CN108567836A - A method of combined extraction separation flavones and polysaccharide from hawthorn skin slag - Google Patents

Abstract

The invention discloses a method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel dregs. The method uses hawthorn peel dregs fermented with hawthorn wine as raw materials, and uses ultrasonic and enzymatic auxiliary technologies to jointly extract flavonoids and polysaccharides from hawthorn peel dregs. polysaccharides, and according to the different characteristics of flavonoids and polysaccharides in hawthorn peel dregs, fixed bed adsorption technology was used to simultaneously separate flavonoids and polysaccharides in hawthorn peel dregs extract, which not only separated and purified hawthorn polysaccharides, but also obtained hawthorn flavonoids extract , realized the comprehensive utilization of hawthorn, and also solved the waste of resources and environmental pollution caused by hawthorn dregs. The method of the present invention has high extraction volume, high purity, high extraction efficiency, no special equipment is needed in the extraction process, and the cost is low. It provides a way for the combined extraction and separation of flavonoids and polysaccharides, and also provides a new way for the utilization of hawthorn peel dregs. It has good social and economic benefits.

Description

A method for joint extraction and separation of flavonoids and polysaccharides from hawthorn peel dregs

technical field

The invention relates to a method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel dregs, belonging to the technical field of extraction technology.

Background technique

Hawthorn (Crataegus pinnatifida Bunge), also known as Shanliguo and Shanlihong, belongs to the genus Hawthorn in the Rosaceae family. It is distributed in Shandong, Shaanxi, Shanxi, Henan, Jiangsu, Zhejiang, Liaoning, Jilin, Heilongjiang, Inner Mongolia, Hebei and other places. Hawthorn fruit can be eaten raw or made into preserved fruit cake, and can be used as medicine after drying. It is a unique tree species with both medicinal and fruit properties in China. It has the functions of lowering blood fat, blood pressure, strengthening the heart, and anti-arrhythmia. It is a good medicine for stagnation, promoting blood circulation and resolving phlegm, and has a good curative effect on diseases such as chest, diaphragm and spleen fullness, hernia, blood stasis, and amenorrhea. Studies have shown that hawthorn contains chemical components such as organic acids, triterpenoids, flavonoids and polysaccharides. Among them, flavonoids and polysaccharides have better functions of lowering blood pressure and blood fat. In addition, hawthorn flavonoids and polysaccharides also have other important functions, such as preventing diabetes and its complications, treating bone disease and osteoporosis, etc. Therefore, it is of great significance to study the flavonoids and polysaccharides of hawthorn.

In recent years, with the improvement of people's living standards and health care awareness, hawthorn fruit wine, fruit vinegar, and fruit juice with health care functions have developed rapidly in the hawthorn processing industry, and a large amount of hawthorn peel dregs have also been produced. The hawthorn bark dregs after the fermentation of hawthorn wine still contains abundant active substances, and because of the lack of effective development and utilization, it causes huge waste of resources and environmental pollution. Therefore, digging out effective extraction and separation methods of functional components can not only improve the comprehensive utilization of hawthorn, but also reduce environmental pollution.

At present, there are few studies on functional substances in hawthorn peel dregs, and they focus on the research on the extraction process of a single functional substance in hawthorn, such as the extraction of flavonoids and triacic acid functional substances. There are very few studies on the extraction technology of hawthorn polysaccharides, and there is no research on the combined extraction and separation technology of functional substances in hawthorn.

Contents of the invention

Aiming at the deficiencies of the prior art, the object of the present invention is to provide a method for jointly extracting and separating flavonoids and polysaccharides from hawthorn skin dregs, which can simultaneously and effectively extract and separate hawthorn flavonoids and polysaccharides.

In order to achieve the above object, the technical solution adopted in the present invention is:

A method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel dregs, comprising the following steps:

(1) Hawthorn skin dregs pretreatment:

After the hawthorn skin dregs fermented with hawthorn wine are filtered, the seeds are removed, and then dried in the shade naturally, and the dried hawthorn skin dregs are put into a grinder to be ground into powder;

(2) Combined extraction by ultrasonic enzymatic method: add 20% ethanol to the hawthorn skin dregs obtained in step (1) according to the ratio of solid to liquid at 1:40 and mix well; then add 0.2-0.3% (m/m) cellulase and 0.2-0.25% (v/v) Tween 80, placed in a 60-70°C water bath for ultrasonic extraction for 30-40 minutes, repeated extraction twice, and centrifuged after extraction to obtain the supernatant and precipitate, the supernatant is hawthorn Extracts of flavonoids and polysaccharides;

(3) Evaporation and concentration: put the hawthorn flavonoids and polysaccharide extract obtained in step (2) on a rotary evaporator, evaporate and concentrate at 50°C, pour it into a petri dish, put it in a -80°C ultra-low temperature refrigerator to freeze overnight, and put Put into freeze-drying machine and freeze-dry, obtain hawthorn flavonoid polysaccharide crude extract;

(4) Separation and purification of hawthorn flavonoids and polysaccharides:

(a1) Hawthorn polysaccharide separation: the hawthorn flavonoid polysaccharide crude extract obtained in step (3) was dissolved in 20% ethanol, adsorbed on AB-8 macroporous resin, eluted with distilled water, collected eluate, and decompressed Concentrate to 1/4 of the volume of the hawthorn eluate to obtain the hawthorn polysaccharide concentrate;

(a2) Purification of hawthorn polysaccharides: add 5 to 6 times the volume of ethanol to the hawthorn polysaccharide concentrate obtained in step (a1) while stirring, put it in a refrigerator at 4°C for 12 hours, then centrifuge to collect the precipitate, and then remove the precipitate After reconstitution with distilled water, add 5-6 times the volume of savage reagent, shake on the shaker, centrifuge, repeat 3 times, combine the supernatant, concentrate under reduced pressure to 1/4 of the volume of the supernatant, and then add 5-6 times the volume of ethanol Dissolve, put it in a refrigerator at 4°C for 12 hours, then centrifuge, and collect the precipitate as hawthorn polysaccharide;

(b1) Separation of hawthorn flavonoids: eluting the macroporous resin eluted with distilled water in step (a1) with 50% ethanol, collecting the eluate, concentrating under reduced pressure to obtain a crude hawthorn flavonoid extract;

(b2) Purification of hawthorn flavonoids: dissolving the crude hawthorn flavonoid extract obtained in step (b2) in ethanol, extracting with 3-5 times the volume of n-hexane, leaving the ethanol phase, and concentrating under reduced pressure to obtain the hawthorn flavonoid extract.

In step (1), the powder is passed through a 60-mesh sieve.

The enzyme activity of cellulase in step (2) is 1000U/mg.

In step (a1), the solid-liquid ratio of the hawthorn flavonoid polysaccharide crude extract and 20% ethanol is 1:2.

In step (a1), the distilled water is 3-5BV, and the elution flow rate is 2ml/min.

Preferably, the distilled water in step (a1) is 3BV.

In step (b1), 50% ethanol is 3-5BV, and the elution flow rate is 2ml/min.

Preferably, the 50% ethanol in step (b1) is 3BV.

Beneficial effects of the present invention:

1. The present invention uses hawthorn peel dregs after fermentation of hawthorn wine as raw materials. Since hawthorn wine has been treated with pectinase during the production process, the pectin in hawthorn peel residue has been enzymatically hydrolyzed. During the extraction process of hawthorn flavonoids and polysaccharides In the middle, no pectinase treatment is needed, which effectively simplifies the extraction process of hawthorn flavonoids and polysaccharides, and also solves the problem of reuse of hawthorn peel dregs.

2. The present invention adopts the joint action of cellulase and Tween 80. Cellulase is a group of compound enzymes that can hydrolyze fibers, destroy plant cell walls, and fully release cell contents, which is beneficial to the extraction of flavonoids and polysaccharides in hawthorn peel dregs. Extraction; Tween 80 can effectively promote the molecular distribution of cellulase, reduce the probability of invalid adsorption, and thus improve the efficiency of enzymatic hydrolysis.

3. The present invention adopts ultrasonic and enzymatic auxiliary technology to jointly extract the flavonoids and polysaccharides in the hawthorn peel dregs, and according to the different characteristics of the flavonoids and polysaccharides in the hawthorn peel dregs, adopts the fixed bed adsorption technology to extract the flavonoids and polysaccharides in the hawthorn peel dregs extract. Synchronous separation with polysaccharides not only separates and purifies hawthorn polysaccharides, but also obtains hawthorn flavonoid extracts, realizes the comprehensive utilization of hawthorn, and also solves the waste of resources and environmental pollution caused by hawthorn peel dregs.

4. The method of the present invention has high extraction volume, high purity and high extraction efficiency. The extraction process does not require special equipment, and the cost is low. It provides a way for the joint extraction and separation of flavonoids and polysaccharides, and also provides a new method for the utilization of hawthorn peel dregs. way, with good social and economic benefits.

Description of drawings

Figure 1 is the result of screening the amount of enzyme added to extract flavonoids from hawthorn peel dregs.

Figure 2 is the result of ethanol concentration screening of flavonoids extracted from hawthorn peel dregs.

Figure 3 is the extraction time screening results of flavonoids in hawthorn peel dregs.

Figure 4 is the extraction temperature screening results of flavonoids in hawthorn peel dregs.

Figure 5 is the screening results of the solid-liquid ratio of flavonoids extracted from hawthorn peel dregs.

Figure 6 is the screening results of the amount of enzyme added for the extraction of polysaccharides from hawthorn peel dregs.

Figure 7 is the ethanol concentration screening results of polysaccharide extraction from hawthorn peel dregs.

Figure 8 is the extraction time screening result of polysaccharide extraction from hawthorn peel dregs.

Fig. 9 is the extraction temperature screening result of polysaccharide extraction from hawthorn peel dregs.

Figure 10 is the screening result of solid-liquid ratio for polysaccharide extraction from hawthorn peel dregs.

Figure 11 shows the effect of Tween 80 on the extraction rate of flavonoids in hawthorn peel dregs.

Figure 12 shows the effect of Tween 80 on the extraction rate of polysaccharides in hawthorn peel dregs.

Figure 13 is the screening result of polysaccharide distilled water elution volume.

Figure 14 is the screening result of ethanol elution volume of flavonoids.

Figure 15 is the screening result of flavone ethanol elution flow rate.

Detailed ways

The specific implementation of the present invention will be described in further detail below in conjunction with the examples.

Example 1

A method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel dregs, comprising the following steps:

(1) Hawthorn skin dregs pretreatment:

After the hawthorn skin dregs fermented by hawthorn wine are filtered with gauze, the seeds are removed, and then naturally dried in the shade, the hawthorn skin dregs after the natural shade drying are put into a pulverizer, ground into powder, and then passed through a 60-mesh sieve;

(2) Combined extraction by ultrasonic enzymatic method: add 20% (v/v) ethanol to the hawthorn peel dregs obtained in step (1) according to the ratio of solid to liquid 1:40 (g/ml) and mix well; then add 0.25% of the mixture (m/m) cellulase (1000U/mg) and 0.2% (v/v) Tween 80, put into 60 ℃ of water baths and extract ultrasonically for 40min, repeat extraction 2 times, extract and centrifuge to obtain supernatant and Precipitation, the supernatant is the extract of hawthorn flavonoids and polysaccharides;

(3) Evaporation and concentration: put the hawthorn flavonoids and polysaccharide extract obtained in step (2) on a rotary evaporator, evaporate and concentrate at 50°C, pour it into a petri dish, put it in a -80°C ultra-low temperature refrigerator to freeze overnight, and put Put into freeze-drying machine and freeze-dry, obtain hawthorn flavonoid polysaccharide crude extract;

(4) Separation and purification of hawthorn flavonoids and polysaccharides:

(a1) Separation of hawthorn polysaccharides: the crude extract of hawthorn flavonoid polysaccharides obtained in step (3) was dissolved in 20% (v/v) ethanol according to the ratio of solid to liquid (1:2 (g/ml), and then applied to AB-8 Macroporous resin adsorption, eluting with 3BV (resin volume) distilled water, the flow rate is 2ml/min, collecting the eluate, concentrating under reduced pressure to 1/4 of the volume of the hawthorn eluate, and obtaining the hawthorn polysaccharide concentrate;

(a2) Hawthorn polysaccharide purification: add 5 times the volume of ethanol to the hawthorn polysaccharide concentrate obtained in step (a1) while stirring, put it in a refrigerator at 4°C for 12 hours, then centrifuge to collect the precipitate, and then wash the precipitate with distilled water After reconstitution, add 5 times the volume of savage reagent to remove the protein in the polysaccharide, shake the shaker for 30 minutes, centrifuge at 4000r/min for 20 minutes, repeat 3 times, combine the supernatant, concentrate under reduced pressure to 1/1 of the volume of the supernatant After 4, add 5 times the volume of ethanol to dissolve, put it in the refrigerator at 4°C for 12 hours, then centrifuge, and collect the precipitate as hawthorn polysaccharide;

(b1) separation of hawthorn flavonoids: the macroporous resin after step (a1) distilled water elution is eluted with 3BV (resin volume) 50% (v/v) ethanol, and the flow rate is 2ml/min, collect the eluent, reduce Concentrate under pressure to obtain the crude extract of hawthorn flavonoids;

(b2) purification of hawthorn flavonoids: dissolve the hawthorn flavonoids crude extract obtained in step (b2) in ethanol, extract with 3 times the volume of n-hexane and leave the ethanol phase to remove the fat-soluble substances in the hawthorn flavonoids crude extract, reduce The hawthorn flavonoids extract is obtained after concentrated under pressure.

Example 2

A method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel dregs, comprising the following steps:

(1) Hawthorn skin dregs pretreatment:

After the hawthorn skin dregs fermented by hawthorn wine are filtered with gauze, the seeds are removed, and then naturally dried in the shade, the hawthorn skin dregs after the natural shade drying are put into a pulverizer, ground into powder, and then passed through a 60-mesh sieve;

(2) Combined extraction by ultrasonic enzymatic method: add 20% (v/v) ethanol to the hawthorn peel dregs obtained in step (1) according to the ratio of solid to liquid 1:40 and mix well; then add 0.2% (m/m) fiber Sulfase (1000U/mg) and 0.25% (v/v) Tween 80 were placed in a 60°C water bath for ultrasonic extraction for 40 minutes, repeated extraction twice, and centrifuged after extraction to obtain a supernatant and a precipitate. The supernatant was It is the extract of hawthorn flavonoids and polysaccharides;

(3) Evaporation and concentration: put the hawthorn flavonoids and polysaccharide extract obtained in step (2) on a rotary evaporator, evaporate and concentrate at 50°C, pour it into a petri dish, put it in a -80°C ultra-low temperature refrigerator to freeze overnight, and put Put into freeze-drying machine and freeze-dry, obtain hawthorn flavonoid polysaccharide crude extract;

(4) Separation and purification of hawthorn flavonoids and polysaccharides:

(a1) Hawthorn polysaccharide separation: the hawthorn flavonoid polysaccharide crude extract obtained in step (3) was dissolved with 20% (v/v) ethanol according to the ratio of solid to liquid at 1:2, and then adsorbed on AB-8 macroporous resin. Elute with 4BV (resin volume) distilled water, the flow rate is 2ml/min, collect the eluate, concentrate under reduced pressure to 1/4 of the volume of the hawthorn eluate, and obtain the hawthorn polysaccharide concentrate;

(a2) Hawthorn polysaccharide purification: add 5 times the volume of ethanol to the hawthorn polysaccharide concentrate obtained in step (a1) while stirring, put it in a refrigerator at 4°C for 12 hours, then centrifuge to collect the precipitate, and then wash the precipitate with distilled water After reconstitution, add 5 times the volume of savage reagent to remove the protein in the polysaccharide, shake the shaker for 30 minutes, centrifuge at 4000r/min for 20 minutes, repeat 3 times, combine the supernatant, concentrate under reduced pressure to 1/1 of the volume of the supernatant After 4, add 5 times the volume of ethanol to dissolve, put it in the refrigerator at 4°C for 12 hours, then centrifuge, and collect the precipitate as hawthorn polysaccharide;

(b1) Separation of hawthorn flavonoids: the macroporous resin eluted with distilled water in step (a1) is eluted with 4BV (resin volume) 50% ethanol, the flow rate is 2ml/min, the eluate is collected, concentrated under reduced pressure, and hawthorn is obtained Crude extract of flavonoids;

(b2) purification of hawthorn flavonoids: dissolve the hawthorn flavonoids crude extract obtained in step (b2) in ethanol, extract with 4 times the volume of n-hexane and leave the ethanol phase to remove fat-soluble substances in the hawthorn flavonoids crude extract, reduce The hawthorn flavonoids extract is obtained after concentrated under pressure.

Example 3

A method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel dregs, comprising the following steps:

(1) Hawthorn skin dregs pretreatment:

After the hawthorn skin dregs fermented by hawthorn wine are filtered with gauze, the seeds are removed, and then naturally dried in the shade, the hawthorn skin dregs after the natural shade drying are put into a pulverizer, ground into powder, and then passed through a 60-mesh sieve;

(2) Combined extraction by ultrasonic enzymatic method: add 20% (v/v) ethanol to the hawthorn skin dregs obtained in step (1) according to the ratio of material to liquid 1:40 and mix well; then add 0.3% (m/m) fiber Sulfase (1000U/mg) and 0.2% (v/v) Tween 80 were placed in a 70°C water bath for ultrasonic extraction for 40 minutes, repeated extraction twice, and centrifuged after extraction to obtain supernatant and precipitate. The supernatant was It is the extract of hawthorn flavonoids and polysaccharides;

(3) Evaporation and concentration: put the hawthorn flavonoids and polysaccharide extract obtained in step (2) on a rotary evaporator, evaporate and concentrate at 50°C, pour it into a petri dish, put it in a -80°C ultra-low temperature refrigerator to freeze overnight, and put Put into freeze-drying machine and freeze-dry, obtain hawthorn flavonoid polysaccharide crude extract;

(4) Separation and purification of hawthorn flavonoids and polysaccharides:

(a1) Hawthorn polysaccharide separation: the hawthorn flavonoid polysaccharide crude extract obtained in step (3) was dissolved with 20% (v/v) ethanol according to the ratio of solid to liquid at 1:2, and then adsorbed on AB-8 macroporous resin. Elute with 5BV (resin volume) distilled water, the flow rate is 2ml/min, collect the eluate, concentrate under reduced pressure to 1/4 of the volume of the hawthorn eluate, and obtain the hawthorn polysaccharide concentrate;

(a2) Hawthorn polysaccharide purification: add 6 times the volume of ethanol to the hawthorn polysaccharide concentrate obtained in step (a1) while stirring, put it in a refrigerator at 4°C for 12 hours, then centrifuge to collect the precipitate, and then wash the precipitate with distilled water After reconstitution, add 6 times the volume of savage reagent to remove the protein in the polysaccharide, shake the shaker for 30 minutes, centrifuge at 4000r/min for 20 minutes, repeat 3 times, combine the supernatant, concentrate under reduced pressure to 1/1 of the volume of the supernatant After 4, add 6 times the volume of ethanol to dissolve, put it in a refrigerator at 4°C for 12 hours, then centrifuge, and collect the precipitate as hawthorn polysaccharide;

(b1) Separation of hawthorn flavonoids: the macroporous resin eluted with distilled water in step (a1) is eluted with 5BV (resin volume) 50% ethanol, the flow rate is 2ml/min, the eluate is collected, and concentrated under reduced pressure to obtain hawthorn Crude extract of flavonoids;

(b2) purification of hawthorn flavonoids: dissolve the hawthorn flavonoid crude extract obtained in step (b2) in ethanol, extract with 5 times the volume of n-hexane and leave the ethanol phase to remove the fat-soluble substances in the hawthorn flavonoid crude extract and reduce The hawthorn flavonoids extract is obtained after concentrated under pressure.

screening experiment

1. Conditions for ultrasonic and enzymatic assisted techniques

(1) Effects of different extraction conditions (without adding Tween 80) on the extraction of flavonoids from hawthorn peel dregs

According to the single factor experiment, the volume fraction of ethanol, the ratio of solid to liquid, the amount of enzyme added, the extraction temperature, and the extraction time were screened, and the optimal extraction process of flavonoids in hawthorn peel dregs can be obtained (Figure 1-5): the volume fraction of ethanol is 20%, the solid-liquid ratio is 1/40 (g/ml), the amount of cellulase is 0.4%, ultrasonic extraction at 60°C for 50 minutes, repeated extraction twice, the extraction rate of flavonoids under these extraction conditions is 5.21%.

(2) Effects of different extraction conditions (without adding Tween 80) on the extraction of polysaccharides from hawthorn peel dregs

According to the single factor experiment, the volume fraction of ethanol, the ratio of solid to liquid, the amount of enzyme added, the extraction temperature, and the extraction time were screened, and the optimal process for extracting polysaccharides from hawthorn peel dregs can be obtained (Figure 6-10): the volume fraction of ethanol is 20%, the solid-liquid ratio is 1/40 (g/ml), the amount of cellulase is 0.4%, ultrasonic extraction at 60°C for 50 min, repeated extraction twice, the extraction rate of polysaccharide under these extraction conditions is 9.18%.

(3) Effect of Tween 80 on the extraction of flavonoids and polysaccharides from hawthorn peel dregs

Under the conditions of 20% ethanol volume fraction, 1/40 (g/ml) solid-liquid ratio, and 60°C ultrasonic conditions, after adding 0.2% Tween 80 to the extract with different enzyme amounts, the results of Tween 80 under different ultrasonic times were detected. The effect on the extraction rate of flavonoids and polysaccharides in hawthorn peel dregs (Figure 11-12): when the addition of cellulase is 0.25%, and the addition of Tween 80 is 0.2% (other extraction conditions remain unchanged), ultrasonic extraction at 60°C After 40 minutes, the extraction rate of flavonoids was 5.19%, and the extraction rate of polysaccharides was 9.22%. This shows that the addition of Tween 80 improves the adsorption efficiency of the enzyme, which can effectively reduce the amount of enzyme and the extraction time.

2. Resin screening

According to the different characteristics of flavonoids and polysaccharides in hawthorn peel dregs, the adsorption of 7 kinds of resins was screened. The macroporous resins have no adsorption to hawthorn polysaccharides, and the hawthorn polysaccharides will stay in the gaps of the macroporous resins. At this time, the hawthorn polysaccharides will be eluted, and the hawthorn flavonoids will be adsorbed on the macroporous resin, thereby achieving the effect of separating the hawthorn polysaccharides. The results are shown in Table 1. The results in Table 1 show that the macroporous resin AB-8 has higher adsorption capacity, adsorption rate and resolution rate of hawthorn flavonoids than other resins, so AB-8 macroporous resin was selected to separate hawthorn flavonoids and polysaccharides.

Table 1. Screening results of resin

resin 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 volume

When the distilled water flowing through is 3BV, the yield of polysaccharide reaches 98.7%. Continue to increase the volume of distilled water eluent, and the yield of polysaccharide is almost unchanged. Therefore, the elution amount of distilled water is the best when the amount of distilled water is 3BV. The results are shown in Figure 13. The purity of hawthorn polysaccharide isolated under this condition was 81.89%, and the yield was 98.7%.

4. Screening of flavonoid eluate concentration

By screening ethanol eluents of different concentrations (Table 2), the flavonoid resolution rate of 50% ethanol eluent was as high as 92.36%, and continuing to increase ethanol concentration AB-8 had little effect on the resolution rate of hawthorn flavonoids, so 50% ethanol was selected as eluent. The flow rate is 2ml/min, and the elution volume is 3BV. The purity of hawthorn flavonoids isolated under this condition was 64.53%, and the yield was 91.58%.

Table 2, concentration screening results of ethanol eluent

AB-8 Resolution (%) water 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 flowing through is 3BV, the yield of flavonoids reaches 91.58%. Continue to increase the volume of ethanol eluent, and the yield of flavonoids is almost unchanged. Therefore, the best elution dosage of 50% ethanol is 3BV, the results are 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 rate of flavonoids reaches 92.36%. Increase the elution flow rate, and the resolution rate decreases. Therefore, the best elution rate of 50% ethanol is 2ml/min. The results are shown in Figure 15 .

The above description is only the best embodiment of the present invention, for those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (8)

1. A method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel residue, is characterized in that, comprises the following steps: (1) Hawthorn skin dregs pretreatment: After the hawthorn skin dregs fermented with hawthorn wine are filtered, the seeds are removed, and then dried in the shade naturally, and the dried hawthorn skin dregs are put into a grinder to be ground into powder; (2) Combined extraction by ultrasonic enzymatic method: add 20% ethanol to the hawthorn skin dregs obtained in step (1) according to the ratio of solid to liquid at 1:40 and mix well; then add 0.2-0.3% (m/m) cellulase and 0.2-0.25% (v/v) Tween 80, placed in a 60-70°C water bath for ultrasonic extraction for 30-40 minutes, repeated extraction twice, and centrifuged after extraction to obtain the supernatant and precipitate, the supernatant is hawthorn Extracts of flavonoids and polysaccharides; (3) Evaporation and concentration: put the hawthorn flavonoids and polysaccharide extract obtained in step (2) on a rotary evaporator, pour it into a petri dish after evaporation and concentration, put it in a -80°C ultra-low temperature refrigerator and freeze it overnight, then put it in the freezer Freeze-drying in a drier to obtain the crude extract of hawthorn flavonoid polysaccharides; (4) Separation and purification of hawthorn flavonoids and polysaccharides: (a1) Hawthorn polysaccharide separation: the hawthorn flavonoid polysaccharide crude extract obtained in step (3) was dissolved in 20% ethanol, adsorbed on AB-8 macroporous resin, eluted with distilled water, collected eluate, and decompressed Concentrate to 1/4 of the volume of the hawthorn eluate to obtain the hawthorn polysaccharide concentrate; (a2) Purification of hawthorn polysaccharides: add 5 to 6 times the volume of ethanol to the hawthorn polysaccharide concentrate obtained in step (a1) while stirring, put it in a refrigerator at 4°C for 12 hours, then centrifuge to collect the precipitate, and then remove the precipitate After reconstitution with distilled water, add 5-6 times the volume of savage reagent, shake on the shaker, centrifuge, repeat 3 times, combine the supernatant, concentrate under reduced pressure to 1/4 of the volume of the supernatant, and then add 5-6 times the volume of ethanol Dissolve, put it in a refrigerator at 4°C for 12 hours, then centrifuge, and collect the precipitate as hawthorn polysaccharide; (b1) Separation of hawthorn flavonoids: eluting the macroporous resin eluted with distilled water in step (a1) with 50% ethanol, collecting the eluate, concentrating under reduced pressure to obtain a crude hawthorn flavonoid extract; (b2) Purification of hawthorn flavonoids: dissolving the crude hawthorn flavonoid extract obtained in step (b2) in ethanol, extracting with 3-5 times the volume of n-hexane, leaving the ethanol phase, and concentrating under reduced pressure to obtain the hawthorn flavonoid extract. 2. The method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel dregs according to claim 1, characterized in that the powder in step (1) passes through a 60-mesh sieve. 3. The method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel dregs according to claim 1, characterized in that the enzyme activity of cellulase in step (2) is 1000U/mg. 4. The method for jointly extracting and separating flavonoids and polysaccharides from hawthorn skin dregs according to claim 1, characterized in that the solid-liquid ratio of the crude extract of hawthorn flavonoid polysaccharides and 20% ethanol in step (a1) is 1:2 . 5. The method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel dregs according to claim 1, wherein the distilled water in step (a1) is 3-5BV, and the elution flow rate is 2ml/min. 6. The method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel dregs according to claim 5, characterized in that, preferably, the distilled water in step (a1) is 3BV. 7. The method for jointly extracting and separating flavonoids and polysaccharides from hawthorn skin dregs according to claim 1, characterized in that, in step (b1), 50% ethanol is 3-5BV, and the elution flow rate is 2ml/min. 8. The method for jointly extracting and separating flavonoids and polysaccharides from hawthorn peel dregs according to claim 7, characterized in that, preferably, the 50% ethanol in step (b1) is 3BV.