Method for one-step conversion and extraction of oleacein from olive leaves by using NADES
By using natural eutectic solvent (NADES) to convert oleacein from olive leaves into oleacein, combined with ultrasonic extraction and macroporous resin separation technology, the problem of low extraction efficiency of oleacein in olive leaves in the prior art is solved, and an efficient and economical oleacein extraction process is achieved.
Patent Information
- Application Number
- CN202510348060.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-24
- Publication Date
- 2025-06-24
AI Technical Summary
The prior art is difficult to efficiently convert and extract oleacein from olive leaves on a large scale, resulting in higher costs.
Natural eutectic solvent (NADES) was used to convert oleacein into oleacein in olive leaves, and the extraction of oleacein was achieved through ultrasonic extraction and macroporous resin separation.
The extraction of high-purity oleacein is realized, with green energy saving, high efficiency and high yield characteristics, which reduces production costs and realizes the recycling and recycling of NADES.
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Figure CN120193025A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of olive leaf extraction, and particularly relates to a method for one-step conversion and extraction of oleacein from olive leaves using NADES. Background Art
[0002] 2-(3,4-dihydroxyphenyl)ethyl (Z)-4-formyl-3-(2-oxoethyl)hex-4-enoate (oleacein, 3,4-DHPEA-EDA), as an important phenolic compound in olive oil, has attracted attention due to its good antioxidant, anti-inflammatory, and cardiovascular protection effects. Oleacein is mainly obtained by stepwise enzymatic hydrolysis of oleuropein as a precursor during the production of olive oil by β-glucosidase and esterase. Currently, the main method for obtaining oleacein is extraction from olive oil. However, due to the high price of olive oil and the relatively low proportion of phenolic components compared to materials such as olive fruits and olive leaves, the cost of batch extraction and separation of oleacein from olive oil has increased significantly.
[0003] Olive leaves contain a large amount of oleuropein (3-14% dry weight) and are inexpensive and easily available, making them a good raw material for preparing oleacein. However, the large-scale conversion and preparation technology has always been a difficult point. In view of this, the present invention proposes a method for one-step promoting the conversion of oleuropein in olive leaves to oleacein and extracting it using Natural Deep Eutectic Solvents (NADES). Summary of the Invention
[0004] Aiming at the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a method for one-step conversion and extraction of oleacein from olive leaves using NADES.
[0005] To achieve the above purpose, the present invention adopts the following technical solutions:
[0006] A method for one-step conversion and extraction of oleacein from olive leaves using NADES, comprising the following steps:
[0007] S1. Pretreat olive leaves and olive fruits respectively to obtain olive leaf powder and olive fruit pulp;
[0008] S2. Mix the olive leaf powder with NADES, homogenize, then add the olive fruit pulp, exhaust air by ultrasonic treatment, introduce CO2, seal and let stand, carry out an enzymatic conversion reaction, and then extract by ultrasonic treatment;
[0009] S3. After extraction, cool the obtained extract, centrifuge, filter by suction, and collect the supernatant;
[0010] S3. Use macroporous resin to separate and recover oleacein and the NADES used in the extraction.
[0011] Preferably, the pretreatment of olive leaves and olives respectively includes:
[0012] Perform microwave short-time sterilization treatment on olive leaves, air-dry them naturally, then crush the dried olive leaves to obtain olive leaf powder, and store it in a light-proof and dry container;
[0013] Prewarm the olives and beat them into olive pulp with a blender, and store it at 4°C.
[0014] Preferably, the synthesis of the NADES includes the following steps:
[0015] Add a hydrogen bond acceptor and a hydrogen bond donor to water, stir and react at 80 - 90°C for 3 - 8 h until a clear, homogeneous and transparent liquid is formed to obtain a natural deep eutectic solvent system (NADES); wherein, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 2:1 - 1:3, and the amount of water used is 20 - 60% of the total mass of the hydrogen bond acceptor and the hydrogen bond donor.
[0016] Preferably, the hydrogen bond acceptor is selected from any one of choline chloride, betaine and L-proline; the hydrogen bond donor is any one of lactic acid, malonic acid, malic acid, tartaric acid, citric acid, glycerol, 1,3-butanediol, urea, xylitol or glucose.
[0017] Preferably, the mass-volume ratio of the olive leaf powder to the NADES is 1 g:15 - 40 mL.
[0018] Preferably, in step S2, the homogenization time is 1 - 5 min.
[0019] Preferably, in step S2, the amount of the olive pulp used is 5 - 20% of the total mass of the olive leaf powder.
[0020] Preferably, in step S2, the conditions for the enzymatic conversion reaction are: standing for 10 - 60 min under the water bath condition of 30 - 50°C.
[0021] Preferably, in step S2, the conditions for the ultrasonic extraction are: the ultrasonic temperature is 25 - 70°C, the ultrasonic power is 140 - 700 W, and the ultrasonic time is 5 - 30 min.
[0022] Preferably, in step S3, the centrifugal force during centrifugation is 8000×g - 10000×g, the centrifugation time is 10 - 25 min, and the cooling temperature is 0 - 4°C.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] The present invention provides a method for one-step conversion and extraction of oleacein from olive leaves using NADES. The extraction of high-purity oleacein is achieved through NADES, which has the characteristics of being green, energy-saving, efficient, and having a high yield. It not only avoids the energy consumption of high-temperature extraction and the extensive use of organic chemical reagents, greatly improves the production efficiency, and is convenient for on-site processing and extraction in olive oil production, but also realizes the recovery and recycling of NADES, enhancing the economy and sustainability of the preparation process. Therefore, it has broad application prospects. Brief Description of the Drawings
[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.
[0026] Figure 1 It is the high-performance liquid chromatography diagram of the extract and oleacein standard product in Example 1;
[0027] Figure 2 It is the ultraviolet-visible absorption spectrum diagram of the extract in Example 1;
[0028] Figure 3 It is the infrared spectrum diagram of the extract in Example 1. Detailed Embodiments
[0029] In the following description, specific details such as specific system structures and technologies are presented for the purpose of illustration rather than limitation, so as to thoroughly understand the embodiments of the present invention. However, those skilled in the art should clearly understand that the present invention can also be implemented in other embodiments without these specific details.
[0030] Example 1
[0031] A method for one-step conversion and extraction of oleacein from olive leaves using NADES, comprising the following steps:
[0032] (1) Pretreat the olive leaves and olive fruits:
[0033] After removing foreign matters such as small branches from the dry olive leaves, perform short-term microwave treatment and then air-dry naturally. Crush the dried olive leaves with a pulverizer to obtain olive leaf powder, and store it in a light-proof and dry container;
[0034] Prewarm the fresh olive fruits and beat them into olive fruit pulp with a blender, and store it at 4°C.
[0035] (2) Synthesis of NADES: Betaine was selected as the hydrogen bond acceptor and glycerol as the hydrogen bond donor. Betaine and glycerol were mixed at a molar ratio of 1:2, then 40% of the total mass of water was added, and finally, it was stirred at 80 °C and 200 rpm for 6 h until a clear, homogeneous and transparent liquid was formed to obtain NADES;
[0036] (3) Transformation and extraction of oleacein: The obtained olive leaf powder was mixed with NADES at a solid-liquid ratio of 1 g:15 mL, homogenized by a homogenizer for 1 min, then 10% of the olive fruit pulp based on the mass of the olive leaf powder was added. After ultrasonic degassing, CO2 was introduced into the bottle, sealed with plastic wrap, and subjected to an enzymatic conversion reaction in a 40 °C water bath for 30 min, and then ultrasonically extracted at 50 °C and 500 W for 10 min. After extraction, the extract was cooled to 4 °C in a water bath, centrifuged at 4 °C and 7000×g for 15 min, and then filtered by suction to collect the supernatant;
[0037] (4) Recovery of macroporous resin: Separated by macroporous resin X-5, the used NADES in the extraction was eluted with water, and the extraction product was eluted with pure ethanol. Finally, after rotary evaporation and freeze-drying, the extract oleacein was obtained.
[0038] The experimental results showed that the yield of oleacein obtained in this example was 45.71 ± 0.78 mg / g of dry olive leaves, and the purity of oleacein in the extract powder could reach 84.3%.
[0039] Example 2
[0040] The steps of this example were basically the same as those of Example 1, except that the hydrogen bond acceptor was changed to choline chloride and the hydrogen bond donor was changed to malic acid, which were mixed at a molar ratio of 1:2, and then 30% of the total mass of water was added.
[0041] The experimental results showed that the yield of oleacein obtained in this example was 10.14 ± 0.31 mg / g of dry olive leaves.
[0042] Example 3
[0043] The steps of this example were basically the same as those of Example 1, except that the hydrogen bond acceptor was changed to L-proline and the hydrogen bond donor was changed to citric acid, which were mixed at a molar ratio of 1:1, and then 40% of the total mass of water was added.
[0044] The experimental results showed that the yield of oleacein obtained in this example was 9.02 ± 0.64 mg / g of dry olive leaves.
[0045] Example 4
[0046] The steps of this example are basically the same as those of Example 1, except that the hydrogen bond acceptor is changed to betaine, the hydrogen bond donor is 1,3-butanediol, and they are mixed in a molar ratio of 1:2, and then 50% of the total mass of water is added.
[0047] The experimental results show that the yield of oleacein obtained in this example is 10.52±1.12 mg / g of dry olive leaves.
[0048] Example 5
[0049] The steps of this example are basically the same as those of Example 1, except that the mass-volume ratio of olive leaf powder to NADES is 1 g:10 mL.
[0050] The experimental results show that the yield of oleacein obtained in this example is 38.63±1.17 mg / g of dry olive leaves.
[0051] Example 6
[0052] The steps of this example are basically the same as those of Example 1, except that the mass-volume ratio of olive leaf powder to NADES is 1 g:40 mL.
[0053] The experimental results show that the yield of oleacein obtained in this example is 44.75±0.76 mg / g of dry olive leaves.
[0054] Example 7
[0055] The steps of this example are basically the same as those of Example 1, except that the ultrasonic time is 5 min.
[0056] The experimental results show that the yield of oleacein obtained in this example is 42.87±0.18 mg / g of dry olive leaves.
[0057] Example 8
[0058] The steps of this example are basically the same as those of Example 1, except that the ultrasonic time is 30 min.
[0059] The experimental results show that the yield of oleacein obtained in this example is 44.85±0.59 mg / g of dry olive leaves.
[0060] Example 9
[0061] The steps of this example are basically the same as those of Example 1, except that the ultrasonic power is 140 W.
[0062] The experimental results show that the yield of oleacein obtained in this example is 42.74±0.80 mg / g of dry olive leaves.
[0063] Example 10
[0064] The steps of this example are basically the same as those of Example 1, except that the ultrasonic power is 700 W.
[0065] The experimental results show that the yield of oleacein obtained in this example is 44.15 ± 0.34 mg / g of dry olive leaves.
[0066] Example 11
[0067] The steps of this example are basically the same as those of Example 1, except that the ultrasonic temperature is 25 °C.
[0068] The experimental results show that the yield of oleacein obtained in this example is 35.88 ± 0.16 mg / g of dry olive leaves.
[0069] Example 12
[0070] The steps of this example are basically the same as those of Example 1, except that the ultrasonic temperature is 70 °C.
[0071] The experimental results show that the yield of oleacein obtained in this example is 41.91 ± 0.47 mg / g of dry olive leaves.
[0072] Comparative Example 1
[0073] The steps of this example are basically the same as those of Example 1, except that olive pulp is not added. The experimental results show that the yield of oleacein obtained in this comparative example is 0.38 ± 0.08 mg / g of dry olive leaves.
[0074] Comparative Example 2
[0075] The steps of this example are basically the same as those of Example 1, except that olive leaf powder is not added. As a blank control, the experimental results show that the yield of oleacein obtained in this comparative example is 0.84 ± 0.12 mg / g of olive pulp.
[0076] Comparative Example 3
[0077] The steps of this example are basically the same as those of Example 1, except that the mass-volume ratio of olive leaf powder to NADES is 1 g: 5 mL. The experimental results show that the yield of oleacein obtained in this comparative example is 31.52 ± 0.54 mg / g of dry olive leaves.
[0078] Comparative Example 4
[0079] The steps of this example are basically the same as those of Example 1, except that the ultrasonic time is 2 min. The experimental results show that the yield of oleacein obtained in this comparative example is 28.96 ± 0.89 mg / g of dry olive leaves.
[0080] Performance Characterization
[0081] (1) High performance liquid chromatography identification: The extract powder in Example 1 was reconstituted with ethanol and then measured using an Agilent 1200 high performance liquid chromatography system. 18 Chromatographic column (4.6mm×250mm, 5μm), mobile phase A was 0.5% phosphoric acid aqueous solution (v / v), mobile phase B was methanol and acetonitrile (1:1, v / v), gradient elution program (52min): 0-40min, 4-50% B; 40-41min, 50-100% B; 41-46min, 100% B; 46-47min, 100-4% B; 47-52min, 4% B. Elution flow rate was 1mL / min; detection temperature was 20℃; injection volume was 10μL; VWD detection wavelength was 280nm. All samples were filtered through a 0.22μm nylon filter before analysis. Weigh 10.0 mg of oleacein and oleuropein standard, dilute to 10 mL, obtain 1.0 mg / mL standard solution, then perform gradient dilution and record the peak area at 280 nm using the above HPLC conditions and methods to make a standard curve of peak area and concentration gradient. Substitute the peak area corresponding to the chromatogram of the sample solution into the calculation to obtain the content. Figure 1 .
[0082] Depend on Figure 1 The results show that the main component of the extract in Example 1 is oleacein (peak at the 32nd minute), and it also contains a small amount of hydroxytyrosol (peak at the 10.9th minute), luteolin (peak at the 27th minute), etc.
[0083] (2) UV-visible absorption spectrum measurement: Take 1 mg / mL aqueous solution of the extract in Example 1 and measure the UV-visible absorption spectrum at 200-600 nm using an ELISA instrument. Use water as a blank control. The results are shown in Figure 2 .
[0084] Depend on Figure 2 From the UV-visible absorption spectrum of the product, it can be seen that there are multiple irregular absorption peaks in the UV absorption range of 200-400nm. The product contains many types of groups, which may come from the E-band and B-band absorption of the benzene ring, phenolic hydroxyl, etc.
[0085] (3) Infrared spectrometry: The extract in Example 1 and the dried potassium bromide powder were thoroughly mixed and ground at a mass ratio of 1:100. The mixed powder was then prepared into a transparent sheet with a thickness of 1 mm using an air compressor. A Nicolet iS50 Fourier transform infrared spectrometer was used at 4000-400 cm -1 Scanning is performed within the range with a scanning resolution of 4cm-1 , using potassium bromide as a blank control. The results are shown in Figure 3 .
[0086] From Figure 3 the results, it can be seen that 3416 cm -1 is the stretching vibration of -OH; 2814 cm -1 is the stretching vibration of -CH3; 1632 cm -1 , 1593 cm -1 is the asymmetric stretching vibration of C=C; 1380 cm -1 , 1351 cm -1 is the in-plane bending vibration of O-H. This compound contains groups such as -CH3, -OH, and C=C, which is consistent with the structure of oleacein.
[0087] (4) Ultra-high performance liquid chromatography-tandem mass spectrometry detection: The phenolic components of the extract were determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-TOF / MS). The ion source was the ESI source, operating in the negative ion mode, with a source voltage of 3 kV, a source temperature of 150 °C, an m / z range of 115 - 1000, a collision energy of 10 - 25 eV, a column temperature of 40 °C, and a flow rate of 0.21 mL / min. Phase A was a 5% aqueous formic acid solution (v / v), and mobile phase B was methanol and acetonitrile (1:1, v / v). The elution gradient was as follows: 0 - 16 min, 4 - 50% B; 16 - 16.4 min, 50 - 100% B; 16.4 - 18.4 min, 100% B; 18.4 - 18.8 min, 100 - 4% B; 18.8 - 20.8 min, 4% B. The injection volume was 1 μL, and all samples were filtered through a 0.22 μm filter before analysis.
[0088] The experimental results showed that the retention time of the target peak was 11.13 min, and the [M-H] - peak was at m / z 319.11680, and the main fragment ion peak was at m / z 195.06534 (C 10 H 11 O4 - ), which was consistent with the structure of oleacein.
[0089] (5) Determination of polyphenol content in the extract by the Folin-Ciocalteu method: 100 μL of the extract was mixed with 500 μL of Folin-Ciocalteu reagent (10%, v / v), and the mixture was allowed to stand and develop color in the dark at 20 - 25 °C for 5 min. Subsequently, 500 μL of Na2CO3 solution (7.5%, w / v) was added and mixed for 20 s. After heating in a water bath at 50 °C for 90 s, it was immediately cooled to 20 - 25 °C and allowed to stand in the dark at 20 - 25 °C for 60 min. Finally, the absorbance of the reaction solution at a wavelength of 765 nm was measured, using gallic acid as the standard control. The results are shown in Table 1.
[0090] Table 1
[0091]
[0092] As can be seen from the results in Table 1, the yield of oleacein in Example 1 was 45.71 ± 0.78 mg / g of dry olive leaves. When olive pulp was not added in Comparative Example 1, the yield of oleacein was only 0.38 ± 0.08 mg / g of dry olive leaves. When olive leaf powder was not added in Comparative Example 2, the yield of oleacein was 0.84 ± 0.12 mg / g of olive pulp. By comparing the above experimental results, it was found that only when both olive leaf powder and olive pulp were added could a higher yield of oleacein be obtained. This was because the olive pulp added in this application was not used as a source of oleacein or oleuropein precursors, but provided corresponding endogenous enzymes such as β-glucosidase and esterase, and its function was to promote the enzymatic reaction of oleuropein in olive leaves to be converted into oleacein.
[0093] In summary, the present invention successfully realizes the one-step promotion of the conversion of oleuropein in olive leaves into oleacein and extraction by using natural deep eutectic solvents (NADES), and has the characteristics of green energy conservation, simplicity and high efficiency, and high yield. It not only avoids the energy consumption of high-temperature extraction and the large use of organic chemical reagents, greatly improves the production efficiency, but also realizes the recovery and recycling of NADES.
[0094] The present invention is not limited to the above specific embodiments. Those of ordinary skill in the art starting from the above concepts and making various changes without creative labor fall within the protection scope of the present invention.
Claims
1. A method for one-step conversion and extraction of oleacein from olive leaves using NADES, characterized in that: The following steps are involved: S1. Pre-treating olive leaves and olive fruits respectively to obtain olive leaf powder and olive pulp; S2, mixing olive leaf powder and NADES homogenously, then adding olive pulp, introducing CO2 by ultrasonic exhaust, sealing and standing to carry out enzymatic conversion reaction, and then ultrasonic extraction; S3. After the extraction is completed, the obtained extract is cooled, centrifuged, filtered, and the supernatant is collected; S3. Use macroporous resin to separate and recover oleacein and NADES.
2. The method of claim 1, wherein the method comprises: The synthesis of the NADES comprises the following steps: A hydrogen bond acceptor, a hydrogen bond donor and water are mixed, and stirred at 80 to 90° C. for 3 to 8 hours until a clear and uniform transparent liquid is formed to obtain NADES; wherein the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 2:1 to 1:3, and the amount of water used is 20 to 60% of the total mass of NADES.
3. The method of claim 2, wherein the method comprises: The hydrogen bond acceptor is selected from any one of choline chloride, betaine and L-proline; the hydrogen bond donor is any one of lactic acid, malonic acid, malic acid, tartaric acid, citric acid, glycerol, 1,3-butanediol, urea, xylitol or glucose.
4. The method of claim 1, wherein the method comprises: The mass volume ratio of the olive leaf powder to NADES is 1 g:15-40 mL.
5. The method of claim 1, wherein the method comprises: In step S2, the amount of olive pulp used is 5-20% of the mass of olive leaf powder.
6. The method of claim 1, wherein the method comprises: In step S2, the enzymatic conversion reaction conditions are: standing in a 30-50° C. water bath for 10-60 min.
7. The method of claim 1, wherein the method comprises: In step S2, the conditions for ultrasonic extraction are: ultrasonic temperature is 25-70°C, ultrasonic power is 140-700W, and ultrasonic time is 5-30min.
8. The method of claim 1, wherein the method comprises: In step S3, the centrifugal force during the centrifugation is 6000×g to 9000×g, the centrifugation time is 10 to 25 min, and the cooling temperature is 0 to 4°C.