Infrared-assisted extraction of salidroside from the root of Rhodiola crenulata with a novel ionic liquid that dissolves cellulose

Abstract

Tetrabutylphosphonium hydroxide (TBPH) aqueous solution, a novel ionic liquid that could dissolve cellulose rapidly at ambient temperature (25 °C), was used for the first time to develop an extraction method for salidroside from Rhodiola crenulata, used as the model sample, with infrared-assisted extraction (IRAE) in this paper. IRAE-TBPH procedures were optimized using a series of single-factor experiments and under optimal conditions, the IRAE-TBPH technique not only took a shorter time (from 1.0 h to 8 min) but also afforded a higher extraction rate of salidroside from the herbs (increased by 15.41–38.65%) compared with other extraction techniques, such as TBPH-based heat reflux extraction (HRE-TBPH), ultrasound-assisted extraction (UAE-TBPH) and conventional solvent (methanol, ethanol and pure water) based IRAE. The results indicated IRAE-TBPH to be a fast and efficient extraction technique. Furthermore, the mechanism of IRAE-TBPH was preliminarily studied by means of the surface structures and chemical compositions of the samples before and after different extraction techniques. On the basis of the destruction of herb surface microstructures, the cellulose dissolving property of TBPH and high efficiency heating of infrared irradiation in the IRAE-TBPH process, the IRAE-TBPH technique eventually achieved the maximum yield value. Therefore, TBPH solution as a novel, effective and alternative solvent with higher extraction efficiency in the IRAE of active compounds from medicinal plants showed a great promising prospect.

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1 Introduction

Infrared-assisted extraction (IRAE), a process of using infrared to achieve high efficiency heating by matching the wavelength of the infrared heaters to the absorption characteristics of the materials, has been increasingly applied for the extraction of active components from medicinal plants during the past few years.^1–5 Our preliminary results have indicated that IRAE could obtain a higher extraction efficiency in a shorter extraction time compared with conventional heat reflux extraction (HRE) and ultrasonic assisted extraction (UAE).^4,5 What's more, unlike microwave-assisted extraction (MAE), the problem of leakage needn't be considered in the process of IRAE as infrared does little harm to human health.²

Traditionally, abundant organic solvents, methanol and ethanol included, were utilized to extract biological active compounds from the herb. Because of the toxicity, volatility and flammability of the organic solvents, the design of safe and environmentally benign extraction solvents and processes has played an increasingly important role in the development of sample pretreatment technologies.

At the same time, because plant cell walls consist of cellulose, hemicellulose, pectin and so on, which represent barriers for the release of intracellular contents, the method applied to facilitate the release of intracellular contents from plant materials has become a hot spot in sample pretreatment research. Although mechanical treatment might give rise to faster and cheaper results, it is not recommended and sometimes almost impossible to process fine powders industrially.⁶ Enzymatic pretreatment of raw material with various enzymes such as cellulases and pectinases, which could interact on the cell wall, breaking down its structural integrity so as to increase cell wall permeability, normally results in a reduction in extraction time and provides increased extraction yield and quality of bioactives from plants.⁵ Although enzymatic hydrolysis procedures are appealing methodologies, they are disadvantageous in that besides the cost of the enzymes, they require a long time to complete the substrate hydrolysis (several hours) before subsequent extraction processes,^7–10 which strongly limits the applicability of the methods.

New and efficient solvents and process technologies are needed to help unlock the promise of sample pretreatment, and in this regard, perhaps the field of ionic liquids (ILs) might actually live up to its tremendous potential as a new class of designer solvents. Interest in ILs stems from this potential application as “green solvents” based on, perhaps, their unique physicochemical properties,¹¹ such as negligible vapor pressure, nonflammability, high thermal stability, and excellent solubility for natural organic compounds, such as cellulose,^12–16 pectin,¹⁷ isoflavones,¹⁸ phenolic compounds¹⁹ and so on.²⁰ Because the wall of a plant cell consists mainly of cellulose, ILs with chloride,^21,22 acetate,^23,24 phosphite and phosphonate anions,²⁵ which have been shown to exhibit a high ability to dissolve cellulose, would likely contribute to the improvement of the extraction efficiency, thus resulting in a higher yield of natural products.

Therefore, cellulose-dissoluble ILs have been proposed as greener and novel alternatives to volatile organic solvents in the extraction of biological active natural products from medicinal plants. Nowadays, it has been demonstrated that tetrabutylphosphonium hydroxide (TBPH) containing 30–50 wt% water could dissolve cellulose rapidly at ambient temperature (25 °C).¹³ TBPH containing water breaks the hydrogen bonding network of cellulose and dissolves cellulose chains at a molecular level.¹³ Despite the extensive research on TBPH,^13,26,27 there is still a lack of information on practical issues related to their application as an extraction solvent for the extraction of bioactive substances from herb samples, such as their efficiency, optimal water content and so on.

The root of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, which belongs to the family Crassulaceae and the genus Rhodiola, is particularly described in the Pharmacopoeia of China and it has been used for treating acute mountain sickness in Tibet since ancient times.²⁸ Although salidroside is found in all species of the genus Rhodiola, R. crenulata contains the highest levels.²⁹ Salidroside has recently attracted significant attention due to its pharmacological properties including anti-asthma,³⁰ anticancer,³¹ hepatoprotective,³² neuroprotective,³³ antioxidative effects³⁴ and so on. Due to these beneficial effects, more attention has been focused on extraction and purification technologies for salidroside.

Up till now, the application of TBPH in the extraction schemes has rarely been described, and no work has been reported on the use of TBPH for the extraction of salidroside from herbal medicines. The purpose of this work therefore, was to study the feasibility of employing TBPH aqueous solution as an alternative and effective solvent for the extraction of salidroside from R. crenulata, used as the model sample. What is more, the conditions of IRAE-TBPH technique were optimized and its mechanism was also investigated.

2 Experimental

2.1 Reagents and materials

The root of R. crenulata was collected in August of 2014 from Cuomei County, Tibet of China, and identified by Professor Jinda Hao from National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences. A voucher specimen has been deposited at the herbarium of National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences. The dried materials were smashed into powder with an electrical disintegrator (DFY-200, Wenling Linda Machinery Co., Ltd., Zhejiang, China), sieved through a 50-mesh sieve, and stored in a desiccator.

Standard of salidroside was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).

TBPH containing 40 wt% water (2.15 mol L⁻¹) was provided by Sigma-Aldrich Co., USA. Methanol of HPLC grade was purchased from Tianjin Shield Specialty Chemical Co., China and ethanol of analytical grade was obtained from Beijing Chemical works, China.

HPLC grade acetonitrile used for mobile phase was purchased from Fisher Scientific, USA. Deionized water was purified by a Milli-Q water purification system (Millipore, MA, USA).

2.2 Extraction procedures

IRAE-TBPH. IRAE experiments were performed with an infrared lamp (Qiyi Lighting Company, Zhejiang, China) which was placed below a round bottom flask as the heat resource to extract salidroside from the sample. A 1 m-long condenser with water circulated through it to prevent losses of the solvents and analytes was connected to the round bottom flask.

0.5 g of sample was put into the round bottom flask with a certain volume of TBPH solution. The effects of TBPH concentration, liquid/solid ratio, extraction time and infrared power on the extraction efficiency of salidroside were systematically investigated by a series of single factor tests.

Control-IRAE (C-IRAE). According to previous reports,^35,36 various solvents with different polarity (pure water, methanol and ethanol) were selected as the reference solvents. The extraction procedures were carried out under the optimized conditions of IRAE-TBPH, except for solvent type.

UAE-TBPH. 0.5 g of sample was introduced into a conical flask and 10 mL 1.61 mol L⁻¹ TBPH aqueous solution was added. Then the mixture was extracted in an ultrasonic bath (KQ-100, Kunshan Ultrasonic Instrument Co., Ltd., China) for 60 min at the ultrasonic power of 100 W.

HRE-TBPH. 0.5 g of sample was put into a round bottom flask and 10 mL 1.61 mol L⁻¹ TBPH aqueous solution was added. Then the flask was placed into water bath with a reflux device, followed by extracting 60 min at 95 °C.

After extraction, all extracts obtained were cooled to ambient temperature and centrifuged at 4000 rpm for 10 min. Then 2 mL of the supernatant was diluted 5 times with the mobile phase before being filtered through a syringe filter (0.45 μm) for subsequent HPLC analysis.

2.3 Chromatography analysis

HPLC analysis was carried out on a Waters Alliance system which consists of a Waters 2695 pump, a Waters 2996 diode array detector (DAD), an auto sampler and a column oven. The chromatographic data were recorded and processed with the Empower^TM 3 workstation software.

Samples were separated on an Ultimate XB-C₁₈ column (250 mm × 4.6 mm i.d., 5 μm) maintained at a temperature of 35 °C. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B) using the following gradient elution program for separation: 0–15 min, 5–10% (B); 15–60 min, 10–28% (B). After a 10 min equilibration period, the column was ready for a new injection.

The flow rate was 1 mL min⁻¹ and the injection volume was 10 μL. The chromatograms were monitored between 190 and 400 nm for peak purity testing, and the peak was recorded using DAD absorbance at 275 nm for salidroside.

The identification of salidroside in samples was carried out by comparing its retention time and UV spectrum with the reference compound. The quantitative analysis of salidroside in the extracts was performed using external standard method by means of calibration curves. All the analyses were repeated triplicate and the level of salidroside was expressed in mg g⁻¹ of the sample.

In Fig. 1, a typical chromatogram of the sample extract is shown, and no effect of TBPH in extract was observed on the retention of salidroside, which was well separated from other compounds in sample.

Fig. 1 Representative HPLC-DAD chromatograms of standard solution of 0.4 mg mL⁻¹ salidroside (a), extract of the root of R. crenulata by TBPH solution (b), with photodiode array spectra taken across the peak of salidroside.

2.4 Statistical methods

All the data were expressed by mean ± standard deviation and SPSS v16.0 software (SPSS Software Products, Chicago, USA) was used for evaluating the statistical differences between groups using one-way ANOVA. p < 0.05 was accepted as statistically significant.

3 Results and discussion

3.1 Optimization of IRAE-TBPH

Previous literature has demonstrated that TBPH containing 30–50 wt% water is the best for dissolving cellulose.¹³ A certain amount of hydroxide concentration should be needed to dissolve cellulose, while an excess amount of hydroxide ion could decrease the stability of the TBP cation.¹³ And because TBPH containing 40 wt% water (2.15 mol L⁻¹) could be commercially available, it was used directly to study the effect of liquid/solid ratio, extraction time and infrared power on the extraction efficiency of TBPH aqueous solution with the univariate method according to previous literature reports.^35,37 Afterwards, the effect of TBPH concentration on the extraction yield of salidroside was further studied in the present work.

3.1.1 Liquid/solid ratio. The liquid/solid ratio is a crucial factor that should be considered to increase the extraction efficiency. Samples were extracted with liquid/solid ratios of 10 : 1, 20 : 1, 30 : 1, 40 : 1, and 50 : 1 mL g⁻¹, respectively, while the irradiation power, extraction time, and TBPH concentration were set at 200 W, 10 min and 2.15 mol L⁻¹, separately.

As shown in Fig. 2a, the salidroside extraction yield increased with the increase of the liquid/solid ratio, reaching the highest yield at a ratio of 20 : 1 mL g⁻¹, and then fell down at the higher ratios. On the one hand, the amount of dissolved cellulose increased with an increasing mass of ionic liquid, which might have increased the permeability of the cell wall thus resulting in higher yield of salidroside. On the other hand, at room temperature ionic liquids were vicious liquid³⁸ and the viscosity of cellulose/IL increased with an increasing mass of ionic liquid, which negatively affected mass transfer and barricaded the penetration of the salidroside into the mixture of the sample/IL. The solvent with low viscosity can more easily diffuse into the pores of plant materials. Therefore, the liquid/solid ratio of 20 : 1 mL g⁻¹ was selected in the subsequent experiments.

Fig. 2 Effect of liquid/solid ratio (a), extraction time (b), infrared power (c) and TBPH concentration (d) on the extraction efficiency of salidroside from the root of R. crenulata in IRAE-TBPH (n = 3).

3.1.2 Extraction time. The choice of proper extraction time is important, which influences the distribution equilibrium between sample matrix and extraction solvent. To optimize the extraction time, extraction was carried out at 200 W with 2.15 mol L⁻¹ TBPH at a phase ratio of 20 : 1 for 4, 6, 8, 10, 12 min, respectively.

As shown in Fig. 2b, the extraction efficiency of salidroside improved greatly as the extraction time increased from 4 to 8 min. After 8 min, no obvious increase in salidroside extraction yield was observed. Considering the improvement of extraction efficiency and the relatively shorter extraction time, 8 min were selected as the appropriate extraction time and used in the following tests.

3.1.3 Infrared power. As is known to all, higher infrared power leads to higher extraction temperature, and the higher the extraction temperature is, the higher the dissolution rates of target compounds will be due to the increasing solubility of most of the solvents. More than this, the viscosity of ILs aqueous solution will decrease and the diffusivity will increase at the high temperature. Thus, the effect of infrared power on the extraction efficiency was investigated by performing IRAE-TBPH with the infrared power of 60, 100, 150, 200, and 250 W, respectively, using 2.15 mol L⁻¹ TBPH at a phase ratio of 20 : 1 for 8 min.

As shown in Fig. 2c, the extraction efficiency of salidroside rose sharply when the infrared power increased from 60 W to 100 W, and then decreased when the infrared power exceeded 100 W. The reason why the infrared power of 150 W showed the lowest extraction yield might be related to the interactions between the variables and the characteristics of the ionic liquids,³⁹ which were different from organic solvents and need to be studied further in follow-up work. The similar phenomenon was observed in Yuan's and Chi's experiments,^39,40 using the ionic liquids based microwave-assisted extraction of podophyllotoxin from Chinese herbal medicine and the ionic liquids based microwave-assisted extraction of lactones from Ligusticum chuanxiong Hort. respectively. In the ionic liquids based microwave-assisted extraction of podophyllotoxin from Chinese herbal medicine by Yuan Y. et al.,³⁹ there were irregular and different changes with increasing temperature in the extraction rates of podophyllotoxin from three herbs, with the extraction yield of podophyllotoxin from Dysosma versipellis at 60 °C lower than that at 50 °C and 70 °C. Thus, in our present work, 100 W was selected as the optimum infrared power for further experiments.

3.1.4 TBPH concentration. Previous literature³⁵ indicated that the concentration of ILs had significant influence on the extraction efficiency. Therefore, the concentration effect of TBPH (from 0.215 to 2.15 mol L⁻¹) on extraction of salidroside from the sample was further studied in the present work. Samples were extracted at a phase ratio of 20 : 1 mL g⁻¹ for 8 min and the infrared power was set at 100 W.

Based on the results in Fig. 2d, the extraction efficiency of salidroside increased obviously when the TBPH concentration increased from 0.215 to 1.61 mol L⁻¹. This increase might be due to the cellulose dissolving property of TBPH.¹³ However, the extraction efficiency fell when the concentration of TBPH further increased. The reason could be that at late stage, the increasing concentration of TBPH resulted in the high viscosity of the solution, which slowed the mass transfer during extraction. Therefore, considering the viscosity and the extraction ability of the TBPH solutions, 1.61 mol L⁻¹ TBPH solution was selected for the extraction of salidroside from the sample.

Based on the above experiment, the optimum IRAE-TBPH conditions for extraction of salidroside from the root of R. crenulata were: extraction solvent 1.61 mol L⁻¹ TBPH, liquid/solid ratio 20 : 1 mL g⁻¹, extraction time 8 min and infrared power 100 W.

3.2 Method validation

After the HPLC gradient conditions were determined, validation tests were performed for linearity, limit of detection (LOD), limit of quantification (LOQ), precision and recovery.

3.2.1 Linearity, LOD and LOQ. Linear regression analysis for salidroside was performed using the external standard method. The standard of salidroside was accurately weighed and dissolved in methanol to make 1.0 mg mL⁻¹ stock solution of salidroside. The stock solution was then diluted with the mobile phase for HPLC to a series of working solutions with final concentrations of 0.05, 0.10, 0.20, 0.30 and 0.40 mg mL⁻¹ respectively.

Under the chromatographic conditions described above, the calibration curves of salidroside, which related the concentrations to the peak areas, exhibited an excellent linear behavior (R² > 0.999) in a relatively wide concentration range of 0.05–0.40 mg mL⁻¹. LOD and LOQ for salidroside, which were calculated at signal to noise ratios (S/N) of 3 : 1 and 10 : 1, respectively, were 0.92 and 3.09 μg mL⁻¹, respectively, which indicated this analytical method was of excellent sensitivity (Table 1).

Table 1 Regression data, LOD and LOQ for salidroside

Analyte	Calibration curve	R^2	Linearity range (mg mL^−1)	LOD (μg mL^−1)	LOQ (μg mL^−1)
Salidroside	y = 3 000 000x − 8176	0.9997	0.05–0.40	0.92	3.09

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3.2.2 Precision and recovery. The precision of developed method was evaluated by intra- and inter-day variations. Intra- and inter-day variability tests were performed by analyzing standard solutions in triplicate for three times within one day and in triplicate on three consecutive days, respectively. The results are summarized in Table 2. The intra-day variations were less than 0.27%, and the corresponding inter-day variations were less than 1.04%, respectively.

Table 2 Precision data of salidroside

Analyte	Concentration (mg mL^−1)	Intra-day (%RSD) (n = 3)	Inter-day (%RSD) (n = 3)
Salidroside	0.05	0.02	1.04
	0.20	0.16	0.83
	0.40	0.27	0.71

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To test the accuracy of the developed chromatographic method, the recovery experiments were performed with the standard-addition method according to Chinese Pharmacopoeia⁴¹ and previous literature reports.^19,42 The amount of salidroside standard which was 80, 100 and 120% of the observed amount in the original samples was prepared, and then they were added to the original samples, respectively. The results in Table 3 show that the recovery of salidroside was in the range of 95.97–99.98% with the relative standard deviation (RSD) lower than 2.05%.

Table 3 Recovery data of salidroside (n = 3)

Analyte	Original (mg)	Added (mg)	Determined (mg)	Recovery (%)	Mean (%)	RSD (%)
Salidroside	0.2161	0.1751	0.3872	97.75	97.90	2.05
	0.2161	0.2173	0.4333	99.98
	0.2161	0.2601	0.4657	95.97

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Therefore, the HPLC-DAD method was precise, accurate, and sensitive enough for quantitative evaluation of salidroside in the root of R. crenulata.

3.3 Comparison of IRAE-TBPH approach with conventional methods

In order to compare the effect of IRAE-TBPH with other traditional extraction methods, parallel experiments were carried out with different extraction methods. The extraction conditions are described elaborately in “2.2 Extraction procedures” section, except for IRAE-TBPH. In IRAE-TBPH, 0.5 g sample was mixed with 10 mL 1.61 mol L⁻¹ TBPH aqueous solution and allowed to extract for 8 min at the infrared power of 100 W. The extraction yield of salidroside obtained from different methods was shown in Table 4.

Table 4 Comparison of extraction efficiency with different extraction methods

Methods	Extraction yields (mean ± SD, mg g^−1, n = 3)
a p < 0.05 compared with IRAE-Methanol, IRAE-Ethanol and IRAE-H2O.b p < 0.05 compared with UAE-TBPH and HRE-TBPH.
IRAE-methanol	17.75 ± 0.23
IRAE-ethanol	17.42 ± 0.00
IRAE-H2O	16.47 ± 0.06
UAE-TBPH	18.16 ± 0.95
HRE-TBPH	19.79 ± 0.05
IRAE-TBPH	22.84 ± 0.15^a^,^b

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The results in Table 4 indicated that the IRAE-TBPH approach could dramatically improve the extraction efficiency of salidroside (15.41–25.74% enhanced) compared with UAE-TBPH and HRE-TBPH. And the total extraction time of IRAE-TBPH (8 min) was significantly shorter than that of UAE-TBPH and HRE-TBPH (1.0 h). This correlates with the unique extraction mechanism of IRAE. During IRAE procedure, infrared directly heat solvent and sample. Therefore, the direct interaction of infrared with TBPH solutions and free water molecules present in the plant cells resulted in the subsequent rupture of the cells and the release of intracellular products into the solvent.

In order to further evaluate the performance of TBPH, the C-IRAE was performed under the same operating conditions, except the extraction solvent. The reference solvents include pure water, methanol and ethanol, the most common and inexpensive solvent. As shown in Table 4, the extraction efficiency of TBPH combined with IRAE was higher than that of the other solvents investigated in the study. Such high effectiveness of IRAE-TBPH resulted from the physiochemical properties of TBPH. The primary cell wall of medicinal plants is made primarily of cellulose. The major action of TBPH is on cell walls. They act on cell wall components, dissolve them in turn, increase the permeability of the cell wall thus resulting in higher yield of the effective constituents. Additionally, methanol and ethanol are volatile and flammable, and they are harmful to environment. Therefore, TBPH solution was a possible alternative solvent in the IRAE of salidroside from the root of R. crenulata.

In conclusion, the results shown above indicated that IRAE-TBPH was a more rapid and effective sample preparation technique compared with all other extraction methods.

3.4 Mechanism of IRAE-TBPH

In order to further elucidate the extraction mechanism, the microstructure and chemical structure of samples before and after different extraction procedures were investigated successively by scanning electron microscopy (SEM) and FT-IR spectroscopy (as shown in Fig. 3 and Fig. 4, respectively). An S-3400N scanning electron microscope (Hitachi, Japan) was used to examine the surface microstructures of samples before and after extraction. FT-IR spectra were recorded in the range of 400–4000 cm⁻¹ on a Vertex 70 FT-IR spectrometer (Bruker, Germany).

Fig. 3 SEM micrographs of the root of R. crenulata before extraction (a), and after extraction by IRAE-methanol (b), IRAE-ethanol (c), IRAE-H₂O (d), UAE-TBPH (e), HRE-TBPH (f) and IRAE-TBPH (g).

Fig. 4 FT-IR spectra of the root of R. crenulata before and after different extraction techniques.

First, the surface structure of the root of R. crenulata before extraction was observed from the SEM image (Fig. 3a). The surface morphology of the samples obtained by IRAE-methanol, IRAE-ethanol and IRAE-H₂O changed moderately and included some disrupted areas (Fig. 3b–d). In contrast, the SEM micrograph of sample extracted by IRAE-TBPH (Fig. 3g) showed a significantly different image, with the larger features completely broken down into much smaller structures, indicating that the cell wall was much more highly dissolved in TBPH compared with that in methanol, ethanol and water.

The microstructures of the samples were not severely damaged after UAE and HRE process (Fig. 3e and f) compared with IRAE-TBPH (Fig. 3g). The results indicated that the extraction efficiency could be significantly enhanced by IRAE-TBPH because of high efficiency heating of infrared irradiation compared with UAE and HRE, and long extraction time were needed in UAE and HRE processes.

As FTIR spectra can provide useful information for identifying the presence of certain function groups or chemical bonds in a molecule or an interaction system, it was applied here to investigate the changes in chemical structures of R. crenulata before and after extraction by various methods, respectively.

Peaks at 1613, 1448, 1380, 1160 and 1030 cm⁻¹ were regarded as the characteristic absorption peaks of carbohydrate compounds, cellulose, hemicellulose and lignin included, the main components of cell wall of plants according to previous literatures.^39,43 Because all the FT-IR spectra in Fig. 4 have been “minimum–maximum” normalized with the workstation software of Vertex 70 FT-IR spectrometer (Bruker, Germany), the changes of absorption intensity of the five peaks could be clearly determined according to the transmittance (%) shown in Fig. 4. The IR spectra of samples after being extracted by different techniques showed that their signal intensity all decreased at 1613, 1448, 1380, 1160 and 1030 cm⁻¹, but the absorption intensity of the residue of the herbs after processing by IRAE-TBPH appeared to be lower than those after processed by IRAE-methanol, IRAE-ethanol, IRAE-H₂O, HRE-TBPH and UAE-TBPH (Fig. 4). These results indicated that the chemical structures of carbohydrate compounds, including cellulose, hemicellulose, and lignin, were broken after extracted by the these methods,⁴³ and the active ingredients presented in the samples had been largely extracted by the IRAE technique.³⁹ Furthermore, there was no obvious change in the absorption bands of herbs before and after extraction with TBPH solutions, which indicated that the chemical structure of the salidroside was not destroyed in these extraction processes.³⁹

4 Conclusion

In the present study, the ionic liquid of TBPH aqueous solution, which is able to dissolve cellulose based on a simple design, was proved to be a possible alternative solvent to conventional potentially toxic organic solvents in the IRAE of salidroside from the root of R. crenulata.

The optimal IRAE-TBPH performance was obtained under operating conditions: infrared power 100 W, extraction time 8 min, and 1.61 mol L⁻¹ TBPH as extraction solvent with a liquid/solid ratio of 20 : 1. The developed HPLC-DAD method revealed a good precision (RSD ≤ 1.04%) and recovery (95.97–99.98%), and was successfully applied for the quantitative determination of salidroside in extracts from R. crenulata.

Compared with other methods (HRE-TBPH, UAE-TBPH, and C-IRAE), the proposed approach provides higher extraction efficiency (15.41–38.65% enhanced) and dramatically reduced extraction time (from 60 min to 8 min). According to the SEM results, the enhanced extraction was mainly based on the destruction of sample microstructures, cellulose dissolving property of TBPH and high efficiency heating of infrared irradiation in IRAE-TBPH process.

The ionic liquid of TBPH aqueous solution was excellent extraction solution and with the development of modern sample preparation techniques, TBPH solution as a novel solvent in the IRAE of useful substances in natural sources showed a great promising prospect.

Acknowledgements

This work was supported by the Specific funds of TCM industry (Grant no. 201407003), National High-tech Research and Development Program (no. SS2014AA022201), National Science Fund for Distinguished Young Scholars (81325023), the central level significant increase or decrease support project, the key project of the National Natural Science Foundation and Jiangsu university collaborative innovation project.

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