource development field, be specifically related to a kind of suillusgranulatus fruitbody polysaccharide SGPII-b with anti-oxidant activity in body and preparation method thereof.
Background technology
Suillusgranulatus [Suillusgranulatus (L.:Fr.) O.Kuntce] has another name called chrysanthemum matsutake, chestnut shell bolete, and classification of fungi belongs to Mycophyta, Basidiomycetes, Agaricales, Boletaceae, Suillus.Suillusgranulatus is a kind of food medicine dual-purpose wild edible fungus, is one of important wild edible fungus in the Northeast.Suillusgranulatus delicious flavour, with antitumor, anti-oxidant, improve the effect such as body immunity.These effects derive from several physiological active substances, as polysaccharide, VITAMIN, mineral substance etc., wherein with polysaccharide component emphasis the most.
There is multiple free radical in living organism, mostly exist with active oxygen form.Can attack body cell, each system function of body be got muddled and injures in-vivo tissue further.At present existing a lot of disease is considered to that free radical is excessive to be caused.As hyperlipidaemia, hypoimmunity, rheumatoid arthritis and acute respiratory distress syndrome etc.Also being considered to base is on the other hand the Main Factors promoting body aging.Due to, the material that therefore can play scavenging(action) to free radical and have an anti-oxidant activity more and more receives the concern of people.Research has confirmed that many edible fungi polysaccharides have anti-oxidant activity, can have scavenging(action), can improve the activity of antioxidase (SOD), GSH-Px etc., thus show antioxidant property various free radical.The research and development of therefore natural antioxidants---edible fungi polysaccharide has broad application prospect in medicines and health protection and food service industry.
Summary of the invention
The present invention is intended to overcome prior art deficiency, and provide a kind of simple to operate, yield is high, suillusgranulatus fruitbody polysaccharide with anti-oxidant activity in obvious body and preparation method thereof.
For solving the problems of the technologies described above, the present invention realizes like this.
Have a suillusgranulatus fruitbody polysaccharide for anti-oxidant activity in body, it is characterized in that, be derived from suillusgranulatus sporophore, Powdered in lark, molecular weight is 25.2kDa; Be made up of rhamnosyl, Fucose, seminose, glucose and semi-lactosi 5 kinds of monose, by mass parts, its proportion of composing is followed successively by: 1:1.37:5.11:16.15:3.59.
The above-mentioned preparation method with the suillusgranulatus fruitbody polysaccharide of anti-oxidant activity in body, can implement as follows successively.
(1) suillusgranulatus sporophore is through cleaning, dries, must extract raw material after pulverizing.
(2) after adding deionized water in extraction raw material, lixiviate; Collected by centrifugation residue, then repeat lixiviate, merging filtrate obtains crude extract.
(3) crude extract is after rotary evaporation concentrates, and adds dehydrated alcohol, stirs and produces precipitation, leaves standstill, centrifugal collecting precipitation, precipitates and obtain Crude polysaccharides CSGP after dehydrated alcohol and washed with diethylether.
(4) Crude polysaccharides CSGP adds Sevag reagent and fully vibrates after being dissolved in deionized water; Centrifugal segregation egg white layer and chloroform layer; In water layer repetitive operation to no longer there is egg white layer, obtained aqueous solution obtains polysaccharide SGP through dialysis and lyophilize.
(5) polysaccharide SGP is dissolved in deionized water, successively carries out elution fractionation with deionized water, 0.5MNaCl and 1.5MNaCl solution; Collect the elutriant of 1.5MNaCl; Elutriant is through dialysis desalination, and lyophilize obtains polysaccharide SGPII.
(6) polysaccharide SGPII is dissolved in deionized water carries out elution fractionation; Collect second component that wash-out flows out; Collect the component obtained and namely obtain through dialysis desalination, lyophilize the suillusgranulatus fruitbody polysaccharide SGPII-b that object product has anti-oxidant activity in body.
As a kind of preferred version, in step of the present invention (2), add 20 ~ 40 times of weight deionized waters in extraction raw material after, 80 ~ 100 DEG C of lixiviate 2 ~ 4h.
Further, in step of the present invention (3), crude extract, after rotary evaporation is concentrated, adds 3 ~ 5 times of volume dehydrated alcohols, and limit edged slowly stirs and produces precipitation, and 2 ~ 6 DEG C leave standstill 10 ~ 20h.
Further, in step of the present invention (1), suillusgranulatus fruit body powder is broken to 40 ~ 100 orders.
Further, in step of the present invention (2), collected by centrifugation speed is 3000 ~ 5000 revs/min; Centrifugal 20 ~ 30 minutes.
Further, in step of the present invention (3), collected by centrifugation speed is 10000 ~ 15000 revs/min; Centrifugal 20 ~ 30 minutes.
Further, in step of the present invention (4), Sevag reagent is prepared 1:4 ~ 5 by volume by propyl carbinol and chloroform; The consumption of Sevag reagent adds by 1:4 ~ 6 of polysaccharide soln volume; Collected by centrifugation speed is 3500 ~ 4500 revs/min; Centrifugal 10 ~ 25 minutes.
Further, in step of the present invention (5), DEAE-cellulose ion exchange column elution fractionation can be adopted.
Further, in step of the present invention (6), agarose gel cl-6b elution fractionation can be adopted.
Anti-oxidant experiment in the body of suillusgranulatus fruitbody polysaccharide.
The principle of the structure of animal model be by certain hour to continuously injecting animals D-semi-lactosi, galactose concentration in cell is increased, under aldose reductase effect, be reduced into melampyrum, the latter can not be deposited in cell by the further metabolism of cell, affects normal osmotic pressure, causes cellular swelling, dysfunction, metabolism disorder; Meanwhile, when D-semi-lactosi is oxidized in vivo, produce a large amount of free radical, exceed the Scavenging activity of body, cause the chain reaction of lipid peroxidation, final body aging.
60 kunming mice (2 monthly ages; Body weight BW20 ± 2g) in be divided into 6 groups at random, often organize 10, comprise normal group, model group, positive controls and 3 polysaccharide experimental group.Normal group presses 10mL/kgBW subcutaneous injection physiological saline every day; Model group, positive controls and 3 polysaccharide experimental group every days, volume injected was equal to normal group all by the normal saline solution of 100mg/kgBW subcutaneous injection D-semi-lactosi.Positive controls adopts gastrogavage to give 100mg/kgBW xitix every day, 3 polysaccharide experimental group adopt gastrogavage to give SGPII-b dosage 100 respectively every day, 200, and 400mg/kgBW, successive administration is after 40 days, and water 12h is can't help in mouse fasting, eyeball gets blood, 4 DEG C, centrifugal 10 minutes of 4000r/min, collects serum to be measured.After getting blood, mouse cervical dislocation is put to death and is got liver homogenate (1g liver adds 9mL physiological saline) 4 DEG C, centrifugal 10 minutes of 4000r/min, is separated supernatant to be measured.Measure superoxide-dismutase (SOD) vigor, catalase (CAT) vigor, Selenoperoxidase (GSH-Px) vigor and mda (MDA) content in serum and liver respectively, the corresponding reagent box that above-mentioned detection all adopts Nanjing to build up Bioengineering Research Institute's production detects.Experimental result is in table 1 and table 2.
Table 1 suillusgranulatus fruitbody polysaccharide is to the impact of SOD, CAT, GSH-Px and MDA level in mice serum.
Note: compare with model group: *, p < 0.05; *, p < 0.01.
Table 2 suillusgranulatus fruitbody polysaccharide is to the impact of SOD, CAT, GSH-Px and MDA level in mouse liver.
Note: compare with model group: *, p < 0.05; *, p < 0.01.
From table 1 and table 2, the blood of model group mouse and liver in the activity of antioxidase (SOD, GSH-Px and CAT) reduce all significantly compared with normal group, MDA also obviously increases compared with normal group, illustrates that aging model successfully builds.The detected result display point handle of polysaccharide experimental group glues lid ox bacillus fruitbody polysaccharide SGPII-b and significantly increases SOD, GSH-Px and CAT vigor also significantly reduces MDA level, and its impact also shows as concentration-dependant.This exterior point handle glues the impact that lid ox bacillus fruitbody polysaccharide SGPII-b shows has similar or better result compared with positive controls (xitix).
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the invention will be further described.Protection scope of the present invention is not only confined to the statement of following content.
Fig. 1 is suillusgranulatus fruitbody polysaccharide DEAE-cellulose ion-exchange chromatography figure.
Fig. 2 is the agarose gel cl-6b tomographic map of suillusgranulatus fruitbody polysaccharide.
Fig. 3 is the monosaccharide composition analysis figure of suillusgranulatus fruitbody polysaccharide SGPII-b.
Fig. 4 is the HPLC figure of suillusgranulatus fruitbody polysaccharide SGPII-b.
Embodiment
Embodiment 1.
The preparation of suillusgranulatus fruitbody polysaccharide SGPII-b.
(1) suillusgranulatus sporophore is through cleaning, dries, is crushed to 80 orders, obtain extraction raw material.
(2) after extraction extraction raw material 1000g adds 25L deionized water, 90 DEG C of lixiviate 3h; 4000 revs/min of centrifugal 20 minutes collecting precipitations, precipitation repeats lixiviate again, and merging filtrate obtains crude extract.
(3) crude extract is concentrated into after 4L through rotary evaporation, add 12L dehydrated alcohol, limit edged slowly stirs and produces precipitation, 4 DEG C of standing 12h, 12000 revs/min of centrifugal 20 minutes collecting precipitations, precipitate and obtain suillusgranulatus sporophore Crude polysaccharides CSGP55.4g after dehydrated alcohols and washed with diethylether.
(4) take Crude polysaccharides 50 grams, be dissolved in 1000 ml deionized water, add 250 milliliters of Sevag reagent (propyl carbinol: chloroform=1:4, v/v), fully concussion 2 hours, 4000 revs/min centrifugal 20 minutes, removes egg white layer and chloroform layer.In water layer repetitive operation to no longer there is egg white layer, obtained aqueous solution obtains suillusgranulatus fruitbody polysaccharide SGP (32.8 grams) through dialysis and lyophilize.
(5) by polysaccharide SGP(5g) be dissolved in 100mL deionized water, adopt exchange column (2.8 × 50cm) classification of DEAE-cellulose ion, use deionized water, 0.5M and 1.5MNaCl eluant solution successively, eluent flow rate is 10 ml/min, a pipe collected by every 20 milliliters of elutriants, detect via Phenol sulfuric acid procedure, after the level lease making deionized water that 1.5M sodium-chlor wash-out obtains is dialysed 24 hours, lyophilize obtains suillusgranulatus fruitbody polysaccharide SGPII (3.6 grams) (Fig. 1).
(6) suillusgranulatus fruitbody polysaccharide SGPII (0.5 gram) is dissolved in 2mL deionized water, adopts agarose gel cl-6b post (2.5 × 90cm) to be separated.Physiological saline is used to carry out wash-out, eluent flow rate is 0.5 ml/min, a pipe collected by every 10 milliliters of elutriants, detect via Phenol sulfuric acid procedure, obtain a and b two fractions, after b level lease making deionized water is dialysed 24 hours, lyophilize obtains suillusgranulatus fruitbody polysaccharide SGPII-b (0.22 gram) (Fig. 2).
1, the purity of suillusgranulatus fruitbody polysaccharide SGPII-b of the present invention and molecular weight determination.
Adopt ShodexKS-802 and KS-804 columns in series, differential refraction detector, by Agilent1100series high performance liquid chromatograph, using the dextran of different relative molecular mass (P-5, P-10, P-20, P-50, P-100, P-200, P-400 and P-800) as standard substance, make typical curve, measure purity and the relative molecular mass of polysaccharide SGPII-b, result as shown in Figure 3.Fig. 3 shows single symmetrical peak, can judge that this polysaccharide is as sterling, and recording suillusgranulatus fruitbody polysaccharide SGPII-b molecular weight according to typical curve calculating is 25.2kDa.
2, the monose composition measuring of suillusgranulatus fruitbody polysaccharide of the present invention.
The 2M trifluoroacetic acid hydrolysis 90min of polysaccharide SGPII-b2mg, 1ml, Rotary Evaporators evaporate to dryness, adds 2ml methyl alcohol, evaporate to dryness, 2 times repeatedly.Residue adds 2ml distilled water, 60mg sodium borohydride reduction 8 hours, adds Glacial acetic acid neutralization; revolve steaming, add methyl alcohol 3ml, 3 times repeatedly; revolve and steam to powder, 110 degree of oven for drying, then add the acetylize of 1ml diacetyl oxide; 100 DEG C of reaction 1h; cooling, then adds 3mL toluene, concentrating under reduced pressure evaporate to dryness; repeat 4-5 time, to remove unnecessary aceticanhydride.Be transferred to separating funnel after being dissolved by product 3mL chloroform after acetylize, add after a small amount of distilled water fully shakes, removing upper water solution, so repeats 4 times.Chloroform layer, with appropriate anhydrous sodium sulfate drying, is settled to 10mL, and GC analyzes.Analytical results is shown in Fig. 4.
GC condition: AgilentGC7890A, Hp-5 (Agilent19091J-413) chromatographic column (30m × 0.25mm × 320 μm); Temperature programming condition is: starting temperature 140 ° of C, are warming up to 200 ° of C/min with 1.5 ° of C/min; Finally be warming up to 250 ° of C with 10 ° of C/min, keep 5min; Injector temperature is 250 ° of C, and detector temperature is 250 ° of C/min, hydrogen flowing quantity 30mL/min, air flow quantity 400mL/min; Carrier gas is N
2, flow velocity is 1mL/min.
Monose group measurement result display suillusgranulatus fruitbody polysaccharide polysaccharide SGPII-b is made up of rhamnosyl, Fucose, seminose, glucose, semi-lactosi 5 kinds of monose, and by mass parts, its proportion of composing is followed successively by: 1:1.37:5.11:16.15:3.59.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. have a suillusgranulatus fruitbody polysaccharide for anti-oxidant activity in body, it is characterized in that, be derived from suillusgranulatus sporophore, Powdered in lark, molecular weight is 25.2kDa; Be made up of rhamnosyl, Fucose, seminose, glucose and semi-lactosi 5 kinds of monose, by mass parts, its proportion of composing is followed successively by: 1:1.37:5.11:16.15:3.59.
2. there is a preparation method for the suillusgranulatus fruitbody polysaccharide of anti-oxidant activity in body as claimed in claim 1, it is characterized in that, implement successively as follows:
(1) suillusgranulatus sporophore is through cleaning, dries, must extract raw material after pulverizing;
(2) after adding deionized water in extraction raw material, lixiviate; Collected by centrifugation residue, then repeat lixiviate, merging filtrate obtains crude extract;
(3) crude extract is after rotary evaporation concentrates, and adds dehydrated alcohol, stirs and produces precipitation, leaves standstill, centrifugal collecting precipitation, precipitates and obtain Crude polysaccharides CSGP after dehydrated alcohol and washed with diethylether;
(4) Crude polysaccharides CSGP adds Sevag reagent and fully vibrates after being dissolved in deionized water; Centrifugal segregation egg white layer and chloroform layer; In water layer repetitive operation to no longer there is egg white layer, obtained aqueous solution obtains polysaccharide SGP through dialysis and lyophilize;
(5) polysaccharide SGP is dissolved in deionized water, successively carries out elution fractionation with deionized water, 0.5MNaCl and 1.5MNaCl solution; Collect the elutriant of 1.5MNaCl; Elutriant is through dialysis desalination, and lyophilize obtains polysaccharide SGPII;
(6) polysaccharide SGPII is dissolved in deionized water carries out elution fractionation; Collect second component that wash-out flows out; Collect the component obtained and namely obtain through dialysis desalination, lyophilize the suillusgranulatus fruitbody polysaccharide that object product has anti-oxidant activity in body.
3. the preparation method with the suillusgranulatus fruitbody polysaccharide of anti-oxidant activity in body according to claim 2, it is characterized in that: in described step (2), add 20 ~ 40 times of weight deionized waters in extraction raw material after, 80 ~ 100 DEG C of lixiviate 2 ~ 4h.
4. the preparation method with the suillusgranulatus fruitbody polysaccharide of anti-oxidant activity in body according to claim 3, it is characterized in that: in described step (3), crude extract is after rotary evaporation is concentrated, add 3 ~ 5 times of volume dehydrated alcohols, limit edged slowly stirs and produces precipitation, and 2 ~ 6 DEG C leave standstill 10 ~ 20h.
5. the preparation method with the suillusgranulatus fruitbody polysaccharide of anti-oxidant activity in body according to claim 4, is characterized in that: in described step (1), suillusgranulatus fruit body powder is broken to 40 ~ 100 orders.
6. the preparation method with the suillusgranulatus fruitbody polysaccharide of anti-oxidant activity in body according to claim 5, it is characterized in that: in described step (2), collected by centrifugation speed is 3000 ~ 5000 revs/min; Centrifugal 20 ~ 30 minutes.
7. the preparation method with the suillusgranulatus fruitbody polysaccharide of anti-oxidant activity in body according to claim 6, it is characterized in that: in described step (3), collected by centrifugation speed is 10000 ~ 15000 revs/min; Centrifugal 20 ~ 30 minutes.
8. the preparation method with the suillusgranulatus fruitbody polysaccharide of anti-oxidant activity in body according to claim 7, is characterized in that: in described step (4), and Sevag reagent is prepared 1:4 ~ 5 by volume by propyl carbinol and chloroform; The consumption of Sevag reagent adds by 1:4 ~ 6 of polysaccharide soln volume; Collected by centrifugation speed is 3500 ~ 4500 revs/min; Centrifugal 10 ~ 25 minutes.
9. the preparation method with the suillusgranulatus fruitbody polysaccharide of anti-oxidant activity in body according to claim 8, is characterized in that: in described step (5), adopts DEAE-cellulose ion exchange column elution fractionation.
10. the preparation method with the suillusgranulatus fruitbody polysaccharide of anti-oxidant activity in body according to claim 9, is characterized in that: in described step (6), adopts agarose gel cl-6b elution fractionation.
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Cited By (3)
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| CN108794649A (en) * | 2018-07-09 | 2018-11-13 | 海南医学院 | A kind of microorganism antioxidant extract and its preparation method and application |
| CN108948222A (en) * | 2018-08-17 | 2018-12-07 | 华南理工大学 | A kind of wrinkle lid wart handle speciosus polysaccharide and its extracting method and application |
| CN112029004A (en) * | 2020-08-03 | 2020-12-04 | 中国农业大学 | Boletus toruloides polysaccharide and extraction method and application thereof |
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| CN108948222A (en) * | 2018-08-17 | 2018-12-07 | 华南理工大学 | A kind of wrinkle lid wart handle speciosus polysaccharide and its extracting method and application |
| CN112029004A (en) * | 2020-08-03 | 2020-12-04 | 中国农业大学 | Boletus toruloides polysaccharide and extraction method and application thereof |
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