Volatile constituents, phenolic compounds, and antioxidant activity of Calamintha...

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Research ArticleReceived: 10 July 2012 Revised: 7 October 2012 Accepted article published: 26 October 2012 Published online in Wiley Online Library: 26 November 2012

(wileyonlinelibrary.com) DOI 10.1002/jsfa.5967

Volatile constituents, phenolic compounds,and antioxidant activity of Calaminthaglandulosa (Req.) BenthamSanja Cavar,∗ Danijela Vidic and Milka Maksimovic

Abstract

BACKGROUND: Calamintha glandulosa (Req.) Bentham is an aromatic perennial plant belonging to the family Lamiaceae, mostlyfound on rocky pastures, dry meadows, and abandoned places of the Mediterranean area. Plants belonging to this genus areknown as highly aromatic and to possess significant antimicrobial and antifungal properties. The aim of this study was toprovide clear picture of the volatiles of this plant species, and, for the first time, to present C. glandulosa from Croatia in termsof its antioxidant activity.

RESULTS: The essential oil and headspace obtained from odorous parts of C. glandulosa were subjected to capillary gaschromatography–mass spectrometry analysis. More than 50 volatile compounds were identified in six samples obtained usingdifferent extraction techniques. The most abundant components in all the samples examined were oxygenated monoterpenes,with piperitone (19.9–59.5%) and piperitenone (7.1–42.6%) as the main representatives. The total phenolic content of extractsobtained by successive Soxhlet extraction was measured, and the scavenging potency of the samples, indicated as IC50 values,were examined using four different spectrophotometric and spectrofluorimetric methods. In all cases the essential oil showedthe lowest antioxidant activity, while the aqueous extract showed the highest. This can be explained by the levels of thephenolic compounds in the samples examined.

CONCLUSIONS: A clear picture of aroma profile of C. glandulosa is presented, and the results obtained differ from thosepublished previously. The high antioxidant potential of C. glandulosa from Croatia was established for the first time. Resultsfrom the present study suggest further analysis on this plant species in order to define its medicinal properties.c© 2012 Society of Chemical Industry

Keywords: Calamintha glandulosa (Req.) Bentham; volatile constituents; piperitone; piperitenone; phenolic compounds; antioxidantactivity

INTRODUCTIONThe genus Calamintha Miller (Eng., Calamint), consists of eightnative species belonging to the Lamiaceae family. It is representedby six extremely polymorphic species in the area of the BalkanPeninsula.1 These are medium-to-large size erect herbaceousperennials or, rarely, annual plant species. Since ancient times thespecies of this genus has been traditionally used as a stomachtonic, and as an antiseptic, antipyretic, diaphoretic, expectorantand sedative.2

Calamintha glandulosa (Req.) Bentham is an aromatic perennialplant, mostly found on rocky pastures, dry meadows, andabandoned places of the Mediterranean and sub-zones. This plantgrows wild along the Adriatic coast and the sub-Mediterraneanpart of Croatia, as well as in Montenegro, Macedonia andGreece. It flowers in July and August. Its notably mint-likecharacter was found to originate from pulegone, menthoneand isomenthone, and this characteristic was proposed tohelp differentiate between Calamintha and Satureja on chemo-taxonomic grounds.3 Moreover, plants belonging to this genusare known to posses great variations in composition of the volatilecompounds, but the major components in the oils generallybelong to the C-3 oxygenated p-menthanes. Generally, three

chemotypes can be distinguished: pulegone, piperitone oxide,and carvone.4,5

Considering the interest in natural products for thecosmetics, perfumery, food and pharmacological industries,deeper knowledge of the potential of different aromatic plantscan lead to results of economic importance. It is known thatCalamintha glandulosa has significant antimicrobial and antifungal

properties,6–8 and it is used in the treatment of several diseases infolk medicine. The interest in phenolic antioxidants has increasedremarkably in the last decade due to their great capacity toscavenge free radicals associated with various human diseases.

In light of the above, it seemed necessary to evaluate the chem-ical composition of the volatile constituents obtained by usingdifferent techniques from C. glandulosa, as well as phenolic contentand its antioxidant activity, using different testing methods.

∗ Correspondence to: Sanja Cavar, University of Sarajevo, Faculty of Science,Department of Chemistry, Zmaja od Bosne 33– 35, 71000 Sarajevo, Bosnia andHerzegovina. E-mail: [email protected]; [email protected]

University of Sarajevo, Faculty of Science, Department of Chemistry, Zmaja odBosne 33-35, 71000 Sarajevo, Bosnia and Herzegovina

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Constituents and antioxidant acitivity of C. glandulosa www.soci.org

MATERIAL AND METHODSGeneralAerial parts of Calamintha glandulosa (Req.) Bentham werecollected at the full flowering stage (September) from plantsgrowing wild on rocky soil near Makarska, Croatia. A voucherspecimen of each plant was deposited at the Faculty of Science,University of Sarajevo.

All applied reagents were of the highest purity available andpurchased from the Sigma-Aldrich Chemical Company (Steinhein,Germany).

Sample preparationAir-dried plant material (10 g) was subjected to hydrodistillation,steam distillation and aqueous reflux extraction for 2 h. Theessential oils were extracted with dichloromethane and dried overanhydrous sodium sulfate.

Moreover, 30 g of air-dried plant material was subjected tosuccessive Soxhlet extraction using five solvents, i.e. petrol ether,chloroform, acetone, absolute ethanol and 96% ethanol for 3 h.Solvents were evaporated under reduced pressure.

Floral and leaf scent, as well as scent from the petrol etherSoxhlet extract, was collected using dynamic headspace sorptionon Drager charcoal tubes (trapping time, 2 h), with coconut shellcharcoal as the sorption agent. Trapped volatiles were eluted withdichloromethane. All samples were stored for 2 days at 4◦C in thedark until analysis.

For the gas chromatography–mass spectrometry (GC-MS) analysis samples of essential oil were dissolved indichloromethane, and for total phenolics and antioxidant assayssamples were dissolved in dimethyl sulfoxide at a concentrationof 10.0 mg mL−1. The percentage content of the oils andextracts was calculated on the basis of the dry weight of plantmaterial.

Trolox was used as a positive probe for antioxidant assays, andthey were prepared in the same manner as tested samples.

Gas chromatography–mass spectrometryGC-MS was carried out on a Hewlett-Packard 6890 Series II gaschromatograph (Santa Clara, USA) fitted with a fused silica HP-5(5% phenyl methyl siloxane) capillary column (30 m × 0.25 mm,0.25 µm film thickness), coupled to a HP 6890 Series II massselective detector. The column temperature was programmedfrom 60◦C to 240◦C at 3◦C min−1, and helium was used ascarrier gas (1.1 mL min−1). Other operating conditions wereas follows: inlet pressure 9.43 psi, injector temperature 250◦C,detector temperature 280◦C, split ratio 1:25, injection volume 1µL. Ionisation of the sample components was performed in the EImode (70 eV), with scan range of 20–555 amu, and scan time of1.60 s.

The linear retention indices for all compounds weredetermined by injection of the hexane solution containingthe homologous series of C8 –C26 n-alkanes.9 The identificationof the volatile constituents was accomplished by the visualinterpretation, comparing their retention indices and massspectra with literature data,10 by computer library search (HPChemstation computer library NBS75K.L, NIST/EPA/NIH MassSpectral Library 2.0 and Mass Finder 4 Computer Softwareand Terpenoids Library), and in our own laboratory database.Compounds concentrations (as % content) were calculated byintegrating their corresponding chromatographic peak areas (TICmode).

High-performance liquid chromatography–electrochemicaldetectionA 1 mL of aqueous extract was decanted and centrifuged(15093 x g), obtaining supernatant which was used for furtheranalysis. HPLC conditions were following: column, ODS HypersilC-18; mobile phase, methanol–acetonitrile–water–acetic acid(20:10:70:1); electrochemical detector with a range of 50 nA;potential, +0.840 V; filter, 0.02 Hz; flow rate, 0.8 mL min−1;temperature, 25◦C. Gallic, chlorogenic, and rosmarinic acids wereused for the preparation of calibration curves.

Total phenolic contentTotal phenolic content of the examined extracts was determinedby a slight modification of the method by Singleton and Rossi.11

A 100 µL of sample solution in various concentrations was dilutedwith 6 mL of distilled water, and the 500 µL of Folin–Ciocalteureagent (2 mol/L), previously diluted two-fold, was mixed. After5–10 min a 1.5 mL of 20% solution of sodium carbonate was added,and the solution obtained was diluted to 10 mL. Prepared sampleswere kept for 2 h at room temperature, and the absorbance wasmeasured at 765 nm. The data were calculated according to astandard curve of gallic acid (0.01–0.20 mg mL−1), and they wereexpressed as gallic acid equivalents (GAE) per gram of dry extract.

Total flavonoid contentTotal flavonoids in the plant extracts examined were determinedby using a slight modification of the method given by Chang etal.12 The principle of method is that aluminium chloride forms acidstable complexes with the C-4 keto group and either the C-3 or C-5hydroxyl group of flavones and flavonols. In addition, aluminiumchloride forms acid labile complexes with the ortho-dihydroxylgroups in the A- or B-ring of flavonoids.

A 0.5 mL of diluted extract solution was mixed with 0.5 mL ofaluminium chloride (2%) and 0.2 mL of potassium acetate (1.0mol L−1). After incubation at room temperature for 30 min, theabsorbance of the reaction mixture was measured at 415 nm. Ablank sample contained 0.5 mL of sample and 0.7 mL of distilledwater. A 0.5 mL sample of aluminium chloride mixed with 0.7mL of distilled water was used to zero the spectrophotometer.The data were calculated according to a standard curve ofquercetin (25–100 µg mL−1), and they were expressed as quercetinequivalents (QE) per gram of dry extract.

1,1-Diphenyl-2-picrylhydrazyl radical-scavenging activityThe ability of the essential oil constituents to donate a hydrogenatom or electron and scavenge the 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical was determined by a slight modification of themethod given by Brand-Williams et al.13 The concentrations of thetested samples ranged from 10.00 to 0.10 mg mL−1. A portion ofsample solution (200 µL) was mixed with 3.0 mL of 5.25 × 10−5 molL−1 DPPH• in absolute ethanol. The decrease in absorbance of thetested mixtures was monitored every minute for 30 min at 517 nmusing a Perkin–Elmer Lambda 25 UV–visible spectrophotometer(Perkin–Elmer, Waltham, USA). Absolute ethanol was used to zerothe spectrophotometer, DPPH• solution was used as blank sample.The DPPH• solution was freshly prepared daily, stored in a flaskcovered with aluminium foil, and kept in the dark at 4◦C beforemeasurements.

The radical-scavenging activity of the tested samples, expressedas the percentage inhibition of DPPH, was calculated according tothe formula: IC (%) = [(A0 − At)/A0] × 100, where IC is the inhibitory

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www.soci.org S Cavar, D Vidic, M Maksimovic

concentration, At is the absorbance value of the tested sample,and A0 is the absorbance value of blank sample, in particular time.The % inhibition after 30 min was plotted against concentration,and the equation for the line was used to obtain the IC50 value. Alower IC50 value indicates greater antioxidant activity.

2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)radical-scavenging activityThe 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)method is based on the reduction of the green ABTS radical cation(7.00 mmol L−1) that was obtained by its oxidation with an equalvolume of potassium persulfate (2.45 mmol L−1),14 for 12–16 h at4◦C in the dark. On the day of analysis the ABTS•+ solution wasdiluted with methanol to an absorbance of 1.00 (± 0.02) at 734nm. After the addition of 100 µL of sample solution to 1.0 mLof ABTS•+ solution, the decrease in absorbance was monitoredevery minute for 7 min at 743 nm using a Perkin–Elmer Lambda25 UV–visible spectrophotometer. Methanol was used to zerothe spectrophotometer; ABTS•+ solution was used as the blanksample.

The radical-scavenging activity of the tested samples, expressedas the percentage inhibition of ABTS•+, was calculated accordingto the formula: IC (%) = [(A0 − At)/A0] × 100, where IC is theinhibitory concentration, and A0 and At are the absorbance valuesof the blank sample and the test sample, at particular times,respectively. The % inhibition after 7 min was plotted againstconcentration, and the equation for the line was used to obtainthe IC50 value. A lower IC50 value indicates greater antioxidantactivity.

Evaluation of reducing powerThe reducing power test is based on reduction of the ferric ionto the ferrous ion by a potent antioxidant. In the presence ofcyanide ions, and adding a new amount of Fe3+, the blue colourof Fe4[Fe(CN)6]3 develops. Reducing power of the samples wasdetermined by using a slight modification of the method given byYen and Duh,15 as described below. A 1.0 mL sample of variousdilutions (from 10.00 to 0.01 mg mL−1) was mixed with 2.50 mL ofphosphate buffer (0.2 mol L−1, pH 6.6) and 2.5 mL of 1% potassiumferricyanide. The mixtures were incubated at 50◦C for 20 min. Afterincubation 2.5 mL of 10% trichloracetic acid was added to themixture, which was then centrifuged at 604 x g for 10 min. Theupper layer (0.5 mL) of solution was mixed with 2.5 mL of distilledwater and 100 µL of 0.1% FeCl3 and the absorbance was measuredat 700 nm. A control sample contained 1.0 mL distilled water, 2.5mL of phosphate buffer, 2.5 mL of 1% potassium ferrocyanide and2.5 mL of 10% trichloracetic acid. A blank sample contained 1.0 mLdistilled water, 2.5 mL of phosphate buffer, 2.5 mL of 1% potassiumferricyanide and 2.5 mL of 10% trichloracetic acid.

The reducing power (RP) of samples was calculated by thefollowing formula: RP (%) = (AB − AA) × 100, where AB isthe absorption of the control sample (100%), and AA is theabsorption of the tested sample. The % inhibition was plottedagainst concentration, and the equation for the line was usedto obtain the RP50 value. The lower RP50 value indicates greaterreducing power.

Oxygen radical absorbance capacity assayValues for the oxygen radical absorbance capacity (ORAC) againstthe hydroxyl radical of the samples were determined by using aslight modification of the method given by Cao et al.16 as described

below. A 100 µL of sample was mixed with 1750 µL of distilledwater (HPLC purity). The diluted sample was mixed with fluoresceindissolved in water (50 µL; 0.64 µmol L−1), and hydrogen peroxide(50 µL; 2.20 mol L−1). The generation of hydroxy radicals wasinduced by adding a solution of CuSO4 (50 µL; 72.0 mmol L−1).The fluorescence intensity of the reaction mixture was measuredusing a Perkin–Elmer Luminescence spectrometer LE 55 withfluorescence filters (excitation, 485 nm; emission, 520 nm). Thedecrease in the fluorescence of the tested mixtures was monitoredevery 5 min for 60 min. A blank sample contained 1850 µL ofdistilled water, 50 µL of fluorescein solution (0.64 µmol L−1), 50 µLof hydrogen peroxide solution (2.20 mol L−1), and 50 µL of CuSO4

solution (72.0 m mol L−1). Trolox (20.0 µmol L−1) was used as apositive probe. The consumption of the fluorescein, associatedwith its incubation in the presence of H2O2 was estimated frommeasurements of the fluorescence (F) and absorbance (A).

Values of (F/F0) or (A/A0) were plotted as a function of time.Integration of the area under the curve (AUC) was performedperiodically in order that (F/F0) or (A/A0) reached a value of0.5. These areas were used to obtain ORAC values, according toformula: ORAC = (AUCA − AUCB)/(AUCC − AUCB) × (MC/MA), whereORAC is the relative ORAC value calculated as Trolox equivalentsper gram of dry extract (µmol TE g−1); AUCA is the area under thecurve of the tested sample, AUCB is the area under the curve of theblank sample, AUCC is the area under the curve of Trolox, MA is themass of the sample, and MC is the molarity of the Trolox. Valuesof AUC for the sample, blank sample and Trolox were calculatedaccording to the equation for calculation of the area of a trapezoid.

Statistical analysisAll measurements were performed in triplicate and results areexpressed as mean ± SD. Moreover, the significance of differencesin the results was obtained, and they are presented in brackets.

RESULTS AND DISCUSSIONIsolation of essential oils and plant extractsIn order to obtain a clear aroma picture of Calamintha glandulosa(Req.) Bentham growing wild on the Adriatic coast (Makarska),six different extraction techniques were employed in this study,i.e. steam distillation, hydrodistillation, aqueous reflux extraction,Soxhlet extraction, and headspace extraction of the petrol etherSoxhlet extract as well as of dry plant material.

Moreover, successive Soxhlet extraction, using five solvents withdifferent polarity, was performed. The percentage content of theoils and extracts was calculated on the basis of the dry weightof plant material and is presented in Table 1. Waste water afterhydrodistillation has also been used for phenolics and antioxidantassays.

Gas chromatography–mass spectrometry analysisIsolated volatiles of C. glandulosa were subjected to detailedGC-MS analysis in order to determine the impact of themethod of isolation as well as the impact of geographicalorigin on volatile constituents. Fifty-two compounds wereidentified in six samples (Table 2). The most abundantcomponents in all samples were oxygenated monoterpenes(75.7–97.5%) with the monoterpenoids piperitone (19.9–40.3%)and piperitenone (10.5–42.5%), which contain a carbonyl group,as major components. The essential oil obtained by steamdistillation showed the highest heterogeneity, with 41 identified

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Table 1. Yields, total phenolic and flavonoid content, and antioxidant activity of essential oils and extracts of Calamintha glandulosa

Sample Yield (%)

Total phenolicsa

(mg GAE g−1)

Total flavonoidsb

(mg QE g−1)

ABTS IC50

(mg mL−1)

DPPH IC50

(mg mL−1)

Reducing power

IC50 (mg mL−1)

ORAC-OH

(mmol TE g−1)

EO-HDc 0.36 — — 4.40 ± 0.09(± 2.04%)

34.29 ± 0.43(± 2.92%)

406.51 ± 6.76(± 1.66%)

4.44 ± 0.32(± 7.21%)

EO-SDd 0.20 — — — — — —

PEe 2.80 29.65 ± 1.46(± 5.02%)k

0.86 ± 0.09(± 10.46%)

1.56 ± 0.08(± 5.13%)

22.72 ± 0.36(± 1.58%)

62.21 ± 1.01(± 1.62%)

1.76 ± 0.16(± 9.09%)

CHf 2.40 171.93 ± 2.27(± 1.32%)

8.80 ± 0.77(± 8.75%)

0.86 ± 0.03(± 3.49%)

3.12 ± 0.05(± 1.60%)

6.00 ± 0.15(± 2.50%)

1.43 ± 0.09(± 6.29%)

ACg 0.96 117.11 ± 7.67(± 6.55%)

12.07 ± 1.34(± 10.94%)

0.86 ± 0.03(± 3.49%)

2.58 ± 0.08(± 3.10%)

5.62 ± 0.08(± 1.42%)

−0.14 ± 0.01(± 7.14%)

100% EtOHh 0.91 128.31 ± 9.91(± 7.72%)

13.96 ± 0.26(± 1.86%)

0.85 ± 0.02(± 2.35%)

2.26 ± 0.06(± 2.65%)

5.21 ± 0.06(± 1.15%)

−1.17 ± 0.03(± 2.56%)

96% EtOHi 1.70 108.20 ± 5.96(± 5.51%)

9.46 ± 0.78(± 8.24%)

0.76 ± 0.05(± 6.58%)

0.79 ± 0.02(± 2.53%)

4.54 ± 0.06(± 1.32%)

−1.80 ± 0.07(± 3.89%)

HD-Aqj — 176.07 ± 6.27(± 3.56%)

0.27 ± 0.02(± 7.41%)

0.51 ± 0.03(± 5.88%)

0.36 ± 0.02(± 5.55%)

2.75 ± 0.04(± 1.45%)

−2.08 ± 0.17(± 8.17%)

Trolox — — — 0.040 ± 0.002(± 5.00%)

0.038 ± 0.003(± 7.89%)

0.96 ± 0.02(± 2.08%)

—

a Total phenolic contant calculated as gallic acid equivalents (GAE) per gram of plant extract;b total flavonoid contant calculated as quercetin acid (QE) equivalents per gram of plant extract;c essential oil obtained by hydrodistillation;d essential oil obtained by steam distillation;e petrolether Soxhlet extract;f chloroform Soxhlet extract;g acetone Soxhlet extract;h absolute ethanol Soxhlet extract;i aqueous ethanol Soxhlet extract;j waste water after hydrodistillation of essential oil;k significance of differences in the results (brackets).ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 1,1-diphenyl-2-picrylhydrazyl; ORAC, oxygen radical absorbance capacity assay;TE, Trolox equivalents.

compounds, while headspace of dry plant material showedthe lowest heterogeneity, with only 11 compounds identified.Similar results were obtained using solvent-free microwaveextraction with a higher composition of lightly oxygenatedmonoterpenes.17 Although it is obvious that the method ofisolation has a high impact on content and composition of volatilecompounds, this specimen of C. glandulosa clearly belongs tothe piperitone/piperitenone chemotype that, so far, has not beenreported in these high concentrations. As indicated above, theliterature reports high intra-specific chemical variations threedifferent chemotypes in of C. glandulosa.4 The most abundantchemotype is pulegone/menthone, found in this species growingwild in Italy,6,7 Turkey,18 Croatia19 and Montenegro,8 then thepiperiton oxide/piperitenone oxide chemotype, also found inCroatia,20 Greece21 and Belgium.3,22

The results presented are in agreement with the known highlycomplex chemical polymorphism of this genus. The observed dif-ferences in essential oil profile of the investigated species confirmthe influence of origin of the plant material, as well as environ-mental conditions on the nature of plant chemical composition.

Phenolic and flavonoid contentPlant extracts obtained by successive Soxhlet extraction, as wellas waste water after hydrodistillation of C. glandulosa werestudied for their contents of total phenolic and flavonoid content.Table 1 shows the total phenol contents that were measured byFolin–Ciocalteu reagent in terms of gallic acid equivalents (GAE),and total flavonoid content measured by a colorimetric method

with aluminium chloride in terms of quercetin equivalents (QE).The results showed that the total phenolic content from differentextracts of C. glandulosa ranging from 29.65 ± 1.46 mg GAE g−1,for the petrol ether extract, to 176.93 ± 6.27 mg GAE g−1, for wastewater after hydrodistillation. The highest amount of flavonoidswas found in the absolute ethanolic Soxhlet extract (13.96 ± 0.26mg QE g−1), while the lowest level of flavonoids was found in thewaste water after hydrodistillation (0.27 ± 0.09 mg QE g−1).

Solvent extractions are the most commonly used proceduresto prepare extracts from plant materials due to their ease of use,efficiency and wide applicability. It is generally known that theyield of a chemical extraction depends on the solvent and itspolarity, extraction time and temperature, sample-to-solvent ratioas well as on the chemical composition and physical characteristicsof the sample. The solubility of phenolics is governed by thechemical nature of the plant sample, as well as the polarityof the solvents used.23 The waste water after hydrodistillationrevealed low yields of phenolics (Table 1). The recovery ofphenolic compounds from plant materials is also influenced bythe extraction time and temperature, which reflects the conflictingactions of solubilisation and analyte degradation by oxidation. Anincrease in the extraction temperature can promote higher analytesolubility by increasing both solubility and mass transfer rate. Inaddition, the viscosity and surface tension of the solvents decreaseat higher temperature, which enables solvents to reach the samplematrices, thus improving the extraction rate. However, manyphenolic compounds are easily hydrolysed and oxidised. Longextraction times and high temperatures increase the chance of the


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Table 2. Volatile constituents of Calamintha glandulosa

Number Retention index Compound EO-SD (%) EO-HD (%) EO-Ra(%) PE (%) PE-HSb(%) HSc (%)

1 900 n-Nonane — — — — 0.3 —

2 904 α-Pinene — — — 0.1 1.6 —

3 937 Sabinene — — — — 1.2 —

4 940 β-Pinene — — — 0.2 2.2 —

5 954 Myrcene — — — 0.1 1.8 —

6 964 3-Octanol 0.4 0.2 — 0.5 2.3 —

7 1003 Limonene — — — 1.5 12.6 1.8

8 1020 Benzene acetaldehyde 0.1 — — — — —

9 1041 γ -Terpinene — — — — 1.2 —

10 1043 (E)-β-Ocimene 0.1 — — 0.2 — —

11 1083 Linalool 0.3 0.2 — — 0.2 —

12 1106 1,3,8-p-Menthatriene 0.1 — — — — —

13 1138 Menthone 8.3 4.7 3.3 12.5 29.1 2.0

14 1147 Isomenthone 0.6 0.4 — 0.6 1.2 2.0

15 1158 trans-Sabinene hydrate 0.3 0.3 — — 0.5 —

16 1173 α-Terpineol 0.2 0.2 — — — —

17 1179 Dihydrocarveol 0.2 — — — — —

18 1198 Shisofuran 0.1 0.1 9.7 — — —

19 1207 8,9-Dehydrothymol 1.3 1.0 — — — —

20 1215 Thymol methyl ether — — 0.8 — — —

21 1222 Pulegone 7.2 9.7 0.6 14.0 13.8 7.2

22 1242 Piperitone 40.3 36.6 26.7 28.0 19.9 59.5

23 1256 Carvone 1.0 1.3 1.9 1.0 0.4 —

24 1263 Thymol — — 0.1 — — —

25 1289 Isopiperitenone — — 0.1 — — —

26 1301 trans-Pinocarvyl acetate 0.1 — — — — 0.5

27 1316 Dihydrocarveol acetate 0.5 0.3 0.1 0.3 0.1 0.7

28 1333 Piperitenone 27.9 42.5 42.6 37.7 10.5 24.1

29 1369 β-Bourbonene 0.4 0.3 — 0.4 0.3 1.0

30 1379 β-Elemene 0.1 0.1 — 0.1 — —

31 1385 8,9-Dehydrothymol acetate 1.2 0.2 — — — —

32 1393 Vanillin 0.1 — 0.8 — — —

33 1404 β-Caryopohyllene 0.5 0.3 — 0.7 0.3 0.9

34 1410 (E)-β-Damascenone 0.1 — — — — —

35 1431 Germacrene D 0.1 — — — — —

36 1447 Geranyl acetone 0.1 — — — — —

37 1450 (E)-β-Farnesene 0.1 0.1 — — — —

38 1469 γ -Muurolene 1.0 0.7 — 1.8 0.2 0.2

39 1479 (E)-β-Ionone 0.1 0.1 — — — —

40 1482 Acetovanillone — — 1.6 — — —

41 1519 Dihydroactinidiolide 0.3 — 0.8 — — —

42 1607 1,10-di-epi-Cubenol 0.2 0.1 — — — —

43 1681 Eudesma-4(15),7-dien-1-β-ol 0.3 — — — — —

44 1848 6,10,14-Trimethylpentadecan-2-one 0.2 0.1 — — — —

45 1925 (5Z,9E)-Farnesyl acetone 0.3 — — — — —

46 1982 n-Hexadecanoic acid 0.7 — — — — —

47 1989 13-epi-Manool 0.2 — — — — —

48 2063 Abietatriene 0.1 0.1 — — — —

49 2254 Eicosanal 0.4 — — — — —

50 2300 Tricosane 0.2 — — — — —

51 2500 Pentacosane 0.2 0.1 0.8 0.1 — —

52 2600 Hexacosane 0.1 — — 0.1 — —

Aliphatic compounds 2.0 0.3 0.8 0.8 2.6 —

Aromatic compounds 0.2 — 3.3 — — —

Monoterpene hydrocarbons 0.2 — — 2.1 20.6 1.8

Oxygenated monoterpenes 89.6 97.5 85.0 94.1 75.7 96.0


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Table 2. continued

Number Retention index Compound EO-SD (%) EO-HD (%) EO-Ra(%) PE (%) PE-HSb(%) HSc (%)

Sesquiterpene hydrocarbons 2.5 1.5 0.8 3.0 0.8 2.1

Oxygenated sesquiterpenes 1.2 0.3 — — — —

Diterpene hydrocarbons 0.1 0.1 — — — —

Oxygenated diterpenes 0.2 — — — — —

Total identified 96.0 99.7 89.9 99.9 99.7 99.9

a Essential oil obtained by aqueous reflux extraction;b headspace sample from petrolether Soxhlet extract;c headspace sample from air-dried plant material.

oxidation of phenolics, which decreases the yield of phenolics inthe extracts.

This sample HD-Aq was subjected to HPLC-ED analysis foridentification of the phenolic acids. It was found that this extractcontains gallic acid (0.024 mg g−1), chlorogenic acid (0.008 mgg−1), and rosmarinic acid (0.054 mg g−1). The results presentedare in agreement with those reported previously.24

Antioxidant activityThe antioxidant activity of C. glandulosa plant extracts has beenevaluated by four methods: DPPH, ABTS, reducing power, andORAC (Table 1). Assessed samples were able to reduce thestable violet DPPH radical to the yellow DPPH-H, reaching 50% ofreduction, with IC50 values ranging from 0.36 ± 0.02 mg mL−1,for waste water after hydrodistillation, to 34.29 ± 0.43 mg mL−1,for hydrodistilled essential oil. Although the DPPH and ABTSmethods are based on the same principle, data obtained from theABTS assay are lower than those obtained from the DPPH assay,but are comparable, reaching IC50 values from 0.51 ±0.03 mgmL−1, for waste water after hydrodistillation, to 4.40 ± 0.09 mgmL−1, for hydrodistilled essential oil. This is probably due to thesteric factors that are one of the major contributors for reducingthe stable DPPH radical. In order to compare the results givenabove, the reducing ability of C. glandulosa plant extracts usinga spectrophotometric method for determination of the ferric ioncontent, which was reduced by the tested samples, was performed.Assayed samples were able to reduce the ferric ions (Fe3+) to thecorresponding ferrous ions (Fe2+), reaching 50% reduction withIC50 values ranging from 2.75 ± 0.04 mg mL−1 for waste waterafter hydrodistillation, to 406.51 ±6.76 mg mL−1 for hydrodistilledessential oil. The ORAC assay is said to be more relevant thanthose methods described above, because it utilises a biologicallyrelevant radical source.16 This method is superior to the othersimilar methods for two reasons. First, the ORAC assay system usesan area-under-curve (AUC) technique and thus combines bothinhibition time and inhibition degree of free radical action by anantioxidant into a single quantity. Second, different free radicalgenerators or oxidants can be used. In this study, the ORAC assayis successfully used to determine the antioxidant behaviour ofC. glandulosa plant extracts, with values in the range of −2.08± 0.17 mmol TE g−1 for waste water after hydrodistillation, to4.44 ± 0.32 mg mL−1 for hydrodistilled essential oil. Negativevalues indicate pro-oxidant behaviour of examined extracts. Thisis probably due to their ability to reduce metal ions that generatefree radicals through the Fenton reaction.25 This behaviour of pro-oxidants is well studied in the case of vitamin C. Besides ascorbate,medically important conditional pro-oxidants included uric acid

and sulfhydryl amino acids, such as homocysteine, and many plantpolyphenolics.26

The results presented are in agreement with those found in theliterature concerning plants belonging to the genus Calamintha

Miller.24,27–29

To conclude, all examined extracts showed potent antioxidantactivity, reducing different radicals, but waste water afterhydrodistillation revealed the highest activity. This is probablydue the high content of phenolic compounds determined in thissample. In general, the antioxidative effectiveness of plant extractsdepends of the content of phenolic compounds and the reactionactivity of the phenol towards the chain-carrying peroxyl radicalsand on the stability of the phenoxyl radical formed in the reaction.It is known that typical phenolics that possess antioxidant activityare phenolic acids30 that are found in this sample.

CONCLUSIONSTo the best of our knowledge, this is the first study providingdata on a detailed GC-MS analysis of volatiles obtained using sixdifferent extraction techniques, and the antioxidant activities of theplant extracts of Calamintha glandulosa from Croatia. The samplesobtained from the Calamintha glandulosa population investigatedare quite interesting from a pharmaceutical standpoint becauseof the antioxidant properties.

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