CN110606900B - 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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- CN110606900B CN110606900B CN201911008949.5A CN201911008949A CN110606900B CN 110606900 B CN110606900 B CN 110606900B CN 201911008949 A CN201911008949 A CN 201911008949A CN 110606900 B CN110606900 B CN 110606900B
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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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- 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 fructus Jujubae antioxidant active polysaccharides PZMP1, PZMP2 and PZMP3 can be obtained by one-time separation and purification by the preparation method of the invention, and the three fructus Jujubae antioxidant active polysaccharides obtained by the separation and purification of the invention not only have high purity, the purity can reach more than 90%, but also have high yield, and the yield is 2.95%, 14.15% and 14.61% based on the fructus Jujubae crude polysaccharide, namely PZMP1, PZMP2 and PZMP3. In addition, the three types of oxidation-resistant active polysaccharides of the jujubes obtained by separation and purification of the invention have strong oxidation resistance, provide raw materials for oxidation-resistant functional foods and promote 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 separation and purification method of 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, and nucleosides. Fructus Jujubae has good medical and health promotion effects, 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), 634.00 ten thousand tons (2013), and accounts for more than 90% 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: in the regions between 37 DEG 41' to 38 DEG 23' and 110 DEG 0' to 110 DEG 45' to 10 ' north latitude, the yellow river coastal region from Jia county pollution-free sources has long cultivation history and strong tree vitality. 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 (21.9 g/100 g), reduced Vc (376.2 mg/100 g), total phenol (541.8 mg GAE eq./100g FW), total flavone (255.5 mg Rutin eq./100g FW), total procyanidin (184.2 mg 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 fructus Jujubae antioxidant active polysaccharides PZMP1, PZMP2 and PZMP3 can be obtained by one-time separation and purification by the preparation method of the invention, and the three fructus Jujubae antioxidant active polysaccharides obtained by the separation and purification of the invention not only have high purity, the purity can reach more than 90%, but also have high yield, and the yield is 2.95%, 14.15% and 14.61% based on the fructus Jujubae crude polysaccharide, namely PZMP1, PZMP2 and PZMP3. In addition, the three types of oxidation-resistant active polysaccharides of the jujubes obtained by separation and purification of the invention have strong oxidation resistance, provide raw materials for oxidation-resistant functional foods and promote 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 polyose 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 PZMP1.
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 using a PBS solution, a 0M NaCl-PBS solution and a 0.2M 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 PZMP2.
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: the method comprises the steps of firstly passing the jujubes polysaccharose through a DEAE Sepharose Fast Flow anion exchange column, sequentially carrying out gradient elution by using a PBS solution, a 0M NaCl-PBS solution, a 0.2M NaCl-PBS solution and a 0.3M NaCl-PBS solution for separation, and purifying through 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 PZMP3.
The gradient elution rate for the above separation is 1.0-2.0mL/min. Preferably, the elution rate is 1.5mL/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 0.8mL/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, the ultrasonic power is 420W, the extraction temperature is 60-80 ℃, and the extraction time is 20-40min. Preferably, the extraction temperature is 70 deg.C, and the extraction time is 30min.
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 20-40min; the volume concentration of the hydrogen peroxide is 30 percent. Preferably, the water bath temperature is 45 ℃, and the water bath time is 30min.
The method for separating and purifying the polysaccharides of the jujubes with the antioxidation 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 an elution rate of 1.5mL/min, and collecting a first crude product of the common jujube antioxidant active polysaccharide, namely ZMP1; 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 oxidation resistant active polysaccharide of the jujubes, wherein the crude product is named as ZMP2; 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 ZMP3; desalting the three crude products of ZMP1, ZMP2 and ZMP3 by dialysis, 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 fructus Jujubae antioxidant active polysaccharide obtained after ZMP1 purification is named PZMP1, the fructus Jujubae antioxidant active polysaccharide obtained after ZMP2 purification is named PZMP2, and the fructus Jujubae antioxidant active polysaccharide obtained after ZMP3 purification is named PZMP3.
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 100 mm) comprises the following steps: the column material is evenly stirred and then injected into a glass column (phi 26mm multiplied by 100 mm) in a glass rod drainage mode, and in the process, the column material is continuously beaten by a aurilave to prevent bubbles from generating, thus obtaining the DEAE Sepharose 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 fructus Jujubae antioxidant active polysaccharides PZMP1, PZMP2 and PZMP3 can be obtained by one-time separation and purification by the preparation method of the invention, and the three fructus Jujubae antioxidant active polysaccharides obtained by the separation and purification of the invention not only have high purity, the purity can reach more than 90%, but also have high yield, and the yield is 2.95% of PZMP1, 14.15% of PZMP2 and 14.61% of PZMP3 based on the fructus Jujubae 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.
(2) According to the method, the polysaccharides of the jujubes are separated by adopting a DEAE Sepharose Fast Flow anion exchange column, and the DEAE Sepharose Fast Flow has stable properties, so that the method is more beneficial to separation of three kinds of polysaccharides with antioxidant activity of the jujubes, and the yield of the three kinds of polysaccharides with antioxidant activity of the jujubes 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 uses the feed-liquid ratio of 23.
Drawings
FIG. 1 shows the three polysaccharides having antioxidant activity of Elaeagnus angustifolia in test example 2 1 H-NMR spectrum; wherein (A) is PZMP1 1 H-NMR spectrum, (B) of PZMP2 1 H-NMR spectrum, (C) of PZMP3 1 H-NMR spectrum.
FIG. 2 shows the results of the three types of polysaccharides having antioxidant activity of Elaeagnus angustifolia in test example 2 13 A C-NMR spectrum; wherein (A) is PZMP1 13 C-NMR spectrum (B) of PZMP2 13 C-NMR spectrum, (C) of PZMP3 13 C-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. A certain mass of jujube powder sample is subjected to ultrasonic assisted extraction for 30min in a water bath at 70 ℃ according to a material-liquid ratio of 23. 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, violently shaking, standing for layering, removing supernate, then adding 30% hydrogen peroxide (volume concentration) which is 0.4 time of the volume of the aqueous solution of the polysaccharides of the jujubes, and carrying out decoloration treatment by keeping the temperature for 30min at the water bath temperature of 45 ℃; and concentrating twice, precipitating with ethanol, washing the precipitate with acetone, diethyl ether and anhydrous ethanol in sequence, redissolving, and freeze-drying to obtain refined polysaccharides of fructus Jujubae.
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 100 mm), 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 ZMP1; 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 oxidation resistant active polysaccharide of the jujubes, wherein the crude product is named as ZMP2; 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 as ZMP3. Desalting the three crude ZMP1, ZMP2 and ZMP3 products for 72h by dialysis (the cut-off molecular weight of a dialysis bag is 3500 Da) respectively to obtain three concentrated solutions of the crude polysaccharides with the antioxidative activity of the jujubes for later use;
(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 100 mm), 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 fructus Jujubae antioxidant active polysaccharide obtained after ZMP1 purification is named PZMP1, the fructus Jujubae antioxidant active polysaccharide obtained after ZMP2 purification is named PZMP2, and the fructus Jujubae antioxidant active polysaccharide obtained after ZMP3 purification is named PZMP3.
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 ℃ according to a material-liquid ratio of 23. 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, violently shaking, standing for layering, removing supernate, then adding 30% hydrogen peroxide (volume concentration) which is 0.4 time of the volume of the aqueous solution of the polysaccharides of the jujubes, and carrying out decoloration treatment by keeping the temperature of a water bath at 50 ℃ for 50 min; 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 jubae polysaccharide in distilled water, sampling 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 100 mm), passing the aqueous solution of the fructus jujubae polysaccharide through the DEAE Sepharose Fast Flow anion exchange column, sequentially eluting one column volume by 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 crude ZMP1 of the fructus jubae antioxidant active polysaccharide; desalting the crude product ZMP1 for 72h by dialysis (the molecular weight cutoff of a dialysis bag is 3500 Da) to obtain a concentrated solution of the crude product of the antioxidant active polysaccharides of the jujubes for later use;
(2) And (3) purification: purifying the concentrated crude ZMP1 of the antioxidant active polysaccharides of the jujubes by a Sephacryl S-300 gel column (phi 26mm multiplied by 100 mm), 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 antioxidant 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. A certain mass of jujube powder sample is subjected to ultrasonic assisted extraction for 30min in a water bath at 70 ℃ according to a material-liquid ratio of 23. 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 100 mm), 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 molecular weight cut-off of a dialysis bag is 3500 Da) 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 by a Sephacryl S-300 gel column (phi 26mm multiplied by 100 mm), eluting the crude ZMP2 by distilled water for one column volume 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 PZMP2.
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 ℃ according to a material-liquid ratio of 23. 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 jujubes polysaccharides:
(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 100 mm), 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 jujubes antioxidant active polysaccharide. Desalting the crude product ZMP3 for 72h by dialysis (the molecular weight cut-off of a dialysis bag is 3500 Da) 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 xylocarpa anti-oxidation active polysaccharide by a Sephacryl S-300 gel column (phi 26mm multiplied by 100 mm), eluting a column volume by distilled water 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 purified xylocarpa anti-oxidation active polysaccharide PZMP3.
Example 5
A separation and purification preparation method of a jujubes 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 60 deg.C, cooling, pulverizing with high speed pulverizer, sieving with 100 mesh sieve, and pulverizing the residue on the sieve again. A certain mass of jujube powder sample is subjected to ultrasonic assisted extraction for 20min in a water bath at 60 ℃ according to a material-liquid ratio of 23. 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, violently shaking, standing for layering, removing supernate, then adding 30% hydrogen peroxide (volume concentration) which is 0.4 time of the volume of the aqueous solution of the polysaccharides of the jujubes, and carrying out decoloration treatment by keeping the temperature of a water bath at 50 ℃ for 50 min; and concentrating twice, precipitating with ethanol, washing the precipitate with acetone, diethyl ether and anhydrous ethanol in sequence, redissolving, and freeze-drying to obtain refined polysaccharides of fructus Jujubae.
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 100 mm), 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 molecular weight cut-off of a dialysis bag is 3500 Da) 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 100 mm), eluting the gel column 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 purified oxidation-resistant active polysaccharides PZMP4 of the jujubes.
The physical and chemical property analysis of the pure jujube polysaccharide product is as follows: PZMP4 yield (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, xylocarpa 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 kinds of the antioxidant polysaccharides of zizyphus jujuba, namely PZMP1, PZMP2 and PZMP3, which were separated and purified in example 1, were 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
The nuclear magnetic analysis is carried out on three kinds of the rosa roxburghii antioxidant active polysaccharides PZMP1, PZMP2 and PZMP3 obtained by separation and purification of the rosa roxburghii polysaccharide in the example 1. Respectively dissolving PZMP1, PZMP2 and PZMP3 in heavy water, and performing one-dimensional nuclear magnetic resonance with 600MHz type nuclear magnetic resonance apparatus 1 H-NMR spectrum, 13 C-NMR spectrum detection. Of PZMP1 1 The H-NMR spectrum is shown in FIG. 1 (A), for PZMP2 1 The H-NMR spectrum is shown in FIG. 1 (B), for PZMP3 1 The H-NMR spectrum is shown in FIG. 1 (C), for PZMP1 13 The C-NMR spectrum is shown in FIG. 2 (A), of PZMP2 13 The C-NMR spectrum of the product is shown in FIG. 2 (B), for PZMP3 13 The C-NMR spectrum is shown in FIG. 2 (C).
By nuclear magnetism one dimension 1 H-NMR spectrum and 13 C-NMR spectroscopic analysis confirmed that the PZMP1 backbone is predominantly composed of 1,3,5-Araf and 1,5-Araf linkages, and that 1,4-Galp may be present in the backbone. The PZMP2 main chain is formed by linking galacturonic acid through 1,4-GalpA and rhamnose through 1,2,4-Rhap, and belongs to type I rhamnogalacturonan polysaccharide. The PZMP3 main chain is formed by galacturonic acid connected through 1,4-GalpA and 1,2,4-GalpA, belonging to HG type galacturonic acid polysaccharide.
The assignment of the glycosidic bond 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 PZMPP 1
TABLE 4 glycosidic bond assignments for PZMPP 2
TABLE 5 glycosidic bond assignments for PZMPP 3
Test example 3 antioxidant Activity of three Aurea jujuba polysaccharides
1. Hydroxyl radical scavenging ability
PZMP1, PZMP2 and PZMP3 obtained by separation and purification in example 1 were prepared into solutions of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL with distilled water, respectively, and 2mL solutions of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL of PZMP1, PZMP2 and PZMP3 were added to FeSO 4 And salicylic acid-ethanol mixed solution (9 mmol/L FeSO) 4 2mL of the solution and 2mL of a 9mmol/L salicylic acid-ethanol solution), and after being uniformly mixed, 8.8mmol/L H are respectively added 2 O 2 After the reaction is finished and the solution is kept still at 37 ℃ for 1h, the absorbance values of PZMP1, PZMP2 and PZMP3 at the concentrations of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL are respectively measured at 510nm, and Vc is used as a positive control. The hydroxyl radical scavenging ability is calculated according to the following formula:
hydroxyl radical clearance (%) = [1- (As-Ab) ]/Ac × 100%
In the formula: as is the absorbance value of the Vc solution; ab is blank absorbance value, i.e. replacing H with distilled water 2 O 2 Absorbance 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 prepared into solutions with the concentration of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL respectively by distilled water, then PZMP1, PZMP2 and PZMP3 are mixed with 1mL of DPPH methanol solution (0.1 mmol/L) respectively in the concentration of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL respectively in the concentration of 1mL, and are protected from light for 30min, 2.0mL of methanol solution is used as a blank, 1mL of DPPH solution and 1mL of methanol solution are mixed as a control, PZMP1, PZMP2 and PZMP3 are respectively measured at 517nm in the blank, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL of methanol solutions respectively, and the absorbance of the blank, PZMP2 and PZMP3 are respectively measured as positive control, absorbance values, and the absorbance of the blank and the positive control are 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 obtained by separation and purification in example 1 were prepared into solutions of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL with distilled water, respectively, and FeCl was added to 1mL of each solution of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL of PZMP1, PZMP2 and PZMP3, respectively 2 Solution (from 2mmol/L FeCl 2 0.1mL of solution and 3.7mL of distilled water), mixing for 30s, adding 0.2mL of 5mmol/L Ferrozine working solution, reacting at room temperature for 10min, and measuring PZMP1, PZMP2 and PZMP3 at 562nm respectively at 0.25, 0.5, respectively,Absorbance values at concentrations of 1.0, 1.5, 2.0, 2.5, 3.0mg/mL, and Vc as a positive control.
The iron ion chelating capacity is calculated as follows:
iron ion chelating ability (%) = [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 water 2 O 2 Absorbance 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 fructus ziziphi spinosae antioxidant active polysaccharides PZMP1, PZMP2 and PZMP3 obtained by separation and purification in the invention are found to have antioxidant activity and are more remarkable than that of PZMP1, PZMP2 and PZMP3, as shown in table 1 of example 5, due to different monosaccharide compositions and proportions of monosaccharides in PZMP1, PZMP2 and PZMP3.
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 (1)
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, leading the fructus jujubae polysaccharide to pass through a DEAE Sepharose Fast Flow anion exchange column, sequentially using a PBS solution and a NaCl-PBS solution for gradient elution separation, and then purifying by a Sephacryl S-300 gel column, wherein the details are as follows:
(1) Separation: enabling the aqueous solution of the common jujube polysaccharide to pass through a DEAE Sepharose Fast Flow anion exchange column, eluting for one column volume by using PBS solution and 0M NaCl-PBS solution in a gradient way under the condition that the pH value is 6.0 at the elution rate of 1.5mL/min, and collecting a first crude product of the common jujube antioxidant active polysaccharide, which is named ZMP1; 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 oxidation resistant active polysaccharide of the jujubes, wherein the crude product is named as ZMP2; 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 ZMP3; desalting the three crude products of ZMP1, ZMP2 and ZMP3 by dialysis, and concentrating for later use;
(2) And (3) purification: purifying the concentrated crude product of the xylocarpa anti-oxidation active polysaccharide ZMP3 by a Sephacryl S-300 gel column, eluting the crude product by distilled water for one column volume under the condition of the flow rate of 0.8mL/min, and then performing reduced pressure concentration and vacuum freeze drying to obtain the purified xylocarpa anti-oxidation active polysaccharide ZMP3; the fructus jujubae polysaccharide is obtained by sequentially performing ultrasonic-assisted hot water extraction, alcohol precipitation and decoloration deproteinization on fructus jujubae; the material-liquid ratio of the ultrasonic-assisted hot water extraction is 23.
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