CN103030704A - Method for preparing efficient oligomerized astragalus polysaccharide by carboxymethylated molecular modification - Google Patents
Method for preparing efficient oligomerized astragalus polysaccharide by carboxymethylated molecular modification Download PDFInfo
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- CN103030704A CN103030704A CN2012105549459A CN201210554945A CN103030704A CN 103030704 A CN103030704 A CN 103030704A CN 2012105549459 A CN2012105549459 A CN 2012105549459A CN 201210554945 A CN201210554945 A CN 201210554945A CN 103030704 A CN103030704 A CN 103030704A
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- carboxymethylation
- acetic acid
- sodium hydroxide
- astragalus polysaccharides
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Abstract
The invention discloses a method for preparing efficient oligomerized astragalus polysaccharide by carboxymethylated molecular modification, which relates to the technical field of preparation and structural improvement of biological active polysaccharides. The preparation method comprises the following steps: carrying out carboxymethylated molecular modification by using a sodium hydroxide-monochloro acetic acid system; using product volume, carboxymethyl substitutivity and immune regulatory function as indexes, using reaction temperature, reagent proportioning and reaction time as factors, and optimizing the optimal condition of the sodium hydroxide-monochloro acetic acid modification for astragalus polysaccharide by an orthogonal test as reaction temperature at 65 DEG C, proportion of sodium hydroxide and monochloro acetic acid: 1.6:1, and reaction time 3.5h; slowly adding astragalus decocted liquor to ethanol to meet the final concentration of 50%, and removing precipitate; adding ethanol into liquid supernatant to meet the final concentration of 60%; taking precipitate to obtain coarse polysaccharide; and modifying purified astragalus polysaccharide by optimized condition. Compared with feeding experiment and research, the growth promoting and immunocompetence enhancing activity of purified astragalus polysaccharide after carboxymethylated molecular modification is the strongest. The oligomerized astragalus polysaccharide has good regulatory activity and resistance to oxidation, and materials and time are saved.
Description
Technical field:
The present invention relates to preparation and the structure of modification technical field of bioactive polysaccharide, be specifically related to the method that a kind of carboxymethylation molecular modification prepares efficient oligomeric astragalus polysaccharides.
Background technology:
Research is in recent years found to contain the plurality of active ingredients such as polysaccharide, saponin, flavones, amino acid in the Radix Astragali, and these activeconstituentss all have enhancing antibody to generate and immunoreactive effect, wherein are reported as many with the research to astragalus polysaccharides.Astragalus polysaccharides is one of main active ingredient of the Radix Astragali, extracts, is isolated through high-tech.
Its immunoregulation activity of the astragalus polysaccharides of extraction and resistance to oxidation are poor at present, the waste starting material, and lose time.
Summary of the invention:
The purpose of this invention is to provide the method that a kind of carboxymethylation molecular modification prepares efficient oligomeric astragalus polysaccharides, it has good regulation activity and resistance to oxidation, and economical with materials is saved time.
In order to solve the existing problem of background technology, the present invention adopts following technical scheme: its preparation method is: adopt sodium hydroxide-Monochloro Acetic Acid system method to carry out the carboxymethylation molecular modification, take product amount, carboxymethyl substitution degree and immunoregulation function as index, take temperature of reaction, reagent proportioning and reaction times as factor, optimizing the top condition that astragalus polysaccharides sodium hydroxide-Monochloro Acetic Acid method modifies with orthogonal test is 65 ℃ of temperature of reaction; The proportioning of sodium hydroxide and Monochloro Acetic Acid 1.6: 1, reaction times 3.5h.Get the Radix Astragali decocting liquid slow ethanol that the adds first time and make final concentration reach 50%, go precipitation; Supernatant liquor adds ethanol again makes final concentration reach 60%, get precipitation and obtain Crude polysaccharides, use the condition modification of optimizing behind the astragalus polysaccharides purifying, by the feeding experiment research and comparison, the gained astragalus polysaccharides is purified and carboxymethylation is modified rear its growth promotion and the enhancing immunocompetence is the strongest.
Carboxymethylation astragalus polysaccharides preparation condition is:
Take by weighing the 10g astragalus polysaccharides of above-mentioned preparation, be added in the 100mL isopropanol solvent, add 1% sodium hydroxide solution 90mL, after stirring makes the alkali lye dissolving, under the rapid stirring condition, (1: 1.2,50mL), the control temperature was 65 ℃ of stirring reactions 3.5 hours to splash into the aqueous isopropanol of Monochloro Acetic Acid by the vacuum dropping funnel.After reaction finishes the upper strata Virahol being inclined and recycles, and lower floor adds 5% hydrochloric acid soln, adds ethanol 50mL, filtration under the rapid stirring, solids is partially dissolved in the water, with the ethanol precipitation, filters, drying gets the white powder solid, i.e. the carboxymethyl astragalus polysaccharides.
The physical and chemical property determining of carboxymethylation astragalus polysaccharides:
One, determination of polysaccharide:
The content of former astragalus polysaccharides is up to 98.46%, and the polysaccharide content of carboxymethylation astragalus polysaccharides all is lower than former astragalus polysaccharides, shows that it is lossy that carboxymethylation is modified for polysaccharide content.By the carboxymethylation polysaccharide yield as can be known top condition be 65 ℃ of temperature of reaction; The proportioning of sodium hydroxide and Monochloro Acetic Acid 1.6: 1, reaction times 3.5h.Its substitution value is 0.82.
The present invention has good regulation activity and resistance to oxidation, and economical with materials is saved time.
Description of drawings:
Fig. 1 is L among the present invention
9(3
4) the quadrature synoptic diagram,
Fig. 2 is that carboxymethylation is modified the synoptic diagram that affects on astragalus polysaccharides content among the present invention,
Fig. 3 is carboxymethylation astragalus polysaccharides infrared spectra synoptic diagram among the present invention,
Fig. 4 be in the present embodiment daily ration carboxymethylation astragalus polysaccharides on the synoptic diagram that affects of growth of meat chicken performance,
Fig. 5 be in the present embodiment daily ration carboxymethylation astragalus polysaccharides on the synoptic diagram that affects of broiler chicken Immune Organs Index,
Fig. 6 is that daily ration carboxymethylation astragalus polysaccharides exempts to exempt from rear different steps serum BSA antibody titer (OD with two to broiler chicken one in the present embodiment
450) affect synoptic diagram,
Fig. 7 be in the present embodiment daily ration carboxymethylation astragalus polysaccharides on the synoptic diagram that affects of broiler chicken blood lymphocytes nutrient solution cytokine,
Fig. 8 be in the present embodiment daily ration carboxymethylation astragalus polysaccharides on the synoptic diagram that affects of broiler chicken periphery Whole blood lymphocyte transformation efficiency,
Fig. 9 be in the present embodiment daily ration carboxymethylation astragalus polysaccharides on the synoptic diagram that affects of broiler chicken spleen antioxidase and lipid peroxide.
Embodiment:
Referring to Fig. 1-Fig. 3, this embodiment adopts following technical scheme: its preparation method is: adopt sodium hydroxide-Monochloro Acetic Acid system method to carry out the carboxymethylation molecular modification, take product amount, carboxymethyl substitution degree and immunoregulation function as index, take temperature of reaction, reagent proportioning and reaction times as factor, optimize proportioning 1: 6 that top condition that astragalus polysaccharides sodium hydroxide-Monochloro Acetic Acid method modifies is 95 ℃ of temperature of reaction, chlorsulfonic acid and pyridine, react 1h with orthogonal test; Get the Radix Astragali decocting liquid slow ethanol that the adds first time and make final concentration reach 50%, go precipitation; Supernatant liquor adds ethanol again makes final concentration reach 60%, get precipitation and obtain Crude polysaccharides, modify with the condition of optimizing behind the astragalus polysaccharides purifying, antiviral and strengthen immunocompetence relatively by series, it was antiviral and to strengthen immunocompetence the strongest after the purified and carboxymethylation of gained astragalus polysaccharides was modified.
Carboxymethylation astragalus polysaccharides preparation condition is:
With temperature of reaction A, sodium hydroxide and Monochloro Acetic Acid mol ratio B and reaction times C are factor, by the orthogonal test L9 (3 of 3 factors, 3 levels
3) design 9 kinds of modification conditions (referring to Fig. 1):
Take by weighing the 10g astragalus polysaccharides of above-mentioned preparation, be added in the 100mL isopropanol solvent, add 1% sodium hydroxide solution 90mL, after stirring makes the alkali lye dissolving, under the rapid stirring condition, (1: 1.2,50mL), the control temperature was 65 ℃ of stirring reactions 3.5 hours to splash into the aqueous isopropanol of Monochloro Acetic Acid by the vacuum dropping funnel.After reaction finishes the upper strata Virahol being inclined and recycles, and lower floor adds 5% hydrochloric acid soln, adds ethanol 50mL, filtration under the rapid stirring, solids is partially dissolved in the water, with the ethanol precipitation, filters, drying gets the white powder solid, i.e. the carboxymethyl astragalus polysaccharides.
The physical and chemical property determining of carboxymethylation astragalus polysaccharides:
One, determination of polysaccharide:
Referring to Fig. 2, the content of former astragalus polysaccharides is up to 98.46%, and the polysaccharide content of carboxymethylation astragalus polysaccharides all is lower than former astragalus polysaccharides, shows that it is lossy that carboxymethylation is modified for polysaccharide content.By the carboxymethylation polysaccharide yield as can be known top condition be 65 ℃ of temperature of reaction; The proportioning of sodium hydroxide and Monochloro Acetic Acid 1.6: 1, reaction times 3.5h.Its substitution value is 0.82.
As shown in Figure 3, on the infrared spectra of carboxymethylation astragalus polysaccharides, 1604,1424, the charateristic avsorption band of COO-appearred in the 1327cm-1 place, the asymmetric stretching vibration absorption peak of C=O that one-COO-is arranged about 1604cm-1,1423, two peaks, 1327cm-1 place be in strong peak, proved the certain own carboxymethylation of astragalus polysaccharides.
This embodiment has good regulation activity and resistance to oxidation, and economical with materials is saved time.
Embodiment:
One, materials and methods:
1.1 experimental animal and material:
The male broiler chicken of 120 AA (Arbor Acres) (1 age in days) are divided into 2 processing at random, and each processes 6 repetitions, and each repeats 10 chickens.Illumination every day 23 hours, free choice feeding is freely drunk water.Raising is undertaken by normal feeding standard.
1.2 test daily ration:
The trophic level preparation that experimental basis daily ration Diet Formula is recommended with reference to NRC (1994), test is prepared by this laboratory with the carboxymethylation polysaccharide.
1.3 sample collecting and analysis:
1.3.1 growth performance:
Test chicken the 21st, 42 ages in days repeat to weigh respectively as unit take each, statistics stage food consumption, mortality ratio and material anharmonic ratio.
1.3.2 blood lymphocytes cytokine secretion:
Gather 21d, the aseptic heparin sodium anticoagulation of 42d is placed with (anticoagulation: lymphocyte separation medium 1: 2) in the centrifuge tube of lymphocyte separation medium in advance along the slow adding of tube wall, the centrifugal 30min of 4800rpm, buffy coat in the middle of drawing, use the cell culture fluid washed twice, and be made into 1 * 10 with the RPMI1640 nutrient solution
7Individual/mL lymphocyte suspension liquid.Above-mentioned lymphocyte is inoculated in 24 porocyte culture plates, and add ConA to make ultimate density be 45ug/mL in every hole, 37 ℃ of 5%CO
2Cultivate 48h under the environment and collect culture supernatant, centrifugal frozen.The mensuration of nutrient solution IL-2, IL-4 concentration adopts measured by radioimmunoassay.
1.3.4 Antioxidant Indexes:
Measure enteron aisle and spleen GSH-Px, SOD, CAT and MDA level.
1.4 data statistics:
8.02 editions statistical softwares of the data SAS carry out data analysis, and numerical value represents with Mean ± SD, ANOVA variance analysis, Duncan ' s multiple comparisons.
Two, results and analysis
2.1 production performance
Referring to Fig. 4, in the present embodiment, add the carboxymethylation polysaccharide in the daily ration full phase broiler chicken body weight gains, food consumption are not made significant difference, add the carboxymethylation polysaccharide in the daily ration full phase broiler chicken body weight gains, food consumption are not made significant difference.
2.2 Immune Organs Index
Referring to Fig. 5, daily ration interpolation CAPS does not reach conspicuous level (P＞0.05) to the impact of 21 ages in days, 42 age in days broiler chicken thymus gland and French capsule index.At 42 ages in days, CAPS group index and spleen index is starkly lower than control group.
2.3 serum BSA antibody titer
Referring to Fig. 6, one exempted from behind the BSA the 7th day, and antibody titer is without significant difference between carboxymethylation APS and the control group; One exempted from rear the 10th day, and carboxymethylation APS interpolation group antibody titer is apparently higher than control group; One exempted from the 14th day, and significant difference disappears again between BSA antibody horizontal group; Two exempted from behind the BSA the 3rd day and the 6th day, and carboxymethylation APS group antibody titer is apparently higher than control group; Exempted from each treatment group significance of difference disappearance the 9th day to two.Antibody titer is the result show, adds carboxymethylation APS in the daily ration and can improve the broiler chicken humoral immune reaction.Annotate: data are with optical density(OD) (OD
450) expression; Different shoulder mark expression significant differences (P＜0.05) in the same column.
2.4 lymphocyte cytokine
Referring to Fig. 7, blood lymphocytes nutrient solution IL-2, IL-4 detected result see Table 5.Control group 21 age in days IL-2 levels significantly are lower than CAPS group (P＜0.0001); During 42 age in days, CAPS group IL-2 level is higher than control group, but without the statistical significance of difference (P=0.1070).During 42 age in days, the CAPS group IL-4 level utmost point is significantly higher than control group (P=0.0004).The cytokine statistics shows: CAPS can obviously strengthen CD4+T emiocytosis IL-2, IL-4.
2.5 lymphocyte transformation rate
Referring to Fig. 8, lymphocyte transformation rate is no matter stimulating former is ConA or LPS, and carboxymethylation APS organizes 21 age in days lymphocyte proliferation activity apparently higher than control group (P＜0.05); At 42 ages in days, the broiler chicken lymphocyte of the carboxymethylation of searching for food APS stimulates through LPS, and its lymphocyte proliferation activity is higher than control group.
2.6 broiler chicken resistance to oxidation index of correlation detected result
Referring to Fig. 9, it is active that CAPS can strengthen 21 age in days broiler chicken spleen GSH-Px.Add CAPS so that broiler chicken spleen 21 age in days SOD activity are improved, but difference does not reach significantly flat accurate.Compare with control group, it is active that CAPS can significantly promote 42 age in days broiler chicken CAT; All there is significant difference in spleen MDA content at 21 ages in days, 42 ages in days between control group and APS group, shows that body adds CAPS and increases antioxidant ability of organism.
Claims (1)
1. the carboxymethylation molecular modification prepares the method for efficient oligomeric astragalus polysaccharides, the preparation method who it is characterized in that it is: adopt sodium hydroxide-Monochloro Acetic Acid system method to carry out the carboxymethylation molecular modification, take product amount, carboxymethyl substitution degree and immunoregulation function as index, take temperature of reaction, reagent proportioning and reaction times as factor, optimizing the top condition that astragalus polysaccharides sodium hydroxide-Monochloro Acetic Acid method modifies with orthogonal test is 65 ℃ of temperature of reaction; The proportioning of sodium hydroxide and Monochloro Acetic Acid 1.6: 1, reaction times 3.5h.Get the Radix Astragali decocting liquid slow ethanol that the adds first time and make final concentration reach 50%, go precipitation; Supernatant liquor adds ethanol again makes final concentration reach 60%, get precipitation and obtain Crude polysaccharides, use the condition modification of optimizing behind the astragalus polysaccharides purifying, by the feeding experiment research and comparison, the gained astragalus polysaccharides is purified and carboxymethylation is modified rear its growth promotion and the enhancing immunocompetence is the strongest.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104892788A (en) * | 2015-06-24 | 2015-09-09 | 河南中烟工业有限责任公司 | Carboxymethylation astragalus polysaccharide, synthesis and purification method and application of carboxymethylation astragalus polysaccharide as tobacco humectant |
| CN105440150A (en) * | 2016-01-07 | 2016-03-30 | 广西医科大学 | Carboxymethyl longan pulp polysaccharide and pharmaceutical application thereof |
| CN106962974A (en) * | 2017-04-21 | 2017-07-21 | 常州市奥普泰科光电有限公司 | A kind of preparation method of improved plant source polysaccharide-based tobacco sense organ humectant |
| CN117717522A (en) * | 2023-12-12 | 2024-03-19 | 佛山科学技术学院 | Carboxymethylated astragalus polysaccharide liposome and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970579A (en) * | 2006-11-28 | 2007-05-30 | 戴甲木 | High substitution degree carboxymethyl indianbread polysaccharide and its preparation method and uses |
| CN101362805A (en) * | 2008-09-24 | 2009-02-11 | 南京农业大学 | Extraction of astragalus polysaccharide and molecular modification method thereof |
-
2012
- 2012-12-05 CN CN2012105549459A patent/CN103030704A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970579A (en) * | 2006-11-28 | 2007-05-30 | 戴甲木 | High substitution degree carboxymethyl indianbread polysaccharide and its preparation method and uses |
| CN101362805A (en) * | 2008-09-24 | 2009-02-11 | 南京农业大学 | Extraction of astragalus polysaccharide and molecular modification method thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104892788A (en) * | 2015-06-24 | 2015-09-09 | 河南中烟工业有限责任公司 | Carboxymethylation astragalus polysaccharide, synthesis and purification method and application of carboxymethylation astragalus polysaccharide as tobacco humectant |
| CN105440150A (en) * | 2016-01-07 | 2016-03-30 | 广西医科大学 | Carboxymethyl longan pulp polysaccharide and pharmaceutical application thereof |
| CN105440150B (en) * | 2016-01-07 | 2018-04-10 | 广西医科大学 | A kind of carboxy methylation Arillus longan polysaccharide and its application in pharmacy |
| CN106962974A (en) * | 2017-04-21 | 2017-07-21 | 常州市奥普泰科光电有限公司 | A kind of preparation method of improved plant source polysaccharide-based tobacco sense organ humectant |
| CN117717522A (en) * | 2023-12-12 | 2024-03-19 | 佛山科学技术学院 | Carboxymethylated astragalus polysaccharide liposome and preparation method thereof |
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Application publication date: 20130410 |