Mechanochemical-Assisted Extraction of Active Alkaloids from Plant

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Mechanochemical Assisted Extraction of Active Alkaloids from Plant with Solid Acids Shu-Ling Wang, Rui Zhang, Meng-Meng Wei, Tian Xie, Yun-Ting Sun, Jin-Dong Hu, Lian-Hui Men, and Jun Cao ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b02902 • Publication Date (Web): 26 Nov 2018 Downloaded from http://pubs.acs.org on November 26, 2018

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ACS Sustainable Chemistry & Engineering

Mechanochemical Assisted Extraction of Active Alkaloids from Plant with Solid Acids Shuling Wang1, Rui Zhang1, Xiaoyu Song1, Mengmeng Wei1, Tian Xie1*, Jun Cao1,2 *

1 Holistic Integrative Pharmacy, Hangzhou Normal University, Hangzhou 311121, PR China 2 College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, PR China

Corresponding author: Tian Xie*, Fax: 86-571-28860237; E-mail: [email protected]. Jun Cao*, Fax: 86-571-28860239; E-mail: [email protected].

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ABSTRACT: In this work, a novel mechanochemical assisted extraction (MCAE) coupled with ultra high performance liquid chromatography (UHPLC) method was developed to extract two alkaloids with solid acids from Stephania tetrandra S. Moore. To further understand the influence of extraction parameters on MCAE process, the response surface methodology was applied for systematic investigation. The results showed that the highest yield of alkaloids was obtained within 15 min of the total extraction time under the optimal conditions: oxalic acid (20 wt.%), grinding plant materials for 5 min, and the liquid/solid ratio of 20 mL/g. Mechanochemical treatment resulted in the destruction of cell walls and increased the total contact surface area due to the smooth surface changing to an open porous structure. Accordingly, the developed extraction method showed the advantages of more ecofriendly, faster, and more efficient than conventional extraction methods. Meanwhile, the extractant was analyzed by UHPLC, the obtained LODs for fangchinoline and tetrandrine were 0.013 and 0.012 μg/mL, and good linearity with correlation coefficient of determination higher than 0.9991. Moreover, spiked recoveries were in the range of 101.03–103.81%. It proves that mechanochemistry processing provides a sensitive, highly effective and environmentally friendly method.

KEYWORDS: Alkaloids, Mechanochemical assisted extraction, Planetary mill, Response surface methodology, Stephania tetrandra S. Moore, Ultra high performance liquid chromatography,


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

INTRODUCTION In recent years, natural products have increasingly been studied because of the

bioactive natural products play an important role in transformation and use of biomass. Different techniques have been reported for the extraction of the biologically active substances, and determination of the chemical structures of natural products from plants. Traditional extraction technologies, such as decoction, percolation, soxhlet extraction,1 and heat reflux extraction,2 and other extraction methods including ultrasound extraction,3 microwave extraction,4 miniaturized solid-phase extraction,5 liquid-solid extraction,6 and so on were reported mainly for the extraction from medicinal plants. Although these methods are relatively safe and simple, they suffer the drawbacks of being time consuming, require a large amount of organic solvent and the extraction efficiency is poor. At the same time, the pharmacological activity of valuable compounds may be decreased and lost in the heating process. Apparently, it's has some risk to environment and not green. Hence, the development of a more efficient and more environmentally friendly extraction technique to prevent pollution and reduce the cost of sample preparation is still need further study, especially for the green development in research and industry. Mechanochemistry is an interdisciplinary science based on chemistry and mechanical engineering, which investigates the chemical or physicochemical changes of substances under high-energy mechanical force.7 Mechanochemical technologies cover a wide range of fields, such as extractive metallurgy, waste management,8 graft modification,9

agriculture,10

and

supramolecular

synthesis.11-12

Recently,

mechanochemical assisted extraction (MCAE) has been introduced as an intensification pretreatment procedure due to its green pollution-free characteristics, which allows the extraction of bioactive compounds for achieving chemical



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processing and transformations.13 The green MCAE method has many advantages, such as increasing the yield of bioactive products, and extracting substances with water at room temperature instead of the organic solvents,14 reducing extraction time and simplifying the purification. Meanwhile, mechanochemical treatment may potentially put the product in a metastable state that releases the accumulated energy to reach a more stable thermodynamic state,15 leading to the conversion of insoluble target compounds into water-soluble salt. Therefore, it is possible to extract and analyze efficiently from a variety of matrices, to fulfill the requirements of green chemistry and green engineering.16 The possible mechanism of MCAE technique is reduced particle size, destroy the cell wall, decompose cellulose,

accelerate the

dissolution kinetics that can increase extraction efficiency and decrease the processing time.17 For example, Xie et al. found that the yield of rutin in Hibiscus mutabilis L was significantly increased by 31.7%.7 Xie et al. reported that the yield of magnolol from Magnolia officinalis produced by mechanochemical extraction was about 7% more than that of heat-reflux extraction.18 Moreover, Xie et al. found that extract the flavonoids from bamboo leaves only require 10 min.19 To date, published reports mainly extract the acidulous compounds including flavonoids, chondroitin sulfate, rutin, kaempferol glycosides and polysaccharides.20 However, the solid reagents used in the MCAE process are solid alkali compounds, such as Na2CO3, NaHCO3 and NaOH. Obviously, it is incapable of extracting alkaline compounds. Consequently, the need for new types solid reagents is crucial for the application of MCAE to extract a great diversity compound. Fangchi, root of Stephania tetrandra S. Moore is one of used extensively traditional medicinal plant listed in the Chinese Pharmacopoeia,21 which has been utilized as an analgesic and diuretic agent and in the treatment of hypertension.22 The Fangchi


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contains various species of bioactive constituents including aporphine, morphine, alkaloids and lignans.23 Tetrandrine (TET) and fangchinoline (FAN) are the main alkaloids, which have broad pharmacological activities such as anti-inflammatory activity, hypotensive,24 anti-fibrotic activity25 and can reverse multidrug resistance.26 It has also been shown to act as a potent calcium channel blocker27 and proved to be a strong anticancer agent based on in vitro and in vivo studies.28 Methods available for extraction alkaloids in Fangchi included conventional heat reflux extraction, ultrasound assisted extraction, and has gained mainly apply, but those methods still require long extraction time and have a low efficiency. Therefore, it’s of great significance to develop a rapid, efficient and eco-friendly technique on the extraction method of Fangchi alkaloids its clinical application. In the present study, a novel based on the MCAE method used solid acid extract main alkaloids FAN and TET in Fangchi combination with UHPLC was applied in order to improve the conventional extraction methods. Box-Behnken experimental design with RSM was implemented in details to obtain the optimal extraction efficiency. The key independent variables have been optimized including the concentration of oxalate acid (X1), liquid/solid ratio (X2) and extraction time (X3). Other significant variables affecting extraction process such as solid acids type, solid acids concentration, milling speed, milling time were also studied. Scanning electron microscopy and infrared spectroscopy were used to characterize the transformation of the structural during the mechanochemical process. In particular, the experimental results were also compared to a conventional extraction process. 

EXPERIMENTAL SECTION Materials and Reagents. The standard FAN and TET were obtained from Shanghai

Winherb Medical Science CO., Ltd. (Shanghai, China) and chemical structures as



showed in Figure 1. The solid acids boric acid (99.5%), succinic acid (99.5%) and citric acid (99%) were purchased from Sinopharm Chemical Reagent CO., Ltd. (Shanghai, China). Oxalic acid (98%) was obtained from Alfa Aesar Chemicals CO., Ltd. (Shanghai, China). Purified water was supplied by Wahaha Group Ltd. (Hangzhou, China) for use throughout the study. Freshly Fangchi was collected from the local supermarket (Hangzhou, China). Methanol and triethylamine (HPLC grade) were provided by Sigma-Aldrich Shanghai Trading Co., Ltd. (Shanghai, China). Mechanochemical Assisted Extraction. The dried plant samples were shredded and

then filtered through a 60 mesh screen. 2.0 g samples with different solid acids were added into planetary ball mill (Focucy F-P400H, China) equipped with four zirconia ball mill drums (weight of balls: 50 g; the volume ratio of load to drum: 1:2; the volume of drum: 100 mL). After grinding at 500 rpm for 10 min, the mixture with an appropriate volume of water was oscillation extracted for 5 min. The extract was then centrifuged at 3000 rpm for 5 min. Next the suspension was collected and filtered through a 0.45 μm membrane filter prior the UHPLC analysis. The analysis and further process as shown in Figure 2. In order to purify and obtain the alkaloids, the supernatant was adjusted the pH to 9 by 0.2 M NaOH. Then concentrated under vacuum by a rotary evaporator to obtain the product. Analytical Conditions. The FAN and TET under investigation were both performed

on an Agilent 1290 series ultra high performance liquid chromatography system (Agilent Technologies Inc., USA) equipped with ultraviolet detector (UV) and autosampler. Chromatographic separation was achieved using an Agilent SB C18 column (50 mm × 4.6 mm i.d, 1.8 μm) and flow rate was constant at 0.5 mL/min. The column oven was maintained at 30 °C. The mobile phase was methanol/water (72: 28,


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v/v) containing 0.05% triethylamine, with 1.0 μL injection volumes. Meanwhile, the detection wavelength was at 282 nm. Qualitative analysis was performed on the Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies, USA) equipped with an ESI source. The operating parameters of the ESI sources were as follows: drying gas (N2) flow rate, 12.0 L/min; drying gas temperature, 350 °C and nebulizer, 45 psig; capillary voltage (Vcap) was 3500 V; fragmentor voltage, 175 V; octapole radio frequency voltage (Oct RFV), 750 V; and skimmer voltage, 65 V. The ESI source was operated in positive ionization mode with the mass range m/z 100–1500. All the data were collected and analyzed by MassHunter software (Agilent Technologies). Preparation of Standard Solution. TET and FAN stock solutions were mixed and

dissolved in methanol to make a final concentration of 1 mg/mL, and stored at 4. 0 °C until use. The standard working solutions were prepared by diluting the stock solutions with methanol. Optimization of Extraction Conditions by Box–Behnken Design (BBD). The Response

Surface Methodology (RSM) was plotted to analyze the interaction of the variables and to attain the optimal conditions29 for the maximum amount of alkaloids extracted. In this study, the statistical analysis was performed using the Design-Expert® Software (v. 8, Stat Ease, USA), and Box-Behnken was selected. In this study, three independent variables of the concentration of oxalate acid (X1), Liquid/solid ratio (X2) and extraction time (X3) were selected for investigation. The total design consisted of 17 experiments with 5 central points, whereas the independent variables and the code levels were shown in Table 1. The experimental data were fitted to a second order polynomial model used in the response surface as follows:



Y  0  



 j j  

j 1



 jj  2j  

j 1

 i j

 ij  iY j

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（1）

where Y is the extracted amount of alkaloid (mg/g), β0 is the intercept constant, and βj; βjj and βij are the regression coefficient for the linear, quadratic and cross-product effects of the X1; X2 and X3 factors, respectively. κ is the number of independent variables (κ=3), while Xi and Xj are levels of the independent variables.



RESULTS AND DISCUSSION Microstructural Characterization by SEM. Scanning electron microscopy (SEM) is

used to describe the microscopic surface morphology of the Fangchi with different pretreatments (Figure 3). Firstly, structures of the untreated sample (Figure 3a) was compared with that of the ground samples (Figure 3b). It could be clearly seen that the Fangchi became the smaller particles after grinding, and the mechanical treatment destroys the plant cell walls. Figure 3c shows the SEM image of the sample that had ground with solid acid for 5 min. As marked in the figure cell wall was found to be completely destroyed, indicating that the majority of bioactive compounds were released. Meanwhile, according to the images shown in Figure 3c, the particles were irregular and the surface was covered with solid acid. See the Supporting Information (Figure S1-S3) for other electron micrographs. Thus, it was conceivable that the use of solid acid not only diminishing of the particle size, but also contributes to decomposition of cell walls and releases more substances. FT-IR Analysis. Fourier Transform Infrared Spectrometer (FT-IR) is an effective

method for analyzing the changes of chemical structure during mechanochemical


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reaction. As showed in Figure 4, IR spectra of the shattered sample and the MCAE treatment sample had similar infrared absorption characteristic peaks but some differences. The intense peaks at 3434.34 cm-1 and 2930.57 cm-1 correspond to the free amide group and the C-H group on unsaturated carbon, respectively. The spectrum of the untreated sample was consistent with the TET on the previous reports.30 In comparison, it was found that there was no absorption at 860.72 cm-1, which could be interpreted as the C-H bending vibration. It also needs to be noticed that the presence of a functional group at 1261.13 cm-1 in the mechanochemical treatment spectrum, showed the participation of the ether oxygens in the formation of alkaloid salts. What’s more, the fingerprints peak appeared at 720.62 cm-1 was also demonstrated that alkaloid might be transformed into the salt form. Effect of Solid Acids type. During the mechanochemical processing, the target

alkaloids will react with solid acids, and insoluble bioactive materials are converted into water-soluble or highly reactive mechanical composites. The type of solid acids depends on the chemical nature of the biologically active substance and the possible reaction between the substance and the solid acids. Hence, different acidic strength acids were investigated to evaluate their performance in the extraction from Fanchi. In this work, oxalic acid, citric acid, succinic acid, and boric acid were used for the mechanochemical reaction at the same condition of solid acids concentration of 15 wt.%; milling speed, 500 rpm; milling time, 10 min; extraction solvent, water; extraction time, 5 min; liquid/solid ratio, 15 mL/g; centrifuge at 3000 rpm for 5 min. Figure 5a shows that milling of the raw material with oxalic acid produced the highest extraction amounts, while boric acid resulted in the lowest extraction amounts. Because of succinic acid and citric acid were both moderate strong acid with similar acidic properties, no significant difference was observed between the succinic acid



and citric acid. In particular, the content of FAN extracted by oxalic acid was four times more than extracted by boric acid. So the main reasons are as follows: firstly, oxalic acid is the strong acid in the organic acid. As acidity increases, the reaction becomes more complete. Secondly, the boric acid is too weak to react with alkaloids which could not be converted to salt completely during the grinding process. What’s more, some significant changes were also been observed, the color of the solution changed from pale yellow to dark yellow. As a result, oxalic acid was selected as the optimal solid acid in the following studies. Effect of Solid Acid Concentration. Solid acid concentration was considered as one of

the foremost parameters that had a remarkable influence on the extraction process. In general, the appropriate amount of solid acids would help enhance the extraction efficiency enormously. In this work, experiments were performed with different oxalic acid concentrations (5.0, 10.0, 15.0, 20.0 wt.%) under the same extraction conditions (milling speed, 500 rpm; milling time, 10 min; extraction solvent, water; extraction time, 5 min; liquid/solid ratio, 15 mL/g; centrifuge at 3000 rpm for 5 min). The results are exhibited in Figure 5b. It was seen from the results, the amount of extraction alkaloids had a slightly increases before the oxalic acid concentration reaching to 20 wt.%, and then decreased with further increase of the oxalic acid concentration. However, in this case is attributed to the kinetics of mechanochemical process. Moreover, the high oxalic acid concentration could cause the excess acidic residues in the extraction matrixes, which resulted in the reduction of extraction efficiency. While oxalic acid concentration increased to 20 wt.%, alkaloids had been transformed into salt completely already and reached the maximum. On the basis of these findings, to obtain high efficiency in the extraction process, 20 wt.% oxalic acid was selected as the optimum solid acid concentration in the following studies.


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Effect of Liquid/Solid Ratio. Liquid/solid ratio was another key factor that affected

extraction yields of alkaloids. The purpose of this study was to determine the appropriate liquid/solid ratio to achieve the higher extraction efficiency. So the liquid/solid ratio was investigated at 5 to 20 mL/g under the same conditions (solid acids concentration of 20 wt.%; milling speed, 500 rpm; milling time, 10 min; extraction solvent, water; extraction time, 5 min; centrifuge at 3000 rpm for 5 min) to find the optimum conditions (Figure 5c). The results indicated that the extraction yields of two alkaloids were improved with the increase of the extraction solvent volume, and the relatively high yields of two alkaloids were observed at the liquid/solid ratio of 15 mL/g, respectively. Moreover, when the liquid/solid ratio was increased to 20 mL/g, the extraction yields decreased slightly. However, it is found that the liquid/solid ratio had a slight effect on the alkaloid extraction. In our study, on the one hand, the content of the sample alkaloid was constant, and the ratio of 15 mL/g was sufficient to extract the alkaloid. On the other hand, in fact, when the mechanochemical reaction was saturated the diffusion rate would decreased. Consequently, the extraction compound had a tendency to stabilize. Therefore, the liquid/solid ratio of 15 mL/g was selected in following experiments. Effect of Milling Speed. It is important to identify optimal ball mill speed, as it is one

of the key factors contributing to extraction efficiency of mechanochemical extraction. To evaluate the different milling speeds (300 rpm, 400 rpm, 500 rpm, 600 rpm), a series of experiments were performed in the same extraction conditions (oxalic acid concentration 20 wt.%; milling time, 10min; extraction solvent, water; extraction time, 5 min; liquid/solid ratio, 15 mL/g; centrifuge at 3000 rpm for 5 min). The results are shown in Figure 5d. It was seen that the highest extracted amount for TET was obtained when the milling speed increased to 400 rpm. In general, this can be



interpreted as the mechanochemical reaction provided a unique force to disrupt the cell membrane and cell walls. Simultaneously higher milling speed accelerated the collision of milling balls and sample to achieve full release of biologically active compounds into the surrounding solvents for extraction. Meanwhile, another phenomenon was that the extracted amount of FAN remained stable almost no more difference, which suggested the milling speed had no significant effect for the extraction of FAN. This possibly due to the content of the FAN in plant material, and excessive speed would agglomerate samples (Figure S1, Supporting Information) so that 400 rpm speed was enough for extraction with higher extraction efficiency. Hence, 400 rpm milling speed was selected for the rest of the experiments. Effect of Milling Time. It is also worth mentioning that the milling time is an

important parameter, which affects the total surface area of the material. Four levels of grinding time (2, 5, 10 and 15 min) on the extraction efficiency were studied under the same conditions (oxalic acid concentration 20 wt.%; milling speed, 400 rpm; extraction solvent, water; extraction time, 5 min; liquid/solid ratio, 15 mL/g; centrifuge at 3000 rpm for 5 min). It could be seen from Figure 5e that extraction amount of alkaloids increased slowly with increasing milling time until to 10 min and then fell down. Thus, the significant impact of milling time was observed. In general, mechanochemical pretreatment not only led to particle size reduction, but also increased the specific surface area. Consequently, Intracellular compounds were released completely and enhanced the potential chemical reactions with solid acids. Nevertheless, if the grinding time was too long, it would result in the recovery of alkaloids decreased and mainly due to their oxidation. Meanwhile, conglomeration might be formed, decreasing the contact surface area and resulted in the lower


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extraction amount. For the consideration of energy and efficiency, 10 min was considered as the optimal milling time. Effect of Extraction Time. In order to identify the minimum time required for the

extraction, the extraction time (2, 5, 10 and 15 min) was assessed under the same conditions (oxalic acid concentration 20 wt.%; milling speed, 400 rpm; milling time, 10 min; extraction solvent, water; liquid/solid ratio, 15 mL/g; centrifuge at 3000 rpm for 5 min). Figure 5f showed that the highest extraction amount was achieved at 5 min. When the extraction time extended from 5 to 15 min, the yield of the TET had a tendency to decrease, and the extraction yield of the FAN remained relatively stable. In general, it was found that extraction efficiency strongly to depend on the extraction solvent diffuse speed into the matrix, increasing extraction time could exert a significant effect on extraction that made target products dissolve into the surrounding solvent completely. However, longer extraction time was not conducive to extraction, the property of the target compound would degrade the extraction efficiency because it was unstable. In addition, longer extraction time required high energy consuming, thus minimize the energy costs of the process should be considered. Therefore, 5 min was selected as the optimum extraction time. Finally, optimum mechanochemical-assisted extraction conditions were as follows: solid acids, oxalic acid (20 wt.%); milling speed, 400 rpm; milling time, 10 min; extraction time, 5 min; liquid/solid ratio, 15 mL/g; centrifuge at 3000 rpm for 5 min. Optimization of Extraction Conditions by RSM. According to the single-factor

experimental results, the three independent variables of the concentration of oxalate acid (15-25 wt.%), liquid/solid ratio (10-20 mL/g) and extraction time (5-15 min) were selected for RSM. The experimental results of 17 runs which performed in random order obtained under different experimental conditions were presented in



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Table 2. Analysis of Variance (ANOVA) was carried out to evaluate the influence of the independent variables and the significance of the interactions. Meanwhile, the accuracy of the model could also be judged according to the importance of different coefficients of the model. The experimental data were analyzed by BBD, and the relevant results of ANOVA for the quadratic regression models were given in Table 3. As showed in Table 3, it could be observed that the obtained data were fitted well with the quadratic model. In order to understand the interaction between independent variables, the p-value was used for the statistical significance of each coefficient. ANOVA revealed that the model was remarkably significant (p < 0.0001) for FAN and TET as evidenced by the high value of the F-test (F=981.14 for FAN and 1489.3 for TET). At the same time, the lack-of-fit value was insignificant as compared to pure error (p > 0.05), further suggesting that the model was statistically accurate. In addition, it also indicated that the experimental and predicted production data fit adequately. The quadratic polynomial equations, described the amount of alkaloids extracted (Y) as a simultaneous determination of solid acid content (X1), liquid/solid ratio (X2) and milling time (X3), were shown in Eqs. (2) and (3), respectively.

Y(FAN) = 9.26 - 0.13X1  0.063X 2 - 0.82X 3 - 0.47X1X 2 - 0.083X1X 3 - 0.091X 2 X 3 2

2

+ 0.1X1 + 0.05X 2 + 0.046X 3

2

(2)

Y(TET) = 10.37 + 0.11X 1 + 0.48X 2 - 1.44X 3 - 0.20X 1 X 2 + 0.25X 1 X 3 - 0.1X 2 X 3 2

2

+ 0.044X 1 + 0.3X 2 + 0.33 X 3

2

(3)

The regression analysis of the data showed the coefficient of determination (R2=0.9992 for FAN and 0.9995 for TET) values were close to 1, which indicated a high degree of correlation between the observed and the predicted value. The high adjusted determination coefficient R2adj (R2adj =0.9982 for FAN and 0.9988 for TET) and predicted determination coefficient R2pred (R2pred =0.9922 for FAN and 0.9947 for TET) also illustrated the model adequately fits the data. Furthermore, the above


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result demonstrated that Eqs. (2) and (3) were suitable to describe the response of the total experiment. Therefore, it could be concluded that the model was statistically reasonable. In order to obtain the optimum values for the target compounds, the threedimensional (3D) response surface described by the regression model was illustrated in Figure 4. At the same time, the statistical significance of the model coefficients was evaluated with a larger F-value and a smaller P-value. And the results were listed in Table 4. Due to the consideration of the above factors, it was shown that the interactive variables (X3 and X1X2) revealed a major effect on the amount of FAN extracted, which was followed by X1, X12, X1, X3X2, X22 and X32. The effect of solid acid content (X1) and liquid/solid ratio (X2) was displayed in Figure 6a, while the milling time was fixed at 0 level of the center value. It could be observed that as the liquid /solid ratio (X2) increases from 14 to 20 mL/g, the amount of extraction increases linearly. However, the interaction effect of solid acid content (X1) with milling time (X3) and the liquid/solid ratio (X2) with milling time (X3) on extraction did not show any significant effect on the extraction (Figure 6b-d). On the other hand, the extracted amount of TET mainly depends on X3, X2 and then on X32, X22, X1, X1X3, X1X2, X2X3 and X12. On the contrary, due to the large molecular size and more hydrophobicity of TET, the liquid/solid ratio and milling time had a significant effect on extraction of TET. As illustrated in Figure 6e, showing a linear increase of extracted amounts associated with an increase of solid acid content. However, increasing milling time from 5 to 15 min showed decreasing trends. This trend was due to the possible formation of aggregates and reduced the contact area then led to a lower extraction volume. Thus it can be seen that the interaction effect of X1X3 was significant. When



two variables were fixed, the high liquid/solid ratio resulted in a high yield of TET, and the interaction effect of X1X2 showed similar trends as observed in the previous single-factor optimization. Similarly, increasing the liquid/solid ratio resulted in a quadratic increase in milling time in the extraction (Figure 6f). Basing on these studies, the software predicted optimum conditions could be modified as follows: solid acid content (17.05 wt.%); liquid/solid ratio, 20 mL/g; milling time, 5 min. In addition, the model gave the largest predicted values of FAN and TET (10.66 mg/g and 13.23 mg/g), slightly higher than that obtained from the curve analysis (10.44 mg/g and 12.87 mg/g). Analysis of Target Compound by Mass Spectrometry. Under positive ion mode, the MS

analysis of the target compound was shown in Figure 7. The [M+H]+ ion at m/z 609 and m/z 623 corresponding to the FAN and TET, respectively. The major ion peaks at m/z 610 [M+2H]+, m/z 611 [M+3H]+ were the result of the reaction of oxalic acid with the tertiary amine base for FAN (Figure 7a). Similarly, the peaks at m/z 624, 625 corresponding to the salt form of TET (Figure 7b). The above results indicated that the mechanochemical reaction with solid acid would occur during the grinding process. As a result, alkaloids could be transformed into a water-soluble salt form and then rapidly released into the water. The Possible Mechanism of MCAE. According to the above SEM, IR and MS results

analysis, which indicate that mechanochemical treatment is essential for microstructure transformation and formation of new compounds to improve extraction efficiency, and the possible processing is shown in Figure 8. After the grinding process, the cell wall and cellulose can be destroyed which resulted from the existence of solid acid that resulted in a significant effect on the extraction yield. Both FAN and TET belonged to tertiary amine alkaloids. In theory, when tertiary amine


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alkaloids were formed into salt, protons mostly bound to nitrogen atoms. Based on SEM and IR results, the presence of solid acid in the MCAE pretreatment was reduced the particle size and made cell breakage, which resulted in the conversion of the alkaloids to the salt form. In addition, by increasing the pH of the extracted sample solution to 9, precipitation was continuously produced. According to MS analysis, the extract was consistent with the standard, indicated that the properties of the target compound had not been changed. Moreover, for other components of the plant, grinding with solid acid also made no difference to its properties (Figure S4). Method Validation and Sample Analysis

To further evaluate the applicability of the method for extraction performance, the extracts require qualitative and quantitative analyzes. In addition, the linear range, correlation coefficients, precision, limit of detection (LOD), and limit of quantification (LOQ) for the validation of the proposed ball milling-assisted extraction method were investigated under the optimal conditions and were listed in Table 5. The linearity of the analytes was examined by analyzing six concentrations of sample solutions in ranges from 0.05-25 μg/mL for FAN and TET under the optimized conditions. Good linearity was observed with the correlation coefficients of the calibration curve for FAN and TET were 0.9999 and 0.9991 respectively, which showed the satisfactory results were achieved (Table 5). The LOD and LOQ for analytes were calculated on the basis of signal-to-noise ratios (S/N) of 3 and 10, respectively. As showed in Table 1, LOD was found to be 0.012 μg/mL, the calculated LOQ value was determined to be 10.043 μg/mL. The intra- and inter-day precision of the proposed method were assessed by relative standard deviation (RSD, %) and listed in Table 5. The RSD values for retention time of intra-day precision were low, ranging from 0.89% to 0.97%, and the inter-day precision were



ranged from 1.48% to 1.87%, respectively. Meanwhile, the RSD values of intra-day and inter-day precision were within the range from 0.62-0.91% and 3.78-4.26% for peak area, respectively, which indicated good accuracy of this method. The developed method was successfully applied to a real sample, which the contents of the targeted compounds of FAN and TET were 10.39 mg/g and 12.87 mg/g, respectively. Typical UHPLC chromatograms demonstrating the separation of two alkaloids are depicted in Figure 9. Accuracy was expressed as a percentage of the recovery. Tests for recoveries were performed in the above mentioned two sample solutions, which were spiked at two different concentrations (1 μg/mL and 10 μg/mL). The FAN and TET recoveries for the spiked sample solutions were 102.12-103.03%, 101.03-103.81%, respectively (Table 6). What’s more, RSDs of repeatability for retention time and peak area were acceptable in the range of 1.05 to 1.11% and 0.75 to 1.39%, respectively. These results demonstrate that analytical method could be used for precise and sensitive measurements. Comparison of The Developed MCAE Method with Other Reported Methods

The proposed method was compared with other reported methods for determination of tetrandrine and fangchinoline, mainly in terms of extraction time, extraction solvent, LOD and extracted amount, and the results are summarized in Table 7. The comparison of different origins and different methods of real samples see the Supporting Information (Table S1-S2). Besides, the MCAE method was also compared with the grinding separately method (Table S3). The published methods included soxhlet extraction, ultrasonic extraction, and heat reflux extraction determination of TET and FAN combination with different detection technology. Under the mechanochemical treatment, however, enabled water to become an extraction solvent, unlike conventional methods used organic solvents. It was well


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known that the consumption of large amounts of organic solvent was dangerous for human health and environment. Additionally, results for ultrasonic extraction showed that a comparatively lower extracted amount was obtained by comparison with this method. This phenomenon may be explained by the mechanical action resulting in particle size reduction and contact surface area increasing, promoting diffusion hindrances to a significant extent.24 Moreover, the reported extraction methods are usually time-consuming. In comparison to other methods, the extraction time was obviously reduced and only required 15 min. Thus, the developed method of mechanochemical-assisted extraction coupled with UHPLC is a quick, reliable and green method for the detection of two alkaloids in this paper. Mechanochemical technology has been developed and widely applied in diverse areas such as waste management,8 mate rials engineering, agriculture,10 synthesis of compounds12 and drug modification.36 It is believed that the green MCAE technique is a valuable method, and has large potential for the replacement of conventional methods for extraction of other natural bioactive compounds. Besides, the developed MCAE method permits a larger industrial applicability, with the advantages of protecting the environment and enhancing the competition of the industry, making it more ecological, economical and innovative. 

Conclusions Mechanochemical-assisted extraction has been successfully developed for

determination of TET and FAN in Fangchi. During the MCAE process, these components could be transformed into water-soluble forms and increase the speed of extraction. When in comparison with conventional ultrasonic extraction and heat reflux extraction method, the optimized MCAE method applies water as extractant solvent offers the significant improvement of extraction yield and reducing the



extraction time. These target compounds content are more than 12.87 mg/g and 10.39 mg/g. Furthermore, the most important advantage of using this convenient method is environmentally friendly, as it very often results in a reduced use of harmful organic solvents and energy. To our best knowledge, this is the first report for the MCAE method to extract alkaloids uses the solid acids. Therefore, it shows that the new method could be an appropriate alternative to efficiently obtain natural products from different sample matrices and remain to be further investigation.


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

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

Supporting Information 1. Additional comparison experiments. 2. Additional characterization images by SEM. 3. Additional Chromatogram image.





Figure Caption Figure 1. The chemical structures of fangchinoline (FAN, a) and tetrandrine (TET,

b). Figure 2. Scheme of mechanochemical assisted extraction method. Figure 3. SEM images of untreated sample without solid acid (a), ball mill sample (b), mechanochemical treatment sample (c). Figure 4. FT-IR spectra of untreated sample without solid acid (a) and mechanochemical treatment sample (b). Figure 5. Effect of different extraction varibles on the alkaloid content in extracts. If not indicated differently the following extraction conditions were applied: oxalic acid (20 wt.%); milling speed, 400 rpm; milling time, 10 min; extraction solvent, water; extraction time, 5 min; liquid/solid ratio, 15 mL/g; centrifuge at 3000 rpm for 5 min. (a) Effect of solid acids. (b) Effect of solid acid concentration. (c) Effect of liquid/solid ratio. (d) Effect of milling speed. (e) Effect of milling time. (f) Effect of extraction time. Figure 6. The three-dimensional (3D) response surface showing the effect of the concentration of oxalate acid (X1), liquid/solid ratio (X2) and extraction time (X3) on the extracted amounts of alkaloids. Figure 7. (+) MS spectra of the extracted solution, FAN (a); TET (b). Figure 8. The procedure of alkaloids reacted with oxalic acid, R1=OH; R2=OCH3. Figure 9. Analytical UHPLC chromatograms of standard sample and extracted by MCAE of FAN and TET.


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

Notes The authors declare no competing financial interest.



AUTHOR INFORMATION

Corresponding Author Tian Xie*, Fax: 86-571-28860237; E-mail: [email protected]. Jun Cao*, Fax: 86-571-28860239; E-mail: [email protected].





ACKNOWLEDGEMENTS This study was supported by the Natural Science Foundation of Zhejiang Province, China (LY16H270018); Key projects of National Natural Science Foundation of China (81730108); Key Project of Zhejiang province Ministry of Science and Technology (2015C03055); Key Project of Hangzhou Ministry of Science and Technology (20162013A07, 20142013A63). Key Laboratory of Elemene Class Anti-cancer Chinese Medicine of Zhejiang Province; Engineering Laboratory of Development and Application of Traditional Chinese Medicine from Zhejiang Province.


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Table 1 Independent Variables and Levels Used for BBD

List

Variables

Level -1

0

1

15

20

25

10

15

20

5

10

15

Oxalate concentration X1

（%，w/w） Liquid /solid ratio

X2

(mL/g) Extraction time

X3

(min)



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Table 2 Box-Behnken Experiment Design with the Independent Variables

Extracted Amount of Alkaloid Run

X1

X2

(mg/g)

X3 FAN

TET

1

20

20

15

8.2

9.9

2

20

20

5

9.9

12.9

3

20

10

15

8.5

9.1

4

20

15

10

8.9

10.4

5

25

20

10

8.6

11.1

6

20

15

10

8.9

10.4

7

15

20

10

9.7

11.2

8

25

10

10

9.4

10.6

9

20

15

10

8.9

10.4

10

25

15

5

10.0

11.8

11

15

15

5

10.4

12.3

12

20

15

10

8.9

10.2

13

20

15

10

8.9

10.3

14

20

10

5

9.8

11.9

15

15

10

10

9.1

9.9

16

25

15

15

8.4

9.6

17

15

15

15

8.8

8.9


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Table 3 Analysis of Variance for the Fitted Quadratic Polynomial Model of Extracted Amounts of Alkaloids Source

Alkaloid

Sum of squares

Degree of freedom

Mean square

F-value

Probability>F

FAN

6.52

9

0.72

981.14

Mechanochemical-Assisted Extraction of Active Alkaloids from Plant

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