This article was downloaded by: [University of Connecticut]On: 11 August 2013, At: 16:10Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Natural Product Research: FormerlyNatural Product LettersPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/gnpl20

Relevance of phenolic diterpeneconstituents to antioxidant activityof supercritical CO2 extract from theleaves of rosemaryChi-Huang Chang a , Charng-Cherng Chyau a , Chiu-Lan Hsieh b ,Yen-Ying Wu c , Yaw-Bee Ker b , Hau-Yang Tsen b & Robert Y. Peng aa Research Institute of Biotechnology, Hungkuang University, 34Chungchie Road, Shalu, ROC, Taichung Hsien 433, Taiwanb Department of Food & Nutrition, Hungkuang University, 34Chungchie Road, Shalu, Taichung Hsien 433, ROC, Taichung Hsien433, Taiwanc Graduate Institute of Biopharmaceutics, Life Science College,National Chiayi University, 300 University Road, ROC, Chiayi,Taiwand Graduate Institute of Chemistry and Biochemistry, NationalChung Cheng University, 168 University Road, ROC, Min-HsiungChiay, TaiwanPublished online: 12 Nov 2007.

To cite this article: Chi-Huang Chang , Charng-Cherng Chyau , Chiu-Lan Hsieh , Yen-Ying Wu , Yaw-Bee Ker , Hau-Yang Tsen & Robert Y. Peng (2008) Relevance of phenolic diterpene constituentsto antioxidant activity of supercritical CO2 extract from the leaves of rosemary, Natural ProductResearch: Formerly Natural Product Letters, 22:1, 76-90, DOI: 10.1080/14786410701591754

To link to this article: http://dx.doi.org/10.1080/14786410701591754

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content

should not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

Natural Product Research, Vol. 22, No. 1, 10 January 2008, 76–90

Relevance of phenolic diterpene constituents to antioxidant

activity of supercritical CO2 extract from the leaves of rosemary

CHI-HUANG CHANGy, CHARNG-CHERNG CHYAU*y, CHIU-LAN HSIEHz,YEN-YING WUx, YAW-BEE KERz, HAU-YANG TSENz and

ROBERT Y. PENGy

yResearch Institute of Biotechnology, Hungkuang University, 34 Chungchie Road, Shalu,Taichung Hsien 433, Taiwan, ROC

zDepartment of Food & Nutrition, Hungkuang University, 34 Chungchie Road, Shalu,Taichung Hsien 433, Taiwan, ROC

xGraduate Institute of Biopharmaceutics, Life Science College, National Chiayi University,300 University Road, Chiayi, Taiwan, ROC

{Graduate Institute of Chemistry and Biochemistry, National Chung Cheng University,168 University Road, Min-Hsiung Chiayi, Taiwan, ROC

(Received 8 March 2007; in final form 21 August 2007)

Isolation of phenolic diterpene constituents from the freeze-dried leaves of Rosmarinusofficinalis has been obtained by supercritical extraction with carbon dioxide. To determine theideal conditions for the maximum yield of extract, nine different conditions using three levels ofpressures (3000, 4000 and 5000 psi) in combination with three temperatures at 40, 60 and 80�C,respectively, in combination with the analyses of the corresponding antioxidant activities andconstituents which existed in extracts has been investigated. The antioxidant activity of eachobtained extract was determined by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicalstest. GC/MS method was used as an alternative to conventional HPLC method for thedetermination of the principal antioxidant constituents in extract, including phenolic diterpenescarnosic acid (CA) and carnosol (CAL). The confirmation of CA and CAL in extract wasforward performed by subjecting HPLC isolates from extract into an ion trap massspectrometer through an electrospray ionization (ESI) interface for MS/MS analysis. Theseresults indicate that an ideal extraction process was obtained at 5000 psi and 80�C with anextraction yield of 4.27% (w/w) and rich in phenolic antioxidants CA and CAL as contents of35.23 and 0.46mg g�1 in extract, respectively.

Keywords: Rosemary; Supercritical fluid extraction (SFE); Antioxidant phenolic diterpenes;HPLC; GC/MS; ESI-MS-MS

1. Introduction

Rosemary (Rosmarinus officinalis L.) has long been well known as a medicinal herbwith anti-inflammatory, antimicrobial and antioxidant activities due to the existence ofrelatively high percentage of phenolic diterpene antioxidants (PDAs) [1–6]. Phenolic

*Corresponding author. Tel.: þ886-4-2631-8652. Fax: þ886-4-2652-5386. Email: ccchyau@sunrise.hk.edu.tw

Natural Product Research

ISSN 1478-6419 print/ISSN 1029-2349 online � 2008 Taylor & Francis

http://www.tandf.co.uk/journals

DOI: 10.1080/14786410701591754

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

compounds such as carnosic acid (CA), carnosol (CAL), rosmanol, epirosmanol and7-methyl-epirosmanol [5,6] have been identified in rosemary extracts. Of thesecompounds, CA and CAL (figure 1) are the principal compounds responsible for theantioxidant properties of fresh rosemary leaves [5]. CA at high concentrations inhibitedthe proliferation (IC50¼�6–7mM), however, it augmented at low concentrations on thedifferentiation of HL-60 and U937 human myeloid leukemia cells [7]. CA was also citedas a lipid absorption inhibitor [8]. Several technologies have been described to obtainthe PDAs from rosemary using both traditional solvents and supercritical fluids [9].To be more effective, sonication-assisted solvent extraction is usually achieved bysonication with one of several common solvents typically including acetone, hexane,methanol or ethanol [9–11]. The extraction efficiency of antioxidants existing inrosemary leaves varied greatly with different extractive methods such as sonicative andsupercritical carbon dioxide (SC-CO2) extractions [9,11–13]. Nonetheless, supercriticalfluid extraction (SFE) has been the object of numerous studies aimed at optimizing theextraction conditions for PDAs [9]. In studies of rosemary extraction, SC-CO2 has beenfound to be the efficient extraction technology with the highest and most reproducible

OH

HO

H

HOOC

Carnosic acid M.W. = 332

OH

HO

H

O

Carnosol M.W. = 330

O

Figure 1. Chemical structure of carnosic acid and carnosol.

Antioxidant activity of supercritical CO2 extract from rosemary leaves 77

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

recovery of CA (35.7� 1.6mg g�1) compared to the common solvent extracts by usingacetone, methanol, hexane or dichloromethane [11].

In a previous report [13], rosemary leaves have been extracted using SFE under

a CO2 pressure ranging between 300 and 350 bar (i.e. 4351–5076 psi) by follow-upwith the analysis of liquid chromatography–electrospray ionization–mass spectro-

metry (LC–ESI–MS). The determination of PDAs in rosemary leaves was carriedout by SFE followed by supercritical fluid chromatography (SFC) separation on

packed capillary columns [14]. Yet, the randomizing resolution of SFC has been

doubted that whether the quantification of antioxidant compounds present in SFEextracts could be precisely estimated. Although the LC/MS analysis of PDAs has

been reported [15], it is more complicated in the selection of ionization methodscompared to that of GC/MS. It is not always a simple way to predict whether

positive or negative ions will be preferentially for the fragmentation pattern (massspectrum or plot of ion abundance versus mass-to-charge ration) [14]. Therefore,

GC/MS analysis is required much more as a method due to its universal detectionability in the ionization of sample. In the course of screening antioxidants of

rosemary extracts, CA concentration has been demonstrated as a relevant indicator

[15]. However, CA has been popularly cited as a rather unstable compound and canbe readily converted into CAL by air oxidation [3,16,17]. A rapid and effective

method with minimum air entrapping extraction process and analytical procedurewas obviously required. It has been indicated that the introduction of SFE

technology as an alternative to conventional procedures, such as solvent extraction,microwave-assisted solvent extraction and sonication extraction method, was

suitable and easily practiced [9]. In this study, SFE with a diversity of pressureand temperature conditions to prepare different extracts from the dried rosemary

leaves were performed for investigating the optimum condition in the preparationsof antioxidant products. Extracted antioxidant constituents in extracts were

identified and confirmed by parallel using of a GC–MS and a LC–MS. In order

to correlate the process conditions with the antioxidant activities and the extractionyield of principal antioxidant constituents (CA and CAL) in fresh rosemary,

antioxidant activities were simultaneously determined by the scavenging capabilityon 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals.

2. Experimental

2.1. Materials and chemicals

Rosemary (R. officinalis) plants were grown in the experimental fields of the

Taichung District Agricultural Research and Extension Station, Changhua, Taiwan.

Fresh rosemary leaves were harvested on November 2004 and were frozen in �80�Cafter harvesting, and lyophilized for 24 h. The lyophilized leaves were ground to a

fine powder (20 mesh) in a comminuted mill (Retsch Ultra Centrifugal Mill andSieving Machine, Type ZM1, Haan, Germany), and the ground sample thus

obtained was stored in dark at �20�C until use. Liquid CO2 (with helium head)was purchased from the Ho Tsun Gas Company (Taichung, Taiwan). Ethanol was

purchased from the Taiwan Tobacco and Liquor Corporation (Taipei, Taiwan).

78 C.-H. Chang et al.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

HPLC-grade acetonitrile, acetone, n-hexane and acetic acid were from Merck(Darmstadt, Germany).

2.2. Supercritical fluid extraction

Ground dried rosemary leaves (5.0 g) were placed into the extraction vessel (10mL) ofthe SFE apparatus (ISCO Model SFX 2–10, Lincoln, NE). Extractions with SC-CO2

(flow rate, 1.0mLmin�1) were operated independently at three levels of 3000, 4000 or5000 psi (corresponding to 207, 276 and 345 bar, respectively) in combination withtemperatures at 40, 60 and 80�C, respectively, for 1 h of static and followed by another1 h of dynamic extractions. Extracted constituents were collected in a 20mL vial thatwas pre-filled with a trapping solvent (10mL of acetone) and maintained at 4�C duringthe extraction step. Extracted sample was evaporated to dryness on a rotary evaporatorat reduced pressure and at 40�C. Obtained concentrates were then weighed and storedat �20�C for further analyses.

2.3. Preparation of phenolic diterpenes by semi-preparative HPLC

A semi-preparative HPLC apparatus equipped with a Hitachi (Tokyo, Japan) L-7100dual pump connected to a Luna C18 (2) (250� 10.00mm i.d.; particle size, 5 mm,Phenomenex, Torrance, CA) column and a L-7455 diode-array detector was used forisolating individual compounds in SFE extracts. The detection was taken at �¼ 284 nm(scanning range �210–400 nm). The effluent from column was collected with a fractioncollector (Isco Retriever 500, Lincoln NE). The mobile phase with a flow rate of4.0mLmin�1 consisted two solvents: A and B. Solvent A was 1% (v/v) acetic acid inHPLC grade water and solvent B was 1% acetic acid in acetonitrile. The columntemperature was maintained at room temperature throughout the test. The mobile phasewas consisted of solvent A and B (60 : 40, v/v) during the initial 5min, then solvent B waslinearly programmed from 40 to 100% within 10min and remained at this percentageduring the next 15min and then returned to the initial condition in 5min. Obtained SFEextracts were dissolved in 5mL of methanol and passed through a 0.2mm filter beforeanalysis of HPLC. Sample size of 500 mL for each extract was injected during HPLCanalysis. Phenolic diterpenes, collected in the semi-preparative HPLC analysis, werethen concentrated and further analysed using the GC–MS and ESI-MS-MS.

2.4. HPLC analysis for phenolic diterpenes

The collected effluents were further analysed on the same HPLC system as describedabove unless fitted with a 20 mL injector loop. An analytical column [250� 4.6mm i.d.,5 mm Luna C18 (2), Phenomenex Co., USA] was operated at a flow rate of 1mLmin�1

for the analysis of the collected compounds in rosemary extracts. Similar gradientconditions were used for the analytical HPLC as described for the previoussemi-preparative HPLC. Separated components from rosemary extracts were measuredby a diode-array detector set at four fixed wavelengths 230, 284, 316 and 365 nm,respectively. PDAs were located and identified in the HPLC/UV patterns by

Antioxidant activity of supercritical CO2 extract from rosemary leaves 79

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

comparison with the spiked authentic samples (A.G. Scientific, San Diego) from theirretention times and UV (210–400 nm) spectra.

2.5. GC/MS identification and quantification

Principal phenolic compounds of rosemary extracts were analysed by GC/MSaccording to the previous report [18] with a slight modification. In brief, GC–MSwas used to identify and quantify the analyte in the SIM mode. 2,3,5-Trimethoxybenzoic acid was used as the internal standard. The following ions were chosen forselective ion monitor (SIM) analyses (quantification ions in italics): CA: 219, 374 andCAL: 243, 372. Quantifications of CA and CAL in extracts were performed fromcalibration curve that generated from authentic carnosic acid (Sigma-Aldrich, St. Louis,USA). A 50 mL of authentic sample at different concentrations (�0–10mgmL�1) orSFE extracts (50mgmL�1) was added with an internal standard solution (50mL of2,3,5-trimethoxy benzoic acid (10mgmL�1), Aldrich Chemicals Company, Inc.Milwaukee, WI), and made to a volume of 100 mL. An aliquot of the mixture wasthen added with 50 mL of derivatizing agent of trimethylanilinium hydroxide (TMAH)(0.1M solution in methanol, Pierce Biotechnology, Inc., Rockfold, IL). After mixing,1 mL of the mixture was measured for the analysis of the phenolic compounds inextracts using a Hewlett-Packard (HP) 5890 gas chromatography (GC) coupled to anHP 5972AMSD mass spectrometer (EI mode, 70 eV). An HP-5MS fused silica capillarycolumn (30m� 0.25mm i.d., 0.25mm film thickness, Hewlett-Packard, Palo-Alto, CA)was adopted and interfaced directly into the ion source of the MSD. Helium (flow rateof 1mLmin�1) was used as the carrier gas. The column temperature was programmedfrom 80 to 280�C at a temperature elevation rate of 10�Cmin�1. The operationaltemperatures for GC injector and GC–MSD interface were held at 280 and 300�C,respectively. The mass scan range was m/z 60–500 for 1 cycle s�1.

Components were identified on the basis of gas chromatographic retention indicesand mass spectra obtained from the authentic compounds. A quantitative method usingan internal standard (2,3,5-trimethoxybenzoic acid) was applied for the simultaneousmeasurement of CA and CAL.

2.6. ESI–MS–MS analyses

The isolated samples from collected peaks in the semi-preparative HPLC chromato-gram were subjected to LC/MS analysis. Analyses were performed on an LCQAdvantage MAX ion trap mass spectrometer with an electrospray ion (ESI) source(Finnigan-Thermo Corporation St. Jose, CA). Sample was dissolved in methanol andinfused into ESI source through a capillary column (ID 100mm; length 30 cm) using asyringe pump at a flow rate of 3 mLmin�1. Nitrogen was used as the nebulizing anddrying gas. The typical operating parameters were as follows: ion spray voltage, 4.5 kV;capillary temperature, 250�C; tube lens offset, 10V; nitrogen sheath gas, 50; andauxiliary gas, 10 (arbitrary units). The ion trap contained helium damping gas whichwas introduced in accordance with the manufacturer’s recommendations. The MS wasprogrammed to perform two scans, a full scan and an MS2 scan. Positive ion massspectra of full scan ESI from the capillary column elute were recorded in range of m/z100–500. The product-ion spectra (MS2) were scanned from 90 to 500 with the collision

80 C.-H. Chang et al.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

energy set at 38V. The precursor ions of CA and CAL were selected at m/z 333[MþH]þ and 331 [MþH]þ, respectively.

2.7. Determination of antioxidant activity by the DPPH assay

The scavenging effect of each extract on the DPPH radicals (Sigma-Aldrich, St. Louis,USA) was assayed according to the procedure described by Shimada et al. [19].An aliquot (4mL) of methanol solution prepared from different extract concentration(0.1–0.5mgmL�1) was added to 1mL of methanolic solution containing a final DPPH(Sigma) radical concentration of 0.2mM prepared daily. After standing for 30min inthe dark, the absorbance of mixture was then measured at 517 nm in a Thermo Biomate5 spectrophotometer (Thermo Electron Corporation, San Jose, CA). Trolox was usedas a control. Measurements were performed in triplicate.

2.8. Statistical analysis

Data were subjected to analysis of variance by using Statistical Analysis System (SASInstitute Inc., Cary, NC) computer package. One way analysis of variance followed byDuncan’s multiple range tests was used to test the significance between the differentvariables studied. A confidence level of p<0.05 was considered statistically significant.

3. Results and discussion

3.1. Yield of rosemary extract by SC-CO2 extraction

Studies have shown that the extract yield from using liquid solvent extractions, onlyacetone extraction provided a comparable result (73% of yield corresponding toSC-CO2 extraction) [11]. Supercritical fluids have very potentially useful physicalproperties, such as low viscosity and high diffusivity into the sample matrix. In thiswork, different extraction conditions of temperature (T) and pressure (P) in SFE werestudied for the investigation of optimum extraction condition on rosemary antioxidantextracts. As shown in table 1, the best extractability reached 4.27� 0.01% under5000 psi at 80�C, comparing to the next 3.27� 0.02% by 5000 psi at 60�C on dry basisand the least with 0.80� 0.01% by 3000 psi at 40�C. Apparently, the extractability wasgreatly influenced by the conditions (P and T) used in extraction. Previous reports

Table 1. Yield of supercritical CO2 extraction for phenolic diterpenes from driedrosemary leaves.

Yield (%, w/w)a

Pressure (psi) 40�C 60�C 80�C

3000 0.80� 0.01Gb 2.41� 0.04D 2.80� 0.03C4000 1.24� 0.01F 2.80� 0.11C 2.89� 0.01C5000 1.47� 0.01E 3.27� 0.02B 4.27� 0.01A

aExtracted from freeze-dried rosemary leaves (5.00 g).bValues with different letters within the same row and column are significantly different (P<0.05). Eachvalue is expressed in mean�SD (n¼ 3).

Antioxidant activity of supercritical CO2 extract from rosemary leaves 81

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

indicated that the optimization of SFE variables seems directly related to pressure andtemperature used for extraction [11]. However, experiments indicated that CA and CAL

were selectively extracted at 250 atm (3626 psi) and 60�C [9]. Whether or not the freeradical scavenging activity was in parallel with the contents of CA and CAL in extracts,

further determination was performed by the assay of DPPH scavenging effect andGC/MS analysis on principle antioxidant components, e.g., CA and CAL.

3.2. Free radical scavenging ability and principal antioxidant phenolic constituentsin SC-CO2 extract

Three levels of pressures (3000, 4000 and 5000 psi) in combination with threetemperatures at 40, 60 and 80�C, respectively, combined with the analyses of the

corresponding antioxidant effects and antioxidant constituents which existed inrosemary leaves, have been tried to find an optimal extraction condition with the

highest antioxidant activity for the application of extraction in rosemary antioxidants.Results from extractabilities of rosemary leaves shown in table 1 were further evaluated

by the assays for scavenging free radicals effect using Trolox as the reference compound(table 2). Rosemary extract at a concentration of 0.01mgmL�1 was not efficient in

scavenging radicals being only 1.51� 0.02% from the extraction condition at 5000 psiand 60�C in comparing to that of Trolox, 61.17� 0.04% (table 2). With extract at

0.10mgmL�1, the scavenging effect was observed reaching the highest level at46.75� 0.01% from sample obtained at 5000 psi, 40�C. Scavenging effects of other

concentration levels (1.0, 3.0 and 5.0mgmL�1) tested mostly showed a plateau withoutsignificant difference (P<0.01) between each other. Similar results have been

comparatively studied to the reference compound Trolox at corresponding amounts.Obviously, higher CO2 pressures (e.g. 5000 psi) in combination with lower temperatures

(e.g. 40�C) were more effective for extraction of the antioxidant compositions fromrosemary. However, the advantage was still limited. In considering the extractability

from dried rosemary leaves and the contents of PDAs, the best compromised extractionwas concluded from the condition at 5000 psi, 80�C (tables 1 and 2). There is a

comparable, yet more vigorous extraction condition (355 bar at 100�C; CO2 density0.72 gmL�1) being reported by Tena et al. [11]. It can be seen that in this study the SFE

condition chosen at 5000 psi and 80�C for rosemary leaves has many advantages beingachieved including, the highest recovery of CA (35.23mg g) (table 3) with the minimum

SD (0.88%) and the cleanest extract that no further cleanup of the preparation isrequired prior to HPLC analysis (table 3).

The comparison of antioxidant effects among extracts (5000psi, 40, 60 and 80�C,

respectively) from various concentrations was expressed as EC50 value (figure 2). Theeffective concentration (EC) at the scavenging ability of DPPH radicals was 50%

obtained from the calculation of data from three levels of concentrations (0.01, 0.10 and1.00mgmL�1) used in tests of scavenging ability of DPPH radicals. A comparison of

the antioxidant effect from three SFE extracts (5000 psi, 40, 60 and 80�C, respectively atconc. 0.10mgmL�1) showed that a highly different activity existed between each other.

Apparently, a combined result from EC50 value and extraction condition at 5000psi and80�C was preferred according to those results of yield (table 1) and the contents of CA

and CAL (table 3).

82 C.-H. Chang et al.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

Table

2.

Scavengingeffect

ofrosemary

extractsfrom

differentsupercriticalCO

2extractionon1,1-diphenyl-2-picrylhydrazyl(D

PPH

. )radicals.

Scavengingeffect

(%)a

3000psi

4000psi

5000psi

Concentration

(mgmL�1)

40�C

60�C

80�C

40�C

60�C

80�C

40�C

60�C

80�C

Trolox

0.01

b2.51�0.08A

bd4.22�0.01B

g7.96�0.01C

e5.80�0.01A

h19.81�0.08D

f7.35�0.25B

d3.93�0.01C

a1.51�0.02A

c2.58�0.02B

61.17�0.04

0.10

ab15.94�0.01E

c33.73�0.01F

a12.05�0.01D

cd34.38�0.01E

bc13.18�0.15C

de42.04�0.01F

e46.75�0.01F

a12.21�0.01D

cd33.12�0.01E

91.52�0.01

1.00

c91.56�0.01H

c91.88�0.01H

b89.53�0.02G

c91.20�0.01H

a88.24�0.01G

c91.75�0.01HI

c91.72�0.01G

c91.53�0.01G

c91.72�0.01G

91.56�0.01

3.00

d91.75�0.01H

c91.66�0.01H

bc91.53�0.01H

d91.95�0.01HI

ab91.20�0.01H

e92.43�0.01I

f93.49�0.01H

bc91.46�0.01G

a91.04�0.01G

91.64�0.01

5.00

ab91.40�0.01H

c92.65�0.01H

ab91.11�0.01H

abc92.69�0.01HI

abc91.88�0.01HI

ab91.95�0.01I

d94.23�0.01I

a90.98�0.01G

bc92.36�0.01H

91.67�0.01

aScavengingeffect

(%)¼[(A

517nm

ofcontrol)�(A

517nm

ofsample)/(A

517nm

ofcontrol)]�

100.

bEach

valueis

expressed

inmean�SD

(n¼3).Meanswithdifferentcapitalletterswithin

acolumnare

significantlydifferent(p<0.01).Meanswithdifferentsm

allletterswithin

araw

are

significantlydifferent(P

<0.01).

Antioxidant activity of supercritical CO2 extract from rosemary leaves 83

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

In studies of the antioxidant activity of rosemary extracts, CA concentration isproved to be the best candidate for the antioxidant activity (R2

¼ 77.3%) fromrosemary extracts when measured with scavenging DPPH radicals abilitycomparing with those of CAL and rosmarinic acid [15]. Notably, CA and CALhave been suggested to account for over 90% of the antioxidant effects of rosemaryextract [16]. In this study, the results showed that the distribution of CA and CALcontents were very similar between two extracts (5000 psi, 40 and 80�C) with ratherequivalent antioxidant activities (tables 2 and 3). However, increasing the pressureat constant temperature can increase the density of supercritical CO2, which furtherincreases the salvation power of the supercritical fluid. Considering to the yield of

Table 3. Contents of carnosic acid and carnosol of rosemary extracts from supercritical CO2

extraction at different extraction conditions.

Amounta (mg g�1 dried leaf)

Pressure (psi) Temperature (�C) Carnosic acid Carnosol

3000 40 12.20� 0.04Db 0.18� 0.02B60 7.16� 0.40E 0.19� 0.03B80 2.79� 0.53G 0.04� 0.02E

4000 40 4.38� 0.07F 0.14� 0.01C60 7.25� 0.42E 0.10� 0.03D80 19.03� 0.03C 0.19� 0.01B

5000 40 38.03� 0.26A 0.17� 0.03B60 32.07� 1.31B 0.53� 0.05A80 35.23� 0.31A 0.46� 0.03A

aSupercritical CO2 extracts from freeze-dried rosemary leaves (5.00 g).bEach value is expressed in mean�SD (n¼ 3). Means with different conditions within a column aresignificantly different ( p<0.05).

Concentration (mg mL−1)

0.01 0.1 1

0

20

40

60

80

100

5000 psi, 40°C5000 psi, 60°C5000 Psi, 80°CTrolox

*

*

EC50

*

Figure 2. Free radicals scavenging effect and the effective concentration at 50% (EC50) scavengingcapability of rosemary extracts (5mgmL�1) obtained at SFE conditions of 5000 psi, 40, 60 and 80�C,respectively. Free radicals: 1,1-diphenyl-2-picrylhydrazyl (DPPH.).

84 C.-H. Chang et al.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

extraction, our result showed in accordance with that of the ideal extractioncondition for rosemary antioxidants was 5000 psi and 80�C and directly related tothe pressure and temperature used in SFE.

3.3. GC–MS identification and quantification of phenolic diterpenes

In a previous report [18], a sensitive and reliable analytical method suitable forquantitative determination of different phenolic xenoestrogens has been developed,which is based on methylation using a methylating agent TMAH and then analysisusing a GC–MS without requiring any clean-up. A similar GC/MS techniquecombining was developed for the analysis of phenolic diterpenes in rosemary extracts.Figure 3(A) and (B) illustrate the characteristic GC–MS fragment spectra of CA andCAL in SC-CO2 extracts of rosemary and has been confirmed by authentic compoundsas shown in figure 4(A). The molecular ions of CA and CAL were detected with m/z at374 and 372, respectively, after the methylation with TMAH agent. Relative intensitiesof CA and CAL, as verified by MS spectra, showed distinctly different ratio betweeneach other. The obtained spectra were also quite different from those spectra detectedby directly introducing the sample into the ionization chamber [20]. They demonstratethe MS data of CA with m/z (%): 286 (100), 230 (48), 243 (24), 287 (22), 204 (20), 271(19), 215 (18), 332 (Mþ, 8), and those of CAL in m/z (%): 286 (100), 215 (50), 204 (22),287 (21), 202 (20), 217 (17), 330 (Mþ, 17), respectively.

Analysis of phenolic diterpenes CA and CAL has been generally accomplished byHPLC with UV or evaporative light scattering detector (ELSD) [3,7–9,11,12]. Furtheridentification of CA and CAL using an LC–MS following a HPLC analysis, includingthe particle beam ionization [9], the atmospheric-pressure chemical ionization [4] andthe ESI [6,11,18,21], have been suggested. However, with regard to the complicatedanalysis techniques used in HPLC and LC/MS, a straight general method from oneinstrument was suggested. Previous reports have indicated that although PDAs havegenerally been identified by combining HPLC data with UV spectra and recordedwith a diode-array detector [4,5,9–11,15,20–25], the different absorption coefficients(response factors) at the detection wavelength in an HPLC analysis may be randomlyvarying, e.g., the response factors of CAL relative to CA at 230 and 280 nm have beenestimated to be 0.92 and 1.36, respectively [24]. A determination of CA and CAL inall of the SFE samples by a universal detector of GC/MS analysis would be inaccordance with requirements. Results from the linear calibration curve of authenticCA obtained from 10–120 mgmL�1 with coefficient of determination (r2) of 0.9809and an equation of Y¼ 3.0456Xþ 0.0277 (data not shown) was found feasible for thequantification of CA as well as CAL owing to the universal detection of GC/MS byelectron ionization technique and the specificity of SIM method. In previous result ofthe PDA fraction in rosemary, data appeared to be very random, which is consideredto be due to rapid and labile auto-oxidation of CA to CAL and then to PDA �- and�-lactones during analysis [20]. A simpler and more rapid methylation procedure forsuch thermolabile compounds during analysis is becoming more necessarily. With asimpler, reliable and a more feasible GC–MS measurement for retention time andcompound structure, it was possible to determine analyte of interest in one run.Figure 4(B) shows total ion chromatograms from GC/MS analysis of SFE samplefrom rosemary leaves and figure 4(C) indicates the selective ion chromatogram of

Antioxidant activity of supercritical CO2 extract from rosemary leaves 85

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

compounds of CA and CAL from the same extract. Obviously, in the determinationof CA and CAL in all of considered SFE sample could be achieved within 20min.Hence, the ions of CA: 219, 374 and CAL: 243, 372 were then chosen for thequantification of these two compounds which existed in extracts. In quantitativeanalysis of principle antioxidant components of rosemary, the extraction yields of

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

200

400

600

800

1000

1200

1400

1600

1800

2000

m/z-->

AbundanceAverage of 12.263 to 12.287 min.: 911016A.D (−)

(a)

(b)

69

83

115 141

165

183

201

213

228

243

271

281296

313

372

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

m/z-->

AbundanceAverage of 12.403 to 12.450 min.: 911016A.D (-)

69

91

105

128129

153165189

219

233 245

259285299

315

374

m/z

Figure 3. Mass spectra from GC/MS analyses of methylated carnosic acid (a) and carnosol (b).

86 C.-H. Chang et al.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

7.00 8.00 9.00 10.00 11.00 12.00 13.000

2000400060008000

100001200014000160001800020000220002400026000280003000032000340003600038000

Time-->

Abundance

2

1

7.00 8.00 9.00 10.00 11.00 12.00 13.00

2000400060008000

1000012000140001600018000200002200024000260002800030000320003400036000380004000042000440004600048000

Time-->

Abundance

7.00 8.00 9.00 10.00 11.00 12.00 13.000

50100150200250300350400450500550600650700750800850900950

100010501100

Time-->

Abundance

Time (min)

(a)

(b)

(c)

1

2

1

2

Figure 4. Total ion chromatograms of authentic compounds (a) and supercritical CO2 extractfrom rosemary leaves (b) and selective ion chromatogram of the same extract (c). 1: Carnosic acid.2: Carnosol.

Antioxidant activity of supercritical CO2 extract from rosemary leaves 87

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

antioxidant compounds such as CA and CAL have been reported in direct proportionto the pressure raised [11]. The presence of CA (35.23mg g�1) in the extract ofrosemary leaves under condition of 5000 psi and 80

�

C (table 3) indicates a comparableresult to that of data with 35.73mg g�1 by Tena et al. [11].

3.4. HPLC analysis and ESI–MS–MS confirmation

By comparing results from the GC/MS analysis of CA and CAL compounds withHPLC analysis, the isolated compounds from semi-preparative HPLC by using diodearray detector (DAD) were investigated (figure 5). Although DAD has beendemonstrated as an effective tool for identifying many organic compounds, thedistinction between the UV spectra of CA and CAL (figure 5) was in an insufficientselectivity and resulted in a limited usage. Therefore, a complementary data from massspectrum was required for confirmation in this work. Figure 6(A) and (B) present theMS/MS analysis of CA and CAL, respectively, in which, the product-ion spectra fromthe protonated molecular ion of m/z 333 and 331 made a distinction between eachother. The relative abundance of spectra confirms the structures of CA as well as CAL.

Experiments in the ionization with particle beam interface (PBI) presented asconventional EI spectra in LC/MS were reported to be lacking in the sensitivityrequired for many applications [14]. The consisting and sufficient ion spectra from GC/EI–MS resulted suitably as mass spectra fingerprints for library searching indicatingthat GC/EI–MS analytical technique was prevailed over LC/MS method. Moreover,the sensitivity from GC/MS detector is usually superior to those of LC/MS detectorsthat resulted from the mobile phase used and suffered from the drawback of the highernoise background. Obviously, in addition to the extraction pressure and temperatureconditions used in SFE, the instrumental analysis method also plays an important rolein the analysis of the active antioxidant composition from dried rosemary leaves.

Figure 5. Semi-preparative high performance liquid chromatogram of rosemary extracts from supercriticalCO2 extraction. The illustration showed UV-Vis spectra of carnosol (1) and carnosic acid (2).

88 C.-H. Chang et al.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

4. Conclusions

The optimum extractability for the active antioxidant constituents (CA and CAL) wasfound to be accessible by SFE with condition at 5000 psi, 80�C and an extraction yield

of 4.27% (w/w) from rosemary leaves. The main advantages of using TMAH derivativereagent for analyses of CA and CAL by GC/MS method such as less operational step

and high resolution in analysis of constituents in rosemary extracts has been expected

as an alternative method for HPLC and especially LC/MS.

Acknowledgements

The authors are grateful to Dr Long-Zen Chang, the Taichung District Agricultural

Research and Extension Station, Taiwan for supplying rosemary plant used in this

Figure 6. MS–MS analyses of product-ion spectra of carnosic acid (A, precursor ion at m/z 333) andcarnosol (B, precursor ion at m/z 331) of rosemary extracts obtained from supercritical CO2 extraction andsemi-preparative HPLC.

Antioxidant activity of supercritical CO2 extract from rosemary leaves 89

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013

study. This work was supported by a research grant of the National Science Council,

the Republic of China (Grant no. NSC 92-2313-B-241-003).

References

[1] R. Inatani, N. Nakatani, H. Fuwa, H. Seto. Agric. Biol. Chem., 46, 1661 (1982).[2] R. Inatani, N. Nakatani, H. Fuwa. Agric. Biol. Chem., 47, 521 (1983).[3] Q. Chen, H. Shi, C.T. Ho. J. Am. Oil Chem. Soc., 69, 999 (1992).[4] M.E. Curvelier, H. Richard, C. Berset. J. Am. Oil. Chem. Soc., 73, 645 (1996).[5] K. Schwarz, W. Ternes. Z. Lebensm. Unters. Forsch., 195, 95 (1992).[6] S.L. Richheimer, M.W. Bernart, G.A. King, M.C. Kent, D.T. Bailey. J. Am. Oil Chem. Soc., 73, 507

(1996).[7] M. Steiner, I. Priel, J. Giat, J. Levy, Y. Sharoni, M. Danilenko. Nutr. Cancer, 41, 135 (2001).[8] K. Ninomiya, H. Matsuda, H. Shimoda, N. Nishida, N. Kasajima, T. Yoshino, T. Morikawa,

M. Yoshikawa. Bioorganic. & Med. Chem. Lett., 14, 1943 (2004).[9] C. Bicchi, A. Binello, P. Rubiolo. Phytochem. Anal., 11, 236 (2000).

[10] S. Albu, E. Joyce, L. Paniwnyk, J.P. Lorimer, T.J. Mason. Ultrason. Sonochem., 11, 261 (2004).[11] M.T. Tena, M. Valcarcel, P.J. Hidalgo, J.L. Ubera. Anal. Chem., 69, 521 (1997).[12] T.C. Matsingou, N. Petrakis, M.Kapsokefalou, A. Salifoglou. J. Agric. Food Chem., 51, 6701 (2003).[13] F.J. Senorans, E. Ibanez, S. Cavero, J. Tabera, G. Reglero. J. Chromatogr. A, 870, 491 (2000).[14] J.M. Halket, D. Waterman, A.M. Przyborowska, R. Patel, P.D. Fraser, P.M. Bramley. J. Experi. Bot.,

56, 219 (2005).[15] C. Wellwood, R.A. Cole. J. Agric. Food Chem., 52, 6101 (2004).[16] O.I. Aruoma, B. Halliwell, R. Aeschbach, J. L’Oligers. Xenobio., 22, 257 (1992).[17] T. Masuda, Y. Inaba, Y. Takeda. J. Agric. Food Chem., 49, 5560 (2001).[18] U. Bolz, W. Korner, H. Hagenmaier. Chemosphere, 40, 929 (2000).[19] K. Shimada, K. Fujikawa, K. Yahara, T. Nakamura. J. Agric. Food Chem., 40, 945 (1992).[20] M.E. Curvelier, C. Berset, H. Richard. J. Agric. Food Chem., 42, 665 (1994).[21] K. Schwarz, W. Ternes. Z. Lebensm. Unters. Forsch., 195, 99 (1992).[22] S. Munne-Bosch, L. Alegre, K. Schwarz. Eur. Food Res. Technol., 210, 263 (2000).[23] P.J. Hidalgo, J.L. Ubera, M.T. Tena, M. Valcarel. J. Agric. Food Chem., 46, 2624 (1998).[24] M.A. Thorsen, K.S. Hildebrandt. J. Chromatogr. A, 995, 119 (2003).[25] E. Ibanez, A. Cifuentes, A.L. Crego, F.J. Senorans, S. Cavero, G. Reglero. J. Agric. Food Chem., 48,

4060 (2000).

90 C.-H. Chang et al.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

onne

ctic

ut]

at 1

6:10

11

Aug

ust 2

013