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Separation and Purification Technology 62 (2008) 480483

Contents lists available at ScienceDirect

Separation and Purification Technology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / s e p p u r

Short communication

Microwave-assisted extraction of chlorogenic acid fromflower buds ofLonicera japonica Thunb.

Bin Zhang a,c, Ruiyuan Yang b, Chun-Zhao Liu a,c,

a National Key Laboratory of Biochemical Engineering, Institute of Process Engineering,

Chinese Academy of Sciences, Beijing 100080, PR Chinab Beijing Pharmaceutical Group Company Limited, Beijing 100020, PR Chinac Graduate School of the Chinese Academy of Sciences, Beijing, 100049, PR China

a r t i c l e i n f o

Article history:

Received 2 October 2007

Received in revised form 12 February 2008

Accepted 14 February 2008

Keywords:

Chlorogenic acid

Heat-flux extraction

Lonicera japonica Thunb.

Microwave-assisted extraction

Scanning electron microscopy

a b s t r a c t

An efficient microwave-assisted extraction (MAE) technique has been developed to recover chlorogenic

acid from flower buds of Lonicera japonica Thunb. The yield of chlorogenic acid rapidly reached 6.14%

within 5 min under the optimal MAE conditions, i.e. 50% ethanol as extraction solvent, 1:10 (w/v) of the

solid/liquid ratio and 60 C of extraction temperature. The MAE showed obvious advantages in terms of

shortduration and highefficiency to recover chlorogenic acidfrom rawplant materialsin comparisonwith

conventional heat-reflux extraction. The mechanism of the enhanced extraction by microwave assistance

was discussed by observing cell destruction of plant material after MAE treatment by scanning electron

microscopy. The results showed that the plant materials were significantly destroyed due to the cell

rupture after MAE treatment.

2008 Elsevier B.V. All rights reserved.

1. Introduction

Flower buds of Lonicera japonica Thunb. are traditionally used

as a herbal medicine in the treatment of a wide range of ailments

including syphilitic skin diseases, tumors, bacterial dysentery,

colds, enteritis, pain, swellings, etc. [1]. Chologenic acid (Fig. 1.),

a major bioactive componentin the flower buds, hasreceivedmore

and more attention because of its antivirus, anticancer and anti-

inflammation activities [24].

Extraction is the first step for preparation of medicine from raw

plant materials and significantly affects the cost of the whole man-

ufactureprocess. Extraction of chologenicacid fromthe flower buds

ofL. japonica is conventionallyperformed by heat-reflux extraction.

The traditional extraction process is time-consuming and labori-

ous, and involves lengthy operation techniques and bulk amount of

organic solvents. Microwave-assisted extraction (MAE) is a process

that uses microwave energy and solvents to extract target com-

pounds from various matrices. The highly localized temperature

and pressure can cause selective migration of target compounds

from the material to the surroundings at more rapid rate and with

similar or better recoveries compared with conventional extrac-

Corresponding author at: National Key Laboratory of Biochemical Engineering,

Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, PR

China. Tel.: +86 10 82622280; fax: +86 10 82622280.

E-mail address: [email protected] (C.-Z. Liu).

tions. MAE has been used for extraction of interested componentsfrom a wide variety of sample matrices and has been used as a

promising alternative sample preparation technique for a number

of applications [58]. Compared to conventional methods, MAE can

considerablyreduce both extraction time and solvent consumption

[9,10].

The objective of this current work was to investigate the fea-

sibility of employing MAE as an efficient technique to recover

chologenic acid from L. japonica flower buds. The optimization of

MAE method was carried out, and the mechanism of the enhanced

extraction by MAE was discussed by observing cell destruction of

plant material by scanning electron microscopy.

2. Materials and methods

2.1. Plant material and chemicals

Dried flower buds of Lonicera japonica Thunb. from local com-

pany in Sichuan province of China were ground into 60 mesh

powders by a mortar, and then were kept at room temperature.

HPLC-grade methanol was purchased from Concord Tech Co. Ltd.

(Tianjin,China).All reagentsused in the experiment were of analyt-

ical grade and purchasedfrom Atoz Fine Chemicals Co. Ltd.(Tianjin,

China). All aqueous solutions were prepared with pure water pro-

duced by Milli-Q system (Bedford, MA, USA).

Chlorogenic acid stock solutions were prepared by dissolving

20mg chlorogenic acid in 10 ml methanoland stored at20 C. The

1383-5866/$ see front matter 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.seppur.2008.02.013
http://www.sciencedirect.com/science/journal/13835866mailto:[email protected]://dx.doi.org/10.1016/j.seppur.2008.02.013http://dx.doi.org/10.1016/j.seppur.2008.02.013mailto:[email protected]://www.sciencedirect.com/science/journal/13835866


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B. Zhang et al. / Separation and Purification Technology 62 (2008) 480483 481

Fig. 1. Chemical structure of chlorogenic acid.

standard chlorogenic acid solutions at the concentration of the cal-

ibration range were prepared by serial dilutions of stock solutions

with methanol.

2.2. Chlorogenic acid extraction

A household microwave oven was modified in our laboratory

with the addition of a magnetic stirrer, water condenser, temper-ature measurement and time controlling for automatic MAE [9].

With ice water running through the condensation pipe of the MAE

system, a given amount (5, 10, 15 and 20 g) of dried plant samples

was mixed with 100ml mixtures of ethanol and water, and then

the suspensions were irradiated automatically with microwave at

a power of 700 W in a pre-setting procedure (a given time of Power

On for heating and a given time of Power Off for cooling) in order

to keep a desired extraction temperature (40 C, 60 C and 80 C).

Heat-reflux extraction using a water-bath was performed with

10 g dried plant samples and 100 ml of mixtures of ethanol and

water in a 250-m flask with a mechanical stirrer andthe extraction

temperature was kept at 60 C.

All samples were centrifuged at 4250gfor 5 min, and filtered

through 0.45m membrane before analysis by high performanceliquid chromatography (HPLC). In the present study, the yield of

chlorogenic acid is defined as follows: Yield of chlorogenic acid

(w/w) = mass of chlorogenic acid in extraction solution/mass of

plant materials100%.

2.3. Analytical method

Quantification of chlorogenic acid was carried out by Agilent

1100 HPLC system equipped with a quaternary pump, an on-line

solvent vacuum degasser, a variable wavelength detector and an

auto sampler with a 20l injection loop. The data were acquired

and processed by Agilent chemstation software. An Alltech C18

column (250 mm4.6mm I.D., 5m) (Deerfield, IL, USA) fitted

with an Alltech C18 guard cartridge (8 mm4.6mm I.D., 5m)was used at a column temperature of 25 C. The mobile phase was

MeOH: 2% acetic acid-water solution (20:80, v/v) at a flow rate of

1 ml/min, and the effluent was monitored at 327nm by UV detec-

tor. Chlorogenic acid standard was supplied by National Institute

for the Control of Pharmaceutical and Biological Product (Beijing,

China) with the purity no less than 98%. The method was validated

to achieve the satisfactory precision and recovery, and the calibra-

tion range is 0.12.0 mg ml1 (correlation coefficient R = 0.9998).

In order to determine accuracy of the MAE procedure, the known

amount (low, medium and high level of 10, 30, 50 mg) of stan-

dard chlorogenic acid dissolved in 100 ml extraction solvent (50%

ethanol) was mixed with 10g of the same batch of plant samples.

The recovery of chlorogenic acid is between 98.62% and 102.37%,

and R.S.D. is between 3.87% and 5.26%. The MAE procedure pro-

Fig.2. Chromatogramof the(A) standardchlorogenicacidand (B)MAE crudeextract

from flower buds ofLonicera japonica Thunb.

vides high recovery and accuracy and is acceptable for the routine

analysis. As shown in Fig. 2, the crude extract of the plant materials

was separated efficiently under the above HPLC conditions.

2.4. Scanning electron micrographs

In order to understand the mechanism of MAE, samples from

different extraction methods were used to take scanning electron

micrographs. After removing the solvent, the remaining plant sam-

ples were plunged in liquid nitrogen and then cut with a cold

knife.The sectionedparticles were fixed on a specimen holderwithaluminum tape and then sputtered with gold in a JEOL JEC-1200

sputter-coater (Tokyo, Japan). All the specimens were examined

with a JEOL JSM-5600 LV scanning electron microscopy (Tokyo,

Japan) under high vacuum condition at an accelerating voltage of

5.0kV (10m, 500 magnification).

3. Results and discussion

3.1. Microwave-assisted extraction of chlorogenic acid

In previous studies, acetone, ethanol, methanol, water and mix-

tures of these solventswere used as extractants for chlorogenicacid

recoveryfrom flower buds ofL. japonica [1113]. Generally, absorp-

tion of the microwave energy increases with the dielectric constant
http://dx.doi.org/10.1016/j.seppur.2008.02.013http://dx.doi.org/10.1016/j.seppur.2008.02.013http://dx.doi.org/10.1016/j.seppur.2008.02.013http://dx.doi.org/10.1016/j.seppur.2008.02.013http://dx.doi.org/10.1016/j.seppur.2008.02.013http://dx.doi.org/10.1016/j.seppur.2008.02.013http://dx.doi.org/10.1016/j.seppur.2008.02.013http://dx.doi.org/10.1016/j.seppur.2008.02.013http://dx.doi.org/10.1016/j.seppur.2008.02.013http://dx.doi.org/10.1016/j.seppur.2008.02.013http://dx.doi.org/10.1016/j.seppur.2008.02.013


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482 B. Zhang et al. / Separation and Purification Technology 62 (2008) 480483

Fig. 3. Effect of extraction solvent of MAE on yield of chlorogenic acid from flower

buds ofLonicera japonica Thunb.

of the molecule, resulting in power dissipated inside the solvent

and plant materials and then generating more effective molecular

movement and heating. As a polar solvent, water can efficientlyabsorb microwave energy and leads to efficient heating. The mix-

turesofwaterandethanolwereselectedforourcurrentstudybased

on our primary experiments and the previous reports [1113]. As

shown in Fig. 3, 50% ethanol extraction solvent gave the best yield

of chlorogenic acid in 5 min at MAE temperature of 60 C. For MAE,

the choice of extraction solvent takes into account not only its solu-

bility for target component but also its ability to absorb microwave

energy.

As shown in Fig. 4, the yield of chlorogenic acid decreased

with the increase of solid/liquid ratios (amount of plant materi-

als/volume of extraction solvent) from 1:20 (g:ml) to 1:5 (g:ml).

Microwave energy was absorbed and dispersed by larger amounts

of plant materials, which was disadvantageous for the extrac-

tion process [14,15]. If the extraction was carried out under lowsolid/liquid ratio, the concentration of chlorogenic acid in extrac-

tion solution was low. This indicated that more energy and time

were needed to condense the extraction solution in later separa-

tion and purification process. Therefore, the solid/liquid ratio of

1:10 (g:ml) was sufficient to reach the high yield of chlorogenic

acid.

The influence of MAE temperature on yield of chlorogenic acid

is shown in Fig. 5. The yield of chlorogenic acid increased rapidly at

Fig. 4. Effect of solid/liquid ratio in MAE on yield of chlorogenic acid from flower

buds ofLonicera japonica Thunb.

Fig. 5. Effect of extraction temperature of MAE on yield of chlorogenic acid from

flower buds ofLonicera japonica Thunb.

60 C and 80 C in 5 min, and then slowed down to reach an equi-

librium concentration. The yield of chlorogenic acid reached 6.14%

and at60 C within 5 min, which was significantly higher than thatat 40 C. There was no obvious difference on yield of chlorogenic

acid between 60 C and 80 C; therefore, the MAE temperature of

60 C was selected suitable for chlorogenic acid extraction from the

raw plant materials.

3.2. Comparison of MAE with conventional heat-reflux extraction

A conventional heat-reflux extraction of chlorogenic acid from

raw plant materials was carried out at 60 C. As show in Fig. 6, the

yield of chlorogenic acid reached 5.19% at an optimal ethanol con-

centration of 50% as extraction solvent within5 min. In comparison

to the heat-flux extraction, the MAE showed obvious advantages

in terms of short duration and high efficiency to extract chloro-

genic acid from the plant materials. This is mainly due to the factthat microwave energy is delivered efficiently to materials through

molecular interaction with the electromagnetic field and offers a

rapid transfer of energy to the extraction solvent and raw plant

materials [16].

The treated plant materials by MAE and heat-reflux extraction

were examined by scanning electron microscopy, and their micro-

graphs are shown in Fig. 7. The change from heat-reflux extraction

sample was notconsiderably differentfrom thatfrom the untreated

Fig.6. Heat-reflux extraction of chlorogenicacid fromflowerbuds ofLonicera japon-

ica Thunb.


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B. Zhang et al. / Separation and Purification Technology 62 (2008) 480483 483

Fig. 7. Scanning electron micrographs of plant materials: (A) untreated sample; (B) heat-reflux extraction sample; and (C) MAE sample.

sample, and only few slight ruptures happened on its surface. The

surface of the MAE sample was greatly destroyed because that

microwave irradiation accelerated cell rupture by sudden temper-

ature rise and internal pressure increase inside the cells of plantsample. Duringthe rupture process, a rapid exudation of thechem-

ical substance within the cells into the surrounding solvents took

place.

4. Conclusions

An efficient microwave-assisted extraction method of chloro-

genic acid from L. japonica flower buds has been developed.

Compared with the conventional heat-reflux extraction, reduced

extraction time and high recovery of chlorogenic acid were

obtainedwith MAE.Under optimalMAE conditions, i.e. 50%ethanol

as extraction solvent, 1:10 (w/v) of solid/liquid ratio and 60 C

of extraction temperature, the yield of chlorogenic acid reached

6.14% within 5 min. The enhanced extraction was related partly to

a greater extent of cell rupture of the plant materials, and this was

observed by scanning electron microscopy.

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