1. Introduction
Populus is a genus of more than 100 species of deciduous plants in the family Salicaceae, These plants, commonly known as poplar, aspen and cottonwood, are native to Europe, Asia and North America [
1,
2]. The poplars are among the most important boreal broadleaf trees, and are almost all grown as ornamental trees and street trees.
Populus alba × P. berolinensis is popular for its rapid growth, adaptability, fine grain and pale color. In China, it has been used widely in forests planted for ecological protection, the Three-North Shelterbelt, agroforestry, industrial plantations, near roads and in garden landscaping [
3]. Wood from
P. alba × P. berolinensis is used in civil construction, furniture, and plant fiber materials. These uses produce large quantities of bark as a side product. Consequently, attention has focused on potential uses for
P. alba × P. berolinensis bark. The bark of
P. alba × P. berolinensis reportedly contains valuable glycosides, such as salicin and rutin [
4,
5], and hyperin [
6], and extraction of these bioactive components from
P. alba × P. berolinensis bark would be useful.
Salicin can be used to treat rheumatic fever and subacute bacterial endocarditis [
7]. It is also used as a traditional analgesic [
8,
9]. Salicin is a prodrug that is gradually converted into salicylic acid after absorption, and is antipyretic without causing gastric injury [
10]. Rutin shows anti-inflammatory and antioxidant activity [
11], can reduce the cytotoxicity of oxidized LDL cholesterol and lower the risk of heart disease [
12], inhibit platelet aggregation [
13], decrease capillary permeability, and improve circulation [
14]. Hyperin is anti-inflammatory [
15,
16], antioxidant
in vivo [
17,
18], antiviral [
19], antidepressant [
20,
21], neuroprotective [
22], vascularprotective [
23] and hepatoprotective [
24]. Because these three natural compounds have many potential applications and economic and environmental value, they are attracting increasing attention.
Conventionally, glycosides are extracted from plant material by heating under reflux (HRE) and Soxhlet extraction. However, the long-term exposure to high temperatures of these methods leads to loss of glycosides because of condensation, degradation, isomerization, or oxidation during extraction. These issues make the method inefficient, require large volumes of toxic organic solvents and result in low recovery of the products. It would therefore be desirable to develop new environmentally friendly methods that can be scaled up for commercial production.
Ionic liquids which are composed of organic cations and inorganic or organic anions are liquid near room temperature. In recent years, ionic liquids have been used as attractive ‘green’ alternatives to conventional volatile organic solvents in various applications, particularly in separation science [
25,
26]. They have been used for a variety of applications, such as alkaloids [
27,
28], metal ions [
29], flavonoids [
30,
31], organic acids [
32], glycosides [
31,
33], lignans [
34] and proanthocyanidins [
35] due to their unique chemical and physical properties, such as negligible vapor pressure, wide liquid range, good stability, tunable viscosity, good miscibility in water and organic solvents, good solubility and extractability for various organic compounds and remarkable advantage of easy to be controlled over conventional solvents [
34]. The results of these studies have demonstrated that the use of ionic liquids as alternative solvents to replace traditional organic solvents in extraction is very promising. Heretofore, however, to our best knowledge, how to separate target analytes from ionic liquids has rarely been reported in the literature.
Compared with conventional extraction, MAE uses less solvent and is faster, while providing equivalent or higher extraction yields for the analytes. The effect of microwave is to diffuse the inner components, change the internal microscopic structure and cause the swelling of cells or the breakdown of cell walls when the temperature rises, which enhances mass transfer of the cell contents. Therefore, MAE is attractive for the extraction of active compounds from herbs. However, the operating temperature for MAE in an open vessel is typically close to the boiling point of the solvent, and salicin, hyperin and rutin are thermo- and oxygen-sensitive and rapidly degrade or oxidize in an open system. Therefore, a new method for the extraction of thermo-sensitive compounds is needed.
To overcome the degradation at high operating temperatures in open MAE, vacuum microwave-assisted extraction (VMAE) has been developed [
36,
37]. Under vacuum, the boiling point of an extraction solvent will be lower than at atmospheric pressure, and the extraction can be performed at lower temperature, which is beneficial for thermo-sensitive compounds. Two major advantages of combining the application of microwave with vacuum techniques are rapid drying due to the ability of microwave to heat solvents instantaneously and homogeneously, and enhanced rate and extent of mass transfer at sub-atmospheric pressure and low temperature. The advantages of VMAE are mainly attributed to the unique extraction mechanism of MAE and the excellent effect of vacuum. In VMAE, because the air in the extraction system is removed, degradation of oxygen-sensitive compounds is avoided or reduced compared to in open MAE [
38,
39], and this can increase the extraction yield [
40].
The objective of this study was to develop an effective and environmentally friendly ionic liquid vacuum microwave-assisted extraction (ILVMAE) method for the extraction of thermo-sensitive and oxygen-sensitive glycosides, salicin, hyperin and rutin from P. alba × P. berolinensis bark. The effects of changing the ionic liquid concentration, bark soaking time, microwave irradiation power and time, liquid/solid ratio, and level of vacuum on the extraction yield were evaluated using a factorial design and response surface methodology (RSM) with a Box-Behnken design (BBD). And the proposed method was validated in stability, repeatability, and recovery experiments. Moreover, the separation of target ingredients from ionic liquid extraction solution using macroporous resin was preliminary attempted. The extraction effect of [C4mim]BF4 after recovery and recycling on the yields of the three target analytes was investigated. The microstructures of unprocessed and processed bark samples were also investigated by scanning electron microscopy (SEM). Hopefully, this work is helpful for the application for the production of salicin, hyperin and rutin from Populus bark and other plants.
3. Experimental Section
3.1. Materials and Chemicals
P. alba × P. berolinensis bark was provided by the Maoershan Experimental Forest Farm of Northeast Forestry University (Harbin, Heilongjiang, China), and authenticated by Prof. Shaoquan Nie from State Engineering Laboratory for Bio-Resource Eco-Utilization (Northeast Forestry University). The bark was dried at room temperature for a month, and then pulverized using a blender and sieved (60–80 mesh) before extraction. The pulverized samples were stored in closed desiccators at 4 °C until use. The same batch of sample was used in all experiments. Reference samples of salicin, hyperin and rutin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methanol used for HPLC analysis was of chromatographic grade and purchased from J&K Chemical Ltd. (Beijing, China). All the other reagents were obtained from Beijing Chemical Reagents Co. (Beijing, China) and were of analytical grade. All ionic liquids ([C4mim]Cl, [C4mim]Br, [C4mim]BF4, [C4mim]NO3, [C4mim]HSO4, [C4mim]ClO4, [C2mim]BF4, [C6mim]BF4, [C8mim]BF4, where C2 = 1-ethyl, C4 = 1-butyl, C6 = 1-hexyl, C8 = 1-octyl, and mim = 3-methylimidazolium) were bought from Shanghai Cheng Jie Chemical Co. LTD. (Shanghai, China) and used without further purification. Deionized water purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA) was used for preparing and diluting all solutions. All of the solvents for HPLC were filtered through a 0.45 µm microporous membrane (Guangfu Fine Chemicals Research Institute, Tianjin, China) and degassed by ultrasonication before use.
D101 macroporous resin (surface area 500–550 m
2/g, average pore diameter 9–10 nm, particle diameter 0.30–1.25 mm, crosslinked polystyrene, non-polar) was purchased from Anhui Sanxing Resin Technology Co., Ltd. (Guzhen, Anhui, China). In order to remove the monomers and porogenic agents trapped inside the pores of macroporous resin during synthesis process, the adsorbent bead was pretreated with the following procedure: first, the resin was soaked in ethanol for 24 h, and then washed with deionized water by circumfluence until there is no residue of ethanol [
45]. The treated resin was stored in a desiccator with deionized water in order to maintain constant moisture content. Prior to use, the resin was wet with ethanol again and then thoroughly replaced with deionized water [
46]. The moisture content of the tested D101 macroporous resin was 66.65%.
3.2. Apparatus
A domestic WP700 microwave-assisted extraction unit (irradiation frequency 2.45 GHz, maximum output power 700 W and with continuously adjustable power; Glanz Electrical Appliance Industrial Co., Ltd., Guangzhou, Guangdong, China) was used for the extraction. The dimensions of the interior cavity of the oven were 215 mm × 350 mm × 330 mm. The microwave was modified in our laboratory with the addition of a water condenser coated with polytetrafluoroethene to prevent microwave leakage. The whole system was run under vacuum. The vacuum in the system was created with a vacuum pump (SHB-IV, Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd., Zhengzhou, Henan, China). The vacuum pump was fitted between the condenser and the flask that was used to collect the crude extract. As shown in
Figure 7, the schematic diagram of the VMAE equipment, some parameters which influence the extraction process, such as the vacuum, microwave irradiation power and time, can be set on the equipment.
3.3. HPLC Analysis and Quantification
3.3.1. Preparation of Standard Solutions of Salicin, Hyperin and Rutin
Standard stock solutions of salicin (14.62 mg/mL), hyperin (2.54 mg/mL) and rutin (2.66 mg/mL) were prepared in methanol. The standard stock solutions were stored at 4 °C, and diluted with methanol to the required concentration before direct analysis by HPLC.
Figure 7.
Schematic diagram of the experimental apparatus.
Figure 7.
Schematic diagram of the experimental apparatus.
3.3.2. Stability Test of Standard Mixtures
Standard mixtures of salicin (14.62 mg/mL), hyperin (2.54 mg/mL) and rutin (2.66 mg/mL) were prepared in 25 mL volumetric flasks using 1ml glass bulb pipettes. For HPLC determination, the mixtures were diluted with methanol. Standard mixtures were stored at room temperature (approximately 18–20 °C) for 4 h in volumetric flask. The stability of the sample at room temperature was evaluated by comparing the assay results of the standard mixtures with that of the freshly thawed standard mixtures. Long-term stability was studied by assaying samples that had been stored at 4–8 °C for a certain period of time (5 days).
3.3.3. HPLC analytical Conditions
The chromatographic system (Waters, Milford, MA, USA) was equipped with Millennium 32 software, a 717 plus autosampler, 1525 pump, 717 automatic column temperature control box and 2487 UV detector. Chromatographic separation was performed on a Kromasil-C18 reversed-phase column (AkzoNobel Pulp and Performance Chemicals AB Separation Products, Bohus, Sweden; 4.6 mm × 250 mm, 5 µm).
The standard solutions of salicin, hyperin and rutin and the extracts were filtered through 0.45 µm membranes before HPLC analysis. The mobile phase used for separation was methanol (A) and deionized water (B). The following gradient elution program was used for separation: 0–15 min, 15% A; 15–18 min, 15%–32% A; 18–68 min, 32% A; and 68–70 min, 32%–15% A. The mobile phase flow rate was 1.5 mL/min, the injection volume was 10 μL, the column temperature was 25 °C and the run time was 70 min. The wavelength used for detection of salicin was 265 nm, and 357 nm was used for hyperin and rutin. Salicin, hyperin and rutin were baseline separated under these conditions. The retention times of salicin, hyperin and rutin were 10.0, 45.1 and 47.8 min, respectively. The chromatographic peaks of the analytes were confirmed by comparing their retention time with reference standards. The results are shown in
Figure 8. No influence attributable to the ionic liquids used were observed on peak resolution, elution order or elution time. The linear equations for the calibration curves for salicin, hyperin and rutin were
Ysalicin = 182369
X – 11025 (
R2 = 0.9999, n = 7),
Yhyperin = 1897587
X + 123321 (
R2 = 0.9997, n = 7), and
Yrutin = 3453739
X + 83323 (
R2 = 0.9997, n = 7), respectively. The calibration curves showed good linearity for salicin between 0.277 and 14.62 mg/mL, hyperin between 0.00124 and 2.54 mg/mL, and rutin between 0.0026 and 2.66 mg/mL.
For HPLC analysis of [C4mim]BF4, acetonitrile–1% volume fraction of acetic acid (20:80, v/v) is used as the mobile phase with 1.0 mL/min flow rate, 10 µL injection volume, and 25 °C column temperature. The absorbance was measured at a wavelength of 210 nm, the retention time is 6.8 min, and the corresponding calibration curve is YIL = 3124560X + 13725 (R2 = 0.9998, n = 7). A good linearity was found in the range of 0.0125–5.0 mg/mL.
3.5. Optimization of ILVMAE by RSM
To further study the interaction between the factors, we optimize the operating condition by Box-Behnken design with three factors applied using Design-Expert 7.0 software [
41] without any blocking. The boundaries of the factors were 10–20 min for the microwave irradiation time, 230–540 W for the microwave irradiation power, and 15–25 for liquid/solid ratio (mL/g). Specific conditions for each experiment are shown in
Table 1.
Figure 8.
HPLC profile of target analytes in an extract obtained using 1 M C4mimBF4 as extraction solvent.
Figure 8.
HPLC profile of target analytes in an extract obtained using 1 M C4mimBF4 as extraction solvent.
3.6. Stability and Repeatability of ILVMAE
The stability test was performed with salicin, hyperin and rutin standards dissolved in 1.0 M [C4mim]BF4 and extracted under the optimum conditions (2 h soak time, 0.08 MPa vacuum, 400 W microwave irradiation power, 20 min microwave irradiation time, liquid/solid ratio of 25:1 mL/g). For intra-day assays, the extract was determined once every hour on the same day. The inter-day experiments were performed by determining the extract once daily within the next five days. The recoveries of salicin, hyperin and rutin were taken as indicators of the stabilities of salicin, hyperin and rutin under these extraction conditions. To determine the reproducibility of this extraction method, bark samples were processed on five consecutive days under the optimum extraction conditions.
3.8. Separation of Salicin, Hyperin and Rutin from Ionic Liquid Extraction Solution
The experiment of separation of target analytes from ionic liquid extraction solution was carried out in a glass column (12 mm × 500 mm) (Tianjin Tianbo Glass Instrument Co., Ltd., Tianjin, China) wet‑packed with 5 g (dry weight basis) D101 macroporous resin [
47,
48,
49]. The bed volume (BV) and the length of the resin were 25 mL and 10 cm, respectively. A 100 mL ionic liquid extraction solution was flowed downward and through the glass column at 1 BV/h, and the target analytes concentration was monitored by HPLC analysis of the effluent liquid collected at 5 mL intervals. The adsorbate-laden columns were first washed with 4 BV deionized water at flow rate of 2 BV/h and then with 60% volume fraction of ethanol at flow rate of 2 BV/h. The target analytes concentrations in the effluent solution were determined by HPLC analysis of the effluent liquid collected at 5 mL intervals. The effluent solution were concentrated and dried under vacuum before further analyses and the recoveries of the target analytes were calculated.
3.9. SEM
The microstructures of unprocessed and processed bark samples were determined by SEM (Quanta 200, FEI, Hillsboro, OR, USA). To provide electrical conductivity, the sample surface was sputter coated with a thin layer of gold (5–10 nm; 10 mA; 30 s) using an SCD 005 sputter coater (KYKY SBC-12, Beijing, China) at room temperature, as used in literature [
50].
3.10. Statistical Analysis
One-way ANOVA was used to determine if differences in the extraction yields were significant. The results of HPLC analysis are expressed as means ± S.D.