Method for extracting tomacoside by using ultrasonic-assisted deep-eutectic solvent

Abstract

The invention discloses a method for extracting tocoside by using an ultrasonic-assisted deep-eutectic solvent, which belongs to the technical field of active substance extraction and comprises the following steps: mixing the deep-eutectic solvent with tomatoes, putting the mixture into a water bath, and carrying out ultrasonic extraction. According to the method, the eutecticevaporate solvent is used as a solvent, the ultrasonic-assisted technology is combined for extracting the tocoside, and the obtained tocoside has a relatively high extraction amount. According to the method for extracting the tocoside through the ultrasonic-assisted deep-eutectic solvent, the used solvent is green and pollution-free, the defects that a traditional organic solvent is toxic, harmful and volatile are overcome, and the safety of a product is guaranteed while the extraction efficiency is guaranteed.

Description

Technical Field

[0001] The present invention belongs to the technical field of active substance extraction, and particularly relates to a method for extracting tomatidine by ultrasonic-assisted deep eutectic solvents. Background Art

[0002] Tomatidine is a glycoside alkaloid containing D-xylose, D-galactose and two molecules of glucose, which exists in solanaceous plants such as eggplant, potato and tomato. It is a bioactive substance with anti-inflammatory, antibacterial, anti-cancer, cholesterol-regulating, neuroprotective and other effects. Studying the extraction of tomatidine is of great significance for the development and utilization of bioactive substances in tomato plants.

[0003] At present, the extraction of tomatidine from tomato plants still uses the solvent extraction method, and the solvents used are organic solvents such as methanol, chloroform, acetic acid, and tetrahydrofuran. This method has disadvantages such as low safety, easy volatility of the solvent, and harm to the environment, which does not meet the development requirements of green chemistry.

[0004] Deep Eutectic Solvents (DES) is a low-melting mixture formed by hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA) through hydrogen bonding, and has similar physical and chemical properties to ionic liquids. As a new type of green solvent, deep eutectic solvents have the advantages of low toxicity, low flash point, low volatility, and environmental friendliness, and can be used as green extraction solvents for many natural active ingredients. At present, it has certain applications in the extraction of active substances such as polyphenols, polysaccharides, and flavonoids, but there is no research on the extraction of tomatidine from tomato plants. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes a method for extracting tomatidine by ultrasonic-assisted deep eutectic solvents. This method has high extraction efficiency and is more green, environmentally friendly and less volatile compared with organic solvents.

[0006] To achieve the above object, the present invention provides a method for extracting tomatidine by ultrasonic-assisted deep eutectic solvents, comprising the following steps:

[0007] Mix the deep eutectic solvent with tomatoes, place it in a water bath, and extract by ultrasound;

[0008] The deep 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, proline-levulinic acid.

[0009] Further, the deep eutectic solvent is choline chloride-levulinic 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 levulinic acid is 1:5.

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

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

[0014] Furthermore, the temperature of the ultrasonic extraction is 30°C to 70°C.

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

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

[0017] Furthermore, after the ultrasonic extraction, filtration is carried out and the clear liquid is taken.

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

[0019] The present invention uses a deep eutectic solvent as the solvent and combines ultrasonic-assisted technology to extract tomatoside, and the obtained tomatoside has a high extraction amount.

[0020] The method for extracting tomatoside by ultrasonic-assisted deep eutectic solvent proposed by the present invention uses a green and pollution-free solvent, overcomes the disadvantages of traditional organic solvents being toxic, harmful and volatile, and ensures the safety of the product while guaranteeing the extraction efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use 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, other drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 It is a comparison diagram of different extraction solvents;

[0023] Figure 2 It is a diagram showing the influence of the ratio of feed liquid on the extraction effect;

[0024] Figure 3 It is a diagram showing the influence of ultrasonic temperature on the extraction effect;

[0025] Figure 4 It is a diagram showing the influence of ultrasonic time on the extraction effect;

[0026] Figure 5 It is a diagram showing the influence of the water content of DES on the extraction effect;

[0027] Figure 6 It is a diagram showing the influence of the molar ratio of choline chloride to levulinic acid on the extraction effect;

[0028] Figures 7 - 12 It is a diagram of the response surface optimization experiment; among them, Figure 7 : water content - molar ratio; Figure 8 : solid - liquid ratio - molar ratio; Figure 9 : temperature - molar ratio; Figure 10 : solid - liquid ratio - water content; Figure 11 : temperature - water content; Figure 12 : temperature - solid - liquid ratio. Detailed implementation manners

[0029] Now, various exemplary implementation manners of the present invention will 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, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0033] Regarding the terms "comprising", "including", "having", "containing", etc. used in this text, they are all open-ended terms, meaning including but not limited to.

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

[0035] In the examples, the eutectic solvents used are all binary eutectic mixtures, which are made by heating and mixing a hydrogen bond acceptor and a hydrogen bond donor.

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

[0037] (1) Weigh accurately the hydrogen bond donor and the hydrogen bond acceptor 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 the basic eutectic solvent prepared in step (2) by volume, add a certain volume of deionized water to obtain a eutectic solvent with a certain water content.

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

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

[0042] Take 5 mL of the test solution and add it to a C18 solid-phase extraction column that has been activated with 5 mL of methanol and 5 mL of aqueous solution successively. Control the flow rate at 1 mL / min. After loading the sample, wash it with 5 mL of water and elute it with 5 mL of methanol solution. The eluate is fixed to 5 mL, filtered through a 0.22 μm organic filter membrane, and then analyzed on the machine.

[0043] (2) Detection conditions

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

[0045] Mass spectrometry conditions: After optimization, the drying gas temperature of the ion source is 350°C; flow rate: 10 L / min; nebulizing gas pressure: 35 psi; sheath gas temperature: 300°C; sheath gas flow rate: 11 L / min; capillary voltage: 4.0 kV; nozzle voltage: 500 V. The multi-reaction monitoring parameters are shown in Table 1.

[0046] Table 1 Multi-reaction monitoring parameters of tomatoside

[0047]

[0048] Liquid chromatography conditions:

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

[0050] Column temperature: 30 °C; Flow rate: 0.3 mL / min; Injection volume: 2 μL; Mobile phase: A is 5 mmol / L ammonium formate solution; B is acetonitrile; The gradient elution program is 68% A held for 2 min, changed to 20% A from 2 - 6 min, held at 20% A from 6 - 10 min, changed from 20% A to 68% A from 10 - 11 min, and 68% A from 11 - 18 min.

[0051] Example 1 Selection of Deep Eutectic Solvent

[0052] In this example, a screening test for extracting tomatoside with different deep eutectic solvents assisted by ultrasound was carried out:

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

[0054] Table 2 Different DES Compositions

[0055]

[0056] The eight prepared different DESs, methanol, and ethanol were mixed with tomato plant powder at a solid - liquid ratio of 1:50 g / ml, and tomatoside was extracted under the conditions of ultrasonic temperature of 50 °C, ultrasonic power of 180 W, and ultrasonic time of 30 min. After ultrasonic extraction, the mixture was centrifuged, and the supernatant was taken and diluted 5 times for measurement.

[0057] Example 2 Optimization of Solid - Liquid Ratio

[0058] This example provides a single - factor optimization experiment for ultrasonic - assisted extraction of tomatoside with DES1.

[0059] Controlling choline chloride: levulinic acid = 1:2 (mol:mol), water content of 20%, ultrasonic temperature of 50 °C, ultrasonic power of 180 W, and ultrasonic time of 30 min, the effects of solid - liquid ratios of 1:30, 1:40, 1:50, 1:60, and 1:70 (g / mL) on the extraction content of tomatoside in tomato plant powder were investigated.

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

[0061] Example 3 Optimization of Ultrasonic Temperature

[0062] This example provides a single - factor optimization experiment for ultrasonic - assisted extraction of tomatoside with DES1.

[0063] Control choline chloride: levulinic acid = 1:2 (mol:mol), water content 20%, solid-liquid ratio 1:50 (g / mL), ultrasonic power 180 W, ultrasonic time 30 min, and investigate the effect on the extraction content of tomatine in tomato plant powder when the ultrasonic temperature is 30, 40, 50, 60, 70 °C respectively.

[0064] The results are as Figure 3 shown. As the temperature increases, the extraction content of tomatine first increases, reaches the maximum value at 40 °C and then tends to be stable. The increase in temperature is beneficial to improving the mass transfer efficiency and extraction efficiency, but too high temperature will also damage the material structure or cause unnecessary waste of resources. Therefore, three levels of 30, 40, and 50 °C are selected for the subsequent response surface optimization experiment.

[0065] Example 4 Optimization of ultrasonic time

[0066] This example provides a single-factor optimization experiment for ultrasonic-assisted extraction of tomatine by DES1.

[0067] Control choline chloride: levulinic acid = 1:2 (mol:mol), water content 20%, solid-liquid ratio 1:50 (g / mL), ultrasonic temperature 50 °C, ultrasonic power 180 W, and investigate the effect on the extraction content of tomatine in tomato plant powder when the ultrasonic time is 10, 20, 30, 40, 50 min respectively.

[0068] The results are as Figure 4 shown. As the ultrasonic time increases, the extraction content of tomatine first increases and then tends to be stable. To ensure the extraction efficiency, the extraction time of 30 min is selected for the subsequent experiment.

[0069] Example 5 Optimization of molar ratio

[0070] This example provides a single-factor optimization experiment for ultrasonic-assisted extraction of tomatine by DES1.

[0071] Control ultrasonic time 30 min, water content 20%, solid-liquid ratio 1:50 (g / mL), ultrasonic power 180 W, ultrasonic temperature 50 °C, and investigate the effect on the extraction content of tomatine in tomato plant powder when the molar ratio of choline chloride: levulinic acid (mol:mol) is 1:1, 1:2, 1:3, 1:4, 1:5 respectively.

[0072] The results are as Figure 5 shown. As the molar ratio changes, the extraction content of tomatine also changes accordingly. The extraction content of tomatine reaches the maximum when the molar ratio of choline chloride and levulinic acid is 1:2. Continuing to increase the molar ratio of levulinic acid has no obvious change in the extraction content of tomatine. Therefore, the molar ratios of 1:1, 1:2, and 1:3 are selected for the response surface optimization in the subsequent experiment.

[0073] Example 6 Optimization of Water Content

[0074] This example provides a single-factor optimization experiment for the extraction of tomatoside by ultrasonic-assisted DES1.

[0075] Controlled choline chloride: levulinic acid = 1:2 (mol:mol), ultrasonic power 180 W, ultrasonic time 30 min, solid-liquid ratio 1:50 (g / mL), ultrasonic temperature 50 °C, and the effects of water contents of 0, 20%, 40%, 60%, and 80% on the extraction content of tomatoside in tomato plant powder were investigated.

[0076] The change in water content will affect the viscosity of the DES system, and the results are as Figure 6 shown. As the water content increases from 0 to 80%, the extraction content of tomatoside first increases and then decreases. The reason may be that appropriate water content will reduce the viscosity of the solvent, which is beneficial to the extraction process, but too high water content will destroy the hydrogen bond interaction in DES and thus reduce the extraction efficiency. Therefore, water contents of 40%, 60%, and 80% were selected for the response surface optimization experiment.

[0077] Example 7 Response Surface Extraction Optimization Experiment

[0078] This example provides a response surface method optimization experiment for the extraction of tomatoside by ultrasonic-assisted DES1.

[0079] According to the results of the single-factor experiment, the effects of molar ratio, water content, solid-liquid ratio, and ultrasonic temperature on the extraction effect of tomatoside were further optimized. Using Design-Expert 13 software, a response surface analysis was performed on the extraction factors of tomatoside to obtain the factor and level table for the response surface analysis, as shown in Table 3.

[0080] Table 3 Factors and Levels for Response Surface Analysis

[0081]

[0082] The results are shown in Table 4, and the quadratic multiple regression equation of the extraction amount of tomatoside with respect to the coded independent variables A molar ratio, B water content, C solid-liquid ratio, and D ultrasonic temperature was 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 shows that the regression model has a relatively high F-value (F = 32.49) and a relatively low P-value (P < 0.0001), indicating that the model is significant. The lack-of-fit term of the equation is not significant (P = 0.8394), and R 2 = 0.97, indicating that the established quadratic regression model can be used to analyze and predict the process conditions for the ultrasonic-assisted DES extraction of tomatoside. The results of the coefficient evaluation and significance test of the regression model (Table 6) show that all linear and quadratic terms have significant effects on the tomatoside extraction yield. Among them, the interaction terms AC and CD have relatively low P-values (P < 0.05), indicating that the molar ratio and solid-liquid ratio, and the solid-liquid ratio and temperature have interactive effects on the extraction effect of tomatoside.

[0094] According to the established model, parameter optimization analysis was carried out, and the optimal conditions for the extraction of tomatoside from tomato plant powder were obtained as follows: the molar ratio of choline chloride to levulinic acid is 1:2.492; the water content is 50.18%; the solid-liquid ratio is 1:65.06 g / mL; the temperature is 47.4 °C; the ultrasonic power is 180 W, and the predicted maximum extraction yield is 1563 mg / kg.

[0095] Example 8

[0096] To verify the prediction results of the above response surface method and for the convenience of actual operation, the optimized extraction process in this example is: the molar ratio is 2:5; the water content is 50%; the solid-liquid ratio is 1:65 g / mL; the temperature is 47 °C. According to the above conditions, ultrasonic-assisted extraction (ultrasonic power is 180 W, ultrasonic time is 30 min) of tomatoside from tomato plant powder was carried out, and the content of the extracted tomatoside was 1551 mg / kg, which is basically consistent with the predicted value.

[0097] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for extracting tomatin using an ultrasound-assisted deep eutectic solvent, characterized in that: The following steps are involved: The deep eutectic solvent was mixed with tomatoes, placed in a water bath, and extracted by ultrasonication; The deep 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.

2. The method for extracting tomatin by using an ultrasound-assisted deep eutectic solvent according to claim 1, characterized in that: The deep eutectic solvent is choline chloride-levulinic acid.

3. The method for extracting tomatin by using ultrasound-assisted deep eutectic solvent according to claim 2, characterized in that: The molar ratio of the choline chloride to levulinic acid is 1:1 to 1:

5.

4. The method for extracting tomatin using an ultrasound-assisted deep eutectic solvent according to claim 3, characterized in that: The molar ratio of the choline chloride to levulinic acid is 1:

5.

5. The method for extracting tomatin by using an ultrasound-assisted deep eutectic solvent according to claim 1, characterized in that: The water content of the low eutectic solvent is 20% to 60%.

6. The method for extracting tomatin by using an ultrasound-assisted deep eutectic solvent according to claim 1, characterized in that: The solid-liquid ratio of the tomato to the low eutectic solvent is 1: (30-70) g / mL.

7. The method for extracting tomatin by using an ultrasound-assisted deep eutectic solvent according to claim 1, characterized in that: The temperature of the ultrasonic extraction is 30°C to 70°C.

8. The method for extracting tomatin by using an ultrasound-assisted deep eutectic solvent according to claim 1, characterized in that: The ultrasonic extraction time is 10 min to 50 min.

9. The method for extracting tomatin by using a deep eutectic solvent with ultrasound assistance according to claim 1, characterized in that: The power of the ultrasonic extraction is 180W.

10. The method for extracting tomatin by using an ultrasound-assisted deep eutectic solvent according to claim 1, characterized in that: After the ultrasonic extraction, the mixture is filtered to obtain a clear liquid.

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