NVEO 2015, Volume 2, Issue 2nveo.org/.../uploads/2019/03/NVEO-2015-Volume-2-Issue-2-Full-Issue… · NVEO 2015, Volume 2, Issue 2 CONTENTS REVIEWS 1. Advanced preparative techniques

NVEO 2015, Volume 2, Issue 2

CONTENTS

REVIEWS

1. Advanced preparative techniques for the collection of pure components from essential oils /

Pages: 1-15

Danilo Sciarrone, Sebastiano Pantò, Francesco Cacciola, Rosaria Costa, Paola Dugo, Luigi

Mondello

2. Models of evaluation of antimicrobial activity of essential oils in vapour phase: a promising

use in healthcare decontamination / Pages: 16-29

Eugene K. Blythe, Nurhayat Tabanca, Betul Demirci, Ulrich R. Bernier, Natasha M.

Agramonte, Abbas Ali, K. Hüsnü Can Başer and Ikhlas A. Khan.

ARTICLES

1. α-Cyclodextrin encapsulation enhances antimicrobial activity of cineole-rich essential oils

from Australian species of Prostanthera (Lamiaceae). / Pages: 30-38

Nicholas Sadgrove, Ben Greatrex and Graham Lloyd Jones

2. Characterization and Antimicrobial Evaluation of the Essential Oil of Pinus pinea L. from

Turkey / Pages: 39-44

Fatih Demirci, Pınar Bayramiç, Gamze Göger, Betül Demirci, Kemal Başer

3. Headspace Solid Phase Microextraction (HS-SPME) and Analysis of Geotrichum fragrans

Volatiles / Pages: 45-51

Gökalp İşcan, Betül Demirci, Fatih Demirci, Kemal Başer

4. Chemical Characterisation of the Essential Oil of Hypericum aviculariifolium Jaub. & Spach

subsp. depilatum (Freyn & Bornm.) Robson var. bourgaei (Boiss.) Robson from Turkey /

Pages: 52-56

Sevim Küçük, Mine Kürkçüoğlu, Yavuz Köse, Kemal Başer

Nat. Volatiles & Essent. Oils, 2015; 2(2): 1-15 Sciarrone et al.

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REVIEW

Advanced preparative techniques for the collection of pure components from essential oils

Danilo Sciarrone1, Sebastiano Pantò1, Francesco Cacciola2, Rosaria Costa1, Paola Dugo1, 3, 4 and Luigi

Mondello1 ,3, 4*

1 Scienze del Farmaco e Prodotti per la Salute Department, University of Messina, Viale Annunziata, 98168 Messina, Italy 2 Scienze dell'Ambiente, della Sicurezza, del Territorio, degli Alimenti e della Salute Department, University of Messina,

Viale F. Stagno d'Alcontres 31, 98166 Messina, Italy 3 University Campus Bio-Medico of Rome, Via Álvaro del Portillo 21, 00128 Roma, Italy 4 Chromaleont s.r.l. c/o Scienze del Farmaco e Prodotti per la Salute Department, University of Messina, viale Annunziata,

98168 Messina, Italy

*Corresponding author. Email: [email protected]

Abstract

Preparative gas chromatography (prep-GC), as opposed to analytical capillary GC, is an analytical technique that allows the separation

and isolation of natural products, specifically volatile pure components from complex matrices. Over the years, different approaches

were used for this purpose, using both mono- and multidimensional systems coupled with different types of collection systems. In

this paper, some of the most relevant results obtained in the isolation of components from essential oils by prep-GC, are reviewed.

Furthermore, the main limitations of prep-GC arising from its daily use and the possible solutions for overcoming drawbacks, are

discussed.

Keywords: Preparative gas chromatography (prep-GC), isolation, multidimensional preparative gas chromatography, Deans switch

Introduction

When Mikhail Tswett, in 1906, gave birth to chromatography, he didn’t mean to develop an analytical

methodology, instead he wanted to isolate specific pigments from plant material. Similarly, all the early

applications of gas chromatography were directed toward preparative purposes, in other words, collection

of chromatographed components. During the years, liquid chromatography demonstrated higher suitability

to preparative approaches rather than GC; the latter, on the other hand, developed successfully in the field

of separation science. Nevertheless, prep-GC wasn’t left apart and many applications of the last decades

related to various topics demonstrate the vitality of a technique, which is still relevant and irreplaceable.

Various are the fields of application of prep-GC: isolation of compounds for flavour and fragrance

manufacturing (Sciarrone et al., 2012; Ledauphin et al., 2004; Marriott, Eyres, & Dufour, 2009; Mason,

Johnson, & Hamming, 1966); environmental chemistry (Meinert et al., 2010; Sansone, Popp, & Rust, 1997);

pharmaceuticals (Codina, Ryan, Joyce, & Richards, 2010); biology and toxicology (Needham et al., 1982;

Smith, Reynolds, Downie, Patel, & Rennie, 1998; Waskell, 1979); radio-carbon analysis (Currie, Eglinton,

Benner, & Pearson, 1997; Ball, Xu, McNichol, & Aluwihare, 2012). As already stated, prep-GC is a convenient

tool for isolation of pure components or specific sample fractions. This can have more than one practical

implication: production of raw material, through collection of the isolated component and exploitation in

chemical formulations; sub-trace level analysis, due to concentration of the target analyte by means of

repetitive injections; iii) enhancement of the identification process, through elimination of impurities and


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further elucidation with different (i.e. spectroscopic) means. Whatever the purpose of the prep-GC analysis,

it is a matter of fact that this technique suffers from limits, which make its exploitation quite challenging.

Such difficulties derive from the optimization of the analytical parameters with peculiar reference to the

instrumental equipment. It is not an easy task to obtain an acceptable amount of a pure component in one

single analysis. In order to isolate single components, sometimes present at low level in complex matrices, it

is necessary to inject sample volumes which are definitely higher than those conventionally used in analytical

gas chromatography. This would cause a column overloading in case of capillary columns. It is for this reason

that wide-bore columns (0.5-0.7 mm I.D.) have been traditionally utilized in prep-GC, thanks to their higher

sample capacity with respect to micro-bore GC columns (0.1-0.25 mm I.D.). Unfortunately, the use of columns

with wide diameter can lead to severe loss in efficiency and resolution, causing coelutions and collection of

impure fractions. In order to face the issue of sample loading, attention has been paid also to the injection

systems: automated samplers with microprocessors, cool injection systems, gas booster sample injection

devices, multi-port valves and expanded vaporizers are some examples of customized approaches (Hua-Li,

Feng-Qing, Wei-Hua, & Zhi-Ning, 2013). The collection systems are critical as well to the success of a prep-GC

process. Also at this level, prep-GC systems show some weakness: inefficient collections are often due to

condensation phenomena occurring in the connection device between the detector outlet and the collector.

Many trapping devices have been designed to improve prep-GC performance: cold-traps are basically used,

with either solid CO2 or liquid nitrogen, depending upon the boiling point of analytes to be trapped. In

addition, thermal gradient traps, electrostatic precipitation, Volmar collectors and potassium bromide traps

have been used (Hua-Li et al., 2013). Many of the drawbacks so far described have been successfully

overcome through the application of multidimensional gas chromatography (MDGC) to prep-GC (Eyres,

Urban, Morrison, Dufour, & Marriott, 2008; Eyres, Urban, Morrison, & Marriott, 2008; Rühle et al., 2009;

Schomburg, Kötter, Stoffels, & Reissig, 1984; Rijks, & Rijkes, 1990; Sciarrone et al., 2012; Sciarrone et al.,

2013; Sciarrone, Pantò, Tranchida, Dugo, & Mondello, 2014). Great part of prep-MDGC applications focused

on the isolation and collection of pure components from essential oils. In particular, the present work gives

an overview on a multidimensional GC system developed by Sciarrone et al. in 2012, and originally applied

to the isolation of the sesquiterpenoid carotol from carrot seed oil. The system consisted of three hyphenated

gas chromatographs, equipped with three Deans-switches and a CO2 cool trap. Successively, the same prep-

MDGC system was applied to the isolation and identification of a misidentified compound from wampee

essential oil. NMR, FTIR, and MS were also used for sure identification of the collected compound (Sciarrone

et al., 2013). A further improvement of the prep-MDGC system was reported by Sciarrone et al., 2014,

through the addition of a liquid chromatographic fourth dimension, which served as a preseparation step for

the successful collection of two sesquiterpenoids from vetiver essential oil. Considerations about the prep-

MDGC system’s troubleshooting, advantages and disadvantages, and positive outcomes from the

applications, are here given, along with a description of the instrumental apparatus.

Materials and Methods

Samples, Chemicals and Sample Preparation

Carrot seed essential oil was provided by Supelco/Sigma-Aldrich (Bellefonte, PA, USA). Clausena lansium

Skeels essential oil was obtained by hydrodistillation of 150 g of fresh leaves, collected in January 2012 from

plants grown in Messina (Italy). Mandarin essential oil was provided by a local industry (Capua, Reggio

Calabria, Italy). Vetiver essential oil (Haitian) was provided by Firmenich SA (Genève, Switzerland).


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n-Hexane (GC grade), n-hexane (LC grade), n-octane, n-nonane, n-decane, n-tetradecane (C14), n-

hexadecane (C16), deuterated chloroform and tert-butyl methyl ether, were supplied by Supelco/Sigma-

Aldrich. Spirogalbanone was provided by L’Oreal (Aulnay-sous-Bois, France).

2-Hexenal, 4-hexen-1-ol, hexanethiol, camphene, methyl octanoate, -terpinene, linalool, camphor, -

heptalactone, 2-decanone, decanal, linalyl acetate, -nonalactone, caryophyllene, caryophyllene oxide and

-bisabolol, were all supplied by Supelco/Sigma-Aldrich. Five different concentrations of standard solutions

were prepared (10, 50, 100, 250 and 500 g/mL), each added with 100 g/mL of n-nonane, as internal

standard.

Two stock solutions, containing respectively 300 mg of C14 and C16 (Sol. A) and 300 mg of caryophyllene

oxide and spirogalbanone (Sol. B), were prepared each in 5 mL of hexane. Vetiver essential oil was diluted

1:5 (v/v) in hexane prior to the injection.

Apparatus and Operational Conditions

Isolation of carotol

A Shimadzu (Kyoto, Japan) GC-2010 gas chromatograph was used in all GC-FID analyses. The column was an

SLB-5ms 30 m × 0.25 mm i.d. × 0.25 μm df (Supelco, Milan, Italy); temperature program, 50−280°C at

5.0°C/min; split/splitless injector (250°C); injection mode, split, 1:100 ratio; injection volume, 1.0 μL; inlet

pressure, 99.5 kPa, carrier gas, He; constant gas linear velocity, 30.0 cm/s. Detector (300 °C) gases: H2, 40

mL/min; air, 400 mL/min; makeup (N2), 40 mL/min; sampling rate, 10 Hz. Data were handled through the use

of GCSolution software (Shimadzu).

Samples were analyzed by GC/MS (EI) on a GCMS-QP2010 Plus system (Shimadzu). GC conditions, in terms

of column type, injection mode, and temperature were the same as for the GC-FID analysis, apart from the

inlet pressure (30.6 kPa) and temperature program: 50−280°C at 3.0°C/min. MS scan conditions: ion source

temperature, 200°C; interface temperature, 250°C; EI energy, 70 eV; scan range, 40−400 m/z. Data handling

was by GCMSsolution software (Shimadzu); identification was supported by the use of a GC/MS mass spectral

database equipped with linear retention indices (FFNSC 2.0, Shimadzu Europe).

For one-dimensional prep-GC analyses, Shimadzu GC2010 equipped with an Equity-5 column, 30 m × 0.53

mm i.d. × 5 μm df (Supelco), combined with a 1 m uncoated column segment of the same i.d. (at the head of

the analytical column), was used; temperature program: 150−280°C (14 min) at 5°C/min; split/splitless

injector (260°C); injection volume, 0.5, 1.0, 2.0, 3.0 μL; injection mode, splitless (1 min), then split 1:10; carrier

gas, He; inlet pressure, 80 kPa, in the constant pressure mode (initial linear velocity, 22 cm/s). The Deans

switch system pressure was 65 kPa (constant). The uncoated columns connected to the FID and collection

system were both 1.0 m × 0.18 mm i.d. Detector (280°C) gases H2, 40 mL/min; air, 400 mL/min; sampling

rate, 5 Hz.

Multidimensional prep-GC analyses were carried out by means of a prep-MDGC instrument (see Figure 1),

consisting of three GC systems (defined as GC1 GC2, and GC3), equipped with three Deans switch transfer

devices, namely, DS1 (between first and second column), DS2 (between the second and third column), and

DS3 (between the third column and the collection system). The MDGC switching elements, located inside the

ovens, were connected to three advanced pressure control systems (APC1, 2, and 3), which supplied carrier

gas (He) at constant pressure. GC1 was equipped with a split/splitless injector and a flame ionization detector

(FID1). Column 1 was the same as for one-dimensional prep-GC. The operational conditions were as follows:

constant inlet pressure 140 kPa (initial linear velocity 17 cm/s); splitless mode (280°C) for 1 min, then 1:10

(gas carrier He); injected volume 3.0 μL. Temperature program: 150−280°C (hold 14 min) at 5°C/min. The


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FID1 (300°C) was connected via a 0.4 m × 0.25 mm i.d. stainless steel uncoated column to the DS1. The APC1

constant pressure was 125 kPa. GC2 was equipped with a split/splitless injector (not used) and a flame

ionization detector (FID2). The transfer line between GC1 and GC2 was maintained at 240°C. The secondary

column was a Supelcowax-10, 30 m × 0.53 mm i.d. × 2 μm df (Supelco) directly connected to the DS1 through

the heated transfer line between GC1 and GC2. Temperature program: 150°C (hold 20 min); 150−240°C at

5°C/min. DS2 was used at the end of the secondary column to direct the effluent either to the FID2 (280°C)

or to the third column. APC2 constant pressure was 95 kPa (initial gas linear velocity, 35 cm/s). The branch

of uncoated column to connect FID2 to the transfer system was 0.5 m × 0.25 mm i.d. GC3 was equipped with

a split/splitless injector (not used) and a flame ionization detector (FID3). The transfer line between GC2 and

GC3 was maintained at 240°C. The third column was a custom-made SLB-IL59 30 m × 0.53 mm i.d. × 0.85 μm

df column (Supelco) directly connected to the DS2 through the heated transfer line between GC2 and GC3.

Temperature program: 150°C (hold 40 min); 150−240°C at 5°C/min. DS3 was used at the end of the third

column to direct the effluent either to FID3 (300°C) or to the collection system (300°C). Connections were

made via two 0.5 m × 0.32 mm i.d. stainless steel uncoated columns. APC3 constant pressure was 35 kPa

(initial gas linear velocity, 70 cm/s). Detector gases and sampling rate conditions were the same as applied in

the one-dimensional experiment. Data were collected by the MDGCsolution software (Shimadzu).

Figure 1. Triple Deans-switch multidimensional prep-GC system scheme. Reprinted with permission from D. Sciarrone, S. Pantò, C. Ragonese, P.Q. Tranchida, P. Dugo, and L. Mondello, Increasing the isolated quantities and purities of volatile compounds by using triple Deans-switch multidimensional preparative gas chromatographic system with an apolar-wax-ionic liquid stationary-phase combination, Analytical Chemistry, 84: 7092-7098. Copyright 2012 American Chemical Society.

Collection system

The simple and low-cost lab- constructed collection system was formed of a heated aluminum block (11 cm

height × 3 cm wide × 1.5 cm deep), equipped with a PT-100 temperature sensor, and was located through

the GC oven roof (Figure 2). The block was characterized by a 0.5 cm diameter hole, which enabled the

introduction of a GC liner and of the collection glass tube. A 90 mm × 0.75 mm i.d. deactivated liner was

located inside the lower part of the block, while a 80 mm × 3.5 mm i.d. glass tube was positioned above the

liner. About 25% of the glass tube was located inside the heated zone, while the remaining part was situated


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outside the block at room temperature, with or without packing material (depending on the specific needs).

The liner and the collection tube were sealed and held in position by using two nuts of appropriate

dimensions; the lower was used to connect the column by using a ferrule for FID detection, while the second

upper one contained a holed rubber septum. The last 5 mm of the uncoated column protruded inside the

glass tube.

Figure 2. Scheme of the collection device installed in the prep-MDGC system. Reprinted with permission from D. Sciarrone, S. Pantò, C. Ragonese, P.Q. Tranchida, P. Dugo, and L. Mondello, Increasing the isolated quantities and purities of volatile compounds by using triple Deans-switch multidimensional preparative gas chromatographic system with an apolar-wax-ionic liquid stationary-phase combination, Analytical Chemistry, 84: 7092-7098. Copyright 2012 American Chemical Society.

As an option, a CO2 cold jet stream, through a 1/8 in. tube, was directed to the empty or packed (10% SP-

2100 on 80/100 Supelcoport) collection vessel to improve the collection of highly volatile components. The

cold jet was switched on 1 min before and turned off 0.5 min after collection. The upper part of the glass

tube was cooled down to −60°C when using CO2, a temperature measured by means of an external PT-100

sensor. After analyte isolation, the collection vessel was removed immediately and flushed four times (in a

1.5 mL vial) with 250 μL of a n-hexane solution, spiked with 100 μg/mL of n-nonane, used as internal standard.

The solution containing both the internal standard and the collected volatile was then analyzed by GC/MS

and by GC-FID for qualitative and quantitative purposes, respectively. Finally, recovery was extrapolated from

a calibration curve, accounting for dilution related to flushing (1 mL of internal standard solution).

For recovery measurement, five-point calibration curves were constructed (n = 3), namely, 10, 50, 100, 250,

and 500 μg/mL, adding n-nonane as internal standard at a fixed concentration of 100 μg mL−1 (regression

coefficients > 0.9985). The concentration levels were selected in order to cover a wide concentration range

for the isolated components, diluted in ≈1 mL of hexane when flushed from the collection tube. Several

representative compounds for a variety of chemical groups were calibrated. To measure the volume of the


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collected solution, the vial was weighed before and after the process (after drying the vial for 30 min at room

temperature).

Wampee essential oil

GC analyses

GC-FID conditions were the same as above reported, with the exception of: oven program rate: 3.0°C/min;

injector temperature and volume: 280°C and 0.2 L; FID hydrogen flow rate: 50.0 mL/min.

GC-MS conditions were the same as reported in section isolation of carotol.

The prep-MDGC system was the same as described above. The collection device was the same as for carotol

experiment, as well as the recovery measurement procedure (Sciarrone et al., 2012). In this last case,

caryophyllene was chosen as representative compound of sesquiterpenes.

NMR analysis

1H, 13C{1H} NMR spectra were run on a Varian 500 spectrometer (operated at 499.74 and 125.73 Mz,

respectively, for the mentioned nuclei), controlled by a VNMRJ (2.2MI version) software package. In order to

attain a profound insight on the molecular structure, beyond the standard 1H and 13C{1H}-APT 1D spectra, 2D 1H–1H homo-nuclear TOCSY and NOESY, along with 1H–13C heteronuclear g-HSQCAD and g-HMBC

experiments, were achieved. These data were all processed and analyzed by the Mestrenova software

package (Mestrelab Research) and the reported chemical shifts at 273 K, are referenced to the solvent (1H,

= 7.26 ppm; 13C, = 77.16, triplet). The low temperature is necessary to prevent decomposition occurring in

chloroform. For elucidation of NMR parameters, readers are referred to Sciarrone et al., 2013.

GC-FTIR analysis

A Shimadzu GC2010 gas chromatograph, equipped with an AOC- 20i series autoinjector, was coupled to a

Bruker Vortex 80 FT-IR system (Bruker Italia, Milan, Italy), by means of a heated transfer line (250°C). An MCT

detector was used, cooled by liquid nitrogen, and operated at a scan velocity of 320 kHz and 4 cm−1 resolution.

The software Opus 7.0, with 3D and chromatography options, was used to acquire the FT-IR data (Bruker).

Two 0.5 m × 0.25 mm ID uncoated columns were used to: connect the analytical column to a capillary GC-

FTIR interface with a solid gold light-pipe (250°C), and from the latter to an FID (280°C), again inside the GC

oven. Two heated transfer lines passed through the GC side wall. The GC method and column were the same

as used in the GC-FID analysis, apart from the injection volume, which was 1 L in the splitless mode.

Vetiver essential oil

LC analyses

The LC preseparation of vetiver oil was performed by using an LC system (Shimadzu, Kyoto, Japan), equipped

with a Model CBM-20A communication bus module, two Model LC-20AD dual-plunger parallel-flow pumps,

a Model DGU-20A online degasser, a Model SPD- 20A UV detector, a Model CTO-20A column oven, and a

Model SIL-20AC autosampler. Five microliters and 50 μL of a vetiver oil solution were injected into a 250 mm

× 4.6 mm ID × 5 μm dp SupelcoSil LC-Si column (Supelco/Sigma-Aldrich, Milan, Italy), operated under the

following gradient conditions: flow rate was 1 mL/min (reduced to 0.35 mL/min during the transfer step):

from 0 to 6 min, the LC effluent was directed to waste; from 6 min to 10 min (1400 μL−100% hexane); and

from 14 min to 18 min (1400 μL−100% MTBE) the LC effluent containing the hydrocarbon and oxygenated

fractions, respectively, were directed to the first GC. Data were acquired by the LCsolution software

(Shimadzu).


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The LC-GC transfer device consisted of a dual-side-port 25-μL syringe (CTC Analytics AG, Zwingen,

Switzerland), controlled by means of a Shimadzu Model AOC- 5000 autosampler. Chromatography band

transfer was achieved, in the stop-flow mode. The lower part of the syringe was connected, via two transfer

lines, to the LC detector exit and to waste. A Teflon plug was located at the end of the syringe plunger; the

latter was characterized by a lower OD, with respect to the barrel ID, thus enabling mobile phase flow inside

the syringe. In the waste mode, the plug was located below both lines and the effluent was directed to waste.

In the cut position, the plug was positioned between the upper and lower line, and the effluent flowed to the

first GC. For more details on the syringe interface, the reader is referred to Sciarrone et al., 2014.

Prep-GC analyses

The configuration of the prep-MDGC system was the same as for previous experiments, with some

modifications (see figure 3). GC1 was equipped with an Optic 3 (ATAS GL International, Eindhoven, The

Netherlands) large volume injector (LVI) and a flame ionization detector (FID1). The LVI temperature program

and flow rate were optimized for each chemical class. LVI conditions for the hydrocarbon fraction: during the

transfer step (4 min) and for the first 0.75 min of the analysis time, the split mode was used (total flow rate

was 230 mL/min, at 45°C), followed by a 1 min splitless period; afterward, the split mode was applied (126

mL/min), heating the injector to 300°C at a rate of 15°C/s. LVI conditions for the oxygenated fraction: during

the transfer step (4 min) and for the first 0.50 min of the analysis time, the split mode was used (total flow

rate was 332 mL/min at 35 °C), followed by a 1 min splitless period; afterward, the split mode was applied

(126 mL/min), heating the injector to 300°C at a rate of 15°C/s. Column 1 was an Equity-5, 30 m × 0.53 mm

ID × 5.0 μm df, preceded by a 1 m segment of uncoated precolumn, with the same ID. Helium was the carrier

gas, having the following pressure conditions: 80 kPa for 0.75 and 0.50 min, for hydrocarbon and oxygenated

compounds, respectively; then to 140 kPa at a rate of 400 kPa/min, with the pressure remaining constant

afterward (initial gas linear velocity ≈ 22 cm/s). Oven temperature program: 45°C for 1.75 min (35°C for 1.50

min in the case of the oxygenated compounds), to 300°C at a rate of 15°C/min. APC1 pressure: 27.5 kPa for

0.75 min (0.50 min in the case of the oxygenated compounds); then to 125 kPa at a rate of 400 kPa/min.

Transfer line between GC1 and GC2 was maintained at 280 °C. The FID1 (330°C) was connected via a 0.25 m

× 0.18 mm ID stainless steel uncoated column to the TD1.

Figure 3. Scheme of the LC-GC-GC-GC preparative system. Reprinted with permission from D. Sciarrone, S. Pantò, P.Q. Tranchida, P. Dugo, and L. Mondello, Rapid isolation of high solute amounts using an online four-dimensional preparative system: normal phase-liquid chromatography cooupled to methyl siloxane-ionic liquid-wax phase gas chromatography, Analytical Chemistry, 86: 4295-4301. Copyright 2014 American Chemical Society.


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The second column was a custom-made ionic liquid one (SLB-IL59) of the following dimensions: 30 m × 0.53

mm ID × 0.85 μm df (Supelco). Oven temperature program for the hydrocarbon fraction: from 50 °C to 100

°C (20.21 min), at a rate of 5 °C/min, then to 240 °C, at a rate of 5 °C/min. Oven temperature program for the

oxygenated fraction: from 50 °C to 150°C (23min), at a rate of 5°C/min,then to 240°C,at a rate of 5 °C/min.

APC2 pressure: 7.8 kPa for 0.75 min (0.50 min in the case of the oxygenated compounds) to 95 kPa at 400

kPa/min. The transfer line between GC2 and GC3 was maintained at 240°C. The uncoated column used to

connect the TD2 to the FID2 was of the same dimensions as that reported for the FID1.

The third column was a Supelcowax-10, 30 m × 0.53 mm ID × 2.0 μm df. Oven temperature program for the

hydrocarbon fraction: from 50°C to 110°C (16.96 min) at a rate of 2°C/min, and then to 240°C at a rate of

5°C/min. Oven temperature program for the oxygenated fraction: from 50°C to 150°C (11.65 min), at a rate

of 2°C/min, then to 240°C, at a rate of 5°C/min. APC3 was maintained off during the LC-GC transfer step, then

to 35 kPa at 400 kPa/min. The connection of the FID3 with the TD3 was realized by means of a 1 m × 0.32

mm ID uncoated column. Detector gases for FID1, FID2, and FID3 (330°C) were as follows: H2, 50.0 mL/min;

air, 400 mL/min; makeup (N2), 40.0 mL/min (sampling rate = 5 Hz). Data were collected by the MDGCsolution

software (Shimadzu). The collection system was a dedicated preparative Brechbühler Prep9000 station

(Brechbühler AG, Switzerland), connected to the Deans switch system (TD3) by means of a flexible heated

transfer line (280°C) containing a 1.4 m branch of uncoated column. The system was equipped with a ten-

position carousel with Carbotrap C (40 mesh) adsorption tubes. The collection system was linked to a vacuum

circuit, isolated by a solenoid valve. During the collection process, the valve was opened, with the effects of

the vacuum enabling a more rapid and effective analyte accumulation; additionally, the condensation of high

boiling components, at the conjunction point between the transfer-line end and the adsorption tube, is

avoided. During normal operation (no collection), the solenoid valve was closed. A scheme of the LC-GC-GC-

GC system is reported in Figure 3.

GC analyses

A Shimadzu GC2010 equipped with an SLB-5ms, 30 m × 0.25 mm ID × 0.25 μm df, was used. Temperature

program: from 100°C to 300°C, at a rate of 5.0°C/min. Injection (280 °C) volume was 0.2 μL, in split mode

(1:100 ratio); carrier gas, He (constant gas linear velocity: 30.0 cm/s). A GCMS-QP2010 Ultra was used for GC-

MS analyses, same conditions as for GC-FID analyses, except: inlet pressure, that was 30.6 kPa; ion source

temperature, 200 °C; interface temperature, 250 °C; mass scan range, 40−400 m/z.

For calculation of LC-GC transfer recovery, normalized peak areas were used. The reference peak area was

determined through the splitless injection of 1.0 μL of the standard solutions (A and B) into GC1. The amounts

of components introduced onto the column were ∼60 μg for each solution. Effectively collected amounts

were measured by means of two five-point calibration curves by choosing caryophyllene as representative

of sesquiterpenes and spirogalbanone for oxygenated sesquiterpenes.

Results and Discussion

Isolation of Carotol

The first application of the prep-MDGC system here described consisted of the isolation of a characteristic

compound from carrot seed oil, namely carotol (Sciarrone et al., 2012). Preliminarily, during method

development, different collection conditions were evaluated. It was demonstrated that recovery values were

dependent on boiling point of analytes, use of packed or empty collection tubes, use of cold CO2. These

parameters were tested on a series of standard compounds, which are natural constituents of carrot seed


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oil. Successively, a one-dimensional prep-GC analysis was carried out, with the purpose of collecting the

compound carotol, which was present at a 30% level in the essential oil. Briefly, in the single dimension prep-

GC configuration, a megabore 5% diphenyl column was used, with a low injection volume (1 L of essential

oil) and a fast oven temperature program. After collection, the fraction was separately injected in a GC-MS

system, the resulting chromatogram being shown in Figure 4.

Figure 4. GC-MS chromatogram of the fraction collected by means of one-dimensional prep-GC. Peak identification: (1) carotol; (2) caryophyllene oxide; (3) and (4) unknowns. Reprinted with permission from D. Sciarrone, S. Pantò, C. Ragonese, P.Q. Tranchida, P. Dugo, and L. Mondello, Increasing the isolated quantities and purities of volatile compounds by using triple Deans-switch multidimensional preparative gas chromatographic system with an apolar-wax-ionic liquid stationary-phase combination, Analytical Chemistry, 84: 7092-7098. Copyright 2012 American Chemical Society.

As can be seen, not only carotol was present in the isolated fraction: other three peaks were detected,

namely, caryophyllene oxide and other two unknown substances. The three peaks accounted for about 25%

of the total fraction. Such a situation was definitely improved through the injection of 3 L of essential oil

into the multidimensional system. As depicted in figure 1, the prep-MDGC configuration involved the use of

three columns, having different selectivities and dimensions. Specifically, a wide-bore column was used in

the first GC oven, for its sample capacity, so to accommodate a higher amount of essential oil. Parameters

such as gas flow rates and pressures were tuned in order to optimize resolution, purity and collection yield

of carotol. The results obtained from this analytical procedure are shown in figure 5, where a GC-FID

chromatogram relative to the collected fraction is depicted. In this case, the fraction isolated contained 99.6%

pure carotol, identified by means of GC-MS with a spectral similarity of 99%. From recovery measurement,

considering the density of the essential oil and volume injection, it came out that for the collection of about

2 mg of pure carotol, at least three prep-MDGC cycles were necessary. Indeed, 2.22 mg of pure carotol were

successfully collected.


10

Figure 5. GC-FID chromatogram of carotol, collected by means of the prep-MDGC instrument. Reprinted with permission from D. Sciarrone, S. Pantò, C. Ragonese, P.Q. Tranchida, P. Dugo, and L. Mondello, Increasing the isolated quantities and purities of volatile compounds by using triple Deans-switch multidimensional preparative gas chromatographic system with an apolar-wax-ionic liquid stationary-phase combination, Analytical Chemistry, 84: 7092-7098. Copyright 2012 American Chemical Society.

Structure Elucidation of a Wampee Essential Oil Compound

Once developed, the prep-MDGC system here described has been applied to the isolation and identification,

by means of spectroscopic techniques, of a compound from Clausena lansium essential oil. Preliminarily, the

hydrodistilled essential oil was investigated by means of conventional GC-FID and GC-MS. One of the last

eluting peaks, in the sesquiterpene region of the chromatogram, couldn’t be assigned, although accounting

for about 10% of the whole sample. The GC-MS library matching procedure returned as best candidate, with

a quite low similarity score (84%), the compound -sinensal, a sesquiterpene previously found in the leaves

of this plant. In order to clarify and possibly confirm what reported in literature, the compound in question

was isolated by exploiting the prep-MDGC system. The relative chromatograms are shown in figure 6. The

use of three different dimensions of selectivity was essential for purification of the analyte to be isolated. As

can be seen in figure 6, the efficiency of the first dimension separation was low, for obvious reasons of sample

overloading. Also, in consideration of the complexity of the matrix and the region of the chromatogram to

be cut, which was crowded of peaks, this first fraction presented numerous coelutions. The two successive

steps of separation, through the use of stationary phases of different selectivity, allowed to obtain a final

collection of a 99.1% pure unknown compound. About 2 mg of analyte were recovered after a 13 hours

period. Successively, the isolated fraction was subjected to further investigation by means of NMR, GC-FTIR

and GC-MS techniques. All the three spectroscopic methodologies confirmed that the unknown analyte

under investigation was (2E, 6E)-2-methyl-6-(4-methylcyclohex-3-enylidene)hept-2-enal.The correspondent

NMR spectrum is shown in figure 7. A further GC-MS analysis of the newly identified component and of pure

-sinensal highlighted differences upon the ion fragment abundances.


11

Figure 6. Prep-MDGC stand-by (upper trace) and cut (lower trace) chromatograms, derived from the analysis of Clausena lansium Skeels essential oil, relative to the first (upper chromatogram), second (middle chromatogram) and third GC dimensions. “Reprinted from Analytica Chimica Acta, 785, D. Sciarrone, S. Pantò, A. Rotondo, L. Tedone, P.Q. Tranchida, P. Dugo, & L. Mondello, Rapid collection and identification of a novel component from Clausena lansium Skeels leaves by means of three-dimensional preparative gas chromatography and nuclear magnetic resonance/infrared/mass spectrometric analysis, pages 119-125, Copyright (2013), with permission from Elsevier”.

Figure 7. 1H spectrum of the unknown compound with the complete assignment, isolated from wampee essential oil. “Reprinted from Analytica Chimica Acta, 785, D. Sciarrone, S. Pantò, A. Rotondo, L. Tedone, P.Q. Tranchida, P. Dugo, & L. Mondello, Rapid collection and identification of a novel component from Clausena lansium Skeels leaves by means of three-dimensional preparative gas chromatography and nuclear magnetic resonance/infrared/mass spectrometric analysis, pages 119-125, Copyright (2013), with permission from Elsevier”


12

Isolation of Sesquiterpenes from Vetiver Essential Oil

An additional dimension of separation, consisting of a liquid chromatograph, was hyphenated to the prep-

MDGC apparatus and applied to the isolation and purification of two sesquiterpenoids from vetiver essential

oil. The extra LC dimension served as a pre-fractionation step of the various chemical groups present in

vetiver essential oil. The goal of this application was basically to develop a preparative methodology for

recovery of pure compounds present at a <10% level. Different parameters, such as LC flow, pressure and

temperature of the GC1 injector, split flow and vent time, were tuned to optimize the LC-GC transfer.

Standard solutions were used for this part of method development. For a detailed description of

troubleshooting related to this issue, the reader is referred to Sciarrone et al., 2014. After optimization, the

final conditions chosen for transferring the sesquiterpene fraction from LC to GC1 dimension were: initial

injector temperature and pressure, 45°C and 80 KPa; LC transfer flow rate, 350 L/min with a split flow at

230 mL/min. Once occurred the LC transfer into the GC system, a pressure and temperature program was

applied to the GC injector. Slight modifications to these conditions were applied to the transfer of the

oxygenated sesquiterpene fraction. After optimization, the real sample of vetiver essential oil was subjected

to the LC-GC-GC-GC preparative analysis. Figure 8 shows an expansion of the monodimensional GC-MS

chromatogram, along with the two traces relative to the fractions isolated by means of the prep-MDGC

system.

Figure 8. GC-MS chromatogram of vetiver essential oil (peak A, amorphene; peak B, -vetivone), and GC-MS chromatograms of the pre-separated LC hydrocarbon (middle trace) and oxygenated sesquiterpene fractions (lower

trace) obtained on an SLB-5ms 30 m × 0.25 mm ID × 0.25 m df. Reprinted with permission from D. Sciarrone, S. Pantò, P.Q. Tranchida, P. Dugo, and L. Mondello, Rapid isolation of high solute amounts using an online four-dimensional preparative system: normal phase-liquid chromatography coupled to methyl siloxane-ionic liquid-wax phase gas chromatography, Analytical Chemistry, 86: 4295-4301. Copyright 2014 American Chemical Society.

As can be seen, vetiver essential oil is a highly complex matrix, characterized by the presence of an abundant

sesquiterpene fraction eluting in the last part of the chromatogram. The LC primary step of separation

resulted to be essential for various reasons: i) reduction of the matrix complexity, through a rough separation

of target fractions; ii) reduction of GC column overloading; iii) decrease of the amount of high boiling point

compounds entering the GC system. For the isolation of 1 mg ca. of -amorphene, seven prep-MDGC

analyses were necessary, each lasting for about 80 min. The purity of this compound was assessed as 90%,

through GC-FID and GC-MS analyses (see Figure 9).


13

Figure 9. GC-MS chromatogram of the compound -amorphene isolated by means of the LC-GC-GC-GC system.

Reprinted with permission from D. Sciarrone, S. Pantò, P.Q. Tranchida, P. Dugo, and L. Mondello, Rapid isolation of high

solute amounts using an online four-dimensional preparative system: normal phase-liquid chromatography coupled to

methyl siloxane-ionic liquid-wax phase gas chromatography, Analytical Chemistry, 86: 4295-4301. Copyright 2014

American Chemical Society.

The same analytical approach was applied to the isolation of another sesquiterpenoid present at higher

amount in vetiver essential oil: -vetivone. In this case, a lower number of prep-MDGC cycles was required

(two prep-cycles) to obtain 1 mg of about 94% purity (see Figure 10).

Figure 10. GC-MS chromatogram of -vetivone isolated by means of the prep-MDGC system. Reprinted with permission

from D. Sciarrone, S. Pantò, P.Q. Tranchida, P. Dugo, and L. Mondello, Rapid isolation of high solute amounts using an online four-dimensional preparative system: normal phase-liquid chromatography coupled to methyl siloxane-ionic liquid-wax phase gas chromatography, Analytical Chemistry, 86: 4295-4301. Copyright 2014 American Chemical Society.


14

In conclusion, the use of a tridimensional prep-GC system has been proven to be an effective approach for

the collection of pure components in the 10-30% range, with high purity degree. Moreover, an additional LC

dimension was of great support in the collection of low concentrated components, in a reasonably short time

of collection. Benefits from the prep-LC-3DGC system were a reduction of GC contamination from dirty

samples (non volatile components) and collection of multiple components in one single run. The versatility

of the system (operated both in an on-line/off-line fashion) makes it an interesting alternative for the

production of pure standard compounds.

REFERENCES

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magnetic resonance spectroscopy. Analytical Chemistry, 80, 6293-6299.

Eyres, G.T., Urban, S., Morrison, P.D., & Marriott, P.J. (2008). Application of microscale-preparative

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pure methylnaphthalenes from crude oils. Journal of chromatography A, 1215 (1-2), 168-176.

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Marriott, P.J., Eyres, G.T., & Dufour, J.P. (2009). Emerging opportunities for flavor analysis through

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Investigation of hyperkeratotic activity of polybrominated biphenyls in firemaster FF-1. Journal of Toxicology

and Environmental Health Part A, 9, 877-887.


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Rijks, J.P.E.M., & Rijks, J.A. (1990). Programmed cold sample introduction and multidimensional preparative

capillary gas chromatography. Part I: introduction, design and operation of a new mass flow controlled

multidimensional GC system. Journal of High Resolution Chromatography, 13, 261-266.

Rühle, C., Eyres, G.T., Urban, S., Dufour, J.-P., Morrison, P.D., & Marriott, P.J. (2009). Multiple component

isolation in preparative multidimensional gas chromatography with characterization by mass spectrometry

and nuclear magnetic resonance spectroscopy. Journal of Chromatography A, 1216, 5740-5747.

Sansone, F.J., Popp, B.N., & Rust, T.M. (1996). Stable carbon isotopic analysis of low-level methane in water

and gas. Analytical Chemistry, 69, 40-44.

Schomburg, G., Kötter, H., Stoffels, D., & Reissig, W. (1984). Automated instrumentation for multidimensional

preparative scale GC (PSGC). Chromatographia, 19, 382-390.

Sciarrone, D., Pantò, S., Ragonese, C., Tranchida, P.Q., Dugo, P., & Mondello, L. (2012). Increasing the isolated

quantities and purities of volatile compounds by using a triple Deans-switch multidimensional preparative

gas chromatographic system with an apolar-wax-ionic liquid stationary-phase combination. Analytical

Chemistry, 84, 7092-7098.

Sciarrone, D., Pantò, S., Rotondo, A., Tedone, L., Tranchida, P.Q., Dugo, P., & Mondello, L. (2013). Rapid

collection and identification of a novel component from Clausena lansium Skeels leaves by means of three-

dimensional preparative gas chromatography and nuclear magnetic resonance/infrared/mass spectrometric

analysis. Analytica Chimica Acta, 785, 119-125.

Sciarrone, D., Pantò, S., Tranchida, P.Q., Dugo, P., & Mondello, L. (2014). Rapid isolation of high solute

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12.

Nat. Volatiles & Essent. Oils, 2015; 2(2): 16-29 Bueno J.

16

REVIEW

Models of evaluation of antimicrobial activity of essential oils in

vapour phase: a promising use in healthcare decontamination

Juan Bueno*

Bioprospecting development and consulting, Bogotá, Colombia


Abstract

Hospital-acquired infections caused by viruses, bacteria and fungi are a constant concern in the health system. In this respect the

decontamination of hospital environments is a control measure which decreases the reservoirs of pathogenic microorganisms and

their transmission. For that reason new products have been developed such as antimicrobial surfaces that prevent microbial

contamination and cleaning vapour systems, in search of a potent disinfection method more friendly to the environment, less toxic,

safer and biodegradable. In this way has been considered the use of microbicidal essential oils (EOs) in vapour-phase as an interesting

alternative in the development of hospital decontamination devices. Although in vitro antimicrobial activity of EOs have been

demonstrated, there are no standardized tests to evaluate these products in vapour-phase, the aim of this review is to present the

different evaluation methods that have been used to establish the activity of the vapours of EOs and other disinfectants, with the

purpose of provide a rational approach to the research, development and implementation of new biocide agents based on this natural

product for cleaning in hospitals and healthcare.

Keywords: Nosocomial infections, decontamination, essential oil, antimicrobial activity, vapour phase

Introduction

Hospital-acquired (nosocomial) infections is a worldwide healthcare problem, with a general prevalence in

developing countries of 15.5 per 100 patients in where patients in intensive care are the most affected with

a global prevalence of 47.9 per 1000 patient-days (Allegranzi et al., 2010). The microorganisms isolated more

often from patients and hospital environments are Staphylococcus aureus, Pseudomonas aeruginosa,

Escherichia coli, Clostridium difficile, Streptococcus species, Enterobacter spp, Acinetobacter spp., Klebsiella

spp., influenza virus and noroviruses (Gadi, Borkow & Monk, 2012). In addition the pharmacological

treatment of nosocomial infection is compounded by the emergence of drug-resistant pathogens (Gady,

Borkow & Gabbay, 2010). Also, microbial colonization and persistence in medical surfaces have been

associated with the existence of intrahospital infections (Tétault et al., 2012; de Abreu et al., 2014). For that

reason the constant cleansing using biocides (antiseptics and disinfectants) reduces the density of

microorganisms in healthcare environments (McCoy et al., 2014). Equally surgical site infections can be

reduced by use of skin antiseptics in a decolonization process or antiseptic wound lavage until 41%

(Hakkarainen et al., 2014; Yokoe et al., 2014).

Between the strategies developed to reduce the environmental microbial burden through biocides is the

vaporous decontamination, which consists in the application of a decontaminant agent in vapour (or gas)

phase to decontaminate confined spaces and medical devices (Kačer et al., 2012). In the use of this

decontamination method, it is very important to take into account that the efficacy depends of the control of

following parameters such as biocide concentration, exposure time, temperature, humidity, and the

contaminant conditions (Fraise, 2013). Vapour phase decontamination and sterilization techniques have been


17

applied in processes as pharmaceuticals manufacturing, equipment cleaning, cleaning healthcare rooms and

foodstuffs (Arlene, Klapes & Vesley, 1990). In this way, biocides such as formaldehyde have been employed,

but it is a human carcinogenic chemical so its use has been restricted (Johnston et al., 2005); currently,

hydrogen peroxide vapour is the most advantageous biodecontamination method with activity against

bacterial endospores, vegetative bacteria, viruses and mycobacteria (Hall et al., 2007).

An interesting choice in the use of biocide in vapour phase are essential oils (EOs) due to their content of

volatile compounds with antimicrobial activity that have been considered as a promising alternative to

biocides and antibiotics. This antimicrobial activity is determined by the synergistic action of functional

groups present in the oil as phenols, aldehydes, ketones, alcohols, ethers and esters (Li et al., 2014). Equally

EOs and their components have shown excellent results against multidrug-resistant (MDR) bacteria such as

methicillin-resistant S. aureus (MRSA), which makes them important for the development of hospital biocides

(Maria, Faleiro & Miguel, 2013). In addition, several studies have confirmed that EOs used in vapour phases

are more potent antimicrobials if used in liquid form between them thyme, citrus oil, Eucalyptus globulus,

Melaleuca alternifolia and lemon grass (Katie, Laird & Phillips, 2012; Nadjib et al., 2014).

Currently, although vapour phase screening platforms for EOs have been described, there are no standardized

tests to evaluate the antimicrobial activity of these vaporized products (Al-Yousef, 2014). The aim of this

review is to present the different evaluation methods that have been used to establish the activity of the

vapours of EOs and other disinfectants, with the purpose of provide a rational approach to the research,

development and implementation of new biocide agents based in this natural product for decontamination

in healthcare.

Vapour-phase Decontamination

The hospital environment is a constant source and reservoir of MDR microorganisms (Radhouani et al., 2014).

Equally, there is an association between contamination of healthcare spaces and the increase of nosocomial

infections (Chemaly et al., 2014). In addition, surfaces, medical equipment and other fomites are frequently

contaminated after contact with patients or contaminated surfaces (Weber et al., 2010). For that reason the

use of environmental disinfection strategies have the ability to reduce transmission (Steinberg et al., 2013).

These strategies have two objectives, first the cleaning and disinfection of hospital rooms to reduce the risk

of acquired pathogens from contaminated surfaces. Second, the disinfection of surfaces to reduce the risk of

contamination and transmission (Donskey, 2013; Weber et al., 2013).

In fact, many methods of disinfection are unable to remove environmental MDR (Tuladhar et al., 2012;

Passaretti et al, 2013). These microorganisms have the ability to persist on inanimate surfaces for days and

months, increasing the risk of transmission (Table 1.)(Kramer et al., 2006; Chemaly et al., 2014). In this way,

hydrogen peroxide vapour (HPV) has been demonstrated to be a microbicidal vapour-phase method that have

the ability of destroy nosocomial pathogens in situ. Equally, HPV have eliminated environmental reservoirs of

MDR more efficiently than other cleaning methods so it is now considered an election in hospital disinfection

(Passaretti et al, 2013).


18

Table 1. Survival time of MDR microorganisms that causing nosocomial infections (Chemaly et al., 2014).

Microorganisms Survival time

Methicillin-resistant Staphylococcus aureus (MRSA) 7 days to >12 month

Vancomycin-resistant Staphylococcus aureus (VRSA) 5 days to >46 months

Pseudomonas aeruginosa 6h to 16 months

Clostridium difficile >5 months (spores)

Acinetobacter baumannii 3 days to 11 months

Norovirus (feline calicivurus) 8h to 7 days

Rotavirus 6-60 days

Antimicrobial Activity of Essential Oils in Vapour Phase

EOs have higher microbicidal potency in vapour phases more than their liquid phases (Imaël Henri Nestor,

Bassolé & Juliani, 2012). Antimicrobial activity of various EOs in vapour phase have been described between

them Eucalyptus (Tyagi et al., 2014), Melaleuca alternifolia (Carson et al., 2006) Cymbopogon citratus (Kumar,

Tyagi & Malik, 2010; 2012) and Thymus vulgaris (Tullio et al., 2007). As well as major compounds as limonene

and citral (Chee et al., 2009; Kumar, Tyagi & Malik, 2010). The antimicrobial action of these vapours depends

of the presence in a gaseous state of functional groups in EOs, as well as their vapour pressure, allowing it to

cross the microbial cell membranes (Belletti et al., 2007). This results in very low concentrations are very

active (1.56–6.25 μg/ml) in inhibit bacterial growth, making them ideal for the development of vaporized

biocides (Reichling et al., 2009).

Between the action mechanisms of these vapours has been found to produce in microbial cells shrinkage and

partial degradation, observed by scanning electron microscopy/atomic force microscopy SEM/AFM of C.

albicans under exposition of Cymbopogon oil in vapour-phase (Kumar, Tyagi & Malik, 2010). Equally, the EOs

have the ability of modulate bacterial resistance mechanims (efflux pumps) by gas contact as oils obtained

from Zanthoxylum articulatum and Hyptis martiusii Engler which makes them useful as adjuvants in antibiotic

therapy and disinfection (Coutinho et al., 2010; Rodrigues et al., 2010; de Oliveira et al., 2014). Also ionized

gaseous species of EOs from orange and thyme generated by candles shown antibacterial effects mediated

by reactive oxygen species (ROS) that induce cell membrane disruption (Gaunt et al., 2005). Other interesting

finding is the concomitant use of EOs in vapour-phase and heat that can produce hyphal damage of

Trichophyton mentagrophytes at 27ºC, fact that can be taken into account when designing an atomiser

(Inouye et al., 2007).

In this order ideas EOs in vapour-phase have been used in formulations for environmental decontamination

as BioScentTM that contains oils of lemongrass and geranium and being dispersed using the ST ProTM machine

(Scent Technologies Ltd) was able to inhibit MRSA after 20 hours of exposure (Doran et al., 2009). Also, Citri-

VTM, that contains oils from orange and bergamot in concentrations of 1:1 v/v had antimicrobial activity

against Enterococcus sp. and S. aureus on stainless steel surfaces as well as bacterial biofilms (Laird et al.,

2012). Which demonstrates the potential of these natural products used as biocides in hospital environments.


19

Methods in Vapour Phase Evaluation

These methods determine the minimal inhibitory dose (MID), which is a measure of the vapour antimicrobial

activity that permits to know vapour concentration and absorption for inhibit microbial species, also MID can

be established in function of time for determine if EOs are more effective in high or low vapour concentration

as well as in short or long exposure time (Inouye et al., 2001).

Disc Volatilization Assay

This assay require a culture agar plate inoculated with 100 μL, of microbial suspension containing 106 colony-

forming unit (CFU)/mL inserted upside down on top of a container (Figure 1.). A paper disc (diameter 6mm)

is deposited at the bottom of the container with 10 μL of different dilutions of EO. The plates inoculated and

the disc should be sealed with parafilm to prevent the steam outlet. Plates should be incubated at 30°C for

24h (also can be observed in function of time) and measure the resulting inhibition (Kumar, Tyagi & Malik,

2012).

A variant of this method for antibiofilm activity of EOs in vapour phase can be developed using a paper disc

(1 cm diameter) soaked with EO or terpene and fixed on the cover lid of 96 multiwell plate with the biofilm.

LIVE/DEAD BacLight Viability kit can be employed for measure the biofilm mass after exposure (Nostro et al.,

2009; Bueno, 2014).

Disc volatilization assay is the simplest assay for antimicrobial evaluation of EOs in vapour-phase and can be

used as primary screening for to choose the most promising oil, terpene or their combinations.

Figure 1. Disc volatilization assay


20

Agar Vapour Assay

Agar vapour assay is other assay useful as primary screening, and have the ability of control the evaporation

reducing the place of exposure. In this technique, a paper disc of 8mm containing 30 μl of different dilutions

of EO is placed over agar medium inoculated with microbial suspension containing 106 CFU/mL surrounded

by a plastic ring and covered by glass in a Petri dish, later the plate should be turned upside down and

incubated at 30°C for 24h (also can be observed in function of time) for measure of inhibition zone and obtain

MID of the EO vapours, the advantage of this method is the control of evaporation and the measure of

antimicrobial activity in a limited space (Figure 2)(Inouye et al., 2006).

Figure 2. Agar vapour assay

Airtight Box

This is another assay that can be useful to determine the antimicrobial activity of the vapours in function of

space using plastic boxes of known volumes. This method uses Petri dishes with culture media or agar plugs

inoculated with 106 CFU/mL of microbial suspension and placed in 1.3 L airtight boxes recovered with

aluminium foil for prevent the plastic absorption of EO and protect boxes of the contamination. EOs in

different dilutions can be inserted in the top of airtight box on filter paper of 9 cm in diameter (Figure 3.). The

boxes should be incubated to 37ºC for 24 h (also can be observed in function of time to 0, 2, 4, 6, 8, 12 and

24 h) and the MID determined in mg of EO/L of air (mg/L) is established where the EO concentration did not

allow microbial growth (Inouye et al., 2000; Inouye et al., 2001a; Inouye et al., 2001b). This assay have the

advantage to permit the use of inoculated material as plastic and steel to evaluate surface decontamination.


21

Figure 3. Airtight box assay

Bio-clean space

After primary screening is necessary to determine the decontamination activity of vapours of EOs in

laboratory spaces as safety cabinets and bio-clean rooms. In this way bio-clean space decontamination is a

useful test. This technique uses Petri dishes inoculated with microorganisms to concentration of 106 CFU/mL

in agar medium or glass, cotton, plastic and steel material introduced in a sealed safety cabinet or in bioclean

room with inlet and outlet fumes (Figure 4.) (Masaoka et al., 1982). With this assay is possible run several

experiments in function of time with various types of microorganisms and require control of vapour pressure,

temperature and relative humidity, after performing exposure, the Petri dishes should be closed and the

materials suspended in sterile saline solution for inoculation in agar plates and incubation, with the aim of

assess the degree of decontamination by oil spray (Moat et al., 2009).


22

Figure 4. Bio-clean room assay in a safety cabinet

AOAC method 961.02 (Germicidal Spray Products as Disinfectants)

This is a standard method used for evaluation of products as a germicidal for disinfecting surfaces such as

glass, metal, plastic and fibres (Pines et al., 2013). Several germicidal spray composition containing EOs have

been evaluated under this technique against reference microorganisms as are Salmonella cholerasuis ATCC

10708, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC 8739,

Streptococcus pneumonia ATCC 49619, and Listeria monocytogenes ATCC 19113. This Association of Official

Analytical Chemists (AOAC) procedure is an accepted method for disinfectant evaluation. First, the

microorganisms grown for 48 h in nutrient broth are inoculated on the test material and exposed to germicidal

spray with EOs by time intervals of 1, 2, 5 and 10 minutes, the germicidal activity must be neutralized using

letheen broth and the microorganisms should be recovered in sterile saline solution (Bowker, 2009). The use

of different materials (carriers) allows evaluate the test substance and viability of microbial cells in a specified

contact time (DeAth, 2008). Germicidal formulations containing thyme oil have been evaluated with this assay

(Samuel, DeAth & Weiss, 2012).


23

Figure 5. AOAC method 961.02 (Germicidal Spray Products as Disinfectants)

Biofilm Surface Decontamination Tests with Vaporized Biocides

Although the polysaccharide matrix components of biofilms decrease gas penetration (Epstein et al., 2011),

it is very important to evaluate the activity of EOs in vapour phase against biofilm formation. A choice is the

use of plastic coupons with biofilms in a isolator chamber joined to a vapour generator for to evaluate surface

decontamination (Eterpi et al., 2011), these coupons can be cultured in a FC270 flow-cell system for flow

biofilm study before the exposure (Bueno, 2014). Equally 96 multiwell plate with a biofilm in a vapour

chamber or airtight box at 37ºC can be used and revealed by the crystal violet method or LIVE/DEAD BacLight

Viability kit to determine the mass of the biofilm (Laird et al., 2012; Bueno, 2014).

ATP Bioluminescence Systems

Currently, it is very important to verify state of disinfection of laboratory and hospitals environments, due to

the little evidence collected about the association between nosocomial infections and hospital surfaces

contamination (Dancer, 2008). In this way the most used technique to assess the hospital cleaning for new

biocidal products is ATP bioluminescence that has been proposed both to monitor the effectiveness of the

cleaning agents as well as cleaning programs (Moore et al., 2010). The method takes a sample from the area

to be tested after disinfection procedure with a swab that is placed in a detection device in presence of

luciferase and luciferin (Dancer, 2014). Later using a luminometer the reaction is revealed and the light

produced is proportional to the amount of ATP, the disadvantages of this method are the variable sensitivity

between the available systems, the inefficacy to detect gram-negative bacteria because performs a

incomplete lysis of this microorganisms and the lectures can be confounded by residues as plastics and

microfibres (Turner et al., 2010; Dancer, 2011; Dancer, 2014).

Conclusions and Perspectives

Cleaning has two purposes, first is maintaining the appearance and function as well as to prevent spoilage;

second is decreasing the microbial burden (Dancer, 2008). The reduction of microbes reduce the risk of

transmission in hospital environments and the association between microbial surface contamination and the

emergence of infectious diseases (Dancer, 2008). For that reason, the development of innovative biocidal


24

products in collaboration with infection control personnel, researchers and industry is necessary (Dancer,

2011).

Although applications in clinical treatment of EOs have been limited, they have been successfully used

topically in creams and lotions as well as in liposomal formulations. Equally, EOs have shown to possess the

ability to enhance penetration of antiseptics and block the resistance mechanisms in MDR microorganisms

(Fortino, Solórzano-Santos & Miranda-Novales 2012).

On the other hand, for the product development of disinfectants and sanitizers agents based on EOs it is

necessary to conduct Human Repeat Insult Patch Test (HRIPT) and the in vitro Dermal Irritection® Assay

according to standardized methods with the aim to obtain safe and sustainable products as is the example of

thyme oil based disinfectants (Bondi, 2011).

Equally, for designing new decontamination systems using EOs is necessary to determinate the physical

chemistry of the decontamination process using this formulae:

mg/liter=P(Mol Wt)(1000 mg/g)/RT

In where the concentration of gas is expressed in mg/liter,

P: represents atmospheric pressure,

Mol Wt: symbolizes molecular weight of the gas,

R: typify ideal gas constant,

T: represents temperature (°K)

With this equation it is possible to determine the gas concentration, the pressure, the relative humidity and

temperature in the decontamination chamber (Hultman et al., 2007).

Finally, the use of aerosol delivery technology for the development of biocides for surface decontamination

is an interesting approach in innovation to increase the antimicrobial potency of EOs because using liquid

droplets suspended in gas can destroy airborne bacteria and spores. This kind of formulations have been

proved in disinfectants such as sodium hypochlorite, peroxyacetic acid and quaternary ammonium (Thorn et

al., 2013).

ACKNOWLEDGMENT

The author acknowledges to Claudia Marcela Montes for the design of the figures contained in this paper.

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30

RESEARCH ARTICLE

α-Cyclodextrin encapsulation enhances antimicrobial activity of cineole-rich essential oils from Australian species of Prostanthera (Lamiaceae)

Nicholas Sadgrove*, Ben Greatrex and Graham Lloyd Jones

Pharmaceuticals and Nutraceuticals Group, Centre for Bioactive Discovery in Health and Ageing, University of New

England Armidale NSW 2351 Australia


Abstract

Highly chemically variable cineole-rich essential oils were produced from cultivated specimens of the genus Prostanthera

(Lamiaceae), currently taxonomically assigned to P. ovalifolia, P. rotundifolia, P. incisa and P. lasianthos. Essential oils were

chemically characterised using GC-MS and NMR. The mean inhibitory concentrations against Gram-positive and Gram-negative

bacterial species were measured using a microtitre plate broth dilution assay. A selection of these oils were further assayed for

antimicrobial activity after being encapsulated at a 1:1 molar ratio using α-cyclodextrin. Cineole-rich essential oils are chemically

differentiated by the character of the sesquiterpene oxides cis-dihydroagarofuran and kessane; and the sesquiterpene alcohols

globulol, prostantherol and ledol. Within the wider context of common essential oils, this selection of essential oils from

Prostanthera demonstrated relatively low inhibitory concentrations (high antimicrobial activity), particularly against Gram-positive

organisms. When some of these oils were encapsulated in α-cyclodextrin the antimicrobial activity was generally enhanced by two

to four-folds. This enhancement may be a result of encapsulation with reduced evaporation during the assay and emulsion

formation which may facilitate delivery to bacterial species. The use of cyclodextrins as a feed and formulation additive should be

considered within the context of the antimicrobial activity of cineole-rich essential oils from Prostanthera.

Keywords: prostantherol, encapsulation, α cyclodextrin, antimicrobial, cineole

Introduction

The genus Prostanthera comprises approx. 100 species (CHAH Australian Plant Census) in the family

Lamiaceae. The number of species is expected to increase dramatically as heterogeneous species

aggregates are revised and new species are subsequently described. By the end of the last century nearly

half of these species had been examined for essential oil character (Baker & Smith, 1912; Dellar, Cole, Gray,

Gibbons, & Waterman, 1994; Lassak, 1980; Southwell & Tucker, 1993). In these previous studies it was

demonstrated that a significant number of species yielded essential oils dominated by 1,8-cineole and a

sesquiterpene alcohol or oxide that gives each chemotype its defining character. Examples of such

characteristic sesquiterpenes include the decahydronapthalenic prostantherol, maaliol or cis-

dihydroagarofuran and the decahydroazulenic kessane, ledol or globulol (Figure 1).


31

Figure 1. Major components in essential oils from species of Prostanthera

The greatest extent of intraspecific variability of essential oils occurs within the P. lasianthos, P. ovalifolia

and P. rotundifolia heterogeneous species aggregates. This variability correlates with morphological

features (I.RH. Telford, 2014 pers. comm.). In light of this, chemotaxonomy has therefore become a

complement to the ongoing taxonomic revision of these three species.

In general, Prostanthera species yield appreciable amounts of essential oil upon hydrodistillation (1-3%

w/w wet leaves). The species P. incisa, P. ovalifolia or P. rotundifolia are generally easily cultivated, and due

to the abundance of 1,8-cineole they could be used in antimicrobial applications. As far as we can tell, only

one previous study has examined antimicrobial activities of compounds from Prostanthera, and focused on

using three isolated sesquiterpenes against phytopathogens (Dellar et al., 1994). In this previous study, no

concentrations higher than 100 µg ml-1 were used, which is relatively low in the context of essential oils. At

these lower concentrations the sesquiterpene alcohol prostantherol inhibited just one of the

phytopathogens, Streptomyces scabies, with a mean inhibitory concentration of 60 µg ml-1.

One of the challenges faced in antimicrobial studies that employ lipophilic compounds such as essential

oils, is in the slowing of evaporation of active volatiles and the formation of a stable solutions or emulsions.

Currently the most widely employed method for emulsifying essential oils in antimicrobial assays is to

thicken the broth with a small amount of agar (Mann & Markham, 1998). It is also possible to emulsify

solutions using common formulations including a mineral oil and surfactants such as polysorbate 80 (Viyoch

et al., 2006). This has substantially enhanced the reproducibility of antimicrobial assays using essential oils,

however in using only agar, complete dispersion is not achieved. This is particularly evident with

sesquiterpene-rich essential oils that can sometimes form aggregates in the agar. In addition, antimicrobial

activities of essential oils in agar emulsions do not necessarily translate to the same activity in a lotion that

uses borax, bees wax or chitosan.


32

There is increasing interest in the use of both lipophilic polymers and cyclodextrins to encapsulate essential

oils to facilitate even dispersal and stability of emulsions. The advantage is that cyclodextrins can be used in

both the antimicrobial assays and the end product, whether it be a cream or feed additive (Karlsen, 2010).

Cyclodextrins are biocompatible, non-toxic and are able to form inclusion complexes that stabilize oil in

water emulsions (Mathapa & Paunov, 2013) and slow the rate of oil evaporation. There is increasing

interest in the use of cyclodextrins to encapsulate essential oils to disperse the oils, which will aid in the

uniformity of emulsions.

In the current study, several cultivated specimens of Prostanthera were sampled for essential oil analysis,

with subsequent antimicrobial studies against Gram-positive and Gram-negative human pathogens, and the

yeast Candida albicans. With the view of subsequent formulation work, essential oils were encapsulated in

α-cyclodextrin as a preliminary attempt to examine for either positive or negative effects on antimicrobial

activity.


Leaf Collection, Essential Oil Production and Characterization

Field specimens of selected Prostanthera specimens were collected and locations are indicated in Table 1.

Cultivated specimens were taken from local gardens with provenances also included in Table 1. Vouchers

from the wild and from garden specimens were lodged with the N.C.W Beadle Herbarium at the University

of New England, Armidale, NSW Australia, labelled numerically with the prefix NJSadgrove. For example,

NJSadgrove239 represents P. sp. aff. ovalifolia, which is cited numerically in the current study as 239.

Essential oils from wild and cultivated specimens were produced using hydrodistillation. Approximately 600

g of leaf was removed from the twig then chopped into 0.5 mm fragments and placed into a 5 L round

bottomed flask with 2.5 L of deionised distilled water (ddH20). Leaves were heated for 4 hr by a 6 L mantle

and the steam/oil mix was condensed and collected in a 500 ml separating funnel then separated from the

hydrosol. Oils were stored away from light at 4˚C until used.

Prior to GC-MS analysis oils were treated with anhydrous sodium sulphate (NaSO4) powder (0.5g in 10ml

oil) for more than 24 hr to remove hydrosol emulsions. Afterwards, oils were dissolved in dichloromethane

(DCM) at a ratio of 1:1000. Analyses were performed using an Agilent Technologies 7890A GC-System

coupled with an Agilent 5975C mass selective detector (insert MSD with triple-Axis detector). An

autosampler unit (Agilent Technologies 7693 – 100 positions) was used to perform the 1 μL injections.

Separation was accomplished with a HP-5MS Agilent column (30m Х 250μm Х 0.25μm). Operating

conditions were as follows: Injector: split ratio 25:1; Temperature: 250˚C; carrier gas: helium, 1.0 mL/min,

constant flow; column temperature, 60˚C (no hold), 5˚C per minute then @ 250 hold 15 minutes. MS was

acquired at -70 eV using a mass scan range of 30 – 400 m/z.

Primary identifications were performed by comparison of mass spectra with an electronic library database

(NIST08) and subsequently confirmed using comparison of temperature programmed retention indices

(IUPAC, 1997) with published values. Most discrepancies in identification were resolved by comparison of

mass spectra with a second and third library (Joulain & Koenig, 1989; Adams, 2007; NIST, 2011).

Quantification was achieved using GC-MS operating software to calculate area under the curve, using data

with a minimum peak area of 0.1%.

Secondary identifications were performed using NMR spectra, by 1H (300 MHz, CDCl3) and 13C NMR (75

MHz, CDCl3), and comparing to published values. Sesquiterpenes which had structures confirmed using

NMR spectra were ledol (Bombarda et al., 2001), globulol (Toyota, Tanaka, & Asakawa, 1999), cis-

dihydroagarofuran/kessane (Southwell & Tucker, 1993) and prostantherol (Dellar et al., 1994).


33

Antibacterial and Anti-Candida activity

Working stocks of all species were maintained on Nutrient Agar (NA) with the exception of Candida

albicans which required Yeast Extract Peptone Agar (YEPA). All growth media were purchased from Oxoid

(Thebarton, South Australia) and prepared as per the manufacturer’s instructions.

Minimum inhibitory concentrations (MIC) of the oils were determined using a micro-titre plate two-fold

broth dilution method (CLSI, 2009; Wiegand, Hilpert, & Hancock, 2008) with the following modifications. Oil

emulsions were prepared by vortexing a measured combination of oil and the appropriate broth with

0.15% w/w agar (Mann & Markham, 1998). Where encapsulation of α-cyclodextrin was used, oils were

mixed at a 1:1 molar ratio with the concentration of α-cyclodextrin not exceeding 50 mg ml-1 and the molar

weight of the oil averaged for the major components. α-Cyclodextrin and the essential oils were dispensed

directly into the sterile broth and irradiated with UV for two hours until encapsulation/complexation had

finished, indicated by a plateau in the development of a translucent emulsion. Most species were assayed

in Tryptone Soya Broth (TSB) containing 0.15% w/w agar, with the exception of C. albicans which required

YEP broth. Broth dilutions were executed using 96-well plates.

Inoculation was achieved by collecting colonies from fresh working stocks and dispensing into 0.9% w/v

NaCl and diluting to match a 0.5McFarland BaSO4 Turbidity Standard (McFarland, 1907) using a

spectrophotometer at 600 nm (or 530nm for C. albicans). To achieve a final inoculation concentration of 5 ×

105 the adjusted saline suspension was diluted into 40 volumes of the appropriate medium and 20µL was

used to inoculate 80µL of media bringing the total volume to 100µL and reducing the essential oil

concentration to the appropriate starting concentration. Following inoculation the 96-well plates were

sealed using parafilm and placed into an incubator at 37˚C for approximately 20 hr before dispensing 40µL

of sterile 0.2 mg/mL p-iodonitrotetrazolium dye and examining for colour changes, which indicated

organism growth. Positive inhibition controls included tetracycline for bacterial growth and nystatin for C.

albicans. Experiments were repeated three or more times and the results are reported as a range.

Free radical (DPPH) Scavenging activity

The method used to assess free radical scavenging activity was the 2,2-diphenyl-1-picrylhydrazyl (DPPH)

method described by Brand-Williams et al. (1995) with the following modifications. Assay volumes were

2mL and kept in 4mL cuvettes, which were sealed with parafilm to slow the evaporation of methanol. Assay

concentrations of DPPH were aimed at 12-15mg/L, however actual concentrations were later determined

using the calculations proposed by Szabo et al. (2007). Spectrophotometer readings were taken at 517nm

after 12 hr.

Results are taken as the quantity of essential oil required to quench 50% of DPPH molecules. Data are firstly

presented as μg/ml of essential oil or control, and secondly presented as a molar ratio. The number of

moles of essential oil was estimated by averaging the molecular weight of the essential oil components.

The molar ratio was produced by dividing number of moles of essential oil or control treatment required to

quench 50% of DPPH, by the number of moles of DPPH quenched. This figure represents the molar quantity

of essential oil or control required to quench 1 mol of DPPH.

Three replicates were prepared for each treatment. Statistical analyses were performed using linear

regression analysis of averaged replicates per treatment. Quantity of essential oil, or positive control,

required to quench 50% of dissolved DPPH was determined from a data range that produced r2 values

above 90% in linear regression analysis. Flanking data was trimmed to include only data with DPPH

concentrations within the approx. range of 80-20%.


34


Due to the inherent complexity in determination of Prostanthera to species level the affiliated taxonomic

classification of specimens included in the current study may be subsequently described. However at

present these species are not up to date. Thus, wherever possible the provenances of cultivated specimens

have been included in Table 1. However, where data related to provenance is lacking, voucher specimens

have been lodged according to collection number with the NE Herbarium, Armidale NSW 2351 Australia. In

conjunction with phytochemical data provided here, the specimens in the current study can be traced to

their most up to date taxon in subsequent investigation. As a general guide, figures depicting some of the

morphological features of the leaves have been provided to scale for select specimens of P. sp. aff.

ovalifolia (Figure 2) and P. sp. aff. rotundifolia (Figure 3) examined in the current study.

The chemical character of specimens chosen for this study demonstrated a high degree of variability in the

composition of the major sesquiterpene components (Table 2). This variability was evident between

species affiliates and also within these apparent species, which demonstrated some degree of correlation

with morphological variants. This corroborates the chemotaxonomic approach as a potential tool to

facilitate taxanomic revision of these heterogeneous species aggregates of Prostanthera.

Table 1. Collection numbers (Coll. no.) of cultivated specimens (except 321). The sp. aff. refers to species most affiliated with. Currently there are no concerns related to the delimitation of P. incisa.

Coll. no. Species Lat Long Location

NJSadgrove321 P. sp. aff. ovalifolia 28°29'09.6'' 153°21'16.9'' Hell’s Hole, Mt Jerusalum NP, Mullumbimby NSW

NJSadgrove393 P. sp. aff. ovalifolia - - Cultivated ex. Blue Mts (Katoomba NSW)

NJSadgrove394 P. sp. aff. rotundifolia - - Cultivated ex. Unknown.

NJSadgrove398 P. sp. aff. ovalifolia - - Cultivated ex. Brundah (near Cowra NSW)

NJSadgrove399 P. sp. aff lasianthos

- -

Cultivated ex. Edgar’s lookout (Wollomombi Falls

Armidale NSW)



NJSadgrove402 P. incisa - - Cultivated ex. Unknown.

NJSadgrove404 P. sp. aff. rotundifolia

- -

Cultivated ex. Unknown. (Resembles Piliga Type; P.

cotinifolia Benth)

NJSadgrove405 P. sp. aff. ovalifolia - - Cultivated ex. Ellenborough Falls, Comboyne NSW

Figure 2 - Morphological variability of P. sp. aff. ovalifolia specimens sampled in the current study. Numbers refer to collection no. of vouchers lodged at the herbarium.


35

Figure 3 - Morphological variability of P. sp. aff. rotundifolia specimens sampled in the current study. Numbers refer to collection no. of vouchers lodged at the herbarium.

The abundance of cis-dihydroagarofuran and kessane is limited to species currently assigned to P.

ovalifolia. However, Prostantherol yields from species currently assigned to P. ovalifolia and P. rotundifolia

but not the others. All specimens collected for the current study yielded appreciable amounts of 1,8-cineole

and a moderate amount of p-cymene.

The antimicrobial activity of the essential oils was generally higher if essential oils were dominated by

approximately equal concentrations of 1,8-cineole and sesquiterpene alcohols, particularly prostantherol.

Table 2. Chemical character of essential oils from species of Prostanthera

Species ‘sp. aff.’

P.

oval

P.

oval

P.

rotua

P.

oval

P.

lasi

P.

rotua

P.

rotua

P.

inci

P.

rotub

P.

ovalc

Voucher 321 393 394 398 399 400 401 402 404 405

Yield % g/g 0.5 1.1 1.0 3.1 0.3 1.3 1.1 1.7 1.3 1.1

Compound Name AI

Pub.

AI

α-Pinene 934 932 - 0.8 1.1 0.5 11.6 - - 1.6 12.2 -

Camphene 950 946 - - 0.7 6.7 0.6 - - 0.5 1.9 -

Sabinene 973 969 - 0.9 - 0.9 1.4 - - 1.7 0.5 -

β-Pinene 979 974 - 0.8 - 1.0 17.7 - - 1.4 1.3 -

α-Phellandrene 1005 1002 - 2.0 - 3.8 0.5 1.0 0.8 3.9 - -

p-Cymene 1025 1020 2.8 9.2 1.9 8.8 2.6 6.3 4.6 10.8 3.3 4.5

Limonene 1029 1024 - - - - 7.2 - 0.9 - 1.5 0.7

1,8-Cineole 1034 1031 13.7 36.1 64.8 15.6 32.6 58.5 49.7 21.4 23.9 7.9

Linalool 1096 1095 - - - 0.5 - - - 1.9 - -

Camphor 1149 1144 - - 5.1 - - - - - 0.8 -

Borneol 1169 1165 - - 4.2 - 0.6 - - 0.5 2.9 -

Terpinen-4-ol 1180 1174 - 0.5 - 0.5 2.0 - - 0.5 - -

α-Terpineol 1192 1186 - - - 0.9 2.0 - - 1.2 - -

Myrtenol 1199 1194 - - - 0.5 0.5 - - 0.5 - -

Myrtenal 1201 1195 - - - 0.5 0.6 - - 0.5 0.5 -

Linalool acetate 1252 1254 - - - 0.6 - - - 4.6 0.5 -

Bornyl acetate 1287 1288 - - 0.8 3.1 0.5 - - - 2.3 -

Isodihydro carveol acetate 1335 1326 - 2.2 - - - - - - - -

α-Terpineol acetate 1349 1346 - - - 0.5 - - - 3.4 - -

Unknown A 1352 - - - - - - 1.2 3.7 - - -

Aromandendrene 1443 1439 - - - - 0.7 - - 0.5 - -

Alloaromadendrene 1462 1458 - 0.5 - 2.2 0.5 - - 2.1 1.0 1.2

Rotundene 1465 1457 - - 0.8 - - - - - - -

4,5-di-epi-Aristolochene 1470 1471 - - - - - - - - 4.8 -

Eremophilene 1487 1486 - - - - - - - - 0.9 -

β-Selinene 1494 1489 - - - - 1.8 - - 1.2 - 0.6


36

Bicyclogermacrene 1504 1500 - 2.1 0.9 1.0 1.5 1.5 - 0.5 6.3 0.7

cis-Dihydroagarofuran 1525 1519 58.7 - - 2.6 - - - - 15.4 50.0

Kessane 1532 1529 17.2 - - 14.2 - - 0.9 7.5 - -

Unknown B 1583 - - 4.1 - - - - - - 4.7 2.3

Spathulenol 1587 1577 - - - 2.2 2.3 - 1.3 0.7 - -

Caryophyllene oxide 1590 1582 - - - - - - 3.3 - - -

Globulol 1590 1590 - - - 3.9 - - - 9.0 - -

Viridiflorol 1601 1592 - - - - - - - - - 1.3

Prostantherol 1602 - 1.3 37.4 1.8 - - 30.0 32.6 - 3.0 12.3

Ledol 1610 1602 5.5 0.6 0.8 11.5 5.0 1.5 1.2 13.7 - 18.0

Prostanthera species were P. incisa, P. ovalifolia, P. lasianthos and P. rotundifolia, a Large round leaves, b Deep,

c Medium incised leaves purple flowers small incised leaves

The antimicrobial activity of cis-dihydroagarofuran and kessane rich oils was generally lower when

compared to the other oils tested (Tables 2 and 4). It is unusual that oils were able to inhibit Pseudomonas

aeruginosa, as this is not common. The radical scavenging activity of a selection of these oils was very low

(Table 3).

All of the oils that were selected for encapsulation with α-cyclodextrin inhibited bacterial species at lower

concentrations relative to concentrations without encapsulation (Table 5). Concentrations depicted in Table

5 are in mg ml-1. Oils were encapsulated with α-cyclodextrin at a 1:1 molar ratio, not exceeding the

solubility of α-cyclodextrin, at approx. 50 mg ml-1. Encapsulation of most oils at this concentration resulted

in a white turbid emulsion. It has been hypothesised that a Pickering emulsion is formed which is stabilized

by a-cyclodextrin micrcrystal precipitation at the oil-water interface (Mathapa & Paunov, 2013).

Table 3. DPPH scavenging ability of Prostanthera essential oils in μg DPPH per one mg of essential oil or positive control. Positive controls in this experiment were ascorbic acid (Vit. C) and Trolox.

Species P. incisa P. sp. aff. ovalifolia P. sp. aff. lasianthos Vit C. Trolox Voucher 402 398 399

DPPH quenched (μg DPPH per mg-1 of essential oil) 3.5 3.8 0.8 2188 4484 Averaged molar ratio w/w (essential oil/DPPH) 683 618 2955 0.5 0.25

Table 4. The mean inhibitory concentrations from essential oils hydrodistilled from cultivated Prostanthera specimens, presented as % v/v of essential oil in agar. Results are presented as a range in some cases. > indicates inhibition not observed.

Species P. oval P. oval P. oval P. oval P. lasi P. inci P. rotua P. ovalb +Control

Voucher - 321 393 398 399 402 404 405 -

S. typhimurium 1 > > 1-1.5 1.5 1.5-3 1 2 0.25

B. subtilis 0.13-0.25 0.13-0.25 0.02-0.03 0.13-0.15 0.75 0.06 0.13-0.15 0.06-0.25 0.13-0.15

K. aurogenes - - - 0.06 0.375 0.3-0.5 - - 0.25-0.7

S. epidermidis 2 > 5 0.13-3 0.13-3 0.13-3 1 2 0.5-0.75

S. aureus 0.13-0.5 0.5-1 0.06-0.13 0.2-0.7 0.2-0.3 0.05-0.5 0.13-0.25 0.06-0.25 0.13-0.25

S. pneumoniae 0.03 - 0.03 0.17 - 0.13 0.03 0.03 0.06

P. aeruginosa 2 > 3 0.5-1.5 0.5 0.5-1.5 1.5 > 0.75-1

C. albicans 0.13 1-3.5 0.06 0.06-0.13 0.5 0.06-0.13 0.13 1 1

a Deep purple flowers small incised leaves, b Medium incised leaves, Bacterial species were Salmonella typhimurium, Bacillus subtilis, Klebsiella aurogenes, Staphylococcus epidermidis, S. aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa and Candida albicans, Prostanthera species were P. incisa, P. ovalifolia, P. lasianthos and P. rotundifolia. +Control – positive (+) control is tetracycline for bacterial species or nystatin for C. albicans in µg ml-1.


37

Table 5. The antimicrobial activity of selected essential oils in mg ml-1 comparing free essential oil (EO) and oil encapsulated with α-cyclodextrin (α-CD).

Species aff. P. oval P. oval P. rot P. rot *Nystatin, Tetracycline Voucher 321 393 401 405

Bacterial species EO α-CD EO α-CD EO α-CD EO α-CD +Control

S. typhimurium >8.3 5 8.3 2.5 8.3 2.5 >8.3 5 0.13

E. coli >8.3 1.3 8.3 5 8.3 2.5 >8.3 5 0.25

B. subtilis 8.3 0.6 0.5 0.2 0.5 0.2 1 0.6 0.25

S. epidermidis >8.3 5 8.3 2.5 8.3 2.5 8.3 5 0.06

S. aureus 2.1 0.6 0.3 0.2 0.3 0.2 1 0.2 0.06

C. albicans 8.3 2.5 2.1 1.3 2.1 1.3 2.1 1.3 1.3*

EO – essential oil, α-CD, α-cyclodextrin encapsulated, Bacterial species were Salmonella typhimurium, Escherichia coli, Bacillus subtilis, Staphylococcus epidermidis, S. aureus, and Candida albicans, Prostanthera species were P. ovalifolia and P. rotundifolia, Positive control - +Control.

In the study of Mathapa that used cyclodextrins to encapsulate n-alkanes or free fatty acids, cyclodextrin

microcrystals were characteristically rod-shaped, formed by threading cyclodextrins over the carbon chains

of the respective lipophilic compound. In the current study, essential oils were presumably formed into

such microcrystals, but their appearances were not examined by the authors. Partial inclusion of oils into

the small α-cyclodextrin core would create an amphiphilic complex around which crystal growth can occur.

Formation of the emulsion took up to 2 hours consistent with the time required for crystal growth.

In the current study the observed Pickering emulsion visibly redissolved after two or three serial dilutions,

which supports the previous proposal of α-cyclodextrin precipitation mechanism of stabilization. The

enhanced antimicrobial activity did not correlate with this visible emulsion, as inhibition concentrations

were measured lower than that required for an emulsion to be observed. The mechanism for the enhanced

antimicrobial activity relative to non-encapsulated oils is not yet clear, but it most likely relates to an

enhanced solubility of sesquiterpenes after formation of an inclusion complex. We did not observed any

change in antimicrobial activity by encapsulation of monoterpenoid essential oils from various chemotypes

of Eremophila longifolia using α- and γ-cyclodextrins (data not shown). The enhanced antimicrobial activity

of purified sesquiterpene alcohols encapsulated in cyclodextrins will be published elsewhere.

At present it is not clear if cyclodextrins are metabolised into a nutrient by bacteria, which could affect the

delivery of antimicrobial compounds to bacterial cell walls, however one study observed increase of growth

vigour of Helicobacter pylori in cultures supplemented with cyclodextrins (Marchini et al., 1995).

Furthermore, the inclusion complexes of synergistic/antagonistic essential oil components may affect their

biotransformation and availability. This may be of particular relevance to the interaction of 1,8-cineole and

the other sesquiterpene alcohols. The small size of 1,8-cineole, which allows inclusion into the cage-like

structures of the cyclodextrins, would counter its higher volatility and maintain its relative abundance

during antimicrobial assays and in topical antimicrobial applications.

ACKNOWLEDGMENT

The authors would like to acknowledge the collaboration of UNE botanist Ian R.H. Telford and John Nevin, Warren Sheather and Maria Hitchcock for access to their cultivated specimens.


38

REFERENCES

Baker, R. T., & Smith, H. G. (1912). On a new species of Prostanthera and its essential oil. Journal and Proceedings of the Royal Society of NSW, 46(1), 103-110.

Bombarda, I., Raharivelomanana, P., Ramanoelina, P. A. R., Faure, R., Bianchini, J.-P., & Gaydou, E. M. (2001). Spectrometric identifications of sesquiterpene alcohols from niaouli (Melaleuca quinquenervia) essential oil. Analytica Chimica Acta, 447, 113-123.

Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und- Technologie, 28, 25-30.

CLSI. (2009). Methods for dilution antimicrobial susceptibility testing for bacteria that grow aerobically; Approved Standard - Eight Edition. M07-A8, 29(2), 1-66.

Dellar, J. E., Cole, M. D., Gray, A. I., Gibbons, S., & Waterman, P. G. (1994). Antimicrobial sesquiterpenes from Postanthera aff. melissifolia and P. rotundifolia. Phytochemistry, 36(4), 957-960.

Karlsen, J. (2010). Chapter 17 - Encapsulation and other programmed release techniques for essential oils and volatile terpenes. In K. Hüsnü Can Baser & B. Gerhard. (Eds.), Handbook of Essential Oils: Science, Technology and Applications. Boca Raton: CRC Press - Taylor and Francis Group.

Lassak, E. V. (1980). New essential oils from the Australian flora (October 1980) - perfumes and flavours symphony of nature. Paper presented at the 8th International Congress of Essential Oils, Fedarom Grasse, France - Paper No. 120 pp. 409-415, Fedarom Grasse, France.

Mann, C. M., & Markham, J. L. (1998). A new method for determining the minimum inhibitory concentration of essential oils. Journal of Applied Microbiology, 84(4), 538-544.

Marchini, A., d'Apolito, M., Massari, P., Atzeni, M., Copass, M., & Olivieri, R. (1995). Cyclodextrins for growth of Helicobacter pylori and production of vacuolating cytotoxin. Archives of Microbiology, 164, 290-293.

Mathapa, B. G., & Paunov, V. N. (2013). Cyclodextrin stabilised emulsions and cyclodextrinosomes. Physical chemistry chemical physics, 15, 17903.

McFarland, J. (1907). Nephelometer: an instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. Journal of the American Medical Association, 14, 1176-1178.

Southwell, I. A., & Tucker, D. J. (1993). cis-Dihydroagarofuran from Prostanthera sp. aff. ovalifolia. Phytochemistry, 22(4), 857-862.

Szabo, M. B., Iditoiu, C., Chambre, D., & Lupea, A. X. (2007). Improved DPPH determination for antioxidant activity spectrophotometric assay. Chemical Papers, 61(3), 214-216.

Toyota, M., Tanaka, M., & Asakawa, Y. (1999). A revision of the 13C NMR spectral assignment of globulol. Spectroscopy, 14, 61-66.

Viyoch, J., Pisutthanan, N., Faikreua, A., Nupangta, K., Wangtorpol, K., & Ngokkuen, J. (2006). Evaluation of in vitro antimicrobial activity of Thai basil oils and their micro-emulsion formulas against Propionibacterium acnes. International Journal of Cosmetic Science, 28(2), 125-133.

Wiegand, I., Hilpert, K., & Hancock, R. E. W. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature Protocols, 3(2), 163-175.

Nat. Volatiles & Essent. Oils, 2015; 2(2): 39-44 Demirci et al.

39

RESEARCH ARTICLE

Characterization and Antimicrobial Evaluation of the Essential Oil

of Pinus pinea L. from Turkey¥

Fatih Demirci1*, Pınar Bayramiç 2, Gamze Göger 3, Betül Demirci 1, 3 and Kemal Hüsnü Can Başer 1,4,5

1Anadolu University, Faculty of Pharmacy, Department of Pharmacognosy, Eskişehir, Turkey 2Ministry of Health, Directorate General of Health Investments, Ankara 3Graduate School of Health Sciences, Anadolu University, 26470-Eskişehir, Turkey 4Badebio Biotechnology Ltd., Eskişehir, Turkey 5King Saud University, College of Pharmacy, Department of Botany and Microbiology, Riyadh, Saudi Arabia


Abstract

Pinus pinea L. is commonly known as Stone or Umbrella Pine, which is a member of the family Pinaceae and grows natively in the

northern Mediterranean and Aegean coastal regions; southern Europe, north Africa, Spain to Turkey. P. pinea is also cultivated for

its edible pine nuts, as ornamental trees and commonly planted in gardens and parks. Its essential oil is used for a variety of skin

complaints, wounds, sores, burns, in herbal steam baths and various inhalers. Air dried needles collected from Ortanca-Muğla were

subjected to water-distillation using a Clevenger-type system. The resulting essential oil was analysed by GC-FID and GC-MS,

simultaneously. Overall, thirty components were characterized. Limonene (54.6 %), α-pinene (4.0 %), myrcene (2.4 %) and α-

phellandrene (2.4 %) were characterized as major constituents. The essential oil was also screened against 8 different human

pathogenic microorganisms, where the minimal inhibitory concentrations (MIC) were determined using a microdilution method. The

oil showed the same inhibitory activity against Escherichia coli, Staphyloccocus aureus, Pseudomonas aeruginosa, Enterobacter

aerogenes, Proteus vulgaris and Salmonella thyphimurium (MIC>0.75 mg/ml). Its antifungal susceptibility against Candida parapsilosis

was relatively more than that of the pathogen Candida albicans with a MIC value of 0.375 mg/ml, when compared with the antifungal

standards.

Keywords Pinus pinea, GC-FID/GC-MS, limonene, antimicrobial

Introduction Pinus pinea L. a member of the Pinaceae family, is commonly known as Stone Pine or Umbrella Pine, which

grows natively in the northern Mediterranean and Aegean coastal regions; southern Europe, north Africa,

Spain to Turkey. P. pinea is also cultivated for its edible pine nuts, as ornamental trees and commonly planted

in gardens and parks. Its essential oil is used for a variety of skin complaints, wounds, sores, burns, in herbal

steam baths and various inhalers.

Phytosterols are triterpenes that are important structural components of plant membranes and they are also

signaling molecules. Sterol and aliphatic alcohol contents and compositions of seed samples for Pinus pinea

are described in literature. β-sitosterol was found as the most abundant (74%) phytosterol whereas

octacosanol and hexacosanol (41%) for aliphatic alcohol, which were described for Pinus pinea seed oils

(Nasri et al, 2007). Resin from P. pinea is a complex mixture of many organic compounds tapped by wounding

its bark. It is also used for timber and resin production and its wood is well known to be stable even at high

humidity.

¥This work was presented at 44th International Symposium on Essential Oils, Budapest, Hungary, 8-12 September 2013.


40

It can be used for construction purposes, furniture making and to a lesser extent for the pulp and paper

industry. Resin also contains turpentine which can be used as an antiseptic, as a remedy for kidney, bladder,

and respiratory problems and also for skin treatments (Nergiz and Dönmez, 2004; Arshad et al, 2010).

Except for these industrial uses, P. pinea is much appreciated for its seed production which is widely used in

food preparation and particularly in cake-pastry. They are commonly added to meat, salads and into bread,

since Pine nuts are a good source of nutrients. They contain vitamins, particularly B1, B2, C. E, K and also

minerals, especially potassium, calcium, iron, magnesium and phosphorus. It is reported that the seeds of P.

pinea show a composition of 5.6 % moisture, 31.1% protein, 47.4% fat, 10.7% carbohydrate and 4.3% ash

Pinus pinea has many fatty acids, such as linoleic acid is the major fatty acid followed by oleic, palmitic and

stearic acids (Nergiz and Dönmez, 2004; Nasri et al, 2005). Additionally, tocopherol and triacylglycerol

contents in P. pinea L. seeds have been reported (Nasri et al, 2009).

In our study, we aimed to evaluate chemical composition of Pinus pinea essential oil. Hence, needles were

distilled by a Clevenger-type apparatus and analysed by GC/GC-MS systems, simultaneously. Limonene (55.0

%) was found the main compound. In vitro antimicrobial activity of the essential oil against 8 different human

pathogenic microorganisms was determined.


Plant Material

Needles (leaves) were collected from Ortanca-Muğla in May of 2009.

Isolation of the Essential Oil

The air dried needles were water distilled for 3 h using a Clevenger-type apparatus. Chemical composition of

the essential oil is shown in Table 1.

GC–MS Analysis

The GC-MS analysis was carried out using an Agilent 5975 GC-MSD system. Innowax FSC column (60 m x 0.25

mm, 0.25 m film thickness) was used with helium as carrier gas (0.8 ml/min). GC oven temperature was

kept at 60°C for 10 min and programmed to 220C at a rate of 4C/min, and kept constant at 220C for 10

min and then programmed to 240°C at a rate of 1°C/min. Split ratio was adjusted at 40:1. The injector

temperature was set at 250C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450. n-

Alkanes were used as reference points in the calculation of the relative retention indices (RRI).

GC-FID Analysis

The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300C. To

obtain the same elution order with GC-MS, simultaneous auto-injection was done on a duplicate of the same

column applying the same operational conditions. Relative percentage amounts (%) of the separated

compounds were calculated from FID chromatograms. The result of the analysis is shown in Table 1.

Identification of Components

Identification of the essential oil components was carried out by comparison of their relative retention times

with those of authentic samples or by comparison of their relative retention indices (RRI) to series of n-

alkanes. Computer matching against commercial (McLafferty and Stauffer, 1989; Koenig et al, 2004) and in-

house “Başer Library of Essential Oil Constituents” built up by genuine compounds and components of known

oils was performed. Additionally, MS literature data (Joulain and Koenig, 1998; ESO 1999) was also used for

the identification of components.


41

Antimicrobial Activity

The essential oil was examined against a panel of 6 different human pathogenic bacterial and 2 Candida

standard strains using the micro-dilution method versus standard antimicrobial agents. In this study

Escherichia coli NRRL B-3008, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 27853,

Proteus vulgaris NRRL B-123, Enterobacter aerogenes NRLL 356, Salmonella typhimurium ATCC 13311,

Candida albicans NRRL Y-12983 and Candida parapsilosis NRRLY 12696 acquired from various culture

collections were used for antimicrobial activity evaluations. The microorganisms were stored at -85°C in

glycerol until inoculation and purity check. Stock solution of the essential oil and the antimicrobial standard

agent were prepared in 25 of % dimethyl sulfoxide (DMSO). The diluted essential oil (200 µL) was added to

wells of row A, while the remaining wells in rows B to H received 100 µl of MHB. Microbial suspensions were

grown overnight in double strength Mueller-Hinton broth (MHB, Merck) standardized to 108cfu /mL for

bacteria and 106cfu/mL for Candida species (corresponding to McFarland no: 0.5) using a turbidometer

(Bioland, Turkey). Each microbial suspension was added to the appropriate well. After incubation at 37°C for

24h the first well without turbidity was determined as the minimal inhibition concentration (MIC, mg/mL).

Antimicrobial standard chloramphenicol and ketoconazole (Sigma–Aldrich) were used for this assay.

Antimicrobial activity results were shown in Table 2.


Water distilled essential oil from the air-dried needles of Pinus pinea L. from Ortanca-Muğla was analysed

both by GC and GC-MS systems, simultaneously. Overall, thirty one components were characterized, where

limonene (54.6 %), β-phellandrene (7.4 %), α-pinene (4.0 %), β-caryophyllene (4.0 %) myrcene (2.4 %) and α-

phellandrene (2.4 %) were identified as major constituents. The essential oil was also screened against 8

different human pathogenic microorganisms, where the minimal inhibitory concentrations were determined

using a microdilution method. The oil showed the same inhibitory activity against Escherichia coli,

Staphyloccocus aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, Proteus vulgaris and Salmonella

thyphimurium (MIC >0.75 mg/ml). Its antifungal susceptibility against Candida parapsilosis was relatively

more than that of the pathogen Candida albicans with a MIC value of 0.375 mg/mL, when compared with

the antifungal standards.

Table 1. Chemical composition of Pinus pinea L. essential oil

RRI Compounds %

1032 α-pinene 4.0

1118 β-pinene 1.7

1132 Sabinene 0.1

1174 Myrcene 2.4

1176 α-Phellandrene 2.4

1203 Limonene 55.0

1210 β-Phellandrene 7.4

1266 (E)-β-ocimene 1.7

1280 p-Cymene 0.2

1290 Terpinolene 0.4

1479

1482

δ-Elemene

longipinene

0.5

1553 Linalool 0.4

1583 Longifolene 1.0

1590 Bornyl acetate 0.3

1612 β-Caryophyllene 4.0


42

RRI: Relative retention indices calculated against n-alkanes, %: percentages were calculated from FID data, tr Trace (< 0.1 %)

According to previous research reports, the chemical composition of Tunisian Pinus pinea essential oils

showed limonene (54.1 %), α-pinene (7.7 %), and β-pinene (3.4 %) as major constituents. Additionally

antifungal activity evaluations on the oil of P. pinea showed that it significantly inhibited the growth of ten

plant pathogenic fungi (Amri et al, 2012). In another previous study, main constituent of the essential oils

from needles, branches and female cones of P. pinea was found as limonene (59.8 %, 62.5 % and 61.6 %,

respectively) in agreement with our findings (Macchioni et al, 2003).

Table 2. Minimum inhibitory concentrations (MICs/mg/ml) obtained from essential of Pinus pinea against 8 different microorganisms

In another report on chemical composition of the essential oils isolated from the needles of Pinus halepensis,

P. canariensis, P. pinaster, P. pinea and P. brutia from Morocco, the most abundant compound in P. pinea oil

was found as α-pinene (37.0 %). Furthermore, the oils as well as the major constituents α-pinene, myrcene

and β-caryophyllene were tested for their inhibitory effect against 21 bacterial strains. Examination of the

antibacterial activity revealed that only P. pinaster and P. pinea oils exhibited a definite activity against all

the organisms tested (Hmamouchi et al, 2001).

Table 1. cont.

RRI Compounds %

1617 6,9-Guaiadiene 0.6

1637 p-Menth-1-en-9-ol 0.5

1596 α-Guaiene 0.9

1687 α-Humulene 0.9

1704 γ-Muurolene 0.2

1706

1707

α-Terpineol

δ- Selinene 1.6

1726 Germacrene D 2.2

1868 (E)-Geranyl acetone 0.7

2103 Guaiol 2.1

2183 Selina-6-en-4-ol 0.7

2185 γ-Eudesmol 0.3

2503 Dodecanoic asit (lauric acid) 2.3

2622 Phytol 1.0

2705 Tetradecanoic acid (myristic acid) 0.7

2931 Hexadecanoic acid (palmitic acid) 1.9

Total 98.1

Microorganism Strains P. pinea essential oil Chloramphenicol Ketoconazole

E. coli NRRL B-3008 >0.75 0.0312 -

S. aureus ATCC 6538 >0.75 0.0156 -

P. aeruginosa ATCC 27853 >0.75 0.5 -

E. aerogenes NRLL 3567 >0.75 0.0312 -

P. vulgaris NRRL B-123 >0.75 0.0078 -

S. typhimurium ATCC 13311 >0.75 0.0039 -

C. albicans NRRL Y-12983 0.375 - 0.125

C. parapsilosis NRRLY 12696 0.75 - 0.125


43

The essential oil of P. pinea was investigated against Citrus pathogens; Botrytis cinerea, Penicillium digitatum

and Geotrichum citri-aurantii and Phytophthora citrophthora according to another previous report (Bouchra

et al, 2003). The acetone, ethyl acetate, and ethanol extracts and essential oils of the twigs and needles of P.

pinea were examined for their inhibitory effects against acetylcholinesterase (AChE), butyrylcholinesterase

(BChE) and antioxidant activity (Üstün et al, 2012).

Another study on the essential oils from the cones and needles of five different Pinus species (P. brutia Ten.,

P. halepensis Mill., P. nigra Arn., P. pinea L. and P. sylvestris (L.) from Turkey evaluated the in vivo wound

healing and anti-inflammatory activities. The essential oils obtained from the cones of Pinus pinea and Pinus

halepensis demonstrated the highest effects on the wound healing activity models (Süntar et al, 2012).

As a conclusion, the essential oil from Pine sp. and in particular P. pinea from Turkey may constitute an

important resource as antimicrobial agents.

ACKNOWLEDGMENT

This work was part of the graduation project 1306S240of Pınar Bayramiç.

REFERENCES

Amri, I., Gargouri, S., Hamrouni, L., Hanana, M., Fezzani, T., Jamoussi, B. (2012). Chemical composition,

phytotoxic and antifungal activities of Pinus pinea essential oil. Journal of Pest Science, 85(2), 199-207.

Arshad, M.A., Khan, M.A., Muhammad, Z., Sarwat, J., Shahzia, S. (2010). Ethnopharmacological application of

medicinal plants to cure skin diseases and in folk cosmetics among the tribal communities of North-West

Frontier Province Pakistan. J Ethnopharmacol 128,322–335.

Bouchra, C., Mohamed, A., Mina, I.H, Hmamouchi, M. (2003). Antifungal activity of essential oils from several

medicinal plants against four postharvest citrus pathogens. Phytopathologia Mediterranea, 42(3), 251-256.

ESO. (1999). The Complete Database of Essential Oils, Boelens Aroma Chemical Information Service, The

Netherlands.

Hmamouchi, M., Hamamouchi, J., Zouhdi, M., Bessiere, J. (2001). Chemical and antimicrobial properties of

essential oils of five Moroccan Pinaceae. Journal of Essential Oil Research, 13(4), 298-302.

Joulain, D., W.A., Koening. (1998). The Atlas of Spectra Data of Sesquiterpene Hydrocarbons, EB-

Verlag,Hamburg.

Koenig, W.A, Joulain, D., Hochmuth, D.H. (2004). Terpenoids and Related Constituents of Essential Oils.

MassFinder 3. Hochmuth DH (ed). Convenient and Rapid Analysis of GCMS, Hamburg, Germany

Macchioni, F., Cioni, P.L., Flamini, G., Morelli, I., Maccioni, S., Ansaldi, M. (2003). Chemical composition of

essential oils from needles, branches and cones of Pinus pinea, P. halepensis, P. pinaster and P. nigra from

central ltaly. Flavour and Fragrance Journal, 18,139-143.

McLafferty, F.W, Stauffer, D.B. (1989).The Wiley/NBS Registry of Mass Spectral Data, J Wiley and Sons: New

York.

Nasri, N., Fady, B., Triki S. (2007). Quantification of Sterols and Aliphatic Alcohols in Mediterranean Stone

Pine (Pinus pinea L.) Populations. Journal of Agriculture and Food Chemistry, 55, 2251-2255.

Nasri, N., Khaldi, A.,Fady, B., Triki S. (2005). Fatty acids from seeds of Pinus pinea L.: Composition and

population profiling. Phytochemistry 66, 1729–1735.


44

Nasri, N., Tlili, N., Ammar, K.M., Khaldi, A.,Fady, B., Triki S. (2009). High tocopherol and triacylglycerol

contents in Pinus pinea L. seeds. International Journal of Food Sciences and Nutrition. 60 (S1),161-169.

Nergiz, C., Dönmez, I. (2004). Chemical composition and nutritive value of Pinus pinea L. seeds. Food

Chemistry. 86, 365–368.

Süntar, I., Tümen, I., Üstün, O., Keleş, H., Küpeli, A. E. (2012). Appraisal on the wound healing and anti-

inflammatory activities of the essential oils obtained from the cones and needles of Pinus species by in vivo

and in vitro experimental models. Journal of Ethnopharmacology, 139(2), 533-54

Üstün, O., Şenol, F.S, Kürkçüoğlu, M, Orhan, I.E, Kartal, M, Baser, K.H.C. (2012). Investigation on chemical

composition, anticholinesterase and antioxidant activities of extracts and essential oils of Turkish Pinus

species and pycnogenol. Industrial Crops and Products, 38, 115-23.

Nat. Volatiles & Essent. Oils, 2015; 2(2): 45-51 İşcan et al.

45

RESEARCH ARTICLE

Headspace Solid Phase Microextraction (HS-SPME) and Analysis of Geotrichum fragrans Volatiles¥

Gökalp İşcan1, 2*, Betül Demirci1, Fatih Demirci1, 3 and K. Hüsnü Can Başer4

1 Department of Pharmacognosy, Anadolu University, Faculty of Pharmacy, 26470, Eskişehir, Turkey. 2 Yunus Emre Vocational School, Anadolu University, Eskişehir, Turkey. 3 Faculty of Health Sciences, Anadolu University, Eskişehir, Turkey. 4 Department of Botany and Microbiology, King Saud University, College of Science, Riyadh, Saudi Arabia.


Abstract

Geotrichum fragrans (syn. Saprochaete suaveolens) can produce fruity aromas (pine-apple like) such as esters and alcohols, when

cultivated in glucose containing liquid media. Consumers’ preferences for the use of natural products especially in foods and

cosmetics have motivated the production of aroma chemicals by biotechnological means. In the present study, the volatile

compounds of the G. fragrans during the 9 days cultivation in liquid media were accumulated by Headspace- Solid Phase

Microextraction (HS-SPME) technique, and, analysed by gas chromatography/flame ionisation detector (GC/FID) and GC coupled to

mass spectrometry (GC/MS), simultaneously. The main volatiles of the G. fragrans culture were determined as ethyl tiglate (8.5-

74.6%), ethyl isovalerate (36.1-41.6%) and methyl isovalerate (0.1-10.9%) during the time course. Daily variation trends of the aroma

volatiles were determined, where ethyl acetate was postulated as an intermediate metabolite in this bioconversion pathway.

Keywords: Geotrichum fragrans, bioconversion, HS-SPME, GC-FID, GC/MS, aroma volatiles

Introduction

Although a trend towards the use of natural ingredients is increasing, most of the aroma compounds are still

produced by chemical synthesis. Consumers’ preferences and demands for natural substances have also

motivated the industry to produce aroma chemicals by biotechnological methods, which include enzymes,

plant cell cultures and fermentation process by using microorganisms (Berger, 2007; De Oliveira et al., 2013).

Today, approximately more than hundred bioflavours are on the market produced by enzymatic or microbial

bioprocesses. Vanillin, γ-decalactone, 2-phenylethanol and raspberry ketone are considered as high value

bioflavour compounds among others (Grondin et al., 2015).

Fungal fermentation is an important tool for the production of new natural flavour substances. It is well-

known that some fungi have remarkable metabolic enzymes and pathways, which are able to synthesize de

novo volatile compounds, which can be directly used in the aroma industry. Among them, Geotrichum is a

genus of yeast like fungi mainly found in soil, water and air. Especially, G. candidum and G. fragrans (also

known as Saprochaete suaveolens and Oidium suaveolens) produce fruity aromas (like pine-apple) such as

esters and alcohols derivatives when cultivated in glucose and l-valine containing media (Goldberg &

Williams, 1991; Neto, Pastore, & Macedo, 2006). Recent publications have outlined the importance and

utilisation of G. fragrans for the production of aroma compounds.

¥This work was presented at 17th BİHAT Symposium, İzmir, Turkey, 2007.


46

Additionally, investigations on alternative wastes as a media and the influence of amino acids, glucose and

fructose in the media for the feasible production of natural aroma compounds were of high interest (De

Oliveira, 2013; Grondin, 2015; Neto, Pastore,& Macedo, 2006; Pinotti, 2006; Midaini, 2006; Damasceno,

Cereda, Pastore, & Oliviera, 2003).

2-Phenylethanol and ethyl tiglate are aroma compounds widely used in food, cosmetics and fragrances

industries, which they can also be produced by fungal fermentation processes. Synthetic ethyl tiglate is used

as raspberry, strawberry, pineapple and rum flavouring additive for beverages, ice cream, candy and liquors.

Ethyl isovalerate is colourless and oily liquid and used as pineapple flavouring additives for beverages,

icecreams, candy, baked goods, chewing gum, gelatine desserts. Ethyl isovalerate is also used in perfumery

or due to its apple-like odour (Berger, 1995 and 2007 Winter, 2009).

In the present study, G. fragrans (NRRL Y-17571) was cultivated in liquid glucose and peptone medium for 9

days. During the fermentation process volatiles were concentrated from the head space of the liquid media

with polymer-coated SPME fiber which then injected into the heated injector of the GC/MS system. Daily

variations of major bioflavours such as ethyl tiglate, ethyl and methyl isovalerate were evaluated.


Microorganism

The strain NRRL Y-17571 of Geotrichum fragrans (syn. Saprochaete suaveolens) used in this study (Figure 1).

The fungi was stored at -85°C in sterile 15% glycerol solutions. Culture media were refreshed on Sabouraud

glucose agar plate (Merck) at 26°C and inoculated in liquid media (containing glucose, peptone, yeast extract,

Na2HPO4 and NaCl) and placed in an orbital shaker (New Brunswick Scientific, USA) operating at 200 rpm and

26°C for 10 days.

Figure 1. Geotrichum fragrans culture and the light microscope image (x400)

Headspace-Solid Phase Microextraction (HS-SPME)

2 x 50 mL liquid culture sample were transferred aseptically into 2 different flasks. Sampling and absorption

process were performed same conditions and times as daily. 100 µm PDMS (Polydimethylsiloxane) coated

SPME fibres inserted in to the head space of the culture flasks that covered with Parafilm for 15 min at 35-

40oC. After the collection process SPME fibres needle (Figure 2.) directly injected into the injector of the GC-

FID and GC/MS systems simultaneously.


47

Figure 2. Analysis of the volatiles by using HS-SPME/GCMS technique

Gas Chromatography and Gas Chromatography-Mass Spectrometry (GC/FID, GC/MS)

The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300C. In

order to obtain same elution order with GC/MS, simultaneous injection was done by using same column and

an appropriate operational condition. Relative percentage amounts of the separated compounds were

calculated from FID chromatograms. The results of analysis are shown in Table 1. The GC/MS analysis was

carried out with an Agilent 5975 GC-MSD system. Innowax FSC column (60m x 0.25mm, 0.25m film

thickness) was used with helium as carrier gas (0.8 ml/min). GC oven temperature was kept at 60C for 10

min and programmed to 220C at a rate of 4C/min, and kept constant at 220C for 10 min and then

programmed to 240C at a rate of 1C/min. The injector temperature was at 250C. MS were taken at 70 eV.

Mass range was from m/z 35 to 450.

Compound Identification

Identification of the volatile components were carried out by comparison of their relative retention times

with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes.

Computer matching against commercial (Wiley GC/MS Library, Adams Library, MassFinder 3 Library)

(McLafferty & Stauffer, 1989; Koenig, Joulain, & Hochmuth, 2004) and in-house “Başer Library of Essential

Oil Constituents” built up by genuine compounds and components of known oils, as well as MS literature

data (Joulain & König, 1998; Adams, 2001) was also used for the identification.


48


The volatile compounds of the G. fragrans were detected during 9 days culture period by using head space

GC/FID and GC/MS systems simultaneously (Table 1.). The main volatiles were ethyl tiglate (8.5-76.4%), ethyl

isovalerate (36.1-41.6%), methyl isovalerate (0.1-11%), 2-methylbutyl 3-methylbutyrate (0.4-10.2%) and

Ethyl 2-methyl-butyrate (1-7.9%) (Figure 3.).

Table 1. Relative percentages and daily variations of volatiles compounds of G. fragrans determined by GC/MS.

Compounds Olfactory note Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Ethyl acetate - 24,7 4,2 1,6 0,8 0,4 0,4 0,7 16,6 2,8

Ethanol - 4,6 8,8 5,2 2,2 1,3 0,2 0,2 - -

Ethyl propionate Pineapple like - - - 0,6 0,6 0,5 - - -

Ethyl isobutyrate Fruity, Citrus like 1,4 2,2 2,5 2,1 1,7 1,1 tr - -

Methyl isovalerate Fruity, estery, harsh 0,3 0,3 0,1 0,2 0,3 1,0 10,9 5,8 7,0

Ethyl butyrate Pineapple 1,5 1,0 1,0 0,4 tr - - - -

Ethyl 2-methyl-butyrate Green apple, raspberry 4,7 7,9 6,5 3,9 2,3 1,0 tr - -

Ethyl isovalerate Banana, apple 37,6 41,6 40,1 40,6 39,0 36,1 tr - -

2-methylpropan-1-ol - - 2,7 0,1 0,1 tr - - - -

Isobutyl 2-methylbutyrate Sweet fruity - 1,0 0,6 0,4 0,3 0,2 - - -

Methyl tiglate Ethereal - - - - - - 0,7 2,1 5,4

Isobutyl isovalerate Apple-strawberry 0,2 3,2 3,1 3,4 4,1 4,5 1,0 - -

1,4-cineole Herbal, minty, camphor 2,5 tr - - - - - - -

Isoamyl alcohol Banana 0,8 5,2 2,5 1,5 0,9 0,4 - - -

1,8-cineole Herbal, eucalyptus 2,4 0,4 0,1 0,1 tr tr - - -

Ethyl tiglate Sweet Fruity, caramel 8,5 12,8 23,2 31,0 33,4 34,5 72,3 66,9 74,6

2-methylbutyl 3-methylbutyrate

Herbal fruity, green apple

0,4 3,3 4,1 5,1 7,3 10,2 2,8 0,6 -

Phenyl ethyl alcohol Floral, Rose like - 0,1 0,7 0,5 0,5 0,9 0,2 tr -

Tr: < 0.1%; -: not detected

Ethyl tiglate production of the fungi was increased during nine days and reached maximum amount at 9th day

(76.4%). First sampling day showed that ethyl isovalerate (37.6) and ethyl acetate (24.7%) were the main

volatiles of the G. fragrans culture. Ethyl acetate and ethanol were decreased day by day. They seemed to

be intermediate metabolites (Figure 4). After the 6th day Isobutyl 2-methyl butyrate, ethyl isovalerate, ethyl

butyrate, 1,8-cineole and 2-methylpropan-1-ol were not detected in the head space of the liquid culture.


49

Figure 3. Volatile compounds produced by G. fragrans

Furthermore, in this present work sterilized molasses, a by-product of the sugar beet industrial refining

process, was also used as fungal liquid culture medium. According to our ongoing studies, molasses can be

utilized as a source of raw material for fermentative media for the production of valuable volatiles, which

showed a similar profile to our glucose-peptone liquid medium results.

G. fragrans is a special fungi strain which produces ester, alcohol and acid-like fruit aromas (Damasceno,

Cereda, Pastore, & Oliviera, 2003). Detected molecules from the culture head-space are well known

flavouring compounds using in food and cosmetic industries. Ethyl isovalerate and especially ethyl tiglate are

widely used in beverages, ice creams, baked goods and desserts as flavouring agents. They are also used as

perfuming agents and precursors for the synthesis of more valuable aroma chemicals.

The use of biotechnology and fungal fermentation processes for the production of natural flavouring

substances is an economic and easy way instead of the chemical synthesis or extraction and purification from

the plants.


50

Figure 4. Daily variations (%) of volatile compounds of G. fragrans

0

20

40

60

80

0 2 4 6 8 10

Ethyl tiglate %

0

10

20

30

0 2 4 6 8 10

Ethyl acetate %

0

10

20

30

40

50

0 2 4 6 8 10

Ethyl isovalerate %

0

5

10

15

0 2 4 6 8 10

2-methylbutyl 3-methylbutyrate %

0

5

10

15

0 2 4 6 8 10

Methyl isovalerate %

0

2

4

6

8

10

0 2 4 6 8 10

Ethyl 2-methyl butyrate %

0

1

2

3

0 2 4 6 8 10

Ethyl isobutyrate %

0

2

4

6

0 2 4 6 8 10

Isoamyl alcohol %

0

1

2

3

4

5

0 2 4 6 8 10

Isobutyl isovalerate %

0

2

4

6

0 2 4 6 8 10

Methyl tiglate %


51

REFERENCES

Adams, R.P. (2001). Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass

Spectroscopy, USA: Allured Publishing.

Berger, R.G. (1995). Aroma Biotechnology, Springer-Verlag Berlin Heidelberg.

Berger, R.G. (2007). Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability, Springer, Berlin.

Damasceno, S., Cereda, M.P., Pastore, G.M., & Oliviera, J.G. (2003). Production of volatile compounds by

Geotrichum fragrans using cassawa waste water as a substrate. Process Biochemistry, 39, 411-414.

Winter, R. (2009). A Consumer's Dictionary of Food Additives, 7th Edition: Descriptions in Plain English of More

Than 12,000 Ingredients both Harmful and Desirable Found in Foods: New York, Three Rivers Press.

De Oliveira, S. M. M., Damasceno Gomes S., Sene, L., Machado Coelho, S. R., Barana, A. C., Cereda, M.P.,

Christ, D., and Piechontcoski, J. (2013). Production of 2-phenylethanol by Geotrichum fragrans,

Saccharomyces cerevisiae and Kluyveromyces marxianus in cassava wastewater. Journal of Food Agriculture

& Environment, 11(2), 158-163.

Goldberg, I., & Williams, R. (1991). Biotechnology and Food Ingredients. USA: Van Nostrand-Reinhold.

Grondin, E., Sing, A. S. C., Caro, Y., de Billerbeck, G. M., François, J. M., and Petit, Thomas. (2015). Physiological

and biochemical characteristics of the ethyl tiglate production pathway in the yeast Saprochaete suaveolens.

Yeast, 32(1), 57-66.

Joulain, D., & König, W. A. (1998). The Atlas of spectra data of sesquiterpene hydrocarbons. Hamburg, E.B.-

Verlag,

Koenig, W. A., Joulain, D., & Hochmuth. D. H. (2004). Terpenoids and related constituents of essential oils,

MassFinder 3”, in Ed. Hochmuth D.H., Convenient and Rapid Analysis of GCMS, Hamburg, Germany.

McLafferty, F. W., & Stauffer, D. B. (1989). The Wiley/NBS registry of mass spectral data; New York, J. Wiley

and Sons.

Mdaini, M., Gargouri, M., Hammami, M., Monser, L., & Hamdi, M. (2006). Production of Natural Fruity Aroma

by Geotrichum candidum. Applied Biochemistry and Biotechnology, 128, 227-235.

Neto, R. S., Pastore, G. M. & Macedo, G.A. (2006). Biocatalysis and Biotransformation Producing γ-

Decalactone. Food Science c, 69(9), 677-680.

Pinotti, T., Carvalho, P. M. B., Garcia, K. M. G., Silva, T. R., Hagler, A. N., Leite, & S. G. F. (2006). Media

components and amino acid supplements influencing the production of fruity aroma by Geotrichum

candidum. Brazilian Journal of Microbiology, 37, 494-498.

Nat. Volatiles & Essent. Oils, 2015; 2(2): 52-56 Küçük et al.

52

RESEARCH ARTICLE

Chemical Characterisation of the Essential Oil of Hypericum

aviculariifolium Jaub. & Spach subsp. depilatum (Freyn &

Bornm.) Robson var. bourgaei (Boiss.) Robson from Turkey

Sevim Küçük1, Mine Kürkçüoğlu2, Yavuz Bülent Köse1 and Kemal Hüsnü Can Başer2, 3

1 Department of Pharmaceutical Botany, Anadolu University, 26470, Eskişehir, TURKEY 2 Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470, Eskişehir, TURKEY 3 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia


Abstract

The genus Hypericum L. is represented by 96 species, 47 taxa being endemic for Turkey. The study material of this present work

Hypericum aviculariifolium Jaub. & Spach subsp. depilatum (Freyn & Bornm.) Robson var. bourgaei (Boiss.) Robson (Clusiaceae) is

also endemic. The essential oil was obtained by hydrodistillation of the aerial parts collected from Antalya, Turkey.

Essential oil was analysed both by GC and GC/MS, simultaneously. Hexadecanoic acid (28.0 %), lauric acid (11.3%), myristic acid (9.7%)

and caryophyllene oxide (8.7 %) were found as the main constituents. To the best of our knowledge, this is the first study on the

essential oil chemistry of this plant.

Keywords: Essential oil, Hypericum, Clusiaceae

Introduction

The genus Hypericum L. is the type genus of Hypericaceae, now usually included as subfamily (Hypericoideae)

in Clusiaceae (Guttiferae) and comprises more than 450 species divided in 36 sections with worldwide

distribution in warm temperate, subtropical and mountainous tropical regions (Robson, 2001). The genus

Hypericum L. is represented by 96 species in Turkey, 47 taxa being endemic. H. aviculariifolium Jaub. & Spach

subsp. depilatum (Freyn & Bornm.) Robson var. bourgaei (Boiss.) Robson (Clusiaceae) is an endemic species

in Turkey (Güner, 2013; Davis 1967). Different parts of Hypericum species are used as appetizer, sedative,

antispasmodic, antidiarrheic, anthelmintic and diuretic in Anatolian folk medicine. H. perforatum is used as

a dye, in flavouring, in food, as a medicine in wound healing, ulcers, the common cold, diabetes mellitus and

as an astringent (Demirezer et al., 2007; Kaçar, 2005; Baytop, 1999; Tuzlacı 2006).

Phytochemical investigations on H. perforatum have shown that it contains flavonols (catechins),

naphthodianthrons (hypericin, pseudohypericin), xanthones, coumarins, glycosides, anthraquinones,

phloroglucinols (hyperforin, adhyperforin), flavonoids (rutin, hyperoside, quercitrin), flavonol glycosides,

lactones, pyrones, lipids, triterpenes, tannins, and essential oils. Hypericine and its derivatives have been

reported to be responsible for antidepressant activity (Nahrstedt, and Butterweck 1997; Lavie, 1995).

The pharmacological activities of Hypericum extracts namely, antidepressive and antiviral activities are

mainly attributed to their flavonoid, hypericin and phloroglucinol contents (Avato, 2005). H. aviculariifolium

Jaub. & Spach subsp. depilatum (Freyn & Bornm.) Robson var. depilatum is another member of Hypericum

genus from Turkish flora which grows wild in some dry stony or rocky and calcareous zones of Turkey (Davis,

1988). H. aviculariifolium subsp. depilatum var. depilatum was reported to have great pharmaceutical


53

potential, with its well-documented contents of hypericin, hyperforin and flavonoids (Cirak, Radusiene,

Janulis, & Ivanauskas, 2007).


Plant Sample

H. aviculariifolium subsp. depilatum var. bourgaei was collected from Aydın, Didim in Turkey on June 16,

2008. Voucher specimens are kept at the Herbarium of Anadolu University, Faculty of Pharmacy Turkey (ESSE

14682).

Isolation of the Essential Oils

Aerial parts of the plant were water distilled for 3h using a Clevenger-type apparatus to yield 1.3% oil on

moisture-free basis.

GC and GC/MS Conditions

The oils were analysed by capillary GC and GC/MS using an Agilent GC-MSD system.

GC/MS: The GC/MS analysis was carried out with an Agilent 5975 GC-MSD system. Innowax FSC column (60m

x 0.25mm, 0.25 m film thickness) was used with helium as carrier gas (0.8 mL/min.). GC oven temperature

was kept at 60 °C for 10 min and programmed to 220 oC at a rate of 4 °C/min, and kept constant at 220 °C for

10 min and then programmed to 240 °C at a rate of 1 °C/min. Split ratio was adjusted 40:1. The injector

temperature was at 250 °C. MS were taken at 70 eV. Mass range was from m/z 35 to 450.

GC

The GC analysis was carried out using an Agilent 6890N GC system. In order to obtain the same elution order

with GC/MS, simultaneous injection was done by using the same column and appropriate operational

conditions. FID temperature was 300 oC.

Identification of Compounds

The components of essential oils were identified by comparison of their mass spectra with those in the Baser

Library of Essential Oil Constituents, Wiley GC/MS Library, Adams Library, MassFinder Library and confirmed

by comparison of their retention indices. Alkanes were used as reference points in the calculation of relative

retention indices (RRI). Relative percentage amounts of the separated compounds were calculated from FID

chromatograms (ESO 2000, 1999; Jennings and Shibamoto, 1980; Joulain and Koenig, 1998; Koenig, Joulain

and Hochmuth, 2004; McLafferty and Stauffer, 1989). The results of analysis are shown in Table 1.


Essential oil was analysed by GC and GC/MS, simultaneously. 27 compounds were identified, representing

92.6 % of the total oil components detected. Hexadecanoic acid (28.0 %), lauric acid (11.3%), myristic acid

(9.7%) and caryophyllene oxide (8.7 %) were the main constituents. Fatty acids and their esters were the

constituents predominated.

In other studies, α-pinene (52.1%), germacrene D (8.5%) and β-pinene (3.6%) have been reported as main

constituents of H. aviculariifolium subsp. depilatum var. depilatum (Yuce & Bagci, 2012). In many studies with

other species belonging to Hypericum, different results have been reported.


54

Major compounds of the volatiles of H. cerastoides (Spach) Robson were reported as α-pinene (58%),

undecane (5%), and β-pinene (3%). The microdistillation of H. montbretii Spach resulted in the

characterization α-pinene (26%), β-pinene (19%), and undecane (5%) as major compounds (Erken et al.,

2001).

Major constituents of H. perforatum L. steam volatiles obtained by microdistillation were identified as α-

pinene (50%) and carvacrol (22%). α-Pinene was reported as the main component in several H. perforatum

essential oils (Erken et al., 2001; Cakir et al., 1997; Nogueira et al., 1999, Weyerstahl et al., 1995). While

other Hypericum species investigated were predominated by monoterpene hydrocarbons, it was interesting

to note the fatty acid dominating composition of the current species.

Table 1. The Composition of the Essential Oil of H. aviculariifolium subsp. depilatum var. bourgaei

RRI: Relative retention indices calculated against n-alkanes, %: percentages were calculated from FID data

RRI Compounds %

1032 α-Pinene 0.9

1048 2-Methyl-3-buten-2-ol 0.2

1065 2-Methyl decane 0.3

1100 Undecane 0.4

1690 Selina-4,11-diene 0.6

1704 γ-Muurolene 0.4

1740 Valencene 0.6

1973 Dodecanol 0.3

2008 Caryophyllene oxide 8.7

2037 Salvial-4(14)-en-1-one 0.7

2071 Humulene epoxide-II 0.4

2123 Salviadienol 1.0

2131 Hexahydrofarnesyl acetone 1.6

2144 Spathulenol 3.3

2243 Torilenol 1.0

2256 Cadalene 0.8

2296 Decanoic acid 1.1

2300 Tricosane 1.1

2324 Caryophylladienol II 0.9

2353 Caryophyllenol I 0.9

2369 Eudesma-4(15), 7-dien-1-ol 2.0

2392 Caryophyllenol II 1.9

2503 Dodecanoic acid 11.3

2696 Tetradecanoic acid 9.7

2700 Heptacosane 2.3

2900 Nonacosane 12.2

2931 Hexadecanoic acid 28.0

Total 92.6


55

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