Determination of Ferulic Acid in Angelica sinensis by Temperature-Controlled Hydrophobic Ionic Liquids-Based Ultrasound/Heating-Assisted Extraction Coupled with High Performance Liquid Chromatography

Hydrophilic ionic liquids are often used to extract the active ingredients of medicinal plants, while hydrophobic ionic liquids are rarely used to directly extract solid samples. In this paper, a simple, novel and efficient temperature-controlled hydrophobic ionic liquids-based ultrasound/heating-assisted extraction (TC-ILs-UHAE) procedure coupled with high-performance liquid chromatography (HPLC) was developed and applied to the determination of ferulic acid (FA) in Chinese herbal medicine Angelica sinensis. During the extraction procedure, hydrophobic ionic liquids (ILs) were dispersed into water to form cloudy solution (fine droplets) with the aid of ultrasound and heating simultaneous. After extraction, phase separation was easily achieved by centrifuging at 0 °C. Among all ILs used, 1-butyl-3-methylimidazolium bis(trifluoromethanesulphonyl)imide ([C4mim]NTf2) exhibited the highest extraction ability and the possible extraction mechanism was discussed. Additionally, the synergistic effect of heating and ultrasound on the extraction efficiency was investigated. Under the optimized conditions, a good linearity was observed with correlation coefficient (r) of 0.9995. The limit of detection of FA (LOD, S/N = 3) was 9.6 µg/L and the spiked recoveries of FA for real samples were in the range of 91.67 to 102.00% with relative standard deviation (RSD) lower than 3.87%. Compared with the traditional extraction methods, the proposed method gave the highest yield of FA and had the shortest extraction time. Therefore, this method is a potential simple, green and highly efficient technique and expected to be applied to the extraction of other bioactive ingredients in medicinal plants.


Introduction
Angelica sinensis (A. sinensis) is the rhizome of Angelica sinensis (Oliv.) Diels (Umbelliferae family). It has been widely used not only as a health food and drug in Asia but also as a dietary supplement in FA in Chinese herbal medicine A. sinensis. Water, methanol and ILs with different anions and cations were selected as extraction solvents and their extraction efficiency were investigated. The possible extraction mechanism was also discussed. Moreover, the synergistic effect of heating and ultrasound on the extraction efficiency was investigated and parameters related to extraction and determination were optimized systematically. Furthermore, the extraction performance of the proposed method was compared with heating reflux extraction and Chinese Pharmacopoeia method for the extraction of FA in real samples.

Equipment
HPLC analysis was performed with a Waters e2695 liquid chromatographic system (Waters, Milford, MA. USA) including a quaternary pump, a 2998 photo-diode array detector (PDA), an automatic sampler and a thermostatic column compartment. A Waters Symmetry C18 column (4.6 mm × 250 mm, particle size 5 µm) was used for the separation of FA extracted in the IL phase. The KQ-500V ultrasonic generator with an electrical power of 500 W was purchased from Kunshan Ultrasonic Instrument Co. Ltd. (Kunshan, China). The centrifugation was carried out on a high-speed freezing Heraeus Multifuge X1R centrifuge from Thermo Scientific (Waltham, MA, USA).

Sample Preparation
All samples were dried in a vacuum oven (Model 1400E, VWR Scientific Products, West Chester, PA, USA) to constant weight at 40 • C, then triturated with a pulverizer and passed through a 40-mesh sieve. After being homogenized, the dried powders of samples were collected and stored in a moisture controlled cabinet.

Temperature-Controlled Hydrophobic IL Based Ultrasound/Heating-Assisted Extraction
Hydrophobic IL, [C 4 mim]NTf 2 (0.80 mL) and ultrapure water (1.20 mL) were added in a calibrated centrifuge tube and mixed at 60 • C in advance. The IL was dissolved completely and mixed with the water entirely under the force of ultrasound and heating. Subsequently, precisely weighed sample powder (50 mg) was added in the centrifuge tube and ultrasonic extracted for 9 min at 60 • C. The resultant mixture was centrifuged at a speed of 11,000 r/min for 5 min at 0 • C. After that, it can be seen from Figure 2 that a sandwich-like structure was formed. The top, middle and bottom phases were water, solid sample and IL phases, respectively. The IL phase was withdrawn using a syringe and filtered through 0.45 µm membrane filter. The clarified filtrate was diluted 5 times by mobile phase before HPLC analysis. A schematic diagram of TC-ILs-UHAE is shown in Figure 3.  Schematic diagram of temperature-controlled hydrophobic ionic liquids-based ultrasound/heating-assisted extraction (TC-ILs-UHAE) method.
The extraction yield of target analyte was determined as follows: yield (mg/g) = mean mass of target analyte in herb samples (mg) mean mass of the herb samples (g) (1) The mean mass of target analyte in herb samples was calculated for 3 subsequent sample determinations under the optimized conditions. The mean mass of the herb samples was the average mass of three samples before extraction.

Conventional Reference Extraction Procedures
HRE and Chinese Pharmacopoeia methods were both selected as the references for extraction of FA from A. sinensis samples. Preliminary experiments shown that the optimal extraction conditions of the two procedures are as follows: In HRE procedure, 200 mg of dried sample powder was added into the mixture of 8.00 mL [C 4 mim] NTf 2 and 8.00 mL ultrapure water in a distillation flask. The sample was then extracted for 60 min under reflux at 80 • C using water bath with magnetic stirring. After that, the sample mixture was transferred into a 15 mL calibrated centrifuge tube and centrifuged at a speed of 11,000 r/min for 5 min at 0 • C. Subsequently, the IL phase was withdrawn using a syringe and filtered through 0.45 µm membrane filter. The clarified filtrate was diluted 5 times by mobile phase before HPLC analysis.
In Chinese Pharmacopoeia method, sample powder (200 mg) and 70% methanol (20 mL) were placed in a distillation flask. The distillation flask was weighed accurately. Then, hot reflux extraction was performed for 30 min. After cooled to the room temperature, the distillation flask was weighed again, and the lost weight was made up with 70% methanol. Subsequently, the sample mixture was shaken and filtered. The obtained filtrate was filtered through a 0.45 µm membrane filter before HPLC analysis.

Scanning Electron Microscopy Analysis (SEM)
Scanning electron microscopy (SEM) imaging was used to characterize the surface morphology of A. sinensis sample 1 before and after extraction. A Phenom XL Scanning Electron Microscope (Thermo Scientific, Waltham, MA, USA) in the secondary electron mode with 15 kV accelerating voltage was used to observe the microstructures of the raw and extracted samples. Before proceeding with imaging procedures, the samples were coated with a thin layer of platinum at room temperature.

HPLC Analysis
The diluted extracts were directly injected into the HPLC system. The mobile phase used was a binary mixture of methanol-0.2% phosphoric acid aqueous solution (32:68, v/v) at a flow rate of 1 mL/min. The injection volume was 10 µL and the column temperature was set at 35 • C. Peak areas of the samples were monitored at 323 nm. Triplicate injections were executed for the analysis of each sample. Chromatograms of FA obtained from standard solution and sample 1 extract are shown in Figure 4.

Optimization of TC-ILs-UHAE
In order to obtain high extraction yields, some important parameters influencing the extraction efficiency of FA were carefully explored by taking sample 1 as a representative. All single factor experiments were performed in triplicate and each datum presented in this work is expressed as mean ± standard deviation (SD).

Selection of the Types of Extraction Solvents
The types of ILs have a significant influence on the extraction efficiency. Yu [30] has reported the extraction of ferulic acid and caffeic acid from aqueous solution by using [C 4 mim]PF 6 and [C 6 mim]PF 6 . In our proposed method, ILs with higher hydrophobicity and lower viscosity is expected to be beneficial to improve the extraction efficiency of FA in the solid herbal samples. Therefore, six kinds of ILs, [C 4 mim]PF 6 , [C 6 mim]PF 6 , [C 8 mim]PF 6 ], [C 4 mim]NTf 2 , [C 6 mim]NTf 2 and [C 8 mim]NTf 2 , were selected to extract FA. This selection enables us to systematically study the influence of the nature of the IL anion and cation on the extraction efficiency of FA. Moreover, pure water and methanol were also selected as the reference extraction solvents for evaluating the extraction efficiency of FA with different solvents. The results shown in Figure 5 indicate that the extraction ability of different extractants follows the order: 6 mim]PF 6 ≈ [C 4 mim]PF 6 > pure water. As can be seen from Figure 5, four interesting phenomena can be obtained: (I) The addition of ILs obviously improves the extraction yield of FA from A. sinensis compared with pure water as solvent. This could be ascribed to the multiple interactions, including hydrophobicity, π-π stacking interaction, dispersion force and hydrogen bonding between FA and imidazolium based ILs [30,31]. (II) For the ILs with the same cation but different anions, the extraction ability of [C n mim]NTf 2 was higher than that of [C n mim] PF 6 (n = 4, 6,8). It is consistent with the order of the anion's hydrophobicity (NTf 2 − > PF 6 − ). This suggests that the hydrophobicity of ILs is one important driving force underlying the extraction process. In addition, ILs with strong hydrophobicity more easily reach phase separation and reduce the solubility in the aqueous phase during centrifugation, which is also beneficial to increase the extraction efficiency. Moreover, the viscosity of extractant has a great influence on extraction efficiency. Low viscosity is not only conducive to the mass transfer rate, but also conducive to better dispersion of IL into the aqueous phase in the process of ultrasound and heating. The viscosities of NTf 2 − based ILs are much lower than those of PF 6 − based ILs [32]. This suggests that viscosity is also a key parameter affecting the extraction efficiency in the proposed method. (III) For the ILs with the same anions but different cations, the hydrophobicity of the ILs increases with prolonging the alkyl chain length of the IL cation. However, as shown in Figure 5, the increase in the alkyl chain length of the IL cation results in the decrease in the extraction ability ([C 4 mim]NTf 2 > [C 6 mim]NTf 2 > [C 8 mim]NTf 2 ), which is opposite to the order of the cation's hydrophobicity. To explain this phenomenon, steric hindrance effect between ILs and FA should be considered. The increase of alkyl chain of ILs leads to the increase of the steric hindrance effect. There are dispersion forces and hydrogen bond forces between ILs and FA, and the increase of steric resistance will reduce these two forces, lowering the extraction ability of ILs. That is to say, there is a competition between hydrophobicity and steric hindrance. Moreover, the viscosity of short chain ILs is relatively low, which is also beneficial to improve the extraction rate of FA. It might be explained as follows: compared with methanol, ILs, especially those containing halogen anions, have shown high capacity for cellulose dissolution, which is conducive to the release of active substances from the cells of medicinal plants [23]. But at the same time, the viscosity of ILs also has a great influence on the extraction rate of FA in the solid samples. Therefore, [C 4 mim]NTf 2 has the highest extraction yield of FA due to its lowest viscosity and the easy formation of homogeneous turbid IL-water solution during the extraction. Due to its acceptable hydrophobicity and low viscosity, [C 4 mim]NTf 2 can be dispersed completely into the aqueous solution during the extraction process and well separated in the centrifugation process. Since [C 4 mim]NTf 2 exhibits the highest extraction ability for FA, it is selected as the extraction solvent in the following studies.

Effect of IL Volume
The effect of different volume of [C 4 mim]NTf 2 on the extraction ability was studied when the total volume of extractant ([C 4 mim]NTf 2 and water) was maintained at 2 mL. The results shown in Figure 6 indicate that the volume of [C 4 mim]NTf 2 is closely related to the extraction yield of FA. With the increase of volume of [C 4 mim]NTf 2 from 0.2 mL to 1.4 mL, the extraction yield of target analyte increases gradually and reaches the maximum extraction yield at 0.8 mL of [C 4 mim]NTf 2 . Then, with the further increase of volume of [C 4 mim]NTf 2 from 0.8 mL to 1.4 mL, the extraction yield of FA actually slightly decreases. This can be explained as follows: as the volume of [C 4 mim]NTf 2 increases, more [C 4 mim]NTf 2 is dispersed into water under the drive of temperature and ultrasound. As a consequence, the interactions among [C 4 mim]NTf 2 and FA are improved, which leads to the increase of extraction yield. However, when the volume of [C 4 mim]NTf 2 is more than 0.8 mL, excess of [C 4 mim]NTf 2 might exceed the target analyte dispersed amount. At the same time, the viscosity of the extractant would increase, which is not beneficial to the mass transfer. Moreover, excessive [C 4 mim]NTf 2 would be adsorbed on the solid sample, which could not only lead to poor infiltration of the solvent into the plant tissues but also make phase separation difficult during the centrifugation, resulting in the decrease of the extraction yield. Thus, 0.8 mL of [C 4 mim]NTf 2 is finally selected for the following experiments.

Effect of Solid-Liquid Ratio
Solid-liquid ratio is also an important factor affecting the extraction efficiency. Therefore, the effect of solid-liquid ratios on the extraction efficiency was investigated by using [C 4 Figure 7 illustrate that the extraction efficiency of FA increases with the increase of solid-liquid ratio from 1:10 (g/mL) to 1:40 (g/mL) and then keeps almost constant with further increasing the solid-liquid ratio. It means that when the solid-liquid ratio is 1:40 (g/mL), the target analytes in the sample are extracted completely by ILs. Therefore, 1:40 (g/mL) is chosen for the optimal solid-liquid ratio to avoid the waste of reagents.

Effect of Extraction Temperature
In the proposed method, temperature is one of the important driving force for the complete dispersion of [C 4 mim][NTf 2 ] into the water and affects the mass transfer. Thus, a series of experiments was designed to investigate the effect of temperature on the extraction efficiency and the results are shown in. It can be observed from Figure 8 that the extraction efficiency of FA increases with the increase of temperatures from 30 • C to 60 • C. When the temperature is in the range of 70 • C to 80 • C, the extraction efficiency of FA is reduced as compared with that at 60 • C. It might be due to the degradation of FA and the increasing solubility of the IL into water, which is negative to phase separation. Therefore, 60 • C is used as the optimum extraction temperature for the future studies.

Effect of Ultrasound Time
Ultrasound time is another leading factor influencing the extraction yield to certain extend. As shown in Figure 9the extraction yield of FA increases with the extension of ultrasound time from 5 to 9 min. When the ultrasound time continues to increase, the extraction efficiency of the FA remains almost unchanged. The rapid extraction may be ascribed to the synergistic effect of ultrasound and heating during the extraction process. Accordingly, the ultrasound time of 9 min is adopted in the following study.

Effect of Centrifugal Speed and Time
In order to achieve better phase separation, centrifugation was performed at 0 • C. At the same time, the effect of centrifugal speed (8000, 9000, 10,000, 11,000, 12,000 and 13,000 rpm) and centrifugal time (2, 3, 4, 5, 6 and 7 min) on the extraction efficiency were investigated. As it can be seen in Figure 10 (a) and (b), the extraction efficiency of FA is the highest and complete phase separation can be achieved when the centrifugal speed is 11,000 rpm and the centrifugal time is 5 min.

Comparison of the Proposed TC-ILs-UHAE with the Conventional Methods
To evaluate the extraction efficiency of different methods, the proposed TC-ILs-UHAE approach under optimal conditions was compared with the optimized conventional methods, including HRE and Pharmacopoeia methods. The results shown in Table 1 indicate that the proposed TC-ILs-UHAE method exhibits the highest extraction efficiency and the shortest extraction time among the three extraction methods. This could be attributed to the synergistic effect of heating and ultrasound in the proposed method. To verify this hypothesis, the SEM images of outer surface of the Angelica sinensis powders before and after different extraction procedures were captured and compared. The results are illustrated in Figure 11a-d.  The raw Angelica sinensis powder in Figure 11a displays a rough and complete surface with numerous protruding blunt horn-like lumps. Figure 11b,c are the images after extraction using HRE and Pharmacopoeia methods, respectively. It can be seen that the surface microstructure of the sample is obviously damaged under the influence of heating. Sample submitted to TC-ILs-UHAE method is damaged thoroughly and presents more fragmented structures and ruptured plant cell under the synergistic effect of heating and ultrasound (Figure 11d). High temperature increases the kinetic energy of molecules and is more conducive to the diffusion of FA. At the same time, ultrasound-treated cells have a more open, fragmented structure, which facilitates the access of the solution into the cellular compartments where the target analyte is located. Although the extraction yield of pharmacopoeia method is similar to that of our proposed method, the extraction time of pharmacopoeia method is longer and environment-hazardous methanol is used as the extraction solvent. Moreover, compared with HRE, much less extraction time and solvent are consumed, and more extraction yield is obtained when the present method is applied. Therefore, the proposed method is considered as a comparatively satisfactory technique.

Analytical Performance
Under the optimized conditions, some parameters such as linearity, limits of detection (LOD, S/N = 3), and reproducibility of the proposed method were investigated. The calibration curve was constructed by plotting the peak areas versus the concentration of FA and obtained by the analysis of six concentrations of the standard FA solutions in triplicate. The calibration equation was y = 38022.5 x + 1829.2 with a correlation coefficient (r) of 0.9995 in the concentration range of 0.25 to 25.00 mg/L. The limit of detection (LOD) based on signal-to-noise ratio of 3 (S/N = 3) is 9.6 µg/L, which indicated the developed method is of satisfactory sensitivity. The reproducibility was investigated by the determination of six replicated extracts of the sample 1 and the relative standard deviation (RSD, n = 6) value was 3.20%.
To test the accuracy of the proposed method, the spiked sample 1 with three levels were determined. The recovery could be calculated as follows: The results shown in Table 2 indicate that good recoveries ranging from 91.67% to 102.00% are obtained and the precision (expressed as RSD, n = 5) is lower than 3.87%.

Analysis of Real Samples
In order to verify the applicability of the proposed method, six batches of A. sinensis samples from different cultivated areas of China were analyzed under the optimal extraction conditions. Results in Table 3 indicate that the content of FA from Gansu province is obviously higher than that from Yunnan province, which suggests that the quality of A. sinensis is related to its cultivated regions.

Conclusions
In the present work, an efficient, environmentally friendly and easily operated TC-ILs-UHAE method using hydrophobic IL [C 4 mim][NTf 2 ] as extraction solvent was developed. The influence of the IL anions and cations on the extraction yield was systematically studied. Among all the ILs used in this work, [C 4 mim]NTf 2 exhibited the highest extraction efficiency due to its relatively higher hydrophobicity and lower viscosity and weaker steric hindrance effect. Compared with the traditional HRE and Pharmacopoeia methods, the present method has higher extraction efficiency and shorter extraction time due to the synergistic effect of simultaneous ultrasound and heating, which was confirmed by SEM images of the samples. Additionally, separation and enrichment could be incorporated into the centrifugation process without extra ice bath and additional salt, which is conducive to saving time and easy operation. Moreover, the present TC-ILs-UHAE method has good analytical performances and has been successfully applied to the analysis of real herbal samples from different cultivated regions. Since hydrophobic ILs were rarely directly used to extract solid samples, this proposed method provides an alternative and promising approach for analysis and separation of active components from other solid natural products.

Conflicts of Interest:
The authors declare no conflict of interest.