Deep Eutectic Solvents Extraction of Polyphenol from the stem of Broccoli :Optimization, Components , and Antioxidant Activity

Deep Eutectic Solvents Extraction of Polyphenol from the stem of Broccoli :Optimization, Components , and Antioxidant Activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Deep Eutectic Solvents Extraction of Polyphenol from the stem of Broccoli :Optimization, Components , and Antioxidant Activity bingqing wang, peiyun chen, huien zhang, Liqing chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4142867/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The stem of broccoli has been reported to contain high levels of polyphenols and other active compounds.To extract polyphenol from broccoli stem more efficiently, a novel procedure of deep eutectic solvent extraction (DESE) was proposed in this paper.The extraction process was optimised by response surface methodology. The optimum extraction parameters to obtain the highest yield of polyphenol 5.10 ± 0.04 mg/g from broccoli stem powder via choline chloride-urea (molar ratio 1:3) were obtained at liquid-solid ratio of 41:1 mL/g, water content of 60%, extraction temperature of 80°C and extraction time of 55 min. The components of the main polyphenol were identified by high-performance liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS/MS).Compositional analysis shows that the extracted polyphenols are rich, including quercetin, isochlorogenic acid, trans-cinnamic acid and sinapinic acid. Furthermore, in vitro tests proved that the extracted polyphenols obtained in (ChCl-urea) DES possessed excellent antioxidant activity.These results provided an effective and feasible method for the extraction and separation of polyphenols in broccoli stalks, providing some technical support and theoretical basis for the green extraction of broccoli waste stalks. Polyphenol deep eutectic solvent extraction broccoli stem antioxidant activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Broccoli (Brassica oleracea var. italica Plenck), a plant belonging to the Bassicaceae family, is a relative of brussels sprouts、cabbage、cauliflower, and kale and an important annual crop in the world(Ilahy et al., 2020 ). Broccoli has gained popularity and as a good source of health promoting compounds owing to its excellent nutritional values(Hang et al., 2022;Takashi et al.,2023;Miyahir et al.,2022)It is a source of diverse nutrients (e.g., protein,dietary fiber,minerals, and vitamins,) and phytochemicals (e.g., glucosinolates and phenolic compounds)(Li et al.2021; Thomas et al. 2018; ).However, the edible part of broccoli accounts for less than half of the whole broccoli, and the broccoli rhizomes account for a large proportion of the weight of the whole broccoli(Le Borja2020). The harvesting of broccoli produces a large quantity of plant by-products, mainly leaves and stems, which make up more than 95% of the material harvested.(Domínguez et al.2011;Borja Borja et al.2020). Recently, numerous studies have explored the use of food by-products as a source of valuable bioactive compounds with potential application in the treatment and prevention of human diseases (Alejandra et al.2022;Gudiño et al. 2022).Among the main components of interest in broccoli by-products are glucosinolates and phenolic compounds, as well as carotenoids, sterols, vitamin C, fibre and mineral elements.Due to the peculiarity of their structure, polyphenols have good antioxidant and free radical scavenging effects. It has good effects in removing free radicals from the human body, defending and treating diseases of the circulatory system, and preventing the deterioration of physiological functions (Pagliarulo 2016; et al. .César et al.2019;Talmaciu et al.2014; Emilio et al.2022).In the research of plant polyphenols, separation and extraction is an important part. Solvents such as water, methanol and its aqueous solutions, ethanol and its aqueous solutions, ethyl acetate, acetone are commonly used for the extraction of plant polyphenols (Gloria et al.2017;Hu et al. 2022 ;Kumar et al.2023). However, these conventional solvents are often flammable, toxic, poorly biodegradable and cannot solve the problem of rapidly extracting products of different polarities and simultaneously maintaining the activity of the products.(Wagare et al.2021;Sara et al. 2018 ). In recent years, deep eutectic solvents (DES) have been found to be composed of hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) in a certain proportion, which can provide or accept external electrons or protons to form hydrogen bonds, why can dissolve a variety of substances. Usually, they are mixtures of non-toxic, cheap and renewable components, which have good properties of ionic liquids, such as excellent solubility, chemical stability and thermal stability, but also have low cost and no pollution(Justyna et al 2022 ;Ashraf et al 2023 ).Currently, some studies have shown that DESs can be used as alternative solvents to extract polyphenols from natural samples(Bener et al 2022 ).However, extraction of polyphenol from broccoli stem has rarely been reported. In the present study, a highly efficient and nonpolluting extraction procedure was developed for the extraction of polyphenols from the discarded stems of broccoli.The optimum extraction conditions were optimised using response surface methodology (RSM) with Box-Behnken design (BBD). The components of the main polyphenol were identified by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS/MS).Moreover, to further understand its potential food applications and health benefits, the in vitro antioxidant activity of the extracted polyphenols obtained in (ChCl-urea) DES was determined by DPPH free radical scavenging ability、ABTS free radical scavenging ability、FRAP total reducing power and ORAC total antioxidant capacity. MATERIALS AND METHODS Materials and Chemical Reagents Fresh broccoli samples were purchased from a local supermarket (Ningbo,China).Standard gallic acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Chemical Corporation (China). Reagents containing folinphenol, citric acid, glucose, sucrose, urea, choline chloride, anhydrous sodium carbonate ,1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2 -Azinobis-(3- ethylbenzthiazoline-6-sulphonate) (ABTS), ORAC total antioxidant assay kit and FRAP antioxidant assay kit were purchased from Sinopharm Group Chemical Reagent Co. Ltd(Shanghai, China)，Other chemicals used throughout the experiment were analytical grade. Extraction Methods Deep Eutectic Solvent Preparation The preparation of DESs was carried out according to literature methods (Jun et al.2018;Li et al.2021;Li et al.2021).Four different types of DESs were prepared by the heating method. A two-component mixture, HBA and HBD, was added to a round-bottomed glass flask and stirred in a constant temperature magnetic stirrer at 80°C until a homogeneous liquid was formed（40-60 min），and a colourless and transparent thick liquid was obtained at room temperature.The composition, molar ratios and codes of DESs used in this study were shown in Table 1. Extraction of polyphenols The stems of fresh broccoli were washed with tap water, cut into 1cm pieces, dried in an oven at 60°C (DHG-9053A, Electric Heating Constant Temperature Blast Drying Oven, Yilin Scientific Instrument Co., Ltd, Shanghai, China) to constant weight and crushed with a food processor (Joyoung Corporation., Ltd, Zhejiang China) and the powder was obtained through the 60-size mesh. The extraction process was performed according to the previously described method(Liu et al.2019;Mondal et al.2019).For all experiments, a quantity of 0.25 g of broccoli stem powder was placed with a certain amount of DES solution, water or70% ethanol, mixed by vortex shaking evenly, and placed in a constant temperature water bath for a certain period of time (5 min shaken by vortexing once)(QL861 Vortex Shaker, Qilin Bell Instrument Manufacturing Co., Ltd.Haimen,China), after constant temperature extraction, the mixture was cooled down to room temperature in a short time, and then the supernatant was collected by centrifugation under 5,000 g for 20 min (LC-180 Centrifuge, Keda Innovation Incorporation, Ltd. China). Total polyphenols content Determination The Folin-Ciocalteu colorimetric method was used to quantify the total polyphenol content（Batool et al.2023）. Gallic acid (Sigma-Aldrich, St-Quentin Fallavier, France) was used for the calibration curve. The results were expressed as mg of gallic acid equivalent (GAE) per gram of dry matter (mg GAE/g DM).The determination of total polyphenols was carried out according to literature methods (Rosa et al.1999 ) (Singleton, Orthofer, & Lamuela-Raventós, 1999).The Folin-Ciocalteu colorimetric method was used with gallic acid as standard.Gallic acid series standard working solution 0, 10, 20, 30, 40, 50 μg/mL were prepared respectively, distilled water (for blank control) into a 25 mL colorimetric tube, add 5.0 mL of Folin-Ciocalteu reagent, shake well. Within 3min~8min of the reaction, add 4.0mL of 7.5% Na2CO3 solution, make up the volume with water and shake well. After reaction at room temperature in the dark for 60 min, the absorbance (A) was measured with a spectrophotometer at a wavelength of 765 nm and the A value was determined for 3 times in parallel.The standard curve for gallic acid is obtained as y=10.8771x+0.0032，R 2 =0.9991. The results were expressed as mg of gallic acid equivalent (GAE) per gram of dry matter (mg GAE/g DM). Total phenolic content（mg GAE/g）= m 1 ×V 1 /V×m×1000 Where m 1 is Content of gallic acid found in standard equation(mg/mL);V 1 is Total volume of extraction(mL)；V is sample volume taken during measurement(mL)；m is the amount of sample(g). Optimization Extraction on the Yield of polyphenols The effects of 4 different DESs on the extraction yield of polyphenols were investigated by single factor test, effects of DES-1 molar ratio (1:2, 1:1, 2:1,3:1 and4:1), water content of DES-1 (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, w/w), extraction time (10, 30, 50, 70 and 90 min), extraction temperature (40, 50, 60, 70, 80 and 90℃) and liquid/solid ratio (10, 20, 30, 40, 50, 60 and 70 v/w) on the extraction efficiency of polyphenols were tested.On the basis of single factorial experiment, extraction time, extraction temperature and liquid-solid ratio were selected as independent variables, and the extraction yield of polyphenols was the response value, Design-Expert 10.0.7 software was used to perform response surface analysis to optimise the extraction conditions. The response surface test factor coding and level design are shown in Table 2, and each experiment was replicated three times. The experimental data obtained from the Box-Behnken design (BBD) were analysed by regression analysis. Under the optimal parameters, the extraction experiment was repeated three times to verify the accuracy of RSM. The polyphenols from broccoli stem were extracted under optimised conditions to determine their structural characterisation and bioactivities. Table 2 Experimental levels of independent variables Variables Units Symbol Variable levels -1 0 1 extraction temperature ℃ A 70 80 90 extraction time min B 40 50 60 liquid–solid ratio C 30:1 40:1 50:1 Component Analysis Chromatographic separation and detection in the samples were analysed using a Waters synpat G2 (UPLC-ESI-QTOF/MS) system (Waters). A reverse-phase ACQUITY UPLC C18 column (100 mm × 2.1 mm × 1.7 μm) with a flow rate of 0.2 mL min-1 was used for the chromatographic separation. A mixture of water (A) and acetonitrile (B) was used as the mobile phase, and the linear gradient programs were as follows: 0-1.0 min, 5% (B); 1.0-12.0 min, 5%→100% (B); 12.0-13.0 min, 100%→100% (B); 13.0-15.0 min, 100%→50% (B); The injection volume was 2 μL. Samples were analysed using ESI negative ionisation modes, scanning the m/z range between 50 and 1200. The desolvation gas flow and temperature were 900 L h-1 and 350 °C, respectively. The mass spectra were acquired using a capillary voltage of 3. 0 kV and cone hole voltage 40 kV. Instrument control and data acquisition were performed using MassLynx software (v 4.1).. Antioxidant Assays DPPH Free Radical Scavenging Assay The antioxidant capacity of extraction of broccoli stem sample was compared with water and 70% ethanol solution. The DPPH free radical scavenging assay was performed as previously reported (Duan et al.2010). Briefly, 1 mL of DES extract of Broccoli stem sample and 3 mL of 50 ug/mL DPPH solution were added to the same test tube with stopper and shaken well.The tubes were left tightly sealed at room temperature for 30 min and anhydrous ethanol was used as reference solution at 517 nm.The absorbance was measured at 517 nm wavelength and measured three times in parallel. The DPPH free radical scavenging rate was calculated using the following equation: DPPH free radical scavenging percentage (%)=[ 1-(A 2 -A 1 )/A 0 ] x 100% Where A 2 is the absorbance of 3ml of DPPH solution after adding 1 mLof extract; A 1 is the absorbance of 1 mL of extract and 3 mL of ethanol; A 0 is the absorbance of 3ml of DPPH solution and 1mL of solvent(DES) ABTS Free Radical Scavenging Assay The ABTS free radical scavenging assay was performed as previously reported, with minor modifications (Meng L. et al. 2019). Briefly, ABTS reserve solution was prepared by mixing 7.4 mmol/L ABTS solution with 4.5 mmol/L potassium persulfate solution for 12-16 h at 25 °C until a dark blue-green colour developed. An aliquot of 200 μL of antioxidant solution at various concentrations was mixed with 200 μL of ABTS solution (1 mM in 95% ethanol). All reaction mixtures contained 1.0 mM ABTS and antioxidant solution at each concentration and the control solution contained no antioxidants. ABTS free radical scavenging rate was calculated by the following equation: ABTS free radical scavenging percentage (%)=[ 1-(A 2 -A 1 )/A 0 ] x 100% Where A 2 is the absorbance of 200μL of ABTS solution after adding 200μLof extract; A 1 is the absorbance of 200μL of extract and 200μL of 95% ethanol; A 0 is the absorbance of 200μL of ABTS solution and 200μL of solvent(DES). FRAP total antioxidant capacity Total antioxidant capacity was determined according to the Total Antioxidant Capacity Assay Kit with FRAP method manual (Yuan Yea, Shanghai). The FRAP working solution with deionised water was prepared from the kit stock solution. 30 uL of ferrous ion standard solution with different gradient concentrations were added to 264 uL of FRAP solution respectively, and 30 uL of deionised water instead of a sample was used as a blank, the mixture was vortexed and incubated at 37°C for 30 min away from light. Absorbance was measured at 593 nm using a spectrophotometer. ORAC total antioxidant capacity The total Oxygen Radical Absorbance Capacity (ORAC) of the extract was evaluated using the test kit (Yuan ye, Shanghai). The fluorescence intensity was followed to monitor the decay of the fluorescence curve. A calibration curve was obtained by plotting the net curve against Trolox solutions in the range 0-40 μM. The equation for the calibration curve was y = 3.943x-2.003, with a good correlation coefficient (r2 = 0.979). Statistical Analysis All determinations were performed at least three times in triplicate and all results are expressed as mean ± standard deviation (SD). Statistical significance between groups was determined by analysis of variance (ANOVA) and least significant difference (LSD) test using SPSS 19. A p < 0.05 was considered statistically significant.In all figures, the error bars correspond to the 95% confidence level. RESULTS AND DISCUSSION Optimization Extraction Effect of Deep Eutectic Solvent System The extraction solvent played a key role in the extraction of natural products.The physicochemical properties of the extraction solvent were essential conditions for this study,which may be due to the permeability of the extractant in the sample matrix,which can be directly determined by them(Xing et al.2022).Table 1 showed the composition,molar ratios and symbols of the DESs used in this study. As a comparative study, the experiment used the four deep eutectic solvents、hot water extraction and 70% ethanol organic solvents. Extraction efficiency varied with different types of extraction solvent. As shown in Figure 1A,the effects of DES-1 on the extraction yields of polyphenols were significantly lower than others(p0.05).DES2 exhibited a significantly higher extraction efficiency than others,reaching 5.051±0.04mg.g -1 . DW.This could be attributed to the low viscosity, relatively large diffusion coefficient, good flowability, better permeability to the cell wall and the presence of a certain viscosity, which promoted the suspension and dispersion of broccoli stem powder in the In solvent, increased the contact area between the two and is conducive to the extraction of polyphenols (Malaeke et al.2018 ). Therefore, a deep eutectic solvent system composed of choline chloride and urea was considered for the follow-up experiments. Table 1 The composition and molar ratios of DESs were used in this study Abbreviation HBA HBD Molar ratio(mol/mol) DES1 Choline chloride Citric Acid 2:1 DES2 Choline chloride Urea 2:1 DES3 Choline chloride Sucrose 2:1 DES4 Choline chloride Glucose 2:1 Effect of Mole Ratio of Deep Eutectic Solvent As shown in Figure 1B, the extraction efficiency of polyphenols was significantly affected by the molar ratio of DES-2. The extraction efficiency increased with the increasing ratio of urea in DES-2, when the molar ratio of urea/choline reached 3:1, the yield of polyphenols was the highest. It may be due to urea improving the viscosity and surface tension of DES-2, the more urea in the system, the stronger the hydrogen bond between urea and choline chloride, the stronger the intermolecular force between urea and choline chloride, the lower the viscosity of DESs and the better the flowability. Since the number of hydrogen bonds formed between urea and choline chloride was limited and had a certain degree of saturation, as the molar ratio of urea/choline chloride continued to increase, the hydrogen bonding force between urea and choline chloride increased, making the viscosity of DESs larger, resulting in poor fluidity, which made the subsequent extraction process difficult. Therefore, the molar ratio of urea-ChCl- in DES used in the subsequent experiments was 2:1. Effect of Water Content of Deep Eutectic Solvent As shown in Figure 1C, the extraction efficiency varied depending on the water content of the DES. The extraction efficiency increased as the water content of DES-2 increased from 10 to 60%, and the yield of polyphenols increased from 13.82±0.05% to 50.83±0.04%. When the water content exceeded 70%, the further increase of the water content resulted in a significant decrease. This phenomenon could be attributed to the fact that increasing the water content in DES-2 from 10 to 60% resulted in a more suitable viscosity and polarity of DES-2 to improve the interactions between polyphenols and DES-2,which obtained a high extraction yield, the interaction force between the deep eutectic solvent and the polyphenols was weakened after the addition of excess water.Therefore,DES-2 with the water content of 60% was considered for the subsequent experiments. Effect of Extraction Time As shown in Figure 1D, the extraction efficiency of ployphenols was affected by the extraction time. With the extension of the extraction time, the extraction efficiency of ployphenols increased from 4.16 to 4.87, but when the extraction time was increased from 50 to 90 min, the extraction efficiency of ployphenols decreased from 4.87 to 3.03.This phenomenon could be attributed to the prolongation of the extraction time, which may destroy the cell structure, increase the permeability and be conducive to ployphenols, but a long-term extraction at high temperature may lead to the decomposition of the extracted polyphenolic compounds. Therefore, an extraction time of 50 min was chosen for the following experiments. Effect of Extraction Temperature As shown in Figure 1E, we can see that the total phenolic yield of broccoli stem increased with the increase of extraction temperature from 40 to 80℃, and reached the maximum polyphenol yield 5.10 at 80℃. However, when the water content exceeded 60%, the further increase of water content resulted in a significant decrease(p<0.05).When the extraction temperature exceeded 80℃, the further increase of temperature resulted in a significant decrease in the extraction efficiency of polyphenols(p<0.05).This phenomenon could be attributed to the high temperature, which not only can reduce the viscosity of DES-2 and increase the diffusion of DES-2, but also accelerate the mass transport of polyphenols ( Zhang et al.2012), which makes polyphenols dissolve more in solvent and improve the extraction yield. However, the high temperature will damage the structure of the polyphenols. Therefore, the extraction temperature was chosen to be 60℃for the following experiments. Effect of Liquid–Solid Ratio The liquid-solid ratio was one of the most important factors influencing the extraction yield of polyphenols. As shown in Figure 1F, the extraction efficiency of polyphenols increased with increasing liquid-solid ratio from 10:1 to 40:1, reaching a maximum of 5.12 mg/g at 40:1 (v/w). However, a further increase in the liquid-solid ratio resulted in a lower extraction efficiency. This phenomenon could be attributed to a large amount of DES-2, which could increase the contact area between the material and the solvent and the concentration gradient becomes larger, which is conducive to the dissolution of polyphenols, but when the solvent was increased too much, the extraction effect on polyphenols is not obvious or even diluted, and the yield decreased.Therefore, 40:1 (v/w) was chosen as the optimal liquid-solid ratio in this experiment. Optimization of the extraction conditions by RSM The RSM-based optimisation was aimed at minimising the use of solvents and broccoli stem powder, saving time and energy during the experiments, and identifying the ideal combination of factors that would result in the maximum extraction of polyphenols. To ensure the fitness of the proposed model at the 95% confidence level, the p-value should be less than 0.05 (p < 0.05) (Temova & Roškar, 2016). Table 3 shows the analysis of variance (ANOVA) for the model.Based on the ANOVA results, the developed model was found to be significant for polyphenol extraction (p < 0.05). The values of coefficient of determination (R 2 ) were 0.9897, together with non-significant lack of fit（p＞0.05），suggesting a good fit, the predicted model seemed to reasonably represent the observed values. The individual factors and the coefficients of the quadratic terms (A 2 、B 2 and C 2 ) were also found to be significant. According to the size of the F-value, the three factors A, B and C can be considered as influencing the yield of broccoli stem polyphenols. The order from largest to smallest was C (liquid/material ratio)>B (extraction time)>A (extraction temperature). Table 3 Analysis of variance (ANOVA). Source df Sum of Squares Mean Square F-Value P-Value Significance Model 9 5.91 0.6568 74.61 ＜0.0001 ** Extraction temperature- 1 0.082 0.082 9.32 0.0185 * Extraction time 1 0.2426 0.2426 27.55 0.0012 ** Liquid–solid ratio 1 0.2824 0.2824 32.08 0.0008 ** Extraction temperature*Extraction time 1 0.0784 0.0784 8.91 0.0204 * Extraction temperature*Liquid–solid ratio 1 1.38 1.38 156.84 ＜0.0001 ** Extraction time*Liquid–solid ratio 1 0.0203 0.0203 2.31 0.1726 Extraction temperature*Extraction temperature 1 1.25 1.25 141.51 ＜0.0001 ** Extraction time*Extraction time 1 0.1507 0.1507 17.12 0.0044 ** Liquid–solid ratio*Liquid–solid ratio 1 2.11 2.11 240.22 ＜0.0001 ** Residual 7 0.0616 0.0088 Lack of Fit 3 0.0505 0.0168 6.03 0.0577 non-significance Pure Error 4 0.0112 0.0028 Cor Total 16 5.97 R-Squared 0.9897 Adj R-Squared 0.9764 Pred R-Squared 0.8619 2D contour plots were obtained using two factors at a time, representing a two-way interaction of the extraction factors, to evaluate their combined effect on the response. From the contour plots as shown in Figure 3, these plots were used as a reference to view the trend of the significant factors and also to study their interactions.It was concluded that the optimised extraction parameters for obtaining maximum extraction were an extraction temperature of 70-90 ℃, an extraction time of 40-60 min and a liquid-solid ratio of 30-50. Compared to the interaction between other factors, the slope change of the response surface between liquid-solid ratio and extraction temperature is steeper, which had a great influence on the extraction amount of broccoli stem polyphenols. These results were consistent with those obtained by ANOVA. A linear equation for the predicted total polyphenols yield of extraction was obtained by multiple regression analysis of the data, which was stated as follows: Y=5.094-0.1013A+0.1741B+0.1879C+0.1400AB+0.5875AC-0.0712BC-0.5439A2-0.1891B 2 -0.7087C 2 where Y was the yield of polyphenols （mg /g Dw），A was the extraction temperature（℃）， B was the extraction time (min)， and C was the Liquid-solid ratio（v/w）. In view of the above results, the selected variables were further optimised using Design-Expert. The software identified the optimal conditions of three parameters for extraction: extraction time (A) of 54.63 min, extraction temperature (B) of 60℃, liquid-solid ratio (C) of 41:1, and the maximum estimated extraction values of 5.12mg.g-1dw. Furthermore, the validity of the model was checked and the experimental value (5.103 ± 0.03 mg/g) was closed with the predicted value (5.12 mg/g). LC-MS Analysis of polyphenols UPLC-Q-TOF/MS was used for further identification of polyphenolic compounds. The identification was done by comparing the masses of molecular ions, fragment ions with the results reported in the literature. The data obtained from the analysis of the molecular ion, fragment ion and retention time of the polyphenol compound peaks by HPLC-ESI-QTOF/MS are shown in Table 4. The four main polyphenols are quercetin, isochlorogenic acid, transcinnamic acid and sinapinic acid.The total ion chromatogram of a broccoli stem extract after (ChCl-Urea) DES and the representative SIM chromatograms of the main polyphenolic compounds are shown in Figure 4 and Figure 5. Table 4 The data obtained from the analysis of the molecular ion and retention time of polyphenol compounds peaks by HPLC-ESI-QTOF/MS Entry Systematic name (Synonym) Rt (min) Formular Mass (Da) [M-H]− 1 Sinapinic acid 18.72 C 22 H 42 O 2 338.57 337.31 2 trans-Cinnamic acid) 4.36 4.71 4.96 C 9 H 8 O 2 148.16 147.04 3 quercetin 2.52 C 15 H 10 O 7 302.24 301.03 4 Isochlorogenic acid 3.69 C 25 H 24 O 12 516.45 515.12 Antioxidant Activity of broccoli extracts broccoli extracts from different solvents Study of the effects of the in vitro antioxidant activity of different extract solvents (DES-2/H2O /70% EtOH). As shown in Figure 6(A), the antioxidant capacity of different solvent extracts is directly proportional to their concentration and increases with increasing concentration. At the mass concentration of 250 μg/ml, the DPPH radical scavenging capacity of 70% EtoH, DES-2 and H2O was 79.48%, 78.12% and 34.39%, respectively. As shown in Figure 4(B), when the different solvent extraction concentrations increased from 20 μg/mL to 250 ug / mL, the clearance of ABTS radical from DES-2 increased from 21.50% to 82.93%, from 70%EtoH increased from 20.37% to 79.63%, and from H2O extract increased from 12.37% to 34.36%, the clearance of ABTS radical of all three extraction solvents were proportional to the concentration. As shown in Figure 6 (C), the Trolox molar equivalence of reduced Fe(III) was achieved as the different solvent extraction concentrations increased from 50 μg/mL to 250 ug/mL. The results showed that DES-2 extract was the best antioxidant, followed by 70% ethanol extract. The water extract has the lowest oxidation, the TE content is only 105 mmol/L at the concentration of 250 u g / mL. As shown in Figure 6 (d), the total antioxidant capacity (TRAC) of the three Trolox at different solvent extraction concentrations increased from 50 μg/mL to 250 ug / mL. DES-2 extract was 3.45 ± 0.1; 3.21 ± 0.2umol TE/L for 70% ethanol and water 1.27 ± 0.2umol TE/L; it is concluded that the antioxidant activity of DES-2 broccoli extract was better than that of the conventional extract. CONCLUSION This study has demonstrated the efficiency of deep eutectic solvents as green alternatives for the extraction of polyphenols from Broccoli Stem.ChCl-urea was selected from a series of solvents for the extraction of total polyphenols from Broccoli Stem. Based on the results of the single factor experiment, RSM was used to identify the main parameters and optimise the extraction conditions.The optimal process conditions for the extraction of Broccoli Stem were determined as the extraction solvent Choline chloride-urea (molar ratio 1:3), water content of 60%, liquid-solid ratio of 41:1 mL/g, extraction temperature of 80°C and extraction time of 55 min, resulting in a maximum yield of 5.13mg.g − 1 dw. In addition, the major polyphenolic composition of ChCl urea extraction from broccoli stem was quercetin, isochlorogenic acid, transcinnamic acid and sinapinic acid. In addition, the in vitro antioxidant activity of was evaluated using multiple radical scavenging assays. The results of bioactivities indicated that the polyphenols of ChCl urea extraction from broccoli stem exhibited excellent antioxidant activity.DPPH radical scavenging capacity and its FRAP total reducing power is higher than that of tranditonal solvent extract. The use of deep eutectic solvents allowed the extraction of a higher yield of polyphenols and improved their biological activities.DES extraction of polyphenols from broccoli stems has the characteristics of simple synthesis, low cost, and good environmental protection extraction effect. Overall, it provides certain theoretical support and technical support for its application and development in the extraction of polyphenols from broccoli waste. Declarations Funding Declaration This work was supported by Ningbo Public Welfare Fund Project(Grant 20211JCGY020055). Data availability The results/data/figures in this manuscript have not been published elsewhere, nor are they under consideration (from you or one of your Contributing Authors) by another publisher.I don't have any research data outside the submitted manuscript file. Declaration of Competing Interest: I declare that the authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Author Contribution Bingqing Wang ,Peiyun Chen,Huien Zhang and Liqing Chen wrote the main manuscript text prepared and Huien Zhang perpared figures 1-3..All authors reviewed the final manuscript. 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Food and Bioprocess Technology,12(01):183-192.DOI：10.1007/s11947-018-2199-x Li C, Huang C, Zhao Y, Zheng C, Su, H, Zhang L, Luo W, Zhao H, Wang S, Huang LJ (2021).Effect of Choline-Based Deep Eutectic Solvent Pretreatment on the Structure of Cellulose and Lignin in Bagasse. Processes 9:384. doi:10.3390/pr9020384 Malaeke H , Housaindokht M R , Monhemi H , et al(2018). Deep eutectic solvent as an efficient molecular liquid for lignin solubilization and wood delignification Journal of Molecular Liquids, 63:193-199.DOI:10.1016/j.molliq.2018.05.001. Ma Z Y et al(2019.)[1] A, L. M. , B, J. Z. A. , C, Y. M. , A, X. S. , A, D. L. ,and A, L. L. , et al.Ma Z Y .Composition and antioxidant activity of anthocyanins from Aronia melanocarpa cultivated in Haicheng, Liaoning, China[J].Food Bioscience, 2019, 30(C): 106102-106102doi:10.1016/j.fbio.2019.100413 Miyahira R F ,de Lima Pena, Fabíola, Fabiano G A ,et al.Changes in Phenolic Compound and Antioxidant Activity of Germinated Broccoli, Wheat, and Lentils during Simulated Gastrointestinal Digestion[J].Plant Foods for Human Nutrition, 2022.DOI:10.1007/s11130-022-00970-7. Mondal M,De S(2019). Purification of Polyphenols from Green Tea Leaves and Performance Prediction Using the Blend Hollow Fiber Ultrafiltration Membrane Food and Bioprocess Technology 12(06):438.DOI：10.1007/s11947-019-02262-6 Pagliarulo C, De Vito V, Picariello G,Colicchio R, Pastore G, Salvatore P, Volpe MG. Inhibitory effect of pomegranate (Punica granatum L.) polyphenol extracts on the bacterial growth and survival of clinical isolates of pathogenic Staphylococcus aureus and Escherichia coli. Food Chem 190:824–831.doi:10.1016/j.foodchem.2015.06.028 Pan X, Xu LM, Meng JL, Chang MC, Cheng YF, Geng XR, Guo DD, Liu RZ(2022).Ultrasound-Assisted Deep Eutectic Solvents Extraction of Polysaccharides From Morchella importuna: Optimization, Physicochemical Properties, and Bioactivities.Frontiers in Nutrition 9:DOI:10.3389/fnut.2022.912014 Rosa M,Lamuela R,Singleton V L , Orthofer R(1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent.Methods in Enzymology 299C(1):152-178.DOI:10.1016/S0076-6879(99)99017-1 Sara C, Cunha J, Fernandes O(2018),Extraction techniques with deep eutectic solvents,TrAC Trends in Analytical Chemistry 105:225-239.doi:10.1016/j.trac.2018.05.001. Talmaciu, A. I., Volf, I., & Popa, V. I. (2015). A comparative analysis of the "green" techniques applied for polyphenols extraction from bioresources. Chem Biodivers, 12：1635–1651. doi：10.1002/cbdv.201400415 Takashi W, Hiroaki K(2023).An approach of shelf-life extension technology focusing on recovery lug of physiological responses: Kinetic analysis for residual effect of modified atmosphere packaging on the color changes of broccoli flower buds,Postharvest Biology and Technology206:112579.doi:10.1016/j.postharvbio.2023.112579. Temova, Ž., & Roškar, R. (2016). Stability-indicating HPLC-UV method for vitamin D3 determination in solutions, nutritional supplements and pharmaceuticals. Journal ofChromatographic Science, 54(7), 1180–1186. doi:10.1093/chromsci/bmw048. Thomas M, Badr A, Desjardins Y, Gosselin A, Angers P(2018)Characterization of industrial broccoli discards (Brassica oleracea var. italica) for their glucosinolate, polyphenol and flavonoid contents using UPLC MS/MS and spectrophotometric methods. Food Chemistry, 245:1204–1211. doi:10.1016/j. foodchem.2017.11.021 Wagare DS., Shirsath S.E., Shaikh M. et al(2021). Sustainable solvents in chemical synthesis: a review. Environ Chem Lett 19: 3263–3282 .doi:10.1007/s10311-020-01176-6 Xing C , Cui W Q , Zhang Y ,et al(2022).Ultrasound-assisted deep eutectic solvents extraction of glabridin and isoliquiritigenin from Glycyrrhiza glabra: Optimization, extraction mechanism and in vitro bioactivities Ultrasonics Sonochemistry 83:105946.DOI:10.1016/j.ultsonch.2022.105946 Zhang Q, De Oliveira Vigier K, Royer S, Jerome F(2012). Deep eutectic solvents:syntheses, properties and applications. Chem Soc Rev. 41:7108–46.doi: 10.1039/c2cs35178a Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4142867","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283370940,"identity":"4c3dae33-a5b6-4f19-be65-8bc8a6c58b4b","order_by":0,"name":"bingqing wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACfvbmA4f//vsvJ8/eQKQWyZ5jiQ942JiNDXsOEKnF4IaPsQFQS2LDjQRibZnBYyYhwcNmzDjz8cYbDDU20QS18Eu3lUkYSPDIsUunFVswHEvLbSBoy5zD2yQSDCSMGWfnmEkwNhwmrMXgRoKZxIEEg8SGm2eI1pJibNhwIAHofR4itYAC+TFjwwFgIAP9kkCMX8BRCdQCjMrDG298qLEhrAXFkRIJpCiHaCFVxygYBaNgFIwMAADNE0DxlvuaJQAAAABJRU5ErkJggg==","orcid":"","institution":"Zhejiang Wanli University","correspondingAuthor":true,"prefix":"","firstName":"bingqing","middleName":"","lastName":"wang","suffix":""},{"id":283370941,"identity":"3d2198f3-da0c-499e-bf1b-d7f2ea705543","order_by":1,"name":"peiyun chen","email":"","orcid":"","institution":"Zhejiang Wanli University","correspondingAuthor":false,"prefix":"","firstName":"peiyun","middleName":"","lastName":"chen","suffix":""},{"id":283370942,"identity":"33f83e1d-a6f4-47d0-a25b-2901987ff966","order_by":2,"name":"huien zhang","email":"","orcid":"","institution":"Zhejiang Wanli University","correspondingAuthor":false,"prefix":"","firstName":"huien","middleName":"","lastName":"zhang","suffix":""},{"id":283370943,"identity":"7f9bc4f3-0415-48b1-bdfe-0b9806423aa4","order_by":3,"name":"Liqing chen","email":"","orcid":"","institution":"Zhejiang Wanli University","correspondingAuthor":false,"prefix":"","firstName":"Liqing","middleName":"","lastName":"chen","suffix":""}],"badges":[],"createdAt":"2024-03-21 10:46:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4142867/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4142867/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53556624,"identity":"42f7a1ec-59c9-4211-ae0f-53a4f42e6804","added_by":"auto","created_at":"2024-03-27 12:43:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110837,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of dfferent extraction parameters in the extracion yield of polyphenols of Broccoli Stem. (A) Types of DESs.(B)mole ratio.(C) Water content.(D)Extraction time.(E)Extraction temperature.(F) ratio of liquild-solid\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4142867/v1/8368429352f7e96abed27cba.png"},{"id":53556622,"identity":"232ce71e-c04b-4328-8d75-a4c6d4a7ae0e","added_by":"auto","created_at":"2024-03-27 12:43:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":146841,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3. Contour plots obtained from the design of experiments approach for maximizing extraction of polyphenols.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4142867/v1/ab7a7495820817f18f3961bd.png"},{"id":53556623,"identity":"0daaa26a-181b-4dfc-a353-ebe64a2ac1c8","added_by":"auto","created_at":"2024-03-27 12:43:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13014,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 4 Total ion chromatogram of an extract of broccoli stem after (ChCl-Urea) DES using UHPLC-QTOF-MS in ESI \u003csup\u003e-\u003c/sup\u003e mode\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4142867/v1/73c466ba99ce1572d48ca26e.png"},{"id":53556625,"identity":"6210a611-0a07-4c5e-b240-43f5589201cb","added_by":"auto","created_at":"2024-03-27 12:43:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60763,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 5 Representative SIM chromatograms obtained for an extract of broccoli stem after (ChCl-Urea) DES using UHPLC-QTOF-MS\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4142867/v1/f0dccd321f87b697ba9364b7.png"},{"id":53556626,"identity":"d7f34187-eccd-41da-80df-2a40aa7af553","added_by":"auto","created_at":"2024-03-27 12:43:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101926,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 6 Effects of different extraction solvents on antioxidant activity\u003c/p\u003e\n\u003cp\u003e（A）the DPPH radical scavenging capacity;（B）the ABTS radical scavenging capacity;（C）Frap;（D）ORAC(Different lower-case letters represent the same concentration there were significant differences between the different groups under the degrees(P\u0026lt;0.05))\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4142867/v1/bbd07680a6464b7ce2f0514b.png"},{"id":54119859,"identity":"2a9b0626-2fd0-4a92-a25d-9af794cdf7ca","added_by":"auto","created_at":"2024-04-04 21:22:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":983693,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4142867/v1/99ec3d79-7166-45e5-b701-1b30fd587538.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Deep Eutectic Solvents Extraction of Polyphenol from the stem of Broccoli :Optimization, Components , and Antioxidant Activity","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eBroccoli (Brassica oleracea var. italica Plenck), a plant belonging to the Bassicaceae family, is a relative of brussels sprouts、cabbage、cauliflower, and kale and an important annual crop in the world(Ilahy et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Broccoli has gained popularity and as a good source of health promoting compounds owing to its excellent nutritional values(Hang et al., 2022;Takashi et al.,2023;Miyahir et al.,2022)It is a source of diverse nutrients (e.g., protein,dietary fiber,minerals, and vitamins,) and phytochemicals (e.g., glucosinolates and phenolic compounds)(Li et al.2021; Thomas et al. 2018; ).However, the edible part of broccoli accounts for less than half of the whole broccoli, and the broccoli rhizomes account for a large proportion of the weight of the whole broccoli(Le Borja2020). The harvesting of broccoli produces a large quantity of plant by-products, mainly leaves and stems, which make up more than 95% of the material harvested.(Dom\u0026iacute;nguez et al.2011;Borja Borja et al.2020).\u003c/p\u003e \u003cp\u003eRecently, numerous studies have explored the use of food by-products as a source of valuable bioactive compounds with potential application in the treatment and prevention of human diseases (Alejandra et al.2022;Gudi\u0026ntilde;o et al. 2022).Among the main components of interest in broccoli by-products are glucosinolates and phenolic compounds, as well as carotenoids, sterols, vitamin C, fibre and mineral elements.Due to the peculiarity of their structure, polyphenols have good antioxidant and free radical scavenging effects. It has good effects in removing free radicals from the human body, defending and treating diseases of the circulatory system, and preventing the deterioration of physiological functions (Pagliarulo 2016; et al. .C\u0026eacute;sar et al.2019;Talmaciu et al.2014; Emilio et al.2022).In the research of plant polyphenols, separation and extraction is an important part. Solvents such as water, methanol and its aqueous solutions, ethanol and its aqueous solutions, ethyl acetate, acetone are commonly used for the extraction of plant polyphenols (Gloria et al.2017;Hu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;Kumar et al.2023). However, these conventional solvents are often flammable, toxic, poorly biodegradable and cannot solve the problem of rapidly extracting products of different polarities and simultaneously maintaining the activity of the products.(Wagare et al.2021;Sara et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn recent years, deep eutectic solvents (DES) have been found to be composed of hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) in a certain proportion, which can provide or accept external electrons or protons to form hydrogen bonds, why can dissolve a variety of substances. Usually, they are mixtures of non-toxic, cheap and renewable components, which have good properties of ionic liquids, such as excellent solubility, chemical stability and thermal stability, but also have low cost and no pollution(Justyna et al \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;Ashraf et al \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).Currently, some studies have shown that DESs can be used as alternative solvents to extract polyphenols from natural samples(Bener et al \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).However, extraction of polyphenol from broccoli stem has rarely been reported. In the present study, a highly efficient and nonpolluting extraction procedure was developed for the extraction of polyphenols from the discarded stems of broccoli.The optimum extraction conditions were optimised using response surface methodology (RSM) with Box-Behnken design (BBD). The components of the main polyphenol were identified by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS/MS).Moreover, to further understand its potential food applications and health benefits, the in vitro antioxidant activity of the extracted polyphenols obtained in (ChCl-urea) DES was determined by DPPH free radical scavenging ability、ABTS free radical scavenging ability、FRAP total reducing power and ORAC total antioxidant capacity.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003ch4\u003eMaterials and Chemical Reagents\u003c/h4\u003e\n\u003cp\u003eFresh broccoli samples were purchased from a local supermarket (Ningbo,China).Standard gallic acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Chemical Corporation (China). Reagents containing folinphenol, citric acid, glucose, sucrose, urea, choline chloride, anhydrous sodium carbonate ,1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2 -Azinobis-(3- ethylbenzthiazoline-6-sulphonate) (ABTS), ORAC total antioxidant assay kit and FRAP antioxidant assay kit were purchased from Sinopharm Group Chemical Reagent Co. Ltd(Shanghai, China)，Other chemicals used throughout the experiment were analytical grade.\u003c/p\u003e\n\u003ch4\u003eExtraction Methods\u003c/h4\u003e\n\u003ch4\u003eDeep Eutectic Solvent Preparation\u003c/h4\u003e\n\u003cp\u003eThe preparation of DESs was carried out according to literature methods (Jun et al.2018;Li et al.2021;Li et al.2021).Four different types of DESs were prepared by the heating method. A two-component mixture, HBA and HBD, was added to a round-bottomed glass flask and stirred in a constant temperature magnetic stirrer at 80\u0026deg;C until a homogeneous liquid was formed（40-60 min），and a colourless and transparent thick liquid was obtained at room temperature.The composition, molar ratios and codes of DESs used in this study were shown in Table 1.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003e\u0026nbsp;Extraction of polyphenols\u003c/h4\u003e\n\u003cp\u003eThe stems of fresh broccoli were washed with tap water, cut into 1cm pieces, dried in an oven at 60\u0026deg;C (DHG-9053A, Electric Heating Constant Temperature Blast Drying Oven, Yilin Scientific Instrument Co., Ltd, Shanghai, China) to constant weight and crushed with a food processor (Joyoung Corporation., Ltd, Zhejiang China) and the powder was obtained through the 60-size mesh.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe extraction process was performed according to the previously described method(Liu et al.2019;Mondal et al.2019).For all experiments, a quantity of 0.25 g of broccoli stem powder was placed with a certain amount of DES solution, water or70% ethanol, mixed by vortex shaking evenly, and placed in a constant temperature water bath for a certain period of time (5 min shaken by vortexing once)(QL861 Vortex Shaker, Qilin Bell Instrument Manufacturing Co., Ltd.Haimen,China), after constant temperature extraction, the mixture was cooled down to room temperature in a short time, and then the supernatant was collected by centrifugation under 5,000 g for 20 min (LC-180 Centrifuge, Keda Innovation Incorporation, Ltd. China). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eTotal polyphenols content Determination\u003c/h4\u003e\n\u003cp\u003eThe Folin-Ciocalteu colorimetric method was used to quantify the total polyphenol content（Batool et al.2023）. Gallic acid (Sigma-Aldrich, St-Quentin Fallavier, France) was used for the calibration curve. The results were expressed as mg of gallic acid equivalent (GAE) per gram of dry matter (mg GAE/g DM).The determination of total polyphenols was carried out according to literature methods (Rosa et al.1999 ) (Singleton, Orthofer, \u0026amp; Lamuela-Ravent\u0026oacute;s, 1999).The Folin-Ciocalteu colorimetric method was used with gallic acid as standard.Gallic acid series standard working solution 0, 10, 20, 30, 40, 50 \u0026mu;g/mL were prepared respectively, distilled water (for blank control) into a 25 mL colorimetric tube, add 5.0 mL of Folin-Ciocalteu reagent, shake well. Within 3min~8min of the reaction, add 4.0mL of 7.5% Na2CO3 solution, make up the volume with water and shake well. After reaction at room temperature in the dark for 60 min, the absorbance (A) was measured with a spectrophotometer at a wavelength of 765 nm and the A value was determined for 3 times in parallel.The standard curve for gallic acid is obtained as y=10.8771x+0.0032，R\u003csup\u003e2\u003c/sup\u003e=0.9991.\u003c/p\u003e\n\u003cp\u003eThe results were expressed as mg of gallic acid equivalent (GAE) per gram of dry matter (mg GAE/g DM).\u003c/p\u003e\n\u003cp\u003eTotal phenolic content（mg GAE/g）= m\u003csub\u003e1\u003c/sub\u003e\u0026times;V\u003csub\u003e1\u003c/sub\u003e/V\u0026times;m\u0026times;1000\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhere m\u003csub\u003e1\u003c/sub\u003eis Content of gallic acid found in standard equation(mg/mL);V\u003csub\u003e1\u003c/sub\u003e is Total volume of extraction(mL)；V is sample volume taken during measurement(mL)；m is the amount of sample(g).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimization Extraction on the Yield of polyphenols\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of 4 different DESs on the extraction yield of polyphenols were investigated by single factor test, effects of DES-1 molar ratio (1:2, 1:1, 2:1,3:1 and4:1), water content of DES-1 (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, w/w), extraction time (10, 30, 50, 70 and 90 min), extraction temperature\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(40, 50, 60, 70, 80 and 90℃) and liquid/solid ratio (10, 20, 30, 40, 50, 60 and 70 v/w) on the extraction efficiency of polyphenols were tested.On the basis of single factorial experiment, extraction time, extraction temperature and liquid-solid ratio were selected as independent variables, and the extraction yield of polyphenols was the response value, Design-Expert 10.0.7 software was used to perform response surface analysis to optimise the extraction conditions. The response surface test factor coding and level design are shown in Table 2, and each experiment was replicated three times. The experimental data obtained from the Box-Behnken design (BBD) were analysed by regression analysis. Under the optimal parameters, the extraction experiment was repeated three times to verify the accuracy of RSM. The polyphenols from broccoli stem were extracted under optimised conditions to determine their structural characterisation and bioactivities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2 Experimental levels of independent variables\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eUnits\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eSymbol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eVariable levels\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eextraction temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e℃\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eextraction time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003emin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eliquid\u0026ndash;solid ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e40:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e50:1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch4\u003eComponent Analysis\u003c/h4\u003e\n\u003cp\u003eChromatographic separation and detection in the samples were analysed using a Waters synpat G2 (UPLC-ESI-QTOF/MS) system (Waters). A reverse-phase ACQUITY UPLC C18 column (100 mm \u0026times; 2.1 mm \u0026times; 1.7 \u0026mu;m) with a flow rate of 0.2 mL min-1 was used for the chromatographic separation. A mixture of water (A) and acetonitrile (B) was used as the mobile phase, and the linear gradient programs were as follows: 0-1.0 min, 5% (B); 1.0-12.0 min, 5%\u0026rarr;100% (B); 12.0-13.0 min, 100%\u0026rarr;100% (B); 13.0-15.0 min, 100%\u0026rarr;50% (B); The injection volume was 2 \u0026mu;L. Samples were analysed using ESI negative ionisation modes, scanning the m/z range between 50 and 1200. The desolvation gas flow and temperature were 900 L h-1 and 350 \u0026deg;C, respectively. The mass spectra were acquired using a capillary voltage of 3. 0 kV and cone hole voltage 40 kV. Instrument control and data acquisition were performed using MassLynx software (v 4.1)..\u003c/p\u003e\n\u003ch4\u003eAntioxidant Assays\u003c/h4\u003e\n\u003ch3\u003eDPPH Free Radical Scavenging Assay\u003c/h3\u003e\n\u003cp\u003eThe antioxidant capacity of extraction of broccoli stem sample was compared with\u003c/p\u003e\n\u003cp\u003ewater and 70% ethanol solution. The DPPH free radical scavenging assay was performed as previously reported (Duan et al.2010). Briefly, 1 mL of DES extract of Broccoli stem sample and 3 mL of 50 ug/mL DPPH solution were added to the same test tube with stopper and shaken well.The tubes were left tightly sealed at room temperature for 30 min and anhydrous ethanol was used as reference solution at 517 nm.The absorbance was measured at 517 nm wavelength and measured three times in parallel. The DPPH free radical scavenging rate was calculated using the following equation:\u003c/p\u003e\n\u003cp\u003eDPPH free radical scavenging percentage (%)=[ 1-(A\u003csub\u003e2\u003c/sub\u003e-A\u003csub\u003e1\u003c/sub\u003e)/A\u003csub\u003e0\u003c/sub\u003e ] x 100%\u003c/p\u003e\n\u003cp\u003eWhere A\u003csub\u003e2\u003c/sub\u003e is the absorbance of 3ml of DPPH solution after adding 1 mLof extract; A\u003csub\u003e1\u003c/sub\u003e is the absorbance of 1 mL of extract and 3 mL of ethanol; A\u003csub\u003e0\u003c/sub\u003e is the absorbance of 3ml of DPPH solution and 1mL of solvent(DES)\u003c/p\u003e\n\u003ch3\u003eABTS Free Radical Scavenging Assay\u003c/h3\u003e\n\u003cp\u003eThe ABTS free radical scavenging assay was performed as previously reported, with minor modifications (Meng L. et al. 2019). Briefly, ABTS reserve solution was prepared by mixing 7.4 mmol/L ABTS solution with 4.5 mmol/L potassium persulfate solution for 12-16 h at 25 \u0026deg;C until a dark blue-green colour developed. An aliquot of 200 \u0026mu;L of antioxidant solution at various concentrations was mixed with 200 \u0026mu;L of ABTS solution (1 mM in 95% ethanol). All reaction mixtures contained 1.0 mM ABTS and antioxidant solution at each concentration and the control solution contained no antioxidants.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;ABTS free radical scavenging rate was calculated by the following equation:\u003c/p\u003e\n\u003cp\u003eABTS free radical scavenging percentage (%)=[ 1-(A\u003csub\u003e2\u003c/sub\u003e-A\u003csub\u003e1\u003c/sub\u003e)/A\u003csub\u003e0\u003c/sub\u003e ] x 100%\u003c/p\u003e\n\u003cp\u003eWhere A\u003csub\u003e2\u003c/sub\u003e is the absorbance of 200\u0026mu;L of ABTS solution after adding 200\u0026mu;Lof extract; A\u003csub\u003e1\u003c/sub\u003e is the absorbance of 200\u0026mu;L of extract and 200\u0026mu;L of 95% ethanol; A\u003csub\u003e0\u003c/sub\u003e is the absorbance of 200\u0026mu;L of ABTS solution and 200\u0026mu;L of solvent(DES).\u003c/p\u003e\n\u003ch3\u003eFRAP total antioxidant capacity\u003c/h3\u003e\n\u003cp\u003eTotal antioxidant capacity was determined according to the Total Antioxidant Capacity Assay Kit with FRAP method manual (Yuan Yea, Shanghai). The FRAP working solution with deionised water was prepared from the kit stock solution. 30 uL of ferrous ion standard solution with different gradient concentrations were added to 264 uL of FRAP solution respectively, and 30 uL of deionised water instead of a sample was used as a blank, the mixture was vortexed and incubated at 37\u0026deg;C for 30 min away from light. Absorbance was measured at 593 nm using a spectrophotometer.\u003c/p\u003e\n\u003ch3\u003e\u0026nbsp;ORAC total antioxidant capacity\u003c/h3\u003e\n\u003cp\u003eThe total Oxygen Radical Absorbance Capacity (ORAC) of the extract was evaluated using the test kit (Yuan ye, Shanghai). The fluorescence intensity was followed to monitor the decay of the fluorescence curve. A calibration curve was obtained by plotting the net curve against Trolox solutions in the range 0-40 \u0026mu;M. The equation for the calibration curve was y = 3.943x-2.003, with a good correlation coefficient (r2 = 0.979).\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eStatistical Analysis\u003c/h4\u003e\n\u003cp\u003eAll determinations were performed at least three times in triplicate and all results are expressed as mean \u0026plusmn; standard deviation (SD). Statistical significance between groups was determined by analysis of variance (ANOVA) and least significant difference (LSD) test using SPSS 19. A p \u0026lt; 0.05 was considered statistically significant.In all figures, the error bars correspond to the 95% confidence level.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003ch3\u003eOptimization Extraction\u003c/h3\u003e\n\u003ch3\u003eEffect of Deep Eutectic Solvent System\u003c/h3\u003e\n\u003cp\u003eThe extraction solvent played a key role in the extraction of natural products.The physicochemical properties of the extraction solvent were essential conditions for this study,which may be due to the permeability of the extractant in the sample matrix,which can be directly determined by them(Xing et al.2022).Table 1 showed the composition,molar ratios and symbols of the DESs used in this study. As a comparative study, the experiment used the four deep eutectic solvents、hot water extraction and 70% ethanol organic solvents.\u003c/p\u003e\n\u003cp\u003eExtraction efficiency varied with different types of extraction solvent. As shown in Figure 1A,the effects of DES-1 on the extraction yields of polyphenols were significantly lower than others(p\u0026lt;0.05).There was no significant difference between DES-3 and DES-4 in the extraction yields of polyphenols(p\u0026gt;0.05).DES2 exhibited a significantly higher extraction efficiency than others,reaching 5.051\u0026plusmn;0.04mg.g\u003csup\u003e-1\u003c/sup\u003e. DW.This could be attributed to the low viscosity, relatively large diffusion coefficient, good flowability, better permeability to the cell wall and the presence of a certain viscosity, which promoted the suspension and dispersion of broccoli stem powder in the In solvent, increased the contact area between the two and is conducive to the extraction of polyphenols (Malaeke et al.2018 ). Therefore, a deep eutectic solvent system composed of choline chloride and urea was considered for the follow-up experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1 The composition and molar ratios of DESs were used in this study\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003eAbbreviation \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\" valign=\"top\"\u003e\n \u003cp\u003eHBA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003eHBD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eMolar ratio(mol/mol)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003eDES1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\" valign=\"top\"\u003e\n \u003cp\u003eCholine chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003eCitric Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e2:1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003eDES2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\" valign=\"top\"\u003e\n \u003cp\u003eCholine chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003eUrea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e2:1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003eDES3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\" valign=\"top\"\u003e\n \u003cp\u003eCholine chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003eSucrose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e2:1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.46938775510204%\" valign=\"top\"\u003e\n \u003cp\u003eDES4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.857142857142854%\" valign=\"top\"\u003e\n \u003cp\u003eCholine chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003eGlucose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e2:1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch4\u003eEffect of Mole Ratio of Deep Eutectic Solvent\u003c/h4\u003e\n\u003cp\u003eAs shown in Figure 1B, the extraction efficiency of polyphenols was significantly affected by the molar ratio of DES-2. The extraction efficiency increased with the increasing ratio of urea in DES-2, when the molar ratio of urea/choline reached 3:1, the yield of polyphenols was the highest. It may be due to urea improving the viscosity and surface tension of DES-2, the more urea in the system, the stronger the hydrogen bond between urea and choline chloride, the stronger the intermolecular force between urea and choline chloride, the lower the viscosity of DESs and the better the flowability. Since the number of hydrogen bonds formed between urea and choline chloride was limited and had a certain degree of saturation, as the molar ratio of urea/choline chloride continued to increase, the hydrogen bonding force between urea and choline chloride increased, making the viscosity of DESs larger, resulting in poor fluidity, which made the subsequent extraction process difficult. Therefore, the molar ratio of urea-ChCl- in DES used in the subsequent experiments was 2:1.\u003c/p\u003e\n\u003ch4\u003eEffect of Water Content of Deep Eutectic Solvent\u003c/h4\u003e\n\u003cp\u003eAs shown in Figure 1C, the extraction efficiency varied depending on the water content of the DES. The extraction efficiency increased as the water content of DES-2 increased from 10 to 60%, and the yield of polyphenols increased from 13.82\u0026plusmn;0.05% to 50.83\u0026plusmn;0.04%. When the water content exceeded 70%, the further increase of the water content resulted in a significant decrease. This phenomenon could be attributed to the fact that increasing the water content in DES-2 from 10 to 60% resulted in a more suitable viscosity and polarity of DES-2 to improve the interactions between polyphenols and DES-2,which obtained a high extraction yield, the interaction force between the deep eutectic solvent and the polyphenols was weakened after the addition of excess water.Therefore,DES-2 with the water content of 60% was considered for the subsequent experiments.\u003c/p\u003e\n\u003ch4\u003eEffect of Extraction Time\u003c/h4\u003e\n\u003cp\u003eAs shown in Figure 1D, the extraction efficiency of ployphenols was affected by the extraction time. With the extension of the extraction time, the extraction efficiency of ployphenols increased from 4.16 to 4.87, but when the extraction time was increased from 50 to 90 min, the extraction efficiency of ployphenols decreased from 4.87 to 3.03.This phenomenon could be attributed to the prolongation of the extraction time, which may destroy the cell structure, increase the permeability and be conducive to ployphenols, but a long-term extraction at high temperature may lead to the decomposition of the extracted polyphenolic compounds. Therefore, an extraction time of 50 min was chosen for the following experiments.\u003c/p\u003e\n\u003ch4\u003eEffect of Extraction Temperature\u003c/h4\u003e\n\u003cp\u003eAs shown in Figure 1E, we can see that the total phenolic yield of broccoli stem increased with the increase of extraction temperature from 40 to 80℃, and reached the maximum polyphenol yield 5.10 at 80℃. However, when the water content exceeded 60%, the further increase of water content resulted in a significant decrease(p\u0026lt;0.05).When the extraction temperature exceeded 80℃, the further increase of temperature resulted in a significant decrease in the extraction efficiency of polyphenols(p\u0026lt;0.05).This phenomenon could be attributed to the high temperature, which not only can reduce the viscosity of DES-2 and increase the diffusion of DES-2, but also accelerate the mass transport of polyphenols ( Zhang et al.2012), which makes polyphenols dissolve more in solvent and improve the extraction yield. However, the high temperature will damage the structure of the polyphenols. Therefore, the extraction temperature was chosen to be 60℃for the following experiments.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eEffect of Liquid\u0026ndash;Solid Ratio\u003c/h4\u003e\n\u003cp\u003eThe liquid-solid ratio was one of the most important factors influencing the extraction yield of polyphenols. As shown in Figure 1F, the extraction efficiency of polyphenols increased with increasing liquid-solid ratio from 10:1 to 40:1, reaching a maximum of 5.12 mg/g at 40:1 (v/w). However, a further increase in the liquid-solid ratio resulted in a lower extraction efficiency. This phenomenon could be attributed to a large amount of DES-2, which could increase the contact area between the material and the solvent and the concentration gradient becomes larger, which is conducive to the dissolution of polyphenols, but when the solvent was increased too much, the extraction effect on polyphenols is not obvious or even diluted, and the yield decreased.Therefore, 40:1 (v/w) was chosen as the optimal liquid-solid ratio in this experiment.\u003c/p\u003e\n\u003ch2\u003eOptimization of the extraction conditions by RSM\u003c/h2\u003e\n\u003cp\u003eThe RSM-based optimisation was aimed at minimising the use of solvents and broccoli stem powder, saving time and energy during the experiments, and identifying the ideal combination of factors that would result in the maximum extraction of polyphenols. To ensure the fitness of the proposed model at the 95% confidence level, the p-value should be less than 0.05 (p \u0026lt; 0.05) (Temova \u0026amp; Ro\u0026scaron;kar, 2016). Table 3 shows the analysis of variance (ANOVA) for the model.Based on the ANOVA results, the developed model was found to be significant for polyphenol extraction (p \u0026lt; 0.05). The values of coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e ) were 0.9897, together with non-significant lack of fit（p＞0.05），suggesting a good fit, the predicted model seemed to reasonably represent the observed values. The individual factors and the coefficients of the quadratic terms (A\u003csup\u003e2\u003c/sup\u003e、B\u003csup\u003e2\u003c/sup\u003e and C\u003csup\u003e2\u003c/sup\u003e) were also found to be significant. According to the size of the F-value, the three factors A, B and C can be considered as influencing the yield of broccoli stem polyphenols. The order from largest to smallest was C (liquid/material ratio)\u0026gt;B (extraction time)\u0026gt;A (extraction temperature).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3 Analysis of variance (ANOVA).\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"97%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003edf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003eSum of Squares\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003eMean Square\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003eF-Value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003eP-Value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003eSignificance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e5.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.6568\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e74.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e＜0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eExtraction temperature-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.082\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.082\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e9.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.0185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eExtraction time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.2426\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.2426\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e27.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.0012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eLiquid\u0026ndash;solid ratio\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.2824\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.2824\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e32.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.0008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eExtraction temperature*Extraction time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.0784\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.0784\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e8.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.0204\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eExtraction temperature*Liquid\u0026ndash;solid ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e156.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e＜0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eExtraction time*Liquid\u0026ndash;solid ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.0203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.0203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e2.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.1726\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eExtraction temperature*Extraction temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e141.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e＜0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eExtraction time*Extraction time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.1507\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.1507\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e17.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.0044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eLiquid\u0026ndash;solid ratio*Liquid\u0026ndash;solid ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e2.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e2.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e240.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e＜0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eResidual\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.0616\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.0088\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eLack of Fit\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.0505\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.0168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e6.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.0577\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003enon-significance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003ePure Error\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e0.0112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.0028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eCor Total\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e5.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eR-Squared\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.9897\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003eAdj R-Squared\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.9764\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.927835051546392%\"\u003e\n \u003cp\u003ePred R-Squared\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e0.8619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.309278350515465%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.587628865979383%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e2D contour plots were obtained using two factors at a time, representing a two-way interaction of the extraction factors, to evaluate their combined effect on the response. From the contour plots as shown in Figure 3, these plots were used as a reference to view the trend of the significant factors and also to study their interactions.It was concluded that the optimised extraction parameters for obtaining maximum extraction were an extraction temperature of 70-90 ℃, an extraction time of 40-60 min and a liquid-solid ratio of 30-50. Compared to the interaction between other factors, the slope change of the response surface between liquid-solid ratio and extraction temperature is steeper, which had a great influence on the extraction amount of broccoli stem polyphenols. These results were consistent with those obtained by ANOVA.\u003c/p\u003e\n\u003cp\u003eA linear equation for the predicted total polyphenols yield of extraction was obtained by multiple regression analysis of the data, which was stated as follows:\u003c/p\u003e\n\u003cp\u003eY=5.094-0.1013A+0.1741B+0.1879C+0.1400AB+0.5875AC-0.0712BC-0.5439A2-0.1891B\u003csub\u003e2\u003c/sub\u003e-0.7087C\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003ewhere Y was the yield of polyphenols\u0026nbsp;（mg /g Dw），A was the extraction temperature（℃），\u0026nbsp;B was the extraction time (min)，\u0026nbsp;and C was the Liquid-solid ratio（v/w）.\u003c/p\u003e\n\u003cp\u003eIn view of the above results, the selected variables were further optimised using Design-Expert. The software identified the optimal conditions of three parameters for extraction: extraction time (A) of 54.63 min, extraction temperature (B) of 60℃, liquid-solid ratio (C) of 41:1, and the maximum estimated extraction values of 5.12mg.g-1dw. Furthermore, the validity of the model was checked and the experimental value (5.103 \u0026plusmn; 0.03 mg/g) was closed with the predicted value (5.12 mg/g).\u003c/p\u003e\n\u003ch4\u003eLC-MS Analysis of polyphenols\u003c/h4\u003e\n\u003cp\u003eUPLC-Q-TOF/MS was used for further identification of polyphenolic compounds. The identification was done by comparing the masses of molecular ions, fragment ions with the results reported in the literature. The data obtained from the analysis of the molecular ion, fragment ion and retention time of the polyphenol compound peaks by HPLC-ESI-QTOF/MS are shown in Table 4. The four main polyphenols are quercetin, isochlorogenic acid, transcinnamic acid and sinapinic acid.The total ion chromatogram of a broccoli stem extract after (ChCl-Urea) DES and the representative SIM chromatograms of the main polyphenolic compounds are shown in Figure 4 and Figure 5.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 4 \u0026nbsp;The data obtained from the analysis of the molecular ion and retention time of polyphenol compounds peaks by HPLC-ESI-QTOF/MS\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"535\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.514018691588785%\"\u003e\n \u003cp\u003eEntry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.570093457943926%\"\u003e\n \u003cp\u003eSystematic name (Synonym)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.962616822429906%\"\u003e\n \u003cp\u003eRt\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(min)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.38317757009346%\"\u003e\n \u003cp\u003eFormular\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.30841121495327%\"\u003e\n \u003cp\u003eMass (Da)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.261682242990656%\"\u003e\n \u003cp\u003e[M-H]\u0026minus;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.514018691588785%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.570093457943926%\"\u003e\n \u003cp\u003eSinapinic acid\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.962616822429906%\"\u003e\n \u003cp\u003e18.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.38317757009346%\"\u003e\n \u003cp\u003eC\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e42\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.30841121495327%\"\u003e\n \u003cp\u003e338.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.261682242990656%\"\u003e\n \u003cp\u003e337.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.514018691588785%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.570093457943926%\"\u003e\n \u003cp\u003etrans-Cinnamic acid)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.962616822429906%\"\u003e\n \u003cp\u003e4.36\u003c/p\u003e\n \u003cp\u003e4.71\u003c/p\u003e\n \u003cp\u003e4.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.38317757009346%\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.30841121495327%\"\u003e\n \u003cp\u003e148.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.261682242990656%\"\u003e\n \u003cp\u003e147.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.514018691588785%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.570093457943926%\"\u003e\n \u003cp\u003equercetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.962616822429906%\"\u003e\n \u003cp\u003e2.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.38317757009346%\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.30841121495327%\"\u003e\n \u003cp\u003e302.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.261682242990656%\"\u003e\n \u003cp\u003e301.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.514018691588785%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.570093457943926%\"\u003e\n \u003cp\u003eIsochlorogenic acid\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.962616822429906%\"\u003e\n \u003cp\u003e3.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.38317757009346%\"\u003e\n \u003cp\u003eC\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.30841121495327%\"\u003e\n \u003cp\u003e516.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.261682242990656%\"\u003e\n \u003cp\u003e515.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch4\u003eAntioxidant Activity of broccoli extracts broccoli extracts from different solvents\u003c/h4\u003e\n\u003cp\u003eStudy of the effects of the in vitro antioxidant activity of different extract solvents (DES-2/H2O /70% EtOH). As shown in Figure 6(A), the antioxidant capacity of different solvent extracts is directly proportional to their concentration and increases with increasing concentration. At the mass concentration of 250 \u0026mu;g/ml, the DPPH radical scavenging capacity of 70% EtoH, DES-2 and H2O was 79.48%, 78.12% and 34.39%, respectively. As shown in Figure 4(B), when the different solvent extraction concentrations increased from 20 \u0026mu;g/mL to 250 ug / mL, the clearance of ABTS radical from DES-2 increased from 21.50% to 82.93%, from 70%EtoH increased from 20.37% to 79.63%, and from H2O extract increased from 12.37% to 34.36%, the clearance of ABTS radical of all three extraction solvents were proportional to the concentration. As shown in Figure 6 (C), the Trolox molar equivalence of reduced Fe(III) was achieved as the different solvent extraction concentrations increased from 50 \u0026mu;g/mL to 250 ug/mL. The results showed that DES-2 extract was the best antioxidant, followed by 70% ethanol extract. The water extract has the lowest oxidation, the TE content is only 105 mmol/L at the concentration of 250 u g / mL. As shown in Figure 6 (d), the total antioxidant capacity (TRAC) of the three Trolox at different solvent extraction concentrations increased from 50 \u0026mu;g/mL to 250 ug / mL. DES-2 extract was 3.45 \u0026plusmn; 0.1; 3.21 \u0026plusmn; 0.2umol TE/L for 70% ethanol and water 1.27 \u0026plusmn; 0.2umol TE/L; it is concluded that the antioxidant activity of DES-2 broccoli extract was better than that of the conventional extract.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study has demonstrated the efficiency of deep eutectic solvents as green alternatives for the extraction of polyphenols from Broccoli Stem.ChCl-urea was selected from a series of solvents for the extraction of total polyphenols from Broccoli Stem. Based on the results of the single factor experiment, RSM was used to identify the main parameters and optimise the extraction conditions.The optimal process conditions for the extraction of Broccoli Stem were determined as the extraction solvent Choline chloride-urea (molar ratio 1:3), water content of 60%, liquid-solid ratio of 41:1 mL/g, extraction temperature of 80\u0026deg;C and extraction time of 55 min, resulting in a maximum yield of 5.13mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003edw. In addition, the major polyphenolic composition of ChCl urea extraction from broccoli stem was quercetin, isochlorogenic acid, transcinnamic acid and sinapinic acid. In addition, the in vitro antioxidant activity of was evaluated using multiple radical scavenging assays. The results of bioactivities indicated that the polyphenols of ChCl urea extraction from broccoli stem exhibited excellent antioxidant activity.DPPH radical scavenging capacity and its FRAP total reducing power is higher than that of tranditonal solvent extract. The use of deep eutectic solvents allowed the extraction of a higher yield of polyphenols and improved their biological activities.DES extraction of polyphenols from broccoli stems has the characteristics of simple synthesis, low cost, and good environmental protection extraction effect. Overall, it provides certain theoretical support and technical support for its application and development in the extraction of polyphenols from broccoli waste.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Ningbo Public Welfare Fund Project(Grant 20211JCGY020055).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results/data/figures in this manuscript have not been published elsewhere, nor are they under consideration (from you or one of your Contributing Authors) by another publisher.I don\u0026apos;t have any research data outside the submitted manuscript file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI declare that the authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBingqing Wang ,Peiyun Chen,Huien Zhang and Liqing Chen wrote the main manuscript text prepared and Huien Zhang perpared figures 1-3..All authors reviewed the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlejandra GA, Marta S\u0026aacute;nchez-Paniagua L\u0026oacute;pez C, Manzanares-Palenzuela L, Araceli Redondo-Cuenca \u0026amp; Beatr\u0026iacute;z L\u0026oacute;pez-Ru\u0026iacute;z(2022)Edible plant by-products as source of polyphenols: prebiotic effect and analytical methods. 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Chem Soc Rev. 41:7108\u0026ndash;46.doi: 10.1039/c2cs35178a\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Polyphenol, deep eutectic solvent, extraction, broccoli stem, antioxidant activity","lastPublishedDoi":"10.21203/rs.3.rs-4142867/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4142867/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe stem of broccoli has been reported to contain high levels of polyphenols and other active compounds.To extract polyphenol from broccoli stem more efficiently, a novel procedure of deep eutectic solvent extraction (DESE) was proposed in this paper.The extraction process was optimised by response surface methodology. The optimum extraction parameters to obtain the highest yield of polyphenol 5.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 mg/g from broccoli stem powder via choline chloride-urea (molar ratio 1:3) were obtained at liquid-solid ratio of 41:1 mL/g, water content of 60%, extraction temperature of 80\u0026deg;C and extraction time of 55 min. The components of the main polyphenol were identified by high-performance liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS/MS).Compositional analysis shows that the extracted polyphenols are rich, including quercetin, isochlorogenic acid, trans-cinnamic acid and sinapinic acid. Furthermore, in vitro tests proved that the extracted polyphenols obtained in (ChCl-urea) DES possessed excellent antioxidant activity.These results provided an effective and feasible method for the extraction and separation of polyphenols in broccoli stalks, providing some technical support and theoretical basis for the green extraction of broccoli waste stalks.\u003c/p\u003e","manuscriptTitle":"Deep Eutectic Solvents Extraction of Polyphenol from the stem of Broccoli :Optimization, Components , and Antioxidant Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-27 12:43:00","doi":"10.21203/rs.3.rs-4142867/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cfa9e325-3ff0-4b8d-95f9-c239ffa733e8","owner":[],"postedDate":"March 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-14T22:27:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-27 12:43:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4142867","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4142867","identity":"rs-4142867","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}