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Separation and Purification Technology 64 (2008) 242–246

Contents lists available at ScienceDirect

Separation and Purification Technology

journa l homepage: www.e lsev ier .com/ locate /seppur

ptimization of supercritical fluid extraction of dl-tetrahydropalmatinerom rhizome of Corydalis yanhusuo W.T. Wang with orthogonal array design

en Liua,∗, Bo Shena, Feng Guoa, Yiling Changb

Department of Bioengineering and Pharmaceutics, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, ChinaDepartment of Pharmaceutics, Zhejiang Pharmaceutical College, Ningbo 315100, China

r t i c l e i n f o

rticle history:eceived 8 May 2008eceived in revised form 2 October 2008ccepted 4 October 2008

eywords:l-Tetrahydropalmatine (dl-THP)

a b s t r a c t

dl-Tetrahydropalmatine (dl-THP), an alkaloid occurring in plant, has been intensively studied due toits pharmacological actions. In this report, dl-THP from rhizome of Corydalis yanhusuo W.T. Wang wasextracted using supercritical fluid extraction (SFE). Determinations of the extracts were performed byhigh-performance liquid chromatography. An orthogonal array design (OAD), OA9(34), was employed foroptimization of supercritical fluid extraction of parameters of the compound. Four factors, namely variousmodifiers, the dynamic extraction time, temperature and pressure of the supercritical fluid, were studied

orydalis yanhusuo W.T. Wangupercritical fluid extractionptimization

and optimized by a three-level OAD. The effects of the parameters on the yield of dl-THP were studiedusing analysis of variance (ANOVA). Temperature had significant effect on the yield of dl-THP, while theother three factors, i.e. various modifiers, dynamic extraction time and pressure, were not identified assignificant factors under the selected conditions, based on ANOVA. The result shown that the optimizationfor the extraction condition of dl-THP was 1,2-propanediol used as modifier of supercritical CO2, extrac-tion time 60 min, temperature 70 ◦C and pressure 200 bar, and that higher yield (1.324 mg/g) was obtained

other

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than that obtained by the

. Introduction

Corydalis yanhusuo W.T. Wang, an herbal medicine, is com-only distributed in East Asia, especially in China. Rhizome

f C. yanhusuo W.T. Wang, also known as “Yan-wu-su” in Chi-ese has been used in traditional Chinese medicine to promotelood circulation, activate vital energy and alleviate pain forreatment of pain in chest, epigastrium and abdomen, amenor-hea and dysmenorrhea, puerperal blood stasis, and pain dueo trauma [1]. The medicinal effects of the herb are attributedo alkaloids, especially dl-tetrahydropalmatine, dl-5,8,13,13a-etrahydro-2,3,9,10-tetramethoxy-6H-dibenzo [a,g] quinolizine, ansoquinoline derivative alkaloid (Fig. 1). In scientific studies, theomponent is found to have pharmacological effects including anal-esics [2], antiepileptogenic and anticonvulsant action [3], decreasen both colonic temperature and release of hypothalamic serotonin4], inhibition of epileptic attack by inhibiting amygdaloid release

f dopamine [5], attenuation of heatstroke-induced hyperthermia,rterial hypotension, cerebral ischemia, brain monoamine over-oad, and cerebral neuronal injury [6], antioxidative activity [7], andnxiolytic-like action [8].

∗ Corresponding author.E-mail address: liu ben0@hotmail.com (B. Liu).

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383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.seppur.2008.10.003

extraction condition.© 2008 Elsevier B.V. All rights reserved.

For conventional extraction methods, such as Soxhlet extraction,here are few adjustable parameters to control the selectivity ofhe extraction processes. Therefore, developing alternative extrac-ion techniques with better selectivity and efficiency are highlyesirable. Consequently, supercritical fluid extraction (SFE) as annvironmentally responsible and efficient extraction technique forolid materials was introduced and extensively studied for theeparation of active compounds from natural product [9]. As a pro-ess, SFE offers numerous potential advantages over conventionalxtraction processes, including reduced extraction time, reducedrganic solvent volume, and more selective extractions [10]. How-ver CO2 is apolar and is an ineffective solvent for extraction of theompounds with higher polarity. To overcome this disadvantage,olar modifiers can be used to increase the overall polarity of theuid phase during extraction. It is considered that the added mod-

fiers often enhance extractions from solid materials by disruptinghe bonding between solutes and the solid matrix [11]. Methanolr ethanol is usually chosen as modifiers for the improvement ofecovery of polar compounds [12], such as flavone [13], isoflavone14] or saponin [15]. 1,2-Propanediol, a polar organic solvent, has

een widely used in food, cosmetics, and pharmaceutical indus-ry, due to its safety use as solvent. However, less report is giveno evaluate its efficiency as a cosolvent used in supercritical fluidO2 extraction for the improvement of extraction yield of polarompound from herbal medicines.

B. Liu et al. / Separation and Purification

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Fig. 1. Structure of tetrahydropalmatine.

Orthogonal array design (OAD) is a fractional factorial designnd a series of trials are assigned by orthogonal array [16]. Theesults of OAD experiments can be treated by analysis of varianceANOVA) and direct observation analysis. The advantages of OADnclude that it may minimize assay numbers and time to keephe experimental cost at a minimum level, and that the optimumarameters obtained from the laboratory can be utilized by largercale of production [17]. This method has been adopted in differentrea for the optimization of the analytical procedure, also in SFEor the optimal procedure of extraction of target compounds fromarious samples [18].

Various approaches are available to determine dl-THP in sam-les. In general, extraction, using organic solvent, of dl-THP fromlant material and biological sample are often used with subse-uent analysis by HPLC [19,20], capillary electrophoresis [21], masspectrometry [22], and LC–MS/MS [23], as well as counter-currenthromatography [24]. However, no report has been done on extrac-ion of the compound from rhizome of C. yanhusuo W.T. Wang usingarious cosolvents in SFE. Therefore, the objective of this study waso develop a SFE method with analysis using HPLC for the content ofl-THP from C. yanhusuo, to optimize a supercritical fluid extractionrocess, and to evaluate the factors, which influence the yield of dl-HP for SEF from the rhizome of C. yanhusuo. The effects of variousodifiers, dynamic extraction time, temperature and pressure on

he extraction efficiencies of dl-THP were studied by a four-factor,hree-level OAD with an OA9(34) matrix without considering thenteractions between the parameters.

. Experimental

.1. Materials

Rhizome of C. yanhusuo W.T. Wang was obtained from Ningboerbs Ltd. (Ningbo, China). dl-THP standard was purchased from

he National Institute for the Control of Pharmaceutical and Biolog-cal Products (Beijing, China). CO2 (food grade) was from Fangxinas Ltd. (Ningbo, China). Acetonitrile of HPLC grade was purchased

rom Tianjin Shield Company (Tianjin, China). 100% methanol, 95%thanol, 100% 1,2-propanediol, phosphoric acid and triethylamineere analytical grade and were purchased from Sinopharm Chem-

cal Reagent Co. Ltd. (Shanghai, China). Celite (Chemical grade) wasrom Fengcheng Chemical Ltd. (Shanghai, China).

.2. Supercritical fluid extraction only

A supercritical fluid extractor Spe-ed SFE-2 (Applied Separation,SA) was used, which operates with two pumps, a master pumptted with a cooling jacket on the pump head and a second pump

Knauer pump, model K-501, Berlin, Germany) for the addition ofrganic modifier. The Sped-ed SFE is a screening system extractornd is capable of pressure up to 680 bar and temperature up to40 ◦C, static and dynamic extraction with flow from 0 to 10 L/mingaseous carbon dioxide at atmospheric pressure) and extraction

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Technology 64 (2008) 242–246 243

essels from 5 mL to 1 L. A metering valve is used to vary flow.ollection is at room temperature and atmospheric pressure. Thextracted analytes are collected in glass vials (30 mL) with a rubberlug at the top. A metal extension to the metering valve is used toierce the rubber plug and allow collection directly in the collec-ion solvent (methanol). A hypodermic needle is pierced throughhe plug and is connected to a flow meter.

Rhizome of C. yanhusuo W.T. Wang was ground into powdersing a herbal pulverizer (FW 100, Tianjin Taisite Instrument Co.td., Tianjia, China) and sieved through a 250-�m filter. For extrac-ion using SFE, a known quantity of grounded sample (1 g) waslaced in a 10 mL of extraction vessel (60 mm × 15 mm, i.d.) andhe void volume was filled with celite. Before the extraction wastarted, the extraction vessel was preheated in the oven for 10 min.he extraction conditions were as follows: extraction time, staticxtraction for 5 min and then dynamic extraction up to 1.5 h; tem-erature, from 50 to 70 ◦C; pressure, from 200 to 400 bar; flow-ratef carbon dioxide (gaseous state), 2 L/min; flow-rate of modifier,.4 mL/min. The extract was collected in a glass vial containing 5 mLf methanol, and then quantitatively transferred to a 25-mL volu-etric flask and made up to the mark with methanol. This solutionas further diluted suitably prior to analysis.

.3. Preparation of medicinal plant extracts with Soxhletxtraction

For Soxhlet extraction, a known quantity of grounded sample1.0 g) was accurately weighed into a thimble and was extractedn a 50-mL extractor with 50 mL of methanol at a syphon ratef 1 cycle/10 min. After extraction with the solvent for 7 h, thextraction solvent was essentially colorless and the extracts wereransferred to a 50-mL volumetric flask and made up to the markith methanol. This solution was further diluted suitably prior to

nalysis.All extracts were filtered through a 0.45-mm membrane filter

efore injecting into the HPLC system.

.4. HPLC conditions

A high-performance liquid chromatography system (Hitachi,apan) equipped with a Hitachi pump (model L-2130) and anv–vis detector (Hitachi, model L-2400) was used. The columnsed for separation was a Diamonsil C18 separation column (5 �m,50 mm × 4.6 mm i.d., Dikma Technologies, Beijing, China). Theobile phase was acetonitrile:water:phosphoric acid (60:40:0.5,

/v/v, pH 6.5 adjusted with triethylamine) at a flow-rate ofmL/min. Detection was at a wavelength of 280 nm. For all exper-

ments, 20 �L of standards and sample extract were injected.A series of standards of dl-THP in the range of 1.7–68 �g/mL were

repared in methanol. Quantification was done using external stan-ard calibration. A linear response with a correlation coefficient of.999 (n = 6) was obtained for the standards.

.5. Experimental design and data analysis

A four-factor, three-level OAD was selected for optimization ofperating condition of dl-THP extraction in SFE. Nine experimentsere performed in triplicate and were run randomized. For evalu-

ting the effects of factors on the SFE efficiency, the selected four

xtraction temperature (C) and extraction pressure (D). Factors andevels tested are reported in Table 1. The data was analyzed usinghe Orthogonality Experiment Assistant software (Sharetop Soft-are Studio, China). The significance level was stated at 95%, with

-value 0.05.

244 B. Liu et al. / Separation and Purification

Table 1The factors and levels of the orthogonal array design.

Level Factors

(A) M (B) t (min) (C) T (◦C) (D) P (bar)

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doevalues of the yield for the factors at each level with standard devi-

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Ethanol 30 50 200Methanol 60 60 3001,2-Propanediol 90 70 400

. Result and discussion

.1. Chromatograms obtained

3 organic solvent modifier systems, i.e. ethanol-modified super-

ritical carbon dioxide, methanol-modified supercritical carbonioxide and 1,2-propanediol-modified supercritical carbon dioxideere used to extract dl-THP from powdered rhizome of C. yan-

usuo in order to evaluate the feasibility of SFE, and the effect of

aobF

Fig. 2. High-performance liquid chromatogram of the extract obtaine

able 2rthogonal array design matrix OA9(34) and experimental results.

rial no. A B C

1 1 11 2 21 3 32 1 22 2 32 3 13 1 33 2 13 3 2

la 0.990 ± 0.062 0.69 ± 0.070 0.822 1.045 ± 0.071 1.051 ± 0.063 1.133 1.088 ± 0.075 1.103 ± 0.075 1.16

a KAi

= ˙(Yield at Ai)/3, the mean values of yield for a certain factor at each level with s

able 3NOVA analysis of four parameters for SFE.

ource Sum of squares (SS) Degrees of freedom (d.f.)

A) Modifiers 0.014 2B) Tirne 0.028 2C) Temperature 0.216 2D) Pressure 0.005 2

Technology 64 (2008) 242–246

elected factors on the yield of dl-THP. Fig. 2 shows a chromatogramf dl-THP extracted by 1,2-propanediol-modified supercritical car-on dioxide at 200 bar, 70 ◦C. Based on the available standard ofl-THP, it is possible to identify the dl-THP peak, which appears atretention time of approximately 10 min. All other extracts show

hromatograms similar with Fig. 2.

.2. Optimization study

The assignment of the experiment and the collected data forl-THP extraction are in Table 2. The data are analyzed using Orthog-nality Experiment Assistant software for the evaluation of theffect of each parameter on the optimization criteria. The mean

tion are also shown in Table 2. Table 3 lists the data of the analysisf variance (ANOVA) table of this experiment. The relationshipsetween the yield of dl-THP and different variables are shown inig. 3.

d with 1,2-propanediol-modified supercritical carbon dioxide.

D Yield (mg/g) ± S.D. (n = 3)

1 0.731 ± 0.0562 1.082 ± 0.0643 1.157 ± 0.0673 1.051 ± 0.0791 1.210 ± 0.0612 0.875 ± 0.0722 1.124 ± 0.0753 0.862 ± 0.0641 1.278 ± 0.085

3 ± 0.064 1.073 ± 0.0677 ± 0.076 1.027 ± 0.0704 ± 0.068 1.023 ± 0.070

tandard deviation.

F-ratio F0.05 p-Value Type of effect

2.8 19 0.24095.6 19 0.1424

43.2 19 0.0209 Significant1 19 –

B. Liu et al. / Separation and Purification Technology 64 (2008) 242–246 245

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lepmdioe0.97 mg/g. However, a further increase in the dynamic extractiontime improves the extraction efficiency only slightly. The differ-ence in yield is about 0.09 mg/g between 30 and 60 min and about0.05 mg/g between 60 and 90 min. Obviously, extraction within

ig. 3. The effect of each parameter on the yield of dl-THP. Error bar shows standardevel of pressure.

.2.1. Effect of various modifiersFor extraction of polar compound like dl-THP, polar solvent is

sed as modifier in supercritical CO2 extraction. Usually, additionf a small amount of a liquid modifier can enhance significantlyhe extraction efficiency and, consequently, reduce the extractionime and improve the recovery of different types of natural productsrom plant materials [9,25,26]. In this paper, various polar solvents,.e. ethanol, methanol and 1,2-propanediol were selected as mod-fiers to evaluate the effect of modifiers on the yield of dl-THP. Its indicated in Fig. 3a that the mean yield of dl-THP is influencedlightly by the change of modifiers. The mean yield varies from.990 to 1.088 mg/g, rising by about 10% when 1,2-propanedioleplaces ethanol. However modifiers was not identified as a sig-ificant factor for extraction of dl-THP, based on ANOVA.

The effect of the three solvents modified supercritical CO2 onhe yield of dl-THP is estimated by the addition of a set of trialsn Fig. 4. The extraction conditions were temperature 60 ◦C, pres-ure 200 bar, dynamic extraction time up to 3 h, and flow-rate ofodifiers 0.4 mL/min. The results clearly show that the yield of

l-THP, after 3 h, obtained using 1,2-propanediol-modified super-ritical CO2, was highest in the three solvent-modified supercriticalO2. As displayed in Fig. 4, it is possible to extract about 95% of dl-HP within 1 h when the three polar solvents are used as modifiers

all yield being based on the recovery obtained after 3 h).

.2.2. Effect of dynamic extraction timeThe yield of extraction can be influenced by extraction time.

horter extraction time could cause incomplete extraction andFv

tions for n = 3. (a) Level of modifiers; (b) level of time; (c) level of temperature; (d)

onger extraction time could be time and solvent wasting. In thisxperiment, a primary extraction step in static mode (5 min) waserformed, which effects a better penetration of the fluid in theatrix than the dynamic mode. This step was followed by a

ynamic extraction to enhance dl-THP solubility in the supercrit-cal fluid. The effect of dynamic extraction time on the extractionf the compound is shown in Fig. 3b. As seen, when the dynamicxtraction time is up to 30 min, the extraction yield attained about

ig. 4. The cummulative yield, with respect to extraction time, of dl-THP usingarious modifiers.

246 B. Liu et al. / Separation and Purification Technology 64 (2008) 242–246

Table 4Extraction yield of dl-THP from the rhizome of Corydalis yanhusuo.

Modifier Extraction condition Yield (mean ± S.D.) (mg/g) (n = 3)

Ethanol Temperature, 70 ◦C; pressure, 200 bar; flow-rate, 2 L/min CO2 and 0.4 mL/min modifier; dynamic extraction time, l h 1.053 ± 0.074

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ethanol Temperature, 70 ◦C; pressure, 200 bar; flow-rate, 2 L/min CO2 an

,2-Propanediol Temperature, 70 ◦C; pressure, 200 bar; flow-rate, 2 L/min CO2 anSoxhlet extractor; extraction solvent methanol, extraction time,

0 min can extract most of dl-THP and that the yield is increasedust a little by prolonging the extraction time. The effect of extrac-ion time on the yield is smaller after 30 min extraction. ANOVAhows that extraction time is not a significant factor for extractionf the compound when extraction time is between 30 and 90 min.

.2.3. Effect of temperatureIncreasing temperature, although causing a decrease in the fluid

ensity, could be responsible for an increase in the solvating powerecause of the increase in the solute vapor pressure. Fig. 3c showshe effect of temperature (50, 60 and 70 ◦C) on the yield of dl-THP.s shown in the figure, the extraction yield increased with increas-

ng temperature. As shown, the change of temperature enhancedhe mean yield from about 0.82 mg/g at 50 ◦C to 1.16 mg/g at 70 ◦Cnd the increase is about 41%. Temperature was appraised as aignificant factor, based on ANOVA with 95% confidence.

.2.4. Effect of pressureSFE was performed at three different pressures (200, 300, and

00 bar) in order to evaluate the effect of pressure on the yieldf dl-THP. Fig. 3d indicates that the yield of dl-THP is influencedlightly, with the mean yield from 1.07 mg/g at 200 bar to 1.02 mg/gt 400 bar. The difference is about 0.05 mg. It is observed that theffect of pressure on the yield of dl-THP is smaller than that of tem-erature when the selected modifiers are used. Increasing pressureoes not improve the yield of the compound extracted between 200nd 400 bar.

The experiment revealed that each of the four factors exertedn influence on the yield of dl-THP in the selected ranges, amonghich temperature was identified as a significant factor. According

o Fig. 4, optimum values of these factors for extraction of dl-THProm the rhizome of C. yanhusuo under the experimental conditionsre: pressure 200 bar, temperature 70 ◦C, extraction time 1 h, and,2-propanediol used as modifier.

In order to compare the yield obtained under the optimumondition with that obtained using different solvent system asell as Soxhlet extraction, the trials were carried out. Table 4

ives the experimental conditions and the results for extrac-ion of dl-THP from the powdered rhizome of C. yanhusuo usinghree solvent systems, i.e. ethanol or methanol or 1,2-propanediol-

odified supercritical CO2, and methanol in a Soxhlet extractor. Aseen from Table 4, the maximum yield (1.324 mg/g) was obtainedith 1,2-propanediol-modified supercritical CO2, while the yield

1.053 mg/g, w/w) obtained with ethanol-modified supercriticalO2 was lowest.

. Conclusions

In the present study, an orthogonal array design OA9(34) wassed to determine the optimum experimental conditions for the

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L/min modifier; dynamic extraction time, l h 1.256 ± 0.056

L/min modifier; dynamic extraction time, 1 h 1.324 ± 0.0671.281 ± 0.076

xtraction of dl-THP from the powdered rhizome of C. yanhusuo.he effects of four factors, namely modifiers, extraction time, tem-erature and pressure on the yield of the compound were observedt pressure from 200 to 400 bar, at temperature from 50 to 70 ◦C,t extraction time from 0.5 to 1.5 h and at ethanol, methanol or 1,2-ropanediol used as modifier. The result shown that the extractionemperature has significant (p < 0.05) effect on the yield of dl-THPuring supercritical extraction, however the other three factorshown some effect but insignificantly (p > 0.05) affected the result.he mean yield, based on the calculation of the orthogonal arrayesign, varied at range from 0.823 to 1.164 mg/g. The optimum val-es of the factors inside the experimental domain considered were:odifier 1,2-propanediol, extraction time 1 h, temperature 70 ◦C

nd pressure 200 bar.

cknowledgement

Financial support of this work by the Ningbo City Educationureau (Project No. Jd070204) is gratefully acknowledged.

eferences

[1] Chinese Pharmacopoeia Committee, Pharmacopoeia of the People’s Repub-lic of China, vol. I, 2005 Chinese ed., Chemical Industry Press, Beijing, 2005,p. 94.

[2] M. Ou, Chinese–English Manual of Common-Used Traditional Medicine,Guangdong Science and Technology Publishing House, Guangzhou, China,1992, p. 76.

[3] M.T. Lin, J.J. Wang, M.S. Young, Neurosci. Lett. 320 (2002) 113–116.[4] M.T. Lin, F.Y. Chueh, M.T. Hsieh, Neurosci. Lett. 315 (2001) 53–56.[5] C.K. Chang, M.T. Lin, Neurosci. Lett. 307 (2001) 163–166.[6] C.K. Chang, F.Y. Chueh, M.T. Hsieh, M.T. Lin, Neurosci. Lett. 267 (1999) 109–112.[7] T.B. Ng, F. Liu, Z.T. Wang, Life Sci. 66 (2000) 709–723.[8] W.C. Leung, H. Zheng, M. Huen, S.L. Law, H. Xue, Prog. Neuropsychopharmacol.

Biol. Psychiatry 27 (2003) 775–779.[9] Q. Lang, C.M. Wai, Talanta 53 (2001) 771–782.10] M. Zougagh, M. Valcarcel, A. Ríos, Trends Anal. Chem. 23 (2004) 399–406.11] J. Porschmann, L. Blasberg, K. Mackenzie, P. Harting, J. Chromatogr. A 816 (1998)

221–232.12] J.A. Mendiola, M. Herrero, A. Cifuentes, E. Ibanez, J. Chromatogr. A 1152 (2007)

234–246.13] Y. Chang, B. Liu, B. Shen, J. Sep. Sci. 30 (2007) 1568–1574.14] T. Bajer, M. Adam, L. Galla, K. Ventura, J. Sep. Sci. 30 (2007) 122–127.15] J.A. Wood, M.A. Bernards, W. Wan, P.A. Charpentier, J. Supercrit. Fluids 39 (2006)

40–47.16] P.J. Oles, J. AOAC Int. 76 (1993) 615–620.17] M. Yesilyurt, Chem. Eng. Process 43 (2004) 1189–1194.18] N. Bahramifar, Y. Yamini, M. Shamsipur, J. Supercrit. Fluids 35 (2005)

205–211.19] J. Ou, L. Kong, C. Pan, X. Su, X. Lei, H. Zou, J. Chromatogr. A 1117 (2006) 163–169.20] Z.C. Fan, C.J. Xie, Z.Q. Zhang, Chromatographia 64 (2006) 577–581.21] Y. Li, S. Cui, Y. Cheng, X. Chen, Z. Hu, Anal. Chim. Acta 508 (2004) 17–22.

23] L. Li, M. Ye, K. Bi, D. Guo, Biomed. Chromatogr. 20 (2006) 95–100.24] Z. Liu, Y. Yu, P. Shen, J. Wang, C. Wang, Y. Shen, Sep. Purif. Technol. 58 (2008)

343–346.25] T.C. Tzeng, Y.L. Lin, T.T. Jong, C.M.J. Chang, Sep. Purif. Technol. 56 (2007) 18–24.26] J.R. Dean, B. Liu, Phytochem. Anal. 11 (2000) 1–6.