extraction of paeonol from jisheng shenqi wan using supercritical fluid extraction

Copyright © 2006 John Wiley & Sons, Ltd. Biomed. Chromatogr. 21: 79–83 (2007)DOI: 10.1002/bmc

Extraction of paeonol from Jisheng Shenqi Wan 79ORIGINAL RESEARCHORIGINAL RESEARCH

Copyright © 2006 John Wiley & Sons, Ltd.

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 21: 79–83 (2007)Published online 2 November 2006 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/bmc.721

Extraction of paeonol from Jisheng Shenqi Wan usingsupercritical fluid extraction

Ben Liu,1* Yiling Chang,2 Hailiang Jiang3 and Bo Shen1

1Department of Bioengineering and Pharmaceutics, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, People’s Republic ofChina

2Department of Pharmaceutics, Zhejiang Pharmaceutical College, Ningbo 315100, People’s Republic of China3Analytical Center, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, People’s Republic of China

Received 18 March 2006; revised 18 May 2006; accepted 20 June 2006

ABSTRACT: Supercritical carbon dioxide with or without methanol and 95% ethanol modifiers was used to extract paeonol fromthe pills of Jisheng Shenqi Wan and high-performance liquid chromatography (HPLC) was used to analyze the extract. The effectof temperature and pressure on the yield of paeonol was tested. The results show that the recovery of paeonol was improved byadding a polar modifier, such as methanol and 95% ethanol in supercritical fluid. The yield obtained after 120 min extraction with95% ethanol modified-supercritical carbon dioxide was the highest (1.51‰, w/w at 60°C and 400 bar), while that obtained withsupercritical carbon dioxide only at 40°C was the lowest from 0.67 to 0.83‰ over a range of 200–600 bar. Copyright © 2006 JohnWiley & Sons, Ltd.

KEYWORDS: paeonol; Jisheng Shenqi Wan; supercritical fluid extraction; sample preparation; quality monitoring

*Correspondence to: Ben Liu, Department of Bioengineering andPharmaceutics, Ningbo Institute of Technology, Zhejing University,Ningbo 315100, China.E-mail: [email protected]

Abbreviations used: SFE, supercritical fluid extraction.

Contract/grant sponsor: Natural Science Funding of Zhejing Pro-vince; Contract/grant number: Y205314.

INTRODUCTION

Jisheng Shenqi Wan, a pill of traditional Chinesemedicinal preparation, is composed of Cortex MoutanRadicis, Radix Rehmanniae, Rhizoma Dioscoreae,Fructus Corni, Rhizoma Alismatis, Poria, RamulusCinnamomi, Radix Aconiti, Semen Plantaginis and Ra-dix Cyathulae, which has the action of warming kidney-yang and activating vital energy, and promoting diuresisand eliminating turgescence (Chinese PharmacopoeiaCommission, 2000). The medicine has been used fortreatment of lumbago, flaccidity of legs, difficulty inurination, and cold sensation over the lower extremitiesfor a long time in China (Ou, 1992). Therefore, JishengShenqi Wan has great commercial interest in the phyto-pharmaceutical market. One of the active components,paeonol (2′-hydroxy-4′-methoxyacetophenone, Fig. 1),in the herbal medicinal preparation has many pharma-cological effects, including anti-inflammatory and anal-gesic effects (Chou, 2003), inhibition of anaphylacticreaction (Kim et al., 2004), inhibition of monoamineoxidase A and B (Kong et al., 2004), anxiolytic-like

effect (Mi et al., 2005) and inhibition of platelet aggre-gation (Doble et al., 2005).

For conventional solvent extraction methods, such asSoxhlet extractor extraction, there are few adjustableparameters to control the selectivity of the extractionprocesses. Therefore, developing alternative extractiontechniques with better selectivity and efficiency arehighly desirable. Supercritical fluid extraction (SFE), anenvironmentally responsible and efficient extractiontechnique for solid materials, was introduced andextensively studied for separation of active compoundsfrom herbs and other plants. Today, SFE has becomean acceptable extraction technique used in differentareas (Zougagh et al., 2004; Lang and Wai, 2001). Tofind the optimal extraction conditions, temperature andpressure optimization and modifier impact study can beconsidered (Klejdus et al., 2005). Various modifiers,such as methanol, ethanol and surfactants have been

Figure 1. Structure of paeonol.


80 B. Liu et al.ORIGINAL RESEARCH

used in attempts to compensate for the poor ability ofsupercritical carbon dioxide to solvate polar organiccompounds and to interrupt strong analyte–matrixinteractions (Porschmann et al., 1998).

Extraction of paeonol from Cortex moutan Radicisand other samples using organic solvents has been pre-viously reported with subsequent analysis being carriedout by capillary electrophoresis (Wu and Sheu, 1996)and high-performance liquid chromatography (Zhaoand Sun, 2003; Wu et al., 2003). Otherwise, microwave-assisted extraction combined with headspace single-drop microextraction of paeonol has been reportedrecently by Deng et al. (2006). An extraction kineticsstudy of paeonol from the cortex of Paeoniasuffruticosa using supercritical carbon dioxide was done(Dean and Liu, 2000). However, no report is availableon the extraction and analysis of paeonol from JishengShenqi Wan using SFE. Therefore, the purposes of thiswork were to develop an SFE method of analysis usingHPLC for the content of paeonol from Jisheng ShenqiWan in order to monitor the quality of the patentmedicines, and to test the effect of some factors, suchas various modifiers, pressure and temperature on theyield of the active component in the samples. Inaddition, SFE was compared with traditional organicsolvent extraction technique.

EXPERIMENTAL

Materials. Jisheng Shenqi Wan (called Jingui Shenqi Wan,batch no 4031496) was provided by Tongrentang T&S Inc.Ltd (Beijing, China). Paeonol standard was purchased fromthe National Institute for the Control of Pharmaceutical andBiological Products (Beijing, China). CO2 (food-grade) wasfrom Fangxin Gas Ltd (Ningbo, China). Methanol (HPLC-grade) was purchased from Tianjin Shield Company (Tianjin,China). Methanol and 95% ethanol were analytical gradeand were purchased from Sinopharm Chemical Reagent Co.Ltd (Shanghai, China). Celite (chemical grade) was fromFengcheng Chemical Ltd (Shanghai, China).

Supercritical fluid extraction system. A supercritical fluidextractor Spe-ed SFE-2 (Applied Separation, USA) was used,which operates with two pumps, a master pump fitted with acooling jacket on the pump head and a second pump (Knauerpump, model K-501, Berlin, Germany) for the addition oforganic modifier. The Spe-ed SFE is a screening systemextractor that allows a modularity of extraction but requiresmore handling of the controls. It is capable of pressure up to680 bar and temperature up to 240°C, static and dynamicextraction with flow from 0 to 10 L/min (gaseous carbondioxide) and extraction vessels from 5 mL to 1 L. A meteringvalve is used to vary flow. Collection is at room tempera-ture and atmospheric pressure. The extracted analytes arecollected in glass vial (30 mL) with a rubber plug at the top.A metal extension to the metering valve is used to piercethe rubber plug and allow collection directly in the collection

solvent (methanol). A hypodermic needle connected to a flowmeter also pierces the plug for the mensuration of flow.

HPLC conditions. A high-performance liquid chromato-graphy system (Knauer, Berlin, Germany) equipped with aKnauer pump (model K-501) and an ultraviolet–visible detec-tor (Knauer, model K2501) was used. The column used forseparation was a Diamonsil C18 separation column (5 µm, 250× 4.6 mm i.d., Dikma Technologies, Beijing, China). Themobile phase was methanol–water (91:9, v/v) at a flow-rateof 1 mL/min. Detection was at a wavelength of 274 nm. Forall experiments, 20 µL of standards and sample extract wereinjected.

Preparation of reference standards. A series of standardsof paeonol in the range 1.16–23.2 µg/mL were prepared inmethanol. Quantification was done using external standardcalibration. A linear response with a correlation coefficient of0.999 (n = 5) was obtained for the standards.

Preparation of medicinal plant extracts. The pills ofJisheng Shenqi Wan were ground into powder using a herbalpulverizer (FW 100, Tianjin Taisite Instrument Co. Ltd,Tianjia, China) and sieved through a 250 µm filter. One partof the powder was mixed with one part of celite for theextraction with SFE. For extraction using SFE, a knownquantity of the mixed sample (2 g) was placed in a 10 mL ofextraction vessel (60 × 15 mm, i.d.) and the void volume wasfilled with celite. Before the extraction was started, theextraction vessel was preheated in the oven for 10 min. Theextraction conditions were as follows: extraction time, staticextraction for 5 min and then dynamic extraction up to120 min; temperature, from 40 to 60°C; pressure, from 200 to400 bar; flow-rate of carbon dioxide (gaseous state), 1 L/min;flow-rate of modifier, 0.4 mL/min. The extract was collectedin a glass vial containing 5 mL of methanol, and then quanti-tatively transferred to a 25 mL volumetric flask and made upto the mark with methanol. This solution was further dilutedsuitably prior to analysis.

For Soxhlet extraction, a known quantity of groundedsample (2.0 g) was accurately weighed into a thimble and wasextracted in a 50 mL of extractor for 6 h with 50 mL of 95%ethanol at a syphon rate of 1 cycle/5 min.

All extracts were filtered through a 0.45 mm membranefilter before injecting into the HPLC system.

RESULTS AND DISCUSSION

Three supercritical fluid systems, i.e. supercritical car-bon dioxide, methanol-modified supercritical carbondioxide and ethanol-modified supercritical carbondioxide, were used to extract paeonol from powderedJisheng Shenqi Wan in order to evaluate the feasibilityof SFE, and the effect of methanol and ethanol usedas modified on the yield of paeonol. The HPLCchromatogram of the extract using 95% ethanol-modified supercritical carbon dioxide is shown in Fig. 2.It is possible to identify the paeonol peak, whichappears at a retention time of approximately 4.2 min.


Extraction of paeonol from Jisheng Shenqi Wan 81ORIGINAL RESEARCH

[Note: all other extracts show chromatograms similarto Fig. 2.]

Figure 3 shows the effect of extraction pressure at40°C on the yield of paeonol using supercritical carbondioxide only with extraction time up to 120 min. It canbe seen that the yield of paeonol is increased from0.67‰ (w/w, 200 bar) to 0.83‰ (w/w, 600 bar). Higherpressure is helpful for obtaining higher yield of theingredient from the powdered pills and the improve-ment of yield by change of pressure over a range from200 to 600 bar was about 25%.

Paeonol is medium–small polar compound and theextraction of the compound using supercritical CO2

only is not complete. As an improved method, polarmodifier should be considered. Usually, addition ofa small amount of a liquid modifier can enhance signifi-cantly the extraction efficiency and, consequently,reduce the extraction time (Lang and Wai, 2001) and

the method can be used to improve yield of someactive ingredients, such as magnolol (Dean et al., 1998)and sinomenine (Liu et al., 2005), from plant materials.Figure 4 shows the effect of extraction pressure at 60°Con the yield of paeonol using methanol-modifiedsupercritical carbon dioxide. It can be seen that theyield, after 120 min, is slightly influenced by change ofpressure from 1.24‰ at 200 bar to 1.31‰ at 400 bar.

The effect of pressure at 60°C on the yield of paeonolwith 95% ethanol-modified supercritical carbon dioxideis shown in Fig. 5. The yield is slightly influenced bychange of pressure from 1.44‰ at 200 bar to 1.51‰ at300 bar. It is noted that higher yield is not obtained byelevation of pressure (400 bar).

The recovery of paeonol over periods of extractionextending to 120 min was shown with supercritical carbondioxide only at 40°C and 600 bar, methanol-modified

Figure 2. High-performance liquid chromatogram of extractobtained with 95% ethanol-modified supercritical CO2.

Figure 3. The yield of paeonol over a range of pressure at40°C using CO2.

Figure 4. The yield of paeonol over a range of pressure at60°C using CO2 + methanol.

Figure 5. The yield of paeonol over a range of pressure at60°C using CO2 + 95% ethanol.


82 B. Liu et al.ORIGINAL RESEARCH

tor are summarized in Table 1. It shows that extractionwith 95% ethanol-modified supercritical CO2 at 60°Cand 400 bar for extraction of 60 min gives higher yieldthan with Soxhlet extraction for 6 h.

CONCLUSION

The study shows that the recovery of paeonol from thepowdered pills of Jisheng Shenqi Wan was influencedby using polar modifiers in supercritical CO2, amongwhich the highest recovery was obtained by using 95%ethanol as modifier at 60°C and 300 bar, while therecovery obtained using methanol as modifier was alsoeffective. The effect of pressure on the yield of paeonolwas tested, which showed that optimal extraction pres-sure was helpful in obtaining higher yield, but theinfluence was less than that using ethanol or methanolmodifier in the experimental conditions. The additionof polar modifier in supercritical CO2 was more neces-sary for exhausting extraction of paeonol from thesamples. Considering extraction time and recovery,supercritical fluid extraction with HPLC determinationis an effective method for monitoring the content ofpaeonol in pills of Jisheng Shenqi Wan.

Acknowledgement

Financial support of this work by the Natural ScienceFunding of Zhejing Province (project no. Y205314) isgratefully acknowledged.

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Table 1. Extraction yield of paeonol from Jisheng Shenqi Wan

Yield (mg/g),Solvent Extraction condition mean ± SD (n = 5)

CO2 Temperature 40°C; pressure 600 bar; 0.76 ± 0.09flow rate 1 L/min CO2;dynamic extraction time, 60 min

CO2 + methanol Temperature 60°C; pressure 400 bar; 1.24 ± 0.14flow rate 1 L/min CO2 and 0.4 mL/minmethanol; dynamic extraction time, 60 min

CO2 + 95% ethanol Temperature 60°C; pressure 300 bar; 1.46 ± 0.12flow rate 1 L/min CO2 and 0.4 mL ethanol;dynamic extraction time, 60 min

95% ethanol Soxhlet extractor; 1.41 ± 0.10extraction time, 6 h

Figure 6. The cummulative extraction yield, with respect toextraction time, of paeonol from Jisheng Shenqi Wan using� supercritical CO2; � methanol-modified supercritical CO2;and � 95% ethanol-modified supercritical CO2.

supercritical carbon dioxide at 60°C and 400 bar and95% ethanol-modified supercritical carbon dioxide at60°C and 300 bar in Fig. 6. It is obvious that improve-ment of recovery of paeonol can be obtained by addingmethanol or 95% ethanol in supercritical carbon dio-xide. The highest recovery was obtained using 95%ethanol as polar modifier, and methanol used as polarmodifier was also effective for the extraction of paeonolfrom the powdered sample. The recovery of paeonolobtained with methanol and 95% ethanol as modifieris about 55 and 80% higher than that obtained withcarbon dioxide only, respectively. As displayed in Fig. 6,it is possible to extract over 95% of paeonol within60 min (all yield being based on the recovery obtainedafter 120 min). The results for extraction of paeonolfrom powdered pills of Jisheng Shenqi Wan usingsupercritical CO2, methanol-modified supercritical car-bon dioxide and ethanol-modified supercritical carbondioxide, as well as using 95% ethanol in Soxhlet extrac-


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extraction of paeonol from jisheng shenqi wan using supercritical fluid extraction

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