CN108567836B - Method for extracting and separating flavone and polysaccharide from hawthorn peel residues in combined manner - Google Patents
Method for extracting and separating flavone and polysaccharide from hawthorn peel residues in combined manner Download PDFInfo
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Abstract
The invention discloses a method for extracting and separating flavone and polysaccharide from hawthorn peel residue in a combined manner, which takes hawthorn peel residue obtained after hawthorn wine fermentation as a raw material, adopts ultrasonic and enzyme method auxiliary technologies to extract the flavone and the polysaccharide in the hawthorn peel residue in a combined manner, and adopts a fixed bed adsorption technology to synchronously separate the flavone and the polysaccharide in an extracting solution of the hawthorn peel residue according to different characteristics of the flavone and the polysaccharide in the hawthorn peel residue, so that hawthorn polysaccharide is separated and purified, hawthorn flavone extract is obtained, the comprehensive utilization of hawthorn is realized, and simultaneously, the resource waste and the environmental pollution caused by the hawthorn peel residue are also solved. The method has the advantages of high extraction amount, high purity, high extraction efficiency, no need of special equipment in the extraction process, low cost, providing a way for combined extraction and separation of flavone and polysaccharide, providing a new way for utilization of hawthorn peel residue, and having good social and economic benefits.
Description
Technical Field
The invention relates to a method for extracting and separating flavone and polysaccharide from hawthorn peel residue in a combined manner, and belongs to the technical field of extraction processes.
Background
Crataegus pinnatifida Bunge, also called Crataegus pinnatifida Bunge, Cr. The hawthorn fruit can be eaten raw or used as a preserved fruit cake, can be used as a medicine after being dried, is a tree species which is a special Chinese medicine and fruit, has the functions of reducing blood fat, blood pressure, strengthening heart, resisting arrhythmia and the like, is also a good medicine for strengthening spleen, stimulating appetite, promoting digestion, removing food stagnation, promoting blood circulation and reducing phlegm, and has good curative effects on chest, diaphragm and spleen fullness, hernia, blood stasis, amenorrhea and other symptoms. Research shows that the hawthorn contains chemical components such as organic acids, triterpenes, flavonoids, polysaccharides and the like. Wherein, the flavone and the polysaccharide have better functions of reducing blood pressure and blood fat. Besides, the hawthorn flavone and polysaccharide also have other important functions, such as prevention of diabetes and complications thereof, treatment of bone diseases, osteoporosis and the like. Therefore, the method has important significance for the research of hawthorn flavone and polysaccharide.
In recent years, with the improvement of living standard and health care consciousness of people, hawthorn fruit wine, fruit vinegar and fruit juice with health care function are rapidly developed in the hawthorn processing industry, and simultaneously, a large amount of hawthorn peel dregs are also generated. The hawthorn peel residue after the hawthorn wine fermentation still contains rich active substances, and huge resource waste and environmental pollution are caused due to the lack of effective development and utilization. Therefore, an effective functional component extraction and separation method is developed, so that the comprehensive utilization of the hawthorn can be improved, and the environmental pollution can be reduced.
At present, the research on functional substances in hawthorn peel residues is less, and the research focuses on the research on the extraction process of a single functional substance in hawthorn, such as the extraction of flavone and tripalmic acid functional substances. The research on the extraction process of the hawthorn polysaccharide is few, and the research on the combined extraction and separation technology of functional substances in hawthorn is not available at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for jointly extracting and separating flavone and polysaccharide from hawthorn peel residues, which can simultaneously and effectively extract and separate the flavone and the polysaccharide of hawthorn.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for extracting and separating flavone and polysaccharide from hawthorn peel residue comprises the following steps:
(1) pretreatment of hawthorn peel residues:
filtering hawthorn peel residues after fermentation of hawthorn wine, removing seeds, naturally drying in the shade, and putting the hawthorn peel residues after natural drying in the shade into a grinder to be ground into powder;
(2) ultrasonic enzyme method combined extraction: adding 20% ethanol into the hawthorn peel residue obtained in the step (1) according to the material-liquid ratio of 1:40, and mixing uniformly; then adding 0.2-0.3% (m/m) cellulase and 0.2-0.25% (v/v) Tween 80, placing the mixture into a water bath at the temperature of 60-70 ℃, ultrasonically extracting for 30-40 min, repeatedly extracting for 2 times, and centrifugally separating after extraction to obtain supernatant and precipitate, wherein the supernatant is the hawthorn flavone and polysaccharide extracting solution;
(3) and (3) evaporation and concentration: placing the hawthorn flavone and polysaccharide extracting solution obtained in the step (2) on a rotary evaporator, evaporating and concentrating at 50 ℃, pouring into a culture dish, freezing in an ultra-low temperature refrigerator at-80 ℃ for overnight, and freeze-drying in a freeze dryer to obtain a hawthorn flavone polysaccharide crude extract;
(4) separating and purifying hawthorn flavone and polysaccharide:
(a1) and (3) separating hawthorn polysaccharide: dissolving the hawthorn flavone polysaccharide crude extract obtained in the step (3) with 20% ethanol, adsorbing with AB-8 macroporous resin, eluting with distilled water, collecting eluate, and concentrating under reduced pressure to 1/4 of hawthorn eluate volume to obtain hawthorn polysaccharide concentrated solution;
(a2) and (3) hawthorn polysaccharide purification: adding 5-6 times of ethanol into the hawthorn polysaccharide concentrated solution obtained in the step (a1) while stirring, standing for 12 hours in a refrigerator at 4 ℃, centrifuging, collecting precipitate, redissolving the precipitate with distilled water, adding 5-6 times of savage reagent, shaking a table, centrifuging, repeating for 3 times, combining supernate, concentrating under reduced pressure to 1/4 times of the volume of the supernate, adding 5-6 times of ethanol for dissolving, standing for 12 hours in a refrigerator at 4 ℃, centrifuging, collecting precipitate, and obtaining hawthorn polysaccharide;
(b1) and (3) separating hawthorn flavone: eluting the macroporous resin obtained by eluting the distilled water in the step (a1) with 50% ethanol, collecting the eluent, and concentrating under reduced pressure to obtain a hawthorn flavone crude extract;
(b2) and (3) hawthorn flavone purification: and (b) dissolving the hawthorn flavone crude extract obtained in the step (b2) in ethanol, extracting with 3-5 times of volume of n-hexane, then keeping an ethanol phase, and concentrating under reduced pressure to obtain the hawthorn flavone extract.
And (2) sieving the powder in the step (1) by a 60-mesh sieve.
The enzyme activity of the cellulase in the step (2) is 1000U/mg.
In the step (a1), the feed-liquid ratio of the hawthorn flavone polysaccharide crude extract to the 20% ethanol is 1: 2.
in the step (a1), the distilled water is 3-5BV, and the elution flow rate is 2 ml/min.
Preferably, the distilled water in step (a1) is 3 BV.
In the step (b1), the 50% ethanol solution is 3-5BV, and the elution flow rate is 2 ml/min.
Preferably, the 50% ethanol in step (b1) is 3 BV.
The invention has the beneficial effects that:
1. the hawthorn peel residue after fermentation of hawthorn wine is used as a raw material, and the hawthorn wine is treated by pectinase in the preparation process, so that pectin in the hawthorn peel residue is subjected to enzymolysis, and the pectinase is not used in the extraction process of hawthorn flavone and polysaccharide, so that the extraction process of hawthorn flavone and polysaccharide is effectively simplified, and the problem of reutilization of the hawthorn peel residue is solved.
2. The invention adopts the combined action of the cellulase and the Tween 80, the cellulase is a group of complex enzymes and can hydrolyze fibers, destroy plant cell walls and fully release cell contents, thereby being beneficial to the extraction of flavone and polysaccharide in hawthorn peel residue; tween 80 can effectively promote cellulase molecule distribution, and reduce the probability of ineffective adsorption, thereby improving the enzymolysis efficiency.
3. The invention adopts the ultrasonic and enzyme method auxiliary technology to extract the flavone and the polysaccharide in the hawthorn peel residue in a combined manner, and adopts the fixed bed adsorption technology to synchronously separate the flavone and the polysaccharide in the hawthorn peel residue extracting solution according to the different characteristics of the flavone and the polysaccharide in the hawthorn peel residue, thereby not only separating and purifying the hawthorn polysaccharide, but also obtaining the hawthorn flavone extract, realizing the comprehensive utilization of hawthorn, and simultaneously solving the problems of resource waste and environmental pollution caused by the hawthorn peel residue.
4. The method has the advantages of high extraction amount, high purity, high extraction efficiency, no need of special equipment in the extraction process, low cost, providing a way for combined extraction and separation of flavone and polysaccharide, providing a new way for utilization of hawthorn peel residue, and having good social and economic benefits.
Drawings
FIG. 1 shows the results of screening the amount of enzymes added for the extraction of flavones from hawthorn peel residue.
FIG. 2 shows the results of ethanol concentration screening of flavone extracted from hawthorn peel residue.
FIG. 3 shows the screening results of the extraction time for extracting flavone from hawthorn peel residue.
FIG. 4 shows the screening results of the extraction temperature for extracting flavone from hawthorn peel residue.
FIG. 5 shows the result of the screening of the ratio of the material to the liquid for extracting the flavone from the hawthorn peel residue.
FIG. 6 shows the results of screening the amount of enzyme added for extracting polysaccharides from the hawthorn peel residue.
FIG. 7 shows the results of ethanol concentration screening of polysaccharide extraction from the hawthorn peel residue.
Fig. 8 shows the result of screening the extraction time for extracting polysaccharides from the hawthorn peel residue.
FIG. 9 shows the results of temperature screening of polysaccharide extraction from the hawthorn peel residue.
FIG. 10 shows the result of the liquid-solid ratio screening of polysaccharide extracted from the hawthorn peel residue.
FIG. 11 shows the effect of Tween 80 on the extraction rate of flavone from hawthorn peel residue.
FIG. 12 is a graph showing the effect of Tween 80 on the extraction rate of polysaccharides from the hawthorn peel residue.
FIG. 13 shows the results of screening the elution volumes of polysaccharide distilled water.
FIG. 14 shows the screening results of the elution volumes of flavonol.
FIG. 15 shows the screening results of the flow rate of flavonol elution.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1
A method for extracting and separating flavone and polysaccharide from hawthorn peel residue comprises the following steps:
(1) pretreatment of hawthorn peel residues:
filtering the hawthorn peel residue after fermentation of the hawthorn wine by using gauze, removing seeds, naturally drying in the shade, putting the hawthorn peel residue after natural drying in the shade into a pulverizer, pulverizing into powder, and sieving with a 60-mesh sieve;
(2) ultrasonic enzyme method combined extraction: adding 20% (v/v) ethanol into the hawthorn peel residue obtained in the step (1) according to a material-liquid ratio of 1:40(g/ml), and mixing uniformly; then adding 0.25% (m/m) cellulase (1000U/mg) and 0.2% (v/v) Tween 80 into the mixture, placing into water bath at 60 deg.C, ultrasonically extracting for 40min, repeating the extraction for 2 times, centrifuging after extraction, to obtain supernatant and precipitate, wherein the supernatant is the extractive solution of hawthorn flavone and polysaccharide;
(3) and (3) evaporation and concentration: placing the hawthorn flavone and polysaccharide extracting solution obtained in the step (2) on a rotary evaporator, evaporating and concentrating at 50 ℃, pouring into a culture dish, freezing in an ultra-low temperature refrigerator at-80 ℃ for overnight, and freeze-drying in a freeze dryer to obtain a hawthorn flavone polysaccharide crude extract;
(4) separating and purifying hawthorn flavone and polysaccharide:
(a1) and (3) separating hawthorn polysaccharide: and (3) adding 20% (v/v) ethanol into the hawthorn flavone polysaccharide crude extract obtained in the step (3) according to a feed-liquid ratio of 1: 2(g/ml), adsorbing with AB-8 macroporous resin, eluting with 3BV (resin volume) of distilled water at a flow rate of 2ml/min, collecting eluate, and concentrating under reduced pressure to 1/4 of fructus crataegi eluate volume to obtain fructus crataegi polysaccharide concentrated solution;
(a2) and (3) hawthorn polysaccharide purification: adding 5 times of ethanol into the hawthorn polysaccharide concentrated solution obtained in the step (a1) while stirring, standing for 12 hours in a refrigerator at 4 ℃, centrifuging, collecting precipitate, redissolving the precipitate by using distilled water, adding 5 times of savage reagent to remove protein in the polysaccharide, shaking by a shaker for 30 minutes, centrifuging for 20 minutes at 4000r/min, repeating for 3 times, combining supernate, concentrating under reduced pressure to 1/4 times of the volume of the supernate, adding 5 times of ethanol to dissolve, standing for 12 hours in a refrigerator at 4 ℃, centrifuging, and collecting precipitate, namely hawthorn polysaccharide;
(b1) and (3) separating hawthorn flavone: eluting the macroporous resin eluted by the distilled water in the step (a1) by using 50% (v/v) ethanol with 3BV (volume of the resin) at the flow rate of 2ml/min, collecting the eluent, and concentrating under reduced pressure to obtain a hawthorn flavone crude extract;
(b2) and (3) hawthorn flavone purification: and (b) dissolving the hawthorn flavone crude extract obtained in the step (b2) in ethanol, extracting by using n-hexane with the volume of 3 times, then keeping an ethanol phase to remove fat-soluble substances in the hawthorn flavone crude extract, and concentrating under reduced pressure to obtain the hawthorn flavone extract.
Example 2
A method for extracting and separating flavone and polysaccharide from hawthorn peel residue comprises the following steps:
(1) pretreatment of hawthorn peel residues:
filtering the hawthorn peel residue after fermentation of the hawthorn wine by using gauze, removing seeds, naturally drying in the shade, putting the hawthorn peel residue after natural drying in the shade into a pulverizer, pulverizing into powder, and sieving with a 60-mesh sieve;
(2) ultrasonic enzyme method combined extraction: adding 20% (v/v) ethanol into the hawthorn peel residue obtained in the step (1) according to the material-liquid ratio of 1:40, and mixing uniformly; then adding 0.2% (m/m) cellulase (1000U/mg) and 0.25% (v/v) Tween 80, placing in 60 deg.C water bath, ultrasonic extracting for 40min, repeating the extraction for 2 times, centrifuging after extraction to obtain supernatant and precipitate, the supernatant is fructus crataegi flavone and polysaccharide extract;
(3) and (3) evaporation and concentration: placing the hawthorn flavone and polysaccharide extracting solution obtained in the step (2) on a rotary evaporator, evaporating and concentrating at 50 ℃, pouring into a culture dish, freezing in an ultra-low temperature refrigerator at-80 ℃ for overnight, and freeze-drying in a freeze dryer to obtain a hawthorn flavone polysaccharide crude extract;
(4) separating and purifying hawthorn flavone and polysaccharide:
(a1) and (3) separating hawthorn polysaccharide: and (3) adding 20% (v/v) ethanol into the hawthorn flavone polysaccharide crude extract obtained in the step (3) according to a feed-liquid ratio of 1: 2, adsorbing with AB-8 macroporous resin, eluting with 4BV (resin volume) of distilled water at the flow rate of 2ml/min, collecting eluate, and concentrating under reduced pressure to 1/4 of hawthorn eluate volume to obtain hawthorn polysaccharide concentrated solution;
(a2) and (3) hawthorn polysaccharide purification: adding 5 times of ethanol into the hawthorn polysaccharide concentrated solution obtained in the step (a1) while stirring, standing for 12 hours in a refrigerator at 4 ℃, centrifuging, collecting precipitate, redissolving the precipitate by using distilled water, adding 5 times of savage reagent to remove protein in the polysaccharide, shaking by a shaker for 30 minutes, centrifuging for 20 minutes at 4000r/min, repeating for 3 times, combining supernate, concentrating under reduced pressure to 1/4 times of the volume of the supernate, adding 5 times of ethanol to dissolve, standing for 12 hours in a refrigerator at 4 ℃, centrifuging, and collecting precipitate, namely hawthorn polysaccharide;
(b1) and (3) separating hawthorn flavone: eluting the macroporous resin eluted by the distilled water in the step (a1) by using 4BV (resin volume) of 50% ethanol at the flow rate of 2ml/min, collecting the eluent, and concentrating under reduced pressure to obtain a hawthorn flavone crude extract;
(b2) and (3) hawthorn flavone purification: and (b) dissolving the hawthorn flavone crude extract obtained in the step (b2) in ethanol, extracting with 4 times of volume of n-hexane, then keeping an ethanol phase to remove fat-soluble substances in the hawthorn flavone crude extract, and concentrating under reduced pressure to obtain the hawthorn flavone extract.
Example 3
A method for extracting and separating flavone and polysaccharide from hawthorn peel residue comprises the following steps:
(1) pretreatment of hawthorn peel residues:
filtering the hawthorn peel residue after fermentation of the hawthorn wine by using gauze, removing seeds, naturally drying in the shade, putting the hawthorn peel residue after natural drying in the shade into a pulverizer, pulverizing into powder, and sieving with a 60-mesh sieve;
(2) ultrasonic enzyme method combined extraction: adding 20% (v/v) ethanol into the hawthorn peel residue obtained in the step (1) according to the material-liquid ratio of 1:40, and mixing uniformly; then adding 0.3% (m/m) cellulase (1000U/mg) and 0.2% (v/v) Tween 80, placing in 70 deg.C water bath, ultrasonic extracting for 40min, repeating the extraction for 2 times, centrifuging after extraction to obtain supernatant and precipitate, the supernatant is fructus crataegi flavone and polysaccharide extract;
(3) and (3) evaporation and concentration: placing the hawthorn flavone and polysaccharide extracting solution obtained in the step (2) on a rotary evaporator, evaporating and concentrating at 50 ℃, pouring into a culture dish, freezing in an ultra-low temperature refrigerator at-80 ℃ for overnight, and freeze-drying in a freeze dryer to obtain a hawthorn flavone polysaccharide crude extract;
(4) separating and purifying hawthorn flavone and polysaccharide:
(a1) and (3) separating hawthorn polysaccharide: and (3) adding 20% (v/v) ethanol into the hawthorn flavone polysaccharide crude extract obtained in the step (3) according to a feed-liquid ratio of 1: 2, adsorbing with AB-8 macroporous resin, eluting with 5BV (resin volume) of distilled water at a flow rate of 2ml/min, collecting eluate, and concentrating under reduced pressure to 1/4 of hawthorn eluate volume to obtain hawthorn polysaccharide concentrated solution;
(a2) and (3) hawthorn polysaccharide purification: adding 6 times of ethanol into the hawthorn polysaccharide concentrated solution obtained in the step (a1) while stirring, standing for 12 hours in a refrigerator at 4 ℃, centrifuging, collecting precipitate, redissolving the precipitate by using distilled water, adding 6 times of savage reagent to remove protein in the polysaccharide, shaking by a shaker for 30 minutes, centrifuging for 20 minutes at 4000r/min, repeating for 3 times, combining supernate, concentrating under reduced pressure to 1/4 times of the volume of the supernate, adding 6 times of ethanol for dissolving, standing for 12 hours in a refrigerator at 4 ℃, centrifuging, and collecting precipitate, namely hawthorn polysaccharide;
(b1) and (3) separating hawthorn flavone: eluting the macroporous resin eluted by the distilled water in the step (a1) by using 5BV (resin volume) of 50% ethanol at the flow rate of 2ml/min, collecting the eluent, and concentrating under reduced pressure to obtain a hawthorn flavone crude extract;
(b2) and (3) hawthorn flavone purification: and (b) dissolving the hawthorn flavone crude extract obtained in the step (b2) in ethanol, extracting with 5 times volume of n-hexane, then keeping an ethanol phase to remove fat-soluble substances in the hawthorn flavone crude extract, and concentrating under reduced pressure to obtain the hawthorn flavone extract.
Screening experiments
1. Conditions of ultrasonic and enzyme method auxiliary technology
(1) Influence of different extraction conditions (without Tween 80) on extraction of flavone from fructus crataegi peel residue
According to a single-factor experiment, the optimal process for extracting the flavone from the hawthorn peel residue is obtained by screening the volume fraction of ethanol, the feed-liquid ratio, the enzyme addition amount, the extraction temperature and the extraction time (shown in figures 1-5): extracting with ethanol at volume ratio of 1/40(g/ml) and cellulase 0.4% at 60 deg.C for 50min, and repeating the extraction for 2 times, wherein the extraction rate of flavone is 5.21%.
(2) Influence of different extraction conditions (without Tween 80) on extraction of polysaccharide from fructus crataegi skin residue
According to a single-factor experiment, the optimal process for extracting the polysaccharide from the hawthorn peel residue is obtained by screening the volume fraction of ethanol, the feed-liquid ratio, the enzyme addition amount, the extraction temperature and the extraction time (shown in figures 6-10): the volume fraction of ethanol is 20%, the feed-liquid ratio is 1/40(g/ml), the addition amount of cellulase is 0.4%, ultrasonic extraction is performed at 60 deg.C for 50min, and the extraction is repeated for 2 times, and the extraction rate of polysaccharide under the extraction condition is 9.18%.
(3) Influence of Tween 80 on extraction of flavone and polysaccharide from fructus crataegi peel residue
Under the ultrasonic conditions of 20 percent of ethanol volume fraction, 1/40(g/ml) of material-liquid ratio and 60 ℃, the influence of Tween 80 on the extraction rate of flavone and polysaccharide in hawthorn peel residue under different ultrasonic time after 0.2 percent Tween 80 is added into the extracting solution with different enzyme addition amounts is detected (figures 11-12): when the addition amount of cellulase is 0.25% and the addition amount of Tween 80 is 0.2% (other extraction conditions are unchanged), ultrasonic extraction is carried out at 60 ℃ for 40min, the extraction rate of flavone is 5.19%, and the extraction rate of polysaccharide is 9.22%. This shows that the adsorption efficiency of the enzyme is improved after adding Tween 80, and the enzyme dosage and the extraction time can be effectively reduced.
2. Screening of resins
According to different characteristics of flavone and polysaccharide in the hawthorn peel residue, the adsorption effect of 7 resins is screened, the macroporous resin has no adsorption to hawthorn polysaccharide, the hawthorn polysaccharide can be retained in gaps of the macroporous resin, the hawthorn polysaccharide can be eluted when the hawthorn polysaccharide is eluted by distilled water, and the hawthorn flavone is adsorbed on the macroporous resin, so that the effect of separating the hawthorn polysaccharide is achieved, and the result is shown in table 1. The results in Table 1 show that the adsorption capacity, adsorption rate and resolution rate of the macroporous resin AB-8 on hawthorn flavone are higher than those of other resins, so that the AB-8 macroporous resin is selected to separate hawthorn flavone and polysaccharide.
TABLE 1 screening results of resins
| Resin composition | Adsorption Capacity (mg/g) | Adsorption Rate (%) | Resolution (%) |
| LS300B | 25.25 | 51.64 | 87.27 |
| LS800 | 24.58 | 50.27 | 88.61 |
| LS803 | 25.62 | 52.39 | 87.27 |
| LX28 | 18.52 | 37.87 | 82.02 |
| LX68M | 19.44 | 39.76 | 78.01 |
| LX32 | 19.33 | 39.52 | 79.61 |
| AB-8 | 28.22 | 57.72 | 90.81 |
3. Screening of polysaccharide distilled water elution volumes
When the distilled water was passed at 3BV, the yield of polysaccharide reached 98.7%, and the polysaccharide yield was almost unchanged by further increasing the volume of the distilled water eluent, so that the amount of distilled water eluent was the best at 3BV, as a result, see FIG. 13. The purity of the hawthorn polysaccharide obtained by separation under the condition is 81.89%, and the yield is 98.7%.
4. Screening of flavone eluent concentration
By screening ethanol eluents with different concentrations (table 2), wherein the flavone resolution of 50% ethanol eluents is as high as 92.36%, and the influence of continuously increasing the ethanol concentration AB-8 on the hawthorn flavone resolution is not great, so 50% ethanol is selected as the eluent. Wherein the flow rate is 2ml/min and the elution volume is 3 BV. Under the condition, the purity of the hawthorn flavone obtained by separation is 64.53%, and the yield is 91.58%.
TABLE 2 results of concentration screening of ethanol eluents
| AB-8 | Resolution (%) |
| Water (W) | 3.19 |
| 20% ethanol | 90.98 |
| 50% ethanol | 92.36 |
| 70% ethanol | 92.75 |
| 100% ethanol | 92.10 |
5. Screening of flavone ethanol eluent volume
When the 50% ethanol is 3BV, the flavone yield reaches 91.58%, and the flavone yield is almost unchanged by continuously increasing the volume of the ethanol eluent, so that the 50% ethanol elution is best when the ethanol eluent is 3BV, and the result is shown in FIG. 14.
6. Screening of flavone ethanol elution flow rate
When the elution flow rate of 50% ethanol is 2ml/min, the resolution of flavone reaches 92.36%, and the elution flow rate is increased and decreased, so that the best elution flow rate of 50% ethanol is 2ml/min, and the result is shown in fig. 15.
The foregoing description is only a preferred embodiment of the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A method for extracting and separating flavone and polysaccharide from hawthorn peel residue in a combined manner is characterized by comprising the following steps:
(1) pretreatment of hawthorn peel residues:
filtering hawthorn peel residues after fermentation of hawthorn wine, removing seeds, naturally drying in the shade, and putting the hawthorn peel residues after natural drying in the shade into a grinder to be ground into powder;
(2) ultrasonic enzyme method combined extraction: adding 20% ethanol into the hawthorn peel residue obtained in the step (1) according to the material-liquid ratio of 1:40, and mixing uniformly; then adding 0.2-0.3% (m/m) cellulase and 0.2-0.25% (v/v) Tween 80, placing the mixture into a water bath at the temperature of 60-70 ℃, ultrasonically extracting for 30-40 min, repeatedly extracting for 2 times, and centrifugally separating after extraction to obtain supernatant and precipitate, wherein the supernatant is the hawthorn flavone and polysaccharide extracting solution;
(3) and (3) evaporation and concentration: placing the hawthorn flavone and polysaccharide extracting solution obtained in the step (2) on a rotary evaporator, evaporating and concentrating, pouring into a culture dish, freezing in an ultra-low temperature refrigerator at-80 ℃ for overnight, and freeze-drying in a freeze dryer to obtain a hawthorn flavone polysaccharide crude extract;
(4) separating and purifying hawthorn flavone and polysaccharide:
(a1) and (3) separating hawthorn polysaccharide: dissolving the hawthorn flavone polysaccharide crude extract obtained in the step (3) with 20% ethanol, adsorbing with AB-8 macroporous resin, eluting with distilled water, collecting eluate, and concentrating under reduced pressure to 1/4 of hawthorn eluate volume to obtain hawthorn polysaccharide concentrated solution;
(a2) and (3) hawthorn polysaccharide purification: adding 5-6 times of ethanol into the hawthorn polysaccharide concentrated solution obtained in the step (a1) while stirring, standing for 12 hours in a refrigerator at 4 ℃, centrifuging, collecting precipitate, redissolving the precipitate with distilled water, adding 5-6 times of savage reagent, shaking a table, centrifuging, repeating for 3 times, combining supernate, concentrating under reduced pressure to 1/4 times of the volume of the supernate, adding 5-6 times of ethanol for dissolving, standing for 12 hours in a refrigerator at 4 ℃, centrifuging, collecting precipitate, and obtaining hawthorn polysaccharide;
(b1) and (3) separating hawthorn flavone: eluting the macroporous resin obtained by eluting the distilled water in the step (a1) with 50% ethanol, collecting the eluent, and concentrating under reduced pressure to obtain a hawthorn flavone crude extract;
(b2) and (3) hawthorn flavone purification: dissolving the hawthorn flavone crude extract obtained in the step (b1) in ethanol, extracting with n-hexane with the volume of 3-5 times, then keeping an ethanol phase, and concentrating under reduced pressure to obtain the hawthorn flavone extract;
in the step (a1), the distilled water is 3-5BV, and the elution flow rate is 2 ml/min; in the step (b1), the 50% ethanol solution is 3-5BV, and the elution flow rate is 2 ml/min.
2. The method for combined extraction and separation of flavone and polysaccharide from hawthorn peel residue as claimed in claim 1, wherein in step (1), the powder is sieved with a 60-mesh sieve.
3. The method for extracting and separating flavone and polysaccharide from hawthorn peel residue in combination as claimed in claim 1, wherein the enzyme activity of cellulase in step (2) is 1000U/mg.
4. The method for extracting and separating flavone and polysaccharide from hawthorn peel residue in a combined manner as claimed in claim 1, wherein the ratio of the hawthorn flavone polysaccharide crude extract to 20% ethanol in step (a1) is 1: 2.
5. the process for the combined extraction and separation of flavone and polysaccharide from hawthorn peel residue as claimed in claim 1, wherein preferably, the distilled water in step (a1) is 3 BV.
6. The process for the combined extraction and separation of flavone and polysaccharide from hawthorn peel residue as claimed in claim 1, wherein the 50% ethanol in step (b1) is preferably 3 BV.
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