CN110606900A - Method for separating and purifying fructus Jujubae polysaccharide with antioxidant effect - Google Patents
Method for separating and purifying fructus Jujubae polysaccharide with antioxidant effect Download PDFInfo
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- CN110606900A CN110606900A CN201911008949.5A CN201911008949A CN110606900A CN 110606900 A CN110606900 A CN 110606900A CN 201911008949 A CN201911008949 A CN 201911008949A CN 110606900 A CN110606900 A CN 110606900A
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- 238000000034 method Methods 0.000 title claims abstract description 35
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- 241001247821 Ziziphus Species 0.000 claims abstract description 122
- 238000000746 purification Methods 0.000 claims abstract description 39
- 238000000926 separation method Methods 0.000 claims abstract description 33
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- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 6
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- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 3
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- 101100130657 Caenorhabditis elegans zmp-1 gene Proteins 0.000 description 2
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- 229910021577 Iron(II) chloride Inorganic materials 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 2
- HEILIGJNYTWOHU-UHFFFAOYSA-N ethanol 2-hydroxybenzoic acid Chemical compound CCO.OC(=O)C1=CC=CC=C1O HEILIGJNYTWOHU-UHFFFAOYSA-N 0.000 description 2
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- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 235000001456 Elaeagnus latifolia Nutrition 0.000 description 1
- 235000007630 Elaeagnus umbellata var parvifolia Nutrition 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 235000009812 Momordica cochinchinensis Nutrition 0.000 description 1
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- 235000013985 cinnamic acid Nutrition 0.000 description 1
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- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
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- 206010016256 fatigue Diseases 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 239000010200 folin Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
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- 230000007365 immunoregulation Effects 0.000 description 1
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- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
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- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
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- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
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- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Sustainable Development (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a method for separating and purifying a jujuba polysaccharide with an antioxidant effect, which comprises the steps of preparing the jujuba polysaccharide and separating and purifying the jujuba polysaccharide, wherein the step of separating and purifying the jujuba polysaccharide comprises the following steps: firstly, the jujubes polysaccharose passes through a DEAE Sepharose Fast Flow anion exchange column, is sequentially subjected to gradient elution by a PBS solution and a NaCl-PBS solution for separation, and is purified by a Sephacryl S-300 gel column. The three woody jujube antioxidant active polysaccharides PZMP1, PZMP2 and PZMP3 can be obtained by one-time separation and purification through the preparation method, the three woody jujube antioxidant active polysaccharides obtained by separation and purification through the preparation method have high purity which can reach over 90 percent and yield which is 2.95 percent of PZMP1, 14.15 percent of PZMP2 and 14.61 percent of PZMP3 based on the woody jujube crude polysaccharide. In addition, the three types of the oxidation-resistant active polysaccharides of the jujubes obtained by separation and purification have stronger oxidation resistance, provide raw materials for oxidation-resistant functional foods and promote the sustainable development of the jujubes.
Description
Technical Field
The invention belongs to the field of deep processing of foods, relates to a polysaccharide extraction technology, and more particularly relates to a method for separating and purifying a jujuba polysaccharide with an antioxidant effect.
Background
Fructus Jujubae (Ziziphus jujuba Mill.) also called fructus Jujubae, fructus Jujubae through, and spine date is the fruit of Ziziphus jujube Mill of Ziziphus of Rhamnaceae. The fructus Jujubae contains water, cellulose, various vitamins, flavonoids, triterpenes, polysaccharides, nucleosides, etc. Fructus Jujubae has good medical care effect, and can be used for treating anorexia, fatigue, anemia, etc. China is the only country in the world with good jujube tree varieties cultivated in large areas. According to the data of the national statistical bureau of the people's republic of China, the current national red date yield is 734.53 ten thousand tons (2014) and 634.00 thousand tons (2013), which account for more than 90 percent of the total world yield. The jujube trees are widely distributed in China, and are mainly distributed in the Xinjiang jujube area and the yellow river riparian drainage area.
Jiaxian jujubes: the cultivation method is characterized in that the cultivation method is between 37 degrees 41 '47' to 38 degrees 23 '34' in northern latitude and 110 degrees 0 '45' to 110 degrees 45 '10' in east longitude, and the cultivation method is from the coastal areas of yellow river without pollution sources in Jiaxian county, the cultivation history is long, and the tree vitality is strong. According to measurement and analysis, the jujubes in Jiaxian county comprise nutrients such as moisture content (68%), titratable acid (0.32%), soluble solid (27.4%), total sugar (21.9g/100g), reduced Vc (376.2mg/100g), total phenol (541.8mg GAE eq./100g FW), total flavone (255.5mg Rutineq./100 g FW), total procyanidine (184.2mg GSPE eq./100g FW) and cinnamic acid (35.2 mu g/100g FW). The polysaccharide is an important bioactive substance in the red dates, and has various biological activities of immunoregulation, oxidation resistance, tumor resistance, liver protection, blood sugar reduction, intestinal flora regulation and the like; however, because the crude polysaccharide component of the jujubes in Jiaxian county is complex and the quality is difficult to effectively control, the research on the jujubes in Jiaxian county, which are varieties with large potential medicinal values, is less at present.
At present, various methods for separating and purifying jujube polysaccharide exist, but the polysaccharide obtained after separation and purification has limited types and low purity and extraction rate, and no jujube antioxidant active polysaccharide with good antioxidant effect is obtained by separating and purifying jujubes in Jiaxian county with complex polysaccharide components.
The invention content is as follows:
the invention aims to provide a method for separating and purifying the momordica polysaccharide with the antioxidation function aiming at the defects. The three woody jujube antioxidant active polysaccharides PZMP1, PZMP2 and PZMP3 can be obtained by one-time separation and purification through the preparation method, the three woody jujube antioxidant active polysaccharides obtained by separation and purification through the preparation method have high purity which can reach over 90 percent and yield which is 2.95 percent of PZMP1, 14.15 percent of PZMP2 and 14.61 percent of PZMP3 based on the woody jujube crude polysaccharide. In addition, the three types of the oxidation-resistant active polysaccharides of the jujubes obtained by separation and purification have stronger oxidation resistance, provide raw materials for oxidation-resistant functional foods and promote the sustainable development of the jujubes.
The technical scheme of the invention is as follows:
the invention provides a method for separating and purifying a jujuba polysaccharide with an antioxidant effect, which comprises the steps of preparing the jujuba polysaccharide and separating and purifying the jujuba polysaccharide, wherein the step of separating and purifying the jujuba polysaccharide comprises the following steps: firstly, the jujubes polysaccharose passes through a DEAE Sepharose Fast Flow anion exchange column, is sequentially subjected to gradient elution by a PBS solution and a NaCl-PBS solution for separation, and is purified by a Sephacryl S-300 gel column. The concentration of the NaCl-PBS solution may be 0M to 0.3M.
Preferably, the method for separating and purifying the momordica polysaccharide with the antioxidation comprises the steps of preparing the momordica polysaccharide and separating and purifying the momordica polysaccharide, wherein the step of separating and purifying the momordica polysaccharide comprises the following steps: firstly, the jujubes polysaccharose passes through a DEAE Sepharose Fast Flow anion exchange column, is sequentially subjected to gradient elution by a PBS solution and a 0M NaCl-PBS solution for separation, and is purified by a Sephacryl S-300 gel column. The antioxidant active polysaccharide component of the jujubes obtained by separation and purification under the gradient elution condition is named as PZMP 1.
Preferably, the method for separating and purifying the momordica polysaccharide with the antioxidation comprises the steps of preparing the momordica polysaccharide and separating and purifying the momordica polysaccharide, wherein the step of separating and purifying the momordica polysaccharide comprises the following steps: firstly, the jujubes polysaccharose passes through a DEAE Sepharose Fast Flow anion exchange column, and is separated by sequentially carrying out gradient elution by using a PBS solution, a 0M NaCl-PBS solution and a 0.2M NaCl-PBS solution, and then purified by a Sephacryl S-300 gel column. The antioxidant active polysaccharide component of the jujubes obtained by separation and purification under the gradient elution condition is named as PZMP 2.
Preferably, the method for separating and purifying the momordica polysaccharide with the antioxidation comprises the steps of preparing the momordica polysaccharide and separating and purifying the momordica polysaccharide, wherein the step of separating and purifying the momordica polysaccharide comprises the following steps: firstly, the jujubes polysaccharose passes through a DEAE Sepharose Fast Flow anion exchange column, and is separated by gradient elution with a PBS solution, a 0M NaCl-PBS solution, a 0.2M NaCl-PBS solution and a 0.3M NaCl-PBS solution in sequence, and then purified by a Sephacryl S-300 gel column. The antioxidant active polysaccharide component of the jujubes obtained by separation and purification under the gradient elution condition is named as PZMP 3.
The gradient elution rate used in the above separation is 1.0-2.0 mL/min. Preferably, the elution rate is 1.5 mL/min.
In the gradient elution, one column volume is eluted by the PBS solution, and one column volume is eluted by NaCl-PBS solution of each concentration.
The pH of the PBS solution was 6.0.
The purification conditions by Sephacryl S-300 gel column are: eluting with distilled water as eluent at a flow rate of 0.6-1.0mL/min, preferably at a flow rate of 0.8 mL/min.
Dialyzing and desalting polysaccharide components obtained by separating DEAE Sepharose Fast Flow anion exchange column, concentrating, and purifying by Sephacryl S-300 gel column; in the dialysis desalting process, the cut-off molecular weight of the dialysis bag is 3500Da, and the dialysis time is 65-80h, preferably 72 h.
The fructus jujubae polysaccharide is obtained by sequentially performing ultrasonic-assisted hot water extraction, alcohol precipitation and decolorization deproteinization on fructus jujubae.
The material-liquid ratio of the ultrasonic-assisted hot water extraction is 23:1, the ultrasonic power is 420W, the extraction temperature is 60-80 ℃, and the extraction time is 20-40 min. Preferably, the extraction temperature is 70 deg.C and the extraction time is 30 min.
The deproteinization treatment adopts papain-8% trifluoroacetic acid method; the decoloring treatment adopts hydrogen peroxide with the volume 0.4 times that of the aqueous solution of the jujube polysaccharide to carry out water bath treatment; the water bath temperature is 40-50 ℃, and the water bath time is 20-40 min; the volume concentration of the hydrogen peroxide is 30 percent. Preferably, the water bath temperature is 45 ℃ and the water bath time is 30 min.
The method for separating and purifying the momordica polysaccharide with the antioxidation effect comprises the following specific steps:
(1) separation: enabling the aqueous solution of the common jujube polysaccharide to pass through a DEAE Sepharose Fast Flow anion exchange column, sequentially eluting a column volume by using PBS (the pH value is 6.0) and 0M NaCl-PBS respectively in a gradient manner at the elution rate of 1.5mL/min, and collecting a first crude product of the common jujube antioxidant active polysaccharide, which is named ZMP 1; then continuously carrying out gradient elution by using 0.2M NaCl-PBS solution for one column volume, and collecting a second crude product of the woody jujube antioxidant active polysaccharide, which is named ZMP 2; finally, continuously carrying out gradient elution by using 0.3M NaCl-PBS solution for one column volume, and collecting a third crude product of the oxidation-resistant active polysaccharides of the jujubes, wherein the third crude product is named ZMP 3; desalting the three crude ZMP1, ZMP2 and ZMP3 by dialysis respectively, and concentrating for later use;
(2) and (3) purification: purifying the three concentrated crude products of the oxidation resistant active polysaccharides of the jujubes by Sephacryl S-300 gel columns respectively, eluting the crude products by distilled water for one column volume under the condition of the flow rate of 0.8mL/min, and then carrying out reduced pressure concentration and vacuum freeze drying to obtain the three purified oxidation resistant active polysaccharides of the jujubes. Wherein, the xylocarpa anti-oxidation active polysaccharide obtained after ZMP1 purification is named as PZMP1, the xylocarpa anti-oxidation active polysaccharide obtained after ZMP2 purification is named as PZMP2, and the xylocarpa anti-oxidation active polysaccharide obtained after ZMP3 purification is named as PZMP 3.
Preferably, the DEAE Sepharose Fast Flow anion exchange column is a phi 26mm multiplied by 100mm anion exchange column; the Sephacryl S-300 gel column is a 26mm × 100mm gel column. The preparation method of the DEAE Sepharose Fast Flow anion exchange column (phi 26mm multiplied by 100mm) comprises the following steps: the column material is injected into a glass column (phi 26mm multiplied by 100mm) in a glass rod drainage mode after being stirred evenly, and is continuously beaten by a ear washing ball in the process to prevent bubbles from being generated, thus obtaining the DEAEPHAROSE Fast Flow anion exchange column. Both the DEAE Sepharose Fast Flow column and Sephacryl S-300 gel column were purchased from GE Healthcare Life Sciences.
According to the invention, a large number of experimental researches confirm that the polysaccharide of the jujubes is separated by using a DEAE Sepharose Fast Flow anion exchange column, three kinds of purified polysaccharides of the jujubes with antioxidant activity can be simultaneously obtained at one time only by using a Sephacryl S-300 gel column for purification, and the polysaccharides with antioxidant activity of the jujubes, which have higher purity and higher yield, can not be obtained by using other anion exchange columns for separation or using other gel columns for purification.
The invention has the beneficial effects that:
(1) the three xylocarpa anti-oxidation active polysaccharides PZMP1, PZMP2 and PZMP3 obtained by separation and purification in one step by the preparation method disclosed by the invention are high in purity which can reach more than 90% and yield, and the yield is high, wherein the yield is 2.95% in PZMP1, 14.15% in PZMP2 and 14.61% in PZMP3 based on the xylocarpa anti-oxidation active polysaccharides. In addition, the three types of the oxidation-resistant active polysaccharides of the jujubes obtained by separation and purification have stronger oxidation resistance, provide raw materials for oxidation-resistant functional foods and promote the sustainable development of the jujubes.
(2) According to the method, the DEAE Sepharose Fast Flow anion exchange column is adopted to separate the jujubes polysaccharides, the DEAE Sepharose Fast Flow has stable properties, the separation of the three jujubes antioxidant active polysaccharides is facilitated, and the yield of the three jujubes antioxidant active polysaccharides is improved; the purification is carried out by adopting a Sephacryl S-300 gel column, the purification range of the Sephacryl S-300 gel column is beneficial to the purification of three jujube polysaccharides, and the purification efficiency is high and reaches more than 90 percent.
(3) The method adopts NaCl-PBS solution as eluent to carry out gradient elution, is more stable than NaCl solution, can effectively prevent the influence of the change of environmental temperature on the property of the extracted polysaccharide component all the year round, and keeps the stability of the property of the separated polysaccharide component of the jujubes. The gradient elution is sequentially carried out by using the PBS solution and the NaCl-PBS solution, so that the oxidation-resistant active polysaccharides of the jujubes can be obtained by one-time separation, the yield of the polysaccharides of the jujubes is improved, the consistency of the polysaccharide structures of the jujubes is ensured, the consumption of the column is reduced, and the service life of the column is prolonged.
(4) The invention carries out ultrasonic-assisted hot water extraction under specific conditions according to the feed-liquid ratio of 23:1, thereby ensuring that each polysaccharide component in the jujubes is extracted to the maximum extent.
Drawings
FIG. 1 shows the three polysaccharides having antioxidant activity of Elaeagnus angustifolia in test example 21H-NMR spectrum; wherein (A) is PZMP11H-NMR spectrum, (B) of PZMP21H-NMR spectrum, (C) of PZMP31H-NMR spectrum.
FIG. 2 shows the results of the three types of polysaccharides having antioxidant activity of Elaeagnus angustifolia in test example 213A C-NMR spectrum; wherein (A) is PZMP113C-NMR spectrum, (B) of PZMP213C-NMR spectrum, (C) of PZMP313C-NMR spectrum.
Detailed Description
Example 1
A separation and purification preparation method of a fructus jujubae polysaccharide comprises the following steps:
1. preparing the fructus jujubae polysaccharide:
(1) ultrasonic-assisted hot water leaching: selecting dried fructus Jujubae from Jiaxian county, drying at 50 deg.C, cooling, pulverizing with high speed pulverizer, sieving with 100 mesh sieve, and pulverizing the residue on the sieve again in the pulverizer. Carrying out ultrasonic-assisted extraction on a certain mass of jujube powder sample in a water bath at 70 ℃ for 30min at a material-liquid ratio of 23:1, wherein the ultrasonic power is 420W. After centrifugal filtration, the obtained clear solution is concentrated to obtain a concentrated solution.
(2) Alcohol precipitation: adding absolute ethyl alcohol into the concentrated solution obtained in the step (1) while stirring until the volume fraction of the ethyl alcohol in the system reaches 85%, standing overnight at normal temperature, performing suction filtration, taking precipitate, and freeze-drying to obtain the crude polysaccharides of the jujubes.
(3) And (3) decoloring and deproteinizing treatment: dissolving the crude polysaccharides of the jujubes prepared in the step (2) in water, firstly carrying out deproteinization treatment by adopting a papain-8% trifluoroacetic acid method, standing and layering after violent shaking, removing supernate, then adding 30% hydrogen peroxide (volume concentration) of 0.4 time of the volume of the aqueous solution of the jujubes polysaccharides, and carrying out decoloration treatment by keeping the temperature for 30min at the water bath temperature of 45 ℃; and then carrying out secondary concentration and alcohol precipitation, washing the precipitate with acetone, diethyl ether and absolute ethyl alcohol in sequence, redissolving, and carrying out freeze drying to obtain the refined polysaccharides of the jujubes.
2. Separating and purifying the fructus jujubae polysaccharide:
(1) separation: weighing 1.0g of the refined jujubes polysaccharide, dissolving the refined jujubes polysaccharide in distilled water, sampling the solution through a 0.45-micron filter membrane, performing primary chromatography by adopting a DEAE Sepharose Fast Flow anion exchange column (phi 26mm multiplied by 100mm), passing the aqueous solution of the jujubes polysaccharide through the DEAE Sepharose Fast Flow anion exchange column, sequentially eluting one column volume by using PBS solution (pH is 6.0) and 0M NaCl-PBS solution respectively in a gradient manner at an elution rate of 1.5mL/min, and collecting a crude product of the first jujubes antioxidant active polysaccharide, which is named as ZMP 1; then continuously carrying out gradient elution by using 0.2M NaCl-PBS solution for one column volume, and collecting a second crude product of the woody jujube antioxidant active polysaccharide, which is named ZMP 2; and finally, continuously performing gradient elution by using 0.3M NaCl-PBS solution for one column volume, and collecting a third crude product of the oxidation-resistant active polysaccharides of the jujubes, wherein the third crude product is named ZMP 3. Desalting the three crude ZMP1, ZMP2 and ZMP3 for 72h by dialysis (molecular weight cut-off of dialysis bag is 3500Da) respectively to obtain three crude polysaccharides concentrate of fructus Jujubae with antioxidant activity;
(2) and (3) purification: the three concentrated crude products of the oxidation resistant active polysaccharides of the jujubes are purified by Sephacryl S-300 gel columns (phi 26mm multiplied by 100mm), and are eluted by distilled water for one column volume under the condition of 0.8mL/min, and then are subjected to reduced pressure concentration and vacuum freeze drying to obtain the three purified oxidation resistant active polysaccharides of the jujubes. Wherein, the xylocarpa anti-oxidation active polysaccharide obtained after ZMP1 purification is named as PZMP1, the xylocarpa anti-oxidation active polysaccharide obtained after ZMP2 purification is named as PZMP2, and the xylocarpa anti-oxidation active polysaccharide obtained after ZMP3 purification is named as PZMP 3.
Example 2
A separation and purification preparation method of a fructus jujubae polysaccharide comprises the following steps:
1. preparing the fructus jujubae polysaccharide:
(1) ultrasonic-assisted hot water leaching: selecting dried jujubes in Jiaxian county, drying the jujubes at 60 ℃, cooling, crushing the jujubes by using a high-speed crusher, sieving the crushed raw materials by using a 100-mesh sieve, and putting the residues on the sieve into the crusher again for crushing again. A certain mass of jujube powder sample is subjected to ultrasonic assisted extraction for 20min in a water bath at 60 ℃ with the material-liquid ratio of 23:1, and the ultrasonic power is 420W. After centrifugal filtration, the obtained clear solution is concentrated to obtain a concentrated solution.
(2) Alcohol precipitation: adding absolute ethyl alcohol into the concentrated solution obtained in the step (1) while stirring until the volume fraction of the ethyl alcohol in the system reaches 85%, standing overnight at normal temperature, performing suction filtration, taking precipitate, and freeze-drying to obtain the crude polysaccharides of the jujubes.
(3) And (3) decoloring and deproteinizing treatment: dissolving the crude polysaccharides of the jujubes prepared in the step (2) in water, firstly carrying out deproteinization treatment by adopting a papain-8% trifluoroacetic acid method, standing and layering after violent shaking, removing supernate, then adding 30% hydrogen peroxide (volume concentration) which is 0.4 time of the volume of the aqueous solution of the jujubes polysaccharides, and carrying out heat preservation for 50min at the water bath temperature of 50 ℃ for carrying out decoloration treatment; and then carrying out secondary concentration and alcohol precipitation, washing the precipitate with acetone, diethyl ether and absolute ethyl alcohol in sequence, redissolving, and carrying out freeze drying to obtain the refined polysaccharides of the jujubes.
2. Separating and purifying the fructus jujubae polysaccharide:
(1) separation: weighing 1.0g of the refined fructus jujubae polysaccharide, dissolving the refined fructus jujubae polysaccharide in distilled water, sampling the solution through a 0.45 mu M filter membrane, performing primary chromatography by adopting a DEAE Sepharose Fast Flow anion exchange column (phi 26mm multiplied by 100mm), passing the aqueous solution of the fructus jubae polysaccharide through the DEAE Sepharose Fast Flow anion exchange column, sequentially eluting one column volume by using PBS solution (pH is 6.0) and 0M NaCl-PBS solution respectively in a gradient manner at an elution rate of 1.0mL/min, and collecting a crude ZMP1 of the fructus jubae antioxidant active polysaccharide; desalting the crude product ZMP1 for 72h by dialysis (the cut-off molecular weight of a dialysis bag is 3500Da) to obtain a concentrated solution of the crude product of the antioxidant activity polysaccharide of the jujubes for later use;
(2) and (3) purification: purifying the concentrated crude ZMP1 of the oxidation-resistant active polysaccharides of the jujubes by a Sephacryl S-300 gel column (phi 26mm multiplied by 100mm), eluting by distilled water for one column volume under the condition of 0.6mL/min flow rate, and then carrying out reduced pressure concentration and vacuum freeze drying to obtain the purified oxidation-resistant active polysaccharides PZMP1 of the jujubes.
Example 3
A separation and purification preparation method of a fructus jujubae polysaccharide comprises the following steps:
1. preparing the fructus jujubae polysaccharide:
(1) ultrasonic-assisted hot water leaching: selecting dried fructus Jujubae from Jiaxian county, drying at 50 deg.C, cooling, pulverizing with high speed pulverizer, sieving with 100 mesh sieve, and pulverizing the residue on the sieve again in the pulverizer. Carrying out ultrasonic-assisted extraction on a certain mass of jujube powder sample in a water bath at 70 ℃ for 30min at a material-liquid ratio of 23:1, wherein the ultrasonic power is 420W. After centrifugal filtration, the obtained clear solution is concentrated to obtain a concentrated solution.
(2) Alcohol precipitation: adding absolute ethyl alcohol into the concentrated solution obtained in the step (1) while stirring until the volume fraction of the ethyl alcohol in the system reaches 85%, standing overnight at normal temperature, performing suction filtration, taking precipitate, and freeze-drying to obtain the crude polysaccharides of the jujubes.
(3) And (3) decoloring and deproteinizing treatment: dissolving the crude polysaccharides of the jujubes prepared in the step (2) in water, firstly carrying out deproteinization treatment by adopting a papain-8% trifluoroacetic acid method, standing and layering after violent shaking, removing supernate, then adding 30% hydrogen peroxide (volume concentration) of 0.4 time of the volume of the aqueous solution of the jujubes polysaccharides, and carrying out decoloration treatment by keeping the temperature for 30min at the water bath temperature of 45 ℃; and then carrying out secondary concentration and alcohol precipitation, washing the precipitate with acetone, diethyl ether and absolute ethyl alcohol in sequence, redissolving, and carrying out freeze drying to obtain the refined polysaccharides of the jujubes.
2. Separating and purifying the fructus jujubae polysaccharide:
(1) separation: weighing 1.0g of the refined jujubes polysaccharide, dissolving the refined jujubes polysaccharide in distilled water, sampling the solution through a 0.45-micron filter membrane, performing primary chromatography by adopting a DEAE Sepharose Fast Flow anion exchange column (phi 26mm multiplied by 100mm), passing the aqueous solution of the jujubes polysaccharide through the DEAE Sepharose Fast Flow anion exchange column, sequentially eluting one column volume by using PBS solution (pH is 6.0), 0M NaCl-PBS solution and 0.2M NaCl-PBS solution respectively in a gradient manner at the elution rate of 2.0mL/min, and collecting the crude ZMP2 of the antioxidant active polysaccharides of the jujujubes. Desalting the crude product ZMP2 for 72h by dialysis (the cut-off molecular weight of a dialysis bag is 3500Da) to obtain a concentrated solution of the crude product of the antioxidant activity polysaccharide of the jujubes for later use;
(2) and (3) purification: purifying the concentrated crude ZMP2 of the xylocarpa anti-oxidation active polysaccharide through a Sephacryl S-300 gel column (phi 26mm multiplied by 100mm), eluting a column volume by using distilled water under the condition of 1.0mL/min, and then carrying out reduced pressure concentration and vacuum freeze drying to obtain the purified xylocarpa anti-oxidation active polysaccharide PZMP 2.
Example 4
A separation and purification preparation method of a fructus jujubae polysaccharide comprises the following steps:
1. preparing the fructus jujubae polysaccharide:
(1) ultrasonic-assisted hot water leaching: selecting dried jujubes in Jiaxian county, drying the jujubes at 60 ℃, cooling, crushing the jujubes by using a high-speed crusher, sieving the crushed raw materials by using a 100-mesh sieve, and putting the residues on the sieve into the crusher again for crushing again. A certain mass of jujube powder sample is subjected to ultrasonic assisted extraction for 20min in a water bath at 60 ℃ with the material-liquid ratio of 23:1, and the ultrasonic power is 420W. After centrifugal filtration, the obtained clear solution is concentrated to obtain a concentrated solution.
(2) Alcohol precipitation: adding absolute ethyl alcohol into the concentrated solution obtained in the step (1) while stirring until the volume fraction of the ethyl alcohol in the system reaches 85%, standing overnight at normal temperature, performing suction filtration, taking precipitate, and freeze-drying to obtain the crude polysaccharides of the jujubes.
(3) And (3) decoloring and deproteinizing treatment: dissolving the crude polysaccharides of the jujubes prepared in the step (2) in water, firstly carrying out deproteinization treatment by adopting a papain-8% trifluoroacetic acid method, standing and layering after violent shaking, removing supernate, then adding 30% hydrogen peroxide (volume concentration) which is 0.4 time of the volume of the aqueous solution of the jujubes polysaccharides, and carrying out heat preservation for 50min at the water bath temperature of 50 ℃ for carrying out decoloration treatment; and then carrying out secondary concentration and alcohol precipitation, washing the precipitate with acetone, diethyl ether and absolute ethyl alcohol in sequence, redissolving, and carrying out freeze drying to obtain the refined polysaccharides of the jujubes.
2. Separating and purifying the fructus jujubae polysaccharide:
(1) separation: weighing 1.0g of the refined jujubes polysaccharide, dissolving the refined jujubes polysaccharide in distilled water, sampling the solution through a 0.45-micron filter membrane, performing primary chromatography by adopting a DEAE Sepharose Fast Flow anion exchange column (phi 26mm multiplied by 100mm), passing the aqueous solution of the jujubes polysaccharide through the DEAE Sepharose Fast Flow anion exchange column, sequentially eluting one column volume by using PBS solution (pH is 6.0), 0M NaCl-PBS solution, 0.2M NaCl-PBS solution and 0.3M NaCl-PBS solution in each gradient at the elution rate of 1.5mL/min, and collecting crude ZMP3 of the jujujubes antioxidant active polysaccharide. Desalting the crude product ZMP3 for 72h by dialysis (the cut-off molecular weight of a dialysis bag is 3500Da) to obtain a concentrated solution of the crude product of the antioxidant activity polysaccharide of the jujubes for later use;
(2) and (3) purification: purifying the concentrated crude ZMP3 of the oxidation-resistant active polysaccharides of the jujubes by a Sephacryl S-300 gel column (phi 26mm multiplied by 100mm), eluting by distilled water for one column volume under the condition of 0.8mL/min flow rate, and then carrying out reduced pressure concentration and vacuum freeze drying to obtain the purified oxidation-resistant active polysaccharides PZMP3 of the jujubes.
Example 5
A separation and purification preparation method of a fructus jujubae polysaccharide comprises the following steps:
1. preparing the fructus jujubae polysaccharide:
(1) ultrasonic-assisted hot water leaching: selecting dried jujubes in Jiaxian county, drying the jujubes at 60 ℃, cooling, crushing the jujubes by using a high-speed crusher, sieving the crushed raw materials by using a 100-mesh sieve, and putting the residues on the sieve into the crusher again for crushing again. A certain mass of jujube powder sample is subjected to ultrasonic assisted extraction for 20min in a water bath at 60 ℃ with the material-liquid ratio of 23:1, and the ultrasonic power is 420W. After centrifugal filtration, the obtained clear solution is concentrated to obtain a concentrated solution.
(2) Alcohol precipitation: adding absolute ethyl alcohol into the concentrated solution obtained in the step (1) while stirring until the volume fraction of the ethyl alcohol in the system reaches 85%, standing overnight at normal temperature, performing suction filtration, taking precipitate, and freeze-drying to obtain the crude polysaccharides of the jujubes.
(3) And (3) decoloring and deproteinizing treatment: dissolving the crude polysaccharides of the jujubes prepared in the step (2) in water, firstly carrying out deproteinization treatment by adopting a papain-8% trifluoroacetic acid method, standing and layering after violent shaking, removing supernate, then adding 30% hydrogen peroxide (volume concentration) which is 0.4 time of the volume of the aqueous solution of the jujubes polysaccharides, and carrying out heat preservation for 50min at the water bath temperature of 50 ℃ for carrying out decoloration treatment; and then carrying out secondary concentration and alcohol precipitation, washing the precipitate with acetone, diethyl ether and absolute ethyl alcohol in sequence, redissolving, and carrying out freeze drying to obtain the refined polysaccharides of the jujubes.
2. Separating and purifying the fructus jujubae polysaccharide:
(1) separation: weighing 1.0g of the refined jujubes polysaccharide, dissolving the refined jujubes polysaccharide in distilled water, sampling the solution through a 0.45-micron filter membrane, performing primary chromatography by adopting a DEAE Sepharose Fast Flow anion exchange column (phi 26mm multiplied by 100mm), passing the aqueous solution of the jujubes polysaccharide through the DEAE Sepharose Fast Flow anion exchange column, sequentially eluting one column volume by using a PBS solution (pH is 6.0), a 0M NaCl-PBS solution, a 0.2M NaCl-PBS solution, a 0.3M NaCl-PBS solution and a 0.4M NaCl-PBS solution respectively at an elution rate of 1.5mL/min, and collecting a crude ZMP4 of the jujubes antioxidant active polysaccharide. Desalting the crude product ZMP4 for 72h by dialysis (the cut-off molecular weight of a dialysis bag is 3500Da) to obtain a concentrated solution of the crude product of the antioxidant activity polysaccharide of the jujubes for later use;
(2) and (3) purification: purifying the concentrated crude ZMP4 of the oxidation-resistant active polysaccharides of the jujubes by a Sephacryl S-300 gel column (phi 26mm multiplied by 100mm), eluting by distilled water for one column volume under the condition of 0.8mL/min flow rate, and then carrying out reduced pressure concentration and vacuum freeze drying to obtain the purified oxidation-resistant active polysaccharides PZMP4 of the jujubes.
The physical and chemical property analysis of the pure jujube polysaccharide product is as follows: yield of PZMP4 (based on crude polysaccharide) 0.76%, molecular weight (kDa): 27.89, polysaccharide purity (%): 90.64. therefore, the polysaccharide has extremely low yield and poor purity, and the product is not suitable for the subsequent oxidation resistance test.
EXAMPLE 6 monosaccharide composition analysis of three polysaccharides having antioxidative Activity of jujubes
The monosaccharide compositions of the three isolated and purified momordica cochinchinensis antioxidant active polysaccharides PZMP1, PZMP2 and PZMP3 of example 1 were determined by a conventional gas chromatography monosaccharide derivatization method, and the results are shown in table 1.
TABLE 1 monosaccharide composition of PZMP1, PZMP2, PZMP3
"-" represents a component containing a slight amount of monosaccharide
Test example 1 test of physicochemical Properties of three kinds of polysaccharides having antioxidative Activity of jujubes
The physicochemical properties of three oleaster antioxidant active polysaccharides, namely PZMP1, PZMP2 and PZMP3, which are separated and purified in example 1 are determined. Wherein, the polysaccharide molecular weight is determined by conventional high performance liquid chromatography gel chromatography, and the polysaccharide purity, the protein content and the total phenol content are respectively determined by conventional phenol-sulfuric acid method, Coomassie brilliant blue method and Folin phenol method. The results of the analysis are shown in Table 2:
TABLE 2 analysis results of physicochemical properties of three kinds of fructus Jujubae polysaccharides with antioxidant activity
Test example 2 Nuclear magnetic analysis of three types of polysaccharides having antioxidant activity of jujubes
Nuclear magnetic analysis was performed on three kinds of the polysaccharides PZMP1, PZMP2 and PZMP3 having antioxidant activity of zizyphus juba obtained by separation and purification of the polysaccharides of zizyphus juba in example 1, respectively. PZMP1, PZMP2 and PZMP3 are respectively dissolved in heavy water, and a 600MHz nuclear magnetic resonance apparatus is adopted to perform one-dimensional reaction1H-NMR spectrum,13C-NMR spectrum detection. Of PZMP11The H-NMR spectrum is shown in FIG. 1 (A), of PZMP21The H-NMR spectrum is shown in FIG. 1 (B), of PZMP31The H-NMR spectrum is shown in FIG. 1 (C), of PZMP113The C-NMR spectrum is as shown in FIG. 2 (A), of PZMP213The C-NMR spectrum is shown in FIG. 2 (B), of PZMP313The C-NMR spectrum is shown in FIG. 2 (C).
By nuclear magnetism one dimension1H-NMR spectrum and13C-NMR spectroscopy confirmed that the PZMP1 backbone is predominantly composed of 1,3,5-Araf and 1,5-Araf linkages, and 1,4-Galp may be present in the backbone. The main chain of PZMP2 is made of galacturonic acidThe trans-1, 4-GalpA is formed by connecting 1,2,4-Rhap with rhamnose and belongs to type I rhamnogalacturonan polysaccharides. The PZMP3 main chain is formed by galacturonic acid connected through 1,4-GalpA and 1,2,4-GalpA, and belongs to HG type galacturonic acid polysaccharides.
The assignment of glycosidic linkages of PZMP1 is shown in table 3, that of PZMP2 is shown in table 4, and that of PZMP3 is shown in table 5.
TABLE 3 glycosidic bond assignments for PZMP1
TABLE 4 glycosidic bond assignments for PZMP2
TABLE 5 glycosidic bond assignments for PZMP3
Test example 3 antioxidant Activity of three Aurea jujuba polysaccharides
1. Hydroxyl radical scavenging ability
PZMP1, PZMP2 and PZMP3 separated and purified in example 1 are respectively prepared into solutions with the concentrations of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL by using distilled water, and 2mL solutions with the concentrations of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL for PZMP1, PZMP2 and PZMP3 are respectively added into FeSO4And salicylic acid-ethanol mixed solution (9 mmol/L FeSO)42mL of solution and 2mL of 9mmol/L salicylic acid-ethanol solution), and after being uniformly mixed, 8.8mmol/L H is respectively added2O22mL of the solution was reacted, and after the reaction was completed and left to stand at 37 ℃ for 1 hour, the concentrations of PZMP1, PZMP2 and PZMP3 at 510nm were measured at 0.25, 0.5, 1.0, 1.5, 2.0, respectively,Absorbance values at concentrations of 2.5, 3.0mg/mL, Vc as a positive control. The hydroxyl radical scavenging ability is calculated according to the following formula:
hydroxyl radical scavenging ratio (%) [ 1- (As-Ab) ]/Ac X100%
In the formula: as is the absorbance value of the Vc solution; ab is blank absorbance value, i.e. replacing H with distilled water2O2Absorbance values of the system of solutions; ac is a contrast absorbance value, namely the absorbance value of a system for replacing the fructus jujubae antioxidant active polysaccharide solution by distilled water. The results of the hydroxyl radical scavenging ability test are shown in table 6:
TABLE 6 hydroxy radical scavenging ability (%)
2. Hydroxyl radical scavenging ability
PZMP1, PZMP2 and PZMP3 separated and purified in example 1 are respectively prepared into solutions with the concentrations of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL by using distilled water, 1mL of solutions with the concentrations of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL of PZMP1, PZMP2 and PZMP3 are respectively mixed with 1mL of a DPPH methanol solution (0.1mmol/L) and are shielded from light for 30min, a blank group is formed by using 2.0mL of the methanol solution as a zero tube, 1mL of the DPPH solution and 1mL of the methanol solution are mixed as a control group, PZMP1, PZMP2 and PZMP3 are respectively measured at the concentrations of 0.25, 0.5, 1.0, 1.5, 2.5, 0.5, 0mg of the PZMP2 and PVc are respectively used as a control group at 517nm, and the absorbance values of the positive control group are respectively measured and calculated according to the formula:
DPPH radical scavenging ratio (%) [ 1- (As-Ac)/Ab ]. times.100%
In the formula: as is the absorbance value of the Vc aqueous solution; ac is the absorbance value of the control group; ab is blank absorbance value.
The DPPH free radical scavenging ability test results are shown in table 7:
TABLE 7 DPPH radical scavenging ability (%)
3. Iron ion chelating ability
PZMP1, PZMP2 and PZMP3 separated and purified in example 1 are respectively prepared into solutions with the concentrations of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL by using distilled water, and 1mL of the solutions with the concentrations of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL of PZMP1, PZMP2 and PZMP3 are respectively added with FeCl2Solution (from 2mmol/L FeCl20.1mL of solution and 3.7mL of distilled water), 0.2mL of 5mmol/L Ferrozine working solution is added after mixing for 30s, the reaction is carried out at room temperature for 10min, and the absorbance values at concentrations of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL of PZMP1, PZMP2 and PZMP3, respectively, are determined at 562nm, and Vc is used as a positive control.
The iron ion chelating capacity is calculated as follows:
iron ion chelating capacity (%) [ 1- (As-Ab) ]/Ac X100%
In the formula: as is the absorbance value of polysaccharide or Vc solution; ab is blank absorbance value, i.e. replacing H with distilled water2O2Absorbance values of the system of solutions; ac is the control absorbance value, i.e., the absorbance value of the system in which the polysaccharide solution was replaced with distilled water.
The iron ion chelating ability is shown in table 8:
table 8 iron ion chelating ability (%)
Through analysis of hydroxyl radical scavenging capacity, hydroxyl radical scavenging capacity and iron ion chelating capacity, the three kinds of antioxidant polysaccharides PZMP1, PZMP2 and PZMP3 obtained by separation and purification in the invention are found to have antioxidant activity and are more remarkable than the antioxidant activity of PZMP1, PZMP2 and PZMP3, and as shown in the table 1 of the example 5, the antioxidant activity is caused by different monosaccharide compositions and proportions of monosaccharides in PZMP1, PZMP2 and PZMP 3.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Claims (10)
1. A method for separating and purifying the fructus jujubae polysaccharide with the antioxidation effect comprises the steps of preparing the fructus jubae polysaccharide and separating and purifying the fructus jubae polysaccharide, and is characterized in that the steps of separating and purifying the fructus jubae polysaccharide are as follows: firstly, the jujubes polysaccharose passes through a DEAE Sepharose Fast Flow anion exchange column, and then gradient elution separation is carried out by using a PBS solution and a NaCl-PBS solution in turn, and then purification is carried out by a Sephacryl S-300 gel column.
2. The method for separating and purifying the polysaccharides from jujubes with anti-oxidation effect according to claim 1, wherein the concentration of the NaCl-PBS solution is 0M to 0.3M.
3. The method for separating and purifying the polysaccharides from zizyphus jujuba as claimed in claim 1, wherein the gradient elution is performed by sequentially gradient elution with PBS solution and 0M NaCl-PBS solution.
4. The method for separating and purifying the polysaccharides from zizyphus jujuba as claimed in claim 1, wherein the gradient elution is performed by sequentially gradient elution with PBS solution, 0M NaCl-PBS solution and 0.2M NaCl-PBS solution.
5. The method for separating and purifying the polysaccharides from zizyphus jujuba as claimed in claim 1, wherein the gradient elution is performed by sequentially gradient elution with PBS solution, 0M NaCl-PBS solution, 0.2M NaCl-PBS solution and 0.3M NaCl-PBS solution.
6. The method for separating, purifying and preparing the jujubes polysaccharide according to any one of claims 1 to 5, wherein the PBS solution elutes one column volume during gradient elution, and NaCl-PBS solution of each concentration elutes one column volume correspondingly.
7. The method for separating, purifying and preparing the polysaccharides from jujubes according to any one of claims 1 to 5, wherein the elution rate is 1.0-2.0 mL/min.
8. The method for separating, purifying and preparing the polysaccharides from jujubes according to any one of claims 1 to 5, wherein the conditions for purifying by Sephacryl S-300 gel column are as follows: eluting with distilled water as eluent at a flow rate of 0.6-1.0 mL/min.
9. The method for separating, purifying and preparing the jujuba polysaccharide according to any one of claims 1 to 5, wherein the jujuba polysaccharide is obtained by sequentially carrying out ultrasonic-assisted hot water extraction, alcohol precipitation and decoloration and deproteinization on jujubes.
10. The method for separating, purifying and preparing the polysaccharides of jujubes according to claim 9, wherein the material-liquid ratio of the ultrasonic-assisted hot water extraction is 23:1, the ultrasonic power is 420W, the extraction temperature is 60-80 ℃, and the extraction time is 20-40 min.
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