Talanta 63 (2004) 659665

Focused microwave-assisted solvent extraction and HPLC determination

of effective constituents in Eucommia ulmodies Oliv. (E. ulmodies)
Hui Li a,c , Bo Chen b , Zhaohui Zhang a , Shouzhuo Yao a,
a

State Key Laboratory of Chemo/Biological Sensing & Chemometrics, Hunan University, Changsha 410082, PR China
b College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China
c College of Resources and Environmental Sciences, Jishou University, Hunan Jishou 416000, PR China
Received 10 July 2003; received in revised form 21 November 2003; accepted 5 December 2003
Available online 8 February 2004

Abstract
A new focused microwave-assisted solvent extraction method using water as solvent has been developed for leaching geniposidic and
chlorogenic acids from Eucommia ulmodies Oliv. The extraction procedures were optimized using a two indexes orthogonal experimental
design and graphical analysis, by varying irradiation time, solvent volume, solvent composition and microwave power. The optimum extraction
conditions were obtained: for geniposidic acid, 50% micorwave power, 40 s irradiation, and 80% (v/v) aqueous methanol as extraction solvent
(20 ml g1 sample); and for chlorogenic acid, 50% micorwave power, 30 s irradiation, and 20% aqueous methanol (20 ml g1 sample). The
composition of the extraction solvent was optimized and can be directly used as the mobile phase in the HPLC separation. Quantification
of organic acids was done by HPLC at room temperature using Spherigel C18 chromatographic column (250 mm 4.6 mm, i.d. 5 m), the
methanol:water:acetic acid (20:80:1.0, v/v) mobile phase and UV detection at 240 nm. The R.S.D. of the extraction process for geniposidic
and chlorogenic acid were 3.8 and 4.1%, respectively.
2004 Elsevier B.V. All rights reserved.
Keywords: Focused microwave-assisted solvent extraction; Geniposidic acid; Chlorogenic acid; Eucommia ulmodies Oliv; HPLC

1. Introduction
The pharmaceutical industry is a considerably attractive
field applying microwave-assisted extraction on dealing with
sample preparation, but so far relatively less papers have
been published in this area [14]. Presently, there have been
developed two types of commercially available microwave
heating system: the closed-vessel system and the open-vessel
system [5]. The former is usually called microwave-assisted
extraction (MAE), by which many compounds with bioactivity can be extracted, such as puerarin from the Chinese
herbal medicine Radix puerariae [6], sulphamethazine from
wine tissue [7], transhinones from sallia miltiorrhiza bunge
[8], glycyrrhizic acid from licorice root [9], and so on. The
latter is also named focused microwave-assisted soxhlet or

Corresponding author. Fax: +86-731-8865515.

E-mail addresses: szyao@hnu.net.cn, lhhndx@263.net (S. Yao).

0039-9140/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2003.12.028

solvent extraction (FMASE), in which the sample is immersed in a microwave-absorbing solvent in an open vessel
and irradiated with focused microwave energy till the completion of extraction. Recently, some improvements and
applications of the FMASE technique have been carried
out. Luque de Castro and coworkers [10] firstly utilized
focused microwave-assisted Soxhlet to extract alkane, polyaromatic hydrocarbons, herbicides from soil matrix. Then,
Garcia-Ayuso et al. combined this extraction system with
Fluorescene detector to allow real-time on-line monitoring
of polyaromatic hydrocarbons extracted from solid matrix [11]. In 2001, Falqui-Cao et al. [12] exploit a novel
method using focused microwave-assisted extraction and
solid phase micro-extraction coupled with high performance
liquid chromatography for the determination of pesticide
residues, and Luque-Garcia and Luque de Castro [13] modified the focused microwave-assisted Soxhlet extractor by
installing a shortened distillation glassware and connecting
a refrigerator to the top of the cartridge vessel, enabling

660

H. Li et al. / Talanta 63 (2004) 659665

the use of water as extractant for leaching herbicides from

soil. However, in spite of these developments, application
of this new FMASE technique for extracting bioactive compounds from natural products or herbal medicines was still
scarce.
E. ulmodies is a precious Chinese material medica, sweet
in taste and warm in nature, and active in nourishing the liver
and kidney, strengthening the bone and muscle, and preventing abortion, etc. [14]. Geniposidic acid and chlorogenic
acid are two important active compounds in E. ulmodies The
former may promote collagen synthesis [15] and improve
the turnover rate of stratum corneum [16], and the latter
has anti-bacterial, phlogosis, mutagenic, oxidant and other
biological activities [17,18]. Some papers dealing with the
extraction of geniposidic and chlorogenic acids from plants
have been published. Extraction under refluxing was widely
used to leach these compounds from plants, such as geniposidic acid from seeds of E. ulmodies [19], chlorogenic
acid from green coffee [20] and sweet potates [21], etc.
One main disadvantage of this method is time-comsuming.
Lyon and Barker applied coventional Soxhlet method to
extract chlorogenic acid from potato leaves [22]. Compared
with the former method, longer time and more solvent were
needed in this approach in spite of the improvement of the
recovery of the extract. Lately, ultrasound-assisted extraction technique has been used by Sun et al. to extract active
compounds from E. ulmodies [23]. Though higher extraction yield could be reached in comparison with coventional
approaches, 45 min was needed to complete the extraction,
and the extract was a mixture of multi-constituents. Comparing with conventional extraction approaches, superiorities from microwave-assisted extraction are obvious. When
tanshinones was leached out from Salvia miltiorrhiza bunge
[24], the extraction efficiency obtained in two minutes using microwave-assisted extraction was the same as that
obtained in 45, 75, 90 and 1440 min using reflux extraction
at heating, ultrasonic-assisted extraction, soxhlet extraction
and extraction at room temperature, respectively. Also, only
1.5 min was needed to complete the procedure when MAE
was used to leach sallidroside and tyrosol from Rhodioda
sachalinensis, but in other methods 30120 min was needed
[25]. To date, study on (focused) microwave-assisted extraction of geniposidic acid and chlorogenic acid from E.
ulmodies has not yet been reported in the literature.
This paper offers a new focused microwave-assisted solvent extraction method using water as solvent for leaching
geniposidic and chlorogenic acids from E. ulmodies. The
extraction procedures were optimized using orthogonal
experimental design and graphical analysis, by varying irradiation time, solvent volume, solvent composition and
microwave power. In present work, a commercially available microwave system is applied and the extraction solvent used in microwave-assisted extraction can be directly
used as mobile phase in HPLC separation with no need of
additional intermittent treatment. The repeatability of the
method was also investigated.

2. Experimental
2.1. Apparatus
The extraction system comprised a commercially available microwave oven (LG electrical equipment Co.,
Ltd., Tianjing, PR China) equipped with a magnetron of
2450 MHz with a nominal maximum power of 700 W. A
1 m-long condensation pipe was connected to the sample
cartridge vessel located at the microwave irradiation zone.
The whole system was open (Fig. 1) and run at atmospheric
pressure.
High performance liquid chromatographer was remodeled from HIC-6A ion chromatographer (Shimadzu Co.,
Ltd., Japan) with a SCL 6B system controller and a SIL-6B
auto-injector connected to an auto-injection pump. LC-10
uv model UV detector, chromatographic work station with
a data auto-collection card, and C18 column (250 mm
4.6 mm, i.d. 5 m) were purchased from Dalian Chromatographic Instrument Co., China.
2.2. Reagents and materials
A.R. grade methanol, glacial acetic acid and redistilled
water were filtrated through a 0.45 m microporous membrane before use. Standards of chlorogenic acid (>95%)
were purchased from Sigma and geniposidic acid (>92%)
from Chemical Research Institute, Hunan Normal University, Changsha, PR China. Dried barks of E. ulmodies came
from the Traditional Chinese Medicine Co., Ltd. (Hunan,
PR China).
2.3. Focused microwave-assisted extraction
0.5 g well grounded sample was placed into the cartridge
vessel, followed by adding appropriate solvent. With ice
water running through the condensation pipe, the sample
was treated under microwave irradiation in an intermittent
way, i.e. irradiationcoolingirradiation. The irradiation

Fig. 1. Schematic diagram of focused microwave-assisted solvent extraction apparatus. (1) Condensation pipe; (2) ice water outlet; (3) ice water
inlet; (4) sample vessel; (5) microwave irradiation system.

H. Li et al. / Talanta 63 (2004) 659665

Table 1
The factors and levels for the orthogonal design

50000

CGA
GA

40000

Levels

Signal

GA: geniposidic acid

30000

CGA: chlorogenic acid

1
2
3

20000
10000
0
0

10

(a)

15

20

25

min

GA

4000

CGA

Signal

3000
GA: geniposidic acid

2000

CGA: chlorogenic acid

1000
0
0

10

(b)

15

661

20

25

30

min

Fig. 2. The chromatograms of geniposidic acid and chlorogenic acid. (a)

Standards; (b) sample.

time was kept for 10 s and 2 min was taken to cool the
sample solution between two irradiations. Then, the resulted solution was cooled off, filtrated through a 0.45 m
microporous membrane, and directly used for HPLC
analysis.
2.4. Chromatographic analysis
HPLC separation was carried out in a C18 column
(250 mm 4.6 mm, i.d. 5 m) at room temperature. Eluent was methanol:water:acetic acid (20:80:1.0, v/v) at
1 ml min1 . Sample volume was 10 l. The elution was
monitored at 240 nm. Calibration curve method was used
for the analysis of chlorogenic acid and geniposidic acid.
Retention times of these two organic acids were 4.398 and
13.504 min, respectively (Fig. 2).

3. Results and discussion

3.1. The preliminary test of the extracting conditions
During focused microwave-assisted extraction of geniposidic and chlorogenic acids from E. ulmodies, there are many

Microwave
power
(%)

Irradiation
time (s)

Solvent volume
(ml g1 sample )

Methanol
concentration in
water (v/v, % )

90
70
50

10
30
50

5.0
10.0
20.0

20
50
80

variables effecting on the extraction efficiency. However,

studying all parameters is time-consuming and unnecessary.
Hence, in this work, only four main factors, i.e. microwave
power, irradiation time, solvent volume and solvent type,
were tested. Because these two components are easily dissolved in water and methanol, methanol:water mixture was
used as extraction solvent in preliminary tests. For each variable, the influence on extraction efficiency was considered
from three levels. Table 1 lists the factors and levels used in
the tests.
In general, a full evaluation of the effect of four factors
from three levels on the extraction efficiency needs 81 (34 )
experiments. In order to reduce the number of experiments,
a L9 (34 ) orthogonal design graph was used (Table 2). By
this way, only nine experiments were needed. The extraction
efficiencies of geniposidic and chlorogenic acids obtained
under orthogonal conditions are also shown in Table 2. To
analyze the influence of each variable on extraction results,
an intuitionstic plot as Fig. 3 was constructed based on the
data in Table 2.
It can be seen from Fig. 3a that, for chlorogenic acid,
the effects of the four factors on extraction efficiency decrease in the order of irradiation time (B), solvent volume
(C), methanol concentration in water (D) and microwave
power (A). When the irradiation time was increased from
10 to 50 s, the extraction efficiency of chlorogenic acid increased at first, then followed by decrease. A similar situation took place when the solvent volume was increased from
5.0 to 20.0 ml g1 sample. However, a different situation
happened with the microwave power and methanol concenTable 2
The results of orthogonal test L9 (34 ) (n = 3)
Test
numbers

Extraction
efficiency of
geniposidic
acid (%)

1
2
3
4
5
6
7
8
9

1
1
1
2
2
2
3
3
3

1
2
3
1
2
3
1
2
3

1
2
3
2
3
1
3
1
2

1
2
3
3
1
2
2
3
1

35.9
50.9
57.6
42.8
50.0
41.5
48.4
51.0
60.0

1.02
0.85
3.22
2.19
1.84
0.55
1.66
1.35
2.15

Extraction
efficiency of
chlorogenic
acid (%)
31.2
60.2
46.3
40.1
56.6
43.7
39.8
47.4
56.1

0.67
3.42
2.53
2.76
1.29
0.46
2.50
1.82
2.11

40
35

(a)

Factors

Extraction
efficienc y(%)

55

Level 1
50

Level 3
45

40

(b)

Level 2

Factors

Fig. 3. The extraction efficiencies of chlorogenic acid (a) and geniposidic

acid (b) under orthogonal conditions (for designations of factors A, B, C
and D, see Table 1).

tration in water: the higher the power or concentration, the

lower the extraction efficiency. Thus, the best conditions,
obtained by preliminary test, for the extraction of chlorogenic acid were: irradiation time = 30 s, solvent volume =
10 ml g1 sample, 20% methanol concentration in water and
50% microwave power.
Different with chlorogenic acid, the influence of these
factors on extraction efficiency of geniposidic acid decrease
in the order of irradiation time (B), solvent volume (C),
microwave power (A) and methanol concentration in water
(D) (Fig. 3b). Analyzing as above, the following conditions
for the extraction of geniposidic acid were selected: 50 s
of irradiation time, 20 ml g1 sample solvent volume, 50%
microwave power and 80% methanol concentration in water.
3.2. Interaction of factors during extraction of effective
constituents from E. ulmodies
Because there might exist some interaction among variables during focused microwave-assisted extraction, the
influence of this interaction on extraction needed to be
considered when the conditions were optimized. For this
purpose, graphs of the following variable pair were constructed: (a) the highest value for two factors; (b) the lowest
value for two factors; (c) the highest and the lowest values
for each pair of factors. In this method, interaction graphs
for all pairs of factors were obtained.
3.2.1. Interaction of irradiation time with the other factors
Fig. 4a shows the influence of the interaction between irradiation time and microwave power on extraction efficiencies

54
50%
48

90%
90%

42
36

50%

54
45

Irradiation time
= 50 s
Irradiation time
= 10 s

50%
50%

90%

36

Irradiation time
= 50 s
Irradiation time
= 10 s

90%

27

(a)

Extraction efficiency of
geniposidic acid (%)

Level 1
Level 2
Level 3

45

Extraction efficiency of
chlorogenic acid (%)

50

Microwavepower(%)

45

20.0

5.0

40
5.0
35

55
50

20.0

Irradiation time
= 50 s
Irradiation time
= 10 s

20.0
5.0

20.0

45
5.0

Irradiation time
= 50 s
Irradiation time
= 10 s

40

(b) Solvent volume (ml/g sample)

Extraction efficiency of
geniposidic acid (%)

Extraction
efficiency(%)

55

Extraction efficiency of Extraction efficiency of

chlorogenic acid (%)
geniposidic acid (%)

H. Li et al. / Talanta 63 (2004) 659665

Extraction efficiency of
chlorogenic acid (%)

662

(c)

50
45

20%
20%

80%

40
80%

35

Irradiation time
= 50 s
Irradiation time
= 10 s

60
57

20%

80%

54
51

20%

80%

Irradiation time
= 50 s
Irradiation time
= 10 s

48
Methanol concertration
in water (v/v %)

Fig. 4. The influence of the interaction of irradiation time with microwave

power (a), solvent volume (b) and methanol concentration in water (c)
on extraction efficiencies of geniposidic acid and chlorogenic acid.

of geniposidic and chlorogenic acid. For both compounds,

at 90% microwave power, the extraction efficiency obtained by irradiating for 10 s is higher than for 50 s. On the
contrary, when 50% microwave power is used, the longer
the irradiation time, the higher the extraction efficiency.
Furthermore, the extraction efficiency of geniposidic acid
obtained with 90% microwave power is dramatically higher

H. Li et al. / Talanta 63 (2004) 659665

3.2.2. The influence of interaction of solvent volume with

microwave power and methanol concentration in water on
extraction process
Interaction of microwave power with solvent volume is
constructed in Fig. 5a. It is found that microwave power has a
weaker effect on the extraction efficiency of geniposidic acid
and 50% microwave power is beneficial for the extraction of
chlorogenic acid. For these two compounds, the larger the
solvent volume, the higher the extraction efficiency at the
same microwave power.
Fig. 5b gives the interaction results between solvent volume and methanol concentration in water. For these two organic acids, when the methanol concentration in water goes
up from 20 to 80%, a similar behavior can be observed except a weaker increase of extraction efficiency of geniposidic

Extraction efficiency of
geniposidic acid (%)

50

20.0

48

20.0

46
44
42
40

Extraction efficiency of
geniposidic acid (%)

Microwave power
= 50%
Microwave power
= 90%

5.0

Extraction efficiency of
chlorogenic acid (%)

38

5.0

68
20.0
66
64
62 5.0
20.0
60
58
5.0
56
(a) Solvent volume (ml/g sample)

Extraction efficiency of
chlorogenic acid (%)

than that obtained with 50% microwave power at 10 s of

irradiation time. Even though in intuitionistic plot, 50%
microwave power may produce a slightly higher extraction
efficiency of geniposidic acid than 90% microwave power,
the interaction between irradiation time and microwave
power indicates that extraction of geniposidic acid with 90%
microwave power at 10 s of irradiation time is more effective. Whereas, for chlorogenic acid, extraction efficiency
gained at 90% microwave power is much lower than that
gained at 50% microwave power when irradiation time
is 50 s.
Interaction graph of irradiation time-solvent volume pair
(Fig. 4b) shows a different behavior for the tested components. When 5.0 ml solvent per gram sample is used, for
geniposidic acid, a 4% higher extraction efficiency can be
produced in 10 s than in 50 s of irradiation time, however,
for chlorogenic acid, a 9% lower extraction efficiency is obtained. Like geniposidic acid, the larger the solvent volume,
the higher the extraction efficiency of chlorogenic acid when
the sample is irradiated for 50 s. But at 10 s irradiation, compared with using 5 ml solvent per gram sample, a 9.6% lower
extraction efficiency is observed when 20.0 ml solvent per
gram sample is used for geniposidic acid; whereas, a 11.9%
higher extraction efficiency can be gained under the same
conditions for chlorogenic acid.
The interaction between irradiation time and methanol
concentration in water is shown in Fig. 4c. It can be found
that when methanol concentration in water is 20%, the extraction efficiency of geniposidic acid hardly changes with
the increase of irradiation time, however, for chlorogenic
acid, a 7.2% higher extraction efficiency can be observed
when the sample is irradiated for 50 s than for 10 s. Using
80% methanol as extracting solvent may produce a 14.1%
increase in the extraction efficiency of geniposidic acid when
irradiation time increases from 10 to 50 s, and reversely,
a 8.0% decrease is observed for chlorogenic acid. In addition, when irradiation time is fixed, a completely opposite change for these two compounds can be observed with
the increase of methanol concentration in water from 20
to 80%.

663

Microwave power
= 50%
Microwave power
= 90%

48
46
44

20%

80%

42
40

20%

80%

Solvent volume =
5.0 ml/g sample
Solvent volume =
20.0 ml/g sample

38
70
65

20%

80%

60
55
50

20%
80%

Solvent volume =
5.0 ml/g sample
Solvent volume =
20.0 ml/g sample

45

(b)

Methanol concentration
in water (v/v %)

Fig. 5. The effect of the interaction of solvent volume with microwave

power (a) and methanol concentration in water (b) on extraction efficiencies of geniposidic acid and chlorogenic acid.

acid under 20 ml solvent per gram sample. The interaction

graph indicates that the optimized solvent volume should be
20 ml g1 sample.
3.2.3. Interaction between methanol concentration in
water and microwave power
The interaction between microwave power and methanol
concentration in water had a great influence on extraction
process (Fig. 6). For geniposidic acid, a 10.3% higher extraction efficiency is gained using 90% than 50% microwave
power in 20% methanol water solution. At the 90% microwave power, the higher the methanol concentration, the
lower the extraction efficiency of geniposidic acid. However,
for chlorogenic acid, the larger the methanol concentration,
the higher the extraction efficiency at these two microwave
powers. A 1.1% higher extraction efficiency of chlorogenic
acid is obtained using 90% microwave power than using
50% one in 20% methanol solution, and reversely, a 1.9%
lower one can be observed in 80% methanol water solution.
By analyzing the influence of interaction of factors on extraction efficiencies of geniposidic acid and chlorogenic acid
in E. ulmodies and the intuitionistic plot of two indexes experimental design, it can be concluded that, 50% microwave

H. Li et al. / Talanta 63 (2004) 659665

Extraction efficiency of Extraction efficiency of
geniposidic acid (%)
chlorogenic acid (%)

664

56
20%
52
48

80%
20%

Microwave power
= 50%
Microwave power
= 90%

80%
44
80%

74
72

80%
70
68

20%

Microwave power
= 50%
Microwave power
= 90%

20%

Methanol concentration
in water (v/v %)

Fig. 6. Effects of the interaction between methanol concentration and

microwave power on extraction efficiency of geniposidic acid and chlorogenic acid.

power and 20 ml solvent per gram sample are more favorable for the extraction of these two constituents.
3.3. The further optimization of methanol concentration
in water and irradiation time
To find out the optimum methanol concentration for
microwave-assisted extraction of geniposidic acid and
chlorogenic acid, extraction was carried out in methanol
water solution of different concentrations (from 0 to 100%).
All the experiments were repeated for three times (n = 3).
Fig. 7 shows that, for geniposidic acid, the extraction efficiency increases when methanol content in water increases
from 0 to 80%, reaching the highest value (54.2%) at 80%,
and then starts to decrease. For chlorogenic acid, the maximum extraction efficiency appears at 20% methanol water
solution.
In the following experiments, by changing the irradiation time from 5 to 70 s, the extraction efficiency of these
two compounds was further investigated. All the extractions

Extraction efficiency ( % )

56

52

48

44

geniposidic acid
chlorogenic acid
40
0

20

40

60

80

100

Methanol concentration in water ( % )

Fig. 7. The influence of methanol concentration in water on extraction of
geniposidic and chlorogenic acids (n = 3).

were repeated for three times. The result shows that, when
irradiation time increases from 5 to 40 s, the extraction efficiency of geniposidic acid increases from 30.4 to 65.5%,
and then decreases slowly, similarly for chlorogenic acid,
the extraction efficiency increases with irradiation time until a maximum value (63.7%) is reached at 30 s irradiation,
followed by slight decrease. Apparently, for these two components, overlong irradiation can lower the extraction efficiency under focused microwave irradiation.
Hence, to effectively extract geniposidic acid and chlorogenic acid from E. ulmodies by focused microwave irradiation, the optimum extraction conditions were: for
geniposidic acid: 50% microwave power, 20 ml solvent per
gram sample, 80% methanol water solution as extraction
solvent and 40 s irradiation; and for chlorogenic acid: 50%
microwave power, 20 ml solvent per gram sample, 20%
methanol as extraction solvent and 30 s irradiation time.
3.4. The effect of acetic acid on focused
microwave-assisted extraction process
Because the ionization of geniposidic and chlorogenic
acids might happen during focused microwave heating process, acetic acid was added into the extraction solvent to
study the influence on extraction efficiencies of the two
tested organic acids. It was found from the results that for
these two compounds, the extraction efficiency increases
with the increase of the content of acetic acid in solvent,
reaching the maximum values at 3.0%, and then decreases.
Therefore, 3.0% acetic acid was the most appropriate for the
extraction of geniposidic and chlorogenic acids.
3.5. The consistency between extraction solvent and
mobile phase
The consistency between the composition of the extraction solvent in focused microwave-assisted extraction and
that of the mobile phase in HPLC separation is helpful for
the pretreatment of the sample. Usually the composition of
the solvent used in the extraction process is different from
that of the mobile phase used in the HPLC separation. Additional treatment is needed for the extraction solvent to be
adjusted to satisfy the HPLC requirement, including evaporation, adding other chemicals, or filtration, centrifugation,
etc. [2628]. In this work, we have succeeded to adjust
the extractant constituents to be directly used as the HPLC
mobile phase.
For quantification of chlorogenic acid and geniposidic
acid by HPLC, mobile phase used was methanol:water:acetic
acid (20:80:1.0, v/v) solvent mixture, where acetic acid
was added to prevent the ionization of organic acids. Furthermore, it is the acetic acid in the extraction solvent that
obviously improve the extraction efficiency of these two
compounds, in correlation to its ionization-inhibition action and polarity change of the solvent. On the other hand,
when the content of acetic acid in the mobile phase was

H. Li et al. / Talanta 63 (2004) 659665

665

Table 3
The repeatability of extraction process
Sample

Extraction efficiencies (%)

Geniposidic acid

1#
2#
3#
4#
5#

Chlorogenic acid

Mean

79.3
79.0
72.8
83.6
75.1

81.0
79.0
73.5
82.5
76.3

80.9
78.4
74.5
83.5
75.4

80.4
78.8
73.6
83.2
75.6

R.S.D. (%)

Mean

R.S.D. (%)

3.8

78.4
81.1
76.0
85.2
84.2

78.1
82.3
75.7
82.8
87.2

77.4
82.0
76.3
83.7
86.3

77.9
81.8
76.0
83.9
85.9

4.1

increased from 1 to 3%, a similar peak separation between

geniposidic acid and chlorogenic acid was observed. Hence,
1% acetic acid in the mobile phase was selected.
3.6. Repeatability
Based on the above tests, the optimum focused microwave-assisted extraction conditions from E. ulmodies were
found to be: for geniposidic acid, 50% microwave power,
80% (v/v) methanol water solution as extraction solvent
(20 ml g1 sample) and 40 s irradiation; and for chlorogenic
acid: 50% microwave power, 20% (v/v) aqueous methanol
(20 ml g1 sample) and 30 s irradiation.
The repeatability of the focused microwave-assisted solvent extraction of geniposidic acid and chlorogenic acid
from E. ulmodies was determined. Five samples with the
same weight (0.5 g) were processed under the conditions
of 50% microwave power, 10 ml methanol:water:acetic acid
(20:80:3.0, v/v) mixture solvent and 30 s irradiation time.
R.S.D. of the focused microwave-assisted extraction of geniposidic and chlorogenic acid were 3.8 and 4.1%, respectively
(Table 3).
The repeatability of chromatographic analysis was also
considered in order to determine the errors. 0.5 g sample
was processed for 30 s at 50% microwave power using 10 ml
solvent as above. The sample was analyzed repeatedly for
five times under the same chromatographic conditions. The
extraction efficiencies of geniposidic acid were 77.3, 75.7,
77.6, 76.2 and 75.7%, and that of chlorogenic acid were 83.3,
81.8, 80.5, 81.7 and 82.6%, respectively. Hence, the R.S.D.
of the chromatographic analysis were 0.9% for geniposidic
acid and 1.3% for chlorogenic acid.

Acknowledgements
Financial supports from China NSF, China MST and Hunan Provincial Department of Science & Technology were
acknowledged.

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