Talanta 80 (2010) 12921297

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Application of ionic liquids based microwave-assisted extraction of three

alkaloids N-nornuciferine, O-nornuciferine, and nuciferine from lotus leaf
Wenyan Ma a , Yanbin Lu b , Ruilin Hu a , Jihang Chen a , Zizhang Zhang a , Yuanjiang Pan a,
a
Department of Chemistry, Zhejiang University, Hangzhou 310027, China
b
College of Food Science and Biological Engineering, Zhejiang Gongshang University, Hangzhou 310035, China

a r t i c l e i n f o a b s t r a c t

Article history: The application of ionic liquids based microwave-assisted extraction (ILMAE) was successfully devel-
Received 25 June 2009 oped for extracting three alkaloids N-nornuciferine, O-nornuciferine, and nuciferine from lotus leaf.
Received in revised form 9 September 2009 Seven kinds of 1-alkyl-3-methylimidazolium with different cations and anions were investigated in this
Accepted 13 September 2009
work and 1.0 M 1-hexyl-3-methylimidazolium bromide ([C6 MIM]Br) solution was selected as solvent. In
Available online 19 September 2009
addition, the microwave parameters including irradiation power, extraction time and solidliquid ratio
were optimized. Compared with the regular MAE and conventional heat-reux extraction (HRE), the
Keywords:
proposed approach exhibited higher efciency (0.943.7% enhanced) and shorter extraction time (from
Ionic liquids
Microwave-assisted extraction
2 h to 2 min), which indicated ILMAE was an efcient, rapid and simple sample preparation technique.
Alkaloids Moreover, the proposed method was validated by the linearity, reproducibility, and recovery experi-
Lotus leaf ments. Good linearity was observed with the regression coefcients (r2 ) between 0.9998 and 0.9999.
The recoveries of all methods were in the range of 94.6% and 105.5% with RSD lower than 6.6%, which
indicated that the proposed method was credible.
2009 Elsevier B.V. All rights reserved.

1. Introduction
Medicinal plants have served as an important source of drugs
for treating diseases since ancient times. In modern pharma-
ceutical industries, despite the remarkable progress in synthetic
organic chemistry of the twentieth century, over 25% of prescribed
medicines in industrialized countries derive directly or indirectly
from plants. This percentage can reach 50% when the over-the-
counter (OTC) market is taken into consideration [1,2]. In recent
years, the validated analytical methods (HPLC, CE, CEC, LC/MSn ,
LC/NMR, LC/SPE/NMR, etc.) [36] are developing rapidly to guar-
antee the safety and efcacy of medicinal products. However, the
importance of sample preparation has always been ignored. The
metabolites extraction from solid plant samples is usually achieved
by using well-established methods such as heat-reux extraction
(HRE) and Soxhlet extraction, which have some drawbacks includ-
ing time-consuming, laborious, and using of a large amount of
toxic and hazardous organic solvents. To overcome the above-
mentioned problems, microwave-assisted extraction (MAE) has
attracted broad interest as an alternative extraction technique with
unique advantages of rapidness, high efciency and low cost [7,8].
Corresponding author. Tel.: +86 571 87951629; fax: +86 571 87951629.
E-mail address: panyuanjiang@zju.edu.cn (Y. Pan).
Ionic liquids (ILs), also known as molten salts, are a new class
of nonmolecular ionic solvents with a melting point xed at or
below 100 C. The potential of ILs in both academic and indus-
trial elds is related to their unique properties of negligible vapor
pressure, high thermal stability, low or virtually no volatility and
ease of handling. ILs are therefore regarded as an attractive alter-
native or replacement to conventional volatile organic solvents
(VOS) [912]. Over the past few years, ILs have attracted exten-
sive attention in modern chemistry. The initial research interests
are focused on organic synthesis [13], catalysis [14], and the pro-
cess of green chemistry [15]. Recently, application of ILs has also
extended into areas of analytical chemistry including electrochem-
istry, separation science, mass spectrometry, and spectroscopy.
Due to the distinct features compared to conventional volatile
organic solvents, sample pretreatment techniques using ILs have
been successfully applied in liquidliquid extraction, liquid-phase
microextraction, solid-phase microextraction and aqueous two-
phase systems extraction [1620]. It has been found that ILs as
solvents and co-solvents can efciently absorb microwave energy
[21]. Therefore, ILs as solvent is of promising potential in the
application of the preparation of various useful substances from
solid samples such as trans-resveratrol from Rhizma Polygoni
Cuspidati [22], three phenolic alkaloids liensinine, isoliensinine,
and neferine from traditional Chinese herbal medicine Lien Tze
Hsin [23], and polyphenolic compounds from medicinal plants
[24].

0039-9140/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2009.09.027
 W. Ma et al. / Talanta 80 (2010) 12921297
Lotus leaf is the leaves of Nelumbo nucifera Gaertn. It is a
traditional Chinese medicine for losing weight and has been
commonly used for clearing heat, removing heatstroke, cooling
blood, and stanching blood [25]. The major phytochemicals present
in lotus leaf are three aporphine alkaloids, N-nornuciferine, O-
nornuciferine, and nuciferine (Fig. 1). Total alkaloids have been
found with biological functions of lowering hyperlipemia [26] and
level of cholesterol [27], resisting karyokinesis as well as antimi-
crobial activity [28]. Recently, nuciferine is newly reported to show
anti-HIV activity [29]. Presently, the alkaloids from this medicinal
plant are mainly extracted by HRE, which is time-consuming and
laborious [3032]. The aim of this work was to develop a rapid,
effective, validated and environmental friendly ionic liquids based
microwave-assisted approach for extraction of three alkaloids from
this herbal medicine, and compare the results with the conven-
tional reference methods. Herein we systematically investigated
the performance of various ILs with different cations and anions as
well as the microwave parameters which demonstrated that the
proposed ILMAE approach has great potential for the ease of qual-
ity assessment of medicinal plants by providing the rapid and facile
alkaloids extraction.
2. Experimental
2.1. Chemicals
All chemical solvents in this study were at least of analytical
reagent grade, while reverse osmosis Milli-Q water (18 M, Milli-
pore, Bedford, MA, USA) was used throughout. Acetonitrile used for
HPLC analysis was of chromatographic grade and purchased from
Merck, Darmstadt, Germany.
Three alkaloids N-nornuciferine, O-nornuciferine, and nucifer-
ine used for HPLC-UV determinations were isolated from lotus
leaf and were conrmed by comparing their 1 H NMR, 13 C NMR,
MS and MS2 with those given in the references [3335]. All ionic
liquids were gained from Chemer, Hangzhou, China, and used as
received.
Lotus leaf was purchased from a local drug store, Hangzhou,
China. All samples were dried, milled, passed through a stainless
steel sieve and stored in closed desiccators at 4 C until use. The
same batch of sample was used through this study to be represen-
tative of variable hardness and density.
Fig. 1. HPLC chromatogram of three alkaloids in the [C6 MIM]Br extract. Inset: HPLC chromatogram of mixture of three standard alkaloids.
1293
2.2. Apparatus
A domestic microwave-assisted extraction unit (Glanz, Shunde,
China) with a 2450 MHz magnetron was used. It was modied in
our laboratory with the addition of a water condenser. The whole
system was run at atmospheric pressure and could be employed at
the maximum power of 700 W.
An Agilent 1100 liquid chromatography equipped with a
G1311A QuatPump, a G1322 Degasser, a G1314A variable wave-
length detector (VWD), a model 7725i injection valve with a 20 L
loop was used for HPLC analysis, and an Agilent ChemStation for
data treatment. An Agilent Zorbax Extend C18 (150 mm 4.6 mm
i.d., 5 m, 120 ) was used as the LC analytical column. Prepara-
tive HPLC was performed using a Varian Prepstar pump equipped
with a Varian 210 UV detector (Varian Co., USA). A reversed-phase
Shim-pack PRC ODS column (250 mm 20 mm, 10 m) was used
for preparation. MS and MS2 gure were obtained from a Bruker
Esquire 3000 mass spectrometer. 1 H and 13 C NMR spectra were
performed in a Bruker Advance 500 NMR spectrometer.
2.3. Ionic liquids based microwave-assisted extraction
Ionic liquid-based microwave-assisted extraction was per-
formed in microwave unit. 0.5 g of dried sample was mixed with
10 mL of different ILs solutions and then the suspension was
extracted by MAE. The optimum anion, cation, concentration of
selected IL, microwave power, irradiation time, and solidliquid
ratio were systematically studied in this work. After irradiation,
the extracts obtained were cooled down to the room temperature,
then diluted to 50 mL with deionized water, and ltrated through
a 0.45 m lter prior to HPLC analysis.
2.4. Conventional reference extraction method
Two HRE methods including acid extraction and organic sol-
vent extraction were selected following the reference methods for
extraction of the three alkaloids from lotus leaf [3032].
The process of acid extraction: 0.5 g sample was mixed with
15 mL hydrochloric acid aqueous solution (pH 2) in a round-bottom
ask (100 mL) and the suspensions were boiled at 100 C for 2 h in
an open system.
1294 W. Ma et al. / Talanta 80 (2010) 12921297

The process of organic solvent extraction: 0.5 g sample was
mixed with 15 mL 95% ethanol in a round-bottom ask (100 mL)
and the suspensions were boiled at 80 C for 2 h in an open system.
After extraction, the obtained extracts were cooled down
to the room temperature, then diluted to 50 mL with identical
solvent, and ltrated through a 0.45 m lter prior to HPLC analy-
sis.
2.5. HPLC analysis and quantication
The diluted extracts were directly injected into the liquid
chromatography. The mobile phase composed of solvent A (0.1%
triethylamine aqueous solution) and solvent B (CH3 CN) was deliv-
ered at a ow rate of 0.8 mL min1 . The gradient elution was
programmed as follows: 05 min, 2030% B; 510 min, 30% B;
1013 min, 3060% B; 1320 min, 6080% B. The UV detection
wavelengths were 280 nm. Each injection volume was 20 L and
the column temperature was ambient. Under these conditions,
the three alkaloids were baseline separated. N-nornuciferine, O-
nornuciferine, and nuciferine were identied by comparing their
retention time with corresponding peaks in the standard solution.
A comparison of the chromatograms of the three alkaloids obtained
from standard solutions with those contained in ILs extract is
shown in Fig. 1.
2.6. Purication of standards of three alkaloids
Dried and powdered lotus leaf (1 kg) was extracted with 16 L of
95% ethanol for 2 h and repeated with three times. Then, the extract
solutions were combined and evaporated under reduced pressure
and 40 C to about 200 mL. The concentrated solution was dissolved
in 2 L 1.0% hydrochloric acid aqueous solution. The supernatant
after centrifugation was adjusted to pH 10 with ammonia, and
then extracted with chloroform (three times). The crude alkaloids
which were obtained after solvent evaporation were dissolved in
the mobile phase solvent, and then were injected into preparative
HPLC. Methanol0.2% ammonia aqueous solution was used as elu-
ent solvents at 6 mL min1 ow rate under 280 nm. Volume ratios
of eluent solvents were 60:40, 65:35, 70:30, for N-nornuciferine,
O-nornuciferine, and nuciferine, respectively.
3. Results and discussion
3.1. Screening of the ILs
The structure of ILs has signicant inuence on its physicochem-
ical properties, which might greatly affect the extraction efciency
of target analytes [23]. The effects of changing the anion and the
alkyl chain length of the cation of 1-alkyl-3-methylimidazolium-

Fig. 2. Effect of ILs anions (a), ILs cations (b), compared [C6 MIM]Br with other solvents (c) and [C6 MIM]Br concentrations (d) on the extraction efciency of target analytes.
Sample: 0.5 g, extractant volume: 10 mL; irradiation power: 280 W; extraction time: 2 min. The extraction efciency is expressed as the observed values of target analytes
and the maximum amount in each curve was taken to be 100%.
 W. Ma et al. / Talanta 80 (2010) 12921297 1295

Table 1
Some physicochemical properties of the studied ILs.

Cation Anion MW Form (25 C) Solubility in H2 O (g/100 mL)

[C2 MIM]Br Br 191 Solid Totally misciblea

[C4 MIM]Cl Cl 174.5 Solid Totally misciblea [10]

[C4 MIM]Br Br 219 Solid Totally misciblea

[C4 MIM]BF4 BF4 225.8 Liquid Totally misciblea [10]

[C4 MIM]PF6 PF6 284 Liquid 1.17a [10]

[C6 MIM]Br Br 247 Liquid Misciblea

[C8 MIM]Br Br 275 Liquid Misciblea

a
Refers to the chemer home page on ionic liquid properties (www.chemer.com).

type ILs (see Table 1) on the extraction efciency were studied in tion efciency of [C4 MIM][PF6 ] is the lowest possibly due to its
this work. hydrophobicity.

3.1.1. Anion effect 3.1.2. Effect of the alkyl chain length

The anion identity is considered to be fore mostly important
and has the most dramatic effect on the properties [10]. It is read-
ily apparent from Table 1 that in the series of ILs studied here,
the identity of the anion greatly inuences the water miscibility.
Therefore, the 1-butyl-3-methylimidazolium ILs with four differ-
ent anions (Cl , Br , BF4 , PF6 ) were studied. Among them, the
former three ILs are hydrophilic and could miscible in any propor-
tion with water, however, [C4 MIM][PF6 ] is relatively hydrophobic
and is only sparingly water soluble. The results obtained were
given in Fig. 2a. Fig. 2a shows that Br is more efcient than oth-
ers. This result indicated the extraction efciency of alkaloids was
mainly anion-dependent, which was similar to the previous stud-
ies [24,36]. It probably because [C4 MIM]Br solution had stronger
multi-interactions including , ionic/chargecharge and hydro-
gen bonding with alkaloids [22]. Besides, [C4 MIM]Br solution had
slightly higher acidity, which might facilitate the extraction of
target alkaloids from lotus leaf [3032]. By contrast, the extrac-
With the same anion of Br , a series of 1-alkyl-3-
methylimidazolium cations including C2 mim+ , C4 mim+ , C6 mim+ ,
and C8 mim+ were evaluated. The results were shown in Fig. 2b.
It showed that extraction efciency increased signicantly when
the alkyl chain length was increased from ethyl to hexyl. However,
increasing alkyl chain length from hexyl to octyl dramatically
decreased the extraction efciency. The length of the alkyl chain
is related with the water miscibility of the Br IL [10], and this
property may be related with the extraction efciency.
Considering the effect of both anion and cation, [C6 MIM]Br was
selected for the subsequent evaluation (Fig. 2c).
3.1.3. Concentration effect
It has been proven that, polar molecule could strongly absorb the
microwave energy [37,38], and therefore water is always selected
as the co-solvent in various MAE process. To nd out the optimum
IL concentration for microwave-assisted extraction of three alka-

Fig. 3. Effect of microwave irradiation power (a), extraction time (b), and solidliquid ratio (c) on the extraction efciency of target analytes with 1.0 M [C6 MIM]Br. Sample:
0.5 g. The extraction efciency is expressed as the observed values of target analytes and the maximum amount in each curve was taken to be 100%.
1296 W. Ma et al. / Talanta 80 (2010) 12921297

loids, extraction was carried out in IL water solution of different

Recovery yields (%)

4.63

6.66
3.03
concentrations (from 0.5 to 2 M). Based on the results in Fig. 2d,

SD
the extraction efciency increased when the IL concentration over
the range of 0.51.0 M. When it further increased, however, slight

Mean

102.3
105.5
99.5
decrease was observed. Finally, 1.0 M of [C6 MIM]Br was selected in
the following experiments.

HRE HCl solution (n = 3)c

Observed values (mg/g)

3.2. Optimization of the MAE parameters

0.0285
0.1027
0.1650
SD
The univariate method was used in all instances to optimize
the three parameters: irradiation power, extraction time and
0.677 solidliquid ratio.
4.763
1.590
Mean

3.2.1. Irradiation power

The irradiation power effect of this method has been examined
as it has been claimed to inuence interactions and equilib-
Recovery yields (%)

3.38
1.17
0.84

rium rates and controls partition of analytes between sample and

SD

extracting phase. To examine the effect of the microwave power on

the extraction efciency, extractions were carried out at 120, 280,
Mean

98.1
102.5
94.6

460, 600, and 700 W, respectively, with a constant irradiation time

of 2 min. From Fig. 3a, irradiation power did not signicantly inu-
ence the extraction efciency. Irradiation power of 280 W (40% of
Observed values (mg/g)

the maximum value) could reach a little higher efciency of three

HRE 95% EtOH (n = 3)c

0.1247
0.0292
0.0573

alkaloids than others.

SD
HRE method

3.2.2. Extraction time

To optimize extraction time, extractions were carried out at
0.825
1.686
5.765
Mean

280 W when microwave irradiation was changed within 4 min. As

shown in Fig. 3b, when the extraction time increased from 0 to
1 min, the extraction efciency of the three alkaloids increased dra-
matically. When the variable was changed from 1 min to 4 min,
Recovery yields (%)

1.48

2.14
2.90
SD

slight improvements were observed. Therefore, 2 min was selected

for further experiments.
Mean

3.2.3. Solidliquid ratio

95.2
99.6
95.1

The solidliquid ratio is a crucial factor that should be studied to

increase the extraction efciency of three alkaloids. Larger solvent
Observed values (mg/g)
b

volumes could make procedure complex and unnecessary waste

MAE 80% EtOH (n = 3)

0.1693
0.0184
0.0554

while smaller ones would make the targets extraction incomplete.

SD

A series of extractions were carried out with different solidliquid

ratios (1:10, 1:20, 1:30, and 1:40 g mL1 ) to evaluate the effect of
See experimental section for operation conditions. The total extraction time is 2 h.

the solidliquid ratio. Fig. 3c indicated that the extraction efciency

0.772

5.264
1.506
Mean

increased obviously with the increase of the solvent volume before

the solidliquid ratio reached 1:30, and then the efciency was
not signicantly improved with the further increase of the solvent
Comparison of extraction efciencies under different extraction methods.

Recovery yields (%)

amount. Thus, a solidliquid ratio of 1:30 was used in the present

1.74
1.86
0.60
SD

study.
Based on the above experiment, the optimum MAE condi-
The same operation conditions except the extracting phase.

tions were found to be: 1.0 M [C6 MIM]Br as extraction solvent,

Mean

microwave irradiation power of 280 W, extraction time of 2 min,

97.3
98.9
99.2

solidliquid ratio of 1:30 (g mL1 ).

a
IL MAE [C6 MIM]Br (n = 3)

Observed values (mg/g)

3.3. Comparison of the proposed ILMAE approach with the

0.0341
0.0928
0.0086

conventional methods
Operation under optimized conditions.
SD
MAE methods

For the comparison of the extraction efciency of ILMAE with

0.973

5.945
1.701

other conventional extraction techniques, HRE including acid

Mean

extraction, organic solvent extraction and regular MAE extraction

were carried out to extract three alkaloids from lotus leaf (Table 2).
From Table 2, the proposed approach not only obviously increased
the extraction efciency (0.943.7% enhanced), but also dramati-
N-nornuciferine
O-nornuciferine

cally reduced the total extraction time (from 2 h to 2 min).

Nuciferine

In order to further demonstrate the use of ILs, the regular MAE

Analytes

process was used to compare with the proposed ILMAE approach.

Table 2

All experiments were carried out under the same MAE conditions
a

except the extractant. 80% ethanol which was optimized as the best
 W. Ma et al. / Talanta 80 (2010) 12921297 1297

Table 3
Calibration graphs of the proposed method.

Analytes Linearities Correlation coefcients Calibration range (mg/L) LOD (g/L) LOQ (g/L)
2
N-nornuciferine y = 49864x + 28.324 R = 0.9999 0.412165.0 13.6 45.3
O-nornuciferine y = 41646x + 36.714 R2 = 0.9998 0.500160.0 6.01 20.1
Nuciferine y = 31173x + 12.888 R2 = 0.9999 1.075344.0 23.6 78.9

The linearity plotting at 280 nm and x was analytes concentration as g mL1 and y was the peak area.

solvent was used in the regular MAE. Results shown in Table 2 indi- Acknowledgements
cated that the extraction efciency of ILMAE was slightly higher
than that of organic solvent (95% EtOH) HRE method, and much Authors thank the National Natural Science Foundation of China
higher than that of regular MAE and acid HRE methods. This showed for Grant 20775069, Ministry of Education of China for Grant NCET-
that, compared with HRE and regular MAE technique, the proposed 06-0520 and Natural Science Foundation of Zhejiang Province for
approach used only small amount of ILs could obtain higher extrac- Grant Z206510.
tion efciency (0.943.7% enhanced) in shorter extraction time
(from 2 h to 2 min), which indicated that the aqueous ILs solutions References
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