Separation and Purication Technology 62 (2008) 480483

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Separation and Purication Technology

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Microwave-assisted extraction of chlorogenic acid from ower buds of Lonicera 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 China b Beijing Pharmaceutical Group Company Limited, Beijing 100020, PR China c Graduate School of the Chinese Academy of Sciences, Beijing, 100049, PR China

a r t i c l e

i n f o

a b s t r a c t
An efcient microwave-assisted extraction (MAE) technique has been developed to recover chlorogenic acid from ower 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 short duration and high efciency to recover chlorogenic acid from raw plant materials in comparison with conventional heat-reux 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 signicantly destroyed due to the cell rupture after MAE treatment. 2008 Elsevier B.V. All rights reserved.

Article history: Received 2 October 2007 Received in revised form 12 February 2008 Accepted 14 February 2008 Keywords: Chlorogenic acid Heat-ux extraction Lonicera japonica Thunb. Microwave-assisted extraction Scanning electron microscopy

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 component in the ower buds, has received more and more attention because of its antivirus, anticancer and antiinammation activities [24]. Extraction is the rst step for preparation of medicine from raw plant materials and signicantly affects the cost of the whole manufacture process. Extraction of chologenic acid from the ower buds of L. japonica is conventionally performed by heat-reux extraction. The traditional extraction process is time-consuming and laborious, 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 compounds 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-

tions. MAE has been used for extraction of interested components from 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 considerably reduce both extraction time and solvent consumption [9,10]. The objective of this current work was to investigate the feasibility of employing MAE as an efcient technique to recover chologenic acid from L. japonica ower 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 ower buds of Lonicera japonica Thunb. from local company 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 reagents used in the experiment were of analytical grade and purchased from Atoz Fine Chemicals Co. Ltd. (Tianjin, China). All aqueous solutions were prepared with pure water produced by Milli-Q system (Bedford, MA, USA). Chlorogenic acid stock solutions were prepared by dissolving 20 mg chlorogenic acid in 10 ml methanol and stored at 20 C. The

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: czliu@home.ipe.ac.cn (C.-Z. Liu). 1383-5866/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2008.02.013

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Fig. 1. Chemical structure of chlorogenic acid.

standard chlorogenic acid solutions at the concentration of the calibration range were prepared by serial dilutions of stock solutions with methanol. 2.2. Chlorogenic acid extraction A household microwave oven was modied in our laboratory with the addition of a magnetic stirrer, water condenser, temperature 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 100 ml 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-reux 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 ask with a mechanical stirrer and the extraction temperature was kept at 60 C. All samples were centrifuged at 4250 g for 5 min, and ltered through 0.45 m membrane before analysis by high performance liquid chromatography (HPLC). In the present study, the yield of chlorogenic acid is dened as follows: Yield of chlorogenic acid (w/w) = mass of chlorogenic acid in extraction solution/mass of plant materials 100%. 2.3. Analytical method Quantication 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 20 l injection loop. The data were acquired and processed by Agilent chemstation software. An Alltech C18 column (250 mm 4.6 mm I.D., 5 m) (Deereld, IL, USA) tted with an Alltech C18 guard cartridge (8 mm 4.6 mm I.D., 5 m) 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 ow rate of 1 ml/min, and the efuent was monitored at 327 nm by UV detector. 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 calibration range is 0.12.0 mg ml1 (correlation coefcient 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 standard chlorogenic acid dissolved in 100 ml extraction solvent (50% ethanol) was mixed with 10 g 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. Chromatogram of the (A) standard chlorogenic acid and (B) MAE crude extract from ower buds of Lonicera 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 efciently 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 samples were plunged in liquid nitrogen and then cut with a cold knife. The sectioned particles were xed on a specimen holder with aluminum 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 5.0 kV (10 m, 500 magnication). 3. Results and discussion 3.1. Microwave-assisted extraction of chlorogenic acid In previous studies, acetone, ethanol, methanol, water and mixtures of these solvents were used as extractants for chlorogenic acid recovery from ower buds of L. japonica [1113]. Generally, absorption of the microwave energy increases with the dielectric constant

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Fig. 3. Effect of extraction solvent of MAE on yield of chlorogenic acid from ower buds of Lonicera japonica Thunb.

Fig. 5. Effect of extraction temperature of MAE on yield of chlorogenic acid from ower buds of Lonicera 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 efciently absorb microwave energy and leads to efcient heating. The mixtures of water and ethanol were selected for our current study based 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 solubility 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 materials/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 extraction process [14,15]. If the extraction was carried out under low solid/liquid ratio, the concentration of chlorogenic acid in extraction solution was low. This indicated that more energy and time were needed to condense the extraction solution in later separation and purication process. Therefore, the solid/liquid ratio of 1:10 (g:ml) was sufcient to reach the high yield of chlorogenic acid. The inuence of MAE temperature on yield of chlorogenic acid is shown in Fig. 5. The yield of chlorogenic acid increased rapidly at

60 C and 80 C in 5 min, and then slowed down to reach an equilibrium concentration. The yield of chlorogenic acid reached 6.14% and at 60 C within 5 min, which was signicantly higher than that at 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-reux extraction A conventional heat-reux 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 concentration of 50% as extraction solvent within 5 min. In comparison to the heat-ux extraction, the MAE showed obvious advantages in terms of short duration and high efciency to extract chlorogenic acid from the plant materials. This is mainly due to the fact that microwave energy is delivered efciently to materials through molecular interaction with the electromagnetic eld and offers a rapid transfer of energy to the extraction solvent and raw plant materials [16]. The treated plant materials by MAE and heat-reux extraction were examined by scanning electron microscopy, and their micrographs are shown in Fig. 7. The change from heat-reux extraction sample was not considerably different from that from the untreated

Fig. 4. Effect of solid/liquid ratio in MAE on yield of chlorogenic acid from ower buds of Lonicera japonica Thunb.

Fig. 6. Heat-reux extraction of chlorogenic acid from ower buds of Lonicera japonica Thunb.

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Fig. 7. Scanning electron micrographs of plant materials: (A) untreated sample; (B) heat-reux 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 temperature rise and internal pressure increase inside the cells of plant sample. During the rupture process, a rapid exudation of the chemical substance within the cells into the surrounding solvents took place. 4. Conclusions An efcient microwave-assisted extraction method of chlorogenic acid from L. japonica ower buds has been developed. Compared with the conventional heat-reux extraction, reduced extraction time and high recovery of chlorogenic acid were obtained with MAE. Under optimal MAE 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. References
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