Method for extracting oleanolic acid using renewable deep eutectic solvent based on hollow microspheres

Abstract

To provide a method for extracting oleanolic acid using renewable deep eutectic solvent based on hollow microspheres.SOLUTION: There is provided a method for extracting oleanolic acid, comprising: heating and dissolving 1, 3, 6, 8-tetracarboxy naphthopyrene by adopting N,N-dimethyl formamide; dropwise adding n-hexane; forming a suspension through pulse ultrasound to synthesize hollow HOF-100 microspheres; synthesizing a deep eutectic solvent composed of ethyl acetate, ethylene glycol, and water; naturally breaking the outer wall of chitin of the grifola frondosa by adopting supercooled liquid nitrogen circulation quenching; freeze-drying and grinding the wall-broken grifola frondosa; mixing the deep eutectic solvent with the wall-broken grifola frondosa freeze-dried powder and the hollow HOF-100 microspheres; selectively extracting and purifying oleanolic acid from grifola frondosa through a high-speed centrifugation one-step method; recovering the hollow HOF-100 microspheres and the deep eutectic solvent; and reusing the recycled hollow HOF-100 microspheres and the deep eutectic solvent for the extraction and purification of oleanolic acid.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention belongs to the technical field of food science and engineering, in particular to the development of a renewable deep food product based on hollow microspheres. This invention relates to a method for extracting oleanolic acid using a eutectic solvent. [Background technology]

[0002] Grifola frondosa, known as Maitake or Chicken of the Forest dosa) is a medicinal and edible fungus belonging to the genus Maitake in the subphylum Basidiomycota, and is widely distributed in Japan, Russia, and It is found in the Changbai Mountains, Sichuan, Zhejiang, and other areas of China. Not only does it have a unique and strong fragrance, but its chemical composition and pharmacological effects have also attracted widespread attention in recent years. It has become a hot spot for research.

[0003] The main active ingredients of Grifola frondosa are mainly polysaccharides, triterpenes, and polyphenos. Their pharmacological effects include immunomodulation, antitumor, antidiabetic, antihyperlipidemic, and antiviral. The triterpene compounds in Grifola frondosa are It is one of the important chemical compounds and has a wide range of biological activities. It is a type of natural terpene compound consisting of six isoprene units and is widely found in nature. It has a variety of biological activities, including anti-inflammatory, anti-cancer, antibacterial, and antiviral effects. Triterpenes from Grifola frondosa may affect cell signaling and inhibit inflammatory responses. They exert antiviral effects by inhibiting viral responses or by directly interacting with viral proteins. The triterpenes of Grifola frondosa are free-radicals. By removing Calcium and inhibiting lipid peroxidation, cells can be protected from oxidative stress. Cut.

[0004] Oleanolic acid from Grifola frondosa is an important pentacyclic triterpene compound. , various biological activities such as anti-inflammatory, antioxidant, antitumor, antiviral, and hepatoprotective effects Oleanolic acid is mainly used in the clinical treatment of acute jaundice type hepatitis and has a protective effect against araniol. It has the effect of lowering oleanolic acid aminotransferase and alleviating jaundice. Collagen-containing fatty acids have also shown potential preventive and therapeutic effects against liver fibrosis, and have been shown to increase the collagen content of the liver. This may have preventive and therapeutic effects on liver cirrhosis. So far, oleanolic acid from Grifola frondosa has been selected. There have been no reports of its effective extraction and purification.

[0005] Solid-liquid extraction is a traditional method for extracting oleanolic acid from Grifola frondosa plant material. The most commonly used solvents in traditional extraction are organic solvents (e.g., methanol). These solvents are widely used, but Grifola frondosa has low selectivity for oleanolic acid and presents risks to health and the environment In 2003, the concept of deep eutectic solvents was first proposed, and hydrogen bonding Deep eutectic solvents are defined as solvents consisting of hydrogen bond acceptors and hydrogen bond donors. It is known for its high activity, as well as for its ability to produce a wide range of compounds through different combination schemes. It can be customized to suit the selectivity of Frondosa for oleanolic acid, making it suitable for food, It has wide application potential in the cosmetics and pharmaceutical industries.

[0006] Hydrogen-bonded Organic Frameworks HOFs) are linked together through intermolecular hydrogen bonds, forming highly HOFs are new porous crystalline materials that form highly crystalline and porous structures. Applications include storage and separation of carbon, multiphase catalysis, biological applications, sensing, and proton conduction. It shows the possibility.

[0007] Therefore, the present invention provides a method for the efficient extraction of oleanolic acid from Grifola frondosa. Extraction of oleanolic acid using a renewable deep eutectic solvent based on hollow microspheres for extraction Suggest a method. Summary of the Invention [Problem to be solved by the invention]

[0008] The object of the present invention is to overcome the drawbacks of the prior art and to obtain the oleanolic acid of Grifola frondosa. The hollow HOF-100 microspheres and deep eutectic solvents are highly selective for the solvents, making them easy to recycle and regenerate. It can be used for multiple efficient extraction of oleanolic acid from Grifola frondosa. Oleanolic acid using a renewable deep eutectic solvent based on hollow microspheres that can be used The present invention provides a method for extracting the above-mentioned compounds. [Means for solving the problem]

[0009] The present invention solves the technical problem by adopting the following technical means.

[0010] A method for the extraction of oleanolic acid using a regenerable deep eutectic solvent based on hollow microspheres, comprising: 1,3,6,8-Tetracarboxynaphthopyrene using N,N-dimethylformamide was heated and dissolved, n-hexane was added dropwise, and a suspension was formed by pulse ultrasonic waves to form hollow HOF- Step S1: synthesizing 100 microspheres; Ethyl acetate is the hydrogen bond acceptor, ethylene glycol is the hydrogen bond donor, and water is the dilution solvent. The solvent was ethyl acetate:ethylene glycol:water in a volume ratio of 1:3 to 5:1. Step S2 of synthesizing a deep eutectic solvent by heating and stirring at 70-80 ° C. with a thermostatic stirrer; The chitinous outer wall of Grifola frondosa was naturally broken down using a supercooled liquid nitrogen circulating quenching method. Step S3 of breaking the mixture, freeze-drying it in a vacuum freeze-dryer, and pulverizing it; Deep eutectic solvent and broken-walled Grifola frondosa freeze-dried powder and hollow HOF-100 microspheres Mix the liquid and solid at a ratio of 30-40 mL: 1 g: 0.1-0.2 g and centrifuge at high speed in one step (high speed centrifugation). Grifola frondosa at 8000-10000 r / min, centrifugation time 20-30 min Selective extraction and purification of oleanolic acid using hollow HOF-100 microspheres and deep eutectic solvent A collecting step S4, and The hollow HOF-100 microspheres and the deep eutectic solvent recovered from step S4 were treated with Grifola et al. Step S5 of reusing for extraction and purification of oleanolic acid from frondosa The above method, comprising:

[0011] The 1,3,6,8-tetracarboxynaphthopyrene is 1,3,6,8-tetracarboxynaphthopyrene. Methoxycarbonylnaphthopyrene, dioxane, potassium hydroxide, and water in a mass ratio of 4 to 6:1 The mixture was stirred at 120°C for 24 hours to give a 1,3,6,8- A tetracarboxynaphthopyrene structural unit is obtained.

[0012] In addition, in the step S1, 1,3,6,8-tetramethyl- ... The concentration of carboxynaphthopyrene is 0.5 to 1.0 g / mL, and the heating and dissolving temperature is 120 to 150°C. The temperature was 150°C, and the volume ratio of n-hexane to N,N-dimethylformamide was 1 The ratio is 2:5.

[0013] In the step S1, the pulse ultrasonic output is 500 to 600 W, and the pulse frequency is The frequency is 2 to 4 Hz, the ultrasonic temperature is 80 to 90°C, and the duration is 1 to 2 hours.

[0014] In step S3, the mass ratio of liquid nitrogen to Grifola frondosa is 8 to 10. :1, and Grifola frondosa was rapidly frozen in liquid nitrogen for 10 minutes and then removed. Place in a 100°C oven and rapidly heat for 10 minutes. Repeat the freezing and heating cycle 3 to 10 times. 5 times, which naturally destroys the chitinous outer wall of Grifola frondosa.

[0015] In the step S3, the conditions for the vacuum freeze-drying are a temperature of −70 to −80° C. Air pressure: 0.1-0.2 mbar, freeze-drying pressure: 5-6 mbar, freeze-drying time: 24 ~36 hours.

[0016] In addition, in the step S3, the crushing conditions are liquid nitrogen and crushed Grifola frondosa. The liquid-solid ratio of the freeze-dried powder is 30-40 mL / g, and the grinding speed is 200-400 r / min.

[0017] In addition, in the step S4, the supernatant obtained by centrifugation is collected by the deep eutectic solution. The hollow HOF-100 microspheres at the bottom were dissolved in ethanol to form a medium for Grifola frondosa. The liquid-solid ratio of ethanol to hollow HOF-100 microspheres was 30-40. The eluate is then evaporated at a pressure of 3-4 mbar for 20-30 min.

[0018] In step S5, hollow HOF-100 microspheres and a deep eutectic solvent are used to form Grifola The number of repeated extractions of oleanolic acid from Frondosa is 8 to 10 times.

[0019] Also, hollow HOF-100 microspheres and oleanolic acid from Grifola frondosa in deep eutectic solvents Grifola frondo was extracted and purified from carboxylic acid, and the extraction and purification process was repeated 8 to 10 times. The extraction rate of oleanolic acid from the soybean can reach 98% of the initial extraction rate. [Effects of the Invention]

[0020] The advantages and benefits of the present invention include: The hollow HOF-100 microspheres and deep eutectic solvent designed in this invention can be recycled. The hollow HOF-100 microspheres used contain oleanolic acid from Grifola frondosa. The deep eutectic solvent can be specifically adsorbed and recovered by centrifugation. It can be used for multiple extractions of oleanolic acid from nudosa while ensuring extraction efficiency. It reduces environmental pollution, cuts costs, and is suitable for industrial applications.

[0021] The present invention uses a combination of hollow HOF-100 microspheres and a deep eutectic solvent to produce Grifola F The oleanolic acid of Grifola frondosa was prepared and compared with that of Grifola frondosa. Conventional purification methods are mainly based on macroporous resins. However, macroporous resins are expensive, require activation before use, and are difficult to operate. It was tedious and its efficiency decreased after multiple uses, increasing purification costs. By designing HOF-100 microsphere material and combining it with a deep eutectic solvent, the ratio of HOF-100 material The surface area can be significantly increased, improving the efficiency of separation and purification.

[0022] This method uses hollow HOF-100 microspheres instead of macroporous resin, and Selective extraction and purification of oleanolic acid from La frondosa. This method is simple and easy to operate. It is convenient, fast, and effective for the extraction and purification of oleanolic acid from Grifola frondosa. The extraction and purification efficiency is significantly improved, and the high efficiency of the extraction and purification effect is maintained even after multiple extractions and purifications. This can be done.

[0023] Hollow HOF-100 microspheres of the present invention and olea from Grifola frondosa in deep eutectic solvent The extraction and purification of nicotinic acid is carried out in the same manner as in the case of Grifola frondosa, even after 8 to 10 times of extraction and purification. The extraction rate of oleanolic acid from Frondosa can reach 98% of the initial extraction rate. Inefficient extraction and purification using macroporous resins, health hazards and environmental pollution caused by organic solvents These problems can be effectively solved, and a green, low-carbon, sustainable development modern society can be built. It is very meaningful to build. [Brief explanation of the drawings]

[0024] [Figure 1] 1 is an SEM image of hollow HOF-100 microspheres of the present invention. [Figure 2] 1 is a nitrogen adsorption / desorption isotherm graph of hollow HOF-100 microspheres of the present invention. [Figure 3] FIG. 1 is a comparison diagram of the effects of extracting and purifying oleanolic acid from Grifola frondosa using different methods of the present invention. [Figure 4] 1 is a temperature change diagram of the Grifola frondosa oleanolic acid, HOF-100 microspheres and deep eutectic solvent system of the present invention. [Figure 5]FIG. 1 is a comparison diagram of the average number of hydrogen bonds in different systems of 5 to 50 ps according to the present invention. [Figure 6] This is a comparison diagram of the extraction rate of oleanolic acid from Grifola frondosa after 10 repeated extractions using different extraction methods of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will now be described in detail with reference to specific examples. However, the following examples are merely illustrative. These are merely illustrative and not restrictive, and the scope of protection of the present invention is not limited thereto. .

[0026] The experimental methods used in the following examples are conventional methods unless otherwise indicated. Materials, reagents and the like used therein are commercially available unless otherwise specified.

[0027] 1,3,6,8-tetramethoxycarbonylnaphthopyrene (analytical reagent), dioxane (analytical reagent) (analytical reagent), potassium hydroxide (analytical reagent), DMF (analytical reagent), ethyl acetate (analytical reagent) , ethylene glycol (analytical reagent), formamide (HPLC grade), sulfuric acid (98% ) was purchased from Shanghai Macklin Biochemical Co., Ltd.; the standard of oleanolic acid was It was purchased from Aladdin Biochemical Technology Co., Ltd. GL323- A 1SCN 0.001g precision electronic balance was purchased from Sartorius, Germany, and The NTZ-IID probe ultrasonic cell disruption device was purchased from Ningbo New Lawn Material Technology Co., Ltd. The Centrifuge 5804 desktop high-speed freezing centrifuge is manufactured by Eppe, Germany. The Agilent 1200 high-performance liquid chromatograph was purchased from Ndorf, USA. It was purchased from Agilent.

[0028] A method for the extraction of oleanolic acid using a regenerable deep eutectic solvent based on hollow microspheres, comprising: 1,3,6,8-Tetracarboxynaphthopyrene using N,N-dimethylformamide was heated and dissolved, n-hexane was added dropwise, and a suspension was formed by pulse ultrasonic waves to form hollow HOF- Step S1: synthesizing 100 microspheres; Ethyl acetate is the hydrogen bond acceptor, ethylene glycol is the hydrogen bond donor, and water is the dilution solvent. The solvent was ethyl acetate:ethylene glycol:water in a volume ratio of 1:3 to 5:1. Step S2 of synthesizing a deep eutectic solvent by heating and stirring at 70-80 ° C. with a thermostatic stirrer; The chitinous outer wall of Grifola frondosa was naturally broken down using a supercooled liquid nitrogen circulating quenching method. Step S3 of breaking the mixture, freeze-drying it in a vacuum freeze-dryer, and pulverizing it; Deep eutectic solvent and broken-walled Grifola frondosa freeze-dried powder and hollow HOF-100 microspheres Mix the liquid and solid at a ratio of 30-40 mL: 1 g: 0.1-0.2 g and centrifuge at high speed in one step (high speed centrifugation). Grifola frondosa at 8000-10000 r / min, centrifugation time 20-30 min Selective extraction and purification of oleanolic acid using hollow HOF-100 microspheres and deep eutectic solvent A collecting step S4, and The hollow HOF-100 microspheres and the deep eutectic solvent recovered from step S4 were treated with Grifola et al. Step S5 of reusing for extraction and purification of oleanolic acid from frondosa The above method, comprising:

[0029] Example 1 A method for the extraction of oleanolic acid using a regenerable deep eutectic solvent based on hollow microspheres, comprising: The process includes the following steps:

[0030] S1: 1,3,6,8-tetramethoxycarbonylnaphthopyrene, dioxane, hydroxide A mixture of sodium and water in a mass ratio of 4:1:1:1 was stirred at 120°C for 24 hours, and 1, 3, and 6 A process for obtaining 8-tetracarboxynaphthopyrene structural units.

[0031] S2: Concentration 0.5 g / mL, temperature 120 °C, volume ratio of n-hexane and DMF added dropwise 1: Dissolve 1,3,6,8-tetracarboxynaphthopyrene in DMF under the conditions of 5 by heating. The ultrasonic output was 500W, the pulse frequency was 2Hz, and the ultrasonic temperature was 90℃ for 2 hours. The hollow HOF-100 microspheres are synthesized by forming a suspension using ultrasonic waves.

[0032] S3: A mixture of ethyl acetate, ethylene glycol, and water in a volume ratio of 1:3:1 is heated at 70°C. and hot stirring to form a deep eutectic solvent.

[0033] S4: Fresh Grifola frondosa was placed in liquid nitrogen and rapidly frozen. The mass ratio of Fola Frondosa is 8:1, and after 10 minutes, remove from the oven and place in a 100°C oven. The freezing and heating cycle was repeated five times for 10 minutes. The chitinous outer wall of Grifola frondosa is then naturally destroyed. The temperature was set to -80°C, the vacuum pressure to 0.1 mbar, and the freeze-drying pressure to 5 mbar. Set the freeze-drying time to 24 hours. Freeze-dried Grifola frondosa in a cryogenic grinder. The liquid-solid ratio of the freeze-dried Grifola frondosa was 40 mL / g, and the grinding speed was The process of obtaining freeze-dried powder of Grifola frondosa was carried out under the conditions of 400 r / min.

[0034] S5: Liquid-solid ratio 30 mL:1 g:0.1 g, high-speed centrifugation speed 8000 r / min, centrifugation time 20 min Under the conditions of , the deep eutectic solvent was broken down into the freeze-dried powder of Grifola frondosa and the hollow HOF-1 00 A process of uniformly mixing with microspheres.

[0035] S6: The supernatant obtained by the centrifugation in step S5 is the recovered deep eutectic solvent. Hollow HOF-100 microspheres in the part were dissolved in ethanol to obtain the oleanolic acid of Grifola frondosa. The ethanol and hollow HOF-100 microspheres were dissolved in ethanol at a liquid-solid ratio of 30 mL / g. The eluted portion was rotary evaporated under a pressure of 3 mbar for 20 min to obtain the Grifola frondosa oleate. A process for obtaining anolic acid.

[0036] S7: Recovered hollow HOF-100 microspheres and deep eutectic solvent from Grifola frondosa A process for reusing the extracted oleanolic acid for extraction and purification (the extraction method is the same as in step S5) ).

[0037] Example 2 A method for the extraction of oleanolic acid using a regenerable deep eutectic solvent based on hollow microspheres, comprising: The specific steps are as follows:

[0038] S1: 1,3,6,8-tetramethoxycarbonylnaphthopyrene, dioxane, hydroxide A mixture of sodium and water in a mass ratio of 5:1:1:1 was stirred at 120°C for 24 hours, and 1, 3, and 6 A process for obtaining 8-tetracarboxynaphthopyrene structural units.

[0039] S2: Concentration 0.75 g / mL, temperature 135 °C, volume ratio of n-hexane to DMF added 1 1,3,6,8-tetracarboxynaphthopyrene was added with DMF under the conditions of 0.5:5. Heat melting, ultrasonic output 550W, pulse frequency 3Hz, ultrasonic temperature 85℃, 1.5 hours Hollow HOF-100 microspheres were synthesized by forming a suspension using pulsed ultrasound under the following conditions: Section.

[0040] S3: A mixture of ethyl acetate, ethylene glycol, and water in a volume ratio of 1:4:1 is heated at 70°C. and hot stirring to form a deep eutectic solvent.

[0041] S4: Fresh Grifola frondosa was placed in liquid nitrogen and rapidly frozen. The mass ratio of Fola Frondosa is 9:1, and after 10 minutes, remove from the oven and place in a 100°C oven. The freezing and heating cycle was repeated four times for 10 minutes. The chitinous outer wall of Grifola frondosa is then naturally destroyed. The temperature was set to -75°C, the vacuum pressure to 0.15 mbar, and the freeze-drying pressure to 5.5 mbar. ar, set the freeze-drying time to 30 hours. Freeze-dried Grifola frondosa at low temperature. Grifola frondosa was placed in a grinder and freeze-dried with liquid nitrogen at a liquid-solid ratio of 35 mL / g. Grifola frondosa freeze-dried powder was obtained under the condition of a grinding speed of 300 r / min. .

[0042] S5: Liquid-solid ratio 35 mL:1 g:0.15 g, high-speed centrifugation speed 9000 r / min, centrifugation time 25 Under the conditions of 1 min, the deep eutectic solvent broke the wall of Grifola frondosa freeze-dried powder and hollow HOF- 100 microspheres and uniformly mixed.

[0043] S6: The supernatant obtained by the centrifugation in step S5 is the recovered deep eutectic solvent. Hollow HOF-100 microspheres in the part were dissolved in ethanol to obtain the oleanolic acid of Grifola frondosa. The liquid-solid ratio of ethanol and hollow HOF-100 microspheres was 35 mL / g. The eluted portion was rotary evaporated under a pressure of 3.5 mbar for 25 min to obtain the Grifola frondosa extract. A process for obtaining oleanolic acid.

[0044] S7: Recovered hollow HOF-100 microspheres and deep eutectic solvent from Grifola frondosa A process for reusing the extracted oleanolic acid for extraction and purification (the extraction method is the same as in step S5) ).

[0045] Example 3 A method for the extraction of oleanolic acid using a regenerable deep eutectic solvent based on hollow microspheres, comprising: The specific steps are as follows:

[0046] S1: 1,3,6,8-tetramethoxycarbonylnaphthopyrene, dioxane, hydroxide A mixture of sodium and water in a mass ratio of 6:1:1:1 was stirred at 120°C for 24 hours, and 1,3,6 A process for obtaining 8-tetracarboxynaphthopyrene structural units.

[0047] S2: Concentration 1.0 g / mL, temperature 120 °C, volume ratio of n-hexane and DMF added dropwise 2: Dissolve 1,3,6,8-tetracarboxynaphthopyrene in DMF under the conditions of 5 by heating. The ultrasonic output was 600W, the pulse frequency was 4Hz, and the ultrasonic temperature was 80℃ for 1 hour. The hollow HOF-100 microspheres are synthesized by forming a suspension using ultrasonic waves.

[0048] S3: A mixture of ethyl acetate, ethylene glycol, and water in a volume ratio of 1:5:1 is heated at 70°C. and hot stirring to form a deep eutectic solvent.

[0049] S4: Fresh Grifola frondosa was placed in liquid nitrogen and rapidly frozen. The mass ratio of Fola Frondosa is 10:1, and after 10 minutes, it is removed and placed in a 100°C oven. The product was then placed in a refrigerator and rapidly heated for 10 minutes, with three freeze-heat cycles. The chitinous outer wall of the Grifola frondosa is allowed to naturally break down. The Grifola frondosa is then vacuum frozen. Place in a dryer, set the temperature to -70°C, vacuum pressure to 0.2 mbar, and freeze-drying pressure to 6 mbar. The freeze-drying time was set to 36 hours. The freeze-dried Grifola frondosa was cryo-pulverized. Grifola frondosa was freeze-dried with liquid nitrogen and crushed to a liquid-solid ratio of 30 mL / g. The process of obtaining freeze-dried powder of Grifola frondosa at a speed of 200 r / min.

[0050] S5: Liquid-solid ratio 40 mL:1 g:0.2 g, high-speed centrifugation speed 1000 r / min, centrifugation time 30 min Under the conditions of , the deep eutectic solvent was broken down into the freeze-dried powder of Grifola frondosa and the hollow HOF-1 00 A process of uniformly mixing with microspheres.

[0051] S6: The supernatant obtained by the centrifugation in step S5 is the recovered deep eutectic solvent. Hollow HOF-100 microspheres in the part were dissolved in ethanol to obtain the oleanolic acid of Grifola frondosa. The liquid-solid ratio of ethanol to hollow HOF-100 microspheres was 40 mL / g. The eluted portion was rotary evaporated under a pressure of 4 mbar for 30 min to obtain the Grifola frondosa oleate. A process for obtaining anolic acid.

[0052] S7: Recovered hollow HOF-100 microspheres and deep eutectic solvent from Grifola frondosa A process for reusing the extracted oleanolic acid for extraction and purification (the extraction method is the same as in step S5) ).

[0053] (Comparative Example 1) Other conditions were the same as in Example 1, except that the extraction solvent was ethanol.

[0054] (Comparative Example 2) The preparation of the freeze-dried powder of broken-walled Grifola frondosa was the same as in Example 1, except for the extraction. Extraction method: liquid-solid ratio 30mL / g, ultrasonic output 500W, extraction temperature 50℃, extraction time 20 minutes Under the conditions, the deep eutectic solvent was uniformly mixed with the freeze-dried powder of Grifola frondosa. The solution is to replace it with an ultrasonic-assisted extraction method.

[0055] (Comparative Example 3) Other conditions were the same as in Example 1, except that LS microspheres were used instead of hollow HOF-100 microspheres. The solution is to use A-21 type macroporous resin.

[0056] The products obtained above or intermediate process products are subjected to detection and analysis based on the following quality indicators: Ta.

[0057] (1) Topography of hollow HOF-100 microspheres As shown in FIG. 1, the hollow HOF-1 obtained in step S2 of Examples 1, 2, and 3 The topography of the 00 microspheres is detected by scanning electron microscope (SEM). Hollow HOF-1 The 00 microspheres were placed in an ion sputtering device and coated with gold-palladium for 5 minutes. After that, it was fixed on a conductive table with conductive adhesive and observed using SEM under a voltage of 3 kV. The surface of the hollow HOF-100 microspheres was found to be smooth, and dense water was found on the surface. This indicates that a bonded organic framework structure was formed.

[0058] (2) Porosity of hollow HOF-100 microspheres As shown in Figure 2, the porosity of the hollow HOF-100 microspheres obtained in step S2 was measured by AUTOS. The nitrogen adsorption / desorption isotherm was measured using an ORB-IQ2-MP specific surface area analyzer and plotted. The specific surface area and pore size of the HOF-100 microspheres can be obtained. , which showed a characteristic type IV hysteresis loop associated with the porous structure. As a result, the specific surface area of ​​the hollow HOF-100 microspheres was 2.46 m 2 / g, pore diameter 190nm there were.

[0059] (3) Yield of oleanolic acid from Grifola frondosa extracted and purified by different methods The oleanolic acid of Grifola frondosa purified and concentrated in step S6 was analyzed by HPLC. Analyzed using a molecular dynamics simulation system, Determination of Oleanolic Acid in Grifola frondosa by HPLC Method: HPLC measurements were performed on an Agilent 1200 HPLC instrument equipped with a diode array detector. Select a ProSher C18 column (4 x 50 mm, 3 μm) and use the mobile phase The solvent was a mixture of formamide and 0.01 mol / L sulfuric acid (volume ratio 10:90). The column temperature was 30°C, the mobile phase flow rate was 0.25 mL / min, and the sample injection volume was 0.5 μL. The detection wavelength of the infrared detector was set at 205 nm.

[0060] Molecular Dynamics Test: In Chem3D software, 1,3,6,8-tetracarboxynaphthopyrene, acetic acid Generates 3D molecular structures such as ethyl, ethylene glycol, and water. The Amorphous Cell module in the s studio 2020 software Construct an amorphous box model using Forcite-Geometry Select optimization, set precision to Fine, and force field to COMPASSII. Optimize the geometry of the solvent model. Select Forcite-Dynamics. , NVT system, van der Waals force cutoff radius 12.5A, hydrogen bond cutoff The radius is set to 2.5A, the temperature is set to 340K, and the molecular dynamics simulation is performed within 50ps. Complete the analysis.

[0061] As shown in FIG. 3, the extraction method of the present invention can effectively extract oleanolic acid from Grifola frondosa. The yield of oleanolic acid from Grifola frondosa in Example 1 can be significantly increased. The yields were 290%, 680%, and 388% higher than those of Comparative Examples 1, 2, and 3, respectively. % higher.

[0062] (4) Temperature change of Grifola frondosa oleanolic acid and five-element eutectic solvent system As shown in Figure 4, Grifola frondosa oleanolic acid and deep eutectic solvent and hollow HO The F-100 system reached a stable temperature after 4 ps.

[0063] (5) Average number of hydrogen bonds in different systems from 5 to 50 ps As shown in FIG. 5, the average number of hydrogen bonds formed in Example 1 (468) was 1.2 times that of Comparative Example 1 (1 65), which is much higher than Comparative Example 2 (81) and Comparative Example 3 (112).

[0064] (6) Grifola frondosa oleanoides after 10 repeated extractions using different extraction methods Yield of carboxylic acid As shown in Figure 6, the hollow HOF-microspheres and deep eutectic solvent of the present invention are recyclable. Even after 10 repeated extractions, the oleanol in Grifola frondosa in Example 1 was The acid yield can reach 98% of the initial extraction rate, and Comparative Example 1 (30%) and Comparative Example 2 (2 This is clearly higher than the yields in Comparative Example 1 (26%) and Comparative Example 2 (26%).

[0065] The present invention also uses hollow HOF-100 microspheres in combination with deep eutectic solvents, Selective preparation of oleanolic acid from Frondosa. Hollow HOF-100 microspheres are The hollow HOF-100 microspheres can specifically adsorb oleanolic acid from Oryza frondosa. The deep eutectic solvent has renewable properties and can be reused. After recovery by centrifugation, It can be used for multiple extractions of oleanolic acid from Fora frondosa, and the actual production It helps to reduce the cost during the production process and is a valuable tool for the application of oleanolic acid from Grifola frondosa. Always meaningful.

[0066] The above is merely a preferred embodiment of the present invention and does not limit the present invention in form or substance. Those skilled in the art will appreciate that some modifications and additions can be made without departing from the method of the present invention. Such improvements and additions should also fall within the scope of protection of the present invention. Anyone skilled in the art can easily implement the methods disclosed above without departing from the spirit and scope of the present invention. Any modifications, adaptations and evolutionary equivalent changes may be made to the technical content of the work. It is an equivalent embodiment of the invention. Also, the above embodiment is added based on the substantial technology of the present invention. Any equivalent modifications, adaptations and evolutions are within the scope of the technical means of the present invention.

[0067] Although embodiments of the present invention have been disclosed for purposes of illustration, it is understood that the spirit and scope of the present invention and the accompanying claims are not to be construed as limiting the scope of the present invention. Various substitutions, changes and modifications can be made without departing from the scope of the claims. It will be understood by those skilled in the art that the scope of the present invention does not extend beyond the examples. It is not limited to the contents shown.

Claims

1. A method for the extraction of oleanolic acid using a regenerable deep eutectic solvent based on hollow microspheres, comprising: 1,3,6,8-tetracarboxynaphthopyrene using N,N-dimethylformamide is heated and dissolved, n-hexane is added dropwise, and a suspension is formed by pulse ultrasonic waves to form hollow HOF- Step S1: synthesizing 100 microspheres; Ethyl acetate is the hydrogen bond acceptor, ethylene glycol is the hydrogen bond donor, and water is the dilution solvent. The solvent was ethyl acetate:ethylene glycol:water in a volume ratio of 1:3 to 5:

1. Step S2 of synthesizing a deep eutectic solvent by heating and stirring at 70-80 ° C. with a thermostatic stirrer; The chitinous outer wall of Grifola frondosa was naturally broken down using the supercooled liquid nitrogen circulating quenching method. Step S3: breaking the mixture, freeze-drying it in a vacuum freeze-dryer, and pulverizing it; The deep eutectic solvent, the broken-walled Grifola frondosa freeze-dried powder, and the hollow HOF-100 microspheres The mixture was mixed with small balls at a liquid-solid ratio of 30-40 mL: 1 g: 0.1-0.2 g, and centrifuged in a single high-speed centrifugation method (high speed Centrifugal speed: 8000-10000 rpm, centrifugation time: 20-30 minutes The hollow HOF-100 microspheres and the pre-extracted oleanolic acid are selectively extracted and purified from the HOF-100 microspheres. A step S4 of recovering the deep eutectic solvent; and The hollow HOF-100 microspheres and the deep eutectic solvent recovered from step S4 are treated with glycerol. Step S5 of reusing for extraction and purification of oleanolic acid from Fora frondosa A method for producing oleanolic acid using a regenerable deep eutectic solvent based on hollow microspheres, comprising: Method for extracting lactic acid.

2. The 1,3,6,8-tetracarboxynaphthopyrene is 1,3,6,8-tetramethoxy Dicarbonylnaphthopyrene, dioxane, potassium hydroxide, and water in a mass ratio of 4 to 6:1:1: The mixture was mixed uniformly in 1, and the reaction mixture was stirred at 120°C for 24 hours to give 1,3,6,8-tetrachloroisothiazolinone. The hollow microspheres according to claim 1, characterized in that carboxynaphthopyrene structural units are obtained. Extraction method of oleanolic acid using renewable deep eutectic solvent based on

3. In the step S1, 1,3,6,8-tetracalcium carbonate is dissolved in N,N-dimethylformamide. The concentration of carboxynaphthopyrene is 0.5 to 1.0 g / mL, and the heating and dissolving temperature is 120 to 150°C. ° C., and the volume ratio of n-hexane to N,N-dimethylformamide to be added dropwise was 1 to 2:

5. The hollow microsphere-based renewable deep eutectic solvent according to claim 1, characterized in that The extraction method of oleanolic acid used.

4. In the step S1, the pulse ultrasonic output is 500 to 600 W, and the pulse frequency is 2 to 4. Hz, ultrasonic temperature is 80 to 90°C, and time is 1 to 2 hours. A method for the extraction of oleanolic acid using a renewable deep eutectic solvent based on hollow microspheres as described in .

5. In the step S3, the mass ratio of liquid nitrogen to Grifola frondosa is 8 to 10:

1. Grifola frondosa was quickly frozen in liquid nitrogen for 10 minutes, then removed and stored for 10 minutes. Place in a 0°C oven and rapidly heat for 10 minutes. Repeat the freezing and heating cycle 3 to 5 times. It is characterized by naturally destroying the chitinous outer wall of Grifola frondosa. Extraction of oleanolic acid using a renewable deep eutectic solvent based on hollow microspheres according to claim 1 How to get out.

6. In the step S3, the conditions for the vacuum freeze-drying are a temperature of −70 to −80° C., a vacuum pressure of 0.1-0.2 mbar, freeze-drying pressure 5-6 mbar, freeze-drying time 24-36 The reproducible deep eutectic solution based on hollow microspheres according to claim 1 is characterized in that the time A method for extracting oleanolic acid using a solvent.

7. In the step S3, the crushing conditions are the liquid nitrogen and the crushed Grifola frondosa. The liquid-solid ratio of the sintered dried powder is 30-40 mL / g, and the grinding speed is 200-400 r / min. The hollow microspheres according to claim 1 are based on olefins containing regenerable deep eutectic solvents. Method for extracting anolic acid.

8. In step S4, the supernatant obtained by centrifugation is the recovered deep eutectic solvent. The hollow HOF-100 microspheres at the bottom were dissolved in ethanol to form the Grifola frondosa The oleanolic acid was dissolved in the ethanol and the hollow HOF-100 microspheres at a liquid-solid ratio of 3. The eluted fraction was rotary evaporated at a pressure of 3-4 mbar for 20-30 minutes. The hollow microspheres according to claim 1 are used to produce a regenerable deep eutectic solvent. A method for extracting oleanolic acid.

9. In step S5, the hollow HOF-100 microspheres and the deep eutectic solvent are mixed to form a Grifola The repeated extraction of oleanolic acid from Frondosa is characterized by the fact that it is 8 to 10 times. oleanol using a renewable deep eutectic solvent based on hollow microspheres according to claim 1 Acid extraction method.

10. The hollow HOF-100 microspheres and the deep eutectic solvent were used to produce olea from Grifola frondosa. Grifola furonic acid was extracted and purified, and even after repeating the extraction and purification process 8 to 10 times, It is noted that the extraction rate of oleanolic acid from the dosa can reach 98% of the initial extraction rate. The hollow microspheres according to claim 9 are characterized by the use of a renewable deep eutectic solvent for oleanolic acid. Method for extracting carboxylic acids.

Images