Separation and Purification Technology 83 (2011) 45–49

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Separation and Purification Technology

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Microwave assisted ionic liquid pretreatment of medicinal plants for fastsolvent extraction of active ingredients

Ronghua Jin a,⇑, Ling Fan b, Xiaoning An c

a State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, Chinab College of Science, South China Agricultural University, Guangzhou 510642, Chinac School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, China

a r t i c l e i n f o

Article history:Received 2 June 2011Received in revised form 28 August 2011Accepted 3 September 2011Available online 21 September 2011

Keywords:Microwave assisted ionic liquidpretreatmentIonic liquid1-N-butyl-3-methylimidazolium chloridePaeono1Cynanchum paniculatum

1383-5866/$ - see front matter � 2011 Elsevier B.V. Adoi:10.1016/j.seppur.2011.09.005

⇑ Corresponding author. Address: State Key LaborSelective Oxidation, Lanzhou Institute of Chemical PSciences, No. 18, Tianshui Middle Road, Lanzhou 74968870; fax: +86 931 4968129.

E-mail address: rh_jin@hotmail.com (R. Jin).

a b s t r a c t

For increasing active ingredients accessibility to the solvents, microwave assisted ionic liquid pretreat-ment (MILP) is introduced to destroy cell walls of medicinal plants before solvent extraction. As a newsample pretreatment method, microwave irradiation on sample pretreatment with ionic liquids (ILs)was investigated. The conditions of MILP including microwave pretreatment time and power, ionic liquid(IL) and sample ratio and particle size were optimized and Cynanchum paniculatum and its active ingre-dient paeonol were chosen as the representative target analyte. Under the optimized conditions, thepaeonol extraction yield by MILP was higher than that of ionic liquid aqueous solution and Soxhletextraction. In addition, the results of bioassay test showed that the antibacterial effect of crude extractby MILP was almost the same with that of soxhlet extraction. Our results demonstrate that sample pre-treatment with ILs and microwave irradiation is a potential alternative method for the pretreatment ofmedicinal plants before solvent extraction.

� 2011 Elsevier B.V. All rights reserved.

1. Introduction

Ionic liquids (ILs) are salts of organic cations that exist as liquidsat a relatively low temperature (<100 �C) [1] and own many attrac-tive properties, such as chemical and thermal stability, non-flam-mability and immeasurably low vapor pressure. Recently, ILshave been in the focus of interest in various applications includingcatalysis [2], synthesis, industrial cleaning, extraction [3] and sep-aration [4,5]. Application of ILs in sample preparation techniques,such as liquid–liquid extraction [6,7], liquid-phase micro-extrac-tion [8], solid-phase micro-extraction [9], and aqueous two-phasesystems extraction [10] has also been published. Following thegeneral research trend devoted to the development of extractionmethods, ILs aqueous solutions as extraction solvents to extract ac-tive ingredients in medicinal plants get considerable concern [11–13]. However, the solubility of target analytes in water limits thescope of the method.

To compensate for lack of more selective solvents, ILs pretreat-ment of medicinal plants was developed to extract active ingredi-ents in medicinal plants. The first use of ionic liquid that destroyedcell wall to improve the release of active ingredient in medicinal

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atory for Oxo Synthesis andhysics, Chinese Academy of

30000, China. Tel.: +86 931

plants was reported [14], but it took a long time (8 h) to do thesample pretreatment in the 1-N-butyl-3-methylimidazolium chlo-ride ([C4mim]Cl). However, dissolution rates could be significantlyimproved by heating in a microwave oven [15]. The dissolution ofcellulose using ILs has been providing a new platform for samplepreparation because the highest concentration of active ingredi-ents in medicinal plants is found in plant cells and the primary cellwall of medicinal plants is made primarily of cellulose. The disso-lution of medicinal plants in ILs results in a significant increase incell wall permeability, which makes active ingredients easier to re-lease. Different solvents in the MILP were used to extract the activeingredients in medicinal plants in view of the solubility of the tar-get analytes until the dissolution of medicinal plants in the[C4mim]Cl reached maximum. The selected solvent would have ahigh selectivity towards the analyte of interest excluding un-wanted matrix components [16]. In addition, the extraction pro-cess was carried out at ambient pressure and temperature.

In this study, [C4mim]Cl was selected as microwave absorptionand cellulose dissolution medium and microwave was used toaccelerate cellulose dissolution. After microwave assisted ILpretreatment, solvents were added into the mixture of sample/ILwith continuous stirring and then filtrated for subsequent analysis.The aim of the current work was to explore the feasibility of MILPof medicinal plants for fast solvent extraction of active ingredientsin medicinal plants and Cynanchum paniculatum was chosen as therepresentative target analyte. This paper gives the details of MILPand the effect of experimental parameters on paeonol extraction

Fig. 1. Chromatograms of paeonol standard solution, IL and extract of Cynanchumpaniculatum by MILP.

46 R. Jin et al. / Separation and Purification Technology 83 (2011) 45–49

yield. Microwave pretreatment time and power, IL and sample ra-tio, particle size and solvents were investigated to assess the effectof experimental conditions in view of paeonol extraction yield.

2. Experimental

2.1. Chemicals

Ethanol, methanol, diethyl ether, acetone and ethyl acetate ob-tained from Nanjing Chemical Reagent Co. Ltd. (Nanjing, China)were of analytical grade. Chromatographic-grade methanol wassupplied by Tianjin Siyou Fine Chemical Co. Ltd. (Tianjin, China).Paeonol standard was purchased from Shanghai Jingchun ReagentCo. Ltd. (Shanghai, China). [C4mim]Cl, mp 63 �C was purchasedfrom Shanghai Cheng Jie Chemical Co. Ltd. (Shanghai, China) andused after drying under vaccum at 70 �C for 24 h.

Dried root of C. paniculatum was purchased from TongrentangMedicine Cooperation in Guangzhou, China. All samples were pul-verized and passed through a stainless steel sieve. Different samplesizes were obtained and stored at room temperature in desiccatoruntil use.

2.2. Apparatus

A domestic microwave oven (MP23C-BF (KA), Midea, Shunde,China) was modified in our laboratory with the addition of amechanical stirrer. The whole system was run at atmospheric pres-sure and could be employed at the maximum power of 800 W.

High performance liquid chromatographic analysis was carriedout on a LC-20A liquid chromatography (Shimadzu, Japan) with aLC-20AT pump, a SPD-20A UV/Vis detector and a CTO-20A columnoven. All separations were performed using a C18 column(250 � 4.6 mm i.d., 5 lm particle size) from Alltech (Deerfield, IL,USA). Data acquisition and processing were accomplished withShimadzu LC solution software.

2.3. Bacterial strains

Escherichia coli and Staphylococcus aureus were used to assessthe antibacterial properties of the test samples, one Gram-positiveand one Gram-negative bacteria. The two bacterial strains weremaintained on Iso-Sensitest agar slopes [CM 471] (Oxoid, UK) atroom temperature. All strains were subcultured every 2 weeks.

2.4. Assessment of inhibition of bacterial growth

The measurement of growth inhibition was carried out in agree-ment with the method of Deans and Ritchie [17] using Iso-Sensi-test agar. Cells from cultures grown on Iso-Sensitest slopes wereinoculated using a sterile loop into fresh Iso-Sensitest broth andincubated overnight at 25 �C (10 mL volume, 108 mL�1 final con-centration). Next, 1.5 lL amounts of each culture were pipettedinto separate sterile Petri dishes to which 10 mL amounts of mol-ten Iso-Sensitest agar (45 �C) were added. Once set, wells of4 mm diameter were made in the centre of each agar plate usinga Pharmacia gel punch (Uppsala, Sweden), into which 15 lL testsubstance was added. The plates were then left undisturbed to al-low diffusion of the sample into the agar, and incubated inverted inthe dark at 25 �C for 48 h. Following this, zones of growth inhibi-tion were measured using Vernier calipers.

2.5. Microwave assisted ionic liquid pretreatment of sample

A half gram of sample (60-mesh) was placed in a 50 mL roundbottom flask and 3.7 g of the [C4mim]Cl was added as a liquid at

70 �C (i.e., above the melting point). Then, the flask was placed inthe microwave oven, irradiated for 60 s with continuous stirringand cooled to room temperature inside the oven. The operationwas repeated several times until the dissolution of sample reachedmaximum.

After the above ionic liquid pretreatment, ethanol (25 mL) wasadded into the mixture with continuous stirring for several min-utes. Then, the suspension was filtered through a sand funnel un-der vacuum at room temperature and diluted to 50 mL withethanol. All extracts were filtered through a 0.45-lm filter beforechromatographic analysis. All extraction experiments were re-peated three times.

2.6. Reference extraction method

2.6.1. Ionic liquid aqueous extraction (ILAE)A half gram of sample (60-mesh) was extracted at the opti-

mized condition of 25 mL of 1.5 mol L�1 aqueous [C4mim]Cl for8 min, 136 W of microwave power.

2.6.2. Soxhlet extraction (SE)A half gram of sample (60-mesh) was extracted at the opti-

mized condition of 25 mL ethanol for 2 h using SE apparatus,95�C of water bath.

2.7. HPLC analysis

The content of paeonol in the filtrate was determined by HPLCwith a UV detector at 274 nm. The chromatographic conditionswere as follows: the mobile phase was methanol/water (45:55,v/v); the flow rate was 1 mL min�1; the column temperature was35 �C and the injection volume was 20 lL. Paeonol was completelyseparated from other compounds. Retention time of paeonol wasabout 7.1 min (Fig. 1). The detector response was linear from4.96 to 19.84 mg mL�1 of paeonol (y = 57826x � 14104,R = 0.9994) and was used to give quantitative data. Chromato-grams of paeonol standard solution, IL and extract of C. paniculatumby MILP were shown in Fig. 1. The results indicated that there wasno interference between IL and paeonol. It is probable that the[C4mim]Cl turn out to be more polar and was eluted faster thanpaeonol.

2.8. GC–MS analysis

GC–MS analysis was performed with a Thermo Finnigan TraceDSQ mass spectrometer coupled to a Thermo Finnigan Trace GCwith a split injector (1:100) and a 0.25-lm BPX5 20 M fused silica

R. Jin et al. / Separation and Purification Technology 83 (2011) 45–49 47

capillary column with a 30 m � 0.25 mm inner diameter was used.The oven temperature was held at 60 �C for 1 min and then in-creased to 280 �C at 10 �C min�1 intervals, with a helium flow rateof 1 ml min�1. The electron ionization-MS voltage was 70 eV, andthe ion source and interface temperature were both 250 �C. Spectrawere recorded and evaluated with Xcalibur software supplied withthe device.

In the present work, the paeonol extraction yield was defined asfollows:

Extraction yield ð%Þ ¼ Quality of paeonol in exactQuality of paeonol in raw material

� 100

Fig. 3. Effect of ionic liquid/sample ratio on paeonol extraction yield. Solvent:25 mL of ethanol, 0.5 g of sample, microwave time 8 min, microwave power 136 W.

3. Results and discussion

3.1. The effect of microwave pretreatment time

Microwave pretreatment time has both positive and negativeinfluence on paeonol extraction yield. It could be observed inFig. 2 that the increase of microwave pretreatment time resultedin the increase of the paeonol extraction yield and reached maxi-mum at 8 min. However, extraction yield decreased with pro-longed microwave pretreatment time. It is because that IL whichwas used as good microwave absorption medium would raise thetemperature of the mixture very rapidly. Additionally, paeonol isa white needle crystal with a relatively low-melting point of 51–52 �C [18] and could be destroyed more and more heavily alongwith the increasing time. Thus, microwave pretreatment timewas set at 8 min in the following experiments.

3.2. The effect of ionic liquid to sample ratio

The ratio of ionic liquid and sample has positive and negativeeffect on the paeonol extraction yield. As shown in Fig. 3, paeonolextraction yield increased with the increase of ionic liquid andsample ratio, reaching the highest yield at a ratio of 7.3:1 (g/g).On the one hand, the amount of dissolved cellulose increased withan increasing mass of ionic liquid, which might have increased thepermeability of the cell wall thus resulting in higher yield of paeo-nol. On the other hand, most room temperature ionic liquids (RTIL)were vicious liquid [19] and the viscosity of cellulose/IL increased

Fig. 2. Effect of microwave pretreatment time on paeonol extraction yield. Solvent:25 mL of ethanol, 0.5 g of sample, microwave power 136 W, ionic liquid/sampleratio: 9.0:1.

with an increasing mass of ionic liquid, which negatively affectedmass transfer and barricaded the penetration of the paeonol intothe mixture of the sample/IL. The solvent with low viscosity canmore easily diffuse into the pores of plant materials [20]. There-fore, the ionic liquid and sample ratio of 7.3:1 was selected inthe subsequent experiments.

3.3. The effect of microwave pretreatment power

The effect of microwave pretreatment power has a negative ef-fect on paeonol extraction yield. Three levels of microwave pre-treatment power, i.e., 136, 264 and 440 W, were selected, whileother parameters such as solvent (25 mL of water), amount of sam-ple (0.5 g), microwave time (8 min) and ionic liquid/sample ratio(7.3:1), were kept constant. The results indicated that extractionyield decreased with an increasing microwave power. ILs areappropriate microwave heating media due to its good absorptionof microwave radiation. However, microwave heating conditionscan be too harsh and must be carefully controlled to avoid thepyrolysis of cellulose in plant samples [21]. Furthermore, paeonolevaporated and/or decomposed at 100–200 �C [22]. Sample wasslightly carbonized when the microwave radiation power was264 W. Therefore, microwave power was set at 136 W in the sub-sequent experiments.

3.4. The effect of particle size

Particle size has both positive and negative influence on thepaeonol extraction yield. The results (Fig. 4) showed that 60-meshsample was optimal for extraction of paeonol. On the one hand, thehigher amount of paeonol was released as the plant cells were de-stroyed by milling and this amount of paeonol was extracted easilyfor direct exposure to the extraction solvent. On the other hand,the amount of dissolved cellulose decreases with increasing DP(degree of polymerization) of the sample [23] thereby it was diffi-cult for ionic liquid to dissolve the larger size of sample. Moreover,small particle size was easily conglomerated by the viscous ionicliquid solution [24]. The residua and extract did not easily separateeach other so that some paeonol extracted out might remain in theresidue. In this work, 60-mesh sample was preferred for the extrac-tion of paeonol.

Fig. 4. Effect of particle size on paeonol extraction yield. Solvent: 25 mL of ethanol,0.5 g of sample, microwave time 8 min, microwave power 136 W, ionic liquid/sample ratio: 7.3:1.

Fig. 5. Effect of solvents on paeonol extraction yield. Solvent: 25 mL of solvent,0.5 g of sample, microwave time 8 min, microwave power 136 W, ionic liquid/sample ratio: 7.3:1.

Table 1Comparison of extraction yields using different extraction methods (averages of thethree replicates).

Extractionmethods

Extraction time (min) Extraction yield (mean ± SD, %)

ILAE 8 1.95 ± 0.5MILP 8 2.51 ± 0.3SE 120 2.49 ± 0.4

Table 2Antimicrobial activity of crude extract of Cynanchum paniculatum with differentextraction methods; well diameter 4.0 mm.

Extraction method Concentration(mg/ml)

Diameter of zones ofinhibition(mean ± SD, mm)

E. coli S. aureus

Soxhlet extraction 1.4871 11.1 ± 0.7 13.2 ± 0.80.7436 10.2 ± 0.1 11.1 ± 0.50.3718 9.2 ± 0.6 10.3 ± 0.60.1859 8.4 ± 0.6 9.3 ± 0.4

Microwave assisted ionic liquidpretreatment

1.4871 12.6 ± 0.6 14.6 ± 0.70.7436 11.4 ± 0.5 13.4 ± 0.60.3718 10.5 ± 0.5 11.1 ± 0.90.1859 9.5 ± 0.4 9.5 ± 0.3

48 R. Jin et al. / Separation and Purification Technology 83 (2011) 45–49

3.5. The effect of different extraction solvents

A correct choice of solvent is fundamental for obtaining an opti-mal extraction process. When selecting solvent, considerationshould be given to the analyte solubility in the solvent [25]. Severalextraction solvents were studied, including diethyl ether(0.2248 g mL�1), acetone (0.3848 g mL�1), methanol (0.3837 gmL�1), ethyl acetate (0.3286 g mL�1), ethanol (0.4887 g mL�1) andwater (0.3498 mg mL�1). The experimental results (Fig. 5) showedthat water, methanol and ethanol could reach almost the sameextraction yield. Water is not as good as the alcohol as an extractingsolvent to get the effective constituents from Chinese herbal medi-cine when they are analyzed since water will leach much starchand produce paste at higher temperature [26]. Additionally, metha-nol is volatile, flammable and harmful to the environment. As etha-nol is non-toxic and environmentally friendly solvent, it was chosento extract paeono1 from C. paniculatum.

3.6. Comparison of extraction methods

The proposed MILP approach under the optimum experimentalconditions was compared with ILAE and SE. The paeonol contentsin the extracts acquired by HPLC analysis are presented in Table 1.It can be seen from Table 1 that the paeonol extraction yield washigher than that of ILAE and SE. In the ILAE and SE processes, thetarget analyte was extracted as a result of diffusion between sam-ple and solvent, so the extraction yield was slow [27].

3.7. Bioassay test

The crude extracts of C. paniculatum inhibited growth of bothGram negative and Gram positive bacteria. As can be seen in Ta-ble 2, the antibacterial effect of the two extraction methods werealmost the same. Therefore, the results in this study can serve asa reference for extraction of effective constituents from medicinalplants.

4. Conclusions

In terms of paeonol extraction yield, significant enhancement ofthe release of paeonol in both rate and yield was achieved. Thiswas mainly due to an increase of cell wall permeability by ILs pre-treatment. The MILP was a possible alternative for extraction ofpaeonol in C. paniculatum samples without removing RTILs in thefiltrate for its subsequent analysis by HPLC. The extraction yieldof paeonol was 2.51 ± 0.3% and the recovery was in the range85.2–126.7% with RSD lower than 3.0%. In conclusion, IL pretreat-ment with microwave irradiation can be used as a potential alter-native method for the pretreatment of medicinal plants beforesolvent extraction. The mechanisms of extraction and the purifica-tion of paeonol in filtrate are in progress.

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