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Accepted Manuscript
Simultaneous extraction, transformation and purification of psoralen from Fig
leaves using pH-dependent ionic liquid solvent based aqueous two-phase system
Tong Wang, Cheng-Bo Gu, Sui-Xin Wang, Ping-Kou, Jiao Jiao, Yu-Jie Fu
PII:
S0959-6526(17)32492-7
DOI:
10.1016/j.jclepro.2017.10.185
Reference:
JCLP 10977
To appear in:
Journal of Cleaner Production
Received Date:
24 August 2017
Revised Date:
16 October 2017
Accepted Date:
17 October 2017
Please cite this article as: Tong Wang, Cheng-Bo Gu, Sui-Xin Wang, Ping-Kou, Jiao Jiao, Yu-Jie
Fu, Simultaneous extraction, transformation and purification of psoralen from Fig leaves using pHdependent ionic liquid solvent based aqueous two-phase system, Journal of Cleaner Production
(2017), doi: 10.1016/j.jclepro.2017.10.185
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ACCEPTED MANUSCRIPT
Highlights.
1、[Bmim]Br was more efficient than ethanol for psoralen extraction from fig leaves.
2、[Bmim]Br-critic acid mixture has enhanced extraction and transformation
efficiency.
3、[Bmim]Br-critic acid single phase system could form ATPS by adjust PH value.
4、The integrated and sustainable ILs-acid based PH-dependent ATPS was efficient.
ACCEPTED MANUSCRIPT
Graphical abstract
ACCEPTED MANUSCRIPT
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Number of words: 6582
2
Simultaneous extraction, transformation and purification of psoralen from Fig leaves
3
using pH-dependent ionic liquid solvent based aqueous two-phase system
4
Tong Wang a,b, Cheng-Bo Gu a,b , Sui-Xin Wang a,b , Ping-Kou a,b, Jiao Jiao a,b, Yu-Jie
5
Fu a,b, *
6
a
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University, Harbin 150040, PR China
8
b
9
Northeast Forestry University, 150040 Harbin, PR China
Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry
Engineering Research Center of Forest Bio-preparation, Ministry of Education,
10
* Corresponding author. Tel./fax: +86-451-82190535.
11
E-mail
address:
yujie__fu@163.com.
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Abstract
13
Recently, integrated and sustainable methods for extraction, transformation and
14
purification of value-added compounds have gained increasing attention. In the
15
present study, ionic liquid (IL)-acid based pH-dependent aqueous two-phase system
16
(ATPS) was developed for simultaneous transformation psoralic acid-glucoside into
17
psoralen, extraction and purification of psoralen from fig leaves for the first time. The
18
cleaner [Bmim]Br-citric acid mixture showed enhanced transformation and extraction
19
efficiency. The extraction yield of psoralen by [Bmim]Br-citric acid mixture was
20
1.45, 2.45 and 3.68 times higher than that of by [Bmim]Br-water, ethanol-critic acid
21
and ethanol, respectively. The cleaner [Bmim]Br-citric acid based ultrasound
22
extraction and transformation process was optimized using response surface method
23
(RSM). Under optimum conditions, the maximum extraction yield of psoralen was
24
31.22mg/g. Subsequently, we investigated and optimized the pH-dependent aqueous
25
two-phase process. Under optimum conditions, the maximum recovery yield of
26
psoralen was 96.34%. These results indicated that our method was efficient and had
27
great potential for producing psoralen from fig leaves in large scale. What's more, this
28
method could also been used to produce value-added compounds from other raw
29
materials.
30
Keywords:
31
Natural products; Extraction; Transformation; Purification; PH-dependent ATPS; Fig
32
leaves.
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34
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38
1.
Introduction
39
In recent years, the demands for natural products sharply increased due to their
40
excellent biological activity and low toxicity compared with their synthetic
41
counterparts (Zhang et al., 2012). The development of integrated, sustainable
42
extraction and purification methods (Cao et al., 2017; Jeong et al., 2017; Lu et al.,
43
2017; Scalia et al., 2016) and the search for cheaper materials are two effective means
44
for meeting rapidly increasing demands, and have become a hot topic in nowadays
45
(Kates et al., 2001).
46
Ficus carica L. is a deciduous tree belonging to Moraceae (Zohary and Spiegel-
47
Roy, 1975). In traditional Chinese medicine, fig leaves have been used to treat many
48
diseases (Lee et al., 2000). Recent studies have shown that fig leaves have many
49
excellent biological activities, such as antioxidant (Konyalιoğlu et al., 2008),
50
antidiabetic (Canal and Perez, 2000; Serraclara et al., 1998), antipyretic (Vikas,2010),
51
anti-inflammatory and antilipemic activity ( Nicotra et al., 2010). Phytochemical
52
studies have shown that psoralic acid-glucoside and psoralen are the two major
53
components in fig leaves, and that the content of psoralic acid-glucoside is about two
54
times of that of psoralen. Psoralen has good pharmacological activity and clinical
55
efficacy, and been used in the clinic. However, psoralic acid-glucoside shows no
56
significant activity, is a precursor of psoralen and can be converted into psoralen by
57
hydrolysis (Hamed et al., 1997; Takahashi et al., 2014). Therefore, we can transform
58
psoralic acid-glucoside into psoralen to increase the yield of psoralen. However, as far
59
as we know, no research reported the extraction, transformation and purification
60
process of psoralen from fig leaves.
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Traditional methods for extraction, transformation and purification of natural
62
products have many disadvantages, including low efficiency, complex operation, and
63
being time-consuming. Moreover, the use of a large amount of volatile and toxic
64
organic solvents caused serious environmental pollution and harm to human beings.
65
To overcome these shortcomings, more and more researchers are using non-volatile
66
ionic liquid solvents (ILs) instead of organic solvents, and developing integrated
67
method for extraction and purification of natural products from natural raw materials.
68
Ionic liquid solvents have gained increasing attention in past years (Capello et al.,
69
2007). ILs are molten organic salts, which are liquid at temperature below 100 ℃
70
(Earle and Seddon, 2009) and possess many excellent properties including negligible
71
volatility, favourable thermal and chemical stabilities, non-flammability, tune ability,
72
and strong solvation ability for most compounds (Ao et al., 2014; Olkiewicz et al.,
73
2015; Rogers, 2007; Rogers and Seddon, 2003). Therefore, the design of new,
74
integrated and sustainable methods for producing natural products from natural
75
materials using ILs is of great significance. In the present study, we aimed to develop
76
a new integrated and sustainable method for simultaneous extraction, transformation
77
and purification of psoralen from fig leaves.
78
A large number of studies have shown that ionic liquids as extraction solvents have
79
higher extraction efficiency, less environmental influence, shorter time and lower
80
energy consumption compared with conventional organic solvents due to their strong
81
ability to dissolve target compounds and to destroy the plant cell wall (Bogdanov and
82
Svinyarov, 2013; Bogdanov et al., 2012; Cláudio et al., 2013; Du et al., 2007; Ma et
83
al., 2011b; Passos et al., 2014; Ressmann et al., 2013; Zhang et al., 2009). Although
84
the extraction of various natural products using ILs has been investigated, for psoralen
85
which belongs to furocoumarin has not been studied. In addition to excellent
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extraction capacity, recent studies have demonstrated that ionic liquids have strong
87
catalytic activity under mild conditions (Siewping et al., 2014). So we hypothesized
88
that IL-acid mixtures could efficiently transform psoralic acid-glucoside into psoralen
89
and extract psoralen from fig leaves simultaneously. For purification target
90
compounds from crude extracts, liquid-liquid extraction (LLE) is a simple, convenient
91
and most commonly used method. Compared with traditional LLE processes, ILs
92
based aqueous two-phase system (ATPS) have many significant advantages, such as
93
no use of toxic and volatile organic solvents, tunable polarity, good selectivity,
94
biocompatibility, enhanced mass transfer rate and high extraction capacity (Freire et
95
al., 2012). As for IL-organic salt ATPS, the pH value has great influence on the phase
96
behavior, extraction selectivity and recovery yields for target compounds. The reason
97
for this was that pH value could influence the form of organic salt. When the pH value
98
was lower than the pKa value of the salt, the salt was in non-ionized form and losed
99
the salting-out ability, thus, the ATPS might become a single-phase system. When the
100
pH value was higher than pKa value of the organic salt, the salt was in ionized form
101
and had strong salting-out ability, thus, promoted the formation of ATPS (Freire et al.,
102
2012; Iqbal et al., 2016; Ventura et al., 2017). So we hypothesized that after
103
simultaneous transformation psoralic acid-glucoside into psoralen and extraction of
104
psoralen using IL-acid mixture, regulate the pH of IL-acid mixture based extracts to
105
convert organic acid from the non-ionized form to ionized form and ATPS would
106
form and compelete the purification of psoralen.
107
Based on mentioned above, we developed and optimized an integrated and
108
sustainable method for simultaneous extraction, transformation and purification of
109
psoralen from fig leaves using pH-dependent IL-acid based ATPS. Ultrasound
110
irradiation is a clean and efficient process intensification method and adopted in this
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study. The details were showed in Fig 1. The cleaner [Bmim]Br (1-Butyl-3-
112
methylimidazolium Bromide)-citric acid mixture was used to simultaneous transform
113
psoralic acid-glucoside into psoralen and extract psoralen from fig leaves in a single-
114
phase system. Adding KOH water solution to make the pH of the single-phase system
115
increase, and to make citric acid change from non-ionized form to ionized form. The
116
salting out ability of citric acid enhanced, and then the ATPS formed to purification of
117
psoralen.
118
2. Materials and Methods
119
2.1. Materials and chemicals
120
Fig leaves were collected from WeiHai, China. The fresh leaves were cleaned and
121
dried to constant weight, pulverized by a disintegrator. The pulverized material was
122
sieved (40 mesh), and stored in darkness at room temperature prior to use.
123
Psoralic acid-glucoside (>98 wt%) and psoralen (>98 wt%) were purchased from
124
Yuanye Chemical Reagent Co. (Shanghai, China). [Emim]Br (>97%), [Bmim]Br
125
(>97 wt%), [Hmim]Br (>98 wt%), [Omim]Br (>98 wt%), [Bmim]BF4 (>98 wt%),
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[Bmim]Cl (99 wt%), [Bmim]HSO4 (>99 wt%) and [Bmim]NO3 (>99 wt%) were
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purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Analytical grade
128
ethanol, citric acid (100%) and potassium hydroxide (100%) were purchased from
129
Tianjin Kemel Reagents Co. (Tianjin, China). Acetonitrile of chromatographic grade
130
was purchased from J & K Chemical Ltd. (China) and 0.45 μm microporous
131
membranes were purchased from Xingya. Shanghai. (China).
132
2.2. Transformation psoralic acid-glucoside into psoralen and extraction of psoralen
133
from fig leaves
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50mg fig leaf powder was added into 1ml 2M [Bmim]Br-citric acid (for qualitative
135
analysis of psoralic acid-glucoside and psoralen, the extraction solvent was ethanol)
136
and put in an ultrasonic bath (KQ-250DB, Kunshan Company of Acoustics, China).
137
Based on our pre-experiments, the conditions for extraction were temperature 60℃,
138
time 30min and ultrasonic power 450W. After transformation and extraction, the
139
extracts were filtered using 0.45um microfiltration and analyzed by HPLC.
140
2.3. Analytical methods
141
2.3.1. Identification of psoralic acid-glucoside and psoralen by LC–DAD–MS/MS.
142
An Agilent 1100 series HPLC (Agilent, San Jose, California, USA) with a diode
143
array detector (DAD) system (Shimadzu, Kyoto, Japan), an API 3000 triple tandem
144
quadrupole MS (Applied Biosystems, Concord, Ontario, Canada) and a Luna C18
145
reversed-phase column (250 × 4.6 mm i.d., 5μm, Phenomenex, Guangzhou) was used
146
for the identification of psoralic acid-glucoside and psoralen. A gradient elution
147
process was used for separation and the details were as follows: 0.1% formic acid
148
aqueous solution (A) and acetonitrile (B) were mobile phase, 0–10 min, 13–16% B;
149
10–11 min, 16–17% B; 11–25 min, 17% B; 25–55 min, 17–65% B. The flow rate and
150
injection volume were 1.0 mL/min and 5μL, respectively. The column temperature
151
was set at 30 ℃. Mass spectra of analyses were performed in the multiple reaction
152
monitoring transitions (MRM) with an electrospray ionization source in the positive
153
ion mode. The optimized conditions were as follows: nebulizing gas (NEB) 8 psi,
154
curtain gas (CUR) 11 psi, collision gas (CAD) 6 psi, ion source temperature 350 ℃,
155
ion spray voltage (IS) 4500 V, focusing potential (FP) 60V and entrance potential
156
(EP) 13 V, declustering potential (DP) 6 V, collision energy (CE) 30 V, and collision
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cell exit potential (CXP) 22 V, MRM (amu): 367.2→115.0 for psoralic acid-glucoside
158
and 187.1-115.0 for psoralen. As shown in Fig 2, A was the MS spectrum of peak 1
159
and B was the MS spectrum of peak 2. By comparing the MS spectrum of A and B
160
with the MS spectrum of psoralic acid-glucoside and psoralen standards and with
161
reference (Ammar et al., 2015; Qiao et al., 2006; Takahashi et al., 2014), peak 1 was
162
identified as psoralic acid-glucoside and peak 2 was identified as psoralen.
163
2.3.2. Quantitative determination of psoralic acid-glucoside and psoralen by HPLC-
164
UV.
165
During the LC-DAD-MS/MS analysis, we carried out full wavelength scanning of
166
psoralic acid-glucoside and psoralen peaks (200-400 nm), comparing the absorption
167
spectra with the standards. The results showed that the spectra were the same as those
168
of the standards and the peaks 1 and 2 have no interference with the absorption of the
169
coexisting peaks, which indicated that the HPLC-UV had very good specificity and
170
could be adopted for quantitative analysis of psoralic acid-glucoside and psoralen
171
Therefore, in the following experiments, we used the HPLC-UV method to
172
quantitatively analyze psoralic acid-glucoside and psoralen in samples. The
173
quantitative analysis method was developed on an Agilent 1200 series liquid
174
chromatography system (Agilent, San Jose, CA) equipped with a G1311A quaternary
175
pump, a G1322A degasser, a 1365B MWD UV detector, and a G1328B manual
176
injector. A Luna C18 reversed-phase column (250 × 4.6 mm i.d., 5μm, Phenomenex,
177
Guangzhou) protected with a guard column. A gradient elution process was used for
178
separation and the details were as follows: 0.1% formic acid aqueous solution (A) and
179
acetonitrile (B) were mobile phase, 0–10 min, 13–16% B; 10–11 min, 16–17% B; 11–
180
25 min, 17% B; 25–55 min, 17–65% B. The flow rate and injection volume were 1.0
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mL/min and 5μL, respectively. The detection wavelength was set at 254 nm.
182
Chromatographic peaks of psoralic acid-glucoside and psoralen were confirmed by
183
comparing retention times with reference standards. The quantification of psoralic
184
acid-glucoside and psoralen were used calibration curves potted by using reference
185
standards. The calibration curves, limits of detection (LOD) and quantification (LOQ)
186
of psoralic acid-glucoside and psoralen were determined as reported previously (Long
187
et al., 2015). These results were shown in Table 1.
188
2.4. Optimization of the operation conditions by response surface methodology (RSM)
189
Box-Behnken design (BBD) was used to optimize the process. Three independent
190
variables were investigated: pH value (1-3), ionic liquid concentration (1.5M-2.5M)
191
liquid solid ratio (10ml/mg-20ml/mg) at three levels (−1, 0, +1). The arrangement 17
192
experimental points were listed in Table 2. Design-Expert Ver. 8.0.6.1 (Stat-Ease,
193
Minneapolis, MN, USA) was used to analyze the results.
194
2.5. Investigation and optimization of the pH-dependent IL-acid based ATPS process.
195
After transformation and extraction, the [Bmim]Br-citric acid based extracts were
196
filtered and the pH value was adjusted by adding appropriate amount of 50% wt
197
aqueous potassium hydroxide solution with aid of a pH meter (PB-21, Sartorius Ag,
198
Germany). When the aqueous two-phase system formed, the volume of the top
199
[Bmim]Br rich phase and the bottom salt rich phase were recorded respectively. The
200
content of psoralen in the two phase was measured by HPLC. To investigate the
201
phase-forming behavior and determine optimal operation conditions, a series of
202
ATPSs with different [Bmim]Br and citric acid contents under different pH value
203
have been used to purify psoralen. The detail composition of the investigated ATPSs
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was shown in table 3.
205
2.6. Statistical analysis
206
All the experiments were performed in triplicates. Results were expressed as means
207
with standard deviations (SD). GraphPad Prism 7.0 (GraphPad Software, San Diego,
208
CA, USA) was used for statistical analysis. The data were compared using one-way
209
analysis of variance (ANOVA) followed by post-hoc Tukey's test t on the level of
210
significance set at p < 0.05.
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3. Results and discussion
212
3.1. Influence of ILs on transformation and extraction
213
The structure of ionic liquid had a great influence on the extraction efficiency of
214
target compounds (Lu et al., 2008). In order to investigate the effect of ionic liquid
215
structure on the extraction efficiency of psoralen, a series of imidazolium based ILs
216
with cations connected with different alkyl side chains combined with different anions
217
were investigated. As shown in Fig 3A, the extraction efficiency for psoralen was
218
[Emim]Br<[Bmim]Br>[Hmim]Br>[Omim]Br. These results indicated that with the
219
increase of side alkyl chain length in imidazolium cation the hydrophobicity increased
220
and hydrophilicity decreased. When the alkyl side chain increased from ethyl to butyl,
221
the increased hydrophobicity enhanced the interaction between IL and psoralen and
222
extraction efficiency increased. With further increased the length of the alkyl side
223
chain to octyl, the steric of IL with psoralen significantly blocked the interaction
224
between them. Thus, the extraction efficiency decreased (Du et al., 2009). The
225
extraction efficiency for psoralen was [Bmim]Br> [Bmim]BF4> [Bmim]HSO4>
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[Bmim]Cl, and the extraction efficiency varied more significantly with the change of
227
anions than that of alkyl chain length of imidazolium cation. These results indicated
228
that for extraction of psoralen from fig leaves using imidazolium based ILs was anion
229
dependent and [Bmim]Br (p < 0.05) was more efficient than others. The reason was
230
possibly that compared with [Bmim]BF4, [Bmim]Cl and [Bmim]HSO4, the interaction
231
between [Bmim]Br and psoralen were enhanced due to its polarity. So [Bmim]Br was
232
selected and used for extraction of psoralen from fig leaves. These results are the
233
same as those of previous studies (Cláudio et al., 2013; Svinyarov et al., 2012).
234
However, no study investigated the extraction mechanism for psoralen from fig
235
leaves.
236
3.2. Influence of pH on transformation and extraction
237
Study has shown that IL-acid mixtures have a stronger catalytic activity than that of
238
simple ionic liquids or acids under milder conditions (Siewping et al., 2014). To
239
confirm our hypothesis that IL-acid mixture could enhance transformation efficiency
240
for psoralic acid-glucoside into psoralen and extraction efficiency for psoralen
241
simultaneously, [Bmim]Br-citric acid mixture was investigated and [Bmim]Br,
242
ethanol and ethanol-acid were selected as reference. As shown in Fig 3B, for
243
[Bmim]Br-citric acid mixture (pH=2), the extraction yield of psoralic acid-glucoside
244
decreased from 9.76mg/g (p < 0.05) to 0.098mg/g (p < 0.01) and that of psoralen
245
increased from 20.89mg/g (p < 0.01) to 30.21mg/g (p < 0.01). The result indicated
246
that there was nearly no psoralic acid-glucoside existed which was thoroughly
247
converted to psoralen, leading to the highest extraction yield of psoralen of 30.21mg/g
248
(p < 0.01) in [Bmim]Br-citric acid mixture (pH=2). For ethanol and ethanol-citric acid
249
(pH=2), the extraction yield of psoralic acid-glucoside decreased from 15.32mg/g (p <
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250
0.05) to 10.11mg/g (p < 0.01) and that of psoralen increased from 8.21mg/g (p < 0.01)
251
to 12.34mg/g (p < 0.05). These results indicated that the addition of citric acid to
252
ethanol promoted the transformation of psoralic acid-glucoside into psoralen,
253
resulting in a decrease in the content of psoralic acid-glucoside and an increase in the
254
content of psoralen. However, in ethanol or ethanol-citric acid mixture, there were
255
still a large number of psoralic acid-glucoside in the extracts, and the extraction
256
efficiency of psoralen was obviously lower than that in [Bmim]Br-citric acid mixture
257
(pH=2). So the addition of citric acid to [Bmim]Br and ethanol could enhance
258
transformation efficiency of psoralic acid-glucoside into psoralen and the extraction
259
efficiency of psoralen simultaneously. The combination of [Bmim]Br with citric acid
260
(pH=2) had much higher extraction and transformation efficiency than that of ethanol-
261
citric acid (pH=2). The variation tendency was intuitively revealed in Fig 4A, B, C, D,
262
in which peak 1 was psoralic acid-glucoside and peak 2 was psoralen. The similar
263
phenomenon has been observed in previously reported research using IL-acid mixture
264
for transformation fructose into 5-Hydroxymethylfurfural (HMF) (Siewping et al.,
265
2014). These results indicated that [Bmim]Br-citric acid mixture is an efficient system
266
and has been selected for simultaneous transformation psoralic acid-glucoside into
267
psoralen and extraction of psoralen from fig leaves. To the best of our knowledge, the
268
use of IL-acid mixture for simultaneous extraction and transformation natural
269
products hasn’t been reported yet.
270
3.2. Optimization of operational conditions of transformation and extraction process
271
Based on our pre-experiments, pH value, ionic liquid concentration and liquid/solid
272
ratio had significant influence on transformation efficiency of psoralic acid-glucoside
273
into psoralen and extraction efficiency of psoralen from fig leaves simultaneously. So
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we investigated these factors based on single factor experiments and then optimized
275
these factors using response surface methodology (RSM).
276
3.2.1. Optimization of transformation and extraction process by single factor
277
experiments
278
PH
of
[Bmim]Br-citric
acid
mixture
could
significantly
influence
the
279
transformation and extraction process. To investigate the influence of pH, the
280
transformation and extraction using [Bmim]Br-citric acid mixture were performed
281
under different pH value (1, 2, 3, 4, 5). As shown in Fig 5A, when the pH value
282
decreased from 5 to 2, transformation and extraction efficiency gradually increased.
283
With further decrease of the pH value to 1, transformation and extraction efficiency
284
decreased. The reason for this phenomenon was probably that the increase of acid
285
intensity increased the catalytic activity of the [Bmim]Br-citric acid mixture,
286
increased the damage to the cell wall of fig leaves and promoted the mass transfer
287
process, thus the extraction efficiency .increased. However, too strong acid could
288
increase the instability and the decomposition of psoralen which caused the extraction
289
yield decrease. Thus, [Bmim]Br-citric acid mixture (pH=2) was most efficient (p <
290
0.01) and selected for simultaneous transformation psoralic acid-glucoside into
291
psoralen and extraction of psoralen from fig leaves.
292
The concentration of ionic liquid could significantly affect the conversion and
293
extraction efficiency. In order to investigate the effect of [Bmim]Br concentration on
294
the conversion and extraction efficiency, [Bmim]Br-citric acid mixtures with different
295
[Bmim]Br concentrations (0.5M, 1M, 1.5M, 2.0M, 2.5M, 3M) were used in the
296
conversion process and extraction. As shown in Fig 5B, when [Bmim]Br
297
concentration increased from 0.5M to 2M, the extraction yield of psoralen increased
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significantly, with further increase of [Bmim]Br concentration, the extraction yield of
299
psoralen decreased. This was because with the increase of [Bmim]Br concentration,
300
the absorption capacity of the [Bmim]Br-citric acid mixture to ultrasound was
301
enhanced, the hydrophobicity was increased, the solubility of the [Bmim]Br-citric
302
acid mixture to psoralen was enhanced, and the mass transfer process was promoted,
303
and the extraction efficiency was increased. With further increased the [Bmim]Br
304
concentration to 3M (p < 0.05) , the viscosity was too high which hindered the mass
305
transfer process, thus the extraction efficiency decreased (Cao et al., 2009). Similar
306
phenomena have been observed in previous studies (Ma et al., 2011a; Ma et al.,
307
2011b).
308
Liquid/solid ratio has great influence on extraction and conversion efficiency.. To
309
investigate the influence of liquid/solid ratio, the transformation and extraction using
310
[Bmim]Br-citric acid mixture were performed under different liquid/solid ratio (5, 10,
311
15, 20, 25, 30). As shown in Fig 5C, when the liquid/solid ratio increased from 5ml/g
312
to 15ml/g, the extraction yield increased obviously. With further increase of
313
liquid/solid ratio, the extraction yield remained unchanged. This was because that
314
when liquid/solid ratio was lower than 15ml/g, although the mass transfer was
315
equilibrium between fig leaves materials and [Bmim]Br-citric acid mixture, there was
316
still plenty of psoralen residue in fig leaves materials. When the liquid/solid ratio
317
reached up to 15ml/g, the residual psoralen in fig leaves materials was negligible after
318
mass transfer process was equilibrium, since the increased liquid volume caused
319
dissolve capacity increase and could thoroughly extract psoralen from fig leaves. So
320
with further increase of the liquid/solid ratio, the extraction yield no longer obviously
321
increased and a liquid/solid ratio 15ml/g (p < 0.05) was selected.
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3.2.2. Optimization of operation conditions of transformation and extraction process
323
by response surface methodology (RSM)
324
Box-Behnken design is a powerful method which has been widely used to optimize
325
the complicated extraction process. Box-Behnken design was implemented using
326
Design-Expert 8.0.6.1 software (Stat-Ease, Minneapolis, MN, USA). The arrangement
327
and results were shown in Table 2. After analysis, a quadratic polynomial equation
328
derived, which was a description of the relationship between psoralen yield and
329
extraction variables:
330
Y=-54.64+10.84X1+48.92X2+3.146X3+0.8650X1X2+0.0450X1X3-0.2600X2X3-2.997
331
X12-11.63X22 -0.0886X32
(4)
332
333
334
Where Y was the extraction yield of psoralen (mg/g), X1 was the pH, X2 was the
ionic liquid concentration (M) and X3 was the liquid/solid ratio (ml/g).
335
The analysis of variance (ANOVA) results were shown in table 4, the Model F-
336
value was 41.92 and “Prob > F” less than 0.0001 indicated the model was significant
337
and the “Lack of Fit F-value” of 1.45 indicated the “Lack of Fit” was not significant,
338
these results indicated the model was successfully fitted. X1, X2, X2X3 X12, X22, X32
339
(p<0.05) were significant model terms which had significant influence on extraction
340
yield of psoralen. The influence of independent variables on extraction yield was
341
clearly shown in Fig 6. As shown in Fig 6A, when the pH was constant, with the ionic
342
liquid concentration increased from 1 to 2, psoralen extraction yield also increased,
343
with further increase of the ionic liquid concentration, the extraction yield decreased
344
significantly, this may be because the ionic liquid concentration was too high which
15
ACCEPTED MANUSCRIPT
345
caused the solvent viscosity increase and the mass transfer rate decrease. In addition,
346
when the ionic liquid concentration was constant, with PH increased from 1 to 2,
347
psoralen extraction yield also increased, with further increase of PH, the extraction
348
yield decreased significantly, this may be because the acid strength affected the
349
conversion yield of psoralic acid-glucoside, but when the acidity was too strong, the
350
degradation of the target compounds enhanced. Similar interaction between
351
liquid/solid ratio and PH (Fig. 6B) and that between liquid/solid ratio and ionic liquid
352
concentration (Fig. 6C) on the extraction yield of psoralen could be easily observed.
353
The optimal extraction conditions provided by the model were pH 2.21, ionic liquid
354
concentration 2.01M and liquid/solid ratio 15.35 ml/g and optimal extraction yield
355
was 30.80 mg/g (p < 0.05). To verify the prediction, the extraction was performed in
356
predicated conditions for three times and the average extraction yield was 31.22 mg/g
357
(RSD<4). These results indicated that RSM could successfully optimize the
358
complicated extraction and transformation process of psoralen from fig leaves.
359
3.3. Investigation and optimization of the purification process
360
3.3.1. Phase forming behavior
361
In order to verify whether [Bmim]Br-citric acid mixture can be converted from
362
single phase into ATPS by regulating pH, KOH solution (50% wt) and water were
363
added to [Bmim]Br-citric acid mixture with aid of a pH meter (PB-21, Sartorius Ag,
364
Germany). The pH value of [Bmim]Br-citric acid mixture was controlled and the
365
composition of [Bmim]Br-citric acid mixture changed constantly. When the ATPS
366
formed, the composition of [Bmim]Br-citric acid mixture were recorded. In the
367
process, we found that when the pH value was lower than 5 the ATPS couldn’t form
368
under any composition of [Bmim]Br-citric acid mixture. When the pH value was 5,
16
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369
ATPS started forming under specific composition of [Bmim]Br-citric acid mixture.
370
With further increase of the pH value, the ATPS more easily formed. Studies have
371
shown that salt which has a higher charge density and anionic valence has stronger
372
hydration
373
form of citric acid converted into C6H7O7-, C6H6O72-, C6H5O73- and salting-out ability
374
increased. Citric acid had 4 pKa values which were 3.05, 4.67, 5.39 and 13.92,
375
respectively. When the pH value was lower than 3.05, C6H8O7 mainly existed in
376
[Bmim]Br-citric acid mixture. The [Bmim]Br-citric acid mixture had no salting-out
377
ability and existed in a single phase system. When the pH was 3.05-4.67, C6H8O7 and
378
C6H7O7- mainly existed, the salting-out ability was too weak to form ATPS. When the
379
pH value was 4.67-5.39, C6H6O72- and C6H5O73- mainly existed and the salting-out
380
ability increased, ATPS started forming. When the pH value was higher than 5.39,
381
C6H5O73- mainly existed, the salting-out ability was the strongest and ATPS more
382
easily formed. These results indicated that [Bmim]Br-citric acid mixture could be
383
converted from single phase into ATPS by regulating pH>5 under certain composition
384
of [Bmim]Br-citric acid mixture.
385
3.3.2. Optimization of ATPS composition for enhanced recovery of psoralen
and salting-out ability [27]. When the pH value increased from 2 to 8, the
386
The composition and pH value had significant influence on the partition of psoralen
387
between the [Bmim]Br rich top phase and salt rich bottom phase. To obtain the
388
highest recovery yield of psoralen, based on the phase forming behavior, a series of
389
[Bmim]Br-citric acid mixtures with different composition under different pH values
390
(table 3) were investigated (temperature 30℃). Under each pH value, the
391
compositions we selected had a tendency of varying from high content of [Bmim]Br
392
(%wt) and low content of citric acid (%wt) to high content of citric acid (%wt) and
17
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393
low content of [Bmim]Br (%wt). So it could adequately reflect the influence of
394
[Bmim]Br-citric acid mixture’s composition on recovery yield of psoralen. As shown
395
in Fig 7A, when the pH value was 5, with the content of citric acid increased from
396
8.05 (wt%) to 12.88 (wt%) and content of [Bmim]Br decreased from 56.59 (wt%) to
397
48.83 (wt%), the recovery yield of psoralen increased from 60.42% to 75.32%. This
398
was because when the pH value was 5, the salting-out ability was weak. [Bmim]Br
399
rich top phase had high polarity due to the presence of plenty of water which was
400
unfavorable for the extraction of psoralen. With the increase of citric acid content, the
401
citrate content in the system increased. More water entered the bottom salt enrichment
402
phase, and the water content in the top [Bmim]Br enrichment phase decreased which
403
made hydrophobic psoralen easier enter the top phase. So the increase of citric acid
404
content could enhance the recovery yield. As shown Fig 7B, C, D, in which the pH
405
value was 6, 7, 8 respectively, the phase-forming area was more broader. So the
406
[Bmim]Br content from 14 (wt%) to 64 (wt%) and citric acid content from 5 (wt%) to
407
37 (wt%) were investigated. The similar phenomenon that with increase of citric acid
408
content the recovery yield was firstly increased and then decreased were observed.
409
This was because with the increase of citric acid content, the citrate content in the
410
system increased. More water entered the bottom salt enrichment phase, and the water
411
content in the top [Bmim]Br enrichment phase decreased which made hydrophobic
412
psoralen easier enter the top phase. However, if the content of citrate in the system
413
was too high, the excess water entered into the bottom phase, resulting in the decrease
414
of the volume of the top phase and the increase of viscosity, so that the distribution of
415
psoralen in the top phase reduced, and the recovery yield decreased. There was a little
416
difference in the extraction yield among these pH values (6, 7, 8). This was because
417
under these pH values, citric acid was in the form of C6H5O73- that had the strongest
18
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418
salting-out ability. The highest recovery yield of psoralen was 96.32% (p < 0.05) at
419
[Bmim]Br content (37.04% wt) and citric acid content (30.21% wt) at pH value 7. The
420
highest recovery yield of psoralen was 94.21% (p < 0.05) at [Bmim]Br content
421
(38.49% wt) and citric acid content (28.01% wt) at pH value 6. The highest recovery
422
yield of psoralen was 96.04% (p < 0.01) at [Bmim]Br content (38.64% wt) and citric
423
acid content (30.32% wt) at pH value 8. So [Bmim]Br content (37.04% wt) and citric
424
acid content (30.21% wt) at pH value 7 was selected.
425
Conclusion
426
In the present study, we reported a new method of simultaneous extraction,
427
transformation and purification of natural products from natural raw materials for the
428
first time which was based on the pH-dependent phase-forming property of IL-organic
429
acid mixture. The new method was called pH-dependent ionic liquid solvent based
430
aqueous two two-phase system. In the process, The extraction yield of psoralen by the
431
cleaner [Bmim]Br-citric acid mixture were 1.45, 2.45 and 3.68 times higher than that
432
of psoralen by [Bmim]Br-water, ethanol-critic acid and ethanol, respectively, which
433
indicated that the cleaner [Bmim]Br–citric acid mixture can enhance efficiency for
434
transformation and extraction of psoralen from fig leaves. The ATPS could form
435
when pH>5 and realize the purification of crude extracts just changing pH. Under the
436
optimal conditions of ATPS, the recovery yield of psoralen was 96.32% which
437
indicated that the pH-dependent ATPS could efficiently purify psoralen from crude
438
extracts. After simultaneous extraction, transformation and purification by the new
439
IL-acid based pH-dependent ATPS method, the extraction yield of psoralen was
440
31.22mg/g with a recovery yield of 96.32%. This approach has a few advantages
441
compared with traditional methods: no harmful organic solvent used, quick, low
19
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442
energy consumption, easy continuous operation and easy scale-up. These advantages
443
indicated the approach has great practicability to replace traditional methods for
444
production of value-added compounds from natural raw materials. However, in terms
445
of large-scale practical applications, there is an important problem, that is, the IL used
446
in present study is relatively expensive. There are two ways to solve this problem. The
447
first is to design and synthesize more benign and cheaper new ILs, the second is to
448
develop efficient methods to recover ILs so that they can be reused. In future work,
449
more benign and safer ionic liquid should be designed and used in the IL-acid based
450
pH-dependent aqueous two-phase system processes for the extraction, transformation
451
and purification of value-added compounds from natural resources. At the same time,
452
various efficient methods for reusing ILs should also be developed.
453
Acknowledgements
454
The authors gratefully acknowledge the financial supports by National Key
455
Research Development Program of China (2016YFD0600805), Fundamental
456
Research Funds for the Central Universities (2572015EA04), Application Technology
457
Research and Development Program of Harbin (2013AA3BS014), and Special Fund
458
of National Natural Science Foundation of China (31270618).
459
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461
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463
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466
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469
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Figure Captions
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Figure. 1. The integrated and sustainable pH-dependent IL-acid based ATPS process for
575
simultaneously transformation psoralic acid-glucoside to psoralen, extraction and
576
purification of psoralen from fig leaves.
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577
Figure. 2. The chromatograms of fig leaves extracts couple by HPLC–DAD-MS/MS in positive
578
ion mode for psoralic acid-glucoside and psoralen: Peak 1: psoralic acid-glucoside,
579
Peak 2: psoralen; MS spectrum of psoralic acid-glucoside (A), MS spectrum psoralen
580
(B).
581
Figure. 3. The effect of ILs on extraction of psoralen from fig leaves (A), the effect of acid on
582
transformation psoralic acid-glucoside into psoralen and extraction psoralen from fig
583
leaves (B), Error bars indicate the SD (n = 3).
584
585
586
587
Figure. 4. The HPLC chromatogram of [Bmim]Br (A), [Bmim]Br+citric acid (B), Ethanol (C)
and Ethanol+citric acid (D) extracts.
Figure. 5. The effect of pH value (A), ionic liquid concentration (B) and liquid/solid ratio (C) on
extraction yields of psoralen from fig leaves.
23
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588
Figure. 6. Response surfaces representations for psoralen: correlative effects of pH value and
589
ionic liquid concentration (A); correlative effects of pH value and liquid/solid ratio
590
(B); correlative effects of ionic liquid concentration and liquid/solid ratio (C).
591
592
Figure. 7. The effect of the pH-dependent IL-acid based ATPS composition on recovery of
psoralen under different pH: pH=5 (A), pH=6 (B), pH=7 (C) and pH=8(D).
24
Fig.1
Fig.2
Fig.3
Fig.4
ACCEPTED MANUSCRIPT
Fig.5
ACCEPTED MANUSCRIPT
Fig.6
Fig.7
Table 1
Calibration curves, LODs and LOQs for psoralic acid-glucoside (PAG), psoralen (PSL).
Analyte
Calibration curve
R2 (n = 8)
Linearity range
(ug/ml)
LOD (ug/mL)
LOQ (ug/mL)
PAG
PSL
Y=68.59X+1.33
Y=81.25X+1.87
0.9997
0.9999
5-100
5-100
0.25
0.21
0.71
0.62
Table 2
Box-Behnken design (BBD) arrangement and results.
Ru
ns
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
a
Factors
X1 (Pa) X2 (ILCb, M) X3 (L/Sc, mL/g)
2
3
2
2
1
1
1
2
2
3
1
2
2
2
3
3
2
2
2.5
2.5
2
2
2.5
2
2
1.5
1.5
1.5
2.5
1.5
2
2
2
2
15
15
10
15
20
15
10
15
10
15
15
20
20
15
10
20
15
pH ; bIonic liquid concentration; c Liquid/solid ratio (mL/g).
Yield
Psoralen
(mg/g)
30.11
26.04
26.39
30.19
24.11
23.15
23.76
31.27
24.31
25.48
24.32
25.45
25.97
30.61
26.32
27.57
31.08
of
Table 3
APTS compositions with different [Bmim]Br-citric acid under different pH value.
pH value
5
6
7
8
components
Composition
1
Composition
2
Composition
3
Composition
4
Composition
5
W1
12.88
10.15
9.38
8.55
8.05
W2
48.83
53.34
54.48
55.78
56.59
W1
36.43
25.22
17.01
10.05
5.74
W2
16.63
28.63
38.49
48.67
58.01
W1
33.17
16.74
2.83
35.01
9.54
W2
15.87
37.04
63.65
14.05
48.99
W1
32.5
22.89
6.37
22.89
4.22
W2
14.06
25.25
53.64
25.25
60.78
W1: Citric acid and its potassium salts (wt%)
W2: [Bmim]Br (wt%)
Table 4
ANOVA analysis results of the quadratic model.
Variables
Model
X1a
X2b
X3c
X1X2
X1X3
X2X3
X12
X22
X32
Lack of fit
R2
Adj-R2
a
Psoralen
F-value
41.92
39.53
24.35
2.10
2.33
0.63
5.27
117.96
110.98
64.41
1.45
0.9818
0.9584
pH ; bIonic liquid concentration; c Liquid/solid ratio (mL/g).
p-value
<0.0001
0.0004
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jclepro, 2017, 185
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