CN108707201B - Arabinogalactan oligosaccharide and preparation and application thereof - Google Patents

Arabinogalactan oligosaccharide and preparation and application thereof Download PDF

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CN108707201B
CN108707201B CN201810413040.7A CN201810413040A CN108707201B CN 108707201 B CN108707201 B CN 108707201B CN 201810413040 A CN201810413040 A CN 201810413040A CN 108707201 B CN108707201 B CN 108707201B
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arabinogalactan
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贺亮
程俊文
王衍彬
张海芸
韦朝阳
魏海龙
柏明娥
童晓青
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Zhejiang Academy of Forestry
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Abstract

The invention discloses arabinogalactan oligosaccharide and preparation and application thereof. The arabinogalactan oligosaccharide polysaccharide consists of polysaccharide with the weight percentage of more than 99 percent, wherein the polysaccharide consists of arabinose with the weight percentage of 3.0 percent and galactose with the weight percentage of 97.0 percent; the weight average molecular weight is 25 KDa. The preparation method comprises the step of directionally degrading arabinogalactan by adopting ultrasonic-assisted free radicals in a mild environment with the pH value of 6.0-8.0 to obtain the arabinogalactan oligosaccharide with the weight-average molecular weight of 25KDa, greatly shortens the degradation time, and solves the problem that the polysaccharide cannot be degraded to a certain fixed molecular weight in the prior art. The arabinogalactan oligosaccharide has obviously enhanced bioactivity and strong anti-oxygen free radical activity, can be directly used as antioxidant or used for preparing antioxidant, and can be used in food additive, health product and medicine fields.

Description

Arabinogalactan oligosaccharide and preparation and application thereof
Technical Field
The invention belongs to the technical field of polysaccharide, and particularly relates to arabinogalactan oligosaccharide and preparation and application thereof.
Background
Arabinogalactans (AG) are a long, highly branched class of hemicelluloses consisting of arabinose and galactose. The main chain is galactan, the branches are mainly arabinose side chains, and the side chains are connected with galactan chains through beta-1, 3 bonds or beta-1, 6 bonds. This sugar is contained in large amounts in the xylem of conifers, in particular up to 25% in larch (Larix). In recent years, researches show that the arabinogalactan is an important bioactive substance and has active functions of immunoregulation, tumor resistance, intestinal function regulation and the like.
Foreign research on arabinogalactans started earlier and was approved by the U.S. FDA in 2002Approved as a food additive. However, the molecular weight of the arabinogalactan is large, the activity is low, and the arabinogalactan can enter an organism to play the bioactivity only by degrading a molecular chain to improve the activity. Foreign research has found that arabinogalactan can be used as a drug carrier, but has weak activity as a functional drug. The research shows that the directional degradation of polysaccharide can improve the bioactivity of polysaccharide, and the degradation of pectin polysaccharide is more, but no report is found on arabinogalactan. In the existing research on the degradation of other polysaccharides, Chinese patent application CN 106478829A discloses a preparation method of hericium erinaceus active polysaccharide, wherein the hericium erinaceus active polysaccharide obtained by ultrasonic degradation is simple and convenient to operate, the molecular weight of the hericium erinaceus polysaccharide can be degraded from 239 ten thousand Da to 20-50-ten thousand Da, and the polysaccharide has the advantages of low viscosity, good solubility and good in vitro immunity. Chinese patent ZL201310085095.7 discloses a method for preparing functional polysaccharide and oligosaccharide with small molecular weight by using lycium barbarum polysaccharide, wherein ultrasonic method and degradation agent are adopted to degrade lycium barbarum polysaccharide, and ethanol with a certain volume is added in the degradation process. Chinese patent ZL03112409.7 discloses a free radical degradation method of polysaccharide, which adopts organic acid and H2O2And a vitamin C degradation system for degrading the polysaccharide, wherein the system can control the reaction activity of oxygen free radicals and reduce the toxicity of the oxygen free radicals; the research shows that the reaction condition of the free radical degradation method is mild, the production cost is low, the production process is feasible, and the molecular weight of the polysaccharide can be controlled within the range of 2KDa-30 KDa. Chinese patent application CN 106046187A discloses a free radical degradation product of Abelmoschus manihot stem and leaf polysaccharide with immunity improving effect and its preparation method, the research adopts H2O2And degrading and purifying a Vc system to obtain a free radical degradation product of the Abelmoschus manihot stem-leaf polysaccharide. Although the effect of degrading polysaccharide by a free radical method is good, the polysaccharide degradation by the free radical method is difficult to control, the amount of free radicals is too much, and the molecular chains of randomly cut polysaccharide are correspondingly increased and irregular; and the molecular weight of the polysaccharide is degraded too much, resulting in polysaccharide activityCannot be fully exerted; however, the amount of free radicals is too small to attack the target site of the polysaccharide chain, and thus polysaccharide having a desired molecular weight cannot be obtained. Therefore, it is difficult to achieve the desired effect by radical degradation of the polysaccharide alone. The existing polysaccharide degradation method can not realize the directional degradation of the polysaccharide to obtain the sugar with a fixed molecular weight at present.
Disclosure of Invention
The object of the present invention is to provide an arabinogalactan oligosaccharide which has biological activity and significantly enhanced activity as compared to arabinogalactan.
The invention also aims to provide a preparation method of the arabinogalactan oligosaccharide, which adopts ultrasonic-assisted free radicals to directionally degrade the arabinogalactan in a mild environment with the pH value of 6.0-8.0 to obtain the oligosaccharide with small molecular weight, can greatly shorten the degradation time, obtains the arabinogalactan oligosaccharide (Oligo-AG) with certain molecular weight after degradation, and obviously enhances the activity of the oligosaccharide with certain molecular weight.
The invention also provides application of the arabinogalactan oligosaccharide, which has a strong oxygen radical scavenging effect, can be used as an antioxidant and can also be used for preparing the antioxidant.
The technical scheme adopted by the invention for solving the technical problems is as follows:
an arabinogalactan oligosaccharide, which consists of more than 99 percent of polysaccharide by weight, wherein the polysaccharide consists of 3.0 percent of arabinose by weight and 97.0 percent of galactose by weight; the weight average molecular weight of the arabinogalactan oligosaccharide is 25 KDa.
The preparation method of the arabinogalactan oligosaccharide comprises the following steps:
(1) and (3) degradation: dissolving arabinogalactan in water to obtain arabinogalactan aqueous solution, adding 0.1-1.6 mmol/L copper acetate (Cu (Ac))2) Adjusting the pH of the aqueous solution to 6.0-8.0 by using 0.1-2.5 mol/L NaOH aqueous solution; preheating at 40-80 deg.c, adding H in 1-6 vol%2O2Mixing the aqueous solution and the mixture; heating and stirring the mixture in ultrasonic waves at 40-80 ℃ for 40-80 min to react; then adding the mass percentage concentration0.1％-0.9％NaHSO3Terminating the reaction by using an aqueous solution; centrifuging to remove the precipitate to obtain a supernatant;
in the presence of H2O2And before ultrasonic degradation, 0.1mmol/L-1.6mmol/L copper acetate aqueous solution is added to mainly play the role of a catalyst, and hydrogen peroxide can quickly generate OH under the catalysis of transition metal to quickly attack sugar chains and capture H atoms connected with carbon to generate hydroxyalkyl radicals, so that the reaction efficiency is greatly improved. Degrading in mild environment with pH value of 6.0-8.0 and assisted by ultrasonic wave2O2Can realize the directional degradation of the arabinogalactan, and then adopts NaHSO with the mass percentage concentration of 0.1-0.9 percent3The aqueous solution is used for stopping the reaction so as to prevent the degradation reaction from being carried out and prevent the polysaccharide from being excessively degraded to obtain the target product.
(2) And (3) dialysis: dialyzing the supernatant obtained in the step (1) in deionized water by using a dialysis bag with the aperture of 4000Da-8000Da, collecting the dialyzed extracting solution, and carrying out vacuum freeze drying to obtain crude polysaccharide;
(3) and (3) purification: dissolving the crude polysaccharide obtained in the step (2) by using deionized water to obtain a crude polysaccharide water solution, purifying by using propylene dextran gel filtration chromatography, detecting polysaccharide peaks of polysaccharide-containing eluent collected by the gel filtration chromatography by using a phenol-sulfuric acid method, and concentrating, dialyzing and freeze-drying the collected polysaccharide-containing eluent with the maximum absorbance to obtain the arabinogalactan oligosaccharide.
In order to achieve better effects of the invention, it is preferable that:
in the step (1), the arabinogalactan can be a commercially available product or prepared by adopting the existing preparation method. For example, arabinogalactans obtained by aqueous extraction and alcohol precipitation can be used.
The arabinogalactan can be arabinogalactan extracted from larch wood. The larch can be northeast larch.
The extraction method of the arabinogalactan comprises the following steps: leaching larch sawdust with hot water at 80-98 deg.C according to a material-to-liquid ratio of 1:10-1:30(m/V, g/mL) for 40-80 min; filtering to obtain primary filtrate, leaching the filter residue with hot water at 80-98 deg.C according to a material-to-liquid ratio of 1:10-1:30(m/V, g/mL) for 40min-80min, filtering to obtain secondary filtrate, collecting all filtrates, and concentrating under reduced pressure at 40-80 deg.C; adding ethanol water solution which accounts for 3-5 times of the volume of the concentrated solution and accounts for 90-95% of the volume fraction of the concentrated solution into the concentrated solution, and standing overnight at 2-6 ℃; centrifuging, collecting precipitate, and drying to obtain arabinogalactan. Wherein the centrifugation condition can be 5000-6000 r/min for 5-10 min.
The weight ratio of the arabinogalactan to the water volume in the aqueous arabinogalactan solution is 0.1g-2.0 g: 100ml-200 ml.
The copper acetate aqueous solution is mainly used as a catalyst, the dosage of the copper acetate aqueous solution is not strictly limited, and the mass ratio of the copper acetate to the arabinogalactan is 1:100-120 in terms of copper acetate.
Said H2O2The amount of the aqueous solution is H2O2Meter, H2O2The mass ratio of the arabinogalactan to the arabinogalactan is 5-10: 100.
In the step (2), the dialysis time in the deionized water is 18-32 h.
In step (3), the concentration of the aqueous solution of the crude polysaccharide is 5mg/mL-20mg/mL, more preferably 5mg/mL-8 mg/mL. During chromatography, the flow rate of the crude polysaccharide water solution is 0.5ml/min-1.2 ml/min.
The conditions of the propylene glucan gel filtration chromatography are as follows: the eluent is 0.05mol/L phosphate buffer solution and 0.15mol/L NaCl aqueous solution, the flow rate is 0.5ml/min, wherein the volume ratio of the phosphate buffer solution to the NaCl aqueous solution is 2-3: 1.
The propylene dextran gel is selected from commercially available products such as SephacrylTmS-100HR, and the like.
The preparation method of the phosphate buffer solution is according to the methods commonly used in the field, and can refer to the Chinese pharmacopoeia of 2005 edition generally.
In the step (3), the dialysis adopts a dialysis bag with the aperture of 4000Da-8000Da to dialyze in the deionized water.
The absorbance was at 510 nm.
The arabinogalactan oligosaccharide has the capability of scavenging oxygen free radicals, and in OH & free radical clearance measurement, under the premise of the same concentration, the OH & free radical clearance of the arabinogalactan oligosaccharide is far greater than that of the arabinogalactan oligosaccharide; the arabinogalactan oligosaccharide has strong anti-oxygen free radical activity, can be directly used as an antioxidant or used for preparing the antioxidant, and can be used in the fields of food additives, health products and medicines.
Compared with the prior art, the invention has the following advantages:
the arabinogalactan oligosaccharide can be fixed to 25KDa in weight average molecular weight, monosaccharide of the polysaccharide part of the arabinogalactan oligosaccharide consists of arabinose and galactose, and the weight percentage ratio of the arabinose to the galactose is 3.0:97.0, so that technical support is provided for the actual application of natural polysaccharide products, and the arabinogalactan oligosaccharide has wide application prospect.
Compared with arabinogalactan, the arabinogalactan oligosaccharide of the invention has obviously enhanced biological activity, obviously enhanced free hydroxyl scavenging capacity and stronger anti-oxygen radical activity, can be directly used as an antioxidant or used for preparing the antioxidant, and can be used in the fields of food additives, health care products and medicines.
The method adopts ultrasonic wave to assist free radicals to directionally degrade arabinogalactan in a mild environment with the pH value of 6.0-8.0 to obtain the low molecular weight oligosaccharide, thereby not only greatly shortening the degradation time, but also obviously reducing the molecular weight of polysaccharide, and being a low-cost, environment-friendly and efficient degradation method.
The method can directionally degrade the arabinogalactan to a fixed molecular weight and find that the activity of the oligosaccharide with the molecular weight is obviously enhanced, thereby solving the problem that the polysaccharide can not be degraded to a certain fixed molecular weight in the prior art.
Drawings
FIG. 1 is a graph showing the absorbance at 510nm of an eluate containing polysaccharides collected from the crude polysaccharides in example 1 by Sephacryl filtration chromatography.
FIG. 2 is a graph showing the results of the absolute molecular mass of degraded arabinogalactan of example 4, wherein the abscissa represents the time to peak (min) and the ordinate represents the corresponding relative value of the apparatus.
FIG. 3 is a GC-MS analysis chromatogram of a monosaccharide standard in example 5, wherein Rham is rhamnose, Fuc is fucose, Ara is arabinose, Xyl is xylose, Man is mannose, Glc is glucose, and Gal is galactose.
FIG. 4 is a GC-MS analysis chromatogram of AG in example 5.
FIG. 5 is a GC-MS analysis chromatogram of Oligo-AG in example 5.
FIG. 6 is a graph comparing the effect of arabinogalactan polysaccharide of example 7 on OH clearance before and after degradation, with polysaccharide concentration (mg/mL) on the abscissa and OH clearance (%) on the ordinate.
FIG. 7 is a graph showing the evaluation of the biological activity of arabinogalactans in example 8.
FIG. 8 is a graph showing the evaluation of the bioactivity of arabinogalactan oligosaccharide in example 8.
Detailed Description
The invention is further illustrated with reference to the following figures and specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Further, it should be understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are also within the scope of the present invention as defined in the appended claims.
Example 1
Extraction of arabinogalactan: weighing a certain amount of larch sawdust in northeast China, and leaching for 80min in hot water at 98 ℃ according to a material-liquid ratio of 1:10(m/V, g/mL); filtering to obtain primary filtrate, extracting the residue with hot water at 98 deg.C according to a material-to-liquid ratio of 1:10(m/V, g/mL) for 80min, and filtering to obtain secondary filtrate; collecting all filtrates obtained by two-time extraction, and concentrating under reduced pressure at 40 deg.C; adding ethanol water solution with volume fraction of 95% and volume of 5 times of the concentrated solution into the concentrated solution, and standing at 6 deg.C overnight; centrifuging at 5000r/min for 10 min; collecting the precipitate, and vacuum drying to obtain arabinogalactan.
Preparation of arabinogalactan oligosaccharide:
(1) and (3) degradation: dissolving 2.0g of the extracted arabinogalactan in 100ml of distilled water, and heating for dissolving; 68.75mL of 1.6mmol/L Cu (Ac) was added2Adjusting the pH of the aqueous solution to 8.0 by using 2.5mol/L NaOH aqueous solution; preheating in 40 deg.C ultrasonic instrument, adding 1.16mL of 6% H2O2Mixing the aqueous solution and the mixture; starting ultrasonic wave, heating and stirring at 40 ℃ for reaction for 80 min; adding NaHSO with the mass percentage concentration of 0.9 percent3The reaction was terminated with an aqueous solution. Centrifuging to remove the precipitate and obtaining the supernatant.
(2) And (3) dialysis: and (2) dialyzing the supernatant obtained in the step (1) in deionized water for 32h by using a dialysis bag with the aperture of 4000Da, removing oligosaccharide, pigment, organic solvent, inorganic salt and the like in the process, collecting the dialyzed extract, and performing vacuum freeze drying to obtain crude polysaccharide.
(3) And (3) purification: dissolving the crude polysaccharide obtained in the step (2) by using deionized water to obtain a crude polysaccharide water solution of 5 mg/ml; subjecting the crude polysaccharide aqueous solution to Sephacryl gel filtration chromatography (Sephacryl)TmS-100HR) is further purified, the specification of a chromatographic column is 2.6cm × 100cm, the sample loading amount is 5ml, the flow rate of a crude polysaccharide aqueous solution is 0.5ml/min, the eluent is 0.05mol/L phosphate buffer solution (pH7.0) +0.15mol/L NaCl aqueous solution (wherein the volume ratio of the phosphate buffer solution to the NaCl aqueous solution is 2:1), the flow rate of the eluent is 0.5ml/min, the polysaccharide peak of the eluent containing polysaccharide collected by gel filtration chromatography is detected by a phenol-sulfuric acid method, the eluent containing polysaccharide and having the maximum absorbance is concentrated, dialyzed by a dialysis bag with the pore diameter of 4000Da and freeze-dried to obtain the arabinogalactan oligosaccharide, which is named as Oligo-AG.
The Abs values (absorbance) of the polysaccharides measured according to the phenol-sulfuric acid method of step (3) were plotted, as shown in fig. 1. Collecting the sample (with Abs value of 1) in the test tube with the highest absorption peak (18 th tube) in FIG. 1, i.e. the eluate containing polysaccharide with the highest absorbance, concentrating, dialyzing, and lyophilizing to obtain the product, i.e. arabinogalactan, named Oligo-AG.
The weight average molecular weight of Oligo-AG was determined to be 25 kDa.
Example 2
Extraction of arabinogalactan: weighing a certain amount of larch sawdust in northeast China, and leaching with hot water at 80 ℃ for 40min according to a material-liquid ratio of 1:30(m/V, g/mL); filtering to obtain primary filtrate, extracting the residue with hot water at 80 deg.C at a ratio of 1:30(m/V, g/mL) for 40min, and filtering to obtain secondary filtrate; collecting all filtrates obtained by two-time extraction, and concentrating under reduced pressure at 80 deg.C; adding ethanol water solution which accounts for 3 times of the volume of the concentrated solution and accounts for 90% of the volume of the concentrated solution into the concentrated solution, and standing overnight at 2 ℃; centrifuging at 6000r/min for 5 min; collecting the precipitate, and vacuum drying to obtain arabinogalactan.
Preparation of arabinogalactan oligosaccharide:
(1) and (3) degradation: dissolving 0.1g of the extracted arabinogalactan in 100ml of distilled water, and heating to dissolve; 45.88mL of 0.1mmol/L Cu (Ac) was added2Adjusting the pH of the aqueous solution to 6.0 by using 0.1mol/L NaOH aqueous solution; preheating in an ultrasonic instrument at 80 ℃, adding 0.69mL of H with the volume fraction of 1 percent2O2Mixing the aqueous solution and the mixture; starting ultrasonic wave, heating and stirring at 80 ℃ for reaction for 40 min; adding NaHSO with the mass percentage concentration of 0.1 percent3The reaction was terminated with an aqueous solution. Centrifuging to remove the precipitate and obtaining the supernatant.
(2) And (3) dialysis: and (2) dialyzing the supernatant obtained in the step (1) in deionized water for 20h by using a dialysis bag with the aperture of 8000Da, removing oligosaccharide, pigment, organic solvent, inorganic salt and the like in the process, collecting the dialyzed extract, and performing vacuum freeze drying to obtain crude polysaccharide.
(3) And (3) purification: dissolving the crude polysaccharide obtained in the step (2) by using deionized water to obtain a crude polysaccharide water solution of 6 mg/ml; subjecting the crude polysaccharide aqueous solution to Sephacryl gel filtration chromatography (Sephacryl)TmS-100HR) is further purified, the specification of a chromatographic column is 2.6cm × 100cm, the sample loading amount is 5ml, the flow rate of a crude polysaccharide aqueous solution is 1.2ml/min, the eluent is 0.05mol/L phosphate buffer solution (pH7.0) +0.15mol/L NaCl aqueous solution (wherein the volume ratio of the phosphate buffer solution to the NaCl aqueous solution is 2:1), the flow rate of the eluent is 0.5ml/min, the polysaccharide peak of the eluent containing polysaccharide collected by gel filtration chromatography is detected by a phenol-sulfuric acid method, the collected eluent containing polysaccharide with the maximum absorbance at 510nm is subjected to concentration, dialysis by a dialysis bag with the aperture of 8000Da and freeze-drying to obtain the polysaccharide AA arabinogalactan oligosaccharide, designated Oligo-AG.
The weight average molecular weight of Oligo-AG was determined to be 25 kDa.
Example 3
Extraction of arabinogalactan: weighing a certain amount of larch sawdust in northeast China, and leaching in hot water at 88 ℃ for 65min according to a material-liquid ratio of 1:20(m/V, g/mL); filtering to obtain primary filtrate, extracting the filter residue with hot water at 88 deg.C according to a material-to-liquid ratio of 1:20(m/V, g/mL) for 65min, and filtering to obtain secondary filtrate; collecting all filtrates obtained by two-time extraction, and concentrating under reduced pressure at 65 deg.C; adding ethanol water solution with volume fraction of 95% 4 times of the volume of the concentrated solution into the concentrated solution, and standing at 4 deg.C overnight; centrifuging at 6000r/min for 6 min; collecting the precipitate, and vacuum drying to obtain arabinogalactan.
Preparation of arabinogalactan oligosaccharide:
(1) and (3) degradation: dissolving 1.2g of the extracted arabinogalactan in 200ml of distilled water, and heating for dissolving; adding 60.06mL of 1.0mmol/L Cu (Ac)2Adjusting the pH of the aqueous solution to 7.0 by using 1.5mol/L NaOH aqueous solution; preheating in 70 deg.C ultrasonic instrument, adding 2.22mL of H with volume fraction of 3%2O2Mixing the aqueous solution and the mixture; starting ultrasonic waves, heating and stirring at 60 ℃ and reacting for 60 min; adding NaHSO with the mass percentage concentration of 0.6 percent3The reaction was terminated with an aqueous solution. Centrifuging to remove the precipitate and obtaining the supernatant.
(2) And (3) dialysis: and (2) dialyzing the supernatant obtained in the step (1) in deionized water for 18h by using a dialysis bag with the pore diameter of 6000Da, removing oligosaccharide, pigment, organic solvent, inorganic salt and the like in the process, collecting the dialyzed extract, and performing vacuum freeze drying to obtain crude polysaccharide.
(3) And (3) purification: dissolving the crude polysaccharide obtained in the step (2) by using deionized water to obtain 8mg/ml crude polysaccharide water solution; subjecting the crude polysaccharide aqueous solution to Sephacryl gel filtration chromatography (Sephacryl)TmS-100HR) of 2.6cm × 100cm, a sample loading of 5ml, a flow rate of the crude polysaccharide aqueous solution of 1.0ml/min, and an eluent of 0.05mol/L phosphate buffer solution (pH7.0) +0.15mol/L NaCl aqueous solution (wherein the volume ratio of the phosphate buffer solution to the NaCl aqueous solution is 3:1) and detecting polysaccharide peak of the collected polysaccharide-containing eluent by using a phenol-sulfuric acid method through gel filtration chromatography, concentrating the collected polysaccharide-containing eluent with the maximum absorbance at 510nm, dialyzing by using a dialysis bag with the pore diameter of 6000Da, and freeze-drying to obtain the arabinogalactan oligosaccharide, wherein the eluent is named Oligo-AG.
The weight average molecular weight of Oligo-AG was determined to be 25 kDa.
The following are examples of the structural identification or performance analysis of Oligo-AG:
example 4: molecular weight detection
The specific test flow is as follows: the sample volume is 200-300 μ L (i.e. 2-3 times the sample ring volume), data is collected for 60min, and the column temperature is room temperature. Preparing 0.1mol/L NaNO3Aqueous solution (containing NaN with mass percent concentration of 0.02 percent)3) As mobile phase, pass through 0.22 μm membrane, ultrasonic degassing for 30 min. 3mg of the sample was weighed and dissolved in 1mL0.1mol/L NaNO3Aqueous solution (containing NaN with mass percent concentration of 0.02 percent)3) In the process, the mixture is dissolved for 4 hours by magnetic stirring and then passes through a 0.22 mu m film. The results of the absolute weight average molecular weight of the polysaccharide after degradation using the crude polysaccharide of example 1 are shown in FIG. 2.
FIG. 2 is a graph showing the results of the absolute molecular weights of the degradation effects of the polysaccharides by ultrasonic-assisted radical degradation of the present invention, and it can be seen from FIG. 2 that the ultrasonic-assisted radical degradation method of the present invention enhances the degradation in the large molecular weight range, can obtain a large number of molecules with a molecular weight of 10kDa, and can achieve a small molecular weight percentage of 7kDa to 25kDa of 60% (calculated according to the peak area ratio). And the activity of the arabinogalactan oligosaccharide with the absolute weight-average molecular mass (Mw) of 25KDa is obviously increased through light scattering measurement, which lays a good foundation for orientation control and application of the arabinogalactan oligosaccharide.
Example 5: and (5) performing GC-MS analysis.
Hydrolysis of polysaccharide: samples of 2mg AG and 2mg Oligo-AG were weighed, respectively, added with 2mol/L aqueous trifluoroacetic acid (TFA), sealed, hydrolyzed in a vacuum oven at 120 ℃ for 4 hours, cooled to room temperature, added with 0.5ml of methanol, and spin-dried at 40 ℃ under reduced pressure for 4 times (2 ml for each addition of methanol) to completely remove TFA.
Preparation of the derivatives: each of the spun-dried standards and samples was opened to the air and placed in a desiccator, the bottom of which was placed a petri dish containing phosphorus pentoxide. The desiccator was sealed, evacuated, and dried overnight.
And (3) derivatization of monosaccharide and sample monosaccharide, namely adding 1ml of hydroxylamine hydrochloride solution into the dried products respectively, sealing and fully shaking the products, and oxidizing the products in a vacuum drying oven at 90 ℃ for 30 minutes. Cooled to room temperature, added with 0.2ml of 1-methylimidazole and 1ml of acetic anhydride and mixed well by shaking. Sealing, and reacting in a vacuum drying oven at 90 deg.C for 30 min. Cooling to room temperature formed the acetylated derivative.
To the acetylated derivative was added 1ml of chloroform (chloroform) and 1ml of water. Fully shaking. Standing for several minutes to be layered, and removing the upper aqueous phase. Then 1ml of water was added, followed by shaking, standing and removal of the aqueous phase. Repeat for 5 times. The chloroform layer was then dehydrated by adding an appropriate amount of anhydrous sodium sulfate. The chloroform layer was aspirated by a syringe with needle, filtered through a 0.45nm organic filter, and placed in a sample bottle for GC detection.
The chromatographic analysis conditions are HP-Innowax capillary column (250 mu m is multiplied by 30m), the liquid film thickness is 0.20 mu m, the chromatographic column temperature is 190 ℃, the vaporization chamber temperature is 280 ℃, the split ratio is 10:1, the detection temperature is 250 ℃ and the sample injection amount is 1 mu L by using an FID detector; the carrier gas was helium, and the gas flow rate was 1 mL/min.
GC-MS analysis of AG and Oligo-AG is as follows:
the GC-MS analysis chromatogram of the monosaccharide standard is shown in FIG. 3, the GC-MS analysis chromatogram of AG in example 1 is shown in FIG. 4, and the GC-MS analysis chromatogram of Oligo-AG in example 1 is shown in FIG. 5.
As can be seen from FIG. 4, AG is composed of polysaccharides with a weight percentage of 99% or more, the monosaccharides of the polysaccharide part are composed of arabinose and galactose, and the weight percentage ratio of arabinose (Ara) to galactose (Gal) is 6.0: 94.0; the AG is a galactose backbone and contains branched polysaccharides.
Corresponding to the monosaccharide standard, Oligo-AG of example 1, 2 or 3 is composed of polysaccharides with weight percentage of 99% or more, monosaccharide of polysaccharide part is composed of galactose and arabinose, mainly galactose, arabinose only occupies a very small amount, and the weight percentage ratio of arabinose to galactose is 3.0:97.0, which indicates that Oligo-AG obtained after AG is degraded is a polysaccharide mainly composed of galactose, and polysaccharide chain branches are very few.
Example 6: determination of hydroxyl radical clearance and comparison of clearance of AG and Oligo-AG for. OH:
the principle is as follows: hydroxyl radical generation using the Fenton reaction: h2O2+Fe2+＝·OH+H2O+Fe3+Salicylic acid is added into a reaction system, hydroxyl radicals generated by Fenton reaction react with the salicylic acid to generate a colored substance with maximum absorption at 510 nm. The absorbance is used to express the hydroxyl radical content.
The method comprises the following specific steps: respectively taking 1mL of 9mmol/L FeSO4Aqueous solution, 1mL of 9mmol/L ethanol salicylic acid solution, 1mL of 8.8mmol/L H2O2Adding 1mL of the prepared sample into the aqueous solution, uniformly mixing, carrying out water bath at 37 ℃ for 30min, using distilled water as a blank, and measuring the absorbance of each solution at 510 nm. The experiment was set up in 3 groups: sample set A1Control group A0Background group A2. The clearance rate calculation method comprises the following steps:
Figure BDA0001648594900000091
in the formula: a. the0Absorbance of the solution without sample; a. the1Is the absorbance of the added sample solution; a. the2The absorbance of the sample itself without the addition of the color developing agent.
0.1mg/mL, 0.3mg/mL, 0.5mg/mL, 0.7mg/mL, 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL of AG aqueous solution and the Oligo-AG aqueous solution obtained in examples 1 to 3 were prepared, respectively, and experiments were performed to compare the removal rates of OH from the samples. The results are shown in FIG. 6.
As can be seen from FIG. 6, the arabinogalactan and arabinogalactan oligosaccharide have certain scavenging effect on hydroxyl radical, and the scavenging effect on hydroxyl radical increases with the increase of polysaccharide concentration in the experimental concentration rangeIs strong. When the mass concentration of the polysaccharide reaches 0.5mg/mL, the clearance rates of AG and Oligo-AG to OH are 37.1 percent and 50.3 percent respectively. Alternatively, IC of Oligo-AG can be obtained by calculation500.497mg/mL, and IC of AG502.058 mg/mL. Under the condition of the same concentration, the OH & free radical clearance rate of the arabinogalactan oligosaccharide is far greater than that of the arabinogalactan. The result shows that Oligo-AG has stronger scavenging action on hydroxyl free radicals, is obviously higher than AG, can be directly used as an antioxidant or used for preparing the antioxidant, and can be used in the fields of food additives, health care products and medicines.
Example 7: evaluation of biological Activity of AG and Oligo-AG
The polysaccharide obtained in example 1 was subjected to Surface Plasmon Resonance (SPR) using a BIAcore 3000 system to determine the binding behavior of galactan and galectin-3 (Gal-3) having different molecular weights. First, Gal-3 was immobilized on a CM5 chip by coupling with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) amine. Samples of arabinogalactan oligosaccharide at different dilution concentrations were loaded at a flow rate of 30. mu.l/min and HBS-EP buffer was separated across the sensor surface. The sensor surface was a surface that was completely regenerated after 3 minutes dissociation by injection of 150mM (mmol/L) lactose.
As can be seen from FIGS. 7 and 8, AG-II (arabinogalactan extracted from larch wood chips is type II arabinogalactan) has a binding strength kd/ka value of 2.58. mu.M (. mu. mol/L); binding strength kd/ka of Oligo-AG was 64.4nM (nmol/L). Compared with arabinogalactan, the binding force between Oligo-AG and Gal-3 of the sample with the weight-average molecular weight of 25KDa obtained by the ultrasonic-assisted free radical method is obviously enhanced, which shows that the bioactivity of Oligo-AG is obviously enhanced relative to AG.
The change of the parameters in the preparation method does not affect the preparation of the arabinogalactan oligosaccharide, so the preparation of the arabinogalactan oligosaccharide can be realized by the combination of any parameter in the preparation method. And will not be described in detail herein.

Claims (9)

1. An arabinogalactan oligosaccharide, which is characterized by consisting of more than 99 percent of polysaccharide by weight, wherein the polysaccharide consists of 3.0 percent of arabinose by weight and 97.0 percent of galactose by weight; the weight average molecular weight of the arabinogalactan oligosaccharide is 25 KDa.
2. The method of producing arabinogalacto-oligosaccharides according to claim 1, comprising the steps of:
(1) and (3) degradation: dissolving arabinogalactan in water to obtain an arabinogalactan aqueous solution, adding a 0.1mmol/L-1.6mmol/L copper acetate aqueous solution, and adjusting pH to 6.0-8.0 with a 0.1mol/L-2.5mol/L NaOH aqueous solution; preheating at 40-80 deg.c, adding H in 1-6 vol%2O2Mixing the aqueous solution and the mixture; heating and stirring the mixture in ultrasonic waves at 40-80 ℃ for 40-80 min to react; then adding NaHSO with the mass percentage concentration of 0.1-0.9%3Terminating the reaction by using an aqueous solution; centrifuging to remove the precipitate to obtain a supernatant;
(2) and (3) dialysis: dialyzing the supernatant obtained in the step (1) in deionized water by using a dialysis bag with the aperture of 4000Da-8000Da, collecting the dialyzed extracting solution, and carrying out vacuum freeze drying to obtain crude polysaccharide;
(3) and (3) purification: dissolving the crude polysaccharide obtained in the step (2) by using deionized water to obtain a crude polysaccharide water solution, purifying by using propylene dextran gel filtration chromatography, detecting polysaccharide peaks of polysaccharide-containing eluent collected by the gel filtration chromatography by using a phenol-sulfuric acid method, and concentrating, dialyzing and freeze-drying the collected polysaccharide-containing eluent with the maximum absorbance to obtain the arabinogalactan oligosaccharide.
3. The method according to claim 2, wherein in the step (1), the arabinogalactan is obtained by water extraction and alcohol precipitation.
4. The method according to claim 2 or 3, wherein in the step (1), the arabinogalactan is an arabinogalactan extracted from larch wood.
5. The method according to claim 4, wherein the arabinogalactan is extracted by a method comprising: leaching larch sawdust with hot water at 80-98 ℃ for 40-80 min according to the material-liquid ratio of 1:10-1: 30; filtering to obtain primary filtrate, leaching the filter residue with hot water at 80-98 deg.C according to a material-liquid ratio of 1:10-1:30 for 40-80 min, filtering to obtain secondary filtrate, collecting all filtrates, and concentrating under reduced pressure at 40-80 deg.C; adding ethanol water solution which accounts for 3-5 times of the volume of the concentrated solution and accounts for 90-95% of the volume fraction of the concentrated solution into the concentrated solution, and standing overnight at 2-6 ℃; centrifuging, collecting precipitate, and drying to obtain arabinogalactan; the unit of the feed-liquid ratio is g/mL.
6. The method according to claim 2, wherein in the step (1), the weight ratio of the arabinogalactan to the water volume in the aqueous arabinogalactan solution is from 0.1g to 2.0 g: 100ml-200 ml.
7. The method according to claim 2, wherein the dialysis time in the deionized water in the step (2) is 18 to 32 hours.
8. The method according to claim 2, wherein in the step (3), the concentration of the aqueous solution of the crude polysaccharide is 5mg/mL to 20mg/mL, and the flow rate is 0.5mL/min to 1.2 mL/min.
9. The method according to claim 2, wherein in the step (3), the conditions for the Sephacryl filtration chromatography are as follows: the eluent is 0.05mol/L phosphate buffer solution and 0.15mol/L NaCl aqueous solution, the flow rate is 0.5ml/min, wherein the volume ratio of the phosphate buffer solution to the NaCl aqueous solution is 2-3: 1.
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