A method for ultrasonic-assisted deep eutectic solvent extraction of tomatidine

Abstract

The application discloses a method for extracting solanigrin by using ultrasonic-assisted deep eutectic solvent, and belongs to the technical field of active substance extraction. The method comprises the following steps: mixing the deep eutectic solvent with tomatoes, and placing the mixture in a water bath for ultrasonic extraction. The method uses the deep eutectic solvent as a solvent, and combines the ultrasonic-assisted technology to extract the solanigrin, so that the obtained solanigrin has a high extraction amount. The method for extracting the solanigrin by using the ultrasonic-assisted deep eutectic solvent uses a green solvent which is free of pollution, and overcomes the defects of traditional organic solvents, such as toxicity, harm and volatility. While the extraction efficiency is ensured, the safety of the product is also ensured.

Description

Technical Field

[0001] This invention belongs to the field of active substance extraction technology, and in particular relates to a method for extracting tomato glycosides using an ultrasound-assisted eutectic solvent. Background Technology

[0002] Tomatine is a glycosidic alkaloid found in nightshade plants such as eggplant, potato, and tomato. It contains D-xylose, D-galactose, and two glucose molecules and is a bioactive substance with anti-inflammatory, antibacterial, anticancer, cholesterol-regulating, and neuroprotective effects. Research on the extraction of tomatine is of great significance for the development and utilization of bioactive substances in tomato plants.

[0003] Currently, the extraction of tomatine from tomato plants still uses solvent extraction, employing organic solvents such as methanol, chloroform, acetic acid, and tetrahydrofuran. This method has drawbacks such as low safety, easy solvent volatility, and environmental harm, and does not meet the requirements of green chemistry development.

[0004] Deep eutectic solvents (DES) are low-melting-point mixtures formed by hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) through hydrogen bonding, exhibiting physicochemical properties similar to ionic liquids. As a novel green solvent, DES possesses advantages such as low toxicity, low flash point, low volatility, and environmental friendliness, making it suitable as a green extraction solvent for many natural active ingredients. Currently, it has some applications in the extraction of polyphenols, polysaccharides, flavonoids, and other active substances, but there is no research on the extraction of tomatine from tomato plants. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for extracting tomato glycosides using an ultrasound-assisted eutectic solvent. This method offers high extraction efficiency and is more environmentally friendly and less volatile compared to organic solvents.

[0006] To achieve the above objectives, the present invention provides a method for extracting tomato glycosides using an ultrasound-assisted eutectic solvent, comprising the following steps:

[0007] The eutectic solvent was mixed with tomatoes and placed in a water bath for ultrasonic extraction.

[0008] The eutectic solvent is selected from one or more of the following solvents: choline chloride-levulinic acid, choline chloride-fructose, choline chloride-ethylene glycol, choline chloride-glycerol, choline chloride-urea, choline chloride-acetamide, betaine-levulinic acid, and proline-levulinic acid.

[0009] Furthermore, the eutectic solvent is choline chloride-acetylpropionic acid.

[0010] Furthermore, the molar ratio of choline chloride to levulinic acid is 1:1 to 1:5.

[0011] Furthermore, the molar ratio of choline chloride to acetylpropionic acid is 1:5.

[0012] Furthermore, the water content of the eutectic solvent is 20% to 60%.

[0013] Furthermore, the ratio of the tomato to the eutectic solvent is 1:(30-70)g / mL.

[0014] Furthermore, the temperature for ultrasonic extraction is 30℃~70℃.

[0015] Furthermore, the ultrasonic extraction time is 10 min to 50 min.

[0016] Furthermore, the power of the ultrasonic extraction is 180W.

[0017] Further, after ultrasonic extraction, the solution is filtered and the clear liquid is collected.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects:

[0019] This invention uses a eutectic solvent as the solvent and combines ultrasonic-assisted technology to extract tomato glycosides, resulting in a high extraction yield of tomato glycosides.

[0020] The ultrasonic-assisted eutectic solvent extraction method for tomato glycosides proposed in this invention uses a green and pollution-free solvent, overcoming the disadvantages of traditional organic solvents that are toxic, harmful, and volatile. This method ensures both extraction efficiency and product safety. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 A comparison chart of different extraction solvents;

[0023] Figure 2 This is a graph showing the effect of the material-to-liquid ratio on the extraction effect;

[0024] Figure 3 This is a graph showing the effect of ultrasonic temperature on the extraction effect;

[0025] Figure 4 This is a graph showing the effect of ultrasound time on the extraction effect;

[0026] Figure 5 The graph shows the effect of DES moisture content on extraction efficiency.

[0027] Figure 6 The graph shows the effect of the molar ratio of choline chloride and levulinic acid on the extraction efficiency.

[0028] Figures 7-12 The experimental plot was optimized for response surface methodology; among which, Figure 7 Moisture content - molar ratio; Figure 8 : Feed-to-liquid ratio - molar ratio; Figure 9 Temperature-molar ratio; Figure 10 : Material-to-liquid ratio - water content; Figure 11 Temperature-moisture content; Figure 12 Temperature-to-liquid ratio. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Unless otherwise specified, all instruments, reagents and consumables used in this invention are purchased from the market.

[0035] In the embodiments, the eutectic solvents used were all two-component eutectic mixtures, which were prepared by heating and mixing hydrogen bond acceptors and hydrogen bond donors.

[0036] The preparation method of the eutectic solvent is as follows:

[0037] (1) Weigh out the exact mass of hydrogen bond donors and hydrogen bond acceptors and mix them;

[0038] (2) Stir in a 60°C water bath until a homogeneous and transparent liquid is formed to obtain the basic eutectic solvent.

[0039] (3) Weigh a certain volume of the basic eutectic solvent obtained in step (2), add a certain volume of deionized water, and obtain a eutectic solvent with a certain water content.

[0040] In this embodiment, after the test solution is extracted, the following method is used for detection:

[0041] (1) Purification conditions of the test solution

[0042] Add 5 mL of the test solution to a C18 solid-phase extraction column that has been activated with 5 mL of methanol and 5 mL of aqueous solution. Control the flow rate at 1 mL / min. After loading the sample, rinse with 5 mL of water and elute with 5 mL of methanol solution. Make up the volume of the eluent to 5 mL, filter it through a 0.22 μm organic filter membrane, and then analyze it.

[0043] (2) Detection conditions

[0044] The detection method was high performance liquid chromatography-tandem mass spectrometry.

[0045] Mass spectrometry conditions: ion source optimized; dry gas temperature 350℃; flow rate 10 L / min; nebulizer gas pressure 35 psi; sheath gas temperature 300℃; sheath gas flow rate 11 L / min; capillary voltage 4.0 kV; nozzle voltage 500 V. Multiple reaction monitoring parameters are shown in Table 1.

[0046] Table 1. Parameters of Multiple Reaction Monitoring for Tomatoside

[0047]

[0048] Liquid chromatography conditions:

[0049] Column: Poroshell 120EC-C18 (3.0mm×100mm, 2.7μm)

[0050] Column temperature: 30℃; flow rate: 0.3 mL / min; injection volume: 2 μL; mobile phase: A is 5 mmol / L ammonium formate solution; B is acetonitrile; gradient elution program: 68% A for 2 min, 2–6 min to 20% A, 6–10 min to 20% A, 10–11 min to 68% A, 11–18 min to 68% A.

[0051] Example 1: Selection of Eutectic Solvent

[0052] This example demonstrates a screening experiment for the extraction of tomatine using different eutectic solvents with ultrasound assistance:

[0053] Eight different eutectic solvents were selected, as shown in Table 2.

[0054] Table 2 Different DES Compositions

[0055]

[0056] Eight different DES, methanol, and ethanol were prepared and mixed with tomato plant powder at a material-to-liquid ratio of 1:50 g / ml. Tomato glycosides were extracted under ultrasonic conditions of 50℃, 180W, and 30 min. After ultrasonic extraction, the mixture was centrifuged, and the supernatant was diluted 5 times before analysis.

[0057] Example 2: Optimization of the feed-liquid ratio

[0058] This embodiment provides a single-factor optimization experiment for ultrasound-assisted DES1 extraction of tomato glycosides.

[0059] The effects of controlling the ratio of choline chloride to levulinic acid to 1:2 (mol:mol), water content of 20%, ultrasonic temperature of 50℃, ultrasonic power of 180W, and ultrasonic time of 30min on the extraction content of tomatine from tomato plant powder were investigated at material-to-liquid ratios of 1:30, 1:40, 1:50, 1:60, and 1:70 (g / mL).

[0060] The results are as follows Figure 2 As shown, with the increase of the solid-liquid ratio, the extraction amount of tomatine first increases and then stabilizes, reaching its maximum value at 1:60 g / mL. Therefore, three levels of 1:50, 1:60, and 1:70 g / mL were selected for subsequent response surface optimization experiments.

[0061] Example 3: Ultrasonic Temperature Optimization

[0062] This embodiment provides a single-factor optimization experiment for ultrasound-assisted DES1 extraction of tomato glycosides.

[0063] The effects of choline chloride:levulinic acid = 1:2 (mol:mol), water content 20%, material-to-liquid ratio 1:50 (g / mL), ultrasonic power 180W, and ultrasonic time 30min on the extraction content of tomatine from tomato plant powder were investigated.

[0064] The results are as follows Figure 3 As shown, the extractable content of tomatine first increases with increasing temperature, reaching a maximum at 40℃ and then stabilizing. Increasing temperature is beneficial for improving mass transfer efficiency and extraction efficiency, but excessively high temperatures can also damage the material structure or cause unnecessary resource waste. Therefore, three levels—30℃, 40℃, and 50℃—were selected for subsequent response surface methodology experiments.

[0065] Example 4: Ultrasound Time Optimization

[0066] This embodiment provides a single-factor optimization experiment for ultrasound-assisted DES1 extraction of tomato glycosides.

[0067] The effects of controlling the ratio of choline chloride to levulinic acid to 1:2 (mol:mol), water content to 20%, material-to-liquid ratio to 1:50 (g / mL), ultrasonic temperature to 50℃, and ultrasonic power to 180W on the extraction content of tomatine from tomato plant powder were investigated.

[0068] The results are as follows Figure 4 As shown, with the increase of ultrasonic time, the content of tomatine extracted first increased and then tended to stabilize. In order to ensure extraction efficiency, the extraction time was selected to be 30 min in subsequent experiments.

[0069] Example 5: Molar ratio optimization

[0070] This embodiment provides a single-factor optimization experiment for ultrasound-assisted DES1 extraction of tomato glycosides.

[0071] The effects of ultrasonic time of 30 min, water content of 20%, material-to-liquid ratio of 1:50 (g / mL), ultrasonic power of 180 W, and ultrasonic temperature of 50 ℃ on the extraction content of tomatine from tomato plant powder were investigated.

[0072] The results are as follows Figure 5 As shown, the extraction content of tomatine changes with the change of molar ratio. The extraction content of tomatine reaches the maximum when the molar ratio of choline chloride to levulinic acid is 1:2. Further increasing the molar ratio of levulinic acid does not significantly change the extraction content of tomatine. Therefore, subsequent experiments selected molar ratios of 1:1, 1:2, and 1:3 for response surface optimization.

[0073] Example 6: Moisture Content Optimization

[0074] This embodiment provides a single-factor optimization experiment for ultrasound-assisted DES1 extraction of tomato glycosides.

[0075] The effects of choline chloride:levulinic acid = 1:2 (mol:mol), ultrasonic power 180W, ultrasonic time 30min, material-liquid ratio 1:50 (g / mL), and ultrasonic temperature 50℃ on the extraction content of tomatine from tomato plant powder were investigated.

[0076] Changes in water content affect the viscosity of the DES system, as shown in the following results. Figure 6 As shown, the extractable content of tomatine first increases and then decreases as the water content increases from 0 to 80%. The reason may be that an appropriate water content will reduce the viscosity of the solvent, which is beneficial to the extraction process. However, excessive water content will disrupt the hydrogen bond interactions in DES, thereby reducing the extraction efficiency. Therefore, response surface methodology experiments were conducted with water contents of 40%, 60%, and 80%.

[0077] Example 7: Response Surface Extraction Optimization Experiment

[0078] This embodiment provides a response surface methodology optimization experiment for ultrasound-assisted DES1 extraction of tomato glycosides.

[0079] Based on the results of the single-factor experiments, the effects of molar ratio, water content, solid-liquid ratio, and ultrasonic temperature on the extraction efficiency of tomatine were further optimized. Response surface methodology (RSM) analysis was performed on the extraction factors of tomatine using Design-Expert 13 software, and the factors and levels of the RSM analysis were obtained, as shown in Table 3.

[0080] Table 3 Factors and levels in response surface analysis

[0081]

[0082] The results are shown in Table 4. The quadratic polynomial regression equations for tomato glycoside extraction amount on the encoded independent variables A (molar ratio), B (moisture content), C (solid-liquid ratio), and D (ultrasonic temperature) were obtained.

[0083] Y=1494+59.08A-71.75B+58.92C+60.58D+2.75AB-25.25AC+9.75AD-7.75BC+17.75BD-26.25CD-53.04A 2 -62.29B 2 -30.54C 2 -29.29D 2 .

[0084] Table 4. Response surface experimental design and its response values

[0085]

[0086]

[0087] The results of the variance analysis of the above regression model are shown in Table 5, and the results of the fitting analysis are shown in Table 6.

[0088] Table 5 Results of response surface variance analysis

[0089]

[0090]

[0091] Table 6. Analysis of Fitting Results

[0092]

[0093] The F-test showed that the regression model had a high F-value (F = 32.49) and a low P-value (P < 0.0001), indicating that the model was significant. The lack-of-fit term in the equation was not significant (P = 0.8394), and R0 was [not specified]. 2 =0.97, indicating that the established quadratic regression model can be used to analyze and predict the process conditions for ultrasound-assisted DES extraction of tomatine. The results of coefficient evaluation and significance testing of the regression model (Table 6) show that all linear and quadratic terms have a significant impact on the extraction yield of tomatine. The interaction terms AC and CD have low p-values ​​(p < 0.05), indicating that the molar ratio and solid-liquid ratio, as well as the solid-liquid ratio and temperature, have interactive effects on the extraction efficiency of tomatine.

[0094] Based on the established model, parameter optimization analysis was conducted, and the optimal conditions for extracting tomatine from tomato plant powder were determined to be: a molar ratio of choline chloride to levulinic acid of 1:2.492; a water content of 50.18%; a solid-liquid ratio of 1:65.06 g / mL; a temperature of 47.4℃; and an ultrasonic power of 180W. The predicted maximum extraction yield was 1563 mg / kg.

[0095] Example 8

[0096] To verify the above response surface methodology prediction results and for ease of practical operation, this embodiment optimized the extraction process as follows: molar ratio 2:5; water content 50%; material-to-liquid ratio 1:65 g / mL; temperature 47℃. Under the above conditions, ultrasonic-assisted extraction of tomatine from tomato plant powder was performed (ultrasonic power 180W, ultrasonic time 30min). The extracted tomatine content was 1551 mg / kg, which is basically consistent with the predicted value.

[0097] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for ultrasonic-assisted deep eutectic solvent extraction of tomatidine, characterized in that, Includes the following steps: The eutectic solvent was mixed with tomatoes and placed in a water bath for ultrasonic extraction. The eutectic solvent is choline chloride-levulinic acid; The molar ratio of choline chloride to levulinic acid is 2:5; The water content of the eutectic solvent is 50%; The ratio of the tomato to the eutectic solvent is 1:65 g / mL; The ultrasonic extraction temperature was 47°C; The ultrasonic extraction time is 30 minutes; The power of the ultrasonic extraction is 180W; After ultrasonic extraction, the solution is filtered and the clear liquid is collected.

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