Optimizing Ultrasonic Extraction and Purification of Nuciferine With Response Surface Method

Abstract

Background

Lotus leaves are obtained from plants belonging to the genus Nelumbo in the Nymphaeaceae family. They serve both as agricultural produce and traditional Chinese medicinal herbs. Nuciferine, an amorphine alkaloid found in lotus leaves, holds significance due to its anti-inflammatory, lipid-lowering, and hypoglycemic properties.

Objective

The factors influencing nuciferine extraction from lotus leaves using the dipping-acid-assisted ultrasonic extraction method (DAUEM) have received limited attention. This study aims to optimize DAUEM conditions (ethanol concentration, solvent-to-material ratio, and ultrasonic extraction time) through a Box-Behnken response surface design.

Methods

Nuciferine purification from lotus leaves is achieved using D101 macroporous adsorptive resin. A single-factor test, with ethanol concentration (V/V), solvent-to-material ratio (V/M), and ultrasonic extraction time as variables, serves as a basis for optimizing the nuciferine extraction process via the response surface methodology.

Results

The highest nuciferine yield (0.1035 ± 0.005)% was obtained using 74% ethanol (V/V), a 26:1 solvent-to-material ratio, and an 82min extraction time. Nuciferine purity reached 67.14% using 70% ethanol as the eluent and high-performance liquid chromatography for determination. DAUEM effectively extracts nuciferine from lotus leaves, with optimization achieved using Box-Behnken response surface methodology. D101 macroporous adsorption resin efficiently separates and purifies nuciferine in lotus leaves.

Conclusion

This experiment demonstrates good precision and accuracy, making it suitable for the extraction, separation, and purification of nuciferine in lotus leaves. The method lays the foundation for the development and utilization of nuciferine.

Keywords

lotus leaf nuciferine response surface methodology ultrasonic extraction purification alkaloids

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 (Figure 1) is an aporphine alkaloid in lotus leaf, and a 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, creams soap, etc. Lotus leaves 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, an 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) was designed for the extraction of nuciferine. The dip method has been widely used in the extraction of traditional Chinese medicine ingredients due to its convenience, cost-saving, and high dissolution rate.^9,10 The 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 be more soluble in polar solutions (such as water, ethanol formic acid, etc). 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 a better effect on the separation and purification of nuciferine in lotus leaves.¹⁵ Consequently, a single-factor test was conducted using nuciferine yield as an indicator and taking ethanol concentration (V/V), the ratio of solvent to material (V/M), and ultrasonic extraction time as the influencing factor. The extraction process of nuciferine was optimized by the 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 providing a theoretical basis for the development and utilization of lotus leaf resources.

Results and Discussion

Response Surface Experimental Results and Its Analysis

RSM was used to optimize the extraction process of nuciferine, the results of the experiment are shown in Table 1. The experimental data in Table 1 were performed regression fitted by Design Expert 8.0 software. The fitted equation was as follows.

\begin{aligned} Y = & 0.11 + 0.004558 X 1 + 0.002363 X 2 + 0.001275 X 3 \\ - & 0.0009 X 1 X 2 + 0.000225 X 1 X 3 - 0.002925 X 2 X 3 \\ - & 0.00684 X 1^{2} - 0.00739 X 2^{2} - 0.006565 X 3^{2} \end{aligned}

Table 1.

The Experimental Results of Response Surface.

	X₁= A:ethanol concentration (V/V)/%		X₂= B: ratio of solventto material (V/M)		X₃= C:ultrasonic extraction time/min		The nuciferineyield (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 for the analysis of variance and significance, and the results are shown in Table 2. The results show that there were significant differences in the model equations (model, F = 18.06, P = .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 the lack of fit of the model (P = .6979 > .05). Nonsignificant 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 >4 was desirable. Your ratio of 11.949 indicates an adequate signal. This model can be used to navigate the design space. The adjusted determination coefficient (adjusted R-squared) was 0.9056 indicating that the model can explain 90.56% of the response value changes. The coefficient of determination (R-squared) was 0.9587 demonstrating 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.

Table 2.

Analysis of ANOVA at Experimental Results of RSM.

Source	Some 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

Abbreviations: ANOVA, analysis of variance; RSM, response surface methodology.

If P was <.05, the difference was significant, and if P was <.01, the difference was extremely significant. It can be seen from Table 2 that the difference in model was extremely significant from the linear term X₁ (P = .001 < .01) and the quadratic term X₁² (P = .0006 < .01), X₂² (P = .0004 < .01), and X₃² (P = .0008 < .01), and was significant from the linear term X₂ (P = .0276 < .05) and the quadratic term X₂X₃ (P = .045 < .05). Through the analysis of P, the order of the main effects of each factor was ethanol concentration (V/V), the ratio of solvent to material (V/M), and ultrasonic extraction time from top to bottom.

The steepness of the response surface reflects the significant degree of interaction between the 2 factors. A large steep surface indicates that the 2 factors were significantly higher, and a small surface steepness suggests that the 2 factors were less significant. To investigate the interaction between the relevant factors, the response surface diagram was plotted by Design Expert 8.0 software for visual analysis (Figure 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 (Figure 2(a) and (b)), indicating that the interaction between ethanol concentration (V/V) and the ratio of solvent to material (V/M) was not significant when ultrasonic extraction time was invariable. Ethanol concentration than the ratio of solvent to the 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 the ratio of solvent to the material. The response surface was flat (Figure 2(c) and (d)) to illustrate that the interaction between ethanol concentration and ultrasonic extraction time was not significant when the 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 enhancement of ethanol concentration (V/V) and ultrasonic extraction time. The response surface was flat (Figure 2(e) and (f)), suggesting that the interaction between the ratio of solvent to material and ultrasonic extraction time was not significant when ethanol concentration (V/V) was unchangeable. The 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 in the ratio of solvent to material and ultrasonic extraction time.

Optimal Process of DAUEM and Its Verification

As can be seen from Figure 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%, the 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 5 measurements was 4.58%, indicating that the extraction process has good precision. The relative error between the mean of 5 measurements and the predicted value of the model was 2.45% <5%, suggesting that the extraction process has good accuracy (Table 3). 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 3.

The Measured and Predicted Response of Nuciferine Yield.

	Measured value of nuciferine yield（%）	Predicted value of nuciferine yield（%）	Relative error (%)
	0.1075	0.1087	2.45
	0.1092
	0.1026
	0.0988
	0.0992
Mean	0.1035
RSD (%)	4.58

Abbreviation: RSD, relative standard deviation.

Optimal Conditions for Purification

The purified chromatogram and elution curve of nuciferine are shown in Figure 3(A) and (B). Compared to unpurified sample chromatograms (Supplemental Figure S7), purified sample chromatograms were clearer and free of impurities (Figure 3(A)). The abscissa was the volume fraction of ethanol concentration (V/V) and the ordinate was the nuciferine purity (Figure 3(B)). 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% to 70% ethanol. Until ethanol concentration (V/V) was 70%, the nuciferine purity reached the maximum. It was explained that 70% ethanol can better elute and dissolve nuciferine. The nuciferine purity was decreased in the range of 80% to 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 from nuciferine was 67.14% using 70% ethanol as an extraction solution.

Method Comparison

The performance of our method was compared with the reported method for extraction and separation of nuciferine (Table 4). Our method was even less for weighing lotus leaf powder, relatively low ethanol concentration, solvent amount, and extraction time than another 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, the percentage of nuciferine was not possible to compare. In addition, compared with the orthogonal method, the response surface method was more flexible and had higher regression accuracy. However, the nuciferine purity in 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 because the extraction and separation methods were different. Therefore, our method provides an economical, easy, and rapid method for extraction and purification of nuciferine. Utilizing ultrasound in our desired extraction solvent produces high-frequency mechanical vibration and thus the cavitation effect, and this cavitation will make the center reaction near the cell damage and destruction, cavitation makes the required cavitation makes the original force between the extract and the raw material natural products weakened, so as to realize the extraction of the required extract. The effect of the ultrasonic extraction method is a high extraction rate, speed, solvent saving, and low temperature. Ultrasonic-assisted extraction compared to the traditional reflux method to improve the extraction rate and shorten the time. In the extraction of natural products, the extraction technology of ultrasound-assisted extraction occupies a great advantage. Therefore, ultrasonic extraction method can economically and efficiently extract nuciferine.

Table 4.

Comparison of Different Methods for Extraction and Purification of Nuciferine.

Extracted substances	Nuciferine	Total alkaloids	Nuciferine	Nuciferine
Extracted method	Heated reflux	Heated reflux	Ultrasonic extraction	Ultrasonic extraction
Optimization method	/	Orthogonal method	Orthogonal method	Response surface method
Weighing of lotus leaf powder (g)	20	100	20	5
Extraction solvent /amount (mL)	50% Ethanol/200	95% Ethanol/2000	90% Ethanol/800	74% Ethanol/130
Extraction time (min)	120	120	20	82
Weighing of crude extraction powder of nuciferine (g)	1.45	/	/	1.25
Percentage of nuciferine (%)	3.196 （ crude extraction powder percent ）	/	1.12 （ crude extraction powder percent ）	0.1035 （ lotus leaf powder percent ）
Purification method	Combination technology of cation exchange resin and silica gel column chromatography	/	/	D101 macroporous resin
The nuciferine purity (%)	94.15	/	/	67.14
Reference	19	16	17	This work

Conclusion

In conclusion, the DAUEM was used for the extraction of nuciferine in lotus leaves. Combining dip with the 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 RSM were ethanol concentration of 74%, the ratio of solvent to material 26:1, and ultrasonic extraction time of 82 min based on the results of a single factor experiment. The nuciferine yield was 0.1035 ± 0.005% under optimal experimental conditions; the RSD of 5 measurements was 4.58% and the relative error between the mean of 5 measurements and the predicted value of the model was 2.45% <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. The prepared resources of nuciferine are abundant because lotus leaves are widely available. This experiment proposed a feasible and cost-effective strategy for the production of nuciferine, which provides a theoretical basis for the development and utilization of lotus leaf resources and lays a foundation for the clinical application and medical research of nuciferine. However, the purity of nuciferine purified by D101 macroporous adsorption resin was not high. Therefore, we will focus on methods that can obtain high-purity nuciferine in the future.

Experimental Section

Chemicals, Solvents, Standards, and Apparatus

The nuciferine standard (Lot No. DSTDH005102) used in this study was purchased in May 2021 from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd, Purity is high-performance liquid chromatography (HPLC) ≥ 98%, stored at 2–8 °C. It is the standard for HPLC test of nuciferine. Lotus leaf powder (Lot No. 20210521) is the powder made from lotus leaves that have been picked and dried in the sun and pulverized in a pulverizer， purchased from Lu'an Danbeier Biotechnology Co., Ltd on May 21, 2021, and was produced in Anhui. Ethanol (C₂H₆O, ≥ 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, analyrically pure) were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. Acetonitrile (chromatographically pure) was purchased from Shanghai Anpu Scientific Instrument Co., Ltd. D101 macroporous adsorptive resin and triethylamine were purchased from Shanghai Macklin Biochemical Co., Ltd.

In this study， the nuciferine ultrasonic extraction process involves dissolving lotus leaf powder in ethanol, followed by infiltration and subsequent ultrasonication using an ultrasonic instrument (model number: KQ5200DE) purchased from Kunshan Ultrasonic Instrument Co., Ltd. Then concentrated with a rotary evaporator (model number, OSB-2200) purchased from Tokyo Physio Equipment Co., Ltd, followed immediately by lyophilization with a freeze dryer (model number, CHRIST) purchased from Shanghai Laijing Scientific Instrument Co., Ltd. In this study, the samples and reagents were diluted and washed with purified water from an Ultra-pure Water Purifier (model number, Milli-Q A10) purchased from Merck & Co Inc. The analysis of nuciferine was performed using the Agilent Technologies LC-1260 High-Performance Liquid Chromatography System (HPLC-DAD) equipped with a diode array detector. A Poroshell 120 EC-C18 reverse-phase column (4.6 × 150 mm, particle size: 4 μm) was employed for the determination of nuciferine content. Additionally, analysis was conducted using a chromatography column from Shanghai Haofu Instrument Co., Ltd (height 400 mm × diameter 20 mm). In this study, an electronic balance from Shanghai Shunyu Hengping Scientific Instrument Co., Ltd (model FA124) was utilized for weighing both samples and reagents. Additionally, a circulating water multipurpose vacuum pump from Shanghai Yukang Science Education Instrument Equipment Co., Ltd (model SHB-HIA) was employed to evacuate the samples.

Experimental Process and Design by DAUEM of Nuciferine

The process of DAUEM was executed. Firstly, lotus leaf powder was precisely weighed at 5 g (m₁). The weight was soaked in ethanol (pH 2.0) for 12 to 24 h to form a dipping substance. Then, the dipping substance was extracted by ultrasound, and let sit for 1 to 2 h to obtain the extracting solution. Lastly, the extracts were filtered, concentrated, and lyophilized to gain crude extraction powder of nuciferine (m₂). The screening process of pH condition factors in the extraction process was presented in Supplemental materials (S1), and the optimal condition was pH 2.0.

The extraction of nuciferine by DAUEM was designed by Box-Behnken RSM. Different ethanol concentrations (V/V), the ratio of solvent to material, and ultrasonic extraction time (3 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 been designed with 3 factors as independent variables and 3 levels based on the results of single-element experiments¹⁷ (Table 5). The experimental procedures and results of the single element were presented in Supplemental materials (Supplemental Figure S2).

Table 5.

Level Table of Experimental Factors in the Response Surface.

Factors (independent variables)	Level
Factors (independent variables)	−1 (Low)	0 (Center)	1 (High)
X₁= A:ethanol concentration (V/V)/%	60	70	80
X₂= B: ratio of solvent to material	20:1	25:1	30:1
X₃= C: ultrasonic extraction time/min	60	80	100

According to the level table of experimental factors (Table 5) and design principle of the response surface, the experimental design of the response surface was placed in Supplemental Table S1.

Determination of Nuciferine

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 Supplemental materials (Supplemental Figure S3).

Crude extraction powder of nuciferine was precisely weighed at 10 mg (m₃), dissolved in methanol, and brought to 10 mL volume (V) to gain a 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 formula¹⁸ (1):

Y (%) = \frac{c \times V \times m_{2} \times 10^{- 3}}{m_{1} \times m_{3}} \times 100 %

(1)

Of these, “Y” was the nuciferine yield, “c” the sample concentration measured by HPLC, “V” the volume of the sample solution, “m₁” the weighing of lotus leaf powder, “m₂” the weighing of crude extraction powder of nuciferine, and “m₃” the weighing that crude extraction powder of nuciferine was weighed.

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 Supplemental Data (Supplemental Figure S4).

Crude extraction powder of nuciferine was weighed at 5 g, 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 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%, and 90%) to gain the nuciferine eluent. Per ethanol concentration (V/V) cleans 4 column volumes and the flow rate was controlled at 10 mL/min. After the nuciferine eluent was collected separately with triangular flasks (numbers 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 was 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):

P (%) = \frac{c \times V \times k \times 10^{- 3}}{m} \times 100

(2)

Of these, “P” was the nuciferine purity, “c” the sample concentration measured by HPLC, “V” the volume of the sample solution, “k” the dilution multiple, and “m” the weighing that the nuciferine purified powder was weighed.

Statistical Analysis

RSM was applied to analyze the optimum condition of DAUEM. Data was analyzed with Design-Expert V 8.0.

Supplemental Material

sj-doc-1-npx-10.1177_1934578X231220119 - Supplemental material for Optimizing Ultrasonic Extraction and Purification of Nuciferine With Response Surface Method

Supplemental material, sj-doc-1-npx-10.1177_1934578X231220119 for Optimizing Ultrasonic Extraction and Purification of Nuciferine With Response Surface Method by Xinshui Ren, Mengdie Wu, Simin Liu and Hongzhi Pan in Natural Product Communications

Footnotes

Author Contributions

XR and MW collected the data, XR and MW analyzed the data and wrote the manuscript, HP managed and directed the trial, and SL gave comments and suggestions to improve the manuscript. All authors read and approved the final manuscript.

Availability of Data and Materials

The datasets used in this study are available from the corresponding author upon reasonable request.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the National Natural Science Foundation of China (81973097).

ORCID iD

Hongzhi Pan

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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