Response surface optimization of ultrasonic extraction conditions of nuciferine and its purification

Response surface optimization of ultrasonic extraction conditions of nuciferine and its purification | 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 Response surface optimization of ultrasonic extraction conditions of nuciferine and its purification Mengdie Wu, Xinshui Ren, Simin Liu, Hongzhi Pan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2257445/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 Lotus leaf is an agricultural product as well as a kind of Chinese herbal medicine. Nuciferine is an amorphine alkaloids in lotus leaf that has anti-inflammatory, lipid-lowering and hypoglycemic effects, so it is greatly significant to extract and purify nuciferine. However, the factors that affect the nuciferine of lotus leaf extraction by dipping-acid-assisted ultrasonic extraction method (DAUEM) are rarely studied. The purpose of this research estimates DAUEM conditions (ethanol concentration, ratio of solution to material, ultrasonic extraction time) on responses by Box–Behnken response surface design. In addition, nuciferine of lotus leaf is purified by D101 macroporous adsorptive resin. A single factor test was conducted using nuciferine yield as an indicator and taking ethanol concentration (V/V), ratio of solution to material (V/M), ultrasonic extraction time as the influencing factor. The extraction process of nuciferine was optimized by response surface method based on the single factor test results. Nuciferine in lotus leaf was separated and purified by D101 macroporous adsorptive resin. The nuciferine yield (0.1035 ± 0.005) % was the largest as using 74% ethanol (V/V), ratio of solution to material 26:1, and the extraction time 82 min. The purity of nuciferine was 67.14% with 70% ethanol as eluent and through high performance liquid chromatography (HPLC) determination. This experiment has good precision and accuracy, so can be used for the extraction, separation and purification of nuciferine in lotus leaf. The method provides a theoretical basis for the development and utilization of nuciferine. Dipping-acid-assisted ultrasonic extraction method (DAUEM) can fully extract nuciferine in lotus leaf. Box-Behnken response surface methodology (RSM) was used to optimize the ultrasonic extraction process. D101 macroporous adsorption resin can admirably separate and purify nuciferine in lotus leaf. Lotus leaf Nuciferine Response surface methodology (RSM) Ultrasonic extraction purification Figures Figure 1 Figure 2 Figure 3 1. Introduction Lotus leaf is an agricultural product as well as a kind of Chinese herbal medicine, which has a wide range of medicinal and food properties [1] . Many studies indicate that lotus leaf contains a variety of chemical components, such as alkaloids [2] , flavonoids [3] , terpenes [4] , etc. Nuciferine (Fig. 1) is an aporphine alkaloids in lotus leaf, and an main ingredient in the quality control of lotus leaf of the Chinese Pharmacopoeia [1] . Modern studies have shown that nuciferine has rich pharmacological effects, such as anti-inflammatory [5] , antioxidant [6] , hypoglycemic [7] and hypolipidemic [8] . Besides, there are many health and wellness products related to nuciferine on the market, such as teas, cream and soap, ect. Lotus leaf are widely distributed and have rich resources in the world. The high yield of nuciferine from lotus leaf could perfect product efficacy and utilization. Therefore, a admirable extraction technique can improve the utilization rate and increase the added value of nuciferine. In this article, dipping-acid-assisted ultrasonic extraction method (DAUEM) were designed for extraction of nuciferine. The dip method has been widely used in the extraction of traditional Chinese medicine ingredients due to its cconvenience, cost saving, and high dissolution rate [9][10] . Ultrasonic method is a means of extraction that can efficiently extract active ingredients, save time and save solvents [11][12] . In this research, combining dip with ultrasonic method can not only increase the efficiency of the extraction of nuciferine, but also not destroy the structure of nuciferine. In addition, alkaline can be dissolved in acidic solutions to form an alkaloid salt. Alkaloid salts can more soluble in polar solutions (such as water, ethanol and formic acid, ect). Therefore, DAUEM was chosen for the extraction of nuciferine. Box-Behnken response surface methodology (RSM) can be used to explore the optimal process parameters of the experiment [13][14] . Meanwhile, studies have shown that D101 macroporous adsorption resin has the better effect on the separation and purification of nuciferine in lotus leaf [15] . Consequently, a single factor test was conducted using nuciferine yield as an indicator and taking ethanol concentration ( V/V ), ratio of solvent to material ( V/M ), ultrasonic extraction time as the influencing factor. The extraction process of nuciferine was optimized by response surface method based on the single factor test results. Nuciferine in lotus leaf was separated and purified by D101 macroporous adsorptive resin. This means provides a theoretical basis for the development and utilization of lotus leaf resources. 2. Experimental Section 2.1 Reagents and apparatus Nuciferine standard was purchased from Chengdu Lemeitian Pharmaceutical Technology Co.,Ltd. Lotus leaf powder was purchased from Luan Danbeier Biological Technology Co., Ltd. Ethanol (C 2 H 6 O, ≥ 99.7%), bismuth potassium iodide (analytically pure) and methanol (chromatographically pure) were purchased from Shanghai Titan Scientific CO.,Ltd. Sodium hydroxide (NaOH, analytically pure), hydrochloric acid (HCl, analytically pure) and acetic acid (AC, analytically pure) were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. D101 macroporous adsorptive resin and triethylamineand were purchased from Shanghai Macklin Biochemical Co., Ltd. Acetonitrile (chromatographically pure) was purchased from Shanghai Anpu Scientific Instrument Co., Ltd. Ultrasonic instrument (model number, KQ5200DE) was purchased from Kunshan Ultrasonic Instrument Co., Ltd. Rotary evaporator (model number, OSB-2200) was purchased from Tokyo Physio Equipment Co., Ltd. Freezing dryer (model number, CHRIST) was purchased from Shanghai Laijing Scientific Instrument Co., Ltd. Ultra-pure Water Purifier (model number, Milli-Q A10) was purchased from Merck & Co Inc. High performance liquid chromatography (model number, LC-1260) and chromatographic column (model number, Poroshell 120, EC-C18, 4µm/4.6*150 mm) was purchased from Agilent Technologies, Inc. Chromatography column (height 400mm*diameter 20mm) was purchased from Shanghai Haofu Instrument Co., Ltd. Circulating water multi-purpose vacuum pump (model number, SHB-HIA) was purchased from Shanghai Yukang Science and Education Instrument Equipment Co., Ltd. Electronic analysis balance (model number, FA124) was purchased from Shanghai Shunyu Hengping Scientific Instrument Co., Ltd. 2.2 Experimental process and design by dipping-acid-assisted ultrasonic extraction method (DAUEM) of nuciferine The process of DAUEM was executed. Firstly, lotus leaf powder was precisely weighed at 5g ( m 1 ). The weight was soaked in ethanol (pH 2.0) for 12 to 24 hours to form dipping-substance. Then, the dipping-substance was extracted by ultrasound, and let it sit for 1 to 2 hours to obtain extracting solution. Lastly, the extracts was filtered, concentrated and lyophilized to gain crude extraction powder of nuciferine ( m 2 ). The screening process of pH condition factors in the extraction process were presented in Supplementary Materials (S2), and the optimal condition was pH 2.0. The extraction of nuciferine by DAUEM had designed by Box-Behnken response surface methodology (RSM). Different ethanol concentrations ( V/V ), ratio of solvent to material and ultrasonic extraction time (three factors) were set separately, and the effects of different extraction conditions on the yield of nuciferine were studied based on single element experiments [16 ] . RSM can be used statistically to optimize experimental conditions. The experiment has designed in three factors as independent variables and three levels based on the results of single element experiments [17 ] (Table 1). The experimental procedures and results of single element were presented in Supplementary Materials (S2). Table 1 Level table of experimental factors in the response surface Factors (independent variables) Level -1 (low) 0 (center) 1 (high) X 1 = A :ethanol concentration (V/V)/% 60 70 80 X 2 = B :ratio of solvent to material 20:1 25:1 30:1 X 3 = C :ultrasonic extraction time/min 60 80 100 According to the level table of experimental factors (Table 1) and design principle of response surface, the experimental design of response surface was placed in Table 2. Table 2 The experimental design of response surface X 1 = A:ethanol concentration (V/V)/% X 2 = B:ratio of solvent to material (V/M) X 3 = C:ultrasonic extraction time/min NO. coded level independent parameters coded level independent parameters coded level independent parameters 1 1 80 0 25 1 100 2 1 80 -1 20 0 80 3 0 70 -1 20 1 100 4 0 70 0 25 0 80 5 0 70 0 25 0 80 6 -1 60 1 30 0 80 7 0 70 0 25 0 80 8 0 70 1 30 -1 60 9 0 70 1 30 1 100 10 0 70 -1 20 -1 60 11 -1 60 0 25 -1 60 12 0 70 0 25 0 80 13 1 80 0 25 -1 60 14 0 70 0 25 0 80 15 -1 60 0 25 1 100 16 -1 60 -1 20 0 80 17 1 80 1 30 0 80 2.3 Determination of nuciferine High performance liquid chromatography (HPLC) was used to determine nuciferine [1] . A standard curve and the linear equation of nuciferine were established by HPLC. The experimental procedures and results of HPLC for nuciferine were presented in Supplementary Materials (S3). Crude extraction powder of nuciferine was precisely weighed at 10 mg ( m 3 ), dissolved in methanol and brought to 10 mL volume ( V ) to gain sample solution of nuciferine. The sample solution was determined according to the HPLC method, and the peak area of the sample solution was brought into the linear equation to calculate the concentration of nuciferine. The nuciferine yield ( Y ) was used to evaluate the amount of nuciferine extracted from lotus leaf powder. The nuciferine yield was calculated by formula [18 ] (1): Of these, “ Y ” was the nuciferine yield, “ c ”was the sample concentration measured by HPLC, “ V ”was the volume of the sample solution, “ m 1 ” was weighing of lotus leaf powder, “ m 2 ” was weighing of crude extraction powder of nuciferine, and “ m 3 ” was weighing that crude extraction powder of nuciferine was weighed. 2.4 Purification of nuciferine D101 macroporous resin has a good effect on alkaloid separation and purification. The preprocessing method of D101 macroporous resin was placed in Supplementary Data (S4). Crude extraction powder of nuciferine was weighed at 5g, and dissolved by ethanol (pH 2.0) to obtain the solution of crude extraction powder of nuciferine. Then, the solution of crude extraction was placed into a pretreated D101 macroporous resin column (diameter 30 mm×length 300 mm). Next, nuciferine was eluted using ethanol solution (pH 2.0) of different concentrations ( V/V , 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) to gain the nuciferine eluent. Per ethanol concentration ( V/V ) cleans four column volumes and the flow rate was controlled at 10 mL/min. After the nuciferine eluent was collected separately with triangular flasks (number 1 through 8 in order), until detected without alkaloid (detection endpoint: add bismuth iodide potassium to the eluent without tangerine precipitation). The nuciferine eluent was concentrated and freeze-dried to obtain the nuciferine purified powder. The nuciferine purified powder was quantified based on “ Determination of nuciferine ”. After the peaking of the nuciferine eluent of ethanol of different concentrations observed, the separation and purification conditions of nuciferine were determined, and the curves of ethanol of different concentrations to the purity of nuciferine were made [19 ] . The nuciferine purity ( P ) was used to estimate the purification efficiency of nuciferine. The nuciferine purity was calculated by formula (2): Of these, “ P ” was the nuciferine purity, “ c ”was the sample concentration measured by HPLC, “ V ”was the volume of the sample solution, “ k ” was dilution multiple, and “ m ” was weighing that the nuciferine purified powder was weighed. 2.5 Statistical analysis RSM was applied to analyze the optimum condition of DAUEM. Data was analyzed with Design-Expert.V 8.0. 3. Results And Discussion 3.1 Response surface experimental results and its analysis RSM was used to optimize the extraction process of nuciferine, the results of experiment were shown in Table 3.The experimental data in Table 3 were performed regression fitted by Design Expert 8.0 software. The fitted equation was as follows. Table 3 The experimental results of response surface X 1 = A:ethanol concentration (V/V)/% X 2 = B:ratio of solvent to material (V/M) X 3 = C:ultrasonic extraction time/min The nuciferine yield (Y) /% NO. coded level independent parameters coded level independent parameters coded level independent parameters 1 1 80 0 25 1 100 0.1002 2 1 80 -1 20 0 80 0.0973 3 0 70 -1 20 1 100 0.0953 4 0 70 0 25 0 80 0.1040 5 0 70 0 25 0 80 0.1059 6 -1 60 1 30 0 80 0.0916 7 0 70 0 25 0 80 0.1089 8 0 70 1 30 -1 60 0.0982 9 0 70 1 30 1 100 0.0961 10 0 70 -1 20 -1 60 0.0857 11 -1 60 0 25 -1 60 0.0890 12 0 70 0 25 0 80 0.1099 13 1 80 0 25 -1 60 0.0984 14 0 70 0 25 0 80 0.1102 15 -1 60 0 25 1 100 0.0899 16 -1 60 -1 20 0 80 0.0870 17 1 80 1 30 0 80 0.0983 This regression model was executed the analysis of variance and significance, and the results were shown in Table 4. The results show that there were significant differences in the model equations (Model, F = 18.06, P = 0.0005). There was only a 0.05% chance that a "Model F-Value" this large could occur due to noise. The difference was not significant in lack of fit of model (P = 0.6979 > 0.05). Non-significant lack of fit is good. The "Lack of Fit F-value" of 0.51 implies the Lack of Fit is not significant relative to the pure error. There is a 69.79% chance that a "Lack of Fit F-value" this large could occur due to noise. "Adeq Precision" measures the signal to noise ratio. A ratio greater than 4 was desirable. Your ratio of 11.949 indicates an adequate signal. This model can be used to navigate the design space. Adjusted determination coefficient (Adj R-squared) was 0.9056 indicate that the model can explain 90.56% the response value changes. Coefficient of determination (R-squared) was 0.9587 demonstrate that the fit of the model was better. The above results indicated that the model was statistically significant and suitable for determining the optimal conditions of extracting nuciferine by DAUEM. If P was less than 0.05, the difference was significant, and if P was less than 0.01, the difference was extremely significant. It can be seen from Table 4 that the difference of model was extremely significant from the linear term X1 (P = 0.001 < 0.01) and the quadratic term X 1 2 (P = 0.0006 < 0.01), X 2 2 (P = 0.0004 < 0.01), and X 3 2 (P = 0.0008 < 0.01), and was significant from the linear term X 2 (P = 0.0276 < 0.05) and the quadratic term X 2 X 3 (P = 0.045 < 0.05). Through the analysis of P, the order of the main effects of each factor was ethanol concentration ( V/V ), ratio of solvent to material ( V/M ), ultrasonic extraction time from top to bottom. Table 4 Analysis of ANOVA at experimental results of RSM Source Sum of Squares df Mean Square F P- value Prob > F Model 9E-04 9 1E-04 18.06 0.0005 X 1 2E-04 1 2E-04 29.01 0.001 X 2 4E-05 1 4E-05 7.69 0.0276 X 3 1E-05 1 1E-05 2.24 0.1781 X 1 X 2 3E-06 1 3E-06 0.56 0.4793 X 1 X 3 2E-07 1 2E-07 0.035 0.8571 X 2 X 3 3E-05 1 3E-05 5.9 0.0455 X 1 2 2E-04 1 2E-04 33.94 0.0006 X 2 2 2E-04 1 2E-04 39.62 0.0004 X 3 2 2E-04 1 2E-04 31.26 0.0008 Residual 4E-05 7 6E-06 Lack of Fit 1E-05 3 4E-06 0.51 0.6979 Pure Error 3E-05 4 7E-06 Cor Total 1E-03 16 The steepness of the response surface reflects the significant degree of interaction between the two factors. A large steep surface indicates that the two factors were significantly higher, and a small surface steepness suggests that the two factors were less significant. In order to investigate the interaction between the relevant factors, the response surface diagram was plotted by Design Expert 8.0 software for visual analysis (Fig. 2). The closer the distance to the contour, the greater the effect on the nuciferine yield, and the farther away, the smaller the effect. The response surface was flat (Fig. 2a ~ b), indicating that the interaction between ethanol concentration ( V/V ) and ratio of solvent to material ( V/M ) was not significant when ultrasonic extraction time was invariable. Ethanol concentration than ratio of solvent to material has a great influence on the nuciferine yield, and the nuciferine yield was increased first and then decreased with the increase of ethanol concentration and ratio of solvent to material. The response surface was flat (Fig. 2c ~ d) to illustrate that the interaction between ethanol concentration and ultrasonic extraction time was not significant when ratio of solvent to material was immobile. Ethanol concentration has a greater influence on the nuciferine yield than ultrasonic extraction time, and the nuciferine yield rises first and then decreases with the enhance of ethanol concentration ( V/V ) and ultrasonic extraction time. The response surface was flat (Fig. 2e ~ f), suggesting that the interaction between ratio of solvent to material and ultrasonic extraction time was not significant when ethanol concentration ( V/V ) was unchangeable. Ratio of solvent to material has a greater influence on the nuciferine yield than ultrasonic extraction time, and the nuciferine yield was strengthened first and then subdued with the increase of ratio of solvent to material and ultrasonic extraction time. 3.2 Optimal process of DAUEM and its verification As can be seen from Fig. 2, there was a maximum value for the nuciferine yield. The optimal process conditions for predicting the extraction of nuciferine were A (ethanol concentration) of 73.28%, B (ratio of solvent to material) of 25.62 : 1, and C (ultrasonic extraction time) of 81.51 min by analyzing the model. At this time, the maximum value of the predicted nuciferine yield was 0.1087%. The above prediction conditions were adjusted to ethanol concentration ( V/V ) of 74%, ratio of solvent to material of 26 : 1, and an ultrasonic extraction time of 82 min based on actual operation. Five parallel experiments were carried out under the above conditions, and the value of nuciferine yield was 0.1035 ± 0.005%. The relative standard deviation (RSD) of five measurements was 4.58%, indicatinng that the extraction process has good precision. The relative error between the mean of five measurements and the predicted value of the model was 2.45% less than 5%, suggesting that the extraction process has good accuracy (Table 5). The above results show that the extraction conditions preferred by the response surface method were accurate and reliable, and the optimization process has practical feasibility and operability. Table 5 The measured and predicted response of nucifeine yield. Measured value of nucifeine yield(%) Predicted value of nucifeine yield(%) Relative error(%) 0.1075 0.1087 2.45 0.1092 0.1026 0.0988 0.0992 Mean 0.1035 RSD (%) 4.58 3.3 Optimal conditions for purification The purified chromatogram and elution curve of nuciferine was shown in Fig. 3A and B. Compared to unpurified sample chromatograms (Fig. S7), purified sample chromatograms were clearer and free of impurities (Fig. 3A). The abscissa was the volume fraction of ethanol concentration (V/V) and the ordinate was the nuciferine purity (Fig. 3B). When the ethanol concentration ( V/V ) was 30%, little nuciferine was eluted. And the nuciferine purity was low, and there were more impurities present at this time. The nuciferine purity was increased in the range of 40–70% ethanol. Until ethanol concentration ( V/V ) was 70%, the nuciferine purity reaches the maximum. It was explained that 70% ethanol can better elute and dissolve nuciferine. The nuciferine purity was decreased in the range of 80–90% ethanol, indicating that higher concentrations of ethanol will elute more impurities. Therefore, 70% ethanol was selected as the best extraction condition for purifying nuciferine in lotus leaves. The purity obtained nuciferine was 67.14% using 70% ethanol as extraction solution. 3.4 Method comparison The performance of our method was compared with the reported method for extraction and separation of nuciferine (Table 6). Our method was even less for weighing of lotus leaf powder, relatively low ethanol concentration, solvent amount and extraction time than other method. The proportion was large for weighing of crude extraction powder of nuciferine and lotus leaf. Due to the different formulas of methods and the lack of relevant calculation parameters, percentage of nuciferine was not possible to compare. In addition, compared with orthogonal method, response surface method was more flexible and has higher regression accuracy. However, the nuciferine purity our work was low compared to other methods, and the separation and purification of nuciferine was later experimental planning and the direction of improvement. This difference may be due to the fact that the extraction and separation method were different. Therefore, our method provides an economical, easy and rapid method for extraction and purification of nuciferine. Table 6 Comparison of different methods for extraction and purification of nuciferine. NO. Extracted substances Extracted method Optimization method Weighing of lotus leaf powder (g) Extraction solvent /amount (mL) Extraction time (min) Weighing of crude extraction powder of nuciferine (g) Percentage of nuciferine (%) Purification method The nuciferine purity (%) Reference 1 nuciferine Heated reflux / 20 50% ethanol/200 120 1.45 3.196(crude extraction powder percent) Combination technology of cation exchange resin and silica gel column chromatography 94.15 [ 19 ] 2 total alkaloids Heated reflux Orthogonal method 100 95% ethanol/2000 120 / / / / [ ] 3 nuciferine Ultrasonic extraction Orthogonal method 20 90% ethanol/800 20 / 1.12(crude extraction powder percent) / / [ ] 4 nuciferine Ultrasonic extraction response surface method 5 74% ethanol/130 82 1.25 0.1035(lotus leaf powder percent) D101 macroporous resin 67.14 this work 4. Conclusion In conclusion, the dipping-acid-assisted ultrasonic extraction method (DAUEM) was used for the extraction of nuciferine in lotus leaves. Combining dip with ultrasonic method can not only increase the efficiency of the extraction of nuciferine, but also not destroy the structure of nuciferine. Furthermore, alkaloid salts formed by dissolving alkaloids in acids can be more easily dissolved in polar solvents. The optimum extraction conditions of nuciferine optimized using the response surface method (RSM) were ethanol concentration of 74%, ratio of solvent to material 26:1 and ultrasonic extraction time of 82 min based on the results of single factor experiment. The nuciferine yield was 0.1035 ± 0.005% under optimal experimental conditions; the relative standard deviation (RSD) of five measurements was 4.58% and the relative error between the mean of five measurements and the predicted value of the model was 2.45% less than 5%, suggesting that the DAUEM has good precision and accuracy. The nuciferine purity purified with D101 macroporous adsorption resin was 67.14%. These results provide a theoretical basis for the development and utilization of nuciferine in lotus leaves. However, the purity of nuciferine purified by D101 macroporous adsorption resin was not high. Therefore, we will focus on method that can obtain high-purity nuciferine in the future. Declarations I am authorized on behalf of all the authors of this article to confirm that no author has any conflict of interest to disclose, all authors have approved the version submitted for publication, the work in this article is original and has not been published previously, and the article is not under consideration by any other journal. Ethical approval and consent to participate Not applicable Competing interests The authors declare that they have no competing interests. Author contributions MW and XRcollected the data, MW and XR analyzed the data and wrote the manuscript, HP managed and directed the trial, HP and SL gave comments and suggestions to improve the manuscript. 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Supplementary Files FigureS2.Effectofethanolconcentrationonthenuciferineyield.tif FigureS5.HPLCchromatogramofnuciferinestandard.tif FigureS6.HPLCchromatogramofnuciferinesample.tif 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Ren","email":"","orcid":"","institution":"Shanghai University of Medicine and Health Sciences, Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xinshui","middleName":"","lastName":"Ren","suffix":""},{"id":151339743,"identity":"683cd3e7-8001-4b2c-835f-74a01cfd9e96","order_by":2,"name":"Simin Liu","email":"","orcid":"","institution":"University of Shanghai for science and technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Simin","middleName":"","lastName":"Liu","suffix":""},{"id":151339745,"identity":"7161e41e-68e9-492e-b4df-438997861192","order_by":3,"name":"Hongzhi Pan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYHACNjBpwMx84MCHH6RpYUs8OLOHJC0MPMaHOdiIUG/OfvbYg487au3N2Xk+HGbgYZDnFzuAX4tlT1664cwzx5ktm3k3HC6wYDCcOTsBvxaDAzlm0rxtx9gMDgO1zOBhSDC4TUjL+TdgLTwGh3keHOZhI0bLDbAtNRJALQzEabGc8cZMcmbbAQODw2wGwECWIOwXc/4cM4mPbXX2BucPP/7w4YeNPL80IYdBqMMwvgR+5Uha6girHAWjYBSMgpELAEg4QyZ8cicJAAAAAElFTkSuQmCC","orcid":"","institution":"Shanghai University of Medicine and Health Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hongzhi","middleName":"","lastName":"Pan","suffix":""}],"badges":[],"createdAt":"2022-11-10 03:44:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2257445/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2257445/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29087007,"identity":"efad2a0b-023d-4fa4-8c58-409f0a37ec1a","added_by":"auto","created_at":"2022-11-15 15:08:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29347,"visible":true,"origin":"","legend":"\u003cp\u003eThe chemical structure of nuciferine.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2257445/v1/11d47e05623eb7708d4577f3.png"},{"id":29087534,"identity":"b52468da-38b6-4a89-a291-f154182ce92a","added_by":"auto","created_at":"2022-11-15 15:16:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":660191,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface analysis of interactions between factors.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2257445/v1/de5f4ea0f882ad038e2cc7bd.png"},{"id":29087535,"identity":"a1a55f97-a6e4-4484-91d2-a47081ff1916","added_by":"auto","created_at":"2022-11-15 15:16:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":102184,"visible":true,"origin":"","legend":"\u003cp\u003eA- HPLC chromatogram of the purified sample, and B-The elution curve of nuciferine.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2257445/v1/5d369802cc058374d7b40914.png"},{"id":29578364,"identity":"e6e8e696-7cb0-4566-8bd4-f8c12a686230","added_by":"auto","created_at":"2022-11-28 06:59:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1224513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2257445/v1/15608e1c-92fc-48fd-878f-96a666ddfe42.pdf"},{"id":29087010,"identity":"9954c6b6-8e81-455d-9112-30aaa68e612c","added_by":"auto","created_at":"2022-11-15 15:08:27","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":53985,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.Effectofethanolconcentrationonthenuciferineyield.tif","url":"https://assets-eu.researchsquare.com/files/rs-2257445/v1/b957f1c142bd309244ac83c5.tif"},{"id":29087533,"identity":"1d1f033d-5662-48d0-acbe-592890aa8f76","added_by":"auto","created_at":"2022-11-15 15:16:27","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":98118,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS5.HPLCchromatogramofnuciferinestandard.tif","url":"https://assets-eu.researchsquare.com/files/rs-2257445/v1/30df645d2d55e850e9922a19.tif"},{"id":29087012,"identity":"73ec570a-e4c1-4f63-8bc4-91cfa6ccd99d","added_by":"auto","created_at":"2022-11-15 15:08:27","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":141431,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS6.HPLCchromatogramofnuciferinesample.tif","url":"https://assets-eu.researchsquare.com/files/rs-2257445/v1/a3fd68c60764cd6ed66e25d1.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Response surface optimization of ultrasonic extraction conditions of nuciferine and its purification","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLotus leaf is an agricultural product as well as a kind of Chinese herbal medicine, which has a wide range of medicinal and food properties\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[1]\u003c/sup\u003e\u003c/strong\u003e.\u0026nbsp;Many studies indicate that lotus leaf contains a variety of chemical components, such as alkaloids\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[2]\u003c/sup\u003e\u003c/strong\u003e, flavonoids\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[3]\u003c/sup\u003e\u003c/strong\u003e, terpenes\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[4]\u003c/sup\u003e\u003c/strong\u003e, etc.\u0026nbsp;Nuciferine\u0026nbsp;(Fig.\u0026nbsp;1)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eis an aporphine alkaloids in lotus leaf, and an main ingredient in the quality control of lotus leaf of the Chinese Pharmacopoeia\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[1]\u003c/sup\u003e\u003c/strong\u003e. Modern studies have shown that nuciferine has rich pharmacological effects, such as anti-inflammatory\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[5]\u003c/sup\u003e\u003c/strong\u003e, antioxidant\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[6]\u003c/sup\u003e\u003c/strong\u003e, hypoglycemic\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[7]\u003c/sup\u003e\u003c/strong\u003e and hypolipidemic\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[8]\u003c/sup\u003e\u003c/strong\u003e. Besides, there are many health and wellness products related to nuciferine on the market, such as teas, cream and soap, ect. Lotus leaf are widely distributed and have rich resources in the world. The high yield of nuciferine from lotus leaf could perfect product efficacy and utilization. Therefore, a admirable extraction technique can improve the utilization rate and increase the added value of nuciferine.\u003c/p\u003e\n\u003cp\u003eIn this article, dipping-acid-assisted ultrasonic extraction method (DAUEM) were designed for extraction of\u0026nbsp;nuciferine. The dip method has been widely used in the extraction of traditional Chinese medicine ingredients due to its cconvenience, cost saving, and high dissolution rate\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[9][10]\u003c/sup\u003e\u003c/strong\u003e. Ultrasonic method is a means of extraction that can efficiently extract active ingredients, save time and save solvents\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[11][12]\u003c/sup\u003e\u003c/strong\u003e. In this research, combining dip with ultrasonic method can not only increase the efficiency of the extraction of nuciferine, but also not destroy the structure of nuciferine. In addition, alkaline can be dissolved in acidic solutions to form an alkaloid salt. Alkaloid salts can more soluble in polar solutions (such as water, ethanol and formic acid, ect). Therefore,\u0026nbsp;DAUEM\u0026nbsp;was chosen for the extraction of nuciferine.\u0026nbsp;Box-Behnken response surface methodology (RSM) can be used to explore the optimal process parameters of the experiment\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[13][14]\u003c/sup\u003e\u003c/strong\u003e. Meanwhile, studies have shown that D101 macroporous adsorption resin has the better effect on the separation and purification of\u0026nbsp;nuciferine\u0026nbsp;in lotus leaf\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e[15]\u003c/sup\u003e\u003c/strong\u003e. Consequently,\u0026nbsp;a\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003esingle factor test was conducted using nuciferine yield as an indicator and taking ethanol concentration (\u003cem\u003eV/V\u003c/em\u003e), ratio of solvent to material (\u003cem\u003eV/M\u003c/em\u003e), ultrasonic extraction time as the influencing factor. The extraction process of nuciferine was optimized by response surface method based on the single factor test results. Nuciferine in lotus leaf was separated and purified by D101 macroporous adsorptive resin. This means provides a theoretical basis for the development and utilization of lotus leaf resources.\u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Reagents and apparatus\u003c/h2\u003e\n \u003cp\u003eNuciferine standard was purchased from Chengdu Lemeitian Pharmaceutical Technology Co.,Ltd. Lotus leaf powder was purchased from Luan Danbeier Biological Technology Co., Ltd. Ethanol (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO, \u0026ge;\u0026thinsp;99.7%), bismuth potassium iodide (analytically pure) and methanol (chromatographically pure) were purchased from Shanghai Titan Scientific CO.,Ltd. Sodium hydroxide (NaOH, analytically pure), hydrochloric acid (HCl, analytically pure) and acetic acid (AC, analytically pure) were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. D101 macroporous adsorptive resin and triethylamineand were purchased from Shanghai Macklin Biochemical Co., Ltd. Acetonitrile (chromatographically pure) was purchased from Shanghai Anpu Scientific Instrument Co., Ltd.\u003c/p\u003e\n \u003cp\u003eUltrasonic instrument (model number, KQ5200DE) was purchased from Kunshan Ultrasonic Instrument Co., Ltd. Rotary evaporator (model number, OSB-2200) was purchased from Tokyo Physio Equipment Co., Ltd. Freezing dryer (model number, CHRIST) was purchased from Shanghai Laijing Scientific Instrument Co., Ltd. Ultra-pure Water Purifier (model number, Milli-Q A10) was purchased from Merck \u0026amp; Co Inc. High performance liquid chromatography (model number, LC-1260) and chromatographic column (model number, Poroshell 120, EC-C18, 4\u0026micro;m/4.6*150 mm) was purchased from Agilent Technologies, Inc. Chromatography column (height 400mm*diameter 20mm) was purchased from Shanghai Haofu Instrument Co., Ltd. Circulating water multi-purpose vacuum pump (model number, SHB-HIA) was purchased from Shanghai Yukang Science and Education Instrument Equipment Co., Ltd. Electronic analysis balance (model number, FA124) was purchased from Shanghai Shunyu Hengping Scientific Instrument Co., Ltd.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Experimental process and design by dipping-acid-assisted ultrasonic extraction method (DAUEM) of nuciferine\u003c/h2\u003e\n \u003cp\u003eThe process of DAUEM was executed. Firstly, lotus leaf powder was precisely weighed at 5g (\u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e). The weight was soaked in ethanol (pH 2.0) for 12 to 24 hours to form dipping-substance. Then, the dipping-substance was extracted by ultrasound, and let it sit for 1 to 2 hours to obtain extracting solution. Lastly, the extracts was filtered, concentrated and lyophilized to gain crude extraction powder of nuciferine (\u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e). The screening process of pH condition factors in the extraction process were presented in Supplementary Materials (S2), and the optimal condition was pH 2.0.\u003c/p\u003e\n \u003cp\u003eThe extraction of nuciferine by DAUEM had designed by Box-Behnken response surface methodology (RSM). Different ethanol concentrations (\u003cem\u003eV/V\u003c/em\u003e), ratio of solvent to material and ultrasonic extraction time (three factors) were set separately, and the effects of different extraction conditions on the yield of nuciferine were studied based on single element experiments\u003csup\u003e\u003cstrong\u003e[16\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e]\u003c/strong\u003e\u003c/sup\u003e. RSM can be used statistically to optimize experimental conditions. The experiment has designed in three factors as independent\u003c/p\u003e\n \u003cp\u003evariables and three levels based on the results of single element experiments\u003csup\u003e\u003cstrong\u003e[17\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e]\u0026nbsp;\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e(Table 1).\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/sup\u003eThe experimental procedures and results of single element were presented in Supplementary Materials (S2).\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eLevel table of experimental factors in the response surface\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eFactors (independent variables)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eLevel\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003cp\u003e(low)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003cp\u003e(center)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e(high)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;\u003cstrong\u003e=\u0026thinsp;A\u003c/strong\u003e:ethanol concentration (V/V)/%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u003cstrong\u003e=\u0026thinsp;B\u003c/strong\u003e:ratio of solvent to material\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30:1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;\u003cstrong\u003e=\u0026thinsp;C\u003c/strong\u003e:ultrasonic extraction time/min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eAccording to the level table of experimental factors (Table 1) and design principle of response surface, the experimental design of response surface was placed in Table 2.\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe experimental design of response surface\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;A:ethanol concentration (V/V)/%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;B:ratio of solvent\u003c/p\u003e\n \u003cp\u003eto material (V/M)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eX\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;C:ultrasonic\u003c/p\u003e\n \u003cp\u003eextraction time/min\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNO.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecoded level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eindependent parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecoded level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eindependent parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecoded level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eindependent parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3 Determination of nuciferine\u003c/h2\u003e\n \u003cp\u003eHigh performance liquid chromatography (HPLC) was used to determine nuciferine\u003csup\u003e\u003cstrong\u003e[1]\u003c/strong\u003e\u003c/sup\u003e. A standard curve and the linear equation of nuciferine were established by HPLC. The experimental procedures and results of HPLC for nuciferine were presented in Supplementary Materials (S3).\u003c/p\u003e\n \u003cp\u003eCrude extraction powder of nuciferine was precisely weighed at 10 mg (\u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e), dissolved in methanol and brought to 10 mL volume (\u003cem\u003eV\u003c/em\u003e) to gain sample solution of nuciferine. The sample solution was determined according to the HPLC method, and the peak area of the sample solution was brought into the linear equation to calculate the concentration of nuciferine.\u003c/p\u003e\n \u003cp\u003eThe nuciferine yield (\u003cem\u003eY\u003c/em\u003e) was used to evaluate the amount of nuciferine extracted from lotus leaf powder. The nuciferine yield was calculated by formula\u003csup\u003e\u003cstrong\u003e[18\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e]\u003c/strong\u003e\u003c/sup\u003e (1):\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"417\" height=\"64\"\u003e\u003c/p\u003e\n \u003cp\u003eOf these, \u0026ldquo;\u003cem\u003eY\u003c/em\u003e\u0026rdquo; was the nuciferine yield, \u0026ldquo;\u003cem\u003ec\u003c/em\u003e\u0026rdquo;was the sample concentration measured by HPLC, \u0026ldquo;\u003cem\u003eV\u003c/em\u003e\u0026rdquo;was the volume of the sample solution, \u0026ldquo;\u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u0026rdquo; was weighing of lotus leaf powder, \u0026ldquo;\u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u0026rdquo; was weighing of crude extraction powder of nuciferine, and \u0026ldquo;\u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e\u0026rdquo; was weighing that crude extraction powder of nuciferine was weighed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4 Purification of nuciferine\u003c/h2\u003e\n \u003cp\u003eD101 macroporous resin has a good effect on alkaloid separation and purification. The preprocessing method of D101 macroporous resin was placed in Supplementary Data (S4).\u003c/p\u003e\n \u003cp\u003eCrude extraction powder of nuciferine was weighed at 5g, and dissolved by ethanol (pH 2.0) to obtain the solution of crude extraction powder of nuciferine. Then, the solution of crude extraction was placed into a pretreated D101 macroporous resin column (diameter 30 mm\u0026times;length 300 mm). Next, nuciferine was eluted using ethanol solution (pH 2.0) of different concentrations (\u003cem\u003eV/V\u003c/em\u003e, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) to gain the nuciferine eluent. Per ethanol concentration (\u003cem\u003eV/V\u003c/em\u003e) cleans four column volumes and the flow rate was controlled at 10 mL/min. After the nuciferine eluent was collected separately with triangular flasks (number 1 through 8 in order), until detected without alkaloid (detection endpoint: add bismuth iodide potassium to the eluent without tangerine precipitation). The nuciferine eluent was concentrated and freeze-dried to obtain the nuciferine purified powder. The nuciferine purified powder was quantified based on \u0026ldquo;\u003cem\u003eDetermination of nuciferine\u003c/em\u003e\u0026rdquo;. After the peaking of the nuciferine eluent of ethanol of different concentrations observed, the separation and purification conditions of nuciferine were determined, and the curves of ethanol of different concentrations to the purity of nuciferine were made\u003csup\u003e\u003cstrong\u003e[19\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e]\u003c/strong\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eThe nuciferine purity (\u003cem\u003eP\u003c/em\u003e) was used to estimate the purification efficiency of nuciferine. The nuciferine purity was calculated by formula (2):\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"262\" height=\"58\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eOf these, \u0026ldquo;\u003cem\u003eP\u003c/em\u003e\u0026rdquo; was the nuciferine purity, \u0026ldquo;\u003cem\u003ec\u003c/em\u003e\u0026rdquo;was the sample concentration measured by HPLC, \u0026ldquo;\u003cem\u003eV\u003c/em\u003e\u0026rdquo;was the volume of the sample solution, \u0026ldquo;\u003cem\u003ek\u003c/em\u003e\u0026rdquo; was dilution multiple, and \u0026ldquo;\u003cem\u003em\u003c/em\u003e\u0026rdquo; was weighing that the nuciferine purified powder was weighed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eRSM was applied to analyze the optimum condition of DAUEM. Data was analyzed with Design-Expert.V 8.0.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv\u003e\n \u003ch2\u003e3.1 Response surface experimental results and its analysis\u003c/h2\u003e\n \u003cp\u003eRSM was used to optimize the extraction process of nuciferine, the results of experiment were shown in Table 3.The experimental data in \u003csup\u003eTable 3\u003c/sup\u003e were performed regression fitted by Design Expert 8.0 software. The fitted equation was as follows.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" width=\"675\" height=\"55\"\u003e\u003c/p\u003e\n \u003ctable border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe experimental results of response surface\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;A:ethanol concentration (V/V)/%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;B:ratio of solvent\u003c/p\u003e\n \u003cp\u003eto material (V/M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eX\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;C:ultrasonic\u003c/p\u003e\n \u003cp\u003eextraction time/min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eThe nuciferine\u003c/p\u003e\n \u003cp\u003eyield (Y) /%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNO.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecoded level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eindependent parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecoded level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eindependent parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ecoded level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eindependent parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0973\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0953\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1040\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1059\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0916\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1089\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0982\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0961\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0857\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0890\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1099\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0984\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1102\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0899\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0870\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 8.9041%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 19.4064%;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0983\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eThis regression model was executed the analysis of variance and significance, and the results were shown in Table 4. The results show that there were significant differences in the model equations (Model, F\u0026thinsp;=\u0026thinsp;18.06, P\u0026thinsp;=\u0026thinsp;0.0005). There was only a 0.05% chance that a \u0026quot;Model F-Value\u0026quot; this large could occur due to noise. The difference was not significant in lack of fit of model (P\u0026thinsp;=\u0026thinsp;0.6979\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Non-significant lack of fit is good. The \u0026quot;Lack of Fit F-value\u0026quot; of 0.51 implies the Lack of Fit is not significant relative to the pure error. There is a 69.79% chance that a \u0026quot;Lack of Fit F-value\u0026quot; this large could occur due to noise. \u0026quot;Adeq Precision\u0026quot; measures the signal to noise ratio. A ratio greater than 4 was desirable. Your ratio of 11.949 indicates an adequate signal. This model can be used to navigate the design space. Adjusted determination coefficient (Adj R-squared) was 0.9056 indicate that the model can explain 90.56% the response value changes. Coefficient of determination (R-squared) was 0.9587 demonstrate that the fit of the model was better. The above results indicated that the model was statistically significant and suitable for determining the optimal conditions of extracting nuciferine by DAUEM.\u003c/p\u003e\n \u003cp\u003eIf P was less than 0.05, the difference was significant, and if P was less than 0.01, the difference was extremely significant. It can be seen from Table 4 that the difference of model was extremely significant from the linear term X1 (P\u0026thinsp;=\u0026thinsp;0.001\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and the quadratic term X\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e (P\u0026thinsp;=\u0026thinsp;0.0006\u0026thinsp;\u0026lt;\u0026thinsp;0.01), X\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e (P\u0026thinsp;=\u0026thinsp;0.0004\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and X\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e (P\u0026thinsp;=\u0026thinsp;0.0008\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and was significant from the linear term X\u003csub\u003e2\u003c/sub\u003e (P\u0026thinsp;=\u0026thinsp;0.0276\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the quadratic term X\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e (P\u0026thinsp;=\u0026thinsp;0.045\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Through the analysis of P, the order of the main effects of each factor was ethanol concentration (\u003cem\u003eV/V\u003c/em\u003e), ratio of solvent to material (\u003cem\u003eV/M\u003c/em\u003e), ultrasonic extraction time from top to bottom. \u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eAnalysis of ANOVA at experimental results of RSM\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSum of\u003c/p\u003e\n \u003cp\u003eSquares\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003edf\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003cp\u003eSquare\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP- value\u003c/p\u003e\n \u003cp\u003eProb\u0026thinsp;\u0026gt;\u0026thinsp;F\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9E-04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1E-04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2E-04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2E-04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0276\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1781\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4793\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8571\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0455\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2E-04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2E-04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0006\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2E-04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2E-04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eX\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2E-04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2E-04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eResidual\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLack of Fit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6979\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePure Error\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCor Total\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1E-03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eThe steepness of the response surface reflects the significant degree of interaction between the two factors. A large steep surface indicates that the two factors were significantly higher, and a small surface steepness suggests that the two factors were less significant. In order to investigate the interaction between the relevant factors, the response surface diagram was plotted by Design Expert 8.0 software for visual analysis (Fig. 2).\u003c/p\u003e\n \u003cp\u003eThe closer the distance to the contour, the greater the effect on the nuciferine yield, and the farther away, the smaller the effect. The response surface was flat (Fig.\u0026nbsp;2a\u0026thinsp;~\u0026thinsp;b), indicating that the interaction between ethanol concentration (\u003cem\u003eV/V\u003c/em\u003e) and ratio of solvent to material (\u003cem\u003eV/M\u003c/em\u003e) was not significant when ultrasonic extraction time was invariable. Ethanol concentration than ratio of solvent to material has a great influence on the nuciferine yield, and the nuciferine yield was increased first and then decreased with the increase of ethanol concentration and ratio of solvent to material. The response surface was flat (Fig.\u0026nbsp;2c\u0026thinsp;~\u0026thinsp;d) to illustrate that the interaction between ethanol concentration and ultrasonic extraction time was not significant when ratio of solvent to material was immobile. Ethanol concentration has a greater influence on the nuciferine yield than ultrasonic extraction time, and the nuciferine yield rises first and then decreases with the enhance of ethanol concentration (\u003cem\u003eV/V\u003c/em\u003e) and ultrasonic extraction time. The response surface was flat (Fig.\u0026nbsp;2e\u0026thinsp;~\u0026thinsp;f), suggesting that the interaction between ratio of solvent to material and ultrasonic extraction time was not significant when ethanol concentration (\u003cem\u003eV/V\u003c/em\u003e) was unchangeable. Ratio of solvent to material has a greater influence on the nuciferine yield than ultrasonic extraction time, and the nuciferine yield was strengthened first and then subdued with the increase of ratio of solvent to material and ultrasonic extraction time.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003ch2\u003e3.2 Optimal process of DAUEM and its verification\u003c/h2\u003e\n \u003cp\u003eAs can be seen from Fig.\u0026nbsp;2, there was a maximum value for the nuciferine yield. The optimal process conditions for predicting the extraction of nuciferine were A (ethanol concentration) of 73.28%, B (ratio of solvent to material) of 25.62 : 1, and C (ultrasonic extraction time) of 81.51 min by analyzing the model. At this time, the maximum value of the predicted nuciferine yield was 0.1087%.\u003c/p\u003e\n \u003cp\u003eThe above prediction conditions were adjusted to ethanol concentration (\u003cem\u003eV/V\u003c/em\u003e) of 74%, ratio of solvent to material of 26 : 1, and an ultrasonic extraction time of 82 min based on actual operation. Five parallel experiments were carried out under the above conditions, and the value of nuciferine yield was 0.1035\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005%. The relative standard deviation (RSD) of five measurements was 4.58%, indicatinng that the extraction process has good precision. The relative error between the mean of five measurements and the predicted value of the model was 2.45% less than 5%, suggesting that the extraction process has good accuracy (Table 5). The above results show that the extraction conditions preferred by the response surface method were accurate and reliable, and the optimization process has practical feasibility and operability.\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 5\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe measured and predicted response of nucifeine yield.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMeasured value of nucifeine yield(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePredicted value of nucifeine yield(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRelative error(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1075\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"6\"\u003e\n \u003cp\u003e0.1087\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"6\"\u003e\n \u003cp\u003e2.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1092\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1026\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0988\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0992\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRSD (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003ch2\u003e3.3 Optimal conditions for purification\u003c/h2\u003e\n \u003cp\u003eThe purified chromatogram and elution curve of nuciferine was shown in Fig.\u0026nbsp;3A and B. Compared to unpurified sample chromatograms (Fig. S7), purified sample chromatograms were clearer and free of impurities (Fig.\u0026nbsp;3A). The abscissa was the volume fraction of ethanol concentration (V/V) and the ordinate was the nuciferine purity (Fig.\u0026nbsp;3B). When the ethanol concentration (\u003cem\u003eV/V\u003c/em\u003e) was 30%, little nuciferine was eluted. And the nuciferine purity was low, and there were more impurities present at this time. The nuciferine purity was increased in the range of 40\u0026ndash;70% ethanol. Until ethanol concentration (\u003cem\u003eV/V\u003c/em\u003e) was 70%, the nuciferine purity reaches the maximum. It was explained that 70% ethanol can better elute and dissolve nuciferine. The nuciferine purity was decreased in the range of 80\u0026ndash;90% ethanol, indicating that higher concentrations of ethanol will elute more impurities. Therefore, 70% ethanol was selected as the best extraction condition for purifying nuciferine in lotus leaves. The purity obtained nuciferine was 67.14% using 70% ethanol as extraction solution.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003ch2\u003e3.4 Method comparison\u003c/h2\u003e\n \u003cp\u003eThe performance of our method was compared with the reported method for extraction and separation of nuciferine (Table\u0026nbsp;6). Our method was even less for weighing of lotus leaf powder, relatively low ethanol concentration, solvent amount and extraction time than other method. The proportion was large for weighing of crude extraction powder of nuciferine and lotus leaf. Due to the different formulas of methods and the lack of relevant calculation parameters, percentage of nuciferine was not possible to compare. In addition, compared with orthogonal method, response surface method was more flexible and has higher regression accuracy. However, the nuciferine purity our work was low compared to other methods, and the separation and purification of nuciferine was later experimental planning and the direction of improvement. This difference may be due to the fact that the extraction and separation method were different. Therefore, our method provides an economical, easy and rapid method for extraction and purification of nuciferine.\u003c/p\u003e\n \u003ctable border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 6\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eComparison of different methods for extraction and purification of nuciferine.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNO.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExtracted substances\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExtracted method\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOptimization method\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWeighing of lotus leaf powder (g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExtraction solvent\u003c/p\u003e\n \u003cp\u003e/amount (mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExtraction time\u003c/p\u003e\n \u003cp\u003e(min)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWeighing of crude extraction powder of nuciferine (g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePercentage of nuciferine (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePurification method\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eThe nuciferine purity (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enuciferine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeated reflux\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50% ethanol/200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.196(crude extraction powder percent)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCombination technology of cation exchange resin and silica gel column chromatography\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003csup\u003e\u003cstrong\u003e[\u003c/strong\u003e19\u003cstrong\u003e]\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etotal alkaloids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeated reflux\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOrthogonal method\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95% ethanol/2000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003csup\u003e\u003cstrong\u003e[\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e]\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enuciferine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUltrasonic extraction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOrthogonal method\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90% ethanol/800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.12(crude extraction powder percent)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003csup\u003e\u003cstrong\u003e[\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e]\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enuciferine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUltrasonic extraction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eresponse surface method\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74% ethanol/130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1035(lotus leaf powder percent)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD101 macroporous resin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ethis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn conclusion, the dipping-acid-assisted ultrasonic extraction method (DAUEM) was used for the extraction of nuciferine in lotus leaves. Combining dip with ultrasonic method can not only increase the efficiency of the extraction of nuciferine, but also not destroy the structure of nuciferine. Furthermore, alkaloid salts formed by dissolving alkaloids in acids can be more easily dissolved in polar solvents. The optimum extraction conditions of nuciferine optimized using the response surface method (RSM) were ethanol concentration of 74%, ratio of solvent to material 26:1 and ultrasonic extraction time of 82 min based on the results of single factor experiment. The nuciferine yield was 0.1035\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005% under optimal experimental conditions; the relative standard deviation (RSD) of five measurements was 4.58% and the relative error between the mean of five measurements and the predicted value of the model was 2.45% less than 5%, suggesting that the DAUEM has good precision and accuracy. The nuciferine purity purified with D101 macroporous adsorption resin was 67.14%. These results provide a theoretical basis for the development and utilization of nuciferine in lotus leaves. However, the purity of nuciferine purified by D101 macroporous adsorption resin was not high. Therefore, we will focus on method that can obtain high-purity nuciferine in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eI am authorized on behalf of all the authors of this article to confirm that no author has any conflict of interest to disclose, all authors have approved the version submitted for publication, the work in this article is original and has not been published previously, and the article is not under consideration by any other journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMW and XRcollected the data, MW and XR analyzed the data and wrote the manuscript, HP managed and directed the trial, HP and SL gave comments and suggestions to improve the manuscript. All authors read and approved the final manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the National Natural Science Foundation of China (81973097).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used in this study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChinese Pharmacopoeia Commission.Chinese pharmacopoeia[M].Beijing:China Medical Science and Technology Press,2020:287.\u003c/li\u003e\n\u003cli\u003eShuhan Zou, Chenhong Wang, Jin Li, Jun He, Xiumei Gao, Yan-xu Chang, Microwave assisted solid phase microextraction for extraction and selective enrichment of four alkaloids in lotus leaf,Sustainable Chemistry and Pharmacy, Volume 18,2020,100345. \u003c/li\u003e\n\u003cli\u003eYuhuan Chen, Qiwen Chen, Xiaozhong Wang, Fan Sun, Yawei Fan, Xiaoru Liu, Hongyan Li, Zeyuan Deng,Hemostatic action of lotus leaf charcoal is probably due to transformation of flavonol aglycons from flavonol glycosides in traditional Chinses medicine,Journal of Ethnopharmacology,Volume 249,2020,112364.\u003c/li\u003e\n\u003cli\u003eKi H K , Sang W C , Shi Y R , et al. 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Guangdong Chemical Industry, 2017,44(13):92-93.\u003c/li\u003e\n\u003cli\u003eZhao Jun, Wang Hongzhang, Qi Xihong, et al Study on the extraction process of lotus leaf alkaloid from lotus leaf [J] Chinese Herbal Medicine, 2003, 34 (010): 916-917.\u003c/li\u003e\n\u003cli\u003eZhou Yun, Luo Jialing, Zhou, et al Optimization of ultrasonic extraction process of pilocarpine from lotus leaf by orthogonal design [J] Guangdong Chemical Industry, 2017, 44 (13): 2.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Materials","content":"\u003cp\u003eThe Supplementary Materials are not available with this version.\u003c/p\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":"Lotus leaf, Nuciferine, Response surface methodology (RSM), Ultrasonic extraction, purification","lastPublishedDoi":"10.21203/rs.3.rs-2257445/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2257445/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLotus leaf is an agricultural product as well as a kind of Chinese herbal medicine. Nuciferine is an amorphine alkaloids in lotus leaf that has anti-inflammatory, lipid-lowering and hypoglycemic effects, so it is greatly significant to extract and purify nuciferine. However, the factors that affect the nuciferine of lotus leaf extraction by dipping-acid-assisted ultrasonic extraction method (DAUEM) are rarely studied. The purpose of this research estimates DAUEM conditions (ethanol concentration, ratio of solution to material, ultrasonic extraction time) on responses by Box\u0026ndash;Behnken response surface design. In addition, nuciferine of lotus leaf is purified by D101 macroporous adsorptive resin. A single factor test was conducted using nuciferine yield as an indicator and taking ethanol concentration (V/V), ratio of solution to material (V/M), ultrasonic extraction time as the influencing factor. The extraction process of nuciferine was optimized by response surface method based on the single factor test results. Nuciferine in lotus leaf was separated and purified by D101 macroporous adsorptive resin. The nuciferine yield (0.1035\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005) % was the largest as using 74% ethanol (V/V), ratio of solution to material 26:1, and the extraction time 82 min. The purity of nuciferine was 67.14% with 70% ethanol as eluent and through high performance liquid chromatography (HPLC) determination. This experiment has good precision and accuracy, so can be used for the extraction, separation and purification of nuciferine in lotus leaf. The method provides a theoretical basis for the development and utilization of nuciferine. Dipping-acid-assisted ultrasonic extraction method (DAUEM) can fully extract nuciferine in lotus leaf. Box-Behnken response surface methodology (RSM) was used to optimize the ultrasonic extraction process. D101 macroporous adsorption resin can admirably separate and purify nuciferine in lotus leaf.\u003c/p\u003e","manuscriptTitle":"Response surface optimization of ultrasonic extraction conditions of nuciferine and its purification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-15 15:08:22","doi":"10.21203/rs.3.rs-2257445/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":"3864acf9-5bba-4267-8578-0ead13504b40","owner":[],"postedDate":"November 15th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-11-28T06:59:28+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-15 15:08:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2257445","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2257445","identity":"rs-2257445","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}