Method for extracting xanthohumol with antibacterial effect from humulus lupulus and application of xanthohumol
By using gradient elution of methyl tert-butyl ether and n-butanol combined with silica gel column chromatography and recrystallization, the problem of extracting and purifying xanthohumol from hops was solved, enabling efficient and low-cost large-scale production.
Patent Information
- Application Number
- CN202511101015.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-07
- Publication Date
- 2025-11-21
AI Technical Summary
Existing technologies are difficult to efficiently extract and purify xanthohumol from hops. The extracts contain a large number of impurities, making purification difficult and unsuitable for mass production.
Gradient elution with methyl tert-butyl ether and n-butanol, combined with silica gel column chromatography and recrystallization, yielded high-purity crude xanthohumol through gradient elution with methyl tert-butyl ether and n-butanol followed by concentration and crystallization.
It achieves efficient extraction and purification of xanthohumol, suitable for large-scale production, with a xanthohumol purity of over 97%, thus reducing production costs.
Smart Images
Figure CN120987744A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant monomer extraction and purification technology, and in particular to a method and application for extracting xanthohumol with antibacterial activity from hops. Background Technology
[0002] Hops, scientifically known as *Hedyotis diffusa*, is also called hops grass, fragrant hops, and hops, and belongs to the genus *Hedyotis diffusa* in the family Cannabisaceae.
[0003] The chemical composition of hops can be summarized into several major components, including resins, volatile oils, polyphenolic compounds, and flavonoids. Among them, hop resins (α-acids and β-acids) are important raw materials required in the beer industry, which can give beer a refreshing bitterness and unique aroma.
[0004] Xanthohumol, chemical name: ((E)-1-[2,4-dihydroxy-6-methoxy-3-(3-methylbut-2-enyl)-phenyl]-3-(4-hydroxyphenyl)-prop-2-en-1-one), molecular formula: C21H22O5, molecular weight: 354.4, is a yellow crystalline powder in high concentrations, soluble in ethanol, methanol, acetone, ethyl acetate, DMSO, etc., slightly soluble in water, and has the following structural formula:
[0005]
[0006] Xanthumol is currently found only in hops in nature. It is an isoprenyl flavonoid, and its content in hops is only 0.1% to 1% of its dry weight. Xanthumol has strong pharmacological activity, inhibiting cancer cell proliferation and preventing cancer cell production. It also has good antioxidant and antibacterial effects, some antiviral activity, and can effectively prevent and treat diabetes. Given the numerous physiological activities of xanthumol, developing new applications for xanthumol in hops in medicine, food, health products, and skin whitening products is of great significance. The prerequisite for developing these applications of xanthumol is how to efficiently extract high-content xanthumol. Given the current limitations of chemical synthesis technology, inventing an efficient and simple method for extracting and purifying high-content xanthumol from hops is an important approach.
[0007] Current methods for extracting xanthohumol from hops result in extracts containing a large number of impurities in addition to xanthohumol, making the purification of xanthohumol quite difficult. This requires the use of high-speed countercurrent chromatography or ultrafiltration and sodium filtration equipment, but these facilities are not suitable for the requirements of mass production processes. Summary of the Invention
[0008] The main objective of this invention is to provide a method for extracting xanthohumol with antibacterial properties from hops, thereby solving the technical problem of the considerable difficulty in extracting xanthohumol from hops in existing methods.
[0009] To achieve the above objectives, the present invention provides a method for extracting xanthohumol with antibacterial activity from hops, comprising the following steps:
[0010] hops were extracted using methyl tert-butyl ether and then defatted.
[0011] The hop extract residue was extracted with n-butanol to obtain an extract;
[0012] The extract was concentrated and subjected to silica gel column chromatography to obtain an elution solution. The silica gel column chromatography employed gradient elution with three sequentially used volume gradients: methyl tert-butyl ether: n-butanol = 4–6:1, methyl tert-butyl ether: n-butanol = 2–3:1, and n-butanol. The eluent was used in increments of 4–10 times the volume of the concentrate, 4–10 times the volume of the concentrate, and 3–6 times the volume of the concentrate.
[0013] The elution solution was concentrated and crystallized, and recrystallized to obtain xanthohumol.
[0014] According to an embodiment of this application, the rinsing agent comprises three sequentially used volume gradients: methyl tert-butyl ether: n-butanol = 5:1, methyl tert-butyl ether: n-butanol = 2.5:1, and n-butanol; the amount of rinsing agent used at one time is 7 times the volume of the concentrate, 7 times the volume, and 5 times the volume;
[0015] According to the embodiments of this application, an elution solution is obtained by collecting 2 / 3 of the eluent after using methyl tert-butyl ether: n-butanol = 2-3:1 and the entire eluent after using n-butanol.
[0016] According to the embodiments of this application, in the step of extracting hops with methyl tert-butyl ether for defatting, the ratio of hops to methyl tert-butyl ether is 1:5 to 8, the extraction temperature is 25 to 50°C, and the extraction time is 1 to 3 hours.
[0017] According to the embodiments of this application, the ratio of hop extract residue to n-butanol is 1:8 to 10, the extraction temperature is 25 to 90°C, and the extraction time is 1 to 3 hours.
[0018] According to an embodiment of this application, the extract is concentrated into a paste.
[0019] According to the embodiments of this application, the conditions for concentrating and crystallizing the elution solution are: negative pressure P ≥ 0.08 MPa, concentration temperature T ≤ 60℃, and crystallization temperature 25~35℃.
[0020] According to an embodiment of this application, the crystallization solvent for recrystallization is n-butanol and water, and the volume ratio of concentrated crystals to n-butanol is 1 / 10 to 15.
[0021] According to the embodiments of this application, the recrystallization conditions are as follows: the volume ratio of concentrated crystals to water is 1 / 1 to 2; ethanol and concentrated crystals are heated and refluxed to dissolve, water is added at 60 to 70°C, and the temperature is lowered to 10 to 20°C for crystallization.
[0022] This application also provides an application of the above-described method for extracting xanthohumol, which has antibacterial properties, from hops.
[0023] In the above-described method for extracting xanthohumol with antibacterial properties from hops, extraction is performed sequentially using methyl tert-butyl ether (MTBE) and n-butanol, followed by gradient elution with a combination of MTBE and n-butanol to obtain a relatively pure crude xanthohumol. This crude product is then concentrated, crystallized, and recrystallized to obtain xanthohumol. This method for extracting xanthohumol with antibacterial properties from hops is simple to operate, does not require high-speed countercurrent chromatography, ultrafiltration, or sodium filtration equipment, and is suitable for large-scale production. Attached Figure Description
[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.
[0025] Figure 1 This is a flowchart of a method for extracting xanthohumol with antibacterial activity from hops according to an embodiment of this application.
[0026] The realization of the objective, functional characteristics and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation
[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0028] It should be noted that all directional indicators (such as up, down, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indicator will also change accordingly.
[0029] Furthermore, in this invention, descriptions involving "first," "second," etc., are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature.
[0030] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.
[0031] This invention provides a method for extracting xanthohumol with antibacterial activity from hops, comprising the following steps:
[0032] S100: Hops are extracted using methyl tert-butyl ether and then defatted.
[0033] In this step, defatting removes most of the soluble volatile oils, such as α-acids and β-acids.
[0034] S200: The hop extract residue was extracted with n-butanol to obtain an extract.
[0035] In this step, xanthohumol is readily soluble in alcohol and almost insoluble in water, so it can be extracted from the residue into n-butanol. This step can remove highly polar impurities (phenolic acids and some glycoside-containing flavonoids) that are not readily soluble in n-butanol but soluble in water, thus facilitating subsequent purification.
[0036] S300: Concentrate the extract and perform silica gel column chromatography to obtain the eluent. Silica gel column chromatography uses a gradient elution with three sequentially used volume gradients: methyl tert-butyl ether: n-butanol = 4–6:1, methyl tert-butyl ether: n-butanol = 2–3:1, and n-butanol. The eluent volume is 4–10 times, 4–10 times, and 3–6 times the volume of the concentrate, respectively. This elution scheme provides better separation of different components in hops compared to petroleum ether / ethyl acetate gradient elution. Collecting the xanthohumol eluent and concentrating it for a single crystallization yields higher yields and purity.
[0037] In this step, the eluent with a ratio of 4 to 6:1 for methyl tert-butyl ether and n-butanol can wash out highly polar volatile oils, while the eluent with a ratio of 2 to 3:1 for methyl tert-butyl ether and n-butanol can wash out small molecule polyphenols such as catechol in the first 1 / 3 part and xanthohumol in the last 2 / 3 part. n-Butanol can wash out xanthohumol.
[0038] S400: Concentrate the elution solution to crystallize and recrystallize to obtain xanthohumol.
[0039] In the above-described method for extracting xanthohumol with antibacterial properties from hops, extraction is performed sequentially using methyl tert-butyl ether (MTBE) and n-butanol, followed by gradient elution with a combination of MTBE and n-butanol to obtain a relatively pure crude xanthohumol. This crude product is then concentrated, crystallized, and recrystallized to obtain xanthohumol. This method for extracting xanthohumol with antibacterial properties from hops is simple to operate, does not require high-speed countercurrent chromatography, ultrafiltration, or sodium filtration equipment, and is suitable for large-scale production.
[0040] In some specific embodiments, the rinsing agent comprises three sequentially used volume gradients: methyl tert-butyl ether: n-butanol = 5:1, methyl tert-butyl ether: n-butanol = 2.5:1, and n-butanol; the amount of rinsing agent used at one time is 7 times the volume of the concentrate, 7 times the volume, and 5 times the volume.
[0041] In some embodiments, two-thirds of the eluent after using methyl tert-butyl ether: n-butanol = 2-3:1 and the remaining eluent after using n-butanol are collected to obtain an eluent solution.
[0042] In some embodiments, the ratio of hop extract residue to n-butanol (g / ml) is 1:8 to 10, the extraction temperature is 25 to 90°C, and the extraction time is 1 to 3 hours.
[0043] In some embodiments, during the defatting step of extracting hops with methyl tert-butyl ether, the ratio of hops to methyl tert-butyl ether (g / ml) is 1:5-8, the extraction temperature is 25-50℃, and the extraction time is 1-3 hours.
[0044] In some embodiments, the extract is concentrated to a viscous consistency.
[0045] In some embodiments, the conditions for concentrating and crystallizing the elution solution are: negative pressure P ≥ 0.08 MPa, concentration temperature T ≤ 60°C, and crystallization temperature 25–35°C.
[0046] In some embodiments, the crystallization solvent for recrystallization is n-butanol and water, and the volume ratio of concentrated crystals to n-butanol is 1 / 10 to 15.
[0047] In some embodiments, the recrystallization conditions are as follows: the volume ratio of concentrated crystals to water is 1 / 1 to 2. Ethanol and concentrated crystals are heated to reflux and dissolved, water is added at 60 to 70°C, and the temperature is lowered to 10 to 20°C for crystallization.
[0048] This application also provides an application of the above-described method for extracting xanthohumol, which has antibacterial properties, from hops.
[0049] Example 1
[0050] Take 5 kg of hop granules and crush them (the measured HPLC content of xanthohumol in hops is 0.32%). Add 40 L of methyl tert-butyl ether and heat at 35 °C with stirring for 2 h. Filter and wash the filter residue with 5 L of methyl tert-butyl ether.
[0051] The filter residue was then extracted with 50 L of n-butanol at 30 °C with stirring for 2 h, filtered, and washed with 5 L of n-butanol. The n-butanol filtrate was concentrated under reduced pressure until no fraction was obtained under conditions of T ≤ 90 °C and vacuum P ≥ 0.08 MPa, yielding 238 g of a yellow-green viscous substance with a xanthohumol content of 7.5%.
[0052] 258g of the yellow-green viscous substance was subjected to silica gel column chromatography and eluted sequentially with methyl tert-butyl ether: n-butanol = 5:1 (1806ml), methyl tert-butyl ether: n-butanol = 3:1 (1806ml), and pure n-butanol = 1032ml. The last two-thirds of the methyl tert-butyl ether: n-butanol = 3:1 eluent and all the n-butanol eluent were collected.
[0053] The product was concentrated under reduced pressure at T≤90℃ and P≥0.08MPa until a large amount of yellow powder precipitated. The concentration was then reduced to 25℃, filtered, and dried under vacuum to obtain 15.2g of crude xanthohumol with an HPLC purity of 92.5%. 15.6g of xanthohumol was added to 156ml of n-butanol, heated to reflux until dissolved, cooled to 65℃, and 16ml of purified water was added. The mixture was then slowly cooled to 15℃ and filtered to obtain 13.1g of xanthohumol with an HPLC purity of 97.8%.
[0054] Example 2
[0055] Take 5 kg of hop granules, crush them, and the HPLC content is 0.30%. Add 35 L of methyl tert-butyl ether, heat at 40 °C and stir for 1 h, filter, and wash the filter residue with 5 L of methyl tert-butyl ether.
[0056] The filter residue was then extracted with 40 L of n-butanol at 35 °C with stirring for 2 h, filtered, and washed with 5 L of n-butanol. The n-butanol filtrate was concentrated under reduced pressure until no fraction was obtained under conditions of T ≤ 90 °C and vacuum P ≥ 0.08 MPa, yielding 249 g of a yellow-green viscous substance with a xanthohumol content of 6.6%.
[0057] 249g of the yellow-green viscous substance was subjected to silica gel column chromatography and eluted sequentially with methyl tert-butyl ether: n-butanol = 5:1 (1743ml), methyl tert-butyl ether: n-butanol = 3:1 (1743ml), and n-butanol = 996ml. The last two-thirds of the methyl tert-butyl ether: n-butanol = 3:1 eluent and all the n-butanol eluent were collected.
[0058] The xanthohumol was concentrated under reduced pressure at T≤90℃ and P≥0.08MPa until a large amount of yellow powder precipitated. The concentration was then reduced to 25℃, filtered, and dried under vacuum to obtain 15.8g of crude xanthohumol with an HPLC purity of 93.4%. 14.8g of xanthohumol was added to 148ml of anhydrous ethanol, heated to reflux until dissolved, cooled to 65℃, and 15ml of purified water was added. The mixture was then slowly cooled to 15℃ and filtered to obtain 13.7g of xanthohumol with an HPLC purity of 98.1%.
[0059] Example 3
[0060] Take 5 kg of hop granules, crush them, and the HPLC content is 0.35%. Add 40 L of methyl tert-butyl ether, heat at 30 °C and stir for 1.5 h, filter, and wash the filter residue with 5 L of methyl tert-butyl ether.
[0061] The filter residue was then extracted with 45 L of n-butanol at 50 °C with stirring for 1 h, filtered, and washed with 5 L of n-butanol. The n-butanol filtrate was concentrated under reduced pressure until no fraction was obtained under conditions of T ≤ 90 °C and vacuum P ≥ 0.08 MPa, yielding 262 g of a yellow-green viscous substance with a xanthohumol content of 8.8%.
[0062] 242g of the yellow-green viscous substance was subjected to silica gel column chromatography and eluted sequentially with methyl tert-butyl ether: n-butanol = 5:1 (1936ml), methyl tert-butyl ether: n-butanol = 2:1 (1694ml), and n-butanol = 1210ml. The last two-thirds of the methyl tert-butyl ether: n-butanol = 3:1 eluent and all the n-butanol eluent were collected.
[0063] The product was concentrated under reduced pressure at T≤90℃ and P≥0.08MPa until a large amount of yellow powder precipitated. The concentration was then reduced to 25℃, filtered, and dried under vacuum to obtain 16.5g of crude xanthohumol with an HPLC purity of 94.3%. 16.5g of xanthohumol was added to 165ml of n-butanol, heated to reflux until dissolved, cooled to 60℃, and 18ml of purified water was added. The mixture was then slowly cooled to 15℃ and filtered to obtain 14.8g of xanthohumol with an HPLC purity of 98.3%.
[0064] The advantage of using methyl tert-butyl ether: n-butanol in this invention is that, compared to existing processes where the eluent is mostly ethanol or methanol:water, resulting in a xanthohumol content of 80%–90%, using methyl tert-butyl ether: n-butanol as the eluent significantly increases the obtained xanthohumol content while maintaining the same yield; the content can reach over 97%, and is comparable to... Figure 1 The solvent used in the eluent can be recovered through degreasing and extraction steps, which greatly improves the solvent recovery rate and reduces industrial costs.
[0065] The above technical solutions of the present invention are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made under the technical concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications in other related technical fields, are included in the patent protection scope of the present invention.
Claims
1. A method for extracting xanthohumol with antibacterial activity from hops, characterized in that, Includes the following steps: hops were extracted using methyl tert-butyl ether and then defatted. The hop extract residue was extracted with n-butanol to obtain an extract; The extract was concentrated and subjected to silica gel column chromatography to obtain an elution solution. The silica gel column chromatography employed gradient elution with three sequentially used volume gradients: methyl tert-butyl ether: n-butanol = 4–6:1, methyl tert-butyl ether: n-butanol = 2–3:1, and n-butanol. The eluent was used in increments of 4–10 times the volume of the concentrate, 4–10 times the volume of the concentrate, and 3–6 times the volume of the concentrate. The elution solution was concentrated and crystallized, and recrystallized to obtain xanthohumol.
2. The method according to claim 1, characterized in that, The rinsing agent comprises three sequentially used volume gradients: methyl tert-butyl ether: n-butanol = 5:1, methyl tert-butyl ether: n-butanol = 2.5:1, and n-butanol; the amount of rinsing agent used at one time is 7 times the volume of the concentrate, 7 times the volume, and 5 times the volume.
3. The method according to claim 2, characterized in that, Collect 2 / 3 of the eluent after using methyl tert-butyl ether: n-butanol = 2-3:1, and the remaining eluent after using n-butanol to obtain the eluent solution.
4. The method according to claim 1, characterized in that, In the step of extracting hops with methyl tert-butyl ether for defatting, the ratio of hops to methyl tert-butyl ether is 1:5-8, the extraction temperature is 25-50℃, and the extraction time is 1-3 hours.
5. The method according to claim 1, characterized in that, The ratio of hop extract residue to n-butanol is 1:8-10, the extraction temperature is 25-90℃, and the extraction time is 1-3 hours.
6. The method according to claim 1, characterized in that, The extract was concentrated into a paste.
7. The method according to any one of claims 1 to 6, characterized in that, The conditions for concentrating and crystallizing the elution solution are: negative pressure P ≥ 0.08 MPa, concentration temperature T ≤ 60℃, and crystallization temperature 25~35℃.
8. The method according to any one of claims 1 to 6, characterized in that, The crystallization solvent for recrystallization is n-butanol and water, and the volume ratio of concentrated crystals to n-butanol is 1 / 10 to 15.
9. The method according to claim 8, characterized in that, The recrystallization conditions are as follows: the volume ratio of concentrated crystals to water is 1 / 1 to 2; ethanol and concentrated crystals are heated and refluxed to dissolve them; water is added at 60 to 70°C; and the temperature is lowered to 10 to 20°C for crystallization.
10. The application of the method for extracting xanthohumol with antibacterial activity from hops as described in any one of claims 1 to 9.