Method for extracting navel orange peel polyphenol by using deep eutectic solvent

Abstract

The invention provides a method for extracting navel orange peel polyphenol by using a deep eutectic solvent, and belongs to the technical field of natural product extraction. The method comprises the following steps: firstly, preparing a deep eutectic solvent consisting of choline chloride and ethylene glycol according to a molar ratio of 1: 2, and then extracting pretreated navel orange peel powder by using the solvent. Through optimization of a response surface method, optimal extraction process parameters are determined as follows: the water content of a solvent is 38%, the material-liquid ratio is 1: 31 g / mL, and the extraction time is 41 min. Under the conditions, the extraction yield of the navel orange peel polyphenol is high, and the extraction process is green and environment-friendly. The obtained polyphenol extract shows excellent scavenging capacity on DPPH free radicals, ABTS cation free radicals and hydroxyl free radicals, is remarkable in antioxidant activity and can be used in the fields of food, health care products, cosmetics and the like.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, and more specifically, to a method for extracting polyphenols from navel orange peel using a deep eutectic solvent. Background Technology

[0002] Navel oranges Citrus sinensis Navel oranges (L. Osbeck), often called the "King of Citrus," belong to the Rutaceae family and the Citrus genus, and are an important economic fruit. The industrial processing and fresh consumption of navel oranges generate a large amount of byproducts—the peels. These peels are usually discarded or landfilled, resulting in a serious waste of biomass resources and environmental pollution. Therefore, realizing the high-value utilization of this abundant resource has become a current focus.

[0003] Studies have confirmed that navel orange peel is an industrial byproduct with important physiological functions, showing broad development prospects in the fields of medicine, food, and cosmetics. The peel contains abundant nutrients, including natural pigments, pectin, vitamins, dietary fiber, soluble sugars, and minerals, especially polyphenols. Polyphenols possess strong antioxidant activity, exhibiting positive effects in anti-cancer, anti-inflammatory, antibacterial, antiviral, and antithrombotic activities, with particularly significant effects in protecting against ultraviolet radiation. Furthermore, polyphenols can effectively inhibit the occurrence and development of various diseases, such as diabetes, neurodegenerative diseases, atherosclerosis, and cardiovascular dysfunction. Therefore, the efficient extraction of these high-value phenolic substances from navel orange peel is of great significance.

[0004] The extraction of bioactive substances from natural products has become a research hotspot in recent years. Currently, various methods exist for extracting plant polyphenols, with the most common method utilizing organic solvents such as methanol, ethanol, and petroleum ether. However, these alcohol or organic solvent extraction methods suffer from significant drawbacks, including high energy consumption, high cost, cumbersome operation, and environmental pollution. More importantly, the extracted system often contains residual organic solvents, which may pose a potential threat to human health and ecosystems through bioaccumulation or environmental migration. Therefore, developing environmentally friendly, low-cost, and more efficient green alternative solvents has become one of the most important themes in the fields of chemistry and food science.

[0005] Deep eutectic solvents, as a novel type of green solvent, are composed of hydrogen bond acceptors and hydrogen bond donors mixed in a certain proportion. They possess numerous advantages, including simple preparation, high safety, low raw material cost, non-toxicity, environmental friendliness, and biodegradability. Studies have shown that deep eutectic solvents exhibit significant targeted extraction advantages for plant secondary metabolites such as flavonoids and phenols, demonstrating enormous application potential in the field of green separation of natural products and becoming a research hotspot in natural product extraction in recent years. Although research on the extraction of plant polyphenols and flavonoids using deep eutectic solvents is increasing both domestically and internationally, a complete study has not yet been reported in existing literature on the systematic application of a specific deep eutectic solvent system to the extraction of polyphenols from navel orange peel, optimization of the extraction process, and evaluation of the antioxidant activity of the extract. This indicates a clear gap in current technology: the lack of an efficient and green deep eutectic solvent extraction method specifically tailored to the characteristics of navel orange peel.

[0006] In summary, current technologies for processing navel orange peel are crude and fail to fully utilize its value; furthermore, traditional organic solvent methods for polyphenol extraction have numerous drawbacks. Although deep eutectic solvents, as an emerging green extraction medium, have been extensively studied, their specific application in the extraction of polyphenols from navel orange peel, and the development of a complete and optimized process system, remains a technological gap. Therefore, there is an urgent need in this field to develop a deep eutectic solvent extraction method specifically for navel orange peel, with optimized process parameters, capable of achieving both high yield and high activity, to solve the aforementioned technical problems. Summary of the Invention

[0007] In view of this, this invention proposes a method for extracting polyphenols from navel orange peel using a deep eutectic solvent to address the problems existing in the prior art. This method innovatively constructs a choline chloride-ethylene glycol binary DES system and optimizes the extraction process of navel orange peel polyphenols using the Box-Behnken response surface methodology. The antioxidant capacity of the polyphenols is explored by combining free radical scavenging kinetics. The research results not only provide a new strategy for the high-value utilization of citrus processing by-products but also establish a theoretical paradigm for the application of DES in the green extraction of natural products.

[0008] To achieve the above objectives, the present invention proposes a deep eutectic solvent for extracting polyphenols from navel orange peel, characterized in that it is composed of choline chloride as a hydrogen bond acceptor and a compound as a hydrogen bond donor, wherein the hydrogen bond donor is selected from polyols, organic acids and urea.

[0009] Furthermore, the hydrogen bond donor is a polyol or an organic acid.

[0010] Furthermore, the polyol is selected from ethylene glycol or glycerol; and / or, the organic acid is selected from lactic acid or malic acid.

[0011] Furthermore, the hydrogen bond donor is ethylene glycol.

[0012] Furthermore, the molar ratio of choline chloride to the hydrogen bond donor is 1:1 to 1:3, and even more specifically 1:2.

[0013] The present invention also provides a method for extracting polyphenols from navel orange peel using a deep eutectic solvent, wherein the navel orange peel powder is extracted using the deep eutectic solvent as described above.

[0014] Further, the method includes the following steps: mixing navel orange peel powder with the deep eutectic solvent with a water content of 30%~50% at a material-to-liquid ratio of 1:25~1:35 g / mL, extracting at 40℃-60℃ for 30-50 minutes, and collecting the supernatant by centrifugation; even further, the deep eutectic solvent is choline chloride-ethylene glycol with a water content of 38%, a material-to-liquid ratio of 1:31 g / mL, an extraction time of 41 minutes, and an extraction temperature of 50℃.

[0015] This invention also provides a polyphenol extract from navel orange peel, which is prepared by the method described above. This extract is rich in polyphenolic compounds, with a polyphenol yield of over 10.27 mg / g, and exhibits significant antioxidant activity.

[0016] The present invention also provides the application of the above-described navel orange peel polyphenol extract in the preparation of antioxidants.

[0017] Furthermore, the antioxidant can be used to scavenge DPPH free radicals, ABTS cation free radicals, and hydroxyl free radicals. Based on its excellent antioxidant properties, the antioxidant is used in food, health products, or cosmetics.

[0018] Compared with existing technologies, the method for extracting polyphenols from navel orange peel based on a deep eutectic solvent provided by this invention exhibits significant advantages in many aspects. The core benefit of this method lies in its green and environmentally friendly characteristics. The selected deep eutectic solvent is composed of safe and renewable components such as choline chloride and ethylene glycol, fundamentally avoiding the toxic residues and environmental pollution problems caused by traditional organic solvent extraction processes, and providing a clean and sustainable technical path for the extraction of natural products.

[0019] In terms of extraction efficiency and economic benefits, this invention achieves highly efficient enrichment of polyphenols in navel orange peel through systematic solvent screening and process optimization. In particular, the choline chloride-ethylene glycol solvent system exhibits excellent targeted dissolution ability for polyphenols. Under optimized and mild process conditions, its polyphenol yield is significantly higher than that of existing technologies such as traditional ethanol extraction, meaning that more target product can be obtained with the same raw material input, effectively reducing the cost per unit output.

[0020] The extraction process developed in this invention possesses high stability and reliability. The mathematical model established using response surface methodology provides accurate predictions, and the optimal process parameter range obtained through optimization is clearly defined. This makes the entire extraction process controllable and highly repeatable, greatly reducing fluctuations and uncertainties in the production process. This lays a solid foundation for this technology to move from the laboratory to large-scale industrial applications.

[0021] Finally, the navel orange peel polyphenol extract obtained by this invention exhibits excellent functional quality. In vitro antioxidant activity evaluation fully confirms that this extract possesses strong scavenging capabilities against DPPH free radicals, ABTS cationic free radicals, and hydroxyl free radicals, and its overall antioxidant activity is far superior to that obtained by traditional solvent extraction methods. This not only enhances the added value of navel orange peel by-products but also provides a reliable raw material guarantee for their application in high-value fields such as functional foods, health products, and cosmetics. In summary, this invention achieves a green and efficient transformation from waste to high-value functional raw materials, combining environmental and economic benefits. Attached Figure Description

[0022] Figure 1 This is a comparison chart of polyphenol extraction yields using different eutectic solvents.

[0023] Figure 2 This is a graph showing the effect of solvent moisture content on the yield of polyphenols from navel orange peel.

[0024] Figure 3 This is a graph showing the effect of the material-to-liquid ratio on the yield of polyphenols from navel orange peel.

[0025] Figure 4 This is a graph showing the effect of extraction temperature on the yield of polyphenols from navel orange peel.

[0026] Figure 5 This is a graph showing the effect of extraction time on the yield of polyphenols from navel orange peel.

[0027] Figure 6 These are response surface plots and contour plots showing the effects of various interaction factors on the yield of polyphenols from navel orange peel. Specifically, A represents the interaction between moisture content and the solid-liquid ratio; B represents the interaction between moisture content and extraction time; and C represents the interaction between the solid-liquid ratio and extraction time.

[0028] Figure 7 This is a graph showing the DPPH free radical scavenging rate. In this graph, A is the standard curve for Trolox's DPPH free radical scavenging; B is the effect of navel orange peel polyphenols on the DPPH free radical scavenging ability.

[0029] Figure 8This is a graph showing the ABTS free radical scavenging rate. 8A is the standard curve for Trolox's ABTS free radical scavenging; B is the graph showing the effect of navel orange peel polyphenols on the ABTS free radical scavenging ability.

[0030] Figure 9 This is a graph showing the scavenging rate of hydroxyl radicals. In this graph, A is the Trolox standard curve for scavenging hydroxyl radicals; B is the graph showing the effect of navel orange peel polyphenols on the scavenging capacity of hydroxyl radicals. Detailed Implementation

[0031] 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.

[0032] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0033] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0034] Experimental materials: Navel oranges: Jiangxi Ganzhou orchard planting base; choline chloride, urea, and Folin-Ciocalteu: analytical grade, Shanghai Maclean Biochemical Technology Co., Ltd.; glycerol, ethylene glycol, lactic acid, malic acid, anhydrous sodium carbonate, and anhydrous ethanol: analytical grade, Tianjin Yongda Chemical Reagent Co., Ltd.; DPPH, ABTS, ferrous sulfate, and salicylic acid: analytical grade, Shanghai Yuanye Biotechnology Co., Ltd.; Trolox: analytical grade, Shanghai Aladdin Biochemical Technology Co., Ltd.

[0035] Experimental apparatus: HH-6 Digital Display Constant Temperature Water Bath: Henan Enge Kiln Machinery Equipment Co., Ltd.; CS-2000Y High-Speed ​​Multifunctional Grinder: Wuhan Haina Electric Co., Ltd.; DHG-9123A Electric Heating Constant Temperature Blower Drying Oven: Shanghai Jinghong Experimental Equipment Co., Ltd.; H1650-W Tabletop High-Speed ​​Refrigerated Centrifuge: Xiangtan Xiangyi Instrument Co., Ltd.; SYNERGY H1 Microplate Reader: Shanghai Aiyan Biotechnology Co., Ltd.

[0036] Example 1: Preparation of Deep Eutectic Solvent (DES) This invention prepared five different deep eutectic solvents for subsequent extraction of polyphenols from navel orange peel. Specific preparation methods are shown in Table 1.

[0037] Table 1 Preparation of eutectic solvents

[0038] The preparation process is as follows: Accurately weigh choline chloride and the corresponding hydrogen bond donor (glycerol, ethylene glycol, lactic acid, malic acid, or urea) according to the molar ratio specified in Table 1, and place them in a round-bottom flask. Place the flask in an oil bath at 80°C and stir continuously with a magnetic stirrer for 1 to 3 hours until the mixture forms a homogeneous, stable, transparent liquid, indicating the formation of a deep eutectic solvent. After cooling, seal and store in a desiccator for later use.

[0039] Example 2: Establishment of a sample pretreatment and polyphenol content determination method 2.1 Sample Pretreatment Clean the fresh Gannan navel orange peels thoroughly, chop them, and dry them in a 60℃ electric constant temperature forced-air drying oven until constant weight. Use a high-speed multi-functional pulverizer to pulverize the dried peels into powder, pass it through a 60-mesh sieve, and obtain uniform and fine navel orange peel powder. Store it in a dark and dry place for later use.

[0040] 2.2 Establishment of a standard curve for polyphenol content determination Accurately pipette 0, 0.5, 1, 2, and 3 mL of gallic acid standard solution (1 mg / mL) into 10 mL volumetric flasks, and dilute to volume with ultrapure water to prepare a series of standard solutions with concentrations of 0, 0.05, 0.10, 0.20, and 0.30 mg / mL. Pipette 0.4 mL of each of the above concentration standard solutions into a 10 mL brown volumetric flask, add 4 mL of ultrapure water, mix well, add 0.6 mL of Folin-Ciocalteu reagent, let stand for 30 s, then add 2.4 mL of 10% Na₂CO₃ solution, and dilute to the mark with ultrapure water and mix well. Place the volumetric flasks in a 40℃ constant temperature water bath and react in the dark for 60 min. Use the 0 mg / mL concentration solution as a blank control, and measure the absorbance of each solution at a wavelength of 765 nm using a microplate reader. Each sample is measured in triplicate, and the average value is taken.

[0041] A standard curve was plotted with gallic acid concentration (X, mg / mL) on the x-axis and absorbance (Y) on the y-axis. The linear regression equation was obtained as: Y = 2.2485X + 0.0483, with a correlation coefficient R0. 2 = 0.9991 > 0.99. The results indicate that gallic acid has a good linear relationship with absorbance in the concentration range of 0–0.30 mg / mL.

[0042] 2.3 Calculation of Polyphenol Yield The yield of polyphenols in the extract was calculated using the following formula:

[0043] Q: Yield of polyphenols from navel orange peel, mg / g; C: Polyphenol concentration in the test solution, mg / mL; P: Dilution factor; V: Total volume of extract, mL; M: Mass of navel orange peel powder, g.

[0044] Example 3: Screening of the optimal eutectic solvent and single-factor experiments 3.1 Optimal DES Selection Accurately weigh 100 mg of navel orange peel powder into a 15 mL centrifuge tube, and add 2.5 mL of five different DES (diethylsaturated emulsions) prepared according to the method in Example 1 with a water content of 30%. Extract in a 60°C water bath for 60 min, then centrifuge at 10000 r / min for 10 min and collect the supernatant. Measure 0.2 mL of the supernatant, determine the absorbance value according to the method in Example 2.2, and calculate the polyphenol yield.

[0045] This invention utilizes choline chloride as a hydrogen bond acceptor and prepares five deep eutectic solvents by varying the types of hydrogen bond donors: choline chloride-glycerol, choline chloride-ethylene glycol, choline chloride-lactic acid, choline chloride-malic acid, and choline chloride-urea. Polyphenols from navel orange peel were extracted under the following conditions: solvent water content 30%, solid-liquid ratio 1:25 g / mL, extraction temperature 60℃, and extraction time 60 min. The polyphenol yields extracted using different deep eutectic solvents were statistically analyzed. The results are as follows: Figure 1 As shown, the extraction yield of choline chloride-ethylene glycol DES was significantly higher than that of the other four DES types. Therefore, choline chloride-ethylene glycol was determined to be the optimal solvent for subsequent extraction experiments.

[0046] 3.2 Single-factor experiment Based on the determination that choline chloride-ethylene glycol is the optimal solvent, the effects of four single factors on polyphenol yield were investigated: solvent moisture content (10%, 20%, 30%, 40%, 50%), solid-liquid ratio (1.5, 2, 2.5, 3, 3.5 mL), extraction temperature (20℃, 40℃, 50℃, 60℃, 70℃, 80℃), and extraction time (20, 30, 40, 50, 60, 70, 80 min). Five levels were set for each factor, with other conditions fixed as follows: moisture content 30%, solid-liquid ratio 1:25 g / mL, extraction temperature 60℃, and extraction time 60 min (unless the factor was a variable). The effects of different factors on the polyphenol extraction rate of navel orange peel were explored.

[0047] (1) Effect of solvent water content: Set the moisture content levels to 10%, 20%, 30%, 40%, and 50%.

[0048] Due to their inherently high viscosity, eutectic solvents typically require the addition of water in practical applications to reduce the system's viscosity and improve extraction efficiency. However, different amounts of water added will have varying effects on the solvent's viscosity, polarity, and surface tension.

[0049] The results are as follows Figure 2 As shown, the yield of polyphenols from navel orange peel exhibits a clear trend of first increasing and then decreasing with increasing moisture content in the eutectic solvent. Specifically, as the moisture content gradually increases from 10% to 30%, the viscosity of the eutectic solvent gradually decreases, while its polarity gradually increases, promoting the dissolution of polyphenols and thus improving the extraction rate of polyphenols from navel orange peel. The polyphenol yield reaches its peak at a moisture content of 40%, at 7.55 mg / g. When the moisture content exceeds 40%, the polyphenol yield gradually decreases. This may be because excessive moisture content leads to decreased system viscosity and excessive polarity, causing the breakage of hydrogen bonds between molecules. The weakened hydrogen bond forces between the solvent and polyphenols result in a lower polyphenol yield from navel orange peel. Therefore, considering all factors, a moisture content of 40% in the eutectic solvent is selected as the optimal process condition. Significance testing using SPSS 26 software shows that moisture content has a significant impact on the extraction yield of polyphenols from navel orange peel. Response surface methodology experiments were then conducted with moisture contents of 30%, 40%, and 50%.

[0050] (2) Effect of material-liquid ratio: Set the feed-to-liquid ratio to 1:15, 1:20, 1:25, 1:30, or 1:35 g / mL.

[0051] The results are as follows Figure 3 As shown, the polyphenol yield initially increased and then decreased with increasing solvent usage. When the solid-liquid ratio gradually increased from 1:15 g / mL to 1:25 g / mL, the increased contact area between the polyphenols in the navel orange peel and the solvent increased the concentration difference between them. This concentration gradient is the driving force for extraction, thus promoting the diffusion of polyphenols from the navel orange peel powder into the solvent, resulting in a higher polyphenol yield. The polyphenol yield peaked at 7.55 mg / g when the solid-liquid ratio reached 1:30 g / mL. Further increases in the solid-liquid ratio thereafter led to a decrease in polyphenol yield. This may be because the navel orange peel powder reached saturation; further increases in solvent could cause the dissolution of polysaccharides, pectin, soluble proteins, and other substances, affecting the dissolution of polyphenols. Additionally, it could cause the adsorbed polyphenols to redissolve in the extract, thus reducing the polyphenol yield and wasting solvent. Considering all factors, the optimal solid-liquid ratio was ultimately chosen to be 1:30 g / mL. The significance test was performed using SPSS 26 software. The results showed that the material-liquid ratio had a significant impact on the extraction yield of polyphenols from navel orange peel. Therefore, the material-liquid ratios of 1:25 g / mL, 1:30 g / mL, and 1:35 g / mL were finally selected for response surface methodology experiments.

[0052] (3) Effect of extraction temperature: The extraction temperature levels are set to 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃.

[0053] The results are as follows Figure 4 As shown, the yield of polyphenols from navel orange peel exhibited a trend of first increasing and then leveling off with increasing temperature. Specifically, when the temperature was between 20℃ and 40℃, the polyphenol yield increased with increasing temperature. This may be because the increase in temperature reduced the viscosity and surface tension of the eutectic solvent, which is conducive to the dissolution of polyphenols. At the same time, it promoted the thermal effect, intensified molecular motion, and increased the diffusion rate and solubility of polyphenols in navel orange peel, thereby improving the extraction efficiency. When the extraction temperature reached 50℃, the polyphenol yield was 7.44 mg / g, while at 60℃, the polyphenol yield was 7.65 mg / g. After that, the increase in polyphenol yield was not significant with further increases in extraction temperature. Moreover, excessively high temperatures not only destroyed the structure of polyphenols, causing polyphenol oxidative degradation, but also increased energy consumption. Therefore, considering energy consumption and the thermal stability of polyphenols, 50℃ was selected as the center point, and three levels of 40℃, 50℃, and 60℃ were selected for response surface methodology experiments.

[0054] (4) The impact of extraction time: The extraction time levels were set to 20, 30, 40, 50, 60, 70, and 80 minutes.

[0055] The results are as follows Figure 5 As shown, the yield of polyphenols from navel orange peel gradually increased with increasing extraction time. The maximum yield (7.50 mg / g) was reached at 40 min. However, further extending the extraction time led to a decrease in polyphenol yield. This may be because, initially, the amount of polyphenols dissolved gradually increased with time, thus increasing the yield; however, due to the instability of polyphenols, excessively long extraction times may damage their structure after reaching dissolution equilibrium. Furthermore, increased impurities can interfere with the dissolution of polyphenols, resulting in a decrease in yield. Therefore, considering all factors, an extraction time of 40 min was selected as the optimal process condition. Significance testing using SPSS 26 software showed that extraction time had a significant impact on the polyphenol yield from navel orange peel. Response surface methodology experiments were then conducted using extraction times of 30 min, 40 min, and 50 min.

[0056] Example 4: Optimization and Validation of Extraction Process Using Response Surface Methodology Based on the results of the single-factor experiments, three factors that significantly affected the yield were selected: moisture content (A), material-to-liquid ratio (B), and extraction time (C). A three-factor, three-level response surface methodology was conducted using Design-Expert 13 software, following the Box-Behnken central composite design principle. The factor and level designs are shown in Table 2.

[0057] Table 2 Factors and Level Design in Response Surface Analysis

[0058] Based on the results of the single-factor experiments, three key influencing factors were selected: moisture content (A), material-to-liquid ratio (B), and extraction time (C). Combining the Box-Behnken experimental design principle, a three-factor, three-level response surface analysis was conducted using Design-Expert 13 software. The experimental scheme and results designed by Box-Behnken Design are shown in Table 3.

[0059] Using Design-Expert 13 software, a multiple regression model was used to fit the data in Table 3. The quadratic regression model equation for polyphenol yield (Y) with three factors—moisture content (A), material-to-liquid ratio (B), and extraction time (C)—was obtained: Y = 11.44 + 0.0786A - 0.2401B + 0.2888C - 0.0218AB + 0.1303AC - 0.0311BC - 3.68A² - 2.95B² - 2.19C². An analysis of variance was performed on the regression equation, and the results are shown in Table 4.

[0060] Analysis of the variance data also revealed that the linear terms A, B, and C, as well as the interaction terms AB, AC, and BC in the regression equation, had no significant effect on the yield of polyphenols from navel orange peel (p > 0.05). However, the quadratic term A... 2 B 2 C 2 All factors had a highly significant impact on polyphenol yield (p<0.001). Further, based on the F-values, the order of influence of each factor on the polyphenol yield of navel orange peel can be inferred as: extraction time (C) > material-to-liquid ratio (B) > moisture content (A). According to the data in Table 4, the P-value (Prob > F) of the regression model is <0.0001, indicating that the regression model reached a highly significant level; meanwhile, the P-value of the lack-of-fit term is 0.4943 > 0.05, indicating that the lack-of-fit term in the regression model is not significant, meaning that the residuals in the model are mainly caused by random error. Furthermore, the coefficient of determination R... 2 =0.9842, adjusted coefficient of determination R 2With an adj value of 0.9638, and values ​​close to 1, the regression model demonstrates a very high degree of fit, explaining up to 96.38% of the data variation. Therefore, this model can be used to analyze and predict the yield of polyphenols from navel orange peel. In conclusion, the model exhibits good fit and small experimental error, making it suitable for analyzing and predicting the process results of deep eutectic solvent extraction of polyphenols from navel orange peel.

[0061] Based on the constructed regression model, interaction surface plots and contour plots (such as...) were drawn to illustrate the interactions between various influencing factors. Figure 6 As shown in the figure, the interactions between moisture content (A) and the solid-liquid ratio (B), moisture content (A) and extraction time (C), and solid-liquid ratio (B) and extraction time (C) are analyzed. The slope and density of contour lines in the response surface plot reflect the strength of the interaction between factors. The steeper the slope of the response surface plot, the more significant the interaction between the two factors; the denser the contour lines (forming an ellipse), the more significant the influence of the factor on the response value. Comparison Figure 6 As shown in AC, the slope of the curve for extraction time is steeper than that for the material-to-liquid ratio and moisture content, indicating that extraction time has a more significant impact on the yield of polyphenols from navel orange peel. When the extraction time is fixed, the slope of the curve for the material-to-liquid ratio is steeper than that for moisture content, indicating that at the same extraction time, the material-to-liquid ratio has a greater impact on the polyphenol yield than moisture content. Furthermore, observing the density of the contour lines reveals that the contour lines for moisture content and extraction time, moisture content and material-to-liquid ratio, and extraction time and material-to-liquid ratio are nearly circular, indicating a certain degree of interaction among the three factors, but their impact on the extraction of polyphenols from navel orange peel is not significant, consistent with the results of the analysis of variance in Table 4.

[0062] Data analysis using Design Expert 13 software revealed the optimal extraction conditions for navel orange peel polyphenols as follows: moisture content 38%, solid-liquid ratio 1:31 g / mL, and extraction time 41 min. Under these optimal conditions, the response surface methodology predicted a theoretical yield of 11.17 mg / g of navel orange peel polyphenols. Three validation experiments were conducted using the optimized conditions, yielding an actual yield of 10.27 mg / g. This yield differs from the theoretical prediction obtained through regression model analysis by only 0.90 mg / g, demonstrating the reliability and accuracy of the regression model and its applicability for regression analysis and parameter optimization in the extraction process of navel orange peel polyphenols.

[0063] Table 3 Response Surface Design Schemes and Results

[0064] Table 4. Analysis of Variance of Regression Model

[0065] ** indicates a highly significant difference (p<0.01); * indicates a significant difference (p<0.05); N indicates no significant difference (p>0.05).

[0066] Comparative Example 1: Ultrasonic-assisted ethanol extraction method To highlight the inventiveness of this invention, a comparative example was provided. 100 mg of navel orange peel powder was accurately weighed and extracted using the ultrasonic-assisted ethanol extraction method optimized in Meng Fangfang's literature (Meng Fangfang. Study on in vitro antioxidant properties of peels from six navel orange varieties in southern Jiangxi [D], 2012). A 60% ethanol solution was used, with a material-to-liquid ratio of 1:30 g / mL, and extraction was performed ultrasonically at 60℃ (250W power) for 40 min. After centrifugation, the supernatant was collected, and the polyphenol yield was determined according to the method in Example 2. The experiment was conducted in three parallel trials, with an average yield of 9.24 mg / g.

[0067] Results Comparison: The polyphenol yield of the method of the present invention (DES extraction) (10.27 mg / g) was significantly higher than that of the traditional ethanol extraction method (9.24 mg / g), with an increase of about 11.15%, which proves the advantage of the present invention in extraction efficiency.

[0068] Example 5: Evaluation of the antioxidant activity of polyphenols in navel orange peel 5.1 DPPH free radical scavenging ability The optimal polyphenol extract obtained in Example 4 was diluted with ultrapure water at concentrations of 0, 4, 8, 12, 16, and 20 times. One mL of sample solution was added to 1 mL of DPPH solution (0.2 mmol / L), shaken well, and reacted in the dark for 30 min. The absorbance (Ax) was then measured at 517 nm. Trolox at different concentrations (0–0.2 mg / mL) was used as a positive control, and its absorbance was measured using the same method.

[0069] The standard curve of Trolox scavenging DPPH free radicals is as follows: Figure 7 As shown in Figure A, the equation y = 363x + 2.49 was obtained by linearly fitting the DPPH radical scavenging rate to the Trolox concentration, with R0... 2 =0.9939, and the two show a good linear relationship.

[0070] like Figure 7As shown in Figure B, using Trolox as a control, the DPPH free radical scavenging ability of the navel orange peel polyphenol solution increased from 0.039±0.002 mg TE / g to 0.18±0.002 mg TE / g with increasing concentration in the range of 1.65 mg / mL to 33 mg / mL. At a concentration of 33 mg / mL, the scavenging rate of the navel orange peel polyphenol extract reached its peak at 0.18 mg TE / g, indicating that the navel orange peel polyphenols had fully reacted with DPPH, and the DPPH free radical scavenging ability had reached saturation. In conclusion, the navel orange peel polyphenol content is positively correlated with the changes in antioxidant activity.

[0071] 5.2 ABTS free radical scavenging ability After diluting the polyphenol extract, take 1 mL of sample solution, add 1 mL of ABTS working solution, shake well, and react in the dark for 10 min. Measure the absorbance (Ax) at 734 nm. Use Trolox at different mass concentrations (0.025–0.05 mg / mL) as positive controls, and measure their absorbance using the same method.

[0072] The standard curve of Trolox scavenging ABTS free radicals is as follows: Figure 8 As shown in Figure A, the linear fit between the ABTS radical scavenging rate and the Trolox concentration yielded the equation y = 1697x + 15.65, R0. 2 =0.9962, and the two show a good linear relationship.

[0073] Depend on Figure 8 According to B, using Trolox as a control, the ABTS radical scavenging rate of navel orange peel polyphenols extracted by the deep eutectic solvent method increased in a dose-dependent manner with increasing mass concentration. Specifically, the ABTS radical scavenging capacity of the polyphenol extract increased significantly between the mass concentrations of 0.21 mg / mL and 0.33 mg / mL, increasing from 0.026 ± 0.0006 mg TE / g to 0.033 ± 0.0004 mg TE / g. Further increasing the concentration of navel orange peel polyphenols to 33 mg / mL resulted in the ABTS radical scavenging rate reaching its maximum value of 0.050 ± 0.0003 mg TE / g, indicating that the navel orange peel polyphenols had fully reacted with ABTS, and their scavenging capacity for ABTS cationic radicals had reached saturation. The results showed that polyphenols extracted by eutectic solvent extraction had a better ABTS scavenging effect than traditional organic reagents, indicating that polyphenols extracted from navel orange peel by eutectic solvent extraction are not only green and environmentally friendly, but also enhance the ability to scavenge ABTS free radicals and improve antioxidant activity.

[0074] 5.3 Hydroxyl radical scavenging ability After diluting the polyphenol extract, take 500 μL of the sample solution and add 500 μL of 9 mmol / L FeSO4, 500 μL of 9 mmol / L salicylic acid-ethanol solution, and 500 μL of 8.8 mmol / L H2O2 sequentially. After mixing, react at 37℃ in the dark for 30 min, and measure the absorbance Ax at 510 nm. Use Trolox at different mass concentrations (0.5–1 mg / mL) as positive controls and measure their absorbance using the same method.

[0075] Using Trolox as a control, a standard curve was plotted based on its concentration versus hydroxyl radical scavenging rate: y = 49x + 15.39 (R²). 2 =0.994), the effect of polyphenols extracted from navel orange peel by deep eutectic solvent extraction on hydroxyl radical scavenging was studied, and the results are shown in the figure. Figure 9 A.

[0076] Depend on Figure 9 As shown in section B, the extract of polyphenols from navel orange peel exhibits scavenging activity against hydroxyl radicals. The scavenging ability of navel orange peel polyphenols increases with increasing concentration. When the concentration of polyphenols in navel orange peel is between 17 mg / mL and 24 mg / mL, the scavenging rate slowly increases from 0.53 ± 0.01 mg TE / g to 0.65 ± 0.09 mg TE / g. With further increases in concentration, the increase in scavenging ability intensifies, reaching a scavenging rate of 1.02 ± 0.11 mg TE / g at a concentration of 33 mg / mL. In conclusion, navel orange peel polyphenols possess excellent scavenging ability against hydroxyl radicals.

[0077] Comparative Example 2: Comparison of antioxidant activity of ethanol extracts The ethanol-derived polyphenol extract obtained in Comparative Example 1 was used to determine its DPPH, ABTS, and hydroxyl radical scavenging capacity (in Trolox equivalents) at the same mass concentration (33 mg / mL) according to the same method as in Example 5.

[0078] Results Comparison: As shown in the table below, the polyphenols extracted by the DES method of this invention are superior to those extracted by the traditional ethanol method in all three free radical scavenging abilities, especially in the hydroxyl free radical scavenging ability, where the advantage is most obvious, with an activity increase of approximately 41.67%. This proves that this invention not only improves extraction efficiency but also significantly enhances the bioactivity of the obtained product, achieving the goal of high-quality utilization.

[0079] Table 5. Comparison of antioxidant activities of polyphenols obtained by different extraction methods (33 mg / mL)

[0080] In summary, this invention uses navel orange peel as the research object. By comparing the polyphenol yields of navel orange peel in five different deep eutectic solvent systems, the choline chloride-ethylene glycol system was determined to be the optimal deep eutectic solvent. Based on the results of four single-factor experiments, moisture content, solid-liquid ratio, and extraction time were identified as the three factors significantly affecting polyphenol yield. The extraction process of navel orange peel polyphenols was optimized using response surface methodology, and the optimal process parameters were determined to be: eutectic solvent moisture content of 38%, solid-liquid ratio of 1:31 g / mL, and extraction time of 41 min. Under these optimal conditions, three verification experiments were conducted, and the actual polyphenol yield was 10.27 mg / g, differing from the theoretical prediction of 11.17 mg / g by only 0.90 mg / g, indicating that the process conditions optimized by response surface methodology have high reliability. The antioxidant activity of the navel orange peel polyphenol extract was studied, and the results showed that it had good scavenging effects on DPPH free radicals, ABTS free radicals, and hydroxyl free radicals. The results of this experiment provide a foundation for the further utilization of navel orange peel resources and the development of functional products.

[0081] 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 deep eutectic solvent for extracting polyphenols from navel orange peel, characterized in that, It consists of choline chloride as a hydrogen bond acceptor and a compound as a hydrogen bond donor, wherein the hydrogen bond donor is selected from polyols, organic acids and urea.

2. The deep eutectic solvent according to claim 1, characterized in that, The hydrogen bond donor is a polyol or an organic acid.

3. The deep eutectic solvent according to claim 2, characterized in that, The polyol is selected from ethylene glycol or glycerol; and / or the organic acid is selected from lactic acid or malic acid.

4. The deep eutectic solvent according to claim 3, characterized in that, The hydrogen bond donor is ethylene glycol.

5. The deep eutectic solvent according to any one of claims 1 to 4, characterized in that, The molar ratio of choline chloride to the hydrogen bond donor is 1:1 to 1:3, preferably 1:

2.

6. A method for extracting polyphenols from navel orange peel using a deep eutectic solvent, characterized in that, The navel orange peel powder was extracted using the eutectic solvent as described in any one of claims 1 to 5.

7. The method according to claim 6, characterized in that, Includes the following steps: Navel orange peel powder is mixed with the deep eutectic solvent with a water content of 30%~50% at a material-to-liquid ratio of 1:25~1:35 g / mL, and extracted at 40℃-60℃ for 30-50 minutes. The supernatant is collected by centrifugation. Preferably, the deep eutectic solvent is choline chloride-ethylene glycol with a water content of 38%, a material-to-liquid ratio of 1:31 g / mL, an extraction time of 41 minutes, and an extraction temperature of 50℃.

8. A polyphenol extract from navel orange peel, characterized in that, The extract is prepared by the method according to claim 6 or 7.

9. The use of the navel orange peel polyphenol extract as described in claim 8 in the preparation of antioxidants.

10. The application according to claim 9, characterized in that, The antioxidants are used in food, health products, or cosmetics.

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