Preparation method of high content naringin and hesperidin extract

Abstract

The application provides a preparation method of high-content naringin and hesperidin extract, and utilizes a microwave-assisted eutectic solvent optimization process to prepare naringin and hesperidin in pericarpium citri reticulatae, and the extraction conditions of the microwave-assisted eutectic solvent are optimized through single-factor experiments and BBD (Box-Behnken Design) response surface experiments, and finally the optimal extraction conditions are obtained as follows: the molar ratio of choline chloride and ethylene glycol is 1:6, the water content is 10%, the solid-liquid ratio is 10 mg / mL, the microwave extraction temperature is 94 DEG C, the time is 41 min, and the power is 400 W. The eutectic solvent is used as the extraction solvent, and the microwave-assisted extraction technology with high energy transmission efficiency and easy to be applied in large scale is combined, so that the efficiency can be improved in future industrial application, the extraction process is more environment-friendly and sustainable, and a new idea is provided for deep processing and utilization of Rutaceae plant resources.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product analysis and preparation, and particularly relates to a preparation method of high-content naringin and hesperidin extract. BACKGROUND

[0002] The information introduced in the background section is intended to improve the understanding of the overall background of the present application and is not necessarily regarded as acknowledging or in any form admitting that this information constitutes the prior art known to those skilled in the art.

[0003] China is the largest citrus fruit producing country in the world, with a total output of about 52 million tons, and the main processed products are juice, cans, etc. More than 10 million tons of waste such as peel and pomace are generated every year, and only 10% of the peel is utilized, resulting in a great waste of resources. How to deeply process the by-products is a bottleneck problem currently faced by the citrus industry.

[0004] Naringin and hesperidin are flavonoids with high biological activity widely existing in natural sources of Rutaceae plants (citrus, grapefruit, lemon, lime, pomelo, etc.). The content in the peel is generally higher than that in the pulp. Literature shows that naringin and hesperidin have biological activities such as antioxidant, anti-inflammatory, hypolipidemic, anti-allergic, immune regulation, etc., and are widely used in food additives, health products, etc., and can be used for developing antioxidant drinks, immune-regulating health products, etc., to meet the needs of consumers for natural and healthy products. If the flavonoid components such as naringin and hesperidin in citrus peel are prepared, it will help the comprehensive utilization of by-products of the citrus industry, realize the deep processing thereof, and improve the economic value thereof.

[0005] At present, naringin and hesperidin are mainly prepared by using natural citrus peel as raw material and adopting organic reagent reflux method. This method generally needs to be refluxed for 2-4 hours, and a large amount of solvent is used, and the extraction temperature is as high as 80℃. Although this method is low in cost and simple in operation, it causes high energy consumption and environmental problems in the operation process, and most importantly, toxic solvent residues are produced in the extract, which limits the application of the extract in related fields. There are also ultrasonic assisted extraction methods, which can extract for 10-40 minutes at room temperature, but the extraction rate is low, and ultrasonic pollution is produced, which is not friendly to the workers.

[0006] Therefore, there is an urgent need for an effective method for preparing naringin and hesperidin extract, which is low in cost, simple in operation and low in energy consumption. SUMMARY

[0007] In order to solve the above problems, the present application provides a safe, efficient and low-cost preparation method of high-content naringin and hesperidin extract.

[0008] The present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing a deep eutectic solvent (DES) composition, wherein the deep eutectic solvent has an optimal composition and ratio and can be used to prepare a high-content naringin and hesperidin extract in the raw material;

[0010] In a second aspect, the present invention provides a method for microwave-assisted extraction, wherein the microwave-assisted extraction conditions are optimized extraction process conditions and can be used in combination with a deep eutectic solvent preparation method to achieve the preparation of a high-content naringin and hesperidin extract in the raw material;

[0011] The specific steps are as follows:

[0012] (1) Preparation of DES composition: Mix A and B in a certain molar ratio, add a certain amount of water, heat in a water bath at 80°C until the solution is clear, and cool to room temperature for use;

[0013] (2) Accurately weigh a certain amount of raw materials, cut them into even-sized pieces with clean scissors, and add a certain amount of DES;

[0014] (3) Add the sample and DES into the digestion tank, set the relevant parameters, and perform microwave extraction.

[0015] (4) The extract was transferred into a centrifuge tube, centrifuged, and the supernatant was taken and made up to volume with chromatographic grade methanol, filtered through a membrane, and measured by high performance liquid chromatography.

[0016] (5) Optimize single-factor processes;

[0017] (6) Based on step (5), the process conditions of the method were optimized using Box-Behnken (BBD) response surface analysis to prepare an extract with high content of naringin and hesperidin. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1 Chromatogram of high-content naringin and hesperidin extract, retention time t = 12.53min for naringin, t = 17.74min for hesperidin

[0020] Figure 2 Effects of DES composition on the extraction rate of naringin and hesperidin from Rutaceae

[0021] Figure 3 Effect of DES molar ratio on the extraction rate of naringin and hesperidin from dried tangerine peel

[0022] Figure 4 Effect of DES water content on the extraction rate of naringin and hesperidin from dried tangerine peel

[0023] Figure 5 Effect of solid-liquid ratio on the extraction rate of naringin and hesperidin from dried tangerine peel

[0024] Figure 6 Effect of extraction power on the extraction rate of naringin and hesperidin from dried tangerine peel

[0025] Figure 7 Effect of extraction time on the extraction rate of naringin and hesperidin from dried tangerine peel

[0026] Figure 8 Effect of extraction temperature on the extraction rate of naringin and hesperidin from dried tangerine peel

[0027] Figure 9 Box-Behnken (BBD) response surface experiment for process optimization DETAILED DESCRIPTION

[0028] Example 1

[0029] (1) Preparation and optimization of DES composition

[0030] Mix A and B in a specific molar ratio, add a certain amount of water, and heat in a water bath at 80°C until the solution is clear and transparent. Cool to room temperature and set aside. The prepared DES is shown in Table 1.

[0031] Using the extraction yields of naringin and hesperidin in the extract as evaluation indicators, the DES combination (Table 1), the molar ratio of A to B (2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8), and the water content (10, 20, 30, 40, 50 wt%) were screened and optimized to determine the optimal extraction solvent. The solid-liquid ratio (10, 30, 50, 70, 90 mg / mL) was also screened.

[0032] Table 1 Composition of DES

[0033]

[0034] (2) Microwave-assisted extraction of naringin from rutin

[0035] Accurately weigh a certain amount of tangerine peel, cut it into even-sized pieces with clean scissors, absorb a certain volume of DES and add it to the digestion tank, set relevant parameters, optimize the process, and perform microwave extraction. (1) Single-factor experimental design

[0036] A single-factor experiment was used, and microwave extraction power (300, 400, 500, 600, 700, 800 W), microwave extraction time (10, 20, 30, 40, 50 min) and microwave extraction temperature (60, 80, 100, 120 ° C) were selected as investigation factors. The extraction rate of naringin and hesperidin in the extract was used as evaluation index to determine the optimal extraction parameter range.

[0037] (3) Box-Behnken (BBD) response surface experiment to optimize the preparation process

[0038] Based on the results of the single-factor experiment, a BBD response surface design was used to further optimize the microwave extraction temperature (A), extraction power (B), and extraction time (C). The experimental factors and levels are shown in Table 2. The BBD was performed using Design-Expert 13 software.

[0039] Table 2 BBD experimental factors and levels

[0040]

[0041] (4) Determination of naringin content in the extract

[0042] The extract was transferred into a centrifuge tube and centrifuged. 1 mL of the supernatant was taken and diluted to 10 mL with chromatographic grade methanol. The supernatant was filtered through a 0.22 μm membrane and analyzed by high performance liquid chromatography. The liquid chromatogram of the naringin extract from Rutaceae is shown in the attached figure. Figure 1 shown.

[0043] Example 2

[0044] (1) Effect of deep eutectic solvent composition on the extraction rate of naringin and hesperidin from tangerine peel

[0045] The effect of DES type on the extraction rate of NAR and HES was investigated under the conditions of DES molar ratio of 1:1, water content of 30wt%, solid-liquid ratio of 50mg / mL, microwave extraction temperature of 100℃, extraction time of 30min, and extraction power of 400W. Figure 2 The results showed that the DES-10 solvent system, i.e. the combination of choline chloride and ethylene glycol, could extract higher amounts of NAR and HES from dried tangerine peel. Therefore, the combination of choline chloride and ethylene glycol was selected as the extraction solvent.

[0046] (2) Effect of DES molar ratio on the extraction rate of naringin and hesperidin from dried tangerine peel

[0047] When the extraction agent was a combination of choline chloride and ethylene glycol with a water content of 30 wt%, a solid-liquid ratio of 50 mg / mL, a microwave extraction temperature of 100 ° C, an extraction time of 30 min, and an extraction power of 400 W, the effects of DES molar ratios (2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8) on the extraction yields of NAR and HES were investigated. Figure 3 The results showed that the molar ratio of 1:6 had a higher extraction rate of NAR and HES from dried tangerine peel, so the molar ratio of choline chloride to ethylene glycol was selected as 1:6.

[0048] (3) Effect of DES water content on the extraction rate of naringin and hesperidin from dried tangerine peel

[0049] Under the conditions of a choline chloride / ethylene glycol molar ratio of 1:6, a solid-liquid ratio of 50 mg / mL, a microwave extraction temperature of 100°C, an extraction time of 30 min, and an extraction power of 400 W, the effect of DES water content (10 wt%, 20 wt%, 30 wt%, 40 wt%, 50 wt%) on the extraction yields of NAR and HES was investigated. Figure 4 The results showed that a 10% water content resulted in the highest extraction rate of NAR and HES from dried tangerine peel. The extraction rate decreased with increasing water content, so a 10% water content was selected. Studies have shown that an appropriate amount of water can promote the penetration of solvents into sample matrices, but excessive water increases the polarity of DES, thereby weakening its intermolecular structure and affecting the solvent's extraction performance.

[0050] (4) Effect of solid-liquid ratio on the extraction rate of naringin and hesperidin from tangerine peel

[0051] Under the conditions of DES molar ratio of 1:1, water content of 10 wt%, microwave extraction temperature of 100 °C, extraction time of 30 min, and extraction power of 400 W, the effects of solid-liquid ratio (10 mg / mL, 30 mg / mL, 50 mg / mL, 70 mg / mL, 90 mg / mL) on the extraction yield of NAR and HES were investigated. Figure 5 The results showed that a solid-liquid ratio of 10 mg / mL resulted in higher extraction yields of NAR and HES from dried tangerine peel. The extraction yields generally decreased with increasing solid-liquid ratios. A higher solid-liquid ratio may result in incomplete contact between the solvent and the sample matrix, leading to incomplete extraction of the target compounds. Therefore, a solid-liquid ratio of 10 mg / mL was selected.

[0052] Example 3

[0053] Optimization of microwave-assisted extraction process conditions

[0054] (1) Effect of extraction power on the extraction rate of naringin and hesperidin in tangerine peel

[0055] Under the conditions of DES molar ratio of 1:1, water content of 10 wt%, solid-liquid ratio of 10 mg / mL, microwave extraction temperature of 100 °C, and extraction time of 30 min, the effects of power (300 W, 400 W, 500 W, 600 W, 700 W, 800 W) on the extraction rate of NAR and HES were investigated. Figure 6 The results showed that when the microwave power was 400W, the extraction rates of NAR and HES were higher, so the microwave power of 400W was selected.

[0056] (2) Effect of extraction time on the extraction rate of naringin and hesperidin in tangerine peel

[0057] Under the conditions of DES molar ratio of 1:1, water content of 10 wt%, solid-liquid ratio of 10 mg / mL, microwave extraction power of 400 W, and extraction temperature of 100 ° C, the effects of extraction time (10 min, 20 min, 30 min, 40 min, 50 min) on the extraction rate of NAR and HES were investigated. Figure 7 The results showed that when the microwave time was 40 min, the extraction rate of NAR and HES in dried tangerine peel was higher, so the extraction time was selected as 40 min.

[0058] (3) Effect of extraction temperature on the extraction rate of naringin and hesperidin in tangerine peel

[0059] Under the conditions of DES molar ratio of 1:1, water content of 10 wt%, solid-liquid ratio of 10 mg / mL, microwave extraction power of 400 W, and extraction time of 40 min, the effects of extraction temperature (60°C, 80°C, 100°C, 120°C) on the extraction yield of NAR and HES were investigated. Figure 8 The results showed that the extraction rate of NAR and HES in dried tangerine peel was higher when the extraction temperature was 100℃, so the extraction temperature was selected as 100℃.

[0060] Example 4

[0061] Box-Behnken (BBD) response surface design

[0062] Table 3 Response surface design scheme and results

[0063]

[0064] Table 4 Analysis of variance of regression model

[0065]

[0066] The results of the model variance analysis are shown in Tables 3 and 4, and the results of the response surface analysis are shown in Appendix Figure 9As shown. Overall, the model's p-value (p<0.0001) was significant, while the p-value for the lack-of-fit term (p>0.05) was insignificant, indicating that the model was established. The R² (coefficient of determination) was 0.9871, indicating a good fit for the regression model. The Adj-R² (adjusted coefficient of determination) was 0.9706, and the Pred-R² (predicted coefficient of determination) was 0.8419, both reaching high levels, with a difference of less than 0.2, indicating that the regression model can fully reflect the process.

[0067] Through data fitting, the regression equations of the total extraction rate of naringin and hesperidin from rutaecarpa and the three factors are as follows:

[0068] Y=45.87-7.75A+0.2704B+0.8779C-2.57AB-0.9280AC-0.0318BC-13.78A2-3.56B2-6.24C2

[0069] Wherein, Y is the total extraction rate of naringin and hesperidin, mg / g; A is the microwave extraction temperature, °C; B is the extraction power, W; C is the extraction time, min.

[0070] The optimal extraction conditions, determined by Design Expert software, were as follows: microwave temperature of 94.045°C, power of 414.579W, and time of 40.922 minutes. Under these optimal conditions, the total extraction yield of NAR and HES from dried tangerine peel reached the maximum predicted value of 47.083 mg / g.

[0071] Due to experimental feasibility and equipment limitations, the optimized conditions were revised to: microwave temperature of 94°C, power of 400 W, and duration of 41 minutes. Three parallel experiments were conducted under these conditions to verify the stability and feasibility of the optimized process. The average total extraction yield of NAR and HES in the DES extract was 46.839 mg / mL, close to the predicted value.

[0072] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned experiments, those skilled in the art may modify the technical solutions described in the aforementioned embodiments or substitute equivalent technical features for some of the technical features. Any modifications, substitutions, and improvements to the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a high-content naringin and hesperidin extract, characterized in that: A high-content naringin and hesperidin extract is prepared using microwave-assisted extraction technology combined with a deep eutectic solvent. The deep eutectic solvent has an optimal composition and ratio for preparing a high-content naringin and hesperidin extract from a raw material. The microwave-assisted extraction technology uses optimized extraction process conditions, which are used in combination with a deep eutectic solvent preparation method to achieve the preparation of a high-content naringin and hesperidin extract from a raw material. The deep eutectic solvent composition is choline chloride and ethylene glycol in a molar ratio of 1:6, and the water content is 10%; The preparation steps of the extract are: (1) Preparation of DES composition: Choline chloride and ethylene glycol were mixed in a certain molar ratio, and a certain amount of water was added. The mixture was heated in a water bath at 80°C until the solution was clear and transparent, and then cooled to room temperature for later use. (2) Accurately weigh a certain amount of raw materials, cut them into even-sized pieces with clean scissors, and add a certain amount of DES; (3) Add the sample and DES into the digestion tank, set the relevant parameters, and perform microwave extraction; (4) Transfer the extract into a centrifuge tube, centrifuge, take the supernatant and dilute to volume with chromatographic grade methanol, filter through a membrane, and perform high performance liquid chromatography determination; (5) Optimize single-factor process; (6) Based on step (5), the process conditions of the method were optimized by Box-Behnken (BBD) response surface experiment to prepare an extract with high content of naringin and hesperidin.

2. The method for preparing a high-content naringin and hesperidin extract according to claim 1, characterized in that: Combined with deep eutectic solvent microwave-assisted extraction technology, the solid-liquid ratio was 10 mg / mL, the microwave extraction temperature was 94°C, the time was 41 min, and the power was 400 W.

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