Talanta 63 (2004) 659665

Focused microwave-assisted solvent extraction and HPLC determinationof 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 Chinac 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 andchlorogenic acids from Eucommia ulmodies Oliv. The extraction procedures were optimized using a two indexes orthogonal experimentaldesign and graphical analysis, by varying irradiation time, solvent volume, solvent composition and microwave power. The optimum extractionconditions 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). Thecomposition of the extraction solvent was optimized and can be directly used as the mobile phase in the HPLC separation. Quantificationof organic acids was done by HPLC at room temperature using Spherigel C18 chromatographic column (250 mm 4.6 mm, i.d. 5m), themethanol: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 geniposidicand 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 attractivefield applying microwave-assisted extraction on dealing withsample preparation, but so far relatively less papers havebeen published in this area [14]. Presently, there have beendeveloped two types of commercially available microwaveheating system: the closed-vessel system and the open-vesselsystem [5]. The former is usually called microwave-assistedextraction (MAE), by which many compounds with bioac-tivity can be extracted, such as puerarin from the Chineseherbal medicine Radix puerariae [6], sulphamethazine fromwine tissue [7], transhinones from sallia miltiorrhiza bunge[8], glycyrrhizic acid from licorice root [9], and so on. Thelatter 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).

solvent extraction (FMASE), in which the sample is im-mersed in a microwave-absorbing solvent in an open vesseland irradiated with focused microwave energy till the com-pletion of extraction. Recently, some improvements andapplications of the FMASE technique have been carriedout. Luque de Castro and coworkers [10] firstly utilizedfocused microwave-assisted Soxhlet to extract alkane, pol-yaromatic hydrocarbons, herbicides from soil matrix. Then,Garcia-Ayuso et al. combined this extraction system withFluorescene detector to allow real-time on-line monitoringof polyaromatic hydrocarbons extracted from solid ma-trix [11]. In 2001, Falqui-Cao et al. [12] exploit a novelmethod using focused microwave-assisted extraction andsolid phase micro-extraction coupled with high performanceliquid chromatography for the determination of pesticideresidues, and Luque-Garcia and Luque de Castro [13] mod-ified the focused microwave-assisted Soxhlet extractor byinstalling a shortened distillation glassware and connectinga refrigerator to the top of the cartridge vessel, enabling

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

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

the use of water as extractant for leaching herbicides fromsoil. However, in spite of these developments, applicationof this new FMASE technique for extracting bioactive com-pounds from natural products or herbal medicines was stillscarce.

E. ulmodies is a precious Chinese material medica, sweetin taste and warm in nature, and active in nourishing the liverand kidney, strengthening the bone and muscle, and prevent-ing abortion, etc. [14]. Geniposidic acid and chlorogenicacid are two important active compounds in E. ulmodies Theformer may promote collagen synthesis [15] and improvethe turnover rate of stratum corneum [16], and the latterhas anti-bacterial, phlogosis, mutagenic, oxidant and otherbiological activities [17,18]. Some papers dealing with theextraction of geniposidic and chlorogenic acids from plantshave been published. Extraction under refluxing was widelyused to leach these compounds from plants, such as geni-posidic acid from seeds of E. ulmodies [19], chlorogenicacid 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 toextract chlorogenic acid from potato leaves [22]. Comparedwith the former method, longer time and more solvent wereneeded in this approach in spite of the improvement of therecovery of the extract. Lately, ultrasound-assisted extrac-tion technique has been used by Sun et al. to extract activecompounds from E. ulmodies [23]. Though higher extrac-tion yield could be reached in comparison with coventionalapproaches, 45 min was needed to complete the extraction,and the extract was a mixture of multi-constituents. Com-paring with conventional extraction approaches, superiori-ties from microwave-assisted extraction are obvious. Whentanshinones was leached out from Salvia miltiorrhiza bunge[24], the extraction efficiency obtained in two minutes us-ing microwave-assisted extraction was the same as thatobtained in 45, 75, 90 and 1440 min using reflux extractionat heating, ultrasonic-assisted extraction, soxhlet extractionand extraction at room temperature, respectively. Also, only1.5 min was needed to complete the procedure when MAEwas used to leach sallidroside and tyrosol from Rhodiodasachalinensis, but in other methods 30120 min was needed[25]. To date, study on (focused) microwave-assisted ex-traction 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 sol-vent extraction method using water as solvent for leachinggeniposidic and chlorogenic acids from E. ulmodies. Theextraction procedures were optimized using orthogonalexperimental design and graphical analysis, by varying ir-radiation time, solvent volume, solvent composition andmicrowave power. In present work, a commercially avail-able microwave system is applied and the extraction sol-vent used in microwave-assisted extraction can be directlyused as mobile phase in HPLC separation with no need ofadditional intermittent treatment. The repeatability of themethod was also investigated.

2. Experimental

2.1. Apparatus

The extraction system comprised a commercially avail-able microwave oven (LG electrical equipment Co.,Ltd., Tianjing, PR China) equipped with a magnetron of2450 MHz with a nominal maximum power of 700 W. A1 m-long condensation pipe was connected to the samplecartridge vessel located at the microwave irradiation zone.The whole system was open (Fig. 1) and run at atmosphericpressure.

High performance liquid chromatographer was remod-eled from HIC-6A ion chromatographer (Shimadzu Co.,Ltd., Japan) with a SCL 6B system controller and a SIL-6Bauto-injector connected to an auto-injection pump. LC-10uv model UV detector, chromatographic work station witha data auto-collection card, and C18 column (250 mm 4.6 mm, i.d. 5m) were purchased from Dalian Chromato-graphic Instrument Co., China.

2.2. Reagents and materials

A.R. grade methanol, glacial acetic acid and redistilledwater were filtrated through a 0.45m microporous mem-brane before use. Standards of chlorogenic acid (>95%)were purchased from Sigma and geniposidic acid (>92%)from Chemical Research Institute, Hunan Normal Univer-sity, Changsha, PR China. Dried barks of E. ulmodies camefrom 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 cartridgevessel, followed by adding appropriate solvent. With icewater running through the condensation pipe, the samplewas treated under microwave irradiation in an intermittentway, i.e. irradiationcoolingirradiation. The irradiation

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

H. Li et al. / Talanta 63 (2004) 659665 661

0 5 10 15 20 25

0

10000

20000

30000

40000

50000

0 5 10 15 20 25 30

0

1000

2000

3000

4000

GA: geniposidic acid

GA

CGA: chlorogenic acid

CGA

GA: geniposidic acidCGA: chlorogenic acid

GA CGA

min

Sign

al

min

Sign

al

(a)

(b)

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 thesample solution between two irradiations. Then, the re-sulted solution was cooled off, filtrated through a 0.45mmicroporous membrane, and directly used for HPLCanalysis.

2.4. Chromatographic analysis

HPLC separation was carried out in a C18 column(250 mm 4.6 mm, i.d. 5m) at room temperature. Elu-ent was methanol:water:acetic acid (20:80:1.0, v/v) at1 ml min1. Sample volume was 10l. The elution wasmonitored at 240 nm. Calibration curve method was usedfor the analysis of chlorogenic acid and geniposidic acid.Retention times of these two organic acids were 4.398 and13.504 min, respectively (Fig. 2).

3. Results and discussion

3.1. The preliminary test of the extracting conditions

During focused microwave-assisted extraction of genipo-sidic and chlorogenic acids from E. ulmodies, there are many

Table 1The factors and levels for the orthogonal design

Levels Microwavepower(%)

Irradiationtime (s)

Solvent volume(ml g1 sample )

Methanolconcentration inwater (v/v, % )

A B C D

1 90 10 5.0 202 70 30 10.0 503 50 50 20.0 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. microwavepower, irradiation time, solvent volume and solvent type,were tested. Because these two components are easily dis-solved in water and methanol, methanol:water mixture wasused as extraction solvent in preliminary tests. For each vari-able, the influence on extraction efficiency was consideredfrom three levels. Table 1 lists the factors and levels used inthe tests.

In general, a full evaluation of the effect of four factorsfrom 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). Bythis way, only nine experiments were needed. The extractionefficiencies of geniposidic and chlorogenic acids obtainedunder orthogonal conditions are also shown in Table 2. Toanalyze the influence of each variable on extraction results,an intuitionstic plot as Fig. 3 was constructed based on thedata in Table 2.

It can be seen from Fig. 3a that, for chlorogenic acid,the effects of the four factors on extraction efficiency de-crease in the order of irradiation time (B), solvent volume(C), methanol concentration in water (D) and microwavepower (A). When the irradiation time was increased from10 to 50 s, the extraction efficiency of chlorogenic acid in-creased at first, then followed by decrease. A similar situa-tion took place when the solvent volume was increased from5.0 to 20.0 ml g1 sample. However, a different situationhappened with the microwave power and methanol concen-

Table 2The results of orthogonal test L9 (34) (n = 3)Testnumbers

A B C D Extractionefficiency ofgeniposidicacid (%)

Extractionefficiency ofchlorogenicacid (%)

1 1 1 1 1 35.9 1.02 31.2 0.672 1 2 2 2 50.9 0.85 60.2 3.423 1 3 3 3 57.6 3.22 46.3 2.534 2 1 2 3 42.8 2.19 40.1 2.765 2 2 3 1 50.0 1.84 56.6 1.296 2 3 1 2 41.5 0.55 43.7 0.467 3 1 3 2 48.4 1.66 39.8 2.508 3 2 1 3 51.0 1.35 47.4 1.829 3 3 2 1 60.0 2.15 56.1 2.11

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

35

40

45

50

55Ex

tract

ion

effi

cienc

y(%)

A B C DFactors

Level 1Level 2Level 3

40

45

50

55

Extra

ctio

neffi

cien

cy(%

)

A B C DFactors

Level 1Level 2Level 3

(a)

(b)

Fig. 3. The extraction efficiencies of chlorogenic acid (a) and geniposidicacid (b) under orthogonal conditions (for designations of factors A, B, Cand D, see Table 1).

tration in water: the higher the power or concentration, thelower the extraction efficiency. Thus, the best conditions,obtained by preliminary test, for the extraction of chloro-genic acid were: irradiation time = 30 s, solvent volume =10 ml g1 sample, 20% methanol concentration in water and50% microwave power.

Different with chlorogenic acid, the influence of thesefactors on extraction efficiency of geniposidic acid decreasein 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 conditionsfor the extraction of geniposidic acid were selected: 50 sof 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 effectiveconstituents from E. ulmodies

Because there might exist some interaction among vari-ables during focused microwave-assisted extraction, theinfluence of this interaction on extraction needed to beconsidered when the conditions were optimized. For thispurpose, graphs of the following variable pair were con-structed: (a) the highest value for two factors; (b) the lowestvalue for two factors; (c) the highest and the lowest valuesfor each pair of factors. In this method, interaction graphsfor all pairs of factors were obtained.

3.2.1. Interaction of irradiation time with the other factorsFig. 4a shows the influence of the interaction between irra-

diation time and microwave power on extraction efficiencies

Extr

actio

n ef

ficie

ncy

of ge

nipo

sidi

c ac

id (%

)

36

42

48

54

Irradiation time = 50 sIrradiation time = 10 s

Extr

actio

n ef

ficie

ncy

of ch

loro

geni

c ac

id (%

)

27

36

45

54Irradiation time = 50 sIrradiation time = 10 s

Extr

actio

n ef

ficie

ncy

of ge

nipo

sidi

c ac

id (%

)

35

40

45Irradiation time= 50 s

Irradiation time= 10 s

Extr

actio

n ef

ficie

ncy

of ch

loro

geni

c ac

id (%

)

40

45

50

55

Irradiation time= 50 sIrradiation time= 10 s

50%

90%50%

90%

50%

90%50%

90%

5.0

5.0

20.0

20.0

5.0

5.0

20.0

20.0

(a)

(b)

Microwave power (%)

Solvent volume (ml/g sample)

Extr

actio

n ef

ficie

ncy

of ge

nipo

sidi

c ac

id (%

)

35

40

45

50

Irradiation time= 50 sIrradiation time= 10 s

Extr

actio

n ef

ficie

ncy

of ch

loro

geni

c ac

id (%

)

48

51

54

57

60

Irradiation time = 50 sIrradiation time = 10 s

20%

20% 80%

80%

20%

20% 80%

80%

Methanol concertrationin water (v/v %)(c)

Fig. 4. The influence of the interaction of irradiation time with microwavepower (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 ob-tained by irradiating for 10 s is higher than for 50 s. On thecontrary, when 50% microwave power is used, the longerthe irradiation time, the higher the extraction efficiency.Furthermore, the extraction efficiency of geniposidic acidobtained with 90% microwave power is dramatically higher

H. Li et al. / Talanta 63 (2004) 659665 663

than that obtained with 50% microwave power at 10 s ofirradiation time. Even though in intuitionistic plot, 50%microwave power may produce a slightly higher extractionefficiency of geniposidic acid than 90% microwave power,the interaction between irradiation time and microwavepower indicates that extraction of geniposidic acid with 90%microwave power at 10 s of irradiation time is more effec-tive. Whereas, for chlorogenic acid, extraction efficiencygained at 90% microwave power is much lower than thatgained at 50% microwave power when irradiation timeis 50 s.

Interaction graph of irradiation time-solvent volume pair(Fig. 4b) shows a different behavior for the tested compo-nents. When 5.0 ml solvent per gram sample is used, forgeniposidic acid, a 4% higher extraction efficiency can beproduced in 10 s than in 50 s of irradiation time, however,for chlorogenic acid, a 9% lower extraction efficiency is ob-tained. Like geniposidic acid, the larger the solvent volume,the higher the extraction efficiency of chlorogenic acid whenthe sample is irradiated for 50 s. But at 10 s irradiation, com-pared with using 5 ml solvent per gram sample, a 9.6% lowerextraction efficiency is observed when 20.0 ml solvent pergram sample is used for geniposidic acid; whereas, a 11.9%higher extraction efficiency can be gained under the sameconditions for chlorogenic acid.

The interaction between irradiation time and methanolconcentration in water is shown in Fig. 4c. It can be foundthat when methanol concentration in water is 20%, the ex-traction efficiency of geniposidic acid hardly changes withthe increase of irradiation time, however, for chlorogenicacid, a 7.2% higher extraction efficiency can be observedwhen the sample is irradiated for 50 s than for 10 s. Using80% methanol as extracting solvent may produce a 14.1%increase in the extraction efficiency of geniposidic acid whenirradiation time increases from 10 to 50 s, and reversely,a 8.0% decrease is observed for chlorogenic acid. In ad-dition, when irradiation time is fixed, a completely oppo-site change for these two compounds can be observed withthe increase of methanol concentration in water from 20to 80%.

3.2.2. The influence of interaction of solvent volume withmicrowave power and methanol concentration in water onextraction process

Interaction of microwave power with solvent volume isconstructed in Fig. 5a. It is found that microwave power has aweaker effect on the extraction efficiency of geniposidic acidand 50% microwave power is beneficial for the extraction ofchlorogenic acid. For these two compounds, the larger thesolvent volume, the higher the extraction efficiency at thesame microwave power.

Fig. 5b gives the interaction results between solvent vol-ume and methanol concentration in water. For these two or-ganic acids, when the methanol concentration in water goesup from 20 to 80%, a similar behavior can be observed ex-cept a weaker increase of extraction efficiency of geniposidic

Extr

actio

n ef

ficie

ncy

ofge

nipo

sidi

c ac

id (%

)

38404244464850

Microwave power= 50%Microwave power= 90%

Extr

actio

n ef

ficie

ncy

of ch

loro

geni

c ac

id (%

)

56586062646668

Microwave power= 50%Microwave power= 90%

Extr

actio

n ef

ficie

ncy

of ge

nipo

sidi

c ac

id (%

)

38

40

42

44

46

48

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

Extr

actio

n ef

ficie

ncy

of ch

loro

geni

c ac

id (%

)

45

50

55

60

65

70

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

(a) Solvent volume (ml/g sample)

(b) Methanol concentration in water (v/v %)

5.0

5.0

20.0

20.0

5.0

5.0

20.0

20.0

20%

20%

80%

80%

20%

20%

80%

80%

Fig. 5. The effect of the interaction of solvent volume with microwavepower (a) and methanol concentration in water (b) on extraction efficien-cies of geniposidic acid and chlorogenic acid.

acid under 20 ml solvent per gram sample. The interactiongraph indicates that the optimized solvent volume should be20 ml g1 sample.

3.2.3. Interaction between methanol concentration inwater and microwave power

The interaction between microwave power and methanolconcentration in water had a great influence on extractionprocess (Fig. 6). For geniposidic acid, a 10.3% higher ex-traction efficiency is gained using 90% than 50% microwavepower in 20% methanol water solution. At the 90% mi-crowave power, the higher the methanol concentration, thelower the extraction efficiency of geniposidic acid. However,for chlorogenic acid, the larger the methanol concentration,the higher the extraction efficiency at these two microwavepowers. A 1.1% higher extraction efficiency of chlorogenicacid is obtained using 90% microwave power than using50% 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 ex-traction efficiencies of geniposidic acid and chlorogenic acidin E. ulmodies and the intuitionistic plot of two indexes ex-perimental design, it can be concluded that, 50% microwave

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

Extr

actio

n ef

ficie

ncy

ofge

nipo

sidi

c ac

id (%

)

44

48

52

56Microwave power = 50%Microwave power = 90%

Extr

actio

n ef

ficie

ncy

of ch

loro

geni

c ac

id (%

)

68

70

72

74Microwave power = 50%Microwave power = 90%

20%

80%20%

80%

20%

20%

80%

80%

Methanol concentrationin water (v/v %)

Fig. 6. Effects of the interaction between methanol concentration andmicrowave power on extraction efficiency of geniposidic acid and chloro-genic acid.

power and 20 ml solvent per gram sample are more favor-able for the extraction of these two constituents.

3.3. The further optimization of methanol concentrationin water and irradiation time

To find out the optimum methanol concentration formicrowave-assisted extraction of geniposidic acid andchlorogenic acid, extraction was carried out in methanolwater 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 effi-ciency increases when methanol content in water increasesfrom 0 to 80%, reaching the highest value (54.2%) at 80%,and then starts to decrease. For chlorogenic acid, the max-imum extraction efficiency appears at 20% methanol watersolution.

In the following experiments, by changing the irradia-tion time from 5 to 70 s, the extraction efficiency of thesetwo compounds was further investigated. All the extractions

0 20 40 60 80 10040

44

48

52

56

geniposidic acid chlorogenic acid

Extr

actio

n ef

ficie

ncy (

% )

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

were repeated for three times. The result shows that, whenirradiation time increases from 5 to 40 s, the extraction ef-ficiency 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 un-til a maximum value (63.7%) is reached at 30 s irradiation,followed by slight decrease. Apparently, for these two com-ponents, overlong irradiation can lower the extraction effi-ciency under focused microwave irradiation.

Hence, to effectively extract geniposidic acid and chloro-genic acid from E. ulmodies by focused microwave ir-radiation, the optimum extraction conditions were: forgeniposidic acid: 50% microwave power, 20 ml solvent pergram sample, 80% methanol water solution as extractionsolvent 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 focusedmicrowave-assisted extraction process

Because the ionization of geniposidic and chlorogenicacids might happen during focused microwave heating pro-cess, acetic acid was added into the extraction solvent tostudy the influence on extraction efficiencies of the twotested organic acids. It was found from the results that forthese two compounds, the extraction efficiency increaseswith 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 theextraction of geniposidic and chlorogenic acids.

3.5. The consistency between extraction solvent andmobile phase

The consistency between the composition of the extrac-tion solvent in focused microwave-assisted extraction andthat of the mobile phase in HPLC separation is helpful forthe pretreatment of the sample. Usually the composition ofthe solvent used in the extraction process is different fromthat of the mobile phase used in the HPLC separation. Ad-ditional treatment is needed for the extraction solvent to beadjusted to satisfy the HPLC requirement, including evapo-ration, adding other chemicals, or filtration, centrifugation,etc. [2628]. In this work, we have succeeded to adjustthe extractant constituents to be directly used as the HPLCmobile phase.

For quantification of chlorogenic acid and geniposidicacid by HPLC, mobile phase used was methanol:water:aceticacid (20:80:1.0, v/v) solvent mixture, where acetic acidwas added to prevent the ionization of organic acids. Fur-thermore, it is the acetic acid in the extraction solvent thatobviously improve the extraction efficiency of these twocompounds, in correlation to its ionization-inhibition ac-tion 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 3The repeatability of extraction process

Sample Extraction efficiencies (%)Geniposidic acid Chlorogenic acid

1 2 3 Mean R.S.D. (%) 1 2 3 Mean R.S.D. (%)1# 79.3 81.0 80.9 80.4 78.4 78.1 77.4 77.92# 79.0 79.0 78.4 78.8 81.1 82.3 82.0 81.83# 72.8 73.5 74.5 73.6 3.8 76.0 75.7 76.3 76.0 4.14# 83.6 82.5 83.5 83.2 85.2 82.8 83.7 83.95# 75.1 76.3 75.4 75.6 84.2 87.2 86.3 85.9

increased from 1 to 3%, a similar peak separation betweengeniposidic 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 micro-wave-assisted extraction conditions from E. ulmodies werefound 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 chlorogenicacid: 50% microwave power, 20% (v/v) aqueous methanol(20 ml g1 sample) and 30 s irradiation.

The repeatability of the focused microwave-assisted sol-vent extraction of geniposidic acid and chlorogenic acidfrom E. ulmodies was determined. Five samples with thesame weight (0.5 g) were processed under the conditionsof 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 geni-posidic and chlorogenic acid were 3.8 and 4.1%, respectively(Table 3).

The repeatability of chromatographic analysis was alsoconsidered in order to determine the errors. 0.5 g samplewas processed for 30 s at 50% microwave power using 10 mlsolvent as above. The sample was analyzed repeatedly forfive times under the same chromatographic conditions. Theextraction 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 geniposidicacid and 1.3% for chlorogenic acid.

Acknowledgements

Financial supports from China NSF, China MST and Hu-nan Provincial Department of Science & Technology wereacknowledged.

References

[1] E.D. Conte, C.Y. Shen, P.W. Perschbacher, D.W. Miller, J. Agric.Food Chem. 44 (1996) 829.

[2] B. Kaufmann, P. Christen, J.L. Veuthey, Phytochem. Anal. 12 (2001)327.

[3] N. Hong, V.A. Yaylayan, G.S. Raghavan, J.R. Park, J.M. Blanger,Nat. Prod. Lett. 15 (2001) 197.

[4] W.H. Ho, S.J. Hsieh, Anal. Chim. Acta. 428 (2001) 111.[5] C.S. Eskilsson, E. Bjorklund, J. Chromatogr. A. 902 (2000) 227.[6] Z.K. Guo, Q.H. Jin, G.Q. Fan, Y.P. Duan, C. Qin, M.J. Wen, Anal.

Chim. Acta 436 (2001) 41.[7] M.H. Akhtar, M. Wong, S.R.H. Crooks, A. Sauve, Food Addit.

Contam. 15 (1998) 542.[8] X.J. Pan, G.G. Niu, H.Z. Liu, J. Biochem. Eng. 12 (2002) 71.[9] X.J. Pan, H.Z. Niu, G.H. Jia, Y.Y. Shu, J. Biochem. Eng. 5 (2000)

173.[10] L.E. Garcia-Ayuso, M. Sanchez, A. Fernandez de Alba, M.D. Luque

de Castro, Anal. Chem. 70 (1998) 2426.[11] L.E. Garcia-Ayuso, J.L. Luque-Garcia, M.D. Luque de Castro, Anal.

Chem. 72 (2000) 3627.[12] C. Falque-Cao, Z. Wang, L. Urruty, J.J. Pommier, M. Montury, J.

Agric. Food Chem. 49 (2001) 5092.[13] J.L. Luque-Garcia, M.D. Luque de Castro, Anal. Chem. 73 (2001)

5903.[14] D. Takeshi, N. Sansci, N. Yoshihisa, Acta Pharmacol. Sin. 22 (2001)

1057.[15] Y. Li, T. Sato, K. Metori, K. Koike, Q. Che, S. Takahashi, Biol.

Pharm. Bull. 21 (1998) 1306.[16] Y. Li, K. Metori, K. Koike, Q. Che, S. Takahashi, Biol. Pharm. Bull.

22 (1999) 582.[17] T. Nakamura, Y. Nakazawa, S. Onizuka, Mutat. Res. 338 (1997) 7.[18] Y.F. Sasaki, A. Chiba, M. Murakam, Mutat. Res. 371 (1996) 203.[19] S.K. Yahara, K. Nakazawa, T.Y. Yoshihisa, N. Toshihiro, Chem.

Pharm. Bull. 38 (1990) 267.[20] R.R. Paris, M. Nishio, C. R. Acad. Hebd. Seances Acad. Sci. D.

270 (1970) 1465.[21] F. Hayase, H. Kato, J. Nutr. Sci. Vitaminol. 30 (1984) 37.[22] G.D. Lyon, H. Barker, Potato Res. 27 (1984) 291.[23] B. Sun, M.J. Peng, X.Y. Yang, Chem. Ind. Forest Prod. 19 (1999) 67.[24] X. Pan, G. Niu, H. Lin, J. Chromatogr. A. 922 (2001) 371.[25] H.B. Li, F. Chen, J. Chromatogr. A. 932 (2001) 91.[26] S.Q. Luo, Chin. Tradition. Herb. Drugs. 20 (1989) 19.[27] X.Y. Qi, W.J. Chen, S.H. Zhang, J. Pharm. Anal. 20 (2000) 22.[28] G. Xiong, J. Liang, S. Zou, Z. Zhang, Anal. Chim. Acta. 371 (1998)

97.

Focused microwave-assisted solvent extraction and HPLC determination of effective constituents in Eucommia ulmodies Oliv. (E. ulmodies)IntroductionExperimentalApparatusReagents and materialsFocused microwave-assisted extractionChromatographic analysis

Results and discussionThe preliminary test of the extracting conditionsInteraction of factors during extraction of effective constituents from E. ulmodiesInteraction of irradiation time with the other factorsThe influence of interaction of solvent volume with microwave power and methanol concentration in water on extraction processInteraction between methanol concentration in water and microwave power

The further optimization of methanol concentration in water and irradiation timeThe effect of acetic acid on focused microwave-assisted extraction processThe consistency between extraction solvent and mobile phaseRepeatability

AcknowledgementsReferences