CN104974271A - Preparation method of rhus verniciflua stoke seed polysaccharide with antioxidant activity - Google Patents
Preparation method of rhus verniciflua stoke seed polysaccharide with antioxidant activity Download PDFInfo
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Abstract
The invention relates to a preparation method of rhus verniciflua stoke seed polysaccharide with antioxidant activity. The rhus verniciflua stoke seed polysaccharide is prepared by taking squeezed rhus verniciflua stoke seed meal as a raw material and is prepared by the following steps: A, degreasing, namely degreasing the rhus verniciflua stoke seed meal by adopting a non-polar solvent to obtain degreased seed meal; B, adding distilled water according to a mass volume ratio of (1:5) to (1:30) (g:mL) to perform extraction, concentrating an extract, adding ethanol to perform ethanol precipitation, centrifuging to obtain a precipitate, and washing for multiple times by adopting an organic solvent; C, performing decolorization and purification by adopting adsorbing materials having different fillers; and D, purifying by adopting a ceramic membrane and ultrafiltration membrane technology, and drying to obtain the rhus verniciflua stoke seed polysaccharide. The preparation method of the rhus verniciflua stoke seed polysaccharide, provided by the invention, is simple and convenient, and the rhus verniciflua stoke seed polysaccharide can be prepared into an antioxidant functional food; and meanwhile, the preparation method provided by the invention can change wastes intro valuables and promote the comprehensive utilization ratio of the rhus verniciflua stoke seed meal.
Description
Technical field
The present invention relates to a kind of preparation method with the paint seed polysaccharide of anti-oxidant activity, refer in particular to the lacquer tree seed dregs of rice polyoses extract with antioxygenation, belong to natural drug, field of health care food.
Background technology
Lacquer tree (
rhus vernicifluastokes), for Anacardiaceae (
anacardiaceae) sumach (
toxicodendron) a kind of deciduous tree.Lacquer tree is the important spy of China with economic forest, and raw lacquer is natural resin coating, have the good reputation of " king of coating ".Mainly be distributed in north latitude 25 ~ 42 °, the mountain area between east longitude 95 ~ 125 °, wherein Qin-ba Mountains and the Yunnan-Guizhou Plateau are lacquer tree distribution area of concentration.Raw lacquer is lacquer tree secretory product, is natural resin coating, have the good reputation of " king of coating ".
Paint seed is the fruit of lacquer tree, and its mesocarp is wax layer, can extract lacquer tree fat, and kernel is seed, can squeeze urushoil, and in the existing lacquer tree resource of China, annual paint seed output is about more than 200 ten thousand tons.At present the extraction of haze tallow mainly concentrated on to the research of paint seed and refine the aspects such as processing, Dong Yanhe etc.
[2-3]have studied single factor test and the orthogonal test of circumfluence distillation lacquer tree fat, optimize the optimised process that lacquer tree fat extracts, and adopting GC-MS stratographic analysis Shaanxi Rhus succedanea and Jiangxi to introduce a fine variety the chemical composition of lacquer tree fat lipid acid in Rhus succedanea, the lacquer tree fat main fatty acid in two places of production consists of brown eleostearic acid, oleic acid and stearic acid etc.And further study the decolor refined technology of lacquer tree fat, the whiteness of the lacquer tree fat adopting the method to obtain reaches 85.2.The lacquer tree seed dregs of rice are the substantial residual thing after paint seed has squeezed, and a large amount of paint seed dregs of rice carry out burning disposal as waste residue, not only cause waste to environment, and resource are not fully used.
Polysaccharide is one of the four large base substances forming life, and has multiple biological activity, about the preparation method of the paint seed polysaccharide with anti-oxidant activity so far there are no report both at home and abroad, also has no corresponding patent and discloses.
Summary of the invention
The object of the invention is to overcome the deficiency in the application of the lacquer tree seed dregs of rice, a kind of preparation method with the paint seed polysaccharide of anti-oxidant activity is provided.
For achieving the above object, the present invention adopts following technical scheme:
The first step
The lacquer tree seed dregs of rice are dry, pulverizing, 1:5 ~ 1:30 (g:mL) in mass ratio, add sherwood oil or normal hexane, in 40 ~ 70 DEG C of heating and refluxing extraction 2 ~ 3 times, each 2h ~ 10h, filters, filtrate merges, reduction vaporization recycling design at 40 ~ 60 DEG C, filter residue, through drying under reduced pressure, obtains the degreasing seed dregs of rice;
Second step
By the degreasing seed dregs of rice, be 1:5 ~ 1:30 (g:mL) by mass volume ratio, add distilled water, normal temperature or heating and refluxing extraction or reduced-pressure backflow extract or ultrasonic-assisted extraction or microwave radiation exaraction 2 ~ 3 times, and each 30min ~ 10h, filters, filtrate merges, concentrating under reduced pressure at 50 ~ 70 DEG C, then through vacuum-drying or lyophilize, obtain crude extract; Concentrated solution is 1 ~ 10:30(v/v by volume) ratio add dehydrated alcohol, carry out alcohol precipitation, centrifugally to be precipitated, precipitation is again through ether, acetone and washing with alcohol 2 ~ 3 times, carry out drying again, be lacquer tree seed dregs of rice Crude polysaccharides, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is 28% ~ 32.5%, and yield is 4.4 ~ 6.2%;
3rd step
By lacquer tree seed dregs of rice Crude polysaccharides, add distilled water, be mixed with the polysaccharide soln of 0.5 ~ 1mg/mL, different adsorption stuffing is adopted to carry out decolouring purifying, the volume ratio of the amount and polysaccharide soln that add adsorption stuffing is 1:20 ~ 50 (g:mL), put into oscillator, rotating speed is 150 ~ 200rpm/min, and temperature is 25 ~ 45 DEG C, time 30min ~ 6h, then filter, removing adsorption stuffing, filtrate carries out vacuum or lyophilize, is lacquer tree seed dregs of rice polyoses extract, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 60%.
4th step
Adopt ceramic membrane and ultrafiltration membrane technique to the further grading purification of lacquer tree seed dregs of rice polyoses extract, ceramic membrane mean pore size is 0.1-1 μm, and ultra-filtration membrane molecular weight cut-off scope is 5000-100000D, and operating pressure is 0.1-0.5Mpa, temperature is 30 ~ 50 DEG C, retain concentrated solution, and with distilled water cleaning, merge washing lotion and concentrated solution, vacuum-drying or lyophilize, obtain painting seed polysaccharide, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 90%;
5th step
Paint seed polysaccharide is carried out antioxidation in vitro test, and the lacquer tree seed dregs of rice polyoses extract of 0.1 ~ 0.5mg/mL is 50 ~ 75.3% to the clearance rate of ABTS free radical, and removing rate to the removing of hydroxyl radical free radical is 52.5 ~ 73.8%.
2. the lacquer tree seed dregs of rice in the first step derive from a kind of of Rhus succedanea, lacquer tree and Burma red paint kind to paint the seed dregs of rice of seed after squeezing.
3. in second step, the temperature of heating and refluxing extraction is 50 ~ 90 DEG C, and the vacuum tightness that reduced-pressure backflow extracts is 0.04 ~ 0.1MPa, temperature 50 ~ 70 DEG C, the power of ultrasound assisted extraction is 120 ~ 600W, temperature is 40 ~ 60 DEG C, and the power of microwave radiation exaraction is 500 ~ 800W, temperature 50 ~ 70 DEG C.
4. the adsorption stuffing in the 3rd step be selected from granulated active carbon, 860021, one or more in DM-2, D941, D101, D845, D296.2. the lacquer tree seed dregs of rice in the first step derive from a kind of of Rhus succedanea, lacquer tree and Burma red paint kind to paint the seed dregs of rice of seed after squeezing.
Beneficial effect of the present invention:
The preparation method of a kind of lacquer tree seed dregs of rice polyoses extract provided by the present invention and its preparation, also there is following characteristics:
(1) operating process of the present invention is simple, and condition is easy to control, can volume facilityization produce;
(2) the paint seed polysaccharide solvability of gained of the present invention is good, has good antioxygenation;
(3) this invention can be turned waste into wealth simultaneously, promotes the added value of the lacquer tree seed dregs of rice, improves the comprehensive utilization ratio of lacquer tree seed.
Accompanying drawing explanation
Fig. 1 is that paint seed polysaccharide is to the restraining effect figure of ABTS free radical;
Fig. 2 is that paint seed polysaccharide is to the restraining effect figure of hydroxy radical qiao.
Embodiment
Following examples are citings more of the present invention, should not be seen as limitation of the invention.
Embodiment 1 Phenol sulfuric acid procedure measures polysaccharide content
Glucose standard curve makes: take glucose 0.01g, surely be dissolved in 50 mL volumetric flasks with distilled water, get 8 10 mL test tube labels (blank, 1, 2, 7), draw Standard glucose solution 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.9 mL, be placed in 1-7 test tube respectively, adding distilled water to final volume is 1.0 mL, add 5%(w/w again) phenol 1.0 mL, concussion shakes up, slowly add the vitriol oil 3.5 mL, rapid concussion shakes up, 250 μ L are drawn to 96 orifice plates after developing the color 25 minutes under room temperature, every sample three holes, 490 nm places measure absorbancy, average, distilled water is blank.Take glucose concn as X-coordinate, absorbance is ordinate zou drawing standard curve; The linear equation obtaining typical curve is y=0.0081x-0.0139(R
2=0.9992) two linear variable displacement good relationship, are shown.
Crude polysaccharides assay: accurately take 0.05 g Crude polysaccharides in 50 mL volumetric flasks, fixed molten with distilled water, draw 0.3 respectively, in 0.5,0.7,0.9 mL solution and 10mL test tube, adding distilled water to final volume is 1.0mL, then adds 5%(w/w) phenol 1.0 mL, concussion shakes up, slowly add the vitriol oil 3.5 mL, rapid concussion shakes up, and draws 250 μ Ls to 96 orifice plates, every sample three holes after developing the color 25 minutes under room temperature, 490 nm places measure absorbancy, average.
Embodiment 2
By dry for the Rhus succedanea seed dregs of rice, pulverize, 1:5 (g:mL), adds sherwood oil in mass ratio, and in 40 heating and refluxing extraction 3 times, each 10h, filters, and filtrate merges, reduction vaporization recycling design at 40 DEG C, and filter residue, through drying under reduced pressure, obtains the degreasing seed dregs of rice; By the degreasing seed dregs of rice, be 1:5 (g:mL), add distilled water by mass volume ratio, extract at room temperature 3 times, each 10h, filter, filtrate merges, concentrating under reduced pressure at 70 DEG C, then through vacuum-drying, obtains crude extract; Concentrated solution is 1:10(v/v by volume) ratio add dehydrated alcohol, carry out alcohol precipitation, centrifugally to be precipitated, precipitation is again through ether, acetone and washing with alcohol 3 times, carry out drying again, be lacquer tree seed dregs of rice Crude polysaccharides, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is 28%, and yield is 4.4%; By lacquer tree seed dregs of rice Crude polysaccharides, add distilled water, be mixed with the polysaccharide soln of 0.5mg/mL; adopt granulated active carbon to decolour, the volume ratio of the amount and polysaccharide soln that add adsorption stuffing is 1:50 (g:mL), puts into oscillator; rotating speed is 150rpm/min, and temperature is 45 DEG C, time 2h; then filter; removing adsorption stuffing, filtrate carries out vacuum, is lacquer tree seed dregs of rice polyoses extract; the content of Crude polysaccharides is measured, polysaccharide content 61.2% with sulfuric acid-phynol method.Adopt ceramic membrane and ultrafiltration membrane technique to the further grading purification of lacquer tree seed dregs of rice polyoses extract, ceramic membrane mean pore size is 0.1-1 μm, and ultra-filtration membrane molecular weight cut-off scope is 5000-100000D, and operating pressure is 0.1-0.5Mpa, temperature is 30 ~ 50 DEG C, retain concentrated solution, and with distilled water cleaning, merge washing lotion and concentrated solution, vacuum-drying or lyophilize, obtain painting seed polysaccharide, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 92%.
Embodiment 3
By dry for the lacquer tree seed dregs of rice, pulverize, 1:30 (g:mL), adds normal hexane in mass ratio, and in 70 DEG C of heating and refluxing extraction 2 times, each 2h, filters, and filtrate merges, reduction vaporization recycling design at 60 DEG C, and filter residue, through drying under reduced pressure, obtains the degreasing seed dregs of rice; By the degreasing seed dregs of rice, be 1:30 (g:mL), add distilled water, heating and refluxing extraction by mass volume ratio, Extracting temperature 50 ~ 90 DEG C, extract 2 times, each 2h, filter, filtrate merges, concentrating under reduced pressure at 50 DEG C, then through lyophilize, obtains crude extract; Concentrated solution is 1:20(v/v by volume) ratio add dehydrated alcohol, carry out alcohol precipitation, centrifugally to be precipitated, precipitation is again through ether, acetone and washing with alcohol 2 times, carry out drying again, be lacquer tree seed dregs of rice Crude polysaccharides, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is 30.5%, and yield is 4.8%; By lacquer tree seed dregs of rice Crude polysaccharides, add distilled water, be mixed with the polysaccharide soln of 1mg/mL, adopt 860021 resins to carry out decolouring purifying, the volume ratio of the amount and polysaccharide soln that add adsorption stuffing is 1:20 (g:mL), puts into oscillator, rotating speed is 200rpm/min, and temperature is 25 DEG C, time 30min, then filter, removing adsorption stuffing, filtrate carries out lyophilize, is lacquer tree seed dregs of rice polyoses extract, the content of Crude polysaccharides is measured, polysaccharide content 60.5% with sulfuric acid-phynol method.Adopt ceramic membrane and ultrafiltration membrane technique to the further grading purification of lacquer tree seed dregs of rice polyoses extract, ceramic membrane mean pore size is 0.1-1 μm, and ultra-filtration membrane molecular weight cut-off scope is 5000-100000D, and operating pressure is 0.1-0.5Mpa, temperature is 30 ~ 50 DEG C, retain concentrated solution, and with distilled water cleaning, merge washing lotion and concentrated solution, vacuum-drying or lyophilize, obtain painting seed polysaccharide, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 91.5%.
Embodiment 4
Red for the Burma lacquer tree seed dregs of rice are dry, pulverize, 1:20 (g:mL), adds sherwood oil in mass ratio, in 50 DEG C of heating and refluxing extraction 3 times, each 5h, filters, and filtrate merges, reduction vaporization recycling design at 50 DEG C, filter residue, through drying under reduced pressure, obtains the degreasing seed dregs of rice; By the degreasing seed dregs of rice, be 1:15 (g:mL) by mass volume ratio, add distilled water, reduced-pressure backflow extracts 3 times, each 1h, and vacuum tightness is 0.04 ~ 0.1Mpa, filters, and filtrate merges, concentrating under reduced pressure at 50 DEG C, then through vacuum-drying, obtains crude extract; Concentrated solution is 1:20(v/v by volume) ratio add dehydrated alcohol, carry out alcohol precipitation, centrifugally to be precipitated, precipitation is again through ether, acetone and washing with alcohol 2 times, carry out drying again, be lacquer tree seed dregs of rice Crude polysaccharides, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is 32.5%, and yield is 6.2%; By lacquer tree seed dregs of rice Crude polysaccharides, add distilled water, be mixed with the polysaccharide soln of 1mg/mL, adopt DM-2 resin to carry out decolouring purifying, the volume ratio of the amount and polysaccharide soln that add adsorption stuffing is 1:30 (g:mL), puts into oscillator, rotating speed is 150rpm/min, and temperature is 30 DEG C, time 1h, then filter, removing adsorption stuffing, filtrate carries out lyophilize, is lacquer tree seed dregs of rice polyoses extract, the content of Crude polysaccharides is measured, polysaccharide content 62.3% with sulfuric acid-phynol method.Adopt ceramic membrane and ultrafiltration membrane technique to the further grading purification of lacquer tree seed dregs of rice polyoses extract, ceramic membrane mean pore size is 0.1-1 μm, and ultra-filtration membrane molecular weight cut-off scope is 5000-100000D, and operating pressure is 0.1-0.5Mpa, temperature is 30 ~ 50 DEG C, retain concentrated solution, and with distilled water cleaning, merge washing lotion and concentrated solution, vacuum-drying or lyophilize, obtain painting seed polysaccharide, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 94.5%.
Embodiment 5
By dry for the lacquer tree seed dregs of rice, pulverize, 1:5 ~ 1:30 (g:mL), adds normal hexane in mass ratio, in 40 ~ 70 DEG C of heating and refluxing extraction 2 ~ 3 times, each 2h ~ 10h, filters, and filtrate merges, reduction vaporization recycling design at 40 ~ 60 DEG C, filter residue, through drying under reduced pressure, obtains the degreasing seed dregs of rice; By the degreasing seed dregs of rice, be 1:5 ~ 1:30 (g:mL) by mass volume ratio, add distilled water, ultrasonic-assisted extraction 2 ~ 3 times, each 30min, ultrasonic power is 120 ~ 600W, filters, and filtrate merges, concentrating under reduced pressure at 50 ~ 70 DEG C, then through vacuum-drying, obtain crude extract; Concentrated solution is 1 ~ 10:30(v/v by volume) ratio add dehydrated alcohol, carry out alcohol precipitation, centrifugally to be precipitated, precipitation is again through ether, acetone and washing with alcohol 2 ~ 3 times, carry out drying again, be lacquer tree seed dregs of rice Crude polysaccharides, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is 30.2%, and yield is 5.21%; By lacquer tree seed dregs of rice Crude polysaccharides, add distilled water, be mixed with the polysaccharide soln of 0.5 ~ 1mg/mL, D941 resin is adopted to carry out decolouring purifying, the volume ratio of the amount and polysaccharide soln that add adsorption stuffing is 1:20 ~ 50 (g:mL), put into oscillator, rotating speed is 150 ~ 200rpm/min, and temperature is 25 ~ 45 DEG C, time 30min ~ 6h, then filter, removing adsorption stuffing, filtrate carries out vacuum or lyophilize, is lacquer tree seed dregs of rice polyoses extract, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 62.3%.Adopt ceramic membrane and ultrafiltration membrane technique to the further grading purification of lacquer tree seed dregs of rice polyoses extract, ceramic membrane mean pore size is 0.1-1 μm, and ultra-filtration membrane molecular weight cut-off scope is 5000-100000D, and operating pressure is 0.1-0.5Mpa, temperature is 30 ~ 50 DEG C, retain concentrated solution, and with distilled water cleaning, merge washing lotion and concentrated solution, vacuum-drying or lyophilize, obtain painting seed polysaccharide, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 95.4%.
Embodiment 6
By dry for the lacquer tree seed dregs of rice, pulverize, 1:5 ~ 1:30 (g:mL), adds normal hexane in mass ratio, in 40 ~ 70 DEG C of heating and refluxing extraction 2 ~ 3 times, each 2h ~ 10h, filters, and filtrate merges, reduction vaporization recycling design at 40 ~ 60 DEG C, filter residue, through drying under reduced pressure, obtains the degreasing seed dregs of rice; By the degreasing seed dregs of rice, be 1:5 ~ 1:30 (g:mL) by mass volume ratio, add distilled water, microwave radiation exaraction 2 ~ 3 times, each 30min ~ 1h, microwave power is 500 ~ 800W, filters, and filtrate merges, concentrating under reduced pressure at 50 ~ 70 DEG C, then through lyophilize, obtain crude extract; Concentrated solution is 1 ~ 10:30(v/v by volume) ratio add dehydrated alcohol, carry out alcohol precipitation, centrifugally to be precipitated, precipitation is again through ether, acetone and washing with alcohol 2 ~ 3 times, carry out drying again, be lacquer tree seed dregs of rice Crude polysaccharides, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is 29.2%, and yield is 6.01%; By lacquer tree seed dregs of rice Crude polysaccharides, add distilled water, be mixed with the polysaccharide soln of 0.5 ~ 1mg/mL, D101 resin is adopted to carry out decolouring purifying, the volume ratio of the amount and polysaccharide soln that add adsorption stuffing is 1:20 ~ 50 (g:mL), put into oscillator, rotating speed is 150 ~ 200rpm/min, and temperature is 25 ~ 45 DEG C, time 4h ~ 6h, then filter, removing adsorption stuffing, filtrate carries out vacuum or lyophilize, is lacquer tree seed dregs of rice polyoses extract, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 63.5%.Adopt ceramic membrane and ultrafiltration membrane technique to the further grading purification of lacquer tree seed dregs of rice polyoses extract, ceramic membrane mean pore size is 0.1-1 μm, and ultra-filtration membrane molecular weight cut-off scope is 5000-100000D, and operating pressure is 0.1-0.5Mpa, temperature is 30 ~ 50 DEG C, retain concentrated solution, and with distilled water cleaning, merge washing lotion and concentrated solution, vacuum-drying or lyophilize, obtain painting seed polysaccharide, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 93.2%.
Embodiment 7
Red for the Burma lacquer tree seed dregs of rice are dry, pulverize, 1:20 (g:mL), adds sherwood oil in mass ratio, in 50 DEG C of heating and refluxing extraction 3 times, each 5h, filters, and filtrate merges, reduction vaporization recycling design at 50 DEG C, filter residue, through drying under reduced pressure, obtains the degreasing seed dregs of rice; By the degreasing seed dregs of rice, be 1:15 (g:mL) by mass volume ratio, add distilled water, reduced-pressure backflow extracts 3 times, each 1h, and vacuum tightness is 0.04 ~ 0.1Mpa, filters, and filtrate merges, concentrating under reduced pressure at 50 DEG C, then through vacuum-drying, obtains crude extract; Concentrated solution is 1:20(v/v by volume) ratio add dehydrated alcohol, carry out alcohol precipitation, centrifugally to be precipitated, precipitation is again through ether, acetone and washing with alcohol 2 times, carry out drying again, be lacquer tree seed dregs of rice Crude polysaccharides, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is 32.5%, and yield is 6.2%; By lacquer tree seed dregs of rice Crude polysaccharides, add distilled water, be mixed with the polysaccharide soln of 1mg/mL, adopt D845 resin to carry out decolouring purifying, the volume ratio of the amount and polysaccharide soln that add adsorption stuffing is 1:30 (g:mL), puts into oscillator, rotating speed is 150rpm/min, and temperature is 30 DEG C, time 6h, then filter, removing adsorption stuffing, filtrate carries out lyophilize, is lacquer tree seed dregs of rice polyoses extract, the content of Crude polysaccharides is measured, polysaccharide content 62.8% with sulfuric acid-phynol method.Adopt ceramic membrane and ultrafiltration membrane technique to the further grading purification of lacquer tree seed dregs of rice polyoses extract, ceramic membrane mean pore size is 0.1-1 μm, and ultra-filtration membrane molecular weight cut-off scope is 5000-100000D, and operating pressure is 0.1-0.5Mpa, temperature is 30 ~ 50 DEG C, retain concentrated solution, and with distilled water cleaning, merge washing lotion and concentrated solution, vacuum-drying or lyophilize, obtain painting seed polysaccharide, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 94.2%.
Embodiment 9
By dry for the lacquer tree seed dregs of rice, pulverize, 1:20 (g:mL), adds sherwood oil in mass ratio, and in 50 DEG C of heating and refluxing extraction 3 times, each 5h, filters, and filtrate merges, reduction vaporization recycling design at 50 DEG C, and filter residue, through drying under reduced pressure, obtains the degreasing seed dregs of rice; By the degreasing seed dregs of rice, be 1:15 (g:mL) by mass volume ratio, add distilled water, reduced-pressure backflow extracts 3 times, each 1h, and vacuum tightness is 0.04 ~ 0.1Mpa, filters, and filtrate merges, concentrating under reduced pressure at 50 DEG C, then through vacuum-drying, obtains crude extract; Concentrated solution is 1:20(v/v by volume) ratio add dehydrated alcohol, carry out alcohol precipitation, centrifugally to be precipitated, precipitation is again through ether, acetone and washing with alcohol 2 times, carry out drying again, be lacquer tree seed dregs of rice Crude polysaccharides, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is 32.5%, and yield is 6.2%; By lacquer tree seed dregs of rice Crude polysaccharides, add distilled water, be mixed with the polysaccharide soln of 1mg/mL, adopt D296 resin to carry out decolouring purifying, the volume ratio of the amount and polysaccharide soln that add adsorption stuffing is 1:30 (g:mL), puts into oscillator, rotating speed is 200rpm/min, and temperature is 45 DEG C, time 2h, then filter, removing adsorption stuffing, filtrate carries out lyophilize, is lacquer tree seed dregs of rice polyoses extract, the content of Crude polysaccharides is measured, polysaccharide content 63.8% with sulfuric acid-phynol method.Adopt ceramic membrane and ultrafiltration membrane technique to the further grading purification of lacquer tree seed dregs of rice polyoses extract, ceramic membrane mean pore size is 0.1-1 μm, and ultra-filtration membrane molecular weight cut-off scope is 5000-100000D, and operating pressure is 0.1-0.5Mpa, temperature is 30 ~ 50 DEG C, retain concentrated solution, and with distilled water cleaning, merge washing lotion and concentrated solution, vacuum-drying or lyophilize, obtain painting seed polysaccharide, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 95.4%.
Embodiment 10
Accurately take Vc 50 mg in 100mL volumetric flask, fixed molten with distilled water, be mixed with the solution of 0.5 mg/mL, then once dilute the solution not obtained six concentration by multiple, be respectively 0.1 mg/mL, 0.05 mg/mL, 0.01 mg/mL, 0.005 mg/mL, 0.002 mg/mL, 0.001 mg/mL, 0.0005 mg/mL, for follow-up mensuration.
Accurately take lacquer tree seed dregs of rice polyoses extract 0.5 g in 100 mL volumetric flasks, fixed molten with distilled water, be mixed with 5 mg/mL, the aqueous solution, then dilute different multiples and obtain the solution of six concentration, be respectively 1 mg/mL, 0.5 mg/mL, 0.1 mg/mL, 0.05 mg/mL, 0.01 mg/mL, 0.005mg/mL are used for subsequent measurements.
A lacquer tree seed dregs of rice polyoses extract is to the scavenging(action) of ABTS free radical
The making of storing solution: by the ABTS solution of 7.4mmol/L and the potassium persulfate solution balanced mix of 2.6mmol/L, at dark place normal temperature standing and reacting 15h, then get the dilution of 1mL solution with methanol, obtaining the absorbancy of solution at 732nm place is 0.74.
Get the above-mentioned storing solution that the lacquer tree polyphenol extract of different concns and polyoses extract 150 μ L add 2.85mL, mix, at dark place normal temperature standing and reacting 2h, survey absorbancy at 732nm place, count A1.Using blank solvent as blank, at dark place normal temperature standing and reacting 2h, survey absorbancy at 732nm place, count A2.
ABTS clearance rate=((A2-A1)/A2) × 100%
In formula, A1-testing sample, the ABTS absorbancy under sample solvent exists system existence.The absorbancy of A2-ABTS under sample solvent exists.
B lacquer tree seed dregs of rice polyoses extract is to the scavenging(action) of hydroxy radical qiao
Be the FeSO of 9mmol/L by the polysaccharide soln of 1mL and 1mL concentration
4solution and 1mL concentration are 9mmol/L Whitfield's ointment ethanolic soln and 1mL concentration is the H of 8.8mmol/L
2o
2solution is water-bath 30min at 37 DEG C, under 510nm, survey light absorption value
a 1; Respectively with distilled water replace polysaccharide soln, 1mL polysaccharide soln mixes with 3mL distilled water, surveys light absorption value under the same conditions
a 0with
a 2, tall bottle with spout jujube polysaccharide calculates, with V by formula the clearance rate of hydroxyl radical free radical
cin contrast.
Clearance rate=((A0-(A1-A2))/A0) × 100%
Testing data as depicted in figs. 1 and 2.
As seen from Figure 1, the Scavenging activity of polysaccharide to ABTS free radical represents with clearance rate, and clearance rate is higher, illustrates that the antioxygenation of polysaccharide is stronger.The lacquer tree seed dregs of rice polyoses extract of 0.1 mg/mL and Vc are respectively 50% and 79.2% to ABTS free radical inhibiting rate, the lacquer tree seed dregs of rice polyoses extract of 0.5mg/mL and Vc are respectively 75.3% and 94.5% to ABTS free radical inhibiting rate, it is 0.025mg/mL to the concentration of the IC50 value Vc of ABTS free radical, and the concentration of lacquer tree seed dregs of rice polyoses extract is 0.1mg/mL.As seen from Figure 2, the paint seed polysaccharide of 0.1 mg/mL and Vc are respectively 52.5% and 70.5% to hydroxy radical qiao inhibiting rate, the paint seed polysaccharide of 0.5mg/mL and Vc are respectively 73.8% and 90.5% to hydroxy radical qiao inhibiting rate, it is 0.05mg/mL to the concentration of the IC50 value Vc of hydroxy radical qiao, and the concentration of lacquer tree seed dregs of rice polyoses extract is 0.95mg/mL.
Claims (4)
1. there is a preparation method for the paint seed polysaccharide of anti-oxidant activity, it is characterized in that being made up of following steps:
The first step
The lacquer tree seed dregs of rice are dry, pulverizing, 1:5 ~ 1:30 (g:mL) in mass ratio, add sherwood oil or normal hexane, in 40 ~ 70 DEG C of heating and refluxing extraction 2 ~ 3 times, each 2h ~ 10h, filters, filtrate merges, reduction vaporization recycling design at 40 ~ 60 DEG C, filter residue, through drying under reduced pressure, obtains the degreasing seed dregs of rice;
Second step
By the degreasing seed dregs of rice, be 1:5 ~ 1:30 (g:mL) by mass volume ratio, add distilled water, normal temperature or heating and refluxing extraction or reduced-pressure backflow extract or ultrasonic-assisted extraction or microwave radiation exaraction 2 ~ 3 times, and each 30min ~ 10h, filters, filtrate merges, concentrating under reduced pressure at 50 ~ 70 DEG C, then through vacuum-drying or lyophilize, obtain crude extract; Concentrated solution is 1 ~ 10:30(v/v by volume) ratio add dehydrated alcohol, carry out alcohol precipitation, centrifugally to be precipitated, precipitation is again through ether, acetone and washing with alcohol 2 ~ 3 times, carry out drying again, be lacquer tree seed dregs of rice Crude polysaccharides, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is 28% ~ 32.5%, and yield is 4.4 ~ 6.2%;
3rd step
By lacquer tree seed dregs of rice Crude polysaccharides; add distilled water; be mixed with the polysaccharide soln of 0.5 ~ 1mg/mL; different adsorption stuffing is adopted to carry out decolouring purifying; the volume ratio of the amount and polysaccharide soln that add adsorption stuffing is 1:20 ~ 50 (g:mL); put into oscillator; rotating speed is 150 ~ 200rpm/min, and temperature is 25 ~ 45 DEG C, time 30min ~ 6h; then filter; removing adsorption stuffing, filtrate carries out vacuum or lyophilize, is lacquer tree seed dregs of rice polyoses extract; measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 60%;
4th step
Adopt ceramic membrane and ultrafiltration membrane technique to the further grading purification of lacquer tree seed dregs of rice polyoses extract, ceramic membrane mean pore size is 0.1-1 μm, and ultra-filtration membrane molecular weight cut-off scope is 5000-100000D, and operating pressure is 0.1-0.5Mpa, temperature is 30 ~ 50 DEG C, retain concentrated solution, and with distilled water cleaning, merge washing lotion and concentrated solution, vacuum-drying or lyophilize, obtain painting seed polysaccharide, measure the content of Crude polysaccharides with sulfuric acid-phynol method, polysaccharide content is greater than 90%;
5th step
Paint seed polysaccharide is carried out antioxidation in vitro test, and the lacquer tree seed dregs of rice polyoses extract of 0.1 ~ 0.5mg/mL is 50 ~ 75.3% to the clearance rate of ABTS free radical, and removing rate to the removing of hydroxyl radical free radical is 52.5 ~ 73.8%.
2. a kind of preparation method with the paint seed polysaccharide of anti-oxidant activity according to claim 1, is characterized in that the lacquer tree seed dregs of rice in the first step derive from a kind of of Rhus succedanea, lacquer tree and Burma red paint kind to paint the seed dregs of rice of seed after squeezing.
3. a kind of preparation method with the paint seed polysaccharide of anti-oxidant activity according to claim 1, it is characterized in that the temperature of heating and refluxing extraction in second step is 50 ~ 90 DEG C, the vacuum tightness that reduced-pressure backflow extracts is 0.04 ~ 0.1MPa, temperature 50 ~ 70 DEG C, the power of ultrasound assisted extraction is 120 ~ 600W, temperature is 40 ~ 60 DEG C, and the power of microwave radiation exaraction is 500 ~ 800W, temperature 50 ~ 70 DEG C.
4. a kind of preparation method with the paint seed polysaccharide of anti-oxidant activity according to claim 1, the adsorption stuffing that it is characterized in that in the 3rd step be selected from granulated active carbon, 860021, one or more in DM-2, D941, D101, D845, D296.
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| CN110538712A (en) * | 2019-09-16 | 2019-12-06 | 河南省予北粮油机械有限公司 | Deparaffinization production method of lacquer seeds |
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