Supercritical carbon dioxide extraction of antioxidant fractions from selected Lamiaceae herbs and their antioxidant capacity

Innovative Food Science and Emerging Technologies 11 (2010) 98–107

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Innovative Food Science and Emerging Technologies j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i f s e t

Supercritical carbon dioxide extraction of antioxidant fractions from selected Lamiaceae herbs and their antioxidant capacity Nada Babovic a,b,⁎, Sonja Djilas c, Milka Jadranin d, Vlatka Vajs d, Jasna Ivanovic a, Slobodan Petrovic a,e, Irena Zizovic a a

University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia Singidunum University, Bulevar Mihaila Pupina 12a, 11000 Belgrade, Serbia University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia d Institute for Chemistry, Technology and Metallurgy, Njegoševa 12, 11000 Belgrade, Serbia e Hemofarm Group, Pharmaceutical and Chemical Industry, Beogradski put bb, 26 300 Vrsac, Serbia b c

a r t i c l e

i n f o

Article history: Received 27 February 2009 Accepted 21 August 2009 Editor proof receive date 5 September 2009 Keywords: Natural antioxidants Antioxidant activity Rosemary Sage Thyme Hyssop Supercritical carbon dioxide extraction

a b s t r a c t Antioxidant fractions from four herb spices belonging to the Lamiaceae family: rosemary (Rosmarinus officinalis), sage (Salvia officinalis), thyme (Thymus vulgaris) and hyssop (Hyssop officinalis) were isolated using supercritical CO2 at 35 MPa and 100 °C. The antioxidant fractions were characterized chemically by HPLC-DAD/ESI-ToF-MS. Antioxidant activity of obtained extracts was determined by measuring their ability to scavenge stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical and reactive hydroxyl radical during the Fenton reaction trapped by 5,5-dimethyl-1-pyroline-N-oxide (DMPO), using electron spin resonance (ESR) spectroscopy. The antioxidant activity of the extracts was compared to the activity of butylated hydroxyanisole (BHA) and Flavor' Plus™ water-soluble rosemary extract. In DPPH radical assay the order from the strongest to the weakest antioxidant activity was: BHA, thyme extract, Flavor' Plus™, rosemary and sage extracts, and hyssop extract, while in hydroxyl radical assay order was: Flavor' Plus™, sage extract, rosemary extract, hyssop extract, BHA and thyme extract. Industrial relevance: Spices and herbs have been used not only for flavoring food but also for improving the overall quality of the product and to extend the shelf-life of foods. The present investigation relates to the field of food additives, and particularly to an antioxidant fractions from four herb spices which belong to the Lamiaceae family: rosemary (Rosmarinus officinalis), sage (Salvia officinalis), thyme (Thymus vulgaris) and hyssop (Hyssop officinalis). Butylated hydroxyanisole (BHA) and Flavor' Plus™ are used in food industry as antioxidants due to their ability to prolong the shelf-life of foodstuffs by protecting them against deterioration caused by oxidation, such as fat rancidity, colour changes, degradation of the flavor and loss of nutrient value. Synthetic antioxidants such as BHA now being replaced by natural antioxidants because of their possible toxicity and due to a suspected action as promoters of carcinogens. The present study confirms that investigated herb spices belonging to the Lamiaceae family present important sources for the production of food additives. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Herbs of the Lamiaceae family (previously known as Labiatae), like rosemary, sage, oregano and thyme are well-known for their antioxidant activity. Antioxidants are important in the food industry not only because of their usefulness as a preservation method but also because of their beneficial effects on human health (Madhavi, Despande, & Salunkhe, 1996). The utilisation of synthetic antioxidants is limited because consumers are increasingly demanding additive-free or natural products (Ahn, Grün, & Fernando, 2002). Therefore, the application of ⁎ Corresponding author. University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia. Tel.: +381 11 3303 795; fax: +381 11 3370387. E-mail address: [email protected] (N. Babovic). 1466-8564/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2009.08.013

spices and herbs as sources of many effective antioxidants is a promising alternative to the use of synthetic antioxidants. The majority of natural antioxidants are phenolic compounds or polyphenols and the antioxidant activity of many natural extracts is due to such phenolic compounds. The leaves of the plant Rosmarinus officinalis L. are commonly used as a spice, flavoring agent, and naturally occurring antioxidant. Phenolic compounds present in rosemary extracts can be classified into three groups: phenolic diterpenes, flavonoids and phenolic acids (Cuvelier, Richard, & Berset, 1996). Among the herbs of the Lamiaceae family, rosemary has been more extensively studied as a natural antioxidant source (Yanishlieva, Marinova, & Pokorný, 2006). Sage, Salvia officinalis L., has been most commonly known not only as a culinary herb for flavoring and seasoning, but has been also of

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great medicinal importance such as anti-lactation, anti-inflammation, anti-sore throat, and anti-dyspepsia (Ninomiya et al., 2004). Together with R. officinalis L., S. officinalis L. has been shown to have the strongest antioxidant activities among herbs. It was previously reported that the antioxidant activity of extract obtained by supercritical carbon dioxide extraction from rosemary and sage leaves was comparable with the activity of synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), without the cytotoxic and carcinogenic risk of synthetic antioxidants (Huang et al., 1994; Ito, Fukushima, Hagiwara, Shibata, & Ogiso, 1983; Djarmati, Jankov, Schwirtlich, Djulinac, & Djordjevic, 1991). Djarmati et al. (1991) reextracted ethanol extract of sage with supercritical carbon dioxide and isolated antioxidant compound (rosmanol-9-ethyl ether) with activity much greater than BHT. Highly active compounds in rosemary and sage extracts were found to be phenolic diterpenes such as carnosol and carnosic acid (Schwarz & Waldemar, 1992), royleanonic acid and 7-methoxyrosmanol (Ninomiya et al., 2004), rosmadial (Nakatani & Inatani, 1983), rosmanol (Inatani, Nakatani, Fuwa, & Seto, 1982; Nakatani & Inatani, 1983), epirosmanol and isorosmanol (Nakatani & Inatani, 1984), methyl carnosate (Cuvelier, Berset, & Richard, 1994), rosmanol-9-ethyl ether (Djarmati et al., 1991) flavonoids such as genkwanin, cirstimaritin and scutellarein and phenolic acids such as rosmarinic acid (Cuvelier et al., 1996; Senorans, Ibanez, Cavero, Tabera, & Reglero, 2000; Cavero et al., 2005). Thyme (Thymus vulgaris L.) has been used commonly as a culinary herb for adding flavor and as cough medicine, to treat dyspepsia and other gastrointestinal disturbances. In particular, thyme is valued for its antiseptic and antioxidant properties (Miura & Nakatani, 1989; Schwarz, Ernst, & Ternes, 1996; Economou, Oreopoulo, & Thomopoulos, 1991), deodorizing (Nakatani, Miura, & Inagaki, 1989) and anti-platelet activity (Okazaki, Kawazoe, & Takaishi, 2002). A recent investigation (Simandi et al., 2001) has demonstrated antioxidant activity of thyme extract obtained by supercritical fluid extraction. Hyssop (Hyssopus officinalis L.) as a food ingredient has its own importance in the flavor industry and also in sauce formulations (Kazazi, Rezaei, Ghotb-Sharif, Emam-Djomeh, & Yamini, 2007). As a medicinal plant, hyssop has also been used as a carminative, emmenagogue, stimulant, stomachic, and tonic. Among the medicinal and aromatic plants, hyssop is a plant that has not been studied very much. Dapkevicius, Venskutonis, van Beek, and Linssen (1998) reported that antioxidant activity of deodorized hyssop extracts was very low in comparison with rosemary, thyme, marjoram and sage. Djarmati et al.(1991) isolated potent antioxidant rosmanol-9-ethyl ether from the alcoholic extract of the hyssop. Supercritical fluid extraction (SFE) with carbon dioxide is considered to be the most suitable method for producing natural antioxidants to be used in the food industry. Carbon dioxide is safe (GRAS), non-toxic, non-carcinogenic, non-flammable, has modest critical conditions (31.1 °C, 7.38 MPa) and inexpensive. This emerging clean technology provides solvent free extracts and the selectivity of the supercritical CO2 can be adjusted by varying temperature and pressure to obtain the fractions consisting of desirable compounds. Antioxidants from rosemary have been extracted and de-aromatized by supercritical CO2 for use in natural supplements and several reports have been published. For instance, Lopez-Sebastian et al. (1998) utilized supercritical CO2 for the deodorization of rosemary extracts obtained by solvent extraction, while Ibanez et al. (1999) fractionated rosemary essential oil and antioxidant fraction. The method describing isolation of antioxidants by SFE at high pressures was patented in USA in 1991 (Nguyen, Frankman, & Evans, 1991). Nguyen et al. (1991) reported extraction of antioxidants by supercritical CO2 from rosemary, sage, oregano and thyme at pressures above 35 MPa and temperatures in range 90–110 °C. At extraction temperature above 110 °C heat damage of the extracted components could occur. Further, the authors reported that it was

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preferred that the supercritical fluid used in process was pure CO2 without the addition of cosolvents such as ethanol or propane. Cosolvents increased the yield by coextraction of other compounds (without antioxidant properties) resulting in reduced antioxidant activity of the obtained extract when compared with the activity of the extract obtained with pure CO2. The aim of the present study was to investigate isolation of antioxidant fractions from rosemary, sage, thyme and hyssop by fractional supercritical carbon dioxide extraction, as well as to determine the antioxidant activity of the obtained extracts by measuring their ability to scavenge DPPH free radical and hydroxyl radical using electron spin resonance (ESR) spectroscopy. Chemical analysis of obtained antioxidant extracts was performed using LC-MS. Carnosol and carnosic acid, among the main compounds present in examined extracts, were quantified with regard to pure standard. Identification of the other compounds was tentative. To the best of our knowledge, there is no data available in the open literature on chemical composition of SFE extracts of investigated plants obtained at 35 MPa and 100 °C. Also there is no data at all on the chemical composition of thyme and hyssop extracts obtained by SFE. 2. Materials and methods 2.1. Samples and chemicals Dried leaves of selected herbs belonging to the Lamiaceae family: rosemary (R. officinalis), sage (S. officinalis), thyme (T. vulgaris) and hyssop (Hyssop officinalis) originated from the southern Balkan region were used for the study. Commercial carbon dioxide (99% purity, Tehno-gas, Novi Sad, Serbia) was used for the SFE. DPPH, DMPO and BHA were from Sigma Chemical Co., St. Louis, USA, and Flavor' Plus™ was from Naturex, France. Methanol for HPLC was from Burdick & Jackson, Mashegon, MI, USA, acetonitrile was from Merck KgaA, Darmstadt, Germany, formic acid was from Lach-Ner, s.r.o., Neratovice, Czech Republic and Milli Q water 18.2 MΩ cm was obtained from purification system. Millipore Simplicity 185 were used for the study. The standard compounds used for the chemical analyses were carnosic acid and carnosol (Sigma Chemical Co. (St. Louis, USA)). These chemicals were of analytical reagent grade. Other used chemicals and solvents were of the highest analytical grade and obtained from “Zorka” Šabac (Serbia). 2.2. Supercritical CO2 extraction In order to isolate antioxidant fractions, method of fractional supercritical extraction with carbon dioxide was applied. Essential oil fraction was extracted first at pressure of 11.5 MPa and temperature of 40 °C. Extraction of antioxidant fraction followed at pressure of 35 MPa and temperature of 100 °C. Extraction conditions (100 °C and 35 MPa) were chosen on the basis of previous investigations. Nguyen et al. (1991) showed that the temperature range 90–110 °C was the most preferable for the isolation of antioxidant fraction from Lamiaceae family species. Further Rižnar, Čelan, Škerget, and Knez (2008) reported that carnosol (from rosemary extract) solubility had the highest value at the pressure of 35 MPa and increased with the temperature increasing from 40 °C to 80 °C. Plant material was ground and sieved. The fraction of the average particle diameter of 0.400 mm was used for further study. The flow rate of SC-CO2 was 0.3 kg/h in all experiments. The initial used mass of the plant samples was 56 g for rosemary, 56.5 g for sage, 54 g for thyme and 55 g for hyssop. Extractions with supercritical carbon dioxide (SC CO2) were performed in the Autoclave Engineers Screening System previously described (Zizovic, Stamenic, Orlovic, & Skala, 2007). The simplified scheme of the laboratory plant is presented in Fig. 1. Liquid CO2 is supplied from the CO2 cylinder by a siphon tube and cooled in a cryostat between the cylinder outlet and

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previous data published by other authors. Their complete identification was not possible since the full scan mass spectra of the chromatographically separated compounds gave only deprotonated [M–H]− ions, and MS/MS experiments were not possible with used instrumentation. For UV–vis identification and quantification of carnosol and carnosic acid, the UV detector was monitored at 240 nm, and peak spectra were recorded between 190 and 450 nm by using a DAD. This data can be very useful to identify compounds of interest. Carnosol and carnosic acid, among the main compounds present in examined extracts, were quantified with regard to pure standard. 2.4. Quantitative analysis of carnosol and carnosic acid by HPLC-DAD Fig. 1. Schematic presentation of the autoclave engineers SFE screening system used in the present study system—T: CO2 storage tank; C: cryostat; LP: high pressure liquid pump; E: extractor vessel; S: separator vessel.

the pump to prevent vaporization. The CO2 is then pumped into the system until the required pressure is obtained. Back pressure regulators are used to set the system pressure (in the extractor and separator). The extractor vessel is filled with the plant material from which a substance is to be extracted. Heaters are supplied on the extractor vessel for temperature elevation. The SC CO2 flows through the extractor and enters the separator vessel. Samples of the extracted substance can be taken by opening the ball valve located at the bottom of the vessel. The CO2 continues to flow out of the separator through the flowmeter/totalizer and out to the atmosphere. 2.3. HPLC-DAD-ESI-ToF-MS analyses of the antioxidant fractions Samples of rosemary, sage, thyme and hyssop extracts were analyzed by HPLC-DAD/ESI-ToF. The samples were prepared at a concentration of 10,000 mg/ml in MeOH, and filtered through a 0.45 μm poly-tetrafluoroethilene (PTFE) filter (Agilent Technologies). The LC/DAD/MS analyses were carried out in an Agilent 1200 HPLC instrument (Agilent Technologies, Waldbronn, Germany) with a binary pump, an autosampler, a column compartment equipped with a Zorbax Eclipse Plus C18 column (1.8 μm, 4.6 × 150 mm, Agilent) and a diode-array detector coupled with a 6210 Time-ofFlight LC-MS system (Agilent Technologies). The mobile phase consisted of water containing 0.2% formic acid (A) and acetonitrile (B). A gradient program was used as follows: initial 0–1.5 min 5% B, 1.5–26 min, linear change from 5% B to 95% B, 26–35 min, maintaining 95% B. The mobile phase flow rate was 1.4 ml/min, the column temperature was set at 40 °C and the injection volume was 5 μl. Spectral data from all peaks were accumulated in the range of 190– 450 nm and chromatograms were recorded at 240 nm. A personal computer system running a MassHunter Workstation software was used for data acquisition and processing. In the atmospheric pressure electrospray ionization (ESI) method, the eluted compounds were mixed with nitrogen in the heated nebulizer interface and polarity was tuned to negative. Adequate calibration of ESI parameters (capillary voltage, gas temperature, nebulizer pressure, and fragmentor voltage) was required to optimize the response and to obtain a high sensitivity of the molecular ion. The selected MS values were: capillary voltage 4000 V, gas temperature 350 °C, drying gas 12 l/min, nebulizer pressure 45 psi, fragmentor voltage 140 V, mass range 100–2000 m/z. Organic acid was added into the mobile phase to ameliorate the peak shape. Different organic acids in different concentrations were tried. The results showed that 0.2% formic acid was suitable. Positive and negative ion modes were tried for phenolic compounds, and the results suggested that negative mode was more sensitive. Compounds were characterized for their retention times (tR), mass spectra and UV spectra, and were tentatively identified based on

Quantitative analyses were carried out with the same 1200 Agilent binary pump system (Waldbron, Germany) used in qualitative analyses. The column, mobile phase, solvent gradient, flow rate and injection volume were also the same as those used in the LC-MS analysis. UV detection was performed at 240 nm. Four calibration curves were done with solutions of known concentrations of standard carnosol and carnosic acid. Samples were prepared at a concentration of 10,000 mg/ml in MeOH, and filtered through a 0.45 μm polytetrafluoroethilene (PTFE) filter (Agilent Technologies). 2.5. DPPH radical assay Blank probe was obtained by mixing 400 μl of 0.4 mM methanolic solution of DPPH and 200 μl of DMF (N, N-dimethylformamide). A volume of × μl of 10 mg/ml DMF solution of investigated antioxidant fractions obtained at 35 MPa and temperatures of 100 °C was added to a mixture of (200×) μl of DMF and 400 μl of 0.4 mM methanolic solution of DPPH radical (probe). The range of concentrations of the investigated extracts was 0.05–0.4 mg/ml for rosemary and sage, 0.01–0.3 mg/ml for thyme and 1.0–20.0 mg/ml for hyssop. The range of investigated concentrations for commercial antioxidants was 0.005–0.2 mg/ml for BHA and 0.01–0.5 mg/ml for Flavor' Plus™. After that the mixture was stirred for 2 min and transferred to a quartz flat cell ER-160FT. The ESR spectra were recorded on an ESR spectrometer Bruker 300E (Rheinstetten, Germany) under the following conditions: field modulation 100 kHz, modulation amplitude 0.256 G, receiver gain 2 · 104, time constant 40.96 ms, conversion time 327.68 ms, center field 3440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW, temperature 23 °C. The AADPPH value of the extract was defined as: AADPPH ð%Þ = 100⋅ðho −hx Þ = ho where ho and hx are the height of the second peak in the ESR spectrum of DPPH radicals of the blank and the probe, respectively. 2.6. Hydroxyl radical assay Hydroxyl radicals were obtained by the Fenton reaction in the system: 0.2 ml 2 mM H2O2, 0.2 ml 0.3 mM FeCl2×4H2O and 0.2 ml 112 mM DMPO as spin trap (blank). The influence of investigated antioxidant fractions of rosemary, sage, thyme and hyssop and commercial antioxidants on the formation and transformation of

Table 1 Obtained extraction yields of antioxidant fractions. Plant material

Pressure, MPa

Temperature, °C

Yield, wt.%

Rosemary Sage Thyme Hyssop

35 35 35 35

100 100 100 100

1.33 1.53 1.58 1.08

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hydroxyl radicals was conducted by adding the DMF solutions of the investigated extracts of rosemary, sage, thyme and hyssop to the Fenton reaction system in the range of concentrations 0.1–3.0 mg/ml for rosemary and hyssop, 0.1–2.0 mg/ml for sage, 0.5–3.0 mg/ml for thyme, 0.1–3.0 mg/ml for BHA and 0.1–1.75 mg/ml for Flavor' Plus™. ESR spectra were recorded after 2.5 min, with the following spectrometer settings: field modulation 100 kHz, modulation amplitude 0.512 G, receiver gain 1 · 104 , time constant 81.92 ms, conversion time 163.84 ms, center field 3440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW, temperature 23 °C. The AAOH value of the extract was defined as: AAOH ð%Þ = 100⋅ðho −hx Þ = ho

where ho and hx are the height of the second peak in the ESR spectrum of DMPO-OH spin adduct of the blank and the probe, respectively.

2.7. Statistical analysis Fig. 2. Yield of antioxidant fraction of rosemary, sage, thyme and hyssop as function of specific amount of solvent (kg CO2/kg herbaceous material) for SFE at 35 MPa and 100 °C.

Origin 6.0 software (OriginLab Corporation, USA) was used for statistic analysis of AADPPH and AAOH data. All measurements were done in triplicate and presented as mean ± standard deviation (SD).

Fig. 3. Chromatographic profiles obtained for rosemary extract, top, ESI negative ionization signal, bottom, DAD signal at 240 nm.

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Fig. 3 (continued).

3. Results and discussion 3.1. Extraction yields The extraction yields of essential oil fractions from rosemary, sage, thyme and hyssop were 0.676% w/w, 2.32% w/w, 0.7541% w/w and 0.651% w/w, respectively. After removing the essential oil fraction which contained the main aroma compounds, SFE of antioxidant fraction commenced. Extraction yields of obtained antioxidant fractions are shown in Table 1. Extraction yield curves are presented in Fig. 2. Cavero et al. (2005) isolated rosemary SFE extract at pressure range of 15 MPa to 35 MPa and temperature range of 40 °C to 60 °C. Extraction yields ranged from 0.03 to 0.13% for the extractions without cosolvent used, and from 3.93 to 6.78% for the extractions in which ethanol was supplied as modifier. Ibanez et al. (1999) obtained rosemary antioxidant fraction at 40 MPa and 60 °C. Obtained yields ranged from 1 to 1.5%. Daukšas, Venskutonis, Povilaityte, and Sivik (2001) reported SFE of sage at the temperature of 100 °C and at 25 and 35 MPa with or without adding 1% or 2% of ethanol as modifier. They found that the pressure between 25 and 30 MPa could be considered as a critical one in terms of solubility of approximately 50% of sage extractives isolated at 35 MPa with CO2 enriched by 1% of ethanol. The yields of the sage extracts at 35 MPa were substantially

increased by using 1% of ethanol (yields ranging from 12.15 ± 0.10 when ethanol not used to 43.99 ± 10.07 g/100 g when ethanol was used), while further adding of ethanol to 2% was not efficient (yield was 21.81 ± 5.82 g/100 g). Dapkevicius et al. (1998) reported that the yields of rosemary, sage, thyme and hyssop extracts obtained by SFE at 30 MPa and 40 °C were 71.5 g/kg, 50.2 g/kg, 54.6 g/kg and 37.1 g/ kg, respectively. Simandi et al. (2001) isolated thyme SFE extract at pressure of 40 MPa and temperature of 60 °C. Obtained yield was 4.92 wt.%. The difference of yields of antioxidant extracts could be explained in terms of different quality of herbs, geographical origin, harvest date, climatic conditions and different extraction operating conditions. 3.2. HPLC-DAD-ESI-ToF-MS analyses of the antioxidant fractions Fig. 3 shows the chromatographic profiles obtained by DAD at 240 nm for rosemary extract. Along with these profiles, signal for ESI in the negative mode is also shown. The contents of carnosol and carnosic acid in different plant extracts have been expressed as g per 100 g of extract and are presented in Table 2. Since the UV detector (in the range of 190–450 nm) has a different response to each compound identified by LC-MS, we have performed

N. Babovic et al. / Innovative Food Science and Emerging Technologies 11 (2010) 98–107 Table 2 Carnosol and carnosic acid content in different plant extracts. Plant extract

Carnosol g/100 g of extract at 240 nm

Carnosic acid g/100 g of extract at 240 nm

Rosemary Sage Thyme Hyssop

3.9368 6.9729 / 7.3341

4.7596 13.7639 / /

semi-quantitative analysis based on ESI(−) peak area. The identified compounds were selected and their relative percentage (referred to the total area of selected compounds based on ESI(−) peak area) is shown in Tables 3–6. Table 3 shows the retention time (tR), absorption maxima of the peaks found, peaks area, the mass spectral data of the rosemary antioxidant fraction and tentative identification on the basis of spectral data and references in the literature (Yesil-Celiktas, Nartop, Gurel, Bedir, & Vardar-Sukan, 2007; Senorans et al., 2000; Zheng, & Wang, 2001; Almela, Sanchez-Munoz, Fernandez-Lopez, Rosa, & Rabe,

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2006; Nakatani & Inatani, 1983; Cuvelier et al., 1994; Richheimer, Bernart, King, Kent, & Beiley, 1996; Houlihan, Ho, & Chang, 1984). By analyzing published phytochemical reports on S. officinalis (Santos-Gomes, Seabra, Andrade, & Fernandes-Ferreira, 2002; Miura, Kikuzaki, & Nakatani, 2002; Ninomiya et al., 2004; Masterova, Misíková, Sirotková, Vaverková, & Ubik, 1996; Tada, Okuno, Chiba, Ohnishi, & Yoshii, 1994; Kavvadias, Monschein, Sand, Riederer, & Schreier, 2003; Fowler, Kilpert, Schueler, Wehrli, & Wyss, 2008) some compounds from sage antioxidant fraction were tentatively identified (Table 4). Peaks 10, (12, 20), 22, 29 and 30 were tentatively identified as paramiltioic acid, salvicanaric acid, royleanone, salviviridinol, α-linolenic acid and α-linoleic acid, respectively. These compounds had been isolated from different Salvia species than S. officinalis (Sun, Luo, Sakai, & Niwa, 1991; Topcu et al., 2008; de Saizieu et al., 2008; Ulubelen et al., 2000; Kilic, Dirmenci, & Goren, 2007; Chicco, D'Alessandro, Hein, Oliva, & Lombardo, 2009). The results of the analysis of thyme antioxidant fraction are presented in Table 5. Peaks 3, 6 and 10 were tentatively identified as naringenin, cirsimaritin and methyl rosmarinate (Fecka, & Turek, 2008). By retrieving the published phytochemical reports on T. vulgaris, peaks 12, (13,14,19,21, 23, 25) and 16, were tentatively identified as

Table 3 Composition of rosemary antioxidant fraction isolated at 35 MPa and 100 °C analyzed by HPLC-DAD/ESI-ToF. Peak no.

Rt (min)

Molecular formula

[M–H]− m/z

Absorbance maxima (nm)

% area

Identificationa

2 3 4 5 6 7 8 11 13 14 15 16 18

12.1 14.0 14.1 14.6 14.8 15.3 15.3 18.7 19.2 19.5 20.1 20.8 22.1

C12H18O3 C17H14O6 C20H26O5 C20H26O5 C20H28O4 C16H12O5 C20H26O5 C20H26O4 C20H26O4 C20H24O5 C20H28O3 C20H28O4 C21H30O4

209 313 345 345 331 283 345 329 329 343 315 331 345

250, 280 250, 282 252sh, 276, 334 252, 282, 312sh 252, 278 254sh, 268, 288sh, 336 252, 270, 334 248, 284 268, 424 254sh, 286 254, 278 242, 286 256sh, 282

0.2 0.5 2.9 0.3 0.5 1.2 0.3 21.8 0.9 2.3 1.8 46.9 12.2

Jasmonic acid Cirsimaritin Rosmanol, isorosmanol, epirosmanol Rosmanol, isorosmanol, epirosmanol Carnosic acid isomer Wogonin, oroxylin A, biochanin A, genkwanin Rosmanol, isorosmanol, epirosmanol Carnosol Carnosol isomer Rosmadial Rosmaridiphenol, cafestol Carnosic acid Methyl carnosate, 12-methoxycarnosic acid

% area: relative percentage (normalized areas (%)). a Identification for carnosol and carnosic acid was performed using standards. For the other compounds identification is tentative.

Table 4 Composition of sage extract analyzed by HPLC-DAD/ESI-ToF. Peak no.

Retention time (min)

Molecular formula

[M–H]− m/z

Absorbance maxima (nm)

% area

Identificationa

2 3 6 11 4 5 8 17 18 10 12 20 13 14 19 21 22 23 24 25 29 30

14.0 14.5 15.2 18.2 14.7 15.1 17.8 19.3 19.8 18.0 18.3 20.1 18.3 18.5 19.9 20.6 21.4 21.9 22.0 23.0 24.0 25.4

C20H26O5 C20H26O5 C20H26O5 C20H26O5 C20H28O4 C16H12O5 C20H24O5 C20H24O5 C20H24O5 C19H24O5 C19H26O5 C19H26O5 C21H28O5 C20H26O4 C20H28O3 C20H28O4 C21H32O4 C21H30O4 C20H30O3 C20H30O3 C18H30O2 C18H32O2

345 345 345 345 331 283 343 343 343 331 333 333 359 329 315 331 347 345 317 317 277 279

244, 284 248, 290, 314 250, 270sh, 288, 336 258, 286 252, 278 254sh, 268, 336 254, 286 242, 286 258, 284, 434 252, 278, 320sh 250sh, 280, 354 280 250sh, 280, 354 240, 284 248, 278 198, 218sh, 232, 286 254sh, 284 272, 298sh 254sh, 280 250, 286, 304sh 378, 304sh 284

9.6 1.3 0.9 1.2 0.4 0.2 0.2 5.6 1.3 0.7 0.4 0.6 1.0 18.5 2.8 26.4 0.4 4.3 6.7 7.1 0.5 0.3

Rosmanol; epirosmanol; isorosmanol; royleanonic acid; epiisorosmanol

Horminone; hydroxyroyleanone Physcion; genkwanin Rosmadial; galdosol; safficinolide

Paramiltioic acid Salvicanaric acid 7-Methoxyrosmanol; epirosmanol methyl ether Carnosol (picrosalvin) Royleanone; 20-deoxocarnosol Carnosic acid (salvin) Salviviridinol 12-O-methylcarnosic acid; methyl carnosate 8,11,13-Abietatriene-11,12,20-triol α-Linolenic acid α-Linoleic acid

% area: relative percentage (normalized areas (%)). a Identification for carnosol and carnosic acid was performed using standards. For the other compounds identification is tentative.

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Table 5 Composition of thyme extract analyzed by HPLC-DAD/ESI-ToF. Peak no.

Retention time (min)

Molecular formula

[M–H]− m/z

Absorbance maxima (nm)

% area

Tentative identification

3 6 7 8 9 10 12 13, 14, 19, 21, 23, 25 16 31

11.9 14.0 14.4 14.5 15.0 15.4 16.6 17.3 17.9 19.8 21.0 21.7 22.0 18.3 25.6

C15H12O5 C17H14O6 C18H16O7 C10H14O2 C18H16O7 C19H18O8 C20H26O4 C20H26O3 C20H26O3 C20H26O3 C20H26O3 C20H26O3 C20H26O3 C20H24O4 C18H32O2

271 313 343 165 343 373 329 313 313 313 313 313 313 327 279

286, 338 262sh, 276, 332 254, 276, 344 200, 226, 274, 344 260sh, 282, 294sh, 332 254, 280, 344 200, 226, 276 254, 280, 344 248, 282 260sh, 278, 364 272, 362 252, 278 258, 278, 358 256, 280, 302sh, 318, 340sh 272, 348

0.3 0.5 1.9 20.3 0.7 2.0 26.4 0.4 8.8 0.2 0.2 8.8 1.9 0.4 0.9

Naringenin Cirsimaritin Cirsilineol Thymohydroquinone, p-cymene-2,3-diol Xanthomicrol Methyl rosmarinate 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl 3,4,4′-trihydroxy-5,5′-diisopropyl-2,2′-dimethyl-3,6-biphenyl

4,4′-dihydroxy-5,5′-diisopropyl-2,2′-dimethyl-3,6-biphenyldione α-Linoleic acid

% area: relative percentage (normalized areas (%)).

Table 6 Composition of hyssop extract analyzed by HPLC-DAD/ESI-ToF. Peak no.

Retention time (min)

Molecular formula

[M–H]− m/z

Absorbance maxima (nm)

% area

Identificationa

1 21 22 23 30 31 33 29

8.9 18.7 18.8 18.9 20.5 20.7 21.0 20.1

C10H16O3 C20H26O4 C18H30O3 C18H30O3 C18H30O3 C18H30O3 C18H30O3 C18H32O3

183 329 293 293 293 293 293 295

248 242, 284 242, 278sh, 358 242, 352 258, 370 278, 356 278, 388 246, 280sh, 348

2.9 10.2 10.9 8.1 0.3 0.5 0.6 4.8

Pinonoc acid Carnosol 9-Oxo-10(E),12(Z)-octadecadienoic acid

32 34 38 40 43

20.8 22.1 24.2 25.6 26.7

C20H28O4 C20H28O4 C18H30O2 C18H32O2 C16H32O2

331 331 277 279 255

276, 270, 266, 266, 268,

366 356 368 364 368

4.9 0.7 12.8 5.0 10.5

Vernolic acid Coronaric acid Dimorphecoloc acid Corialic acid 13(S)-Hydroxyoctadeca-9Z,11E-dienoic acid Marrubiin, horminone, glaucocalyxin A Linolenic acid Linoleic acid Palmitic acid Ethyltetradecanoate

% area: relative percentage (normalized areas (%)). a Identification for carnosol was performed using standards. For the other compounds identification is tentative.

3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl, 3,4,4′trihydroxy-5,5′-diisopropyl-2,2′-dimethyl-3,6-biphenyl and 4,4′-dihydroxy-5,5′-diisopropyl-2,2′-dimethyl-3,6-biphenyldione, respectively (Miura, Inagaki, & Nakatani, 1989; Nakatani et al, 1989; Okazaki et al., 2002; Dapkevicius et al., 2002; Haraguchi et al., 1996). Peaks 7, 8, 9 and 31 were tentatively identified as cirsilineol, thymohydroquinone, xanthomicrol and 9,12-octadecadienoic acid (Horwath, Grayer, KeithLucas, & Simmonds, 2008; Takeuchi, Lu, & Fujita, 2004; Guillen, & Manzanos, 1998). The HPLC-DAD/ESI-TOF results of antioxidant fraction of H. officinalis are summarized in Table 6. One of the peaks was identified as carnosol. Other peaks couldn't been tentatively identified because of the lack of literature data. Also, further confirmation is needed for these peaks because of the lack of standards. 3.3. DPPH radical antioxidant activity of extracts The DPPH radical antioxidant activity (AADPPH) of the studied extracts (antioxidant fractions) and commercial antioxidants is presented in Fig. 4(A, B). The EC50 value (concentration of an antioxidant needed to decrease the radical concentration by 50%) is a parameter widely used to measure the free radical scavenging activity (Cuvelier, Richard, & Berset, 1992). A lower EC50 indicated a higher antioxidant activity. The EC50 values of investigated extracts

are presented in Table 7. It is evident that the interaction of a potential antioxidant with DPPH radical depends on the type and concentration of the investigated extract. As can be seen from Fig. 4 and Table 7 the order from the strongest to the weakest antioxidant activity was: BHA, thyme extract, Flavor' Plus™, rosemary and sage extracts and hyssop extract. As shown in Fig. 4(A) the concentrations of BHA, thyme extract, rosemary extract, sage extract and Flavor' Plus™ which were required for totally reducing the DPPH radicals molecules were 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.4 mg/ml and 0.5 mg/ml, respectively. The concentration of hyssop extract which was required for totally reducing of DPPH radicals molecules was 20 mg/ml (Fig. 4(B)). For example, AADPPH of thyme antioxidant fraction at a concentration of 0.3 mg/ml was 100%, which was comparable to BHA and Flavor' Plus™, whereas AADPPH of rosemary and sage antioxidant fractions were only 73.97% and 71.69%, respectively. Hyssop antioxidant fraction at a concentration less than 2.5 mg/ml did not show any AADPPH. At the concentration less than 0.1 mg/ml besides BHA and Flavor' Plus™ only thyme antioxidant fraction showed AADPPH. Dapkevicius et al. (1998) reported that rosemary, sage and thyme extracts obtained by SFE possessed high antioxidant activity, while hyssop extracts showed low antioxidant activity which is in accordance with the results obtained in DPPH radical assay. Recent investigation (Yesil-Celiktas, Bedir, & Vardar-Sukan, 2007) has demonstrated different parameters of antioxidant activity obtained

N. Babovic et al. / Innovative Food Science and Emerging Technologies 11 (2010) 98–107

105

BHT (butylated hydroxytoluene) activity. Among rosemary's and sage's antioxidant compounds carnosic acid is believed to possess the highest antioxidant activity (Schwarz & Waldemar, 1992; Okamura, Fujimoto, Kuwabara, & Yagi, 1994; Cuvelier et al., 1996; Richheimer et al., 1996; Cavero et al., 2005). As shown in Table 2, carnosic acid was found in rosemary and sage extracts in amounts of 4.7596 and 13.7639 g/100 g of extract, respectively. Carnosic acid wasn't found in thyme and hyssop extracts. The results obtained by DPPH radical assay indicate that biphenyl compounds appeared to be the main compounds responsible for the antioxidant activity of thyme extracts. Thyme extract showed antioxidant activity similar to Flavor' Plus™ and showed much stronger antioxidant activity compared to the rosemary and sage extracts. A biphenyl compound isolated from thyme has been reported to be a potential antioxidant (Miura et al., 1989; Nakatani et al, 1989; Haraguchi et al., 1996; Dapkevicius et al., 2002). The antioxidant activity of phenolic compounds is mainly due to their redox properties, simultaneous hydrogen atom donation to free radicals, electron transfer and metal chelating (Calliste, Trouillas, Allais, Simon, & Duroux, 2001). In the case of investigated extracts antioxidant activity on DPPH radical is suggested to be via hydrogen atom donation as the predominant mode. 3.4. Hydroxyl radical antioxidant activity of extracts

Fig. 4. (A) The antioxidant activity (AADPPH, %) of different concentrations of rosemary, sage and thyme antioxidant fractions obtained at 35 MPa and 100 °C, Flavor' Plus™ and BHA on DPPH radicals. (B) The antioxidant activity (AADPPH, %) of different concentrations of hyssop antioxidant fraction obtained at 35 MPa and 100 °C, on DPPH radicals.

The antioxidant activity of the rosemary, sage, thyme and hyssop fractions was investigated by the ability of the fractions to scavenge hydroxyl radicals. This ability is very important because of the fact that hydroxyl radicals were mentioned as the major active oxygen species causing lipid oxidation (Milic, Djilas, & Canadanovic-Brunet, 1998). To test the reactions of hydroxyl radicals with investigated extracts, the Fenton reaction (Fe2+ + H2O2 → Fe3+ + −\OH + •OH) was used as a source of hydroxyl radicals. Using a spin trap, such as DMPO, it is possible to convert reactive hydroxyl radicals to stable nitroxide radicals (DMPO-OH adducts). The influence of the different concentrations of the studied extracts (antioxidant fractions) and commercial antioxidants on formation and transformation of hydroxyl radical produced in Fenton reaction is presented in Fig. 5. The EC50 values of investigated extracts, BHA and Flavor' Plus™ are presented in Table 7. As can be seen from Fig. 5 and Table 7 the order from the strongest to the weakest antioxidant activity was: Flavor' Plus™, sage extract, rosemary extract, hyssop extract, BHA and thyme extract. As can be seen from Fig. 5 the concentrations of Flavor' Plus™, sage,

from rosemary SFE extracts harvested from different locations in Turkey at different times of the year. The authors also confirmed that the rosemary samples harvested from different locations showed considerable differences in antioxidant activity as well as in carnosol and carnosic acid content. Topal, Sasaki, Goto, and Otles (2008) investigated antioxidant properties of essential oils from nine spices of Turkish plants using DPPH radical assay. Rosemary supercritical extract was reported to have strong antioxidant activity which was slightly higher than the

Table 7 EC50 values of extracts and commercial antioxidants. Sample

Rosemary extract Sage extract Thyme extract Hyssop extract BHA Flavor' PlusTM

EC50 (mg/ml) DPPH radical assay

Hydroxyl radical assay

0.23 0.23 0.08 6.14 0.03 0.09

1.03 0.59 1.75 1.3 1.51 0.35

Fig. 5. The antioxidant activity (AAOH, %) of different concentrations of rosemary, sage, thyme and hyssop antioxidant fractions obtained at 35 MPa and 100 °C, Flavor' PlusTM and BHA on DMPO-OH spin adduct.

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N. Babovic et al. / Innovative Food Science and Emerging Technologies 11 (2010) 98–107

hyssop, thyme, rosemary and BHA which were required for the complete elimination of hydroxyl radicals were 1.75 mg/ml, 2.0 mg/ ml, 3.0 mg/ml, 3.0 mg/ml, 3.0 mg/ml and 3.0 mg/ml, respectively. For example, AAOH of sage antioxidant fraction at a concentration of 2.0 mg/ml was 100%, which was comparable to Flavor' Plus™, whereas AAOH of hyssop, thyme, rosemary and BHA were only 64.84%, 76.74%, 76.85% and 67.33% respectively. Thyme antioxidant fraction at a concentration less than 1.0 mg/ml did not show any AAOH. AAOH of hyssop, thyme, rosemary and BHA at a concentration of 2.5 mg/ml were 92.37%, 86.05%, 81.25% and 79.3% respectively. Polyphenols with o-dihydroxyl groups might exert their protective effects through chelation of metal ions in the course of the Fenton reaction (Rice-Evans, Miller, & Paganga, 1996). According to this fact it can be concluded that the high contents of carnosic acid in sage and rosemary extract and carnosol in hyssop extract are responsible for better hydroxyl radical antioxidant activity of these extracts comparing to thyme extract and synthetic antioxidant BHA. 4. Conclusion The obtained results of antioxidant investigation confirmed that examined SFE extracts had remarkable antioxidant activities against DPPH and hydroxyl radicals. It was shown that in DPPH radical assay the order from the strongest to the weakest antioxidant activity was: BHA, thyme extract, Flavor' Plus™, rosemary and sage extracts and hyssop extract. Thyme extract showed the highest antioxidant activity among the examined extracts, similar to Flavor' Plus™. Hyssop extract showed much weaker antioxidant activity compared to the other extracts. Antioxidant activity of the other extracts was comparable to the activity of BHA. The order from the strongest to the weakest antioxidant activity in hydroxyl radical assay was: Flavor' Plus™, sage extract, rosemary extract, hyssop extract, BHA and thyme extract. In hydroxyl radical assay all examined SFE extracts were comparable. The highest carnosol and carnosic acid content were detected for sage antioxidant fraction. Further research is needed in order to obtain more reliable results on the determination of chemical composition of antioxidant fractions especially in the case of thyme and to elucidate the compounds that contributed to the strong antioxidant activity of thyme extract. This investigation showed that antioxidant fraction from rosemary and sage had the highest amounts of phenolic diterpenes, indicating their potential use in the food industry as nutritional supplements, functional food components or food antioxidants. Acknowledgements The authors are grateful for the financial supports from the Ministry of Science and Environmental Protection of the Republic of Serbia (EUREKA project E!3490 HEALTHFOOD). References Ahn, J., Grün, I. U., & Fernando, L. N. (2002). Antioxidant properties of natural plant extracts containing polyphenolic compounds in cooked ground beef. Journal of Food Science, 67, 1364−1369. Almela, L., Sanchez-Munoz, B., Fernandez-Lopez, J. A., Rosa, M. J., & Rabe, V. (2006). Liquid chromatograpic-mass spectrometric analysis of phenolics and free radical scavenging activity of rosemary extract from different raw material. Journal of Chromatography A, 1120, 221−229. Calliste, C. A., Trouillas, P., Allais, D. P., Simon, A., & Duroux, J. L. (2001). Free radical scavenging activities measured by electron spin resonance spectroscopy and B16 cell antiproliferative behaviors of seven plants. Journal of Agricultural & Food Chemistry, 49, 3321−3327. Cavero, S., Jaime, L., Martin-Alvarez, J. P., Javier Senorans, F., Reglero, G., & Ibanez, E. (2005). In vitro antioxidant analysis of supercritical fluid extracts from rosemary (Rosmarinus officinalis L.). European Food Research & Technology, 221, 478−486. Chicco, A. G., D'Alessandro, M. E., Hein, G. J., Oliva, M. E., & Lombardo, Y. B. (2009). Dietary chia seed (Salvia hispanica L.) rich in α-linolenic acid improves adiposity and normalises hypertriacylglycerolaemia and insulin resistance in dyslipaemic rats. British Journal of Nutrition, 101, 41−50. doi:10.1017/S000711450899053X.

Cuvelier, M. E., Berset, C., & Richard, H. (1994). Antioxidant constituents in sage (Salvia offcinalis). Journal of Agricultural & Food Chemistry, 42, 665−669. Cuvelier, E. M., Richard, H., & Berset, C. (1996). Antioxidant activity and phenolic composition of pilot-plant and commercial extracts of sage and rosemary. Journal of American Oil Chemists Society, 73, 645−652. Cuvelier, M. E., Richard, H., & Berset, C. (1992). Comparison of the antioxidative activity of some acid-phenols: Structure–activity relationship. Bioscience Biotechnology and Biochemistry, 56, 324−327. Dapkevicius, A., van Beek, T. A., Lelyveld, G. P., van Veldhuizen, A., de Groot, A., Linssen, J. P. H., et al. (2002). Isolation and structure elucidation of radical scavengers from Thymus vulgaris leaves. Journal of Natural Products, 65, 892−896. Dapkevicius, A., Venskutonis, R., van Beek, T. A., & Linssen, J. P. H. (1998). Antioxidant activity of extracts obtained by different isolation procedures from some aromatic herbs grown in Lithuania. Journal of the Science & Food Agriculture, 77, 140−146. Daukšas, E., Venskutonis, P. R., Povilaityte, V., & Sivik, B. (2001). Rapid screening of antioxidant activity of sage (Salvia officinalis L.) extracts obtained by supercritical carbon dioxide at different extraction conditions. Nahrung/Food, 45, 338−341. de Saizieu, A., Fowler, A., Goralczyk, R., Kilpert, C., Schueler, G., & Wehrli, C. (2008). Dietary or pharmaceutical compositions containing tricyclic diterpenes and derivatives thereof for the treatment of depression. PCT Patent WO/2008/061754. Djarmati, Z., Jankov, M. R., Schwirtlich, E., Djulinac, B., & Djordjevic (1991). A high antioxidant activity of extracts obtained from sage by supercritical CO2 extraction. Journal of American Oil Chemists Society, 68, 731−734. Economou, K. D., Oreopoulo, V., & Thomopoulos, C. D. (1991). Antioxidant properties of some plant extracts of the Labiatae family. Journal of American Oil Chemists Society, 68, 109−113. Fecka, I., & Turek, S. (2008). Determination of polyphenolic compounds in commercial herbal drugs and spices from Lamiaceae: Thyme, wild thyme and sweet marjoram by chromatographic techniques. Food Chemistry, 108, 1039−1053. Fowler, A., Kilpert, C., Schueler, G., Wehrli, C., & Wyss, A. (2008). Dietary and pharmaceutical compositions comprising a sage extract containing a mixture of tricyclic diterpenes and their derivatives and their uses. PCT Patent WO/2008/061756. Guillen, M. D., & Manzanos, M. J. (1998). Composition of the extract in dichloromethane of the aerial parts of a Spanish wild growing plant Thymus vulgaris L. Flavour & Fragrance Journal, 13, 259−262. Haraguchi, H., Saito, T., Ishikawa, H., Date, H., Kataoka, S., Tamura, Y., et al. (1996). Antiperoxidative components in Thymus vulgaris. Planta Medica, 62, 217−221. Horwath, A. B., Grayer, R. J., Keith-Lucas, D. M., & Simmonds, M. S. J. (2008). Chemical characterisation of wild population of Thymus from different climatic regions in southeast Spain. Biochemical Systematics & Ecology, 36, 117−133. Houlihan, C. M., Ho, C., & Chang, S. S. (1984). Elucidation of the chemical structure of a novel antioxidant, rosmaridiphenol, isolated from rosemary. Journal of American Oil Chemists Society, 61, 1036−1039. Huang, M. T., Ho, C. T., Wang, Z. Y., Ferraro, T., Lou, Y. R., Stauber, K., et al. (1994). Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid. Cancer Research, 54, 701−708. Ibanez, E., Oca, A., de Murga, G., Lopez-Sebastion, S., Tabera, J., & Reglero, G. (1999). Supercritical fluid extraction and fractionation of different preprocessed rosemary plants. Journal of Agricultural & Food Chemistry, 47, 1400−1404. Inatani, R., Nakatani, N., Fuwa, H., & Seto, H. (1982). Structure of a new antioxidative phenolic diterpene isolated from rosemary (Rosmarinus offcinalis L.). Agricultural & Biological Chemistry, 46, 1661−1666. Ito, N., Fukushima, S., Hagiwara, A., Shibata, M., & Ogiso, T. (1983). Carcinogenicity of butylated hydroxyanisole in F344 rats. Journal of the National Cancer Institute, 70, 343−352. Kavvadias, D., Monschein, V., Sand, P., Riederer, P., & Schreier, P. (2003). Constituents of sage (Salvia officinalis) with in vitro affinity to human brain benzodiazepine receptor. Planta medica, 69, 113−117. Kazazi, H., Rezaei, K., Ghotb-Sharif, J. S., Emam-Djomeh, Z., & Yamini, Y. (2007). Supercritical fluid extraction of flavors and fragrances from Hyssopus officinalis L. cultivated in Iran. Food Chemistry, 105, 805−811. Kilic, T., Dirmenci, T., & Goren, A. C. (2007). Chemotaxonomic evaluation of species of Turkish Salvia: Fatty acid composition of seed oils. II. Records of Natural Products, 1(1), 17−23. Lopez-Sebastian, S., Ramoas, E., Ibanez, E., Bueno, J. M., Ballester, L., & Reglero, G. (1998). Dearomatization of antioxidant rosemary extracts by treatment with supercritical carbon dioxide. Journal of Agricultural & Food Chemistry, 46, 13−19. Madhavi, D. L., Despande, S. S., & Salunkhe, D. K. (1996). Food antioxidants (pp. 1). New York: Marcel Dekker. Masterova, I., Misíková, I. E., Sirotková, L., Vaverková, S., & Ubik, K. (1996). Royleanones in the roots of Salvia officinalis L. of domestic provenance and their antimicrobial activity. Ceská a Slovenská farmacie, 45, 242−245. Milic, B. Lj., Djilas, S. M., & Canadanovic-Brunet, J. M. (1998). Antioxidative activity of phenolic compounds on the metal-ion break-down of lipid peroxidation system. Food. Chemistry, 61, 443−447. Miura, K., Inagaki, T., & Nakatani, N. (1989). Structure and activity of new deodorant biphenyl compounds from thyme (Thymus vulgaris L.). Chemical & Pharmaceutical Bulletin, 37, 1816−1819. Miura, K., Kikuzaki, H., & Nakatani, N. (2002). Antioxidative activity of chemical components from sage (Salvia officinalis L.) and thyme (Thymus vulgaris L.). Journal of Agricultural & Food Chemistry, 50, 1845−1851. Miura, K., & Nakatani, N. (1989). Antioxidative activity of flavonoids from thyme (Thymus vulgaris L.). Agricultural & Biological Chemistry, 53, 3043−3045. Nakatani, N., & Inatani, R. (1983). A new diterpene lactone, rosmadial, from rosemary (Rosmarinus offcinalis L.). Agricultural and Biological Chemistry, 47, 353−358. Nakatani, N., & Inatani, R. (1984). Two antioxidative diterpenes from rosemary (Rosmarinus offcinalis L.) and a revised structure for rosmanol. Agricultural & Biological Chemistry, 48, 2081−2085.

N. Babovic et al. / Innovative Food Science and Emerging Technologies 11 (2010) 98–107 Nakatani, N., Miura, K., & Inagaki, T. (1989). Structure of new deodorant bipheny1 compounds from thyme (Thymus vulgaris L.) and their activity against methyl mercaptan. Agricultural & Biological Chemistry, 53, 1375−1381. Ninomiya, K., Matsuda, H., Shimoda, H., Nishida, N., Kasajima, N., Yoshino, T., et al. (2004). Carnosic acid, a new class of lipid absorption inhibitor from sage. Bioorganic & Medicinal Chemistry Letters, 14, 1943−1946. Nguyen, U., Frankman, G., & Evans, D. A. (1991). United States Patent 5,017,397. Okamura, N., Fujimoto, Y., Kuwabara, S., & Yagi, A. (1994). High-performance liquid chromatographic determination of carnosic acid and carnosol in Rosmarinus officinalis and Salvia officinalis. Journal of Chromatography A, 679, 381−386. Okazaki, K., Kawazoe, K., & Takaishi, Y. (2002). Human platelet aggregation inhibitors from thyme (Thymus vulgaris L.). Phytotherapy Research, 16, 398−399. Rice-Evans, C. A., Miller, J., & Paganga, G. (1996). Structure–antioxidant activity relationships of flavonoids and phenolic acids. Free radical in Biology and Medicine., 20, 933−956. Richheimer, S. L., Bernart, M. W., King, G. A., Kent, M. C., & Beiley, D. T. (1996). Antioxidant activity of lipid-soluble phenolic diterpenes from rosemary. Journal of American Oil Chemists Society, 73, 507−514. Rižnar, K., Čelan, Š., Škerget, M., & Knez, Ž. (2008). Solubility of carnosoic acid and carnosol from rosemary extract in supercritical CO2. Acta Chimica Slovenica, 55, 516−520. Santos-Gomes, P. C., Seabra, R. M., Andrade, P. B., & Fernandes-Ferreira, M. (2002). Phenolic antioxidant compounds produced by in vitro shoots of sage (Salvia officinalis L.). Plant Science, 162, 981−987. Schwarz, K., Ernst, H., & Ternes, W. (1996). Evaluation of antioxidative constituents from thyme. Journal of the Science & Food Agriculture, 70, 217−223. Schwarz, K., & Waldemar, T. (1992). Antioxidative constituents of Rosmarinus officinalis and Salvia officinalis. Journal Zeitschrift für Lebensmitteluntersuchung und—Forschung A, 195, 99−103. Senorans, F. J., Ibanez, E., Cavero, S., Tabera, J., & Reglero, G. (2000). Liquid chromatographic-mass spectrometric analysis of supercritical-fluid extracts of rosemary plants. Journal of Chromatography A, 870, 491−499.

107

Simandi, B., Hajdu, V., Peredi, K., Czukor, B., Nobik-Kovacs, A., & Kery, A. (2001). Antioxidant activity of pilot-plant alcoholic and supercritical carbon dioxide extracts of thyme. European Journal of Lipid Science & Technology, 103, 355−358. Sun, X. R., Luo, H. W., Sakai, T., & Niwa, M. (1991). Paramiltioic acid, a novel rearranged norabietanoid from salvia paramiltiorrhiza. Tetrahedron Letters, 32, 5797−5800. Tada, M., Okuno, K., Chiba, K., Ohnishi, E., & Yoshii, T. (1994). Antiviral diterpenes from Salvia officinalis. Phytochemistry, 35, 539−541. Takeuchi, H., Lu, Z. G., & Fujita, T. (2004). New monoterpene glucoside from the aerial parts of thyme (Thymus vulgaris L.). Bioscience Biotechnology & Biochemistry, 68, 1131−1134. Topal, U., Sasaki, M., Goto, M., & Otles, S. (2008). Chemical compositions and antioxidant properties of essential oils from nine species of Turkish plants obtained by supercritical carbon dioxide extraction and steam distillation. International Journal of Food Sciences and Nutrition, 59, 619−634. Topcu, G., Turkmen, Z., Schilling, J. K., Kingston, D. G. I., Pezzuto, J. M., & Ulubelen, A. (2008). Cytotoxic activity of some Anatolian Salvia extracts and isolated abietane diterpenoids. Pharmaceutical Biology, 46, 180−184. Ulubelen, A., Oksuz, S., Kolak, U., Bozok-Johansson, C., Celik, C., & Voelter, W. (2000). Antibacterial diterpenes from the roots of Salvia viridis. Planta medica, 66, 458−462. Yanishlieva, V. N., Marinovaa, E., & Pokorný, J. (2006). Natural antioxidants from herbs and spices. European Journal of Lipid Science & Technology, 108, 776−793. Yesil-Celiktas, O., Bedir, E., & Vardar-Sukan, F. (2007). In vitro antioxidant activities of Rosmarinus officinalis extracts treated with supercritical carbon dioxide. Food Chemistry, 101, 1457−1464. Yesil-Celiktas, O., Nartop, P., Gurel, A., Bedir, E., & Vardar-Sukan, F. (2007). Determination of phenolic content and antioxidant activity of extracts obtained from Rosmarinus officinalis' calli. Journal of Plant Physiology, 164, 1536−1542. Zheng, W., & Wang, S. Y. (2001). Antioxidant activity and phenolic compounds in selected herbs. Journal of Agricultural and Food Chemistry, 49, 5165−5170. Zizovic, I., Stamenic, M., Orlovic, A., & Skala, D. (2007). Supercritical carbon dioxide extraction of essential oils from plants with secretory ducts: Mathematical modelling on the micro-scale. Journal of Supercritical Fluids, 39, 338−346.