A method for jointly extracting total flavonoids, polyphenols and 10-HDA from honeycomb

Abstract

This invention relates to a method for the combined extraction of total flavonoids, polyphenols, and 10-HDA from honeycomb, addressing the technical problems of cumbersome and unsuitable industrial production processes for extracting total flavonoids from honeycomb, and the imperfections in the simultaneous extraction of total flavonoids, polyphenols, and 10-HDA. The method includes: 1. Obtaining an oily extract and a solid extraction residue after CO2 supercritical extraction of honeycomb raw materials; 2. Extracting total flavonoids from the oily extract; obtaining a precipitate and filtrate from the solid extraction residue; concentrating and drying the filtrate to obtain an extract containing polyphenols; 3. Obtaining a 10-HDA extract from the precipitate.

Description

Technical Field

[0001] This invention relates to a method for the combined extraction of total flavonoids, polyphenols and 10-HDA, and more specifically to a method for the combined extraction of total flavonoids, polyphenols and 10-HDA from honeycomb. Background Technology

[0002] A honeycomb is the dwelling place of a bee colony for living and reproducing. It consists of combs, each of which hangs parallel to the others in the honeycomb space. Each comb is formed by tens of thousands of cells tightly connected together and is built by worker bees using beeswax secreted from their wax glands. Countless combs are tightly connected to form the honeycomb. The honeycomb not only contains a large amount of solid beeswax, but also sugars, phenolic acids, proteins, flavonoids, and various trace elements such as Fe, Ga, K, and Cu.

[0003] Honeycomb contains abundant honeycomb flavonoids and honeycomb polyphenols. Honeycomb flavonoids have various medicinal functions, and many studies have confirmed that they have antibacterial and antioxidant effects. They are often referred to as scavengers of most oxygen free radicals. Honeycomb polyphenols are a general term for polyhydroxyphenolic compounds, which are secondary metabolites. Studies have shown that phenolic compounds have functions such as lowering blood lipids, preventing arteriosclerosis, reducing cholesterol, anti-tumor, and anti-inflammatory effects. They can inhibit auto-oxidation and scavenge hydroxyl, DPPH free radicals, and superoxide anions.

[0004] 10-Hydroxy-2-decenoic acid, abbreviated as 10-HDA, also known as royal jelly acid, is a special and highly effective bioactive substance found in royal jelly, generally accounting for about 1.4% to 2.0% of the total composition. In recent years, scholars both domestically and internationally have conducted in-depth research on 10-HDA from various aspects, including its sources, properties, structure, pharmacology, and assay methods. 10-HDA is an unsaturated fatty acid. It is a white crystalline solid at room temperature, with the molecular formula C2. 10 H 18 O3, with a molecular weight of 186.25, is stable and readily soluble in methanol, ethanol, chloroform, and ether; slightly soluble in acetone; and sparingly soluble in water. It possesses immune-enhancing, antibacterial, anti-inflammatory, anti-ulcer, anti-radiation, and lipid-lowering effects. 10-HDA is primarily extracted from royal jelly, which yields high levels of 10-HDA and is easy to extract. However, royal jelly, the raw material for 10-HDA extraction, is difficult to obtain and has high costs.

[0005] Chinese patent CN111789870A discloses a response surface methodology-based ultrasonic-assisted extraction method for honeycomb flavonoids, honeycomb flavonoids, and their effects. This patent utilizes ultrasonic extraction technology to explore the optimal process for ultrasonic extraction of honeycomb flavonoids and to study the antioxidant activity of honeycomb flavonoids. Based on single-factor experiments and using the Box-Behnken principle of response surface methodology, a predictive model is constructed to further optimize the extraction process and promote the development and utilization of honeycomb resources. However, this method only determines the extraction factors during the extraction of flavonoids from honeycomb and does not conduct subsequent purification experiments. Furthermore, ultrasonic extraction is cumbersome and time-consuming, making it unsuitable for large-scale industrial production.

[0006] Chinese patent CN103783349A discloses a process for preparing honeycomb polyphenol extract microcapsules by spray drying. This patent belongs to the field of microcapsule preparation. While it explores the powder spray drying of honeycomb water extract, it does not conduct more in-depth research on honeycomb polyphenols. Summary of the Invention

[0007] To address the technical problems of cumbersome extraction of total flavonoids from honeycomb, unsuitability for industrial production, and imperfections in the simultaneous extraction of total flavonoids, polyphenols, and 10-HDA, this invention proposes a method for the combined extraction of total flavonoids, polyphenols, and 10-HDA from honeycomb.

[0008] The technical solution provided by this invention is as follows:

[0009] A method for the combined extraction of total flavonoids, polyphenols and 10-HDA from honeycomb, characterized by comprising the following steps:

[0010] S1. Take the dried honeycomb raw material, crush it, and extract it using the supercritical CO2 method. The extraction entrainer is ethanol with a volume fraction of 90% to 95%. After the extraction is completed, separate it using a separation vessel to obtain the oily extract and solid extraction residue after supercritical CO2 extraction.

[0011] This step uses ethanol with a volume fraction of 90%–95% as the entrainer in the CO2 supercritical extraction. This is because using an ethanol entrainer with a volume fraction greater than 90% allows for better separation of total flavonoids and polyphenols, resulting in a reduction of flavonoids in the solid extraction residue and an increase in the flavonoid content in the oil extract. When the volume fraction of the ethanol entrainer is higher than 95%, it does not produce better results but instead wastes ethanol resources and increases the cost of industrial production.

[0012] S2. Concentrate the oily extract obtained in step S1 to an oil paste with a specific gravity of 1.15 to 1.25. Add petroleum ether to the oil paste at a volume of 4 to 6 times that of the oily extract to dissolve it. After complete dissolution, obtain an oil paste solution. Add methanol aqueous solution to the oil paste solution at a volume of 8 to 10 times that of the oily extract, stir and extract. Take the methanol phase extract, concentrate and dry it to obtain the total flavonoid extract.

[0013] Add 50-70% ethanol (volume fraction) to the solid extraction residue obtained in step S1 at a ratio of 1g:(8-12)ml, heat and extract multiple times, concentrate the obtained ethanol extract to a specific gravity of 1.15-1.20, and obtain concentrated ethanol extract; add 1-3 times the amount of water to the concentrated ethanol extract for dilution, then add 1-3% chitosan clarifying agent (mass fraction), stir and clarify at 60-70℃ for 10-30min, let it stand at room temperature for 20-24h to settle, filter to obtain precipitate and filtrate, concentrate and dry the filtrate to obtain an extract containing polyphenols;

[0014] S3. Add 6-9 times the amount of ethyl acetate to the precipitate from step S2, heat to dissolve for 1-3 hours, filter, and concentrate the filtrate to a specific gravity of 1.15-1.20 to obtain a concentrated paste. Mix the concentrated paste with silica gel at a mass ratio of 1:(2-3) and pack the sample silica gel with blank silica gel at a mass ratio of 1:(8-10) using a wet packing method to obtain a silica gel chromatography column. Elute the silica gel chromatography column with an eluent, monitor the eluent using thin-layer chromatography, collect the eluent containing 10-HDA, concentrate the eluent, allow it to stand and crystallize, filter, rinse the crystals and vacuum dry to obtain the 10-HDA extract.

[0015] Furthermore, in step S2, the oil extract concentration conditions are: vacuum degree of -0.08Mpa to -0.06Mpa, and temperature of 65℃ to 75℃;

[0016] A methanol-water solution with a volume fraction of 75%–85% was used for the extraction of total flavonoids; the extraction temperature was 20℃–30℃, the extraction time was 1h–2h, and the number of extractions was 2–4.

[0017] The conditions for concentrating and drying the methanol phase extract were: vacuum degree of -0.08 MPa to -0.06 MPa and temperature of 55℃ to 65℃.

[0018] Further, in step S1, the CO2 supercritical extraction method is specifically as follows: the crushed honeycomb raw material is added to the extraction vessel, and an ethanol entrainer is added. The extraction temperature is set to 47℃~53℃, the extraction pressure is 28Mpa~32Mpa, and the extraction time is 3h~5h for CO2 supercritical extraction.

[0019] Furthermore, in step S1, the separation using a separation vessel specifically means: separation is performed using a separation vessel;

[0020] Furthermore, in step S1, the separation vessel is used with a separation temperature of 43℃~46℃ and a separation pressure of 4Mpa~6Mpa.

[0021] Furthermore, in step S2, the multiple heating extractions specifically involve: an extraction temperature of 70℃~90℃, an extraction time of 1.5h~2.5h, and 2 to 4 extractions.

[0022] The filtrate concentration and drying process specifically involves the following steps: the vacuum degree for concentration and drying is -0.08 MPa to -0.06 MPa, and the temperature is 65℃ to 75℃.

[0023] Furthermore, in step S3, the ethyl acetate is heated to a temperature of 50-70°C;

[0024] The eluent is a mixture of petroleum ether and ethyl acetate in volume ratios of 20:1, 10:1, 8:2, and 7:3, or a mixture of n-hexane and ethyl acetate in volume ratios of 20:1, 10:1, 8:2, and 7:3, used for sequential elution.

[0025] The silicone is 100 mesh to 160 mesh;

[0026] The eluent concentration is specifically carried out at a concentration temperature of 50℃~60℃ and a concentration vacuum pressure of -0.09Mpa~-0.07Mpa.

[0027] The vacuum drying process specifically involves a drying temperature of 50℃~60℃ and a drying vacuum pressure of -0.09Mpa~-0.07Mpa.

[0028] Further, step S1 specifically involves: taking dried honeycomb raw material and crushing it, adding the crushed honeycomb raw material to the extraction vessel, and adding ethanol entrainer with a volume fraction of 90%. The extraction temperature is set to 50℃, the extraction pressure to 30 MPa, and the extraction time to 4 hours. After extraction, separation is performed using a separation vessel with a separation temperature of 45℃ and a separation pressure of 5 MPa to obtain the oily extract and solid extraction residue after CO2 supercritical extraction.

[0029] Step S2 specifically involves: concentrating the oily extract obtained in step S1 under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 70°C to a paste with a specific gravity of 1.20; dissolving the paste in petroleum ether at a volume ratio of 5 times that of the oily extract; obtaining a paste solution after complete dissolution; adding an 80% (v / v) methanol aqueous solution to the paste solution at a volume ratio of 10 times that of the oily extract; stirring and extracting at a temperature of 25°C for 1.5 hours, for a total of 3 extractions; concentrating and drying the methanol phase extract under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 60°C to obtain the total flavonoid extract;

[0030] Add 60% ethanol (volume fraction) to the solid extraction residue obtained in step S1 at a ratio of 1g:10ml, heat to 80℃ and extract three times, each extraction time being 2h. The obtained ethanol extract is concentrated to a specific gravity of 1.18 under vacuum of -0.08Mpa to -0.06Mpa and temperature of 70℃ to obtain concentrated ethanol extract. Dilute the concentrated ethanol extract with twice the volume of water, then add 2% chitosan clarifying agent (mass fraction), stir and clarify at 65℃ for 20min, and let it stand at room temperature for 22h to settle. Filter to obtain precipitate and filtrate. Concentrate and dry the filtrate under vacuum of -0.08Mpa to -0.06Mpa and temperature of 60℃ to obtain an extract containing polyphenols.

[0031] Step S3 specifically involves adding 8 times the amount of ethyl acetate to the precipitate from step S2, heating and dissolving it at 60°C for 2 hours, filtering, and concentrating the filtrate to a specific gravity of 1.18 under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 55°C to obtain a concentrated paste. The concentrated paste is then mixed with silica gel at a mass ratio of 1:2. The silica gel containing the sample and the blank silica gel are then wet-packed at a mass ratio of 1:9 to obtain a silica gel chromatography column. A petroleum ether:ethyl acetate ratio of 2:1 is used. Mixtures of 0:1, 10:1, 8:2, and 7:3 were sequentially used to elute a silica gel column. The eluent was monitored by thin-layer chromatography, and the eluent containing 10-HDA was collected. The eluent was concentrated at 55°C and a vacuum pressure of -0.09 MPa to -0.07 MPa. After crystallization, the eluent was filtered, washed with petroleum ether, and then dried under vacuum at 55°C and a vacuum pressure of -0.09 MPa to -0.07 MPa to obtain the 10-HDA extract.

[0032] The beneficial effects of this invention are:

[0033] 1. The method for jointly extracting total flavonoids, polyphenols and 10-HDA from honeycomb provided by the present invention adopts supercritical CO2 extraction and separates total flavonoids, polyphenols and 10-HDA from honeycomb by selecting 90% to 95% ethanol entrainer. The method is simple to operate, uses less solvent, and operates at a low temperature, which greatly reduces the extraction cost.

[0034] 2. This invention provides a convenient, quick, and environmentally friendly method for further processing the oily extract and solid residue after CO2 supercritical extraction. The resulting products have high content of total flavonoids, polyphenols, and 10-HDA, and high recovery rate, making them suitable for industrial production.

[0035] 3. The method for jointly extracting total flavonoids, polyphenols and 10-HDA from honeycomb provided by the present invention has a simple extraction and purification process for total flavonoids, polyphenols and 10-HDA, and a short process flow, thereby achieving efficient industrial production.

[0036] 4. The method for jointly extracting total flavonoids, polyphenols, and 10-HDA from honeycomb provided by this invention involves the extraction and purification of 10-HDA, thus utilizing the trace amounts of 10-HDA in the original honeycomb and increasing the utilization rate of effective substances in the honeycomb. Furthermore, the extraction and purification of 10-HDA from honeycomb in this patent provides technical support for the subsequent preparation of high-content 10-HDA. Detailed Implementation

[0037] The following is a detailed description with reference to specific examples.

[0038] Example 1

[0039] Dry honeycomb raw material was crushed and added to the extraction vessel along with 90% ethanol entrainer. The extraction temperature was set at 50℃, the extraction pressure at 30 MPa, and the extraction time at 4 hours. After extraction, the mixture was separated in a separation vessel at a separation temperature of 45℃ and a separation pressure of 5 MPa to obtain the oily extract after CO2 supercritical extraction and the solid residue in the extraction vessel.

[0040] The obtained oily extract was concentrated to a paste with a specific gravity of 1.20 under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 70°C. Petroleum ether was added to the paste at a volume of 5 times that of the oily extract for dissolution. After complete dissolution, an oily solution was obtained. An 80% (v / v) methanol aqueous solution was added to the oily solution at a volume of 10 times that of the oily extract. The mixture was stirred and extracted at a temperature of 25°C for 1.5 hours, and the extraction was repeated 3 times. The methanol phase extract was concentrated and dried under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 60°C to obtain a total flavonoid extract with a content of 51.37%.

[0041] Add 60% ethanol (v / v) to the solid residue obtained by supercritical extraction at a ratio of 1g:10ml, heat to 80℃ and extract three times, each time for 2 hours. The obtained ethanol extract is concentrated to a specific gravity of 1.18 under vacuum of -0.08MPa to -0.06MPa and temperature of 70℃ to obtain concentrated ethanol extract. Dilute the concentrated ethanol extract with twice the volume of water, then add 2% chitosan clarifying agent, stir and clarify at 65℃ for 20 minutes, and let it stand at room temperature for 22 hours. Filter to obtain precipitate and filtrate. Concentrate and dry the filtrate under vacuum of -0.08MPa to -0.06MPa and temperature of 60℃ to obtain polyphenol extract with a content of 31.43%.

[0042] Eight times the volume of ethyl acetate was added to the precipitate, and the mixture was heated at 60°C for 2 hours to dissolve. The solution was then filtered, and the filtrate was concentrated to a specific gravity of 1.18 under a vacuum of -0.08 MPa to -0.06 MPa at 55°C to obtain a concentrated paste. The concentrated paste was mixed with silica gel at a mass ratio of 1:2 and thoroughly stirred. The silica gel containing the sample was then packed with blank silica gel at a mass ratio of 1:9 using a wet packing method to obtain a silica gel chromatography column. Petroleum ether:ethyl acetate ratios of 20:1, 10:1, and 8:2 were used. A 7:3 mixture was used to elute a silica gel column. The eluent was monitored by thin-layer chromatography, and the eluent containing 10-HDA was collected. The eluent was concentrated at 55°C and a vacuum pressure of -0.09 MPa to -0.07 MPa. After standing and crystallization, the eluent was filtered. The crystals were washed with petroleum ether and then dried under vacuum at 55°C and a vacuum pressure of -0.09 MPa to -0.07 MPa to obtain a 10-HDA crystalline extract with a content of 27.62%.

[0043] Example 2

[0044] Dry honeycomb raw material was crushed and added to an extraction vessel, along with ethanol entrainer with a volume fraction of 95%. The extraction temperature was set at 47℃, the extraction pressure at 32 MPa, and the extraction time at 3 hours. After extraction, the mixture was separated in a separation vessel at a separation temperature of 44℃ and a separation pressure of 4 MPa to obtain an oily extract and a solid residue after CO2 supercritical extraction.

[0045] The obtained oily extract was concentrated to a paste with a specific gravity of 1.25 under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 65°C. Petroleum ether was added to the paste at a volume of 6 times that of the oily extract for dissolution. After complete dissolution, a paste solution was obtained. A 75% (v / v) methanol aqueous solution was added to the paste solution at a volume of 8 times that of the oily extract. The mixture was stirred and extracted at 20°C for 1 hour, and the extraction was repeated 4 times. The methanol phase extract was then concentrated and dried under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 65°C to obtain a total flavonoid extract with a content of 50.86%.

[0046] Add 50% ethanol (v / v) to the solid residue obtained by supercritical extraction at a ratio of 1 g: 8 ml, and extract twice at 90°C for 2.5 h each time. The ethanol extract is concentrated to a specific gravity of 1.20 under vacuum of -0.08 MPa to -0.06 MPa and at 65°C to obtain concentrated ethanol extract. Dilute the concentrated ethanol extract with an equal volume of water, then add 3% chitosan clarifying agent. Stir and clarify at 60°C for 30 min, then let stand at room temperature for 20 h to settle. Filter to obtain precipitate and filtrate. Concentrate and dry the filtrate under vacuum of -0.08 MPa to -0.06 MPa and at 60°C to obtain a polyphenol extract with a content of 31.85%.

[0047] Add 6 times the volume of ethyl acetate to the precipitate, heat at 50℃ for 3 hours to dissolve, filter, and concentrate the filtrate to a specific gravity of 1.15 under vacuum of -0.08 MPa to -0.06 MPa at 50℃ to obtain a concentrated paste. Mix the concentrated paste with silica gel at a mass ratio of 1:2. Pack the silica gel containing the sample and the blank silica gel at a mass ratio of 1:8 using a wet packing method to obtain a silica gel chromatography column. Use n-hexane:ethyl acetate ratios of 20:1, 10:1, and 8:2. A 7:3 mixture was used to elute a silica gel column. The eluent was monitored by thin-layer chromatography, and the eluent containing 10-HDA was collected. The eluent was concentrated at 50°C and a vacuum pressure of -0.09 MPa to -0.07 MPa. After standing and crystallization, the eluent was filtered. The crystals were washed with petroleum ether and then dried under vacuum at 50°C and a vacuum pressure of -0.09 MPa to -0.07 MPa to obtain a 10-HDA crystalline extract with a content of 28.45%.

[0048] Example 3

[0049] Dry honeycomb raw material was crushed and added to an extraction vessel, along with ethanol entrainer with a volume fraction of 92%. The extraction temperature was set at 53℃, the extraction pressure at 28 MPa, and the extraction time at 5 h. After extraction, the mixture was separated in a separation vessel at a separation temperature of 43℃ and a separation pressure of 5 MPa to obtain an oily extract and a solid residue after CO2 supercritical extraction.

[0050] The obtained oily extract was concentrated to a paste with a specific gravity of 1.15 under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 70°C. Petroleum ether was added to the paste at a volume of 4 times that of the oily extract for dissolution. After complete dissolution, an oily solution was obtained. An 80% (v / v) methanol aqueous solution was added to the oily solution at a volume of 9 times that of the oily extract. The mixture was stirred and extracted at a temperature of 30°C for 1.5 hours, and the extraction was repeated twice. The methanol phase extract was concentrated and dried under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 55°C to obtain a total flavonoid extract with a content of 51.14%.

[0051] Add 60% ethanol (v / v) to the solid residue obtained by supercritical extraction at a ratio of 1 g: 12 ml, and extract three times at 80°C for 1.5 h each time. The ethanol extract was concentrated to a specific gravity of 1.16 under vacuum of -0.08 MPa to -0.06 MPa and at 75°C to obtain a concentrated ethanol extract. Dilute the concentrated ethanol extract with three times the volume of water, then add 2% chitosan clarifying agent. Stir and clarify at 70°C for 10 min, then let it stand at room temperature for 24 h to settle. Filter to obtain precipitate and filtrate. Concentrate and dry the filtrate under vacuum of -0.08 MPa to -0.06 MPa and at 75°C to obtain a polyphenol extract with a content of 30.73%.

[0052] Add 7 times the volume of ethyl acetate to the precipitate, heat at 70℃ for 1 h to dissolve, filter, and concentrate the filtrate to a specific gravity of 1.20 under vacuum of -0.08 MPa to -0.06 MPa and at 605℃ to obtain a concentrated paste. Mix the concentrated paste with silica gel at a mass ratio of 1:3. Pack the silica gel containing the sample and the blank silica gel at a mass ratio of 1:10 using a wet packing method to obtain a silica gel chromatography column. Use petroleum ether:ethyl acetate ratios of 20:1, 10:1, and 8:1. 2. A 7:3 mixture was used to elute a silica gel column sequentially. The eluent was monitored by thin-layer chromatography, and the eluent containing 10-HDA was collected. The eluent was concentrated at 50°C and a vacuum pressure of -0.09 MPa to -0.07 MPa. After standing and crystallization, the eluent was filtered. The crystals were washed with petroleum ether and then dried under vacuum at 50°C and a vacuum pressure of -0.09 MPa to -0.07 MPa to obtain a 10-HDA crystalline extract with a content of 29.22%.

[0053] Example 4

[0054] Dry honeycomb raw material was crushed and added to an extraction vessel, along with ethanol entrainer with a volume fraction of 94%. The extraction temperature was set at 52℃, the extraction pressure at 30 MPa, and the extraction time at 3 hours. After extraction, the mixture was separated in a separation vessel at a separation temperature of 46℃ and a separation pressure of 6 MPa to obtain an oily extract and a solid residue after CO2 supercritical extraction.

[0055] The obtained oily extract was concentrated to a paste with a specific gravity of 1.22 under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 75°C. Petroleum ether was added to the paste at a volume of 5 times that of the oily extract for dissolution. After complete dissolution, an oily solution was obtained. An 85% methanol aqueous solution was added to the oily solution at a volume of 10 times that of the oily extract. The mixture was stirred and extracted at a temperature of 28°C for 2 hours, and the extraction was repeated 3 times. The methanol phase extract was concentrated and dried under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 58°C to obtain a total flavonoid extract with a content of 51.71%.

[0056] The solid residue obtained from supercritical fluid extraction was added with 70% ethanol at a ratio of 1 g: 9 ml, and the mixture was heated to 70°C for extraction four times, each extraction lasting 2 hours. The resulting ethanol extract was concentrated to a specific gravity of 1.15 under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 70°C to obtain a concentrated ethanol extract. The concentrated ethanol extract was diluted with twice the volume of water, and then 1% chitosan clarifying agent was added. The mixture was stirred and clarified at 70°C for 30 minutes, and then allowed to stand at room temperature for 21 hours. The precipitate and filtrate were obtained by filtration. The filtrate was concentrated and dried under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 65°C to obtain a polyphenol extract with a content of 31.46%.

[0057] Nine times the volume of ethyl acetate was added to the precipitate, and the mixture was heated at 60°C for 3 hours to dissolve it. The mixture was then filtered, and the filtrate was concentrated to a specific gravity of 1.15 under a vacuum of -0.08 MPa to -0.06 MPa at 60°C to obtain a concentrated paste. The concentrated paste was mixed with silica gel at a mass ratio of 1:3 and stirred thoroughly. The silica gel containing the sample was then packed into a silica gel column using a wet packing method with blank silica gel at a mass ratio of 1:9. A hexane:ethyl acetate ratio of 20:1, 10:1, and 8:2 was used. A 7:3 mixture was used to elute a silica gel column. The eluent was monitored by thin-layer chromatography, and the eluent containing 10-HDA was collected. The eluent was concentrated at 60°C and a vacuum pressure of -0.09 MPa to -0.07 MPa. After standing and crystallization, the eluent was filtered. The crystals were washed with petroleum ether and then dried under vacuum at 60°C and a vacuum pressure of -0.09 MPa to -0.07 MPa to obtain a 10-HDA crystalline extract with a content of 26.92%.

Claims

1. A method for co-extraction of total flavonoids, polyphenols and 10-HDA from honeycomb, characterized in that, Includes the following steps: S1. Take the dried honeycomb raw material and crush it. Add the crushed honeycomb raw material to the extraction vessel and add ethanol entrainer with a volume fraction of 90% to 95%. Set the extraction temperature to 47℃ to 53℃, the extraction pressure to 28 MPa to 32 MPa, and the extraction time to 3h to 5h for CO2 supercritical extraction. After the extraction is completed, use a separation vessel to separate the oily extract after CO2 supercritical extraction and the solid extraction residue in the extraction vessel. The separation temperature of the separation vessel is 43℃~46℃, and the separation pressure is 4Mpa~6Mpa; S2. The oily extract obtained in step S1 is concentrated to an oil paste with a specific gravity of 1.15 to 1.25 under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 65°C to 75°C. Petroleum ether is added to the oil paste at a volume of 4 to 6 times that of the oily extract to dissolve it. After complete dissolution, an oil paste solution is obtained. A methanol aqueous solution with a volume fraction of 75% to 85% is added to the oil paste solution at a volume of 8 to 10 times that of the oily extract. The mixture is stirred and extracted at a temperature of 20°C to 30°C for 1 to 2 hours, and the extraction is repeated 2 to 4 times. The methanol phase extract is concentrated and dried to obtain the total flavonoid extract. Add 50-70% ethanol (volume fraction) to the solid extraction residue obtained in step S1 at a ratio of 1g:(8-12)ml, heat at 70℃-90℃, extract for 1.5h-2.5h, and extract 2-4 times. Concentrate the obtained ethanol extract to a specific gravity of 1.15-1.20 under vacuum of -0.08Mpa to -0.06Mpa and temperature of 65℃-75℃ to obtain concentrated ethanol extract. Dilute the concentrated ethanol extract with 1-3 times the amount of water, then add 1-3% chitosan clarifying agent (mass fraction), stir and clarify at 60-70℃ for 10-30min, let it stand at room temperature for 20-24h to settle, filter to obtain precipitate and filtrate, concentrate and dry the filtrate to obtain extract containing polyphenols. S3. Add 6-9 times the amount of ethyl acetate to the precipitate from step S2, heat to dissolve for 1-3 hours, filter, and concentrate the filtrate to a specific gravity of 1.15-1.20 to obtain a concentrated paste. Mix the concentrated paste with silica gel at a mass ratio of 1:(2-3) and pack the sample silica gel with blank silica gel at a mass ratio of 1:(8-10) using a wet packing method to obtain a silica gel chromatography column. Elute the silica gel chromatography column with an eluent, monitor the eluent using thin-layer chromatography, collect the eluent containing 10-HDA, concentrate the eluent, allow it to stand and crystallize, filter, rinse the crystals and vacuum dry to obtain the 10-HDA extract. The temperature at which ethyl acetate is heated and dissolved is 50-70℃; the eluent is a mixture of petroleum ether and ethyl acetate in volume ratios of 20:1, 10:1, 8:2, and 7:3, or a mixture of n-hexane and ethyl acetate in volume ratios of 20:1, 10:1, 8:2, and 7:4, used for sequential elution. The silicone is 100-160 mesh.

2. The method for jointly extracting total flavonoids, polyphenols and 10-HDA from honeycomb according to claim 1, characterized in that: In step S3, the eluent concentration specifically involves a concentration temperature of 50℃~60℃ and a concentration vacuum pressure of -0.09Mpa~-0.07Mpa. The vacuum drying process specifically involves a drying temperature of 50℃~60℃ and a drying vacuum pressure of -0.09Mpa~-0.07Mpa.

3. The method for jointly extracting total flavonoids, polyphenols and 10-HDA from honeycomb according to claim 2, characterized in that: Step S1 is as follows: Take the dried honeycomb raw material and crush it. Add the crushed honeycomb raw material to the extraction vessel and add ethanol entrainer with a volume fraction of 90%. Set the extraction temperature to 50℃, the extraction pressure to 30 MPa, and the extraction time to 4 hours. After the extraction is completed, use a separation vessel for separation. The separation vessel is set to a separation temperature of 45℃ and a separation pressure of 5 MPa to obtain the oily extract after CO2 supercritical extraction and the solid extraction residue in the extraction vessel. Step S2 specifically involves: concentrating the oily extract obtained in step S1 under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 70°C to a paste with a specific gravity of 1.20; dissolving the paste in petroleum ether at a volume ratio of 5 times that of the oily extract; obtaining a paste solution after complete dissolution; adding an 80% (v / v) methanol aqueous solution to the paste solution at a volume ratio of 10 times that of the oily extract; stirring and extracting at a temperature of 25°C for 1.5 hours, for a total of 3 extractions; concentrating and drying the methanol phase extract under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 60°C to obtain the total flavonoid extract; Add 60% ethanol (volume fraction) to the solid extraction residue obtained in step S1 at a ratio of 1g:10ml, heat to 80℃ and extract three times, each extraction time being 2h. The obtained ethanol extract is concentrated to a specific gravity of 1.18 under vacuum of -0.08Mpa to -0.06Mpa and temperature of 70℃ to obtain concentrated ethanol extract. Dilute the concentrated ethanol extract with twice the volume of water, then add 2% chitosan clarifying agent (mass fraction), stir and clarify at 65℃ for 20min, and let it stand at room temperature for 22h to settle. Filter to obtain precipitate and filtrate. Concentrate and dry the filtrate under vacuum of -0.08Mpa to -0.06Mpa and temperature of 60℃ to obtain an extract containing polyphenols. Step S3 specifically involves adding 8 times the amount of ethyl acetate to the precipitate from step S2, heating and dissolving it at 60°C for 2 hours, filtering, and concentrating the filtrate to a specific gravity of 1.18 under a vacuum of -0.08 MPa to -0.06 MPa and a temperature of 55°C to obtain a concentrated paste. The concentrated paste is then mixed with silica gel at a mass ratio of 1:

2. The silica gel containing the sample and the blank silica gel are then wet-packed at a mass ratio of 1:9 to obtain a silica gel chromatography column. A petroleum ether:ethyl acetate ratio of 2:1 is used. Mixtures of 0:1, 10:1, 8:2, and 7:3 were sequentially used to elute a silica gel column. The eluent was monitored by thin-layer chromatography, and the eluent containing 10-HDA was collected. The eluent was concentrated at 55°C and a vacuum pressure of -0.09 MPa to -0.07 MPa. After crystallization, the eluent was filtered, washed with petroleum ether, and then dried under vacuum at 55°C and a vacuum pressure of -0.09 MPa to -0.07 MPa to obtain the 10-HDA extract.