Ionic liquid-based enzyme-assisted extraction of chlorogenic acid from Flos Lonicera Japonicae
© The Author(s) 2017
Received: 20 September 2017
Accepted: 14 October 2017
Published: 24 October 2017
In recent years, ionic liquids and enzymes have been widely used in the separation and extraction processes of natural products. Chlorogenic acid (CGA) has important biological and pharmacological activities. It is significant to develop a green and efficient method to extract GCA from Flos Lonicera japonica (FLJ) by integrating the advantages of the ionic liquids and enzymes.
The optimal type of enzyme and ionic liquid was screened. Pectinase in [C6mim] Br aqueous phase was demonstrated to be an ideal combination. The parameters including extraction time, extraction temperature, pH, enzyme amount, and IL concentration were optimized systematically. Scanning electronic microscopy of FLJ samples demonstrated that pectinase and ionic liquid disposal both obviously facilitated the extraction process by destroying the structure of cell wall. Circular dichroism spectroscopy showed that ionic liquid enhanced the activity of the pectinase by altering its secondary structure.
Flos Lonicera japonica (FLJ) is widely distributed in China and officially listed in the Chinese pharmacopoeia (Xiang and Ning 2008). It was reported to have various biological activities such as antiviral, antioxidant, and anti-inflammatory and widely used in treating exopathogenic wind-heat, epidemic febrile diseases and some infectious diseases such as SARS coronavirus and swine H1N1 flu virus (Shang et al. 2011; Wang and Weller 2006). Chlorogenic acid (CGA) is the major bioactive components of FLJ, and had been reported to have various biological and pharmacological activities such as antioxidant, anti-inflammatory, and anti-carcinogenic activities (Wu et al. 2012; Shin et al. 2015). A series of methods had been reported for the extraction bioactive CGA from plant materials. In conventional methods, CGA extraction was mainly achieved by water extraction (WE) (Zheng et al. 2006; Wu et al. 2006) or refluent ethanol extraction (REE) (Lan and Zhang 2005). Some disadvantage of these methods were fraction of oxidation, hydrolysis, and ionization due to excessive extraction time and relatively higher temperature (Duarte et al. 2010). Nowadays, microwave-assisted extraction (MAE) (Zhang et al. 2008) and ultrasound-assisted extraction (UAE) (Hu et al. 2009) had been developed as the feasible extraction methods. Although these two extraction methods greatly lessen the extraction time, it still can’t meet the satisfaction of green chemistry for the consumption of organic solvents (Zhang et al. 2011).
In this work, we come up with a method to use ionic liquid aqueous phase to enhance the penetrability of the solvent over the extraction process and utilize the characteristics of enzymatic hydrolysis on cell wall to enhance efficiency and extraction yield of CGA from FLJ. The optimized ILEAE method was investigated and the extraction mechanism by ILEAE was discussed by scanning electron microscopy and circular dichroism spectroscopy.
Reagents and materials
Flos Lonicera japonica was obtained from Xinyi Honeysuckle Agricultural Development Co., Ltd. (Xin yi, China). FLJ was crushed and dried in the hot air oven before extraction. The standard sample of CGA was purchased from Aladdin (Shanghai, China) and its purity was ≥ 98%. All ionic liquids were bought from Shanghai Cheng Jie Chemical Co., Ltd. (Shanghai, China). Cellulase, pectinase, dextranase, and xylanase were purchased from Jiangsu Rui Yang Biotechnology Co., Ltd. (Jiangsu China). Other reagents were analytical grade and used without further treatment. Deionized water was used to prepare the sample solutions.
The chromatographic system (Dalian, China) consisted of ultimate 3000 autosampler, ultimate 3000 pump, and ultimate 3000 variable wavelength detector. Chromatographic separation was performed on a Amethyst C18-H (4.6 mm × 250 mm, 5 μm) reversed-phase column (Sepax Technologies, Inc.). For HPLC analysis, gradient elution was performed using methanol–water as the mobile phase. CGA was separated by UV detector at a wavelength of 327 nm. Injection volume was 10 ml, flow rate was 1.0 ml/min and column temperature was maintained at 25 °C. The CGA standard curve showed a good linear relationship within the scope of 0.01–1 mg/ml. The standard formula for extraction yield of CGA was \(y = 0. 9 9 9x + 1. 2 9 6\) (R = 0.999, x: concentration, g/ml; y: the peak area).
Optimization of extraction systems
In the preliminary work, the components of extraction solution had been found having significant influence on extraction yield of GCA. Then, several experiments were carried out to investigate on solution components.
Screening the enzyme type
Calculated amounts of cellulase, pectinase dextranase, and xylanase were weighed 20 mg, respectively and an amount of 0.25 mol/l [C4mim]Br solvent was added to attain concentration of 0.5 mg/ml. The ionic liquid solvent pH 4.0 was prepared for further usage. 2.0 g dried samples were put into 40 ml of the enzymatic ionic liquid solvent and then put in a shaking bath at temperature of 40 °C for 2 h, individually. After the reaction, the extract was filtrated and diluted for subsequent HPLC analysis.
Screening the ionic liquid type
In the original step, five different anions including Br−, Cl−, NO3−, BF4 −, and PF6 − and cations including [C2mim]+, [C4mim]+, [C6mim]+, [C8mim]+, and [C10mim]+ with different length of carbon chains of glyoxaline were examined. Enzyme solution was added into 0.25 M ionic liquid solution to attain a concentration of 0.5 mg/ml, respectively. The ionic liquid solvents were prepared to adjust and attain solutions with pH of 4.0. 2.0 g dried samples were combined with 40 ml of the selected four enzymatic ionic liquid solution, individually and reacted under mixing 150 rpm (on a shaking bath) at temperatures of 40 °C for 2 h. After the reaction, the extract was filtrated and diluted for the further HPLC analysis.
Determination of pectinase activity
Ionic liquid-based enzyme-assisted extraction (ILEAE)
Pectinase was put into ionic liquids to attain IL-enzyme aqueous solution of concentrations (0.25–2.0 mg/ml). IL solutions were prepared to adjust pH values (3.0–5.0) for attaining the proper pH of extraction solution. 2.0 g dried sample was put into the IL-enzyme aqueous solvent and then it was put on a shaking bath for 0–4 h at 20–70 °C. After ILEAE, the extracts were filtrated and then diluted through a 0.45 μm filter for further HPLC analysis. All experimental steps were performed in duplicate.
Scanning electron micrographs (SEM)
TM3000 benchtop scanning electron microscopy (Hitachi Co., Ltd. Japan) was applied to examine the effect of IL-enzyme solutions on the structural changes of the plant cells after treatment. The samples were fixed on adhesive tape and examined under high vacuum condition at a voltage of 15.0 kV (10 μm, 1500 magnification).
Circular dichroism (CD) spectroscopy
Circular dichroism (CD) spectra were recorded on a JASCO-J810 Spectropolarimeter (Jasaco Co., Ltd. Japan) in a cell with 1 cm light path length at 25 °C. The scanning wavelength was 200–270 nm and the scanning rate was set at 50 nm/min. The CD spectra were expressed in terms of the average residue molar ellipticity in units of deg cm2 dmol−1. After obtaining CD spectra, the secondary structure of lipase was calculated using CDNN (version 2.1) which was distributed by Applied Photophysics.
Results and discussion
Sieving of enzyme and ionic liquid type
Optimization of the ILEAE process
In the initial process, we investigated the enzyme types and ionic liquid types. Subsequently, important factors affecting extraction yield including extraction time, extraction temperature, pH, enzyme concentration, and IL concentration were studied.
Effect of extraction time
Therefore, 40 min was chosen as appropriate time for subsequent experiments.
Effect of extraction temperature
To select a proper extraction temperature, we did a series of experiments to complete extraction process. The influence of extraction time on CGA extraction yield was studied under the conditions of 0.5 mg/ml pectinase and 0.25 M [C4mim]Br treatment, pH 3.0, and extraction time 40 min, As shown in Fig. 3b, the extraction yield reached the maximum at the optimum temperature of 40 °C and decreased with the increase of temperature. It may be the effect of temperature on the catalytic activity of the enzyme resulting in a decrease in the extraction rate of GCA.
Effect of pH value
Holding the conditions of 0.5 mg/ml pectinase and 0.25 M [C4mim]Br treatment, extraction time 40 min and extraction temperature 40 °C, the influence of pH value on CGA extraction yield was investigated. As shown in Fig. 3c, pectinase showed ability to collapse within a wide pH range. It can be noticed that the extraction yields varied unregularly in the range of pH 3–5. This result showed the activity of pectinase was susceptible to solvent pH. The extraction yield of CGA reach peak value around pH 4.0.
Effect of enzyme concentration
As shown in Fig. 3d, the influence of different enzyme concentrations on the extraction yields was investigated under the conditions of 0.25 M [C4mim]Br treatment, extraction time 40 min, pH 4.0, and temperature 40 °C. At the concentration of 1 ml/mg, the extraction yield reached the maximum, then the extraction yield of CGA was almost unchanged. Higher concentrations than 1 mg/ml couldn’t increase extraction yields. Thus, 1 mg/ml was chosen as appropriate concentration for the extraction process.
Effect of IL concentration
As shown in Fig. 3e, under the conditions of 1 mg/ml pectinase treatment, pH 4.0, extraction time 40 min, and temperature 40 °C, the influence of different ionic liquid concentration on the yields of CGA was evaluated. Concentration of 0.75 mol/l gave a peak extraction yield about 6.06%, indicating that a concentration of 0.75 mol/l offered adequate amounts of pectinase for the disruption of cell wall. Higher concentrations than 0.75 mol/l did not increase yields. Thus, 0.75 mol/l was chosen as appropriate concentration for the extraction process.
Comparison with the reference methods
Comparison of ILEAE with the reference and conventional methods
Optimized extraction parameters
CGA yield (%)
Liquid ratio 10 ml/g, extraction temperature 75 °C, extraction time 1 h
2.29 (Yu et al. 2010)
Liquid ratio 15 ml/g, extraction temperature 70 °C, extraction time 1 h
3.8 (Li et al. 2011)
Ethanol concentration 80% (v/v),extraction temperature 70 °C, extraction time 80 min
3.63 (Li and Hou 2013)
Ethanol concentration 75% (v/v), flocculation time 24 min, reflux time 1.5 h
3.81 (Xiao and Li 2011)
Soaking time 12 h, ultrasonic time 45 min, ethanol concentration 60%
5.62 (Lu et al. 2015)
Microwave power 300 W, microwave time 3 min, extraction temperature 60 °C, ethanol concentration 70% (v/v), extraction time 20 min
5.16 (Cao et al. 2008)
Dosage of pectinase 0.5%, operating temperature 45 °C, extraction time 120 min
0.75 M [C6mim]Br, extraction time 40 min, extraction temperature 70 °C, pH 4.0, 1 mg/ml pectinase
Scanning electron micrographs (SEM) of different samples
Circular dichroism (CD) spectroscopy analysis
The percentage of secondary structure elements and enzyme activity
Random coil (%)
Enzyme activity (u/mg)
Aqueous solution pectinase
In this work of ILEAE, pectinase was successfully combined with ionic liquids for the extraction of CGA from FLJ. As far as we know, this is far-reaching significance investigation on developing a method for extraction of CGA from FLJ using ionic liquid-based enzyme-assisted solvent as extraction medium. From the final test results, the CGA yield was enhanced obviously with the addition of pectinase and ionic liquids. Compared to reported extraction methods, the optimal ILEAE method reduced the extraction time significantly and offered higher extraction yield. By taking into account the practical application, the ILEAE showed great prospect and provided new ideas for the extraction and separation of other natural products.
YS wrote the article; YS and SD conducted the experiment; YH conceived the research; YH and HH provided experimental materials and guidance. All authors read and approved the final manuscript.
The authors thank anonymous reviewers for their insightful comments and careful corrections. We also acknowledge the support from the self-owned research project from key laboratory of material- oriented chemical engineering (Grant No. ZK201603), National Natural Science Foundation of China (21676143) and Qing Lan Project of Jiang Su Province and Program for Innovative Research Team in University of Jiangsu Province.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included in the main manuscript file.
Consent for publication
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The self-owned research project from key laboratory of material-oriented chemical engineering (Grant No. ZK201603), National Natural Science Foundation of China (21676143) and Qing Lan Project.
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