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Article Cite This: ACS Omega 2019, 4, 6000−6009
http://pubs.acs.org/journal/acsodf
Decreased Biomass Recalcitrance Effect and Enhanced Hydrolysis Using Ionic Liquids: Toward Improvements in Isofraxidin Extraction Yu Xia, Wei Li, Zhijun Zhang, Sha Luo, Chunhui Ma,* and Shouxin Liu* Key Laboratory of Bio-based Material Science and Technology, Ministry of Education, College of Material Science and Engineering, Northeast Forestry University, Harbin 150040, China
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S Supporting Information *
ABSTRACT: A microwave-assisted ionic liquid (IL) treatment was used to efficiently extract isofraxidin from the Acanthopanax senticosus root and simultaneously hydrolyze isofraxidin glucoside into free-state isofraxidin. The optimized parameters for the extraction and hydrolyses of 10.0 g of A. senticosus root required microwave irradiation (400 W, 100 °C) time of 20 min and 2.0 g of the optimum catalyst ([C4mim][HSO4]). This yielded 52 μg g−1 of free-state isofraxidin. During the extraction, the IL decreased the biomass recalcitrance effect by removing cellulose, hemicellulose, and lignin to varying degrees. Microwave-assisted heating (MAH) resulted in “hotspots” within the lignocellulosic feedstocks, causing swelling and fragmentation that occurred within the biomass, which disrupted the lignocellulosic structure and allowed the efficient extraction of isofraxidin. To optimize the hydrolysis and extraction of the desired material, IL aqueous solutions that contained different mole fractions of water were examined using FTIR and Raman spectroscopy. IL solutions with high water content did not form ion clusters, and so these solutions had low viscosity and high diffusivity, which was beneficial for the extraction process. Besides, a mechanism for the hydrolysis of isofraxidin glucoside with Brønsted acid ILs was proposed.
1. INTRODUCTION Room-temperature ionic liquids (ILs) that are composed of large and asymmetric organic cations and organic or inorganic anions are desirable solvents. They exhibit a number of advantages, such as being liquid at temperatures below 100 °C, having a negligible vapor pressure, being nonflammable, and being thermally and chemically stable.1,2 ILs can be varied by changing the cations and anions, which is a significant advantage for the extraction, separation, and analysis of valuable compounds from biomass. Therefore, the polarity and affinity of an IL can be tailored by manipulating the cation/anion chemical structure, allowing them to dissolve both polar and nonpolar solutes.3−5 ILs can also be used as an acid/base catalyst by selecting a potential acid/alkaline site while avoiding the disadvantages of homogeneous acidic or basic catalysts (e.g., hydrochloric acid and sodium hydroxide), which include difficulties associated with recovery after use and secondary pollution from materials that are not neutralized following the hydrolysis reaction.6 Acanthopanax senticosus (Rupr. et Maxim.) Harms is widely cultivated in the far eastern region of Russia and Northeast Asian countries and is known as a powerful tonic and a medicinal herb. These herbal plants have attracted attention in the past few years because they exhibit strong bioactivity and appear to be harmless with minimal side effects. A. senticosus improves the body’s hematopoietic ability, protects human bone marrow mesenchymal cells, scavenges free radicals, and repairs oxidative damage.7−9 The isofraxidin compound in A. senticosus roots exists in two states: (i) free-state isofraxidin, © 2019 American Chemical Society
which exhibits antibacterial, antioxidant, antidepressive, and antiinflammatory effects,10,11 and (ii) isofraxidin glucoside, which exists because the glycosidation of isofraxidin at the seven-site hydroxyl group (Figure 3) occurs easily in plants. A number of reports have suggested that the principal active component of A. senticosus roots is free-state isofraxidin and the pharmacological activities of isofraxidin glucoside are significantly lower.12 It has been suggested that isofraxidin glucoside could be hydrolyzed using an acidic catalyst to increase the extraction of the desired material, without inducing further structural changes.13 As the glucoside hydrolysis reaction is endothermic, we examined the use of microwave-assisted heating (MAH) to provide heat for the hydrolysis of isofraxidin glucoside. MAH is an alternative to conventional heating (green heating), can increase reaction yields, reduces reaction times, and avoids damage to active components that can occur during traditional, high-temperature heating methods.14,15 As ILs are polar molecules that are composed of ions, they can effectively dissipate microwave energy.16 Therefore, in our work, the acidic ILs cooperate with the microwave magnetic field for the hydrolysis of isofraxidin glucoside from A. senticosus. Biomass recalcitrance derives from the structure of the plant cell wall, which is composed of cross-linked materials such as cellulose, lignin, and hemicelluloses to form a rigid and Received: January 18, 2019 Accepted: March 19, 2019 Published: March 28, 2019 6000
DOI: 10.1021/acsomega.9b00168 ACS Omega 2019, 4, 6000−6009
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four different Brønsted acid ILs ([C4mim][NO3], [C4mim][CH3SO3], [C4mim][pTSA], and [C4mim][HSO4]) were examined and compared with the inorganic acid HCl as a reference. The IL was judged on the efficiency of the hydrolysis, which involved mixing the A. senticosus extracts (100 mL) with the ILs (1 g) or HCl and subjecting them to microwave irradiation (500 W) for 30 min. The efficiencies of the hydrolysis using different catalysts are shown in Figure 2.
compact structure. The stiff molecules and close-chain packing that occurs via numerous intermolecular and intramolecular hydrogen bonds protect isofraxidin glucoside from hydrolysis using the ILs, and they also affect the energy dissipation and efficiency when extracting isofraxidin from A. senticosus.17,18 Loosening and separating cell wall biomass components is the basis for the extraction of isofraxidin; however, traditional methods to decrease the biomass recalcitrance effect are energy-intensive processes that generate waste. For example, enzymatic hydrolysis suffers from low rates and expensive enzymes, and diluted acid requires harsh conditions, such as high pressures and elevated temperatures (usually ≥200 °C).19 Recently, ILs have been recognized as energy-efficient solvents that can dissolve lignocellulosic materials and overcome the physical and biochemical barriers of the hydrolysis reaction.20 Thus, we examined the ability of ILs to both catalysis and hydrolysis of isofraxidin glucoside and decreased the biomass recalcitrance effect by reducing the crystallinity of cellulose and increasing its porosity and surface area for the efficient extraction of isofraxidin. Therefore, understanding the dispersion states and behavior of the ILs in aqueous solutions that contained different mole fractions of water was essential to this work and was analyzed using FTIR and Raman spectroscopy.
Figure 2. Hydrolysis efficiency of isofraxidin glucoside using different IL catalysts.
The ILs exhibited significantly improved hydrolysis efficiencies when compared with HCl as a catalyst (average hydrolysis efficiency of free-state isofraxidin = 45 μg g−1). The catalytic activity of the ILs was consistent with their acidity, and the Brønsted acidity of the ILs is dependent on the anion, and so the catalytic activity of the ILs depended on the anions.22,23 The IL that had the highest acidity ([C4mim][HSO4]) has the highest catalytic activity (Table 1). Two grams of [C4mim]-
2. RESULTS AND DISCUSSION 2.1. Detection of Isofraxidin by HPLC−MS. As shown in Figure 1, an acetonitrile/water/acetic acid (20:80:1, v/v/v)
Table 1. Determination of Acidity by Hammett Acidity Functions parameters
[C4mim] [NO3]
[C4mim] [CH3SO3]
[C4mim] [pTSA]
[C4mim] [HSO4]
I IH+ H0 pH
0.72 0.28 −4.11 5.3
0.78 0.22 −3.97 3.67
0.86 0.14 −3.74 2.46
0.62 0.38 −4.32 0.48
[NO3], [C4mim][CH3SO3], [C4mim][pTSA], and [C4mim][HSO4] each was dissolved in 100 mL of water. The acidity of IL solution was measured with a pH meter, and the results are shown in Table 1. Besides, the acidity of the IL was also necessary to weaken the intermolecular and intramolecular hydrogen bonds of the cellulose chains. This decreased the biomass recalcitrance effect of the A. senticosus roots cell walls and allowed for the efficient extraction of isofraxidin. Therefore, [C4mim][HSO4] was selected as the catalyst for the hydrolysis of isofraxidin glucoside because it led to the most efficient hydrolysis of isofraxidin (average hydrolysis efficiency of isofraxidin = 57 μg g−1). The hydrolysis mechanism is shown in Figure 3. The glycosidic bond was protonated by the H3O+ ion hydrolyzed from the [C4mim][HSO4] anion, which was then cleaved to give the aglycone and the carbocation intermediate. The carbocation intermediate was solvated in water, and the hydrogen ion was removed to form the sugar molecule.24 2.3. Present State of [C4mim][HSO4] in Solutions by FTIR and Raman Analysis. Water was required for the extraction process, and different mole fractions of water in
Figure 1. (a) MS. (b) HPLC chromatogram. (c) Structure of the isofraxidin standard. (d) HPLC chromatogram of A. senticosus.
mobile phase was used at a flow rate of 1.0 mL min−1, a 5 μL injection volume, and a column temperature of 25 °C. The absorbance was measured at 344 nm, and the run time was 35 min. These parameters resulted in a retention time for isofraxidin of 9.4 min. The calibration curve for the detection of isofraxidin was Y = 16322X + 4.3242 (r = 0.9998). A good linear region was found between 1.95 and 31.25 μg mL−1. 2.2. Screening of the Optimum Acidic IL Solution. Acidic ILs are divided into Lewis acids and Brønsted acids. Compared with Lewis acid ILs, Brønsted acid ILs are stable, have high catalytic activity, are easy to separate, have been used in a wide range of applications, and have been proven useful in many catalytic reactions. Critically for our purposes, water can be used in the Brønsted acid IL reaction systems.21 Therefore, 6001
DOI: 10.1021/acsomega.9b00168 ACS Omega 2019, 4, 6000−6009
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Figure 3. Mechanism of the [C4mim][HSO4] acid-catalyzed hydrolysis of isofraxidin glucoside.
aqueous IL solutions strongly affected the microstructure and solvent properties of the IL, such as self-aggregation, density, polarity, surface tension, and, particularly, viscosity, which significantly affects the diffusivity of the IL. Infrared absorption spectroscopy can provide information on the interactions between water and the ILs by examining the OH stretching modes of water (3000−3800 cm−1). If significant H bonding occurs between the acidic protons of [C4mim][HSO4], imidazolium ring, and water, then the imidazolium ν(C−H) stretching region that is characteristic of the ILs (3200−3000 cm−1) is expected to exhibit a shift. However, the changes that were observed in the spectra of [C4mim][HSO4] (Figure 4a) in this region (between water mole fractions of 0.2 to 0.8) were negligible. As water was added into [C4mim][HSO4], the antisymmetric (3756 cm−1) and symmetric (3657 cm−1) bands from the water merged to produce a broad band with the peak at 3400 cm−1 because of H-bonding effects. Fermi resonance between antisymmetric and symmetric bands further complicated the structure of this broad band. The position of the broad band indicated that the water molecules were not associated into clusters or pools of water and could be viewed as “free” water molecules that were interacting with the HSO4− anion via H bonding. This unique phenomenon that was observed in the aqueous [C4mim][HSO4] solutions was attributed to the formation of chain-like structures (i.e., anion···water···anion···). As a result, the hydrogen bond interaction between ion pairs was weakened, but the number of hydrogen bonds between water and anions increased with increasing water content.25 The Raman spectra of a series of [C4mim][HSO4] aqueous solutions (different mole fractions of water) are shown in Figure 4b. The Raman bands below 3200 cm−1 were caused by the imidazolium ring C−H stretching and other ν(C−H) stretching modes of the IL. There were no significant changes in these bands upon going from a water mole fraction of 0.2 to 0.8, which indicated that the anions were primarily responsible for the solubility and miscibility of water in the IL, while the cations played a secondary role. A weak, broad band that was characteristic of O−H stretching vibrations (3470 cm−1) was present in the Raman spectra of the mixtures. The amplitude of
Figure 4. (a) FTIR and (b) Raman spectra of [C4mim][HSO4] aqueous solution.
this peak increased as the water content increased. As the higher-frequency components can be attributed to weakly hydrogen-bonded water molecules, the change of this peak indicated that the strong H-bonding network of water was largely disrupted and the number of the free O−H increases. This Raman band corresponds to the “liquid-like peak” as the relative enhancement of this peak correlates with the more disordered structure of water and can occur when differentsized cages are formed from different conformers of an IL and H2O molecules that are trapped in the inhomogeneous regions.26 6002
DOI: 10.1021/acsomega.9b00168 ACS Omega 2019, 4, 6000−6009
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content had high diffusivity, which was beneficial for the extraction process.27 2.4. Single Factor of the Hydrolytic Process with [C4mim][HSO4]. 2.4.1. Effect of Microwave Time on Hydrolysis Efficiency. The hydrolysis was optimized by varying the amount of time that the samples were exposed to the microwave irradiation (400 W) up to a maximum of 40 min, as shown in Figure 6a. The heating curves of different powers are shown in Figure S2 (Supporting Information). As the microwave treatment time was increased from 0 to 20 min, the efficiency of hydrolysis increased dramatically. Only slight improvements were observed from 20 to 40 min of treatment time. Additionally, longer extraction times during MAH might result in isomerization of free-state isofraxidin in the extracts. Thus, a microwave exposure time of 20 min was chosen as it gave high yields, had relatively low-energy consumption, and should minimize isomerization. 2.4.2. Effect of Temperature on Hydrolysis Efficiency. The temperature of the reaction system has a great influence on the reaction speed and hydrolysis rate. As shown in Figure 6b, when the temperature is changed from 60 to 100 °C, the hydrolysis yield increased. This is due to the hydrolysis reaction, which is an endothermic reaction, and heating can greatly promote the efficiency of hydrolysis reaction. However, when the temperature reaches 110 °C, the average hydrolysis yield decreased. Therefore, 100 °C was chosen as the optimum temperature. 2.4.3. Effect of Microwave Power on Hydrolysis Efficiency. As ILs have a very high capacity for absorbing microwave energy, the microwave irradiation power was optimized to ensure maximum reaction efficiency, to avoid isomerization of
Analysis of the aqueous ILs using FTIR and Raman spectroscopy indicated that the water molecules tended to interact with the anions first and then with the cations and the H2O molecules primarily bridged the anions and cations at low water mole fraction (
