Green extraction method of polyphenol compounds in olive oil

Abstract

The present invention belongs to the field of green sample pretreatment, specifically relating to a method for the green and rapid extraction of polyphenolic compounds from olive oil based on a water-based deep eutectic solvent-liquid microextraction technique. The present invention discloses a green extraction method for polyphenolic compounds from olive oil, comprising the following steps: mixing a hydrogen bond acceptor reagent with water and then sonicating to obtain a water-based deep eutectic solvent; vortex-mixing olive oil, n-hexane, and the water-based deep eutectic solvent, then sonicating and centrifuging to separate the mixture into an upper oil layer and a lower deep eutectic solvent layer; and extracting the deep eutectic solvent layer as an extract; the extract containing polyphenolic compounds.

Description

Technical Field

[0001] The present invention belongs to the field of green sample pretreatment, and specifically relates to a green and rapid method for extracting polyphenol compounds in olive oil based on a water-based deep eutectic solvent-liquid microextraction technology. Background Art

[0002] Olive oil is a woody oil extracted from the fruit of the olive tree, a member of the Oleaceae family. It is the primary source of fat in the healthy Mediterranean diet. Olive oil has a complex nutritional profile, with variations in chemical composition depending on its origin, climate, variety, maturity, and pressing method. Generally speaking, olive oil is composed primarily of 98% saponifiable fraction and 2% unsaponifiable fraction. Saponifiable fractions primarily consist of unsaturated fatty acids, accounting for approximately 90% of the total fatty acids. The unsaponifiable fraction primarily consists of various trace components, including polyphenols, triterpenoids, phytosterols, squalene, vitamins, carotenoids, and volatile compounds. Epidemiological and experimental studies have demonstrated that olive oil has neuroprotective, immunomodulatory, anti-inflammatory, antioxidant, antibacterial, anticancer, hypoglycemic, and cardiovascular disease-preventing properties. It is now widely used in food, medicine, daily cosmetics, and skincare, and is highly sought after by consumers.

[0003] Polyphenols are representative nutritional components in olive oil and the material basis for its diverse active functions. Their content and type are important indicators for evaluating olive oil quality. Therefore, accurate and efficient analysis of olive oil polyphenols is essential for evaluating olive oil quality. Chromatography is the primary method for analyzing the profiles of olive oil polyphenols. Ultra-high performance liquid chromatography, with its advantages of efficient separation and high sensitivity, is a powerful tool for studying olive oil polyphenols. However, due to the low concentration of polyphenols in olive oil and the complex and viscous matrix, effective separation and extraction methods are crucial for detection and analysis. Currently, the main methods for extracting phenolic compounds from olive oil are liquid-liquid extraction and solid-phase extraction. Preliminary experiments conducted by the applicants compared the extraction efficiency of these two methods. The results showed that while these methods can effectively extract polyphenols, they are cumbersome, time-consuming, and require large amounts of organic solvents. This not only increases experimental costs, but also poses health risks to researchers, causes environmental pollution, and increases safety risks. Therefore, developing a simple, rapid, efficient, and green method for extracting polyphenols from olive oil is a prerequisite for achieving the green development goal of analytical chemistry.

[0004] Since Abbott et al. first discovered deep eutectic solvents in 2004, they have attracted widespread attention in the field of separation technology as a new type of green and environmentally friendly solvent. Deep eutectic solvents are a type of low-melting mixture composed of hydrogen bond donors and hydrogen bond acceptors. The components interact with each other through hydrogen bonds, electrostatic forces, and van der Waals forces. Hydrogen bond acceptors mainly include quaternary ammonium salts and quaternary phosphonium salts, while hydrogen bond donors are mainly carboxylic acids, alcohols, amines, and carbohydrates. Their components are safe and biodegradable. As an environmentally friendly solvent, it has the characteristics of simple preparation, low cost and easy availability, strong extraction ability for compounds of different polarities, high extraction efficiency, and reusability. It can be used as a substitute for toxic and volatile organic reagents.

[0005] Deep eutectic solvents have a designable molecular structure. Their molecular composition and molar ratio can significantly affect the solvent's polarity, viscosity, and pH. Different deep eutectic solvents are suitable for extracting different compounds. Literature reports that choline chloride-based deep eutectic solvents have a much higher extraction rate for polyphenols than traditional solvents. García et al. used a choline chloride-type low eutectic solvent to extract phenolic compounds in olive oil. Specifically, choline chloride was mixed with xylitol and 1,2-propylene glycol respectively, and stirred in an 80°C water bath until a uniform colorless liquid was formed, which was the low eutectic solvent and used as the subsequent extraction solvent; 14 g of the low eutectic solvent was added to 14 g of the olive oil sample and stirred in a 40°C water bath for 1 hour. During the water bath process, the sample was vortexed for 1 minute every 15 minutes. After the water bath, it was centrifuged at 1200G for 10 minutes, the upper oil layer was collected and extracted again, the lower extracts were combined, and the residual oil was removed with 60 mL of n-hexane, and the n-hexane was removed by nitrogen blowing; the obtained extract was eluted with an aqueous ion exchange resin XAD-16 column, 100 mL of deionized water was added to elute the low eutectic solvent, and then 100 mL of methanol was added to elute the polyphenol part, the methanol filtrate was collected, the methanol was evaporated under reduced pressure at 35°C, the residue was re-dissolved with 2 mL of methanol, and analyzed by high performance liquid chromatography. Results showed that choline chloride-xylitol and choline chloride-1,2-propylene glycol deep eutectic solvents were more efficient at extracting polyphenols from olive oil than a methanol-water (80:20, v:v) mixture. Paradiso et al. and Shabani et al. used lactic acid and glucose to prepare deep eutectic solvents for the extraction of polyphenols from olive oil, and measured total phenol content using UV spectrophotometry and electrochemical methods, respectively. Although the above-mentioned deep eutectic solvents have been successfully used to extract polyphenol compounds in olive oil, there are still certain limitations in their application: (1) The polyphenol compounds in olive oil are of various types, complex structures, and the polarity of different compounds varies greatly. It has been reported that the extraction efficiency of low-polarity phenolic compounds by low eutectic solvents is poor; (2) Due to the low content of phenolic compounds in the oil and the low volatility of low eutectic solvents, it is difficult to use traditional methods to concentrate samples. The literature uses an offline solid phase column to remove the low eutectic solvent, which greatly increases the pretreatment time; (3) The high viscosity and low volatility of low eutectic solvents can easily lead to instrument contamination and reduced sensitivity, hindering their direct application in instrumental analysis such as chromatography and mass spectrometry. Studies have used pure water dilution to reduce their concentration, but this method can also lead to a decrease in the concentration of target compounds. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a simple, rapid and green method for extracting polyphenol components in olive oil.

[0007] To solve the above technical problems, the present invention provides a green extraction method for polyphenol compounds in olive oil, comprising the following steps:

[0008] 1) Preparation of water-based deep eutectic solvent:

[0009] A hydrogen bond acceptor reagent was mixed with water at room temperature and sonicated (50 kHz for 10 min) until a clear and homogeneous solution was formed, thereby preparing a water-based deep eutectic solvent (WDES);

[0010] 2) Extraction:

[0011] 2.1) Weigh 1.5 g (accurate to 0.001 g) of olive oil into a 10 ml plastic centrifuge tube, add 1-2 ml of n-hexane, mix evenly (vortex to mix thoroughly for about 1 min), then add (quickly add) 200-400 μl of a water-based deep eutectic solvent, vortex for 1-3 min, sonicate at 50 kHz for 10-20 min at room temperature, then centrifuge at 10000 ± 1000 rpm for 10 ± 2 min. After centrifugation, the mixture is separated into an upper oil layer and a lower deep eutectic solvent layer (lower clear layer);

[0012] 2.2) Taking the deep eutectic solvent layer as an extract; the extract contains polyphenol compounds (polyphenol components).

[0013] Note: After centrifugation, the upper solution is discarded and the collected low eutectic solvent layer (lower clear liquid) is used to determine the content of polyphenol compounds (polyphenol components) by ultra-high performance liquid chromatography.

[0014] As an improvement of the green extraction method of polyphenol compounds in olive oil of the present invention:

[0015] Repeat step 2.1) 1 to 3 times using the upper oil layer obtained after centrifugation instead of 1.5 g of olive oil. Combine the deep eutectic solvent layers obtained by extraction to obtain a combined extract containing polyphenol compounds (polyphenol components).

[0016] As a further improvement of the green extraction method of polyphenols in olive oil of the present invention:

[0017] The olive oil is extra virgin olive oil.

[0018] As a further improvement of the green extraction method of polyphenols in olive oil of the present invention:

[0019] When the hydrogen bond acceptor reagent is choline chloride (preferred), betaine, lactic acid, citric acid, urea, tetramethylammonium bromide;

[0020] When the hydrogen bond acceptor reagent is choline chloride, the molar ratio of choline chloride to water is 1:2-5;

[0021] When the hydrogen bond acceptor reagent is betaine, the molar ratio of betaine to water is 1:5;

[0022] When the hydrogen bond acceptor reagent is lactic acid, the molar ratio of lactic acid to water is 1:5;

[0023] When the hydrogen bond acceptor reagent is citric acid, the molar ratio of citric acid to water is 1:10;

[0024] When the hydrogen bond acceptor reagent is urea, the molar ratio of urea to water is 1:5;

[0025] When the hydrogen bond acceptor reagent is tetramethylammonium bromide, the molar ratio of tetramethylammonium bromide to water is 1:10.

[0026] As a further improvement to the green extraction method of polyphenol compounds in olive oil of the present invention: when the hydrogen bond acceptor reagent is choline chloride, the molar ratio of choline chloride to water is 1:3.

[0027] As a further improvement to the green extraction method of polyphenol compounds in olive oil of the present invention: in step 2), the amount of the water-based deep eutectic solvent used is 300 μL (ie, according to a solid-liquid ratio of 1.5 g / 300 μL).

[0028] As a further improvement of the green extraction method of polyphenols in olive oil of the present invention:

[0029] In the step 2), 1 mL of n-hexane and 300 μL of a water-based deep eutectic solvent were added, and the vortex time was 2 min and the ultrasonic time was 15 min.

[0030] The present invention also provides a method for detecting the content of polyphenol compounds (polyphenol components) in the extract obtained by any of the above methods, comprising the following steps:

[0031] Take 100 μl of the extract / combined extract, dilute 10 times with acetonitrile-water mixture as a reconstitution solution, pass through a 0.22 μm organic filter membrane, and then perform ultra-high performance liquid chromatography for detection;

[0032] In the acetonitrile-water mixture, the volume ratio of acetonitrile:water is 2-5:8-5 (e.g., 2:8, 3:7, and 5:5, v / v);

[0033] Ultra-high performance liquid chromatography conditions: ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm); column temperature, 30°C; injection volume, 10 μl; flow rate, 0.3 mL min -1Mobile phase: 0.1% formic acid in water (A)-acetonitrile (B); elution gradient: 0-4 min, 5%-5% B; 4-8 min, 5%-10% B; 8-10 min, 10%-15% B; 10-14 min, 15%-20% B; 14-16 min, 20%-30% B; 16-20 min, 30%-40% B; 20-21 min, 40%-70% B; 21-25 min, 70%-90% B; 25-26 min, 90%-100% B; 26-30 min, 100%-100% B; 30-31 min, 100%-5% B; 31-35 min, 5%-5% B. UV detection wavelengths were 220 nm and 340 nm, FLD excitation wavelength Em was 278 nm, and emission wavelength Ex was 340 nm.

[0034] As an improvement to the content detection method of the present invention: in the acetonitrile-water mixture, the volume ratio of acetonitrile to water is 3:7.

[0035] The technical advantages of the present invention over García are: (1) the preparation process of the low co-solvent is simple and does not require heating; (2) the sample extraction process is simple, does not require heating, and is time-saving. After extraction, no polyphenol component recovery and concentration steps are required, and the amount of organic solvent used is small; (3) it has a good recovery rate for compounds of different polarity, especially for low-polarity compounds, such as hydroxytyrosol and tyrosol, the extraction efficiency is higher than that of the methanol-water (8:2, v:v) group.

[0036] The method for extracting olive oil polyphenols of the present invention has the following technical advantages:

[0037] 1) The method for extracting olive oil polyphenol components established in the present invention can extract polyphenol compounds from the complex olive oil system and has the characteristics of simple, rapid and pollution-free operation.

[0038] 2) The present invention can be used to quickly extract olive oil polyphenol compounds, providing a basis for further research on their analysis and application.

[0039] 3) The detection method established by the present invention is a dual-detector multi-channel mode, which can achieve the requirements of interference-free, rapid and synchronous determination of multiple components.

[0040] The present invention prepares a deep eutectic solvent using choline chloride and other solvents as hydrogen bond acceptors and water as a hydrogen bond donor. The deep eutectic solvent is combined with ultrasound-assisted liquid-liquid microextraction to achieve a pretreatment process that integrates separation, purification, and enrichment of polyphenol components in olive oil. Ultra-high performance liquid chromatography is then used to qualitatively and quantitatively analyze the olive oil polyphenols. Currently, no methods for extracting olive oil polyphenols based on water-based deep eutectic solvent-liquid microextraction have been reported. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0042] Figure 1 is the chromatogram of the polyphenol compound standard;

[0043] Figure 1 middle:

[0044] 1. Hydroxytyrosol; 2. Tyrosol; 3. Vanillic acid; 4. Caffeic acid; 5. Vanillin; 6. p-Coumaric acid; 7. Ferulic acid; 8. Resveratrol; 9. Oleuropein; 10. Luteolin; 11. Pinoresinol; 12. Apigenin. DETAILED DESCRIPTION

[0045] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0046] Example 1: A green extraction method for polyphenol components (polyphenol compounds) in olive oil, comprising the following steps:

[0047] 1) At room temperature, choline chloride and water were mixed in a molar ratio of 1:3 and sonicated at 50 kHz for 10 minutes until a clear and homogeneous solution was formed to prepare a water-based deep eutectic solvent.

[0048] 2) Extraction:

[0049] 2.1) Accurately weigh 1.5 g (accurate to 0.001 g) of virgin olive oil sample into a 10 ml plastic centrifuge tube, add 1 ml of n-hexane, vortex (at 2500 rpm) for about 1 min to mix thoroughly, then quickly add 300 μl of a water-based deep eutectic solvent, vortex (at 2500 rpm) for 2 min, sonicate at 50 kHz for 15 min at room temperature, and then centrifuge at 10,000 rpm for 10 min. After centrifugation, the mixture is separated into an upper oil layer and a lower deep eutectic solvent layer, and the lower layer is used as the extract.

[0050] 2.2) Repeat step 2.1) above using the upper oil layer obtained by centrifugation instead of the above 1.5 g virgin olive oil sample, repeating the extraction 2 times, i.e., repeating the extraction 3 times in total;

[0051] The deep eutectic solvent layers obtained from the three extractions were combined to obtain a combined extract.

[0052] The virgin olive oil used in this invention is commercially available. Because virgin olive oil contains very low levels of certain polyphenolic compounds, a certain amount of low-content polyphenolic standard was added to the virgin olive oil to facilitate comparison of the extraction methods. The specific polyphenolic compounds and their addition amounts are: vanillic acid: 4 μg / g; caffeic acid: 12.5 μg / g; vanillin: 12.5 μg / g; p-coumaric acid: 12.5 μg / g; ferulic acid: 12.5 μg / g; resveratrol: 12.5 μg / g; oleuropein: 25 μg / g; luteolin: 25 μg / g; pinoresinol: 2.5 μg / g; and apigenin: 5 μg / g. For example, 4 μg of vanillic acid was added to every gram of virgin olive oil.

[0053] The following Experiments 1 and 2 were carried out using the extra virgin olive oil to which the polyphenolic compounds were added.

[0054] Experiment 1

[0055] 1) Setting up the experimental and control groups:

[0056] The virgin olive oil to which polyphenols had been added was used as the test sample. 100 μl of the combined extract obtained was diluted 10-fold with an acetonitrile-water mixture (3:7, v / v) according to the method described in Example 1, i.e., 900 μl of the acetonitrile-water mixture was added. The extract was then filtered through a 0.22 μm organic filter membrane to form the experimental group. Ultra-high performance liquid chromatography was then performed.

[0057] Using the same virgin olive oil supplemented with polyphenols as described above, the olive oil polyphenol extraction method described in COI / T.20 / Doc No. 29-2009 was used as the test sample, using 80% methanol as the extraction solution. Specifically, the following method was used: the "water-based deep eutectic solvent" in Example 1 was replaced with "80% (volume %) methanol" in a volume of 3.75 mL. The extract was vortexed (at 2500 rpm) for 2 minutes, sonicated at 50 kHz for 15 minutes at room temperature, and then centrifuged at 10,000 rpm for 10 minutes. After centrifugation, the extract separated into a lower oil layer and an upper methanol layer. The upper methanol layer was used as the control extract. 1875 μl of the control extract was nitrogen-dried, reconstituted with 1 mL of a 3:7, v / v acetonitrile-water mixture, filtered through a 0.22 μm organic filter membrane, and analyzed by ultra-high performance liquid chromatography.

[0058] The extraction efficiency of the method was evaluated by the UPLC peak area ratio of the corresponding phenolic compounds in the experimental group and the control group. When the ratio was greater than 1, it indicated that the extraction efficiency of the method of the present invention was superior to that of the traditional method.

[0059] 2) Ultra-performance liquid chromatography (UPLC) conditions:

[0060] ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm); column temperature 30°C; injection volume 10 μl; flow rate 0.3 mL min -1 Mobile phase: 0.1% formic acid water (A)-acetonitrile (B); Elution gradient: 0-4 min, 5%-5% B; 4-8 min, 5%-10% B; 8-10 min, 10%-15% B; 10-14 min, 15%-20% B; 14-16 min, 20%-30% B; 16-20 min, 30%-40% B; 20-21 min, 40%-70% B; 21-25 min, 70%-90% B; 25-26 min, 90%-100% B; 26-30 min, 100%-100% B; 30-31 min, 100%-5% B; 31-35 min, 5%-5% B.

[0061] UV detection wavelengths are 220nm and 340nm, FLD excitation wavelength Em 278nm, emission wavelength Ex 340nm. See the standard sample chromatogram for details. Figure 1 .

[0062] The experimental group was subjected to ultra-performance liquid chromatography (UPLC), and the directly obtained data are shown in Table 1 , which are the peak areas of the compounds described in “Example 1”;

[0063] The control group was subjected to ultra-performance liquid chromatography (UPLC), and the directly obtained data are shown in Table 1, which is the peak area of ​​the compound described in "80% methanol".

[0064] The “extraction efficiency” in Table 1 was calculated according to formula (1).

[0065]

[0066] A s is the peak area of ​​polyphenol compounds in the experimental group, A c is the peak area of ​​the corresponding polyphenolic compound in the control group.

[0067] The present invention was used to extract 12 polyphenolic compounds from olive oil samples and assayed using UPLC. The results are shown in Table 1. Compared with a control group using 80% methanol as the extraction solution, the extraction efficiency of the 12 polyphenols in olive oil prepared with a deep eutectic solvent prepared with choline chloride-water (1:3 molar ratio) ranged from 0.98 to 1.71. With the exception of vanillin, the extraction efficiencies of the other 11 polyphenolic compounds were all higher than those of the conventional control group (80% methanol).

[0068] Table 1 Extraction efficiency of 12 polyphenol components in olive oil samples

[0069]

[0070]

[0071] Experiment 2

[0072] The 1:3 molar ratio of choline chloride to water in step 1) of Example 1 was changed to choline chloride to water 1:2, 1:5, betaine to water 1:5, lactic acid to water 1:5, citric acid to water 1:10, urea to water 1:5, and tetramethylammonium bromide to water 1:10, respectively, to prepare a water-based deep eutectic solvent, and the resulting mixture was used as the extractant; in step 2) of Example 1, the material-liquid ratio was changed to 1.5 g / 200 μL (i.e., the amount of virgin olive oil after the addition of polyphenol compounds remained unchanged, still 1.5 g, and the amount of water-based deep eutectic solvent was changed to 200 μL), the volume of n-hexane was changed to 3 mL, the ultrasonic time was 15 min, and the number of extractions was 1. The rest was the same as in Example 1.

[0073] Using virgin olive oil supplemented with polyphenols as the test sample, the extract obtained above was tested according to the following experimental method: The ratio of acetonitrile to water in the dilution solution in Experiment 1 was changed to 2:8, and all other conditions were the same as in Experiment 1. The control group was set up in the same manner as in Experiment 1.

[0074] The final results are shown in Table 2. Taking 80% methanol extraction as the control group, the extraction efficiency of the 12 polyphenolic compounds according to the method of the present invention was higher than that of the choline chloride-water 1:2 group (experimental group 6); compared with the choline chloride-water 1:5 group (experimental group 7), the betaine-water 1:5 group (experimental group 1), the lactic acid-water 1:5 group (experimental group 2), the citric acid-water 1:10 group (experimental group 3), the urea-water 1:5 group (experimental group 4), and the tetramethylammonium bromide-water 1:10 group (experimental group 5), except for hydroxytyrosol and tyrosol, the extraction efficiency of the polyphenolic compounds of the present invention was significantly higher than or equivalent to the above experimental groups.

[0075] Table 2 Extraction efficiency of polyphenols in olive oil by different deep eutectic solvents

[0076]

[0077]

[0078]

[0079] Comparative Example 2-1: The volume of n-hexane in Example 1 was changed from 1 mL to 2 mL; the rest was the same as Example 1.

[0080] Comparative Example 2-2: The volume of n-hexane in Example 1 was changed from 1 mL to 3 mL; the rest was the same as Example 1.

[0081] Comparative Example 3-1: The material-liquid ratio in Example 1 was changed from 1.5 g / 300 μL to 1.5 g / 200 μL (ie, the water-based deep eutectic solvent was 200 μL), and the rest was the same as Example 1.

[0082] Comparative Example 3-2: The material-liquid ratio in Example 1 was changed from 1.5 g / 300 μL to 1.5 g / 400 μL, and the rest was the same as Example 1.

[0083] Comparative Example 4-1: The vortex time after adding the water-based deep eutectic solvent in Example 1 was changed from 2 min to 1 min, and the rest was the same as Example 1.

[0084] Comparative Example 5-1: The ultrasonic time in Example 1 was changed from 15 min to 10 min, and the rest was the same as Example 1.

[0085] Comparative Example 5-2: The ultrasonic time in Example 1 was changed from 15 min to 20 min, and the rest was the same as Example 1.

[0086] The virgin olive oil to which polyphenol compounds were added was used as the test sample, and the combined extracts obtained according to the methods described in Comparative Examples 2-1 to 5-2 were tested according to the method described in Experiment 1.

[0087] Comparative Example 6-1: Extra virgin olive oil to which polyphenol compounds were added was used as the test sample. The dilution solution in Experiment 1 was changed from "acetonitrile:water = 3:7" to "acetonitrile:water = 2:8". The rest was the same as Experiment 1.

[0088] Comparative Example 6-2: Extra virgin olive oil with added polyphenols was used as the test sample. The dilution solution in Experiment 1 was changed from "acetonitrile:water = 3:7" to "acetonitrile:water = 5:5". The rest was the same as Experiment 1.

[0089] The comparison of the above test results is shown in Table 3. The control group is the same as Experiment 1. The extraction efficiency of polyphenol compounds in Example 1 is significantly higher than that of the above comparative examples, except for oleuropein (Comparative Examples 2-1, 2-2, and 3-1).

[0090] Table 3 Extraction rate of polyphenol compounds in olive oil by different extraction methods

[0091]

[0092]

[0093] Extraction efficiency

[0094]

[0095]

[0096] Using virgin olive oil as the test sample, the combined extracts obtained according to the method described in Example 1 were subjected to the Folin phenol colorimetric method to determine the total polyphenol content.

[0097] Comparative Example 7-1: Using virgin olive oil as the test substance, the water-based deep eutectic solvent of Example 1 was replaced with choline chloride-xylitol (2:1 ratio), with all other modifications being identical to Example 1. Due to the high viscosity of this solvent, direct HPLC analysis was not possible. Therefore, the total polyphenol content of the combined extracts was determined using the folin-phenol colorimetric method.

[0098] Comparative Example 7-2: Using virgin olive oil as the test substance, the "water-based deep eutectic solvent" of Example 1 was replaced with "choline chloride-1,2-propylene glycol = 1:1," with all other modifications remaining the same as in Example 1. Due to the high viscosity of this solvent, direct HPLC analysis was not possible. Therefore, the total polyphenol content of the combined extracts was determined using the folin-phenol colorimetric method.

[0099] Two replicates were set for each group, and the results are shown in Table 4. It can be seen from the results that the total polyphenol content extracted in Example 1 is significantly higher than that in the comparative example.

[0100] Table 4 Total polyphenol content of olive oil by different extraction methods

[0101]

[0102] Experiment 3: Using refined olive oil with an extremely low polyphenol content as a matrix, a quantitative amount of 12 polyphenol standards were added thereto, and extraction was performed according to the method described in Example 1. The combined extract was tested according to the method described in Experiment 1. The results are shown in Table 5. The 12 polyphenol compounds in olive oil were extracted (extraction) using the method of Example 1, and the recovery rate was 78.3%-115.2%, indicating that the present invention has a good extraction efficiency for the 12 polyphenol compounds in olive oil.

[0103] Table 5 Recovery rates of 12 polyphenol compounds in olive oil

[0104]

[0105] Comparison of the results shows that the technical solution of the present invention using a water-based deep eutectic solvent as an extractant can improve the extraction rate of olive oil polyphenol compounds.

[0106] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A method for detecting the content of polyphenols in olive oil, characterized in that: The method for extracting polyphenolic compounds from olive oil comprises the following steps: 1) Preparation of water-based deep eutectic solvent: A hydrogen bond acceptor reagent is mixed with water at room temperature and ultrasonicated until a clear and homogeneous solution is formed, thereby preparing a water-based deep eutectic solvent; The hydrogen bond acceptor reagents are choline chloride, betaine, lactic acid, citric acid, urea, or tetramethylammonium bromide; 2) Extraction: 2.1) Weigh 1.5 g of olive oil into a centrifuge tube, add 1–2 ml of n-hexane, mix well, then add 200–400 μl of a water-based deep eutectic solvent. Vortex for 1–3 min, sonicate at 50 kHz for 10–20 min at room temperature, and then centrifuge at 10,000 ± 1,000 rpm for 10 ± 2 min. After centrifugation, the mixture will separate into an upper oil layer and a lower deep eutectic solvent layer. 2.2) taking the deep eutectic solvent layer as an extract; the extract contains polyphenol compounds; The polyphenolic compounds are hydroxytyrosol, tyrosol, vanillic acid, caffeic acid, vanillin, p-coumaric acid, ferulic acid, resveratrol, oleuropein, luteolin, pinoresinol, and apigenin; The method for determining the content of polyphenols in olive oil is: Take 100 μl of the extract, dilute it 10 times with acetonitrile-water mixture as a reconstitution solution, pass it through a 0.22 μm organic filter membrane, and then perform ultra-high performance liquid chromatography for detection; In the acetonitrile-water mixture, the volume ratio of acetonitrile:water = 3:7; Ultra-high performance liquid chromatography conditions: ACQUITY UPLC BEH C18 column, 2.1 × 100 mm, 1.7 μm; column temperature, 30°C; injection volume, 10 μl; flow rate, 0.3 mL min -1 Mobile phase: A: 0.1% formic acid in water, B: acetonitrile; elution gradient: 0-4 min, 5%-5% B; 4-8 min, 5%-10% B; 8-10 min, 10%-15% B; 10-14 min, 15%-20% B; 14-16 min, 20%-30% B; 16-20 min, 30%-40% B; 20-21 min, 40%-70% B; 21-25 min, 70%-90% B; 25-26 min, 90%-100% B; 26-30 min, 100%-100% B; 30-31 min, 100%-5% B; 31-35 min, 5%-5% B; UV detection wavelengths were 220 nm and 340 nm, FLD excitation wavelength Em 278 nm, emission wavelength Ex 340 nm.

2. The method for detecting the content of polyphenol compounds in olive oil according to claim 1, wherein: Repeat step 2.1 above using the upper oil layer obtained after centrifugation instead of 1.5 g of olive oil) for 1 to 3 times, and combine the obtained deep eutectic solvent layers to obtain a combined extract, which contains polyphenol compounds.

3. The method for detecting the content of polyphenol compounds in olive oil according to claim 2, wherein: The olive oil is extra virgin olive oil.

4. The method for detecting the content of polyphenols in olive oil according to any one of claims 1 to 3, wherein: When the hydrogen bond acceptor reagent is choline chloride, the molar ratio of choline chloride to water is 1:2~5; When the hydrogen bond acceptor reagent is betaine, the molar ratio of betaine to water is 1:5; When the hydrogen bond acceptor reagent is lactic acid, the molar ratio of lactic acid to water is 1:5; When the hydrogen bond acceptor reagent is citric acid, the molar ratio of citric acid to water is 1:10; When the hydrogen bond acceptor reagent is urea, the molar ratio of urea to water is 1:5; When the hydrogen bond acceptor reagent is tetramethylammonium bromide, the molar ratio of tetramethylammonium bromide to water is 1:

10.

5. The method for detecting the content of polyphenol compounds in olive oil according to claim 4, wherein: When the hydrogen bond acceptor reagent is choline chloride, the molar ratio of choline chloride to water is 1:

3.

6. The method for detecting the content of polyphenol compounds in olive oil according to claim 5, wherein: In step 2), the amount of the water-based deep eutectic solvent used is 300 μL.

7. The method for detecting the content of polyphenol compounds in olive oil according to claim 6, wherein: In step 2), 1 mL of n-hexane and 300 μL of a water-based deep eutectic solvent were added, and the vortex time was 2 min and the ultrasonic time was 15 min.

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