BE1030422B1 - METHOD FOR EXTRACTION AND PURIFICATION OF 8-PRENYLNARINGENIN FROM HOPS - Google Patents

Abstract

The present invention provides a method for the extraction and purification of 8-prenylnaringenin from hops, comprising the following steps: S1, first hop extract with supercritical carbon dioxide to obtain the first hop extract; S2, The first hop extract is extracted using an extractant to obtain an extraction solution; The extractant consists of water and a first solvent, the first solvent being selected from methanol, ethanol, acetone and ethyl acetate and the volume ratio of water and first solvent being 1:0.05 to 1; S3, A second extraction of the extract is performed with an extractant to obtain a second extract; The extractant consisted of one or two of n-butanol, isobutanol, ethyl acetate, n-hexane, cyclohexane and petroleum ether, mixed in a volume ratio of 0.5:0.1 to 5; S4, The second extract was purified by column chromatography to obtain 8-prenyl naringenin. A highly pure 8-prenyl-naringenin was isolated and purified from hops.

Description

1 BE2022/5985

METHOD FOR EXTRACTION AND PURIFICATION OF

8-PRENYLNARINGENIN FROM HOPS

Technical part

The present invention relates to the field of biopharmaceuticals and more particularly to a method for the extraction and purification of 8-prenylnaringenin from hops.

Background Technology 8-Prenylnaringenin (8PN) is an estrogenic isopentenyl flavonoid found in hops and has been described in the literature as the most promising phytoestrogen ever discovered. In vitro and in vivo studies have shown that SPN has a strong estrogenic effect at low doses and is more potent than others

Phytoestrogens such as soy isoflavones and chrysin, which have been shown to be effective in...

have been shown to provide relief from menopause symptoms in women. In addition to its estrogenic effect, 8-Prenyl Naringenin also has other effects, such as: b.

Cancer prevention, prevention of osteoporosis, inhibition of vascular hyperplasia and

Antioxidation.

At present, studies at home and abroad focus on the extraction and

Isolation of oleoresin and xanthohumol from hops, but none have been reported yet

Studies conducted on the extraction of the beneficial ingredient 8-prenylnaringenin from hops; In particular, the highly pure 8-prenylnaringenin is difficult to extract from hops using current processes.

Considering this, it is necessary to have a method of extraction and

Purification of 8-prenylnaringenin from hops to extract and purify 8-prenylnaringenin from hops with high purity and an industrial

to achieve production.

Content of the invention

The main aim of the present invention is to provide a method for

To provide extraction and purification of 8-prenylnaringenin from hops aimed at the industrial production of 8-prenylnaringenin with high purity from hops

2 BE2022/5985 aims.

In order to achieve the above purpose, the present invention provides

A method for the extraction and purification of 8-prenylnaringenin from hops that includes the following steps:

S1, A first extraction of the hop material with supercritical carbon dioxide to obtain a first hop extract residue.

S2, extraction of the first hop extract using an extractant to obtain an extract; the extractant comprises water and a first solvent, the first solvent being selected from methanol, ethanol, acetone,

ethyl acetate, wherein the water and the first solvent are mixed in a volume ratio of 1:0.05 to 1;

S3, A second extraction of the extract is performed with an extractant, producing a second extract; The extractant consists of one or both of n-butanol, isobutanol, ethyl acetate, n-hexane, cyclohexane,

petroleum ether mixed in a volume ratio of 0.5:0.1 to 5;

S4, The second extract is purified by column chromatography to produce 8-prenyl

to receive naringenin.

Furthermore, the hops are crushed to 20-120 meshes in stage S1.

Furthermore, in step S1, the first extraction is carried out at 10 to 60 °C.

Furthermore, the first extraction takes between 2 and 10 hours.

In step S2, the extraction process is followed by another step: concentrating the volume of the mixture of the extractant and the first hop extract to 0.1 to 0.9 times the original volume at 40 to 90 ° C to obtain the extract .

Furthermore, the column chromatography method in step S4 includes in particular:

S41, upsampling of the second extraction solution;

S42, elution with a first aqueous ethanol solution of 0-50% to obtain a first

to obtain eluent;

S43, elution of the first eluate with a second aqueous ethanol solution of 50-100% to obtain the second eluate;

S44, concentration of the second eluate at 40-90°C until ethanol-free.

3 BE2022/5985

In step S4, the first filtration process is followed by decolorization; The

Discoloration procedures include in particular:

S51, vacuum drying of the first filtered product to the first dried

to receive product.

S52, dissolving the dried product at 30-90°C in a third aqueous

Ethanol solution with a concentration of 50-100% in a ratio of 1:5-20 by mass of the dried product to the third aqueous ethanol solution to one

to obtain decolorizing solution;

S53, adding 0.5-10% of the volume of the decolorizing solution to activated carbon,

Decolorization at 30-90°C for 0.5-4 hours and filtering, concentrating, obtaining the

Decolorization concentrate, cooling and crystallization, recrystallization to obtain the 8-prenylnaringin.

Further, the exothermic crystallization process in step S53 particularly includes: crystallization of the decolorized concentrate after the decolorization process at 4 to 10 ° C for 2 to 48 hours and second filtration to obtain the crystalline product.

Furthermore, the recrystallization process in step S53 particularly includes:

Dissolving the crystalline product of the exothermic crystallization process at 30-90°C in 50-100% of a third high concentration of ethanolic water in a ratio of 1:5-20, based on the mass of the crystalline product, to the third high concentration of ethanolic water to one

to obtain redissolution solution;

The redissolution solution is crystallized at 4-10 °C for 2 to 48 hours and filtered a third time to obtain the 8-prenylnaringenin.

The second and third filtration steps are followed by vacuum drying of the second and third filtration products.

Useful effects achieved by the invention: (1) The invention is based on a first extraction of the hop material with supercritical carbon dioxide to obtain a first hop extract; The

Extraction and purification of 8-prenylnaringenin from hops was carried out by the

Extraction of the first hop extract, the second extraction and column chromatography.

The purity of 8-prenylnaringenin obtained through the above extraction and

4 BE2022/5985

Purification process was high and impurities were low. (2) In the first extraction, pure carbon dioxide was used for the first extraction, which consisted of water and a first solvent from the group methanol,

Ethanol, acetone or ethyl acetate, and in the second extraction one or both of n-butanol, isobutanol, ethyl acetate, n-hexane, cyclohexane or petroleum ether.

These solvents have small residual amounts and are not toxic to the

Environment.

Description of the attached drawings

To view the technical solutions in the embodiments or in the state of the art

To illustrate the technique of the invention more clearly, there follows a brief description of the accompanying drawings, which are included in the description of the embodiments or the

State of the art can be used, whereby it is obvious that the

Drawings in the following description only show some embodiments of the

Invention and that other drawings do not involve any creative work by a person of ordinary skill based on those depicted in those drawings

structures can be preserved. Fig. 1 shows a special embodiment of the

Invention.

Figure 1 shows a schematic diagram of a method for extraction and

Purification of 8-prenylnaringenin from hops in a specific embodiment of the present invention.

The objects, functional features and advantages of the present invention are described in more detail in connection with the exemplary embodiments and with reference to the accompanying drawings.

Specific implementation

What follows is a clear and complete description of the technical solutions in the

Embodiments of the present invention in conjunction with those attached

Drawings, it being clear that the embodiments described are only some of the

Embodiments of the present invention and not all of them.

Based on the embodiments of the present invention, all other embodiments that can be achieved by a person skilled in the art without any creative activity also fall within the scope of the present invention.

It should be noted that the following embodiments and features of the

Embodiments can be combined with one another, provided they do not contradict each other. It should also be understood that the terms used in the 5 embodiments of the invention are intended to indicate specific

Describe embodiments and are not intended to limit the scope of

to limit invention.

Unless otherwise defined, all are technical and scientific

Terms used in the present invention, the prior art and the invention documented herein, and all methods, devices and

Prior art materials similar to those described in the embodiments of

Invention are described, are similar or equivalent, can also be used

Implementation of the invention can be used.

If a range of values is given in the examples, unless otherwise stated in the present invention, the two end points can be any

Value range and each of the values between the two end points can be selected.

Unless specific conditions are specified in the following examples, test procedures generally correspond to conventional conditions or the conditions recommended by the relevant manufacturer. The ones in the following

Materials or reagents required for the implementation are commercially available unless otherwise stated.

It should be noted that the solvents according to the invention, such as the first solvents (methanol, ethanol, acetone and ethyl acetate) and the

Solvent of the extractants (n-butanol, isobutanol, ethyl acetate, n-hexane,

Cyclohexane and petroleum ether) are commercially available reagents with original purity if no concentration is specified.

Referring to Figure 1, the present invention provides a method

Extraction and purification of 8-prenylnaringenin from hops is available to achieve industrial production of 8-prenylnaringenin with high purity, which includes the following steps:

S1, A first extraction of the hop material with supercritical carbon dioxide to obtain a first hop extract residue.

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Supercritical carbon dioxide is carbon dioxide at a temperature above the critical temperature Tc=31.26°C and a pressure above the critical pressure

PC=7.4 MPa. Specifically, the hops can be in the first extraction tank of the supercritical

Carbon dioxide plant with pure carbon dioxide extracted to the first

To obtain hop extract.

S2, The first hop extract is extracted using an extractant to obtain an extraction solution; The extractant consists of water and a first solvent, the first solvent being selected from methanol,

Ethanol, acetone and ethyl acetate and the volume ratio of water and first

Solvent is 1:0.05 to 1;

S3, A second extraction of the extract is performed with an extractant to obtain a second extract; The extractant consisted of one or two of n-butanol, isobutanol, ethyl acetate, n-hexane, cyclohexane and petroleum ether, mixed in a volume ratio of 0.5:0.1 to 5; After a second extraction step, a second extraction is carried out, which contains the active ingredient 8-prenyl

Naringenin containing, further extracted, and the foreign fraction is discarded; It is advantageous to obtain a highly pure 8-prenylnaringenin after the remaining steps.

S4, The second extract was purified by column chromatography to obtain 8-prenyl

to receive naringenin.

The step described above allows the extraction of 8-prenylnaringenin from hops with high purity and few impurities.

During the first extraction in step S1, pure carbon dioxide is used for the first

Extraction used in the extraction in step S2 includes the extractant

Water and a first solvent, the first solvent being one of methanol,

is ethanol, acetone or ethyl acetate, and in the second extraction in step S3, the extractant comprises one or both of n-butanol, isobutanol, ethyl acetate, n-hexane, cyclohexane or petroleum ether. These solvents have low

residual amounts and are not toxic to the environment.

In step S1, the hops are crushed to 20-120 mesh. The crushed one

Hops (20-120 mesh) can be mixed with pure carbon dioxide to im

Extraction tank of the supercritical carbon dioxide system to carry out an extraction reaction and obtain the first hop extract.

7 BE2022/5985

In addition, in step S1, the first extraction is carried out at 10-60°C. This

Temperature range facilitates the extraction of pure carbon dioxide from the hops.

In addition, the duration of the first extraction is between 2 and 10 hours. With an extraction time of less than 2 hours, a maximum extraction of the first

Hop residue not possible; If the extraction time is too long, more than 10

Hours later, the first hop extract becomes supersaturated.

In step S2, the extraction process continues: concentrating the volume of the mixture of extractant and first hop extract to 0.1-0.9 times the original volume at 40-90°C to obtain an extract.

Specifically, the extract is mixed with water and one of the substances methanol, ethanol,

Acetone or ethyl acetate mixed in a ratio of 1:0.05 to 1. The extract will be 2 to 4

Extracted times at 10 to 60 °C with stirring, each for 0.5 to 5 hours. —2 times, filter, combine the filtrates and reduce the filtrate

Pressure at 40 to 90 ° C to 0.2 to 2 times the volume of the first hop extract.

Furthermore, the process for elution and removal of impurities in

Step S4:

S41, sampling of the second extraction solution;

S42, elution of the product of column chromatography with a first aqueous

Ethanol solution of 0-50% to obtain the first eluate.

S43, The first eluate was eluted with 50-100% of the second ethanol solution to obtain the second eluate.

S44, The second eluate is concentrated at 40-90°C until it is free of ethanol.

Specifically, the extract is placed on a resin column and eluted with 0-50% of the first aqueous ethanol to remove some impurities and the first

to obtain eluate; The second eluate was eluted with 50-100% ethanol in water to obtain the second eluate; The second eluate is collected and concentrated at 40-90 ° C under reduced pressure until free of ethanol, filtered and dried in vacuo.

In step S4, the first filtration process is followed by decolorization, Der

Decolorization process includes:

S51, vacuum drying of the first filtered product to the first dried

8 BE2022/5985

to receive product.

S52, dissolving the dried product with a third aqueous ethanol solution of 50-100% at 30-90°C in a ratio of 1:5-20 by mass of the dried

product to the third aqueous ethanol solution to obtain a decolorizing solution.

Note that the third ethanol solution may have the same concentration as the second ethanol solution used in step S43 above.

S53, To the decolorization solution, 0.5-10% of the volume of the decolorization solution is added to activated carbon, decolorized and filtered at 30-90°C for 0.5-4 hours, concentrated to obtain the decolorization concentrate, cooled and crystallized and recrystallized, to obtain 8-prenyl naringenin.

Specifically, the first dried product was in 50-100% aqueous

Ethanol solution dissolved in a ratio of 1:5-20 w/w at 30-90°C with stirring, 0.5-10% of the decolorization volume of activated carbon added, at 30-90°C for 0.5-4

Decolorized for hours, passed through a precision filter and the resulting filtrate was concentrated under reduced pressure at 40-90 ° C to 0.2-1 of the decolorized liquid volume.

In step S53, the method of crystallization by cooling further includes:

After the decolorization process, the decolorized concentrate is crystallized at 4 to 10 °C for 2 to 48 hours and then filtered to obtain the crystalline product.

The decolorization concentrate is crystallized at 4 to 10 °C for 2 to 48 hours and then filtered to obtain yellowish crystals.

Further, the recrystallization process in step S53 includes: dissolving the crystalline product from the cold crystallization process at 30-90° C. with 50-100% of a third high concentration of ethanol in water, with a

Mass to volume ratio of 1:5-20 between the crystalline product and the third high concentration of ethanol in water to obtain a redissolution solution.

The redissolution solution crystallizes at 4-10°C for 2 to 48 hours and is then filtered to obtain 8-prenylnaringenin.

Specifically, the crystalline product of the cold crystallization process was dissolved in water at 30-90 ° C with 50-100% of a third high concentration of ethanol, where

9 BE2022/5985 the mass-volume ratio between the crystalline product and the third high

Ethanol concentration in water was 1:5-20 to obtain a redissolution solution. The crystals were precipitated at 4 to 10 °C for 2 to 48 hours, after complete crystallization, the product is filtered and dried under vacuum to obtain white crystals.

The second and third filtration steps are followed by vacuum drying of the second and third filtration products.

In particular, the vacuum-dried end product is crushed in order to...

To obtain end product 8-prenylnaringenin.

To further understand the present invention, examples are given:

example 1

A method for the extraction and purification of 8-prenylnaringenin from hops, comprising: 1. Take 2 kg of raw hops, crush it to 40-60 mesh, put it in a supercritical carbon dioxide extraction kettle and leave the supercritical

Carbon dioxide extraction system run for 4 hours to extract the hops.

After the extraction is complete, 1.95 kg of first hop extract is obtained. 2. The 1.95 kg of the first hop extract was placed in an extraction tank, 16 L of 40% aqueous methanol was added and extracted at 50°C for 2 hours.

The extracts were extracted twice, combined, concentrated to 0.5 L at 50 ° C under reduced pressure, extracted with 2 L ethyl acetate for 1 hour, allowed to stand for 0.5 hour, the ethyl acetate layer was separated and the aqueous layer was washed three times with a second extraction of 2 L ethyl acetate, the combined ethyl acetate extraction layers were concentrated under reduced pressure until the absence of ethyl acetate, completely dissolved by adding 50 ml ethanol, placed on a D101 resin column, with 2x water, 2x 20%

Ethanol in water, washed 2x 40% ethanol in water to remove impurities, then eluted 1x with 90% ethanol, and the 90% ethanol eluate was collected. 3. The powder was recovered under reduced pressure to 25 mL at 50°C and dried under vacuum to obtain 150 mg of light yellow powder. The 8-prenylnaringenin content was measured at 65.23%, and the 150

10 BE2022/5985 mg of the light yellow powder were dissolved in 20 ml of anhydrous ethanol and passed

Add 0.1 g of activated carbon to decolorize for 1 hour, filter through a 0.45 µm membrane and concentrate the filtrate to 10 ml at 50 °C under reduced pressure, 24

Placed in the refrigerator at 4°C for hours, filtered and dried under vacuum to obtain 85 mg of 8-prenylnaringenin, which contained 89.77% 8-prenylnaringenin and was slightly yellow in color. The resulting 85 mg of light yellow powder was dissolved in mL of anhydrous ethanol and crystallized at 4 °C for 24 h. The

Crystals were filtered and dried under vacuum to obtain 72 mg of 8-prenyl-naringenin, which contained 98.70% 8-prenyl-naringenin and was white in color. 10 Example 2

A method for the extraction and purification of 8-prenylnaringenin from hops, comprising: 1. Take 1 kg of raw hops, crush it to 40-60 mesh, put it in a supercritical carbon dioxide extraction kettle and leave the supercritical

Carbon dioxide extraction system run for 4 hours to extract the hops.

After the extraction is complete, 0.92 kg of the first hop extract is obtained. 2. The 0.92 kg of the first hop extract was placed in an extraction tank, 8L of 60% aqueous methanol was added and extracted at 50°C for 2 hours.

The extracts were extracted twice, combined, concentrated to 0.3 L at 50°C under reduced pressure, extracted with 1 L ethyl acetate for 1 hour, allowed to stand for 2 hours, the ethyl acetate layer was separated and the aqueous layer was extracted once with a second extraction extracted from 1 L ethyl acetate.

The ethyl acetate extracted layers were combined and reduced under reduced pressure

Pressure concentrated until free of ethyl acetate and completely with 80 mL

Ethanol dissolved, added to an AB-8 resin column, with 0.5x water, 1x 20%

Ethanol in water, eluted 1x 60% ethanol in water to remove impurities, then eluted 1x with 80% ethanol and collected the 80% ethanol eluate. 3. The powder was recovered under reduced pressure to 25 mL at 90°C and dried under vacuum to obtain 160 mg of light yellow powder. The 8-prenylnaringenin content was measured to be 72.2%, and the 160 mg of light yellow powder was dissolved in 10 mL of anhydrous ethanol and steeped

11 BE2022/5985

Adding 0.2 g of activated carbon for 1 hour, decolorized, filtered through a 0.45 µm membrane and concentrated the filtrate to 30 mL at 50 °C under reduced pressure. 24

Placed in the refrigerator at 4°C for hours, filtered and dried under vacuum to obtain 92 mg of 8-prenylnaringenin, which contained 91.6% 8-prenylnaringenin and was slightly yellow in color. The resulting 92 mg of light yellow powder was dissolved in 30 mL of anhydrous ethanol and crystallized at 4 °C for 24 h. The

Crystals were filtered and dried under vacuum to obtain 77 mg of 8-prenyl-naringenin, which contained 98.05% 8-prenyl-naringenin and was white in color.

Example 3

A method for the extraction and purification of 8-prenylnaringenin from hops, comprising: 1. Take 10 kg of raw hops, crush it to 40-60 mesh, put it in a supercritical carbon dioxide extraction kettle and leave the supercritical

Carbon dioxide extraction system run for 4 hours to extract the hops.

After the extraction is complete, 9.58 kg of first hop extract is obtained. 2. The 9.58 kg of the first hop extract was placed in an extraction tank, 80 L of 60% aqueous methanol was added and extracted at 50°C for 2 hours.

The extracts were extracted twice, pooled, The extract was extracted twice at 50°C for 2 h. The extract was concentrated to 3 L at 50 °C under reduced pressure, extracted with 6 L of ethyl acetate for 1 h and allowed to stand for 2 h. The

Ethyl acetate layer was separated and the aqueous The aqueous layer was extracted a second time with 6 L of ethyl acetate and the ethyl acetate extracted layer was combined. The layer was concentrated under reduced pressure until free of ethyl acetate and 640 mL of ethanol was added to dissolve completely, placed on a D101 resin column, with 3x water, 3x 20% ethanol in water, 2x 60 % ethanol in water to remove impurities, then eluted once with 95% ethanol and collected the 95% ethanol eluate. 3. The powder was recovered under reduced pressure to 450 mL at 40°C and dried under vacuum to obtain 1863 mg of light yellow powder. The content of 8-prenylnaringenin was measured to be 70.08%, and the 1863 mg of the light yellow powder was dissolved in 150 mL of anhydrous ethanol and percolated

Add 4.5 g of activated carbon for 1 hour to decolorize through a 0.45 µm membrane

12 BE2022/5985 filtered and the filtrate concentrated to 160 mL at 80 ° C under reduced pressure, 24

Placed in the refrigerator at 4°C for hours, filtered and dried under vacuum to obtain 865 mg of 8-prenylnaringenin, which contained 90.66% 8-prenylnaringenin and was slightly yellow in color. The resulting 865 mg of light yellow powder was dissolved in 100 mL of anhydrous ethanol and crystallized at 4 °C for 24 hours. The

Crystals were filtered and dried under vacuum to obtain 689 mg of 8-prenyl-naringenin, which contained 98.79% 8-prenyl-naringenin and was white in color.

Example 4

In comparison to Example 1, only the first solvent and that were used in step 2

Extractant changed. Add 1.95 kg of the first hop extract to the extraction tank, add 16 L of 30% aqueous acetone, extract for 2 hours at 50°C, extract twice, the

Combine extracts, concentrate under reduced pressure to 0.5 L at 50°C, add 2 L of a mixture of hexane and cyclohexane in a volume ratio of 0.5:1, 1

Extract for hour, let stand for 0.5 hour, separate The ethyl acetate layer and the aqueous layer were extracted three times with 2 L of ethyl acetate, the ethyl acetate extracted layer was combined and concentrated under reduced pressure until no more ethyl acetate was present, 50 mL of ethanol was added To completely dissolve them, the column was loaded onto the D101 resin, the

Impurities were removed by washing with 2x water, 2x 20% ethanol in water, 2x 40% ethanol in water, and then eluted 1x with 90% ethanol and the 90% ethanol eluate was collected.

The procedure from step 3 of Example 1 was repeated to obtain 312 mg of 8-prenylnaringin, which was measured to be 98.30% white.

Example 5

In comparison to Example 1, only the first solvent and that were used in step 2

Extractant changed.

That is, 1.95 kg of the first hop extract after defatting was put into one

Extraction tank given and 16 | Added 10% aqueous ethyl acetate and 2

Extracted at 50 °C for hours, and, The extract was extracted twice and concentrated to 0.5 L at °C under reduced pressure. 2 L of petroleum ether were added and extracted for 1 hour. After a standing time of 0.5 h, the

13 BE2022/5985

Ethyl acetate layer separated and the aqueous layer three times with a second

Extraction of 2 L ethyl acetate, and combine the layers extracted with ethyl acetate, concentrate under reduced pressure to ethyl acetate, add 50 mL ethanol, dissolve completely, place on a D101 resin column, with 2x water, 2x 20% ethanol in water, Wash 2x 40% ethanol in water

To remove impurities, then elute 1x with 90% ethanol, the 90%

Collect ethanol eluate.

The procedure from step 3 of Example 1 was repeated to obtain 529 mg of 8-prenylnaringin, which was measured to be 99.20% white.

Comparison ratio 1

In comparison to Example 1, only the first solvent and that were used in step 2

Extractant changed.

In other words: 1.95 kg of the first hop extract after defatting was placed in an extraction tank, a mixture of n-butanol and petroleum ether in a volume ratio of 1:5 was added in XXL, extracted at 50 ° C for 2 hours, extracted twice, the extracts were combined, concentrated to 0.5 L at 50 ° C under reduced pressure, 2 L of ethyl acetate was added, extracted for 1 hour, allowed to stand for 0.5 hour, the ethyl acetate layer was separated, the water layer was extracted three times with ethyl acetate. The aqueous layer was three times with 2 L

Ethyl acetate extracted, combined with the ethyl acetate extracted layer, concentrated under reduced pressure to ethyl acetate, completely dissolved by adding 50 mL of ethanol, placed on a D101 resin column, with 2x water, 2x 20%

Ethanol in water, washed 2x 40% ethanol in water to remove impurities, then eluted 1x with 90% ethanol and collected the 90% ethanol eluate.

Repeat step 3 of Example 1 to obtain 8-prenylnaringin.

Comparison ratio 2

In comparison to Example 1, only the first solvent and that were used in step 2

Extractant changed.

In other words, 1.95 kg of the first hop extract after defatting was placed in an extraction tank, a 1:1 mixture of hexane and

Petroleum ether XXL added, extracted for 2 hours at 50 ° C, extracted twice,

14 BE2022/5985 the extracts were combined, concentrated at 50 ° C under reduced pressure to 0.5 L, 2 L of ethyl acetate was added, extracted for 1 hour, left to stand for 0.5 hour, the ethyl acetate layer was separated, the aqueous layer was added three times

Ethyl acetate extracted and the aqueous layer extracted twice with ethyl acetate. The aqueous layer was extracted three times with ethyl acetate 2L, the extracted layer was combined with ethyl acetate, concentrated under reduced pressure to ethyl acetate, completely dissolved by adding 50 mL of ethanol, placed on a D101 resin column, with 2 times water, 2 times 20 % ethanol in water, washed twice with 40% ethanol in water to remove impurities, then once with 90%

Ethanol eluted and the 90% ethanol eluate was collected.

Repeat step 3 of Example 1 to obtain 8-prenylnaringin.

With the present invention, the extraction and purification of 8-prenylnaringenin from hops with high purity and few impurities has been achieved; In addition, the solvent residue that occurs during the extraction and

Cleaning costs are low and have no toxic effects on the environment.

In summary, the above-mentioned technical solutions of the present invention are only preferred embodiments of the present

Invention and do not serve to limit the scope of patent protection of the present

to limit invention. All equivalent structural transformations associated with the

Contents of the description and the attached drawings within the scope of the technical

Concept of the present invention or applied directly/indirectly in other related technical areas are within the scope of

Patent protection of the present invention included.

Claims (10)

15 BE2022/5985 patent claims 1. A method for the extraction and purification of 8-prenylnaringenin from hops, characterized in that it comprises the following steps: S1, first hop extract with supercritical carbon dioxide to obtain the first hop extract; S2, First hop extract using an extractant to obtain an extract; the extractant comprises water and a first solvent, the first solvent being selected from methanol, ethanol, acetone, ethyl acetate, the water and the first solvent being mixed in a volume ratio of 1:0.05 to 1; S3, A second extraction of the extract is performed with an extractant, producing a second extract; The extractant consists of one or both of n-butanol, isobutanol, ethyl acetate, n-hexane, cyclohexane, petroleum ether, mixed in a volume ratio of 0.5:0.1 to 5; S4, A second extract is purified by column chromatography to obtain 8-prenyl naringenin. 2. A method for the extraction and purification of 8-prenylnaringenin from hops according to claim 1, characterized in that in step S1 the hop material is crushed into 20-120 mesh hops. 3. A method for the extraction and purification of 8-prenylnaringenin from hops according to claim 1, characterized in that in step S1 the first extraction is carried out at 10-60 ° C. 4. Fine method for the extraction and purification of 8-prenylnaringenin from hops according to claim 1, characterized in that the first extraction lasts between 2 and 10 hours. 5. A method for extraction and purification of 8-prenylnaringenin from hops according to claim 1, characterized in that in step S2 the extraction process is followed by: concentrating the volume of the mixture of the extractant and the first hop extract to the 0.1 to 0.9 times the original volume at 40 to 90°C to obtain the extract. 16 BE2022/5985 6. A method for the extraction and purification of 8-prenylnaringenin from hops according to claim 1, characterized in that in step S4 the column chromatography method particularly comprises: S41, upward sampling of the second extraction solution; S42, elution with a first aqueous ethanol solution of 0- 50% to obtain a first eluent; S43, elution of the first eluate with a second aqueous ethanol solution of 50-100% to obtain the second eluate; S44, concentration of the second eluate at 40-90°C until ethanol-free. 7. A method for the extraction and purification of 8-prenylnaringenin from hops according to claim 1, characterized in that in stage S4 the first filtration process is followed by decolorization; The decolorization process includes in particular: S51, vacuum drying the first filtered product to obtain the first dried product; S52, dissolving the dried product at 30-90°C in a third aqueous ethanol solution with a concentration of 50-100% in a ratio of 1:5-20 by mass of the dried product to the third aqueous ethanol solution to obtain a decolorizing solution ; S53, adding 0.5-10% of the volume of the decolorizing solution to activated carbon, decolorizing at 30-90°C for 0.5-4 hours and filtering, concentrating, obtaining the decolorizing concentrate, cooling and crystallizing, recrystallizing to obtain the 8- to receive prenylnaringin. 8. A method for the extraction and purification of 8-prenylnaringenin from hops according to claim 7, characterized in that in step S53 the method of cooling and crystallization particularly comprises: crystallization of the decolorized concentrate after the decolorization process at 4 to 10 ° C for 2 to 48 hours and second filtration to obtain the crystalline product. 9. A method for the extraction and purification of 8-prenylnaringenin from hops according to claim 7, characterized in that in step S53 the recrystallization process particularly comprises: dissolving the crystalline product of the exothermic crystallization process at 30-90 ° C in 50-100% third high 17 BE2022/5985 Concentration of ethanolic water in a ratio of 1:5-20, based on the mass of the crystalline product, to the third high concentration of ethanolic water to obtain a redissolution solution; The redissolution solution is crystallized at 4-10 °C for 2 to 48 hours and filtered a third time to obtain the 8-prenylnaringenin. 10. A method for the extraction and purification of 8-prenylnaringenin from hops according to one of claims 8 to 9, characterized in that the second filtration and the third filtration are followed by vacuum drying of the second and third filtration products.