An extraction method of oleuropein based on deep eutectic solvent and ultrasonic coupling

Abstract

The application discloses an extraction method of oleuropein based on deep eutectic solvent and ultrasonic coupling, which comprises the following steps: drying, crushing and sieving of olea leaf to obtain olea leaf powder; at least one of sucrose, ethylene glycol, glycerol, urea and citric acid is used as a hydrogen bond donor to prepare a deep eutectic solvent with choline chloride as a hydrogen bond acceptor, and the water content is adjusted; the olea leaf powder is mixed with the deep eutectic solvent to perform ultrasonic-assisted extraction to obtain an extraction liquid; and the extraction liquid is subjected to solid-liquid separation, dilution and filtration to obtain an oleuropein extract. The extraction method has high extraction efficiency and is green and environment-friendly: the deep eutectic solvent is coupled with ultrasonic assistance, the extraction amount is significantly better than that of a traditional organic solvent method, and the solvent is biodegradable, non-volatile and non-residual, and meets the requirements of green chemistry.

Description

Technical Field

[0001] This invention relates to the field of olive bitter glycoside extraction technology, specifically to an olive bitter glycoside extraction method based on deep eutectic solvent and ultrasonic coupling. Background Technology

[0002] Against the backdrop of global efforts towards efficient utilization and green transformation of agricultural resources, olive oil, as a typical Mediterranean advantageous crop, has formed an industrial system with significant economic value. Currently, the EU accounts for approximately 68% of global olive oil production, with a planting area of ​​about 4 million hectares in the Mediterranean region. In China, the olive oil industry has developed rapidly, with a planting area exceeding 130,000 hectares and maintaining an annual growth rate of approximately 12%. During olive oil processing, a large amount of byproducts are generated simultaneously, with olive leaves accounting for about 5% to 10% of the total raw materials. However, for a long time, these byproducts have been mostly processed in a low-value manner or even discarded. However, research shows that olive leaves are rich in phenolic active substances such as oleuropein and hydroxytyrosol, possessing significant antioxidant, anti-inflammatory, and antibacterial activities, making them a natural functional resource with great development potential. Currently, the high-value utilization rate of global olive byproducts is still less than 1%, resulting in both resource waste and environmental burden. There is an urgent need to develop efficient and green extraction and conversion strategies to enhance their value.

[0003] Traditional extraction methods, such as organic solvent reflux and maceration, are widely used in laboratories and industries for the development and utilization of oleuropein from olive leaves. However, their inherent drawbacks are becoming increasingly apparent. These methods typically rely on large amounts of organic solvents, posing a risk of solvent residue, and are accompanied by long extraction times and high energy consumption. Furthermore, high temperatures or prolonged treatments can easily lead to the degradation of heat-sensitive components such as oleuropein, thus affecting product quality and bioactivity. In addition, traditional methods still have limitations in selectivity and mass transfer efficiency, making it difficult to meet the development needs of green and refined processing of natural products. Therefore, constructing environmentally friendly and efficient extraction systems has become an important research direction. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for extracting oleuropein based on deep eutectic solvent and ultrasonic coupling. By constructing a choline chloride deep eutectic solvent system and combining it with ultrasonic enhancement technology, efficient and green extraction of oleuropein is achieved.

[0005] This invention discloses a method for extracting oleuropein based on deep eutectic solvent and ultrasonic coupling, comprising the following steps: Olive leaves are dried, pulverized, and sieved to obtain olive leaf powder; Ethylene glycol was used as a hydrogen bond donor and choline chloride as a hydrogen bond acceptor to prepare a deep eutectic solvent, and the water content was adjusted. The olive leaf powder was mixed with the deep eutectic solvent and subjected to ultrasonic-assisted extraction to obtain an extract. The extract was subjected to solid-liquid separation, dilution, and filtration to obtain oleuropein extract.

[0006] Furthermore, the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:1 to 1:3.

[0007] Furthermore, the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:2.5.

[0008] Furthermore, the water content of the deep eutectic solvent is 10% to 40%.

[0009] Furthermore, the water content of the deep eutectic solvent is 20%.

[0010] Furthermore, the material-to-liquid ratio of the extract is 1:30 to 1:50 (g / mL).

[0011] Furthermore, the material-to-liquid ratio of the extract is 1:41 (g / mL).

[0012] Furthermore, the extraction temperature is 20~50℃, and the ultrasonic time is 20~60min.

[0013] Furthermore, the ultrasonic time is 52 minutes and the extraction temperature is 33°C.

[0014] Furthermore, the olive leaves are selected from one of the following: Ezhi No. 8, Miracle, Alposana, Bean, and Corati.

[0015] The beneficial effects of this invention are as follows: 1. The extraction method in this invention has high extraction efficiency and is environmentally friendly: it uses deep eutectic solvent coupled with ultrasound assistance, and the extraction yield is significantly better than that of traditional organic solvent methods. Moreover, the solvent is biodegradable, non-volatile, and leaves no residue, which meets the requirements of green chemistry.

[0016] 2. The extraction method in this invention has mild conditions and good product stability: the extraction temperature is low and the time is short, which effectively avoids the degradation of heat-sensitive components; the hydrogen bond network of the deep eutectic solvent has a protective effect on oleuropein, making its stability under heat and light conditions better than that of traditional solvent systems.

[0017] 3. The extraction method in this invention has a clear synergistic mechanism and is easy to purify: It reveals for the first time the multi-scale synergistic enhancement mechanism of ultrasonic cavitation and deep eutectic solvent hydrogen bond network at the level of cell structure destruction and molecular recognition, providing a theoretical basis for process scale-up; combined with macroporous resin, high-purity products can be obtained quickly.

[0018] 4. The extract obtained by the extraction method of the present invention has excellent biological activity and wide applicability: the obtained oleuropein has good antioxidant, antibacterial and potential lipid-lowering activities, and can be applied to functional foods, pharmaceuticals and cosmetics; and can effectively distinguish the content differences of different varieties of olive leaves, which is suitable for resource evaluation and high-value utilization. Detailed Implementation

[0019] The specific embodiments of the present invention will be described in further detail below. Example

[0020] 1. Preparation of olive leaf powder In this embodiment, the olive leaf raw material was selected from the Ezhi-8 variety of olive, collected in Wanzhou District, Chongqing, China, in early August 2024. To minimize the degradation of heat-sensitive phenolic active substances after collection, the fresh leaves were dried in a 35 ℃ constant temperature forced-air drying oven for 7 days until constant weight. After drying, the sample was pulverized using a high-speed pulverizer and passed through a 60-mesh sieve (approximately 0.25 mm) to ensure uniform particle size. The resulting powder was placed in sealed polyethylene bags and stored in a refrigerator at 4 ℃, protected from light, for later use.

[0021] 2. Preparation of deep eutectic solvents Weigh appropriate amounts of raw materials according to a choline chloride to ethylene glycol molar ratio of 1:2.5, place them in a sealed beaker, and continuously stir magnetically under a constant temperature water bath at 80℃ until a homogeneous and transparent liquid is formed, indicating that the deep eutectic solvent DES system has been successfully constructed. To reduce the viscosity of the system and improve mass transfer performance, add 20% deionized water (by volume) to the resulting solution, mix well, and store in a desiccator in a sealed, light-protected container for later use, thus obtaining the deep eutectic solvent.

[0022] 3. Ultrasonic-assisted extraction Accurately weigh 1.00 g of olive leaf powder and add it to the pre-prepared DES solution at a material-to-liquid ratio of 1:41 (g / mL). After thorough mixing, wrap the container with aluminum foil to protect it from light. Place the mixture in an ultrasonic water bath and extract at 33℃ for 52 min. The temperature control accuracy of the circulating water bath is ±1.0℃ to obtain the extract.

[0023] 4. After extraction, the extract was centrifuged at 4℃ and 12840×g for 15 min, the supernatant was collected and diluted twice with 40% methanol, filtered through a 0.45μm filter membrane to obtain oleuropein extract, and stored at −20℃ for analysis. Example

[0024] 1. Preparation of olive leaf powder In this embodiment, the olive leaf raw material used was from the Qiji variety of olive, collected in Wanzhou District, Chongqing, China, in early August 2024. To minimize the degradation of heat-sensitive phenolic active substances after collection, the fresh leaves were dried in a 35°C constant-temperature forced-air drying oven for 7 days until constant weight. After drying, the sample was pulverized using a high-speed pulverizer and passed through a 60-mesh sieve (approximately 0.25 mm) to ensure uniform particle size. The resulting powder was placed in sealed polyethylene bags and stored in a refrigerator at 4°C, protected from light, for later use.

[0025] 2. Preparation of deep eutectic solvents Weigh appropriate amounts of raw materials according to a choline chloride to ethylene glycol molar ratio of 1:1, place them in a sealed beaker, and continuously stir magnetically under a constant temperature water bath at 80℃ until a homogeneous and transparent liquid is formed, indicating that the deep eutectic solvent DES system has been successfully constructed. To reduce the viscosity of the system and improve mass transfer performance, add 40% deionized water (by volume) to the resulting solution, mix well, and store in a desiccator in a sealed, light-protected container for later use, thus obtaining the deep eutectic solvent.

[0026] 3. Ultrasonic-assisted extraction Accurately weigh 1.00 g of olive leaf powder and add it to the pre-prepared DES solution at a material-to-liquid ratio of 1:30 (g / mL). After thorough mixing, wrap the container with aluminum foil to protect it from light. Place the mixture in an ultrasonic water bath and extract at 20℃ for 20 min. The temperature control accuracy of the circulating water bath is ±1.0℃ to obtain the extract.

[0027] 4. After extraction, the extract was centrifuged at 4℃ and 12840×g for 10 min, the supernatant was collected and diluted twice with 40% methanol, filtered through a 0.45μm filter membrane to obtain oleuropein extract, and stored at −20℃ for analysis. Example

[0028] 1. Preparation of olive leaf powder In this embodiment, the olive leaf raw material was selected from the Arbosana variety of olive, collected in Wanzhou District, Chongqing, China, in early August 2024. To minimize the degradation of heat-sensitive phenolic active substances after collection, the fresh leaves were dried in a 35 ℃ constant-temperature forced-air drying oven for 7 days until constant weight. After drying, the sample was pulverized using a high-speed pulverizer and passed through a 60-mesh sieve (approximately 0.25 mm) to ensure uniform particle size. The resulting powder was placed in sealed polyethylene bags and stored in a refrigerator at 4 ℃, protected from light, for later use.

[0029] 2. Preparation of deep eutectic solvents Weigh appropriate amounts of raw materials according to a choline chloride to ethylene glycol molar ratio of 1:1, place them in a sealed beaker, and continuously stir magnetically under a constant temperature water bath at 80℃ until a homogeneous and transparent liquid is formed, indicating that the deep eutectic solvent DES system has been successfully constructed. To reduce the viscosity of the system and improve mass transfer performance, add 10% deionized water (by volume of the total system) to the resulting mixture, mix well, and store in a desiccator in a sealed, light-protected container for later use, thus obtaining the deep eutectic solvent.

[0030] 3. Ultrasonic-assisted extraction Accurately weigh 1.00 g of olive leaf powder and add it to the pre-prepared DES solution at a material-to-liquid ratio of 1:50 (g / mL). After thorough mixing, wrap the container with aluminum foil to protect it from light. Place the mixture in an ultrasonic water bath and extract at 50℃ for 60 min. The temperature control accuracy of the circulating water bath is ±1.0℃ to obtain the extract.

[0031] 4. After extraction, the extract was centrifuged at 4℃ and 12840×g for 20 min. The supernatant was collected and diluted twice with 40% methanol. After filtration through a 0.45μm filter membrane, the oleuropein extract was obtained and stored at −20℃ for analysis. Example

[0032] 1. Preparation of olive leaf powder In this embodiment, the olive leaf raw material was selected from the Douguo variety of olive, collected in Wanzhou District, Chongqing, China, in early August 2024. To minimize the degradation of heat-sensitive phenolic active substances after collection, the fresh leaves were dried in a 35 ℃ constant temperature forced-air drying oven for 7 days until constant weight. After drying, the sample was pulverized using a high-speed pulverizer and passed through a 60-mesh sieve (approximately 0.25 mm) to ensure uniform particle size. The resulting powder was placed in sealed polyethylene bags and stored in a refrigerator at 4 ℃, protected from light, for later use.

[0033] 2. Preparation of deep eutectic solvents Weigh appropriate amounts of raw materials according to a choline chloride to ethylene glycol molar ratio of 1:1.5, place them in sealed beakers, and continuously stir magnetically under a constant temperature water bath at 80℃ until a homogeneous and transparent liquid is formed, indicating that the deep eutectic solvent DES system has been successfully constructed. To reduce the viscosity of the system and improve mass transfer performance, add 10% deionized water (by volume of the total system) to the resulting mixture, mix well, and store in a desiccator in a sealed, light-protected container for later use, thus obtaining the deep eutectic solvent.

[0034] 3. Ultrasonic-assisted extraction Accurately weigh 1.00 g of olive leaf powder and add it to the pre-prepared DES solution at a material-to-liquid ratio of 1:50 (g / mL). After thorough mixing, wrap the container with aluminum foil to protect it from light. Place the mixture in an ultrasonic water bath and extract at 50℃ for 60 min. The temperature control accuracy of the circulating water bath is ±1.0℃ to obtain the extract.

[0035] 4. After extraction, the extract was centrifuged at 4℃ and 12840×g for 20 min. The supernatant was collected and diluted twice with 40% methanol. After filtration through a 0.45μm filter membrane, the oleuropein extract was obtained and stored at −20℃ for analysis. Example

[0036] 1. Preparation of olive leaf powder In this embodiment, the olive leaf raw material was selected from the Coratina variety of olive, collected in Wanzhou District, Chongqing, China, in early August 2024. To minimize the degradation of heat-sensitive phenolic active substances after collection, the fresh leaves were dried in a 35 ℃ constant-temperature forced-air drying oven for 7 days until constant weight. After drying, the sample was pulverized using a high-speed pulverizer and passed through a 60-mesh sieve (approximately 0.25 mm) to ensure uniform particle size. The resulting powder was placed in sealed polyethylene bags and stored in a refrigerator at 4 ℃, protected from light, for later use.

[0037] 2. Preparation of deep eutectic solvents Weigh appropriate amounts of raw materials according to a choline chloride to ethylene glycol molar ratio of 1:2, place them in a sealed beaker, and continuously stir magnetically under a constant temperature water bath at 80℃ until a homogeneous and transparent liquid is formed, indicating that the deep eutectic solvent DES system has been successfully constructed. To reduce the viscosity of the system and improve mass transfer performance, add 10% deionized water (by volume of the total system) to the resulting mixture, mix well, and store in a desiccator in a sealed, light-protected container for later use, thus obtaining the deep eutectic solvent.

[0038] 3. Ultrasonic-assisted extraction Accurately weigh 1.00 g of olive leaf powder and add it to the pre-prepared DES solution at a material-to-liquid ratio of 1:50 (g / mL). After thorough mixing, wrap the container with aluminum foil to protect it from light. Place the mixture in an ultrasonic water bath and extract at 50℃ for 60 min. The temperature control accuracy of the circulating water bath is ±1.0℃ to obtain the extract.

[0039] 4. After extraction, the extract was centrifuged at 4℃ and 12840×g for 20 min. The supernatant was collected and diluted twice with 40% methanol. After filtration through a 0.45μm filter membrane, the oleuropein extract was obtained and stored at −20℃ for analysis.

[0040] The oleuropein content of the oleuropein extracts obtained in Examples 1 to 5 was determined by high-performance liquid chromatography (HPLC). The chromatographic analysis used an HPLC system equipped with a UV detector, a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm), and a mobile phase of methanol-water (40:60, v / v) in isocratic elution mode. The chromatographic conditions were set as follows: flow rate 1.0 mL / min, column temperature 40 ℃, detection wavelength 280 nm, and injection volume 10 μL. A series of solutions with concentrations ranging from 62.5 to 2000 μg / mL were prepared using oleuropein standards, and a linear regression equation between peak area and concentration was established for quantitative analysis. The oleuropein content in the samples was calculated based on the standard curve, and the extraction rate was converted according to the oleuropein concentration, total volume, dilution factor, and sample mass in the extract. The results are expressed in mg / g.

[0041] Comparative Example 1 80% methanol Miracle 37.92±0.35 Example 1 DES-2 Ezhi No. 8 72.35±0.87 Example 2 DES-2 Miracle 61.69±0.47 Example 3 DES-2 Al-Posana 69.31±1.82 Example 4 DES-2 Douguo 50.46±1.19 Example 5 DES-2 Corrati 69.69±0.87 As can be seen from the table above, compared with the same variety (Miracle), the extraction rate of the DES-2 system (61.69%) is much higher than that of the traditional 80% methanol system (37.92%), an increase of about 62.7%.

[0042] The optimal variety in the DES-2 system: Ezhi No. 8 has the highest extraction rate of 72.35 mg / g.

[0043] This invention constructs a deep eutectic solvent system with choline chloride and ethylene glycol as the core, and combines it with ultrasonic enhancement technology to achieve efficient and green extraction of oleuropein from olive leaves. Results show that choline chloride and ethylene glycol form a stable deep eutectic structure through Cl⁻···H–O directional hydrogen bonds and multiple non-covalent interactions. This structure plays a key role in reducing system viscosity and enhancing intermolecular interactions. Under optimal conditions, the extraction yield of oleuropein is significantly higher than that of the traditional methanol system. Kinetic analysis shows that the extraction process conforms to a second-order kinetic model, controlled synergistically by desorption and intraparticle diffusion. Structural characterization and theoretical calculations further reveal that ultrasonic cavitation and the hydrogen bond network of the deep eutectic solvent form a multi-scale synergistic enhancement mechanism at the macroscopic structural disruption and molecular recognition levels, thereby significantly reducing mass transfer resistance and improving dissolution efficiency. The obtained oleuropein exhibits good stability under heat and light conditions, and its purity is significantly improved after purification with macroporous resin. It also exhibits excellent antioxidant, antibacterial, and potential lipid-lowering activities. It should be noted that this study is mainly based on laboratory-scale validation, and its scale-up behavior in complex industrial systems, long-term cycling stability, and in vivo functional mechanisms still require further systematic investigation. Overall, this study systematically elucidates the intrinsic mechanisms of ultrasound-assisted deep eutectic solvent extraction of oleuropein from three levels: solvent construction mechanism, mass transfer enhancement mechanism, and functional validation. This provides important theoretical basis and technical support for the high-value utilization of olive by-products and the development of green extraction technologies.

[0044] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for extracting oleuropein based on deep eutectic solvent and ultrasonic coupling, characterized in that, Includes the following steps: Olive leaves are dried, pulverized, and sieved to obtain olive leaf powder; Ethylene glycol was used as a hydrogen bond donor and choline chloride as a hydrogen bond acceptor to prepare a deep eutectic solvent, and the water content was adjusted. The olive leaf powder was mixed with the deep eutectic solvent and subjected to ultrasonic-assisted extraction to obtain an extract. The extract was subjected to solid-liquid separation, dilution, and filtration to obtain oleuropein extract.

2. The extraction method according to claim 1, characterized in that, The molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:1 to 1:

3.

3. The extraction method according to claim 2, characterized in that, The molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:2.

5.

4. The extraction method according to claim 1, characterized in that, The water content of the deep eutectic solvent is 10%~40%.

5. The extraction method according to claim 4, characterized in that, The water content of the deep eutectic solvent is 20%.

6. The extraction method according to claim 1, characterized in that, The material-to-liquid ratio of the extract is 1:30 to 1:50 (g / mL).

7. The extraction method according to claim 1, characterized in that, The material-to-liquid ratio of the extract is 1:41 (g / mL).

8. The extraction method according to claim 1, characterized in that, The extraction temperature is 20~50℃, and the ultrasonic time is 20~60min.

9. The extraction method according to claim 1, characterized in that, The ultrasonic time was 52 minutes, and the extraction temperature was 33°C.

10. The extraction method according to claim 1, characterized in that, The olive leaves are selected from one of the following: Ezhi No. 8, Miracle, Alposana, Bean, and Corati.