Lipase-catalyzed Synthesis of Feruloylated Lysophospholipid in Toluene-Ionic Liquids and Its Antioxidant Activity.

In this study, Novozym 435-catalyzed interesterification of ethyl ferulate (EF) with phosphatidylcholine (PC) in a two-phase system consisting of an ionic liquid (IL) and toluene was optimized to prepare feruloylated lysophospholipids (FLPs). Optimum conditions for the interesterification process were found to be [Bmim][Tf2N]/toluene ratio of 1:1 (v/v), solvent volume of 4 mL, molecular sieves (4 Å) concentration of 80 mg/mL, reaction temperature of 55°C, substrate molar ratio of 5:1 (PC/EF), Novozym 435 concentration of 50 mg/mL. Under these conditions, two FLPs products (1-FLP and 2-FLP) with total conversion rate of 50.79% were obtained. Because the formation of 1-FLP was significantly higher than 2-FLP, 1-FLP was purified and characterized by LC-MS and NMR. In addition, 1-FLP showed DPPH scavenging activity comparable with those of EF and BHT. Therefore, this study provides a good method for transformation of ferulic acid to improve its solubility and promote its application as functional ingredient in the food and pharmaceutical industries.

incorporated with bioactive compounds such as FA 11 13 . In our previous study, feruloylated lysophospholipids FLPs were synthesized from phosphatidylcholine and ethyl ferulate 14 . In another study, 1-4-hydroxy-3-methoxy cinnamoyl-2-acyl-sn-glycerol-3-phosphocholine could be synthesized using chemoenzymatic method and showed high antioxidant activity 15 . However, chemical synthesis of FLPs is very difficult because FA and phospholipids are sensitive to heat and oxidation. On the other hand, since the mild reaction conditions as well as no waste release, enzymatic synthesis approaches of FLPs are good compared to chemical synthesis 16,17 .
Because of the steric hindrance of the phosphate group in the glycerol backbone of phospholipid molecular and the benzene ring in the FA molecule, there is a technical problem for incorporation of FA molecular into phospholipid molecular. Another issue is the high polarity of FA and phospholipid, which result in low solubility and mass transfer limitation of reaction system. Although solvents with higher polarity such as DMSO could increase the FA solubility and reduce the mass transfer resistance, biocatalysts in such solvents tend to be partially or completely deactivated. In addition, some organic solvents are toxic and difficult to be recovered or recycled, which limit their application in the organic synthesis reactions. Therefore, it is essential to find an appropriate and safe reaction media with good solubility for reaction substrate and avoid deactivation of biocatalyst to increase the conversions of products.
Ionic liquids ILs are salts consisting a mixture of cations and anions with melting points near room temperature. Ionic liquids have been used in homogeneous and heterogeneous catalysis and biocatalysis 18,19 . Ionic liquids are known as green solvents , and their physical properties such as polarity, hydrophobicity, and hydrogen-bond basicity could be adjusted through a rational design and combination of cations and anions 9,20 . These tunable properties are very important for biocatalytic systems. Physicochemical properties of ILs such as melting temperature, polarity, and hydrophobicity can be fine-tuned by simply changing the structure or nature of the cation or anion, which lead to new solvents 21,22 . The properties and development of ILs make them valuable reaction media for production of target chemicals. Recently, using ILs as medium for the enzymatic production of FA derivatives have attracted increasing interest 6,23 . However, to the best of our knowledge, there are no studies available about application of ILs as reaction media for lipase-catalyzed synthesis of feruloylated lysophospholipids FLPs .
Therefore, the aim of the present study was to optimize biocatalytic process to prepare FLPs from ethyl ferulate EF and phosphatidylcholine PC by lipase-catalyzed synthesis in ILs as a reaction medium. Mass spectroscopy MS and nuclear magnetic resonance spectroscopy NMR were used to study the chemical structure of FLPs. Antioxidant activity of FLP product was also determined by DPPH radical scavenging assay in comparison with BHT and EF.

Isolation of 1,2-diacyl Phosphatidylcholine
Egg phosphatidylcholine PC, purity ≥ 90 was isolated from crude egg yolk according to a method 24 with some modifications. About 300 mL egg yolk was extracted by 3-fold volume of acetone for seven times and the mixture was filtered. The solid-residue was then extracted by 3-fold volume of ethanol for six times. The obtained liquid extract was bleached by aluminium oxide for three times. The extract was filtered and the filtrate was concentrated by rotary evaporation. The purity of isolated PC was determined by HPLC AOCS Ja 7b-91 . The PC isolate was stored at 20 for further analysis.

Lipase-catalyzed Synthesis of FLPs
The interesterification reaction was performed in a 10 mL screw capped vial. Phosphatidylcholine PC, 0.25 mmol was dissolved in toluene and Novozym 435 was added at concentration of 50 mg/mL. The mixture was then incubated at 55 for 1.5 h under shaking. Ionic liquid was then added to hydrolyzed PC in toluene at ionic liquid/ toluene ratios of 3:1, 2:1, 1:1, 1:2, and 1:3 v/v . EF of 0.05 mmol was added to the mixture. The reaction conditions, including solvent volume in range from 2 to 7 mL, molecular sieves dried in muffle for 2 h at 350 in range from 0 to 120 mg/mL, and reaction temperature in range from 45 to 70 were optimized. The interesterification reactions were carried under shaking at 250 rpm for 5 days. Samples were withdrawn at known time intervals for further analy-sis. Optimum enzyme concentration and substrate molar ratio were chosen based on optimization process performed in our previous study 14 . All experiments were performed in triplicate and the data were expressed as mean standard deviation.

Puri cation and Quantitative Analysis by RP-HPLC
After interesterification reactions, the enzyme was filtered out. The reaction products were extracted by 3-fold volume of acetone, then extracted by 3-fold volume of hexane and concentrated under vacuum. The residue was separated and purified through flash column chromatography using benzene/chloroform/hexane 5:3:2, v/v/v as the mobile phase. All experiments were performed in triplicate and the data were expressed as mean standard deviation.
The analysis and calculation of Feruloylated lysophospholipids FLPs were performed using RP-HPLC according to our previous study 15 with some modifications. The temperature of Zorbax SB-C18 column 250 4.6 mm i.d., particle size 5 μm was set at 30 with a UV/DAD detector at 325 nm. The mobile phase was a binary solvent system of water containing 3 acetic acid solvent A and acetonitrile solvent B at a flow rate of 1 mL/min. The gradient was operated as follows: 0 min: 90 A and 10 B; 0-10 min: changed to 30 A and 70 B; 10-20 min: maintained 30 A and 70 B; 20-20.5 min: changed to 90 A and 10 B. samples of 100 μL each were withdrawn from the reaction mixture, blown by nitrogen, and diluted by methanol. The injection volume was 10 μL. The conversions of FLPs were calculated in terms of the molar percentage interesterification. The relative conversion values of FLP products was calculated based on LC peak areas/total peak area. The total conversions were obtained as sum of 1-FLP and 2-FLP.

Identi cation of interesteri cation Products by LC-MS
The interesterification products were determined by HPLC-ESI-MS analysis. The mass spectrum was obtained by mass spectrometry Agilent LC-QQQ6460, USA with positive and negative electron spray ionization mode. The MS conditions were as follows: 3.5 kV capillary voltage; 30 V cone voltage; 40 L/Hr cone gas flow; 150 source block temperature; 300 dissolution temperature. The conditions of negative ion mode were: 2.9 kV capillary voltage; 30 V cone voltage; 40 L/Hr cone gas flow; 130 source block temperature; 300 dissolution temperature. The mass scan spectra were recorded in the range of m/z 100-800.

Identi cation of interesteri cation Products by NMR
The identification of 1-FLP was confirmed by NMR analysis which included one-dimensional NMR 1 H, 13

Determination of water content
Water content of ionic liquid and toluene was determined using coulometric Karl-Fisher analysis 915 KFTi-Touch, Metrohm with Hydranal AG-H/methanol as solvent.
A known amount of sample was put into the reaction bottle and titrated with Karl-Fischer reagent to end point and methanol sample was titrated as blank. The volume of the Karle Fischer reagent for titration was recorded as V mL , and the sample weight was recorded as m g . The titrimetric titration T of Karl-Fischer reagent was 4.0402 mg/ mL. The water content mg/g was calculated as flows: The water content of Novozym 435, PC, and EF was determined by drying in an oven at 105 until constant weight.

Antioxidant activity
Antioxidant activity of the 1-FLP was evaluated by free radical scavenging activity DPPH assay as described in previous studies 15,25 with some modification. Solutions of DPPH and the test samples with different concentration were prepared in methanol. For the test, 2 mL methanolic solution of 1-FLP with different concentrations 5, 20, 30, 50, 75, 100, 150, 200, 400, and 600 μg/mL and 2 mL methanolic solution of DPPH 0.2 mmol/L were put into the sample tube. Positive controls, BHT and EF, were also run. The mixtures were vortexed and kept in the dark for 30 min, then the absorbance was measured at 517 nm A i . The absorbance of methanolic solution of DPPH at the same wavelength was recorded as A o , and the absorbance of methanolic solution of 1-FLP was recorded as A j . The DPPH inhibition percentage was calculated as follows: The inhibition 1

Statistical analysis
All experiments performed in triplicate were statistically analyzed. The differences of mean values were determined using analysis of variance ANOVA method and standard deviations were calculated to verify the results reliability. Significance was determined at a 95 level of probability.

FLPs identi cation
HPLC analysis was employed to identify the reaction substrates and the new products in terms of retention time, which produced fluorescence absorption under UV light 325 nm . Ferulic acid FA and ethyl ferulate EF were eluted with relative retention times of 7.384 and 11.252 min, respectively. The chromatogram for the substrates and interesterification products is shown in Fig. 1. There are two new peaks #1 and #2 at retention time of 5.250 and 5.434 min were found after the interesterification reaction. These two peaks could be attributed to interesterification products identified as 1-feruloyl-lysophosphatidylcholine 1-FLP and 2-feruloyl-lysophosphatidylcholine 2-FLP , respectively. The molecular structure of 1-FLP and 2-FLP products is shown in Scheme 1.
The purified FLPs were identified by LC-MS. Electrospray ionization-mass spectroscopy ESI-MS in the positive and negative ion mode. Low energy bombardment was used to characterize the molecular structure of the products Fig. 2 . LC-MS spectrum with #3 and #4 showed major ion peaks at m/z of 193.0 and 221.2 in the negative ion mode corresponding to M-H ions of FA and EF, respectively. However, LC-MS spectrum of #1 and #2 peaks was appeared at m/z of 434.0 in the positive ion mode. Fragmentation of peak #1 and #2 produced a fragment ion at m/z 177.0 M H-OH , which was identified as fragmentation of peak #3 FA, m/z 194 and #4 EF, m/z 222 . On the other hand, new peaks of #1 and #2 could be attributed to new products resulting from the esterification reaction, identified as 1-FLP and 2-FLP.
Because 1-FLP was formed in higher amount compared with 2-FLP, 1-FLP was characterized by NMR. The purified 1-FLP was identified by 1 H and 13 C NMR, using DEPT, HSQC and the HMBC spectrum. The DEPT spectrum was used to determine the carbon types in the compound, whether they are primary carbon CH 3 , secondary carbon CH 2 , tertiary carbon CH , or quaternary carbon C . The HSQC and HMBC spectrum were used to test the one-toone correspondence and remote triple correlation between C atom in 13 C NMR and H atom in 1 H NMR, respectively. Relevant circumstances of HMBC correlations of H to C are shown in Fig. 3. According to the results of LC-MS, one-dimensional NMR 1 H, 13 C and DEPT and doubled NMR HSQC and HMBC , the molecular structure of the main product was confirmed as 1-FLP.

Effect of solvent system
According to our previous study 14 , toluene was chosen as the best solvent medium for lipase-catalyzed synthesis of FLPs. Considering the steric hindrance of benzene ring in the FA and phosphate group in the phospholipid, two- step routes hydrolysis and interesterification was adopted to facilitate the FA molecular into PC molecular. A main problem in the interesterification reaction is the high polarity of FA and PC, which lead to lower solubility in conventional organic solvents and influence the mass transfer in the reaction system. However, the suitable ionic liquids ILs could not only enhance the solubility of FA at higher concentration, but also create a favorable microenvironment for the enzyme to maximize the conversions of the products.
Because of the high viscosity of ILs, which was the most drawback for application in the organic synthesis, the conversions of FLPs in the single ILs were found to be lower than 5 . However, when toluene was added to Bmim Tf 2 N , FLPs conversion increased to be 28.78 compared with 18.01 in toluene only Table 1 . This could be attributed to that the addition of toluene as co-solvent with ILs reduced the viscosity and increased the solubility of the substrate 26 . Therefore, the two-phase system, consisting of ILs and toluene, was chosen as reaction medium for the further experiments. The high viscosity results in an in-crease in the resistance to mass transfer and reaction. The increase of pyridinium-ionic liquids viscosity is in agreement with the increase in alkyl side chains of cationic nitrogen. The viscosity of IL is expressed by B -coefficient Table 1 . Conversions of FLPs was also determined at different ILs/toluene ratios Table 1 . All chosen ILs belong to imidazolium-type, the conversion of FLPs varied significantly with the anions and the cations.
The FLPs conversion in Bmim BF 4 and Bmim PF 6 is lower because of their high viscosity or B -coefficient Table 1 . The high viscosity of ILs can be attributed to the interaction of molecules through electric charges and van der Waals forces. The conversion of FLPs in ILs was in the order Bmim Tf 2 N Emim Tf 2 N Omim PF 6 Hmim BF 4 . Highest conversion rate of 28.78 was obtained with Bmim Tf 2 N after 5 days. In the practical applications, although IL containing Tf 2 N with stronger van der Waals forces than BF 4 and PF 6 , but it showed lower viscosity. This may be attributed to the weak hydrogen bonds force of Tf 2 N , which makes the viscosity more decrease compared with the increase caused by van der Waals forces.
Other physicochemical properties of ILs such as polarity, hydrophobicity, nucleophilicity, H-bond basicity, and kosmotropicity/chaotropicity also influence lipase activity and conversions of FLP products. In this study, Log P, Log S and the solvatochromic polarity scales such as E T N and Kamlet-Taft scales were used to quantify the polarity of ILs. The E T N Scale or Solvatochromic polarity Scale is a normalized polarity scale, which sets tetramethylsilane as 0.0 and water as 1.0 27 . The E T N , Log P, and Log S values of the used ILs are summarized in Table 1 28 30 . From Table 1, it can be seen that the correlation between IL polarity and conversions was not clearly established for Novozym 435-catalyzed synthesis of FLPs. This result is consistent with previous studies 31,32 . Based on these results, Bmim Tf 2 N was chosen as the optimal reaction media for Novozym  435-catalyzed synthesis of FLPs.

Effect of molecular sieves concentration
In enzymatic reactions, minimal amount of water is necessary for the enzyme to form and maintain active conformation or the loosening up of the rigid structure 15 . On the other hand, an excess of water would inhibit the interesterification reaction and promote the hydrolysis of acylated products 33 . It was found that minimal amount of water was necessary for maintaining enzyme active in imidazolium based ILs 34 . Moreover, the relation between water quantity and the reaction rate in ILs showed a bell shape, which was similar to behavior in common organic solvents. It was also found that the water absorbed by Bmim Tf 2 N was equivalent to that of organic solvents 35 . The water content in the interesterification reaction was studied and the results are shown in Fig. 4. It should be noted that water is not a product of the interesterification reaction. The Novozym 435 as catalyzer was found to contain about 1.00 0.19 w/w water and the reaction substrates PC and EF provide about 2 mg of water PC 1 -1.2 , EF 3 , w/w . However, water content was found to be 0.03 0.04 w/w for toluene and 0.85 0.14 w/w for Bmim Tf 2 N . The water content was needed for hydrolysis of phosphatidylcholine in the first step. However, excessive water is not preferred in the interesterification reaction in the second step. Therefore, the molecular sieves were used to control the water content during the interesterification. From Fig. 4, it can be seen that the water content decreased as 4 Å molecular sieves concentration increased. The decrease in water content increased the FLPs conversion. These results are in good agreement with findings of previous studies 36 . However, the water content does not reflect the actual amount of water available for enzyme molecules. This because some water molecules may be attracted by solvent molecules such as through hydrogen bonds with ILs and molecular sieves. Novozym 435-catalyzed resulted in highest conversion rate of FLPs in Bmim Tf 2 N /toluene at molecular sieves concentration of 80 mg/mL Fig. 4 . However, conversion of FLPs decreased with the increase in molecular sieves concentration higher than 80 mg/mL. These results are in good agreement with previous studies 6,15 . Therefore, 80 mg/mL was chosen as optimal molecular sieve concentration for further experiment.  3.4 Effect of IL/toluene volume ratio As mentioned above, Bmim Tf 2 N /toluene was chosen as optimal reaction medium for lipase-catalyzed synthesis of FLPs. Addition of organic co-solvent to ILs system could increase the solubility of the substrate and reduce viscosity of ILs 25 . This may be attributed to that when the organic solvent is added into ILs, the hydrogen bonds or the ion-dipole interaction between organic solvent and cation/anions of ILs would be formed, then the ionic hydrogen bonds was weaken and the ionic mobility increased, which lead to a reduction in viscosity of ILs.
The influence of varying Bmim Tf 2 N /toluene volume ratios on the lipase-catalyzed synthesis of FLPs was investigated and the results are shown in Fig. 5. Maximum conversion rate of 28.78 was obtained at Bmim Tf 2 N / toluene volume ratio of 1:1. However, total conversion of FLPs decreased as IL/toluene volume ratio increased from 1:1 to 3:1. In other words, solvent with higher amount of IL content leads to a decrease in conversions of FLPs, which can be attributed to the high viscosity of IL. Also, the decrease of IL content leads to a decrease in conversions of FLPs, which may be attributed to compromise of the favorable microenvironment in ILs for the enzyme compared with organic solvent. These results are in agreement with a previous study on lipase-catalyzed esterification of ferulic acid with lauryl alcohol in IL-hexane mixture 6 .

Effect of temperature
Due to the high melting point and polarity of the reac-tion substrates PC and EF , the solubility of the substrates and the mass transfer are important and should be considered. Reaction temperature not only improve the rate of molecular movement and the mass transfer, but also influence the stability and activity of the lipases. Moreover, the increase of reaction temperature could reduce the viscosity of ILs and affect the thermodynamic equilibrium of lipasecatalyzed reactions 6 . Also, choosing suitable temperature for maximum production of a targeted product is economically important. The effect of temperature in range from 45 to 70 on the conversion of FLPs in Bmim Tf 2 N / toluene was studied and the results are shown in Fig. 6.
The conversion of FLPs increased as the reaction temperature increased from 45 to 55 , which can be attributed to the reduction in viscosity of the reaction system and the increase in transfer rate of the reaction substrates. However, no further increase in conversion of FLPs was found as temperature increased from 55 to 65 . On the other, a decrease in conversion of FLPs was found as temperature increased from 65 to 70 , which can be attributed to the deactivation of Novozym 435. Therefore, the optimal reaction temperature for conversion of FLPs in the Bmim Tf 2 N /toluene was chosen to be 55 in further experiments.

Effect of total solvent volume
Based on the Michaelis-Menten equation, solvent volume could affect the mass transfer and the reaction rate 34 . The effect of total Bmim Tf 2 N /toluene volume in range from 2 to 7 mL on the conversion of FLPs was investigated and  the results are shown in Fig. 7. An increase in total conversions of FLPs products was found as solvent volume increased and maximum conversion of 50.79 was obtained at volume of 4 mL. However, the conversion rate decreased as solvent volume increased from 4 to 7 mL. This may be associated with low vapor pressure and low volatility of ILs. Therefore, solvent volume of 4 mL was chosen as optimal for further experiments.

Antioxidant activity
Antioxidant activity of 1-FLP was investigated by DPPH radical scavenging activity assay and the results are shown in Fig. 8. It was found that the DPPH scavenging activity increased significantly as the concentration of 1-FLP increased. A maximum DPPH scavenging activity of 89.26 was found at 1-FLP concentration of 150 μg/mL. Several studies have found antioxidant activity for phenolipids and could be attributed to the hydroxyl and methoxy functional groups in phenolic acid 14 . It should also be noted that 1-FLP showed antioxidant activity close to those of EF and BHT. These results indicate that the antioxidant activity of feruloyl group in the glycerol backbone of phospholipids was maintained after interesterification. Therefore, feruloylated lysophospholipids has the potential for application in the food, cosmetic, and pharmaceutical industries.

Conclusion
Novozym 435-catalyzed interesterification of ethyl ferulate EF with phosphatidylcholine PC in Bmim Tf 2 N and toluene mixture was optimized to prepare feruloylated lysophospholipids FLPs . Tow FLPs products 1-FLP and 2-FLP with total conversion rate of 50.79 were obtained after optimization of reaction conditions. The optimum conditions for Novozym 435-catalyzed interesterification of EF with PC were found to be 1:1 v/v for Bmim Tf 2 N /toluene ratio, 4 mL for solvent volume, 80 mg/mL for molecular sieves 4 Å concentration, and 55 for temperature. 1-FLP was purified and characterized by LC-MS and NMR. Also, 1-FLP product showed DPPH scavenging activity almost equivalent to those of EF and BHT. Suitability of the prepared feruloylated lysophospholipids for applications in the food and pharmaceutical industries as well as their potential health benefits need to be investigated in future studies.