NVEO 2015, Volume 2, Issue 4...NVEO 2015, Volume 2, Issue 4 CONTENTS 1. Influence of growing...

NVEO 2015, Volume 2, Issue 4

CONTENTS

1. Influence of growing location and variety on the essential oil content of Melissa officinalis L.

and Thymus vulgaris L. / Pages: 1-3

Éva NÉMETH-ZÁMBORINÉ, Zsuzsanna PLUHÁR, Dóra SZABÓ, Katarzyna SEIDLER-

LOSYKOWSKA, Krisztina SZABÓ

2. Volatile compounds of Peucedanum chryseum (Boiss. Et Heldr.) Chamberlain Fruits . / Pages:

4-10

Hale AĞALAR, Mine KÜRKÇÜOĞLU, Ahmet DURAN, Özlem ÇETİN, Kemal BAŞER

3. Cytotoxicity and Acetylcholinesterase inhibitory and PRAP activities of the essential oils of

selected Tanacetum L. species. / Pages: 11-16

Kaan POLATOĞLU, Hüseyin SERVİ, Yasemin YÜCEL YÜCEL, Ayşe NALBANTSOY

4. Essential oil composition of leaves of Pistacia lentiscus L. growing wild in Valencia (Spain) /

Pages: 17-26

Juan LLORENS-MOLINA, Sandra VACAS GONZÁLEZ, Josep Sabater MARTÍNEZ

5. Selected Essential Oils in Cosmetic Emulsions: Process Oriented Stability Studies and

Antimicrobial Activity / Pages: 27-39

Daria KACZMARCZYK, Daniel STRUB, Anna POLOWY, Kazimiera WILK, Stanisław LOCHYŃSKI

6. Erratum – “Essential Oils of Anatolian Apiaceae: A Profile” / Pages: 1-50

Kemal BAŞER

Nat. Volatiles & Essent. Oils, 2015; 2(4): 1-3 Németh-Zámboriné et al.

1

SHORT COMMUNICATION

Influence of growing location and variety on the essential oil content of Melissa officinalis L. and Thymus vulgaris L.

Éva Németh-Zámboriné1*, Zsuzsanna Pluhár1, Dóra Szabó1, Katarzyna Seidler-Losykowska2,

Krisztina Szabó1

1 Szent István University, Department of Medicinal and Aromatic Plants, Villányi Str. 35-43. H-1118 Budapest, Hungary 2 Institute of Natural Fibres and Medicinal Plants, ul. Wojska Polskiego 71B, PL- 60-630 Poznan, Poland

*Corresponding author. Email: [email protected]

Abstract

Five lemon balm and four thyme varieties were grown at two locations in open field plots. A significant, however opposite effect of

the habitat was detected on the essential oil content of both species. The accumulation of essential oil of lemon balm was superior

in Budapest compared to Poznan (means of 0.185 0nd 0.105%, respectively). In thyme, higher contents were produced in Poznan

than in Budapest (means of 3.542 and 2.782%, respecitively). The size of the response was not uniform among cultivars. More than

three-fold increase was measured in Melissa officinalis ’Lorelei’ and ’Soroksári’. In case of Thymus vulgaris, essential oil content of

’Varico 3’, showed largest difference (45%) between the two locations .

Keywords: lemon balm, thyme, habitat, cultivar, environment

Introduction

Lemon balm and thyme are important and popular MAP species widely utilized and processed all over the

world. In the practise, the production of these species is going on under diverse ecological conditions.

Besides, several selected cultivrs and accessions are already available, however, there are hardly any data on

their cultivation value. The goal of our investigation was studying the effect of growing location, as complex

environment on these species and their intraspecific varieties.

Materials and Methods

Experimental sites

The experiment was installed at two different growing locations: Budapest (4754’N, 1914’E) and Poznan

(5242’N, 1689’E). Distance in a straight line between these sites is 570 km. Main differences of weather

conditions were registered in rainfall (Sum in Poznan 165 mm and in Budapest 330 mm during the season).

The temperature was slightly lower in Poznan while among the soil characteristics the largest difference was

measured concerning the pH (Table 1.).

Table 1. Marginal temperature values and soil characteristics of the experimental locations

Location TEMPERATURE (C) SOIL

minimum

maximum

mean pH humus

%

NO3-N

(mg/kg)

P2O

5

(mg/kg)

K2O (mg/kg)

Ca % Mg (mg/kg)

Budapest 5.0 35.3 20.2 6.91 2.58 7.73 1260 189 0.51 32.1

Poznan 6.2 34.5 18.5 5.18 0.87 8.46 395 131 1.36 27.4


2

Plant material

Melissa officinalis L.: Varieties: ’Lorelei’, ’Lemona’, ’Soroksári’, ’Quedlinburger Niederliegende’, ’Gold Leaf’’

Thymus vulgaris L.: Varieties ’French Summer’, ’Sloneczko’, ’Standard Winter’, ’Varico 3’

Seeds were obtained from genebank collections and seedlings raised in greenhouse. Planting of 50-50

seedlings/genotype was carried out at the beginning of June 2014, to a spacing of 40x 25 cm. Sampling

happened at the beginning of September by cutting the vegetative shoots above the woodened part in three

bulk replications.

Isolation of essential oil

The herb was dried at room temperature, then cleaned and the leaf fraction was investigated. Fifty g of each

sample was hydrodistilled for three hours in a Clevenger-type apparatus. The essential oil content was

calculated as volume (mL) of essential oil per 100 g of dried weight (three hours, at 105°C).

Results and Discussion

The essential oil content of both species was in the range which is known to be characteristic for the species

(Atti-Santos, Pansera, Paroul, Atti-Serafini, & Moyana, 2004; Oniga, Vlase, Toiu, Benedec, & Duda, 2010;

Seidler-Lozykowska, Bocianowsky, & Król, 2013; Stahl-Biskup, & Sáez, 2003). However, a large intraspecific

variability could be established: there are up to seven fold differences among lemon balm varieties and up

to 2.3 fold ones among the thyme accessions (Tables 2 and 3.).

The responses of the two experimental species on the growing habitat concerning essential oil accumulation

level were opposite ones. For each lemon balm variety we got a better essential oil content at the plots in

Budapest. The increase is 76% as a mean and is significant (p


3

Standard W 2.459b 0.062 2.740c 0.041

MEAN 2.782 3.542

Different letters represent significant differences in the coloumns

In thyme, we detected a significant difference (p

Nat. Volatiles & Essent. Oils, 2015; 2(4): 4-10 Ağalar et al.

4

RESEARCH ARTICLE

Volatile compounds of Peucedanum chryseum (Boiss. et Heldr.)

Chamberlain Fruits

Hale Gamze Ağalar1*, Mine Kürkçüoğlu1, Ahmet Duran2, Özlem Çetin2, K. Hüsnü Can Başer1,3

1 Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University 26470 Eskisehir, Turkey 2 Department of Biology, Faculty of Education, Selçuk University 42090 Meram, Konya, Turkey 3 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia

*Corresponding author: [email protected]

Abstract

In this study, we report the isolation of volatile constituents of Peucedanum chryseum (Boiss. et Heldr.) Chamberlain fruits collected

from Honaz, Denizli in Turkey. The volatile constituents obtained from whole fruits by micro-hydrodistillation were analyzed by GC

and GC/MS, simultaneously. Thirty nine components of the whole fruits were characterized representing 99.4% of the sample. The

major components of the whole fruits were α-pinene (72.8%) and β-pinene (20.4%). Monoterpene hydrocarbons were observed as

the main constituents in the sample.

Keywords: Peucedanum chryseum; Gas Chromatography; Gas Chromatography/Mass Spectroscopy; pinene; monoterpene

Introduction

Peucedanum L. belongs to the family Apiaceae and comprises about 200 species, which occur largely in

Europe, Asia and Mediterranean region (Zargari, 1993). Davis, (1972) reported that the genus Peucedanum

was represented in Turkey by 16 taxa of which 5 were endemic. But recent studies have shown that

Peucedanum species in the Turkish flora are represented by 23 taxa of which 9 are endemic (Davis, 1972;

Duran, 2014). The genus Peucedanum is rich in aromatic plants, but taxonomically many of its species are

still unresolved. Because characterization of each Peucedanum species by anatomical and morphological

features is difficult, chemical analysis of essential oils has played an important role in identifying

chemotaxonomic markers (Skalicka-Wozniak, Los, Glowniak, & Malm, 2008; Sarkhail, 2014).

Fruits of several members of the family Apiaceae are used as carminatives as they are rich in essential oil and

have an antispasmodic effect. Many of these species are also important as spice or condiment (Heinrich,

Barnes, Gibbons, & Williamson, 2004; Tanker, Koyuncu, & Coşkun, 2007). Some Peucedanum species have

been used in local medicine to treat various conditions, including sore throat, coughs, colds, headaches,

asthma, angina, cramps, epilepsy, gastrointestinal disorders, rheumatism, gout, and cardiovascular

problems, and as a chemopreventive agent and antifebrile (Sarkhail, 2014). Because of extensive uses in folk

medicine, the phytochemical studies are still promising for these species.

Coumarins (Chen et al., 1996; Urbain, Marston, & Hostettman, 2005; Skalicka-Wozniak, Mroczek, Garrard, &

Glowniak, 2009; Yu et al., 2012) and essential oils (Fraternale, Giamperi, Ricci, & Manunta, 2000; Rustaiyan,

Komeilizadeh, Mojab, & Khazaie, 2001; Cisowski, Sawicka, Mardarowicz, Asztemborska, & Luczkieewicz,

2001; Masoudi, Akhgar, & Rustaiyan, 2004; Skalicka-Wozniak, Los, Glowniak, & Malm, 2009) are considered

to be the main constituents in nearly all Peucedanum plants and can be responsible for many of their

biological and pharmacological activities. According to the published data, P. chryseum was studied for its

fatty acid composition by Akpınar et al. (2012). But the volatile composition of P. chryseum has never been


5

studied before. Our results can be useful for the chemotaxonomic point of view of Peucedanum species

(Akpinar et al. 2012; Sarkhail, 2014).


Plant Material

The fruits of P. chryseum (2 g) were collected from Honaz, Denizli, Menteş Village in Turkey on 17th July 2008

(GPS data: 37 44 147 N, 29 20 026 E). The plant was identified by one of us (AD). Voucher specimens are kept

at the Herbarium of Selçuk University, Konya, Turkey (AD 8068).

Isolation of the volatile compounds

The whole fruits were subjected to micro-hydrodistillation. Micro-hydrodistillation method which is

applicable to very small amounts of plant material (


6

Identification of the volatile compounds

The components of the sample were identified by comparison of their mass spectra with those in the Baser

Library of Essential Oil Constituents, Adams Library (Adams, 2007), MassFinder Library (Hochmuth, 2008),

Wiley GC/MS Library (McLafferty & Stauffer, 1989) and confirmed by comparison of their retention indices.

These identifications were accomplished by comparison of retention times with authentic samples or by

comparison of their relative retention index (RRI) to a series of n-alkanes. Alkanes (C8-C21) were used as

reference points in the calculation of relative retention indices (RRI) (Curvers, Rijks, Cramers, Knauss, &

Larson, 1985). Relative percentage amounts of the separated compounds were calculated from FID

chromatograms.


Thirty eight components of the whole fruits were characterized representing 99.4% of the sample. The major

components were α-pinene (72.8%) and β-pinene (20.4%). The detailed information is given in Table 1. Table

1 summarizes the identified compounds with their retention indices and relative percentages of the total

volatile compounds. Monoterpene hydrocarbons were observed as the main constituents in the sample.

Literature research indicated that the essential oil composition of the genus Peucedanum is important to

reveal chemotaxonomic variations. In P. scoparium oil collected from Sanandaj to Divandareh, province of

Kordestan, 33 components, which represented about 99.3% of the total composition of the oil, were

identified. The oil of P. scoparium consisted mainly of 12 monoterpene hydrocarbons (86.8%), 11 oxygenated

monoterpenes (8.3%) and 10 sesquiterpenes (4.2%). α-Pinene (39.6%), β-pinene (23.9%) and β-phellandrene

(9.5%) were the major components (Masoudi, Akhgar, & Rustaiyan, 2004). An analysis of leaf and fruit

essential oils of P. austriacum from Serbia by GC and GC/MS resulted in identification of 141 different

components, representing 93.6% and 96.1% of the total oils. The most abundant class of compounds in fruit

essential oil was monoterpenoids (64.0%) and in the leaf essential oil sesquiterpenoids (82.0%). The major

contributors of fruit essential oil were β-phellandrene (45.2%) and α-pinene (10.1%). The major constituents

of leaf essential oil were caryophyllene oxide (23.1%), germacrene D (12.2%) and (E)-caryophyllene (10.2%)

(Jovanovic et al. 2013). The oils constituents from rhizomes, leaves and fruits of P. petiolare (DC.) Boiss.

collected from Tehran province have been studied, the major constituents of rhizome oil were found to be

β-bisabolene (31.3%), (E)-sesquilavandulol (20.5%), geranyl acetate (5.7%), citronellyl acetate (5.2%) and

sabinene (5.2%); the main components of leaf oil were sabinene (42.3%), α-pinene (42.6%) and limonene

(2.6%) and in the fruits, α-pinene (47.3%) and sabinene (45.9%) were the major volatile compounds (Mirza,

Navaei, & Dini, 2005). Rustaiyan et al. (2001) reported essential oil of the aerial parts of P. petiolare. Among

the ten components (97.6%) which he identified in this oil, sabinene (57.8%) and δ-3-carene (36.2%) were

found to be the major constituents (Rustaiyan et al. 2001). A very recent study reported the chemical

composition of P. longifolium Waldst. & Kit. essential oil and headspace (HS) volatiles obtained from the fresh

aerial parts collected from Serbia. The major contributors of the essential oil were found as β-elemene

(24.7%), (E)-β-ocimene (11.7%), cis-linalool oxide (furanoid) (5.7%), (Z)-β-ocimene (5.6%) and α-humulene

(5.2%). MS analysis gave the identification of thirty-one components, representing 98.7% of the total

volatiles. The main constituents were: (E)-β-ocimene (28.5%), (Z)-β-ocimene (11.9%), α-pinene (8.9%),

myrcene (8.5%), p-cymene (6.4%), limonene (6.3%), sabinene (6.0%) and α-phellandrene (5.6%) (Jovanovic,

Zlatkovic, Jovanovic, Petrovic & Stojanovic, 2015). Bartnik et al. (2015) recently isolated two compounds,

guaia-1(10),11-diene and guaia-9,11-diene, from the fruits of P. tauricum M. B. (Bartnik & Komsta, 2015). The

herbal parts of Peucedanum luxurians which is endemic to Armenia was found to be rich in trans-α-farnesene,


7

germacrene D, α-zingiberene and β-caryophyllene while the fruits were rich in α-phellandrene, γ-elemene,

β-curcumene (Widelski, Grzegorczyk, Malm, Chinou, & Glowniak, 2015).

Kapetanos et al. (2008) reported that the essential oil compositions of six Peucedanum species from Central

Balkans. In all Peucedanum species, the dominant component was (-)-α-pinene (4.0 – 38.7%). P. alsaticum L.,

P. austriacum (Jacq.) Koch, and P. oreoselinum (L.) Moench are characterized by the presence of (+)-α-pinene

(15.0, 8.0, and 15.2%, resp.). Only P. cervaria (L.) Cuss stands out from the other species of the genus

Peucedanum, with a domination of (+)-limonene (11.5%), p-cymene (7.7%), and α-phellandrene (3.5%).

According to their results, monoterpene hydrocarbons were identified as the main components for these

species (Kapetanosa, Kariotia, Bojovic, Marinc, Veljic, & Skaltsa, 2008).

Some Peucedanum species growing in Turkey were investigated for their essential oil compositions. Tepe et

al., (2011) revealed the esssential oil compositions of aerial parts from P. longifolium (Waldst. & Kit.) and P.

palimbioides (Boiss.) collected from Karabuk and Ankara, Turkey, respectively. The P. longifolium essential

oil was found to be rich in sesquiterpenoids. The major volatiles were 8-cedren-13-ol (33.7%), myristicin

(8.0%), germacrene-D (7.7%) and -3-carene (6.4%). The essential oil of P. palimbioides was found to be rich

in monoterpenes with major compounds followed by α-pinene (35. 5%), (E)-9-octadecenoic acid (23.6%) and

β-pinene (20.2%) (Tepe, Akpulat, & Sokmen, 2011).

Based on the literature search, monoterpene hydrocarbons are the main components of Peucedanum oils

occurring in many Peucedanum species, including P. officinale, P. schotti, P. scoparium, P. petiolare P.

alsaticum, Peucedanum austriacum, Peucedanum oreoselinum, Peucedanum longifolium and Peucedanum

cervaria, α-pinene is the major component of essential oils. However, some species are dominated by other

monoterpenes or sesquiterpenes (Skalicka-Wozniak, Los, Glowniak, & Malm, 2008; Tepe et al., 2011;

Sarkhail, 2014). According to our results, the fruit oils of P. chryseum obtained by micro-hydrodistillation are

rich in α-pinene and β-pinene.

Table 1. Composition of the volatile compounds of P. chryseum (Boiss. et Heldr.) Chamberlain

Retention

time RRI

Volatile

Compounds

A

%

Method of

identification

9.0 1032 α-Pinene 72.8 tR , MS

10.8 1076 Camphene 1.0 tR , MS

12.7 1118 β-Pinene 20.4 tR , MS

15.4 1174 Myrcene 0.6 tR , MS

16.2 1188 α-Terpinene 0.2 tR , MS

17.0 1203 Limonene 1.5 tR , MS

17.6 1218 β-Phellandrene 0.6 tR , MS

18.8 1246 (Z)-β-Ocimene 0.1 tR , MS

19.2 1255 γ-Terpinene 0.4 tR , MS

19.7 1266 (E)-β-Ocimene < 0.1 MS,

20.4 1280 p-Cymene 0.1 tR , MS

20.9 1290 Terpinolene 0.2 tR , MS

23.2 1348 6-Methyl-5-hepten-2-one < 0.1 MS,

24.6 1382 cis-Alloocimene 0.1 MS,

27.2 1452 p-Cymenene (dehydro-p-cymene) < 0.1 MS,

27.7 1466 α-Cubebene < 0.1 MS,


8

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31.8 1586 Pinocarvone < 0.1 MS,

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TOTAL 99.4


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11

RESEARCH ARTICLE

Cytotoxicity and Acetylcholinesterase inhibitory and PRAP activities of the essential oils of selected Tanacetum L. species.

Kaan Polatoğlu1*, Hüseyin Servi2, Yasemin Yücel Yücel3, Ayşe Nalbantsoy4

1 Department of Analytical Chemistry, Istanbul Kemerburgaz University, Faculty of Pharmacy, 34217, Istanbul, TURKEY 2 Department of Organic Chemistry, Yıldız Technical University, Graduate School of Science, 34210, Istanbul, TURKEY 3 Department of Biochemistry, Istanbul Kemerburgaz University, Faculty of Pharmacy, 34217, Istanbul, TURKEY 4 Department of Bioengineering, Ege University, Faculty of Engineering, 35100, Izmir, TURKEY


Abstract

Essential oils obtained from flowers of Tanacetum mucroniferum Hub. - Mor. & Grierson, leaves of Tanacetum densum (Labill.)

Heywood ssp. eginense Heywood, stems of Tanacetum densum (Labill.) Heywood ssp. sivasicum Hub. - Mor. & Grierson were

investigated for their cytotoxicity, AChE inhibitory and PRAP activities. We have previously investigated chemical composition of

these oils and we have also suspected that these oils also do have strong AChE inhibitory activities due to the insecticidal activities

which we have reported for the other members of this genus. Highest activities were observed for pure oils: T. mucronifeum,

T.densum ssp. sivasicum and T. densum ssp. eginense were 100.00±0.0%; 100.00±0.0% and 91.31±3.30% respectively (n=3). The

observed activities were concentration dependent. Compared with the positive control α-tocopherol (3.735±0.063 AU; n=3) in same

concentrations, all of the investigated oils showed low PRAP activity at the highest concentration (10 mg/mL). Highest activity was

observed for T. mucroniferum (1.319±0.017 AU; n=3) at 10 mg/mL concentration.

Keywords: In vitro cytotoxicity, AChE Inhibition, PRAP Activity, T. mucroniferum, T. densum ssp. sivasicum, T.densum ssp. eginense,

Introduction

Previously, we have investigated essential oil composition of Tanacetum densum (Labill.) Heywood ssp.

sivasicum Hub. - Mor. & Grierson, T. densum (Labill.) Heywood ssp. eginense Heywood and Tanacetum

mucroniferum Hub. - Mor. & Grierson, (Polatoglu et. al., 2009; 2012a; 2012b). The essential oils of T. densum

ssp. sivasicum contained 1,8 cineole (21.1% and 28.3%), camphor (19.2% and 16.4%) and borneol (5.8% and

6.4%) main components in flower and stem oils respectively (Polatoglu et. al., 2009). The essential oils of T.

densum ssp. eginense have camphor (30.9%, 25.7%, 27.7%), 1,8 cineole (12.4% flower oil), camphene (10.6%,

%7.0, flower and leaf oils), bornyl acetate, (9.4%, 11.8%, stem and leaf oils), α-pinene (7.0%, %5.3, flower

and leaf oils), borneol (5.1%, 5.2%, stem and leaf oils), neodihydrocarveol (5.1%, flower oil) as well as an

unidentified compound (11.5%, 27.2%, 20.5%) in flower, stem and leaf oils respectively (Polatoglu et. al.,

2012a). T. mucroniferum flower essential oil contained 1,8-cineole 21.9 % and camphor 6.4 % as the main

components (Polatoglu et. al., 2012b). The essential oils of T. densum were evaluated for its antimicrobial

activity against selected pathogens but no significant activity was observed. The oils of this species were also

evaluated for their general toxicity using Vibrio fischerii toxicity test. As a result, all of the oils inhibited the

growth of this organism which suggests potential cytotoxicity (Polatoglu et. al., 2012a). The flower oil of T.

mucroniferum was evaluated for its radical scavenging activity against DPPH radical but the oil produced low

scavenging activity when compared with the positive control at the same concentration (Polatoglu et. al.,

2012b). Pyrethrins are one of the most important commercially produced natural insecticides which are

produced from Tanacetum (Pyrethrum) cinerariifolium. The activity of pyrethrins manifests itself acting on


12

the nervous system of the insect species (Isman, 2006; Wandahwa et al., 1996). Unfortunately, beside T.

cinerariifolium there are very few reports on the insecticidal activity of other Tanacetum species especially

on the essential oils. Previously, insecticidal activity of T. vulgare on moths and mites (Chiasson et al., 2001;

Gabel and Thiery, 1994; Palsson et al., 2008) and insecticidal activity of T. abrotanifolium, T. macrophyllum

on stored product pest Sitophilus granarius (Polatoglu et. al., 2015a, 2015b) were reported. The concerns

related to food safety and enviromentally friendly agricultural methods prompted acception of certain

regulations in agricultural production. The European Pesticide Regulation (EC) No. 1107/2009 promotes the

use of less harmfull substances in pest management. The acceptance of this regulation by the European

Union made a huge impact on pesticide industry and provided attention to the natural substances that could

be used in pest management (Villaverde et al., 2014).

The purpose of this study is to evaluate the essential oils of T. mucroniferum, T.densum ssp. eginense and T.

densum ssp. sivasicum for their acetylcholinesterase (AChE) inhibition, cytotoxic activity and PRAP

(Phosphomolybdenum Reducing Antioxidant Power) activity. AChE inhibition of the monoterpenes were

previously correlated with their insecticidal activity (Lopez and Pascual-Villalobos, 2010). Therefore, with this

report we intend to provide initial information on their potential insecticidal activity as well as their safety.


Plant Materials

Plant materials were collected during their flowering period. T. mucroniferum was collected in July 2008 from

Erzincan-Sakaltutan, Turkey at 2333 m altitude, voucher specimens have been deposited at the Herbarium

of the Faculty of Science, Istanbul University (Voucher no. ISTE 85425 identified Prof. Dr. Neriman Özhatay).

T. densum ssp. sivasicum and T. densum ssp. eginense were collected in July 2002 from Böğrüdelik Village

(1800 m) and Mt. Tecer (1900 m) Sivas Turkey, voucher specimens have been deposited at the Herbarium of

the Faculty of Science, Istanbul University (Voucher no. ISTE 80539 and ISTE 80538, respectively, identified

by Prof. Dr. Kerim Alpınar).

Essential oil isolation

Plant materials (100 g each) were subjected to hydrodistillation for 4 h using a Clevenger- type apparatus to

produce the oils. Essential oil yields obtained from flowers, T. mucroniferum, leaves of T. densum ssp.

eginense, stems of T. densum ssp. sivasivum 0.1; 0.45; 0.1 (v/w) respectively. Oils were kept in amber bottles

in -20⁰C until the biological activity assays were conducted.

AChE inhibition assay

The inhibitory effect of the essential oils were determined with the previously described protocol (Ellman et

al., 1961). The assay solution contained 240 μL, 1.25 mM 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), 192 μL

acetylthiocholine iodide (AChI), 1200 μL, 100 mM Tris–HCl buffer pH 8.0 and 20 μL essential oil. The blank

solution contained 20 μL of buffer solution instead of the essential oil. Galanthamine hydrobromide (from

Lycoris sp.), was used as a positive control in the assay. The calibration curve obtained by testing AChE

inhibition of different concentrations of galanthamine was constructed the data points were fitted with

logarithmic function to obtain the calibration curve y = 24.968ln(x) + 32.003, that have R² = 0.9934. The

calibration curve is given in Figure 1. Reactions were started by adding 0.0325 U/mL of AChE (electric eel)

into the reaction mixture. The reaction was monitored for 2 min at 412 nm wavelength using a

spectrophotometer (Carry 60 single beam spectrophotometer, Agilent Technologies, USA). The enzymatic


13

activity was calculated as the percentage of the reaction rate in accordance with the activity obtained from

the blank. The data obtained from the linear section of the initial 60 s were used in the calculation of the

activities. The AChE inhibition was calculated by the subtraction of the ratio of the sample activity versus

blank activity from 100. The results of the experiments were given as mean ± standard deviation of three

parallel experiments. The results of the AChE inhibition tests are given in Table 1.

PRAP (phosphomolybdenum reducing antioxidant power) assay

The antioxidant activity of the essential oils were determined with a previous spectrophotometric protocol

(Falcioni et al., 2002). Stock solutions of 10% (w/v) phosphomolybdic acid, 1, 5, 10 mg/mL essential oils,

positive controls: α-topocherol and BHT were prepared. As a negative control, essential oil free methanol

was used. Phosphomolybdic acid solution (200 μL) and samples (200 μL) were mixed and then incubated at

80 °C for 30 min. After all of the samples and controls were cooled to room temperature, they were measured

for their absorbance at 600 nm (Agilent, Carry 60 UV-Vis. Spectrophotometer). Increased absorbance of the

reaction meant increased reducing power of the compounds when compared to blank control. The results of

the experiments were given as mean ± standard deviation of five parallel experiments. The results of the

PRAP tests are given in Table 2.

Cell lines and maintenance

HeLa (human cervix adenocarcinoma), A-549 (human alveolar adenocarcinoma), MCF-7 (human breast

adenocarcinoma), CaCo-2 (human colon colorectal adenocarcinoma), mPANC96 (human pancreas

adenocarcinoma), PC-3 (human prostate adenocarcinoma), U87MG (human glioblastoma-astrocytoma) and

as a normal cell line HEK293 (human embryonic kidney cells) were used for testing cytotoxicity. All cell lines

were purchased from ATCC. The cell lines were maintained in DMEM Ham’s F12 medium supplemented with

10% FBS, 1% L-glutamine, 1% gentamycine and 1 mM HEPES in a humidified atmosphere with 5% CO2, at

37°C. The cells were subcultured twice a week and cells in the exponential growth phase was used in the

experiments.

Cytotoxicity assay

Cytotoxicity of the oils were determined using a modified MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-

tetrazoliumbromide)] assay (Mosmann T., 1983), which detects the activity of mitochondrial reductase of

viable cells. The assay principle is based upon the cleavage of MTT that forms formazan crystals by cellular

succinate-dehydrogenases in viable Adding dimethyl sulfoxide to wells helps formazan crystals to be

resolved. For this purpose, all cell lines were cultivated for 24 h in 96 well microplates with an initial

concentration of 1x105 cells//well in a humidified atmosphere with 5% CO2, at 37°C. Then, the cultured cells

were treated with different dilutions of the extracts (0.5, 5, 50 μg/ml) followed by incubation for 48 h at 37

°C. Parthenolide was used as positive control. The optical density of the dissolved material was measured at

570 nm (reference filter, 620 nm) with UV-visible spectrophotometer (Thermo Multiskan Spectrum)

(Nalbantsoy et al., 2008).

Cytotoxicity was expressed as mean percentage increase relative to the unexposed control ± standard

deviation. Control values will be set at 0% cytotoxicity. Cytotoxicity data (where appropriate) will be fitted to

a sigmoidal curve and a four parameters logistic model used to calculate the IC50, which is the concentration

of extracts causing 50% inhibition in comparison to untreated controls. The mean IC50 is the concentration of

agent that reduces cell growth by 50% under the experimental conditions and is the average from at least

three independent measurements that will be reproducible and statistically significant. The IC50 values were


14

reported at ±95% confidence intervals (±95% CI). This analysis was performed with Graph Pad Prism (San

Diego, USA).


All of the investigated pure oils showed very high AChE inhibition. The highest inhibitions were observed for

T. densum ssp. sivasicum (100 ± 0.00) and T. mucroniferum (100 ± 0.00). However, when dilutions were

tested, these oils produced lower inhibition. T. densum ssp. sivasicum oil afforded higher activity at 10 mg/mL

application doses. The AChE inhibition of T. densum ssp. sivasicum oil at 10 mg/mL corresponds to activity of

0.529 μm galanthamine. The oil of T. densum ssp. eginense produced lower activity than the other oils. The

T. densum ssp. sivasicum oil that have produced high activity contains 1,8-cineole (28.3%), camphor (16.4%)

and borneol (6.4%) as major components (Polatoglu et. al., 2009). Similarly, T. mucroniferum oil have 1,8-

cineole (21.9%) and camphor (6.4%) as major component but have smaller amounts of borneol (1.7%)

(Polatoglu et. al., 2012b). The oil of T. densum ssp. eginense contains camphor (27.7%), an unknown

compound (20.5%), camphene (7.0%), α-pinene (5.3%) and borneol (5.2%) (Polatoglu et. al., 2012a). The

observed activity of the oils could be associated with 1,8 cineole and camphor which were reported to have

high AChE inhibition (Perry et. al., 2000). None of the oils showed any cytotoxic activity against the tested

cell lines. All of the oils produced very low PRAP activity, the highest activity was observed for T.

mucroniferum oil which produced 1.32 ± 0.02 AU at 10 mg/mL concentration. This activity is lower than the

activity of the positive control at 1 mg/mL (1.46 ± 0.33 AU). The oils did not show considerable cytotoxic

activity and PRAP activity, however they showed very high AChE inhibition. As suggested before, in most

cases the insecticidal activity is related with AChE inhibition (Lopez and Pascual-Villalobos, 2010); therefore,

we believe these oils do possess strong chance to have high insecticidal activity but low toxicity. The results

of this study also suggest essential oils of other members of genus Tanacetum have high potential for

insecticidal activity. This is the first report on AChE inhibitory, PRAP and cytotoxic activities of the essential

oils of T. densum ssp. sivasicum, T. densum ssp. eginense, T. mucroniferum.

Table 1. AChE Activity of investigated Tanacetum essential oils.

Concentration T. densum ssp. sivasicum

T.densum ssp. eginense

T. mucroniferum α-Tocopherolb Blankc

Pure Oila 100 ± 0.00b 91.31 ± 3.30 100 ± 0.00

10 mg/mL 16.09 ± 1.51 0.00 ± 0.00 13.81 ± 2.97 3.74 ± 0.06 0.15 ± 0.01

5 mg/mL 0.00 ± 0.00 0.00 ± 0.00 5.80 ± 1.98 3.64 ± 0.19

1 mg/mL 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 1.46 ± 0.33 aThe results were given as mean ± standard deviation of three parallel experiments. bPositive control. cMethanol was used as the negative control.

Table 2. PRAP Activity of investigated Tanacetum essential oils.

Concentration T. densum ssp. sivasicum

T.densum ssp. eginense

T. mucroniferum α-Tocopherolb Blankc

10 mg/mL 0.52 ± 0.00a 0.42 ± 0.15 1.32 ± 0.02 3.74 ± 0.06 0.15 ± 0.01

5 mg/mL 0.51 ± 0.24 0.22 ± 0.01 0.73 ± 0.07 3.64 ± 0.19

1 mg/mL 0.29 ± 0.04 0.13 ± 0.01 0.20 ± 0.04 1.46 ± 0.33 aThe results were given as mean ± standard deviation of three parallel experiments. bPositive control. cMethanol was used as the negative control.


15

Figure 1. The calibration Curve of AChE inhibition of Galanthamine.

ACKNOWLEDGMENT

This research was financially supported by the projects TOVAG 111O138 of TÜBİTAK and İstanbul Kemerburgaz University

-BAP.

REFERENCES

Chiasson, H., Belanger, A., Bostanian, N., Vincent, C., Poliquin, A., (2001). Acaricidal properties of Artemisia absinthium and Tanacetum vulgare (Asteraceae) essential oils obtained by three methods of extraction. Journal of Economic Entomology, 94(1), 167–171.

Ellman, G. L., Courtney, K. D., Andres Jr., V., Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7, 88–95.

Falcioni, G., Fedeli, D., Tiano, L., Calzuola, I., Mancinelli, L., Marsili, V., Gianfranceschi, G., 2002. Antioxidant activity of wheat sprouts extracts in vitro: inhibition of DNA oxidative damage. Journal of Food Science, 67, 2918–2922.

Gabel, B., Thiery, D. (1994). Nonhost plant odor (Tanacetum-vulgare, ASTERACEA) affects. The reproductive-behavior of Lobesia-botrana DEN-ET-SCHIFF (LEPIDOPTERA, TORTRICIDAE). Journal of Insect Behaviour, 7(2), 149–157.

Isman, M. B. (2006). Botanical insecticides deterrents, and repellents in modern agriculture and an increasingly regulated world. Annual Review of Entomology, 51, 45–66.

Lopez, M. D., Pascual-Villalobos, M. J. (2010). Mode of inhibition of acetylcholinesterase by monoterpenoids and implications for pest control. Industrial Crops and Products, 31, 284–288.

Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65, 55–63.

Nalbantsoy, A., Ayyıldız Tamis, D., Akgun, I. H., Ozturk Yalcın, T., Deliloğlu Gürhan, I., Karaboz, I. (2008). Antimicrobial and Cytotoxic Activities of Zingiber officinalis Extracts. Journal of Pharmaceutical Sciences, FABAD, 33, 76–85.

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

80,0

90,0

100,0

0 2 4 6 8 10

Per

cen

t A

Ch

E in

hib

itio

n

Galanthamine concentration (μM)


16

Palsson, K., Jaenson, T. G. T., Baeckstrom, P., Borg-Karlson, A. K. (2008). Thick repellent substances in the essential oil of Tanacetum vulgare. Journal of Medical Entomology, 45(1), 88–93.

Perry, N. S., Houghton, P. J., Theobald, A., Jenner, P., Perry, E. K. (2000). In-vitro Inhibition of Human Erythrocyte Acetylcholinesterase by Salvia lavandulaefolia Essential Oil and Constituent Terpenes. Journal of Pharmacy and Pharmacology, 52(7), 895-902.

Polatoglu, K., Başer, K. H. C., Gören, N., Demirci, B. (2009). The Essential Oil Composition of Tanacetum densum (Labill.) Heywood ssp. sivasicum Hub.-Mor. & Grierson from Turkey. Journal of Essential Oil Research, 21, 200-202.

Polatoglu, K., Demirci, B., Demirci, F., Gören, N., Başer, K. H. C. (2012a). Composition of Tanacetum densum (Labill.) Heywood ssp. eginense Heywood from Turkey. Records of Natural Products, 6(4), 402-406.

Polatoglu, K., Şen, A., Kandemir, A., Gören, N. (2012b). Essential oil composition and DPPH scavenging activity of endemic Tanacetum mucroniferum Hub. –Mor. & Grierson from Turkey. Journal of Essential Oil Bearing Plants, 15(1), 66-74.

Polatoğlu, K., Karakoç, Ö. C., Yücel, Y. Y., Demirci, B., Gören, N., Başer, K. H. C. (2015a). Composition, insecticidal activity and other biological activities of Tanacetum abrotanifolium Druce. essential oil. Industrial Crops and Products, 71, 7-14.

Polatoğlu, K., Karakoç, Ö. C., Demirci, B., Gören, N., & Can Başer, K. H. (2015b). Sitophilus granarius L.(Coleoptera) Toxicity and Biological Activities of the Essential Oils of Tanacetum macrophyllum (Waldst. & Kit.) Schultz Bip. Journal of oleo science, 64(8), 881-893.

Villaverde, J. J., Sevilla-Morán, B., Sandín-Espâna, P., López-Goti, C., Alonso-Prados, J. L. (2014). Biopesticides in the framework of the European Pesticide Regulation (EC) No. 1107/2009. Pest Management Science, 70, 2–5.

Wandahwa, P., Van Ranst, E., Van Damme, P., (1996). Pyrethrum (Chrysanthemum cinerariaefolium Vis.) cultivation in West Kenya: origin, ecological conditions and management. Industrial Crops and Products, 5, 307–322.

Nat. Volatiles & Essent. Oils, 2015; 2(4): 17-26 Llorens-Molina et al.

17

RESEARCH ARTICLE

Essential oil composition of leaves of Pistacia lentiscus L.

growing wild in Valencia (Spain)

Juan A. Llorens-Molina1*, Sandra Vacas González2 and Josep Sabater Martínez3

1 Mediterranean Agroforestry Institute, Universitat Politècnica de València, Camí de Vera s/n, 46022, Valencia SPAIN 2 Centre for Agricultural Chemical Ecology, (Mediterranean Agroforestry Institute), Universitat Politècnica de València. 3 School of Agricultural Engineering and Environment, Universitat Politècnica de València.


Abstract

Pistacia lentiscus L. grows wild in a wide range of habitats in Mediterranean countries. The essential oil (EO) composition of its

aerial parts has been extensively studied showing noticeable differences according to plant organ (leaves and twigs or fruits),

geographical origin and harvest time. In this study, EO composition of leaves of samples coming from two representative habitats

from Valencia (Spain) -siliceous (Segart) and calcicolous (Xeraco) soils-were determined by GC/MS and GC/FID. Monoterpene

hydrocarbons were found as the major fraction (57.5 %) in calcicolous soil, whereas sesquiterpene hydrocarbons were in siliceous

soil (39.9 %). Oxygenated monoterpenes reached a significantly higher amount in leaves from siliceous location (11.2 %) as against

the calcicolous one (6.7 %). The rate of oxygenated sesquiterpenes was also higher in siliceous soil (15.2 % in contrast to 1.6 % in

calcicolous one. These results lead to consider soil parameters as hypothetical source of EO composition variability being the target

for further researches.

Keywords: Pistacia lentiscus, essential oil, leaves, siliceous soil, calcicolous soil

Introduction

Pistacia lentiscus L is a termophilous evergreen shrub growing wild in a wide range of habitats throughout

the Mediterranean countries and Canary Islands. It is abundant in sunny scrublands of Eastern and

Southern Iberian Peninsula, sharing their habitats with species such as Chamaerops humilis L., Quercus

coccifera L., Rhamnus lycioides L., etc., being adapted to a varied range of substrates (Del Campo et al.,

2015). It has been used in folk medicine as antihypertensive, while its resin from stems (gum mastic) is used

for the treatment of digestive diseases (Vidrich et al., 2004). More recently, antimicrobial, antifungicidal,

antioxidant and other pharmacological activities have been reported (Nahida et al., 2012)

The essential oil (EO) composition of its aerial parts (leaves and twigs, fruits and galls) has been extensively

studied. Recent data from many studies carried out in different Mediterranean countries, detailing the

major components for each type of terpenic components, are displayed in Table 1. A wide range of

chemical variability affecting both major compounds and total amounts of terpene classes can be noted.

Generally, monoterpene hydrocarbons is the major fraction, mainly in ripe fruits (96.3 – 98.8 %) as reported

by Boelens & Jiménez, (1991) and Vidrich et al., (2004).


18

Table 1. Chemical composition of Pistacia lentiscus EO (leaves, twigs and fruits) from literature

References.

Plant material and harvest

stage

Sampling location Major compounds (> 5 %) by hydrodistillation (% peaks area) Monoterpenes Sesquiterpenes

No oxig. Oxig. No oxig. Oxig.

Boelens & Jiménez (1991), October (fruiting)

Spain (Sevilla) Leaves: -pinene (11.20 %), -myrcene (19.25 %), terpinen-4-ol (8.41 %), -terpineol (6.68 %), -caryophyllene (8.60 %), germacrene-D (6.35 %)

Unripe fruits: -pinene (21.65 %), -myrcene (54.46 %)

Ripe fruits: -pinene (10.50 %), -myrcene (72.25 %), limonene (6.70 %)

50.3 %

90.0 % 96.3 %

19.41 %

1.83 % 0.68 %

24.54 %

2.39 % 1.01 %

1.65 %

0.07 % 0.03 %

Fernández et al. (2000). Air-dried aerial parts. Fruiting

Spain (Jaén) -pinene (13.0 %), limonene, -phellandrene (5.4 %), -caryophyllene (6.9 %) 33.4 % 7.3 % 24.8 % 13.3 %

Zrira et al. (2003), Air dried aerial parts (leaves and twigs). Monthly sampling, december to june

Moroccoa

Mehdia Chaouen Oulmes

Terpinen-4-ol (4.8-7.5 %), limonene (6.7-8.1 %), -pinene (4.8-7.5 %), caryophyllene oxide (6.5-

10.3 %), sabinene (3.3-6.2 %), -terpinene (5.1-6.0 %)

Terpinen-4-ol (32.7-43.8 %), -pinene (7.1-13.5 %), bornyl acetate (6.8-10.3 %), -caryophyllene (4.3-5.2 %), sabinene (4.2-5.4 %)

-pinene (16.5-38.5 %), myrcene (10.2-11.5 %), limonene (6.8-9.8 %)

26.8-41.2 %

20.0-45.6 % 41.9-75.8 %

19.1-25.2 %

39.5-55.0 % 0.8-3.1 %

6.1-11.8 %

9.0-17.0 % 4.4-8.8 %

6.7-10.8 %

1.2-1.6 % 0.1-0.6 %

Vidrich et al.(2004) Seasonal sampling: Winter, spring, summer, autumn (fruiting) over 2 years

Italy (Tuscany)a Leaves: -pinene (16.1-25.3 %), germacrene-D (9.6-14.3 %), terpinen-4-ol (7.6-12.7 %), -

caryophyllene (5.2-8.7 %), -myrcene (4.6-8.3 %), -pinene (4.2-6.8 %), -cadinene (2.7-5.2 %), -phellandrene (2.4-5.0 %)

Fruits: -pinene (7.5-11.2 %), -myrcene (68.2-71.2 %), limonene (9.6-19.7 %)

56.9-64.4 %

97.8-98.8 %

7.8-12.9 %

0.2-1.3 %

21.0-29.1 %

0.7 %

- -

Kivçak et al. (2004) Twigs and leaves, separately. February

Çeşme-Ardiç (Turkey)

Twigs: Sabinene (23.2 %); -pinene (19.4 %); germacrene-D (14.1 %); limonene (6.9 %); -phellandrene (6.5 %); terpinen-4-ol (5.7 %) Leaves: terpinen-4-ol (29.2 %); p-cymene (7.1 %)

49.0 % 12.8 %

6,6 %

36.0 %

21.4 % 15.0 %

4.9 %

15.20 %

Ben Douissa et al. (2005), fresh leaves

Tunisia (Zaghouan) -pinene (16.8 %), 4-terpineol (11.9 %)-phellandrene (8.9 %), sabinene (5.7 %), -terpinene (5.5 %),

59.5 %

16.4 %

12.7 %

-

Tahar Dob et al. (2006). Air dried aerial parts

Algeria Algiers Tizi-Ozou Oran

Longifolene (12.8 %), -cadinene (6.2 %), (E)--terpineol (5.0 %)

Longifolene (16.4 %), (E)--terpineol (15.6 %), terpinen-4-ol (7.0 %), -muurolene (5.7 %)

pinene (19.0 %), (E)--terpineol (13.1 %), sabinene (12.6 %), -pinene (6.5 %), (E)--ocimene (5.5 %), longifolene (5.2 %)

13.6 % 13.5 %

56.3 %

20.0 % 3.6 %

23.1 %

35.5 % 29.9 %

10.9 %

14.9 % 5.1 %

3-0 %

Barra et al. (2007) Fresh aerial parts. Full flowering

Sardinia (Italy) Torre delle Stele Orroli Villaputzu

-pinene (20.40 %), -myrcene (18.29 %), p-cymene (14.79 %), terpinen-4-ol (14.17 %)

-pinene (19.15 %), -myrcene (19.36 %), terpinen-4-ol (15.07 %) p-cymene (9.92 %)

Terpinen-4-ol (19.74 %), -pinene (14.81 %), p-cymene (14.79 %), -myrcene (10.84 %), -

70.01 % 68.54 %

20.33 % 21.46 %

4.99 % 6.81 %

t t


19

Oristano Alghero

terpinene (6.56 %), -terpinene (5.12 %),

-pinene (21.55 %), terpinen-4-ol (19.84 %), p-cymene (14.79 %), phellandrene (10.84) %,

phellandrene (5.39 %),-terpinene (5.15 %)

Terpinen-4-ol (28.29 %), -pinene (22.59 %), sabinene (8.13 %), p-cymene (16.22 %)

57.24 %

67.42 % 58.88 %

24.2 %

23.73 % 34.31 %

12.87 %

6.30 % 3.26 %

2.75 % t

0.36 %

Gardeli et al. (2008) Air dried leaves. Before flowering, flowering, fruiting

Greece (Zakynthos)a

-pinene (9.4-24.9 %), myrcene (0.1-33.1 %), sabinene (1.0-6.7 %), -pinene (2.0-6.9 %), limonene (9.0-17.8 %), terpinen-4-ol (2.7-13.5 %), germacrene D (2.7-13.5%)

45.0-68.3%

13.3-23.1 %

9.2-28.1%

0.5-1.6 %

Lo Presti et al. (2008) Leaves and twigs, Vegetative stage

Italy (Sicily)

Leaves: germacrene-D (18.61 %), -caryiophyllene (14.68 %), -cadinene (11.13 %), epi--muurolol

(5.79 %), -cadinol (5.36 %)

Twigs: germacrene-D (16.77 %), sabinene (12.19 %), -phellandrene (7.56 %)

3.05 %

34.7 %

0.97 %

0.81 %

67.08 %

38.20 %

15.61 %

10.75 %

Mecherara-Idjeri et al. (2008) Leaves and fruits (air dried for 2-3 days). September-november

Algeriab

Leaves (17 locations) Fruits (9 locations)

Leaves: limonene (0.1-43.8 %), -pinene (0.5-34.2 %), myrcene (0.1-33.1 %), -pinene (0.2-21.8 %),

-phellandrene (tr-14.1 %), sabinene (0.1-11.7 %), p-cymene (0.1-7.7 %), camphene (tr-7.1 %),

terpinen-4-ol (0.2-7.0 %), germacrene D (2.7-13.5%), germacrene-D (0.0 – 10.3 %), -caryophyllene (0.7-2.4 %).

Fruits: myrcene (0.9-69.7 %), -pinene (9.4-51.5 %), limonene (0.8-24.1 %), sabinene (0.1-11.6 %),

-pinene (2.8-6.8 %), camphene (0.5-5.8 %)

2.1-76.1 % 70.4-94.0 %

0.7-12.5 % 1.1-12.2 %

6.5-45.4 % 1.7-9.4 %

1.8-25.9 % 0.4-4.7 %

Amhamdi et al. (2009). Leaves. February

Morocco (Tafoghalt)

-myrcene (39.2 %), limonene (10.3 %), -gurgujene (7.8 %)

57.7 %

2.6 %

26.1%

1.6 %

Djenane et al. (2011) Shopping precedence

France (biological cultive)

-myrcene (15.18 %), 1,8-cineol (15.02 %), terpinen-4-ol (6.41 %), -pinene (5.54 %), -pinene (5.10 %)

40,21 %

33,42 %

8,10 %

-

Ismail et al. (2012) Fresh leaves. October

Tunisia (Korbous)

pinene (20.6 %), limonene (15.3 %), -pinene (9.6 %), germacrene-D (8.4 %), terpinen-4-ol (8.2 %)

63.9 %

11.7 %

19.6 %

3.7 %

Hafsé et al. (2013) Dried leaves and twigs

Morocco (Taounate)

Leaves: myrcene (25.3 %), limonene (15.7 %), terpinen-4-ol (9.2 %)

Twigs: myrcene (34.1 %), limonene (9.6 %), -gurgujene (9.6 %), terpinen-4-ol (9.2 %)

57.6 % 60.0 %

16.8% 14.4 %

10.8% 26.4 %

1.3% 2.9 %

Haloui et al., (2015) Fresh leaves and twigs. May (flowering stage)

Morocco (Taounate)

Leaves: Tricyclene (7.71 %), terpinen-4-ol (7.44 %), sabinene (6.96 %), -caryophyllene (6.62 %), caryophyllene oxide (6.05 %), p-cymene (5.04 %)

Twigs: -pinene (19.24 %), tricyclene (8.16 %), (E)--ocimene (6.90 %), -caryophyllene (6.18 %), germacrene-D (5.17), 3-carene (5.18 %).

33.52 %

48.12 %

16.12 %

4.53 %

18.93 %

24.48 %

8.77 %

6.97 %

a Values range over sampling period is displayed b Values range from sampling location


20

The major part of studies displayed in Table 1 are focused on fresh or dried leaves and twigs. Monoterpene

hydrocarbons are considered as the predominant fraction in some studies (20.0 – 75.8 %) (Boelens &

Jiménez, 1991; Fernandez, Camacho, Fernández, Altarejos & Pérez, 2000; Zrira et al., 2003; Vidrich et al.,

2004; Ben Douissa et al., 2005; Dob, Dahmane & Chelghoum (2006); Barra, Coroneo, Dessi, Cabras &

Angioni (2007).; Gardeli et al., 2008; Amhamdi, Aouinti, Wathelet & Elbachiri, (2009); Djenane, Yangüela,

Montañés, Djerbal & Roncalés (2011); Ismail, Lamia, Mohsen & Bassem, (2012); Hafsé, Benbrahim, Saidi, &

Farah, (2013); Haloui et al., 2015). Nevertheless, strong variability can be observed according to the harvest

time (Zrira, Elamrani & Benjilali, (2003); Vidrich et al., 2004; Gardeli, Vassiliki, Athanasios, Kibouris &

Komaitis, (2008) and the place of sampling, even when it is carried out in close locations in the same

country (Zrira et al., 2003; Dob et al., 2006; Mecherara-Idjeri, Hassani, Castola & Casanova, 2008a). Indeed,

samples containing up to 82.69 % of sesquiterpene fraction have been reported (Lo Presti et al., 2008) and

oxygen-containing monoterpenes in leaves fluctuate between 0.97 % (Lo Presti et al., 2008) and 55.0 %

(Zrira et al., 2003). Small amounts of diterpenes (dimyrcene isomers) have also been reported by Boelens &

Jiménez (1991) (0.47 %, 0.53 %, 0.81 % in leaves, unripe and ripe fruits, respectively) and Lo Presti et al.

(2008) (0.16 % in twigs). This strong chemical variability agrees with the high degree of morphological and

physiological plasticity related to its environmental tolerance and therefore, its wide range of habitats in

which it grows (Nahum, Inbar, Ne’eman & Ben-Shlomo, (2008).

The aim of this work is to extend the knowledge of EO profiles of Pistacia lentiscus leaves coming from

Eastern Iberian Peninsula. Given that no references about type of soil are available in the above mentioned

literature, a first approach to this source of variability may be interesting. For this purpose, two different

sampling areas according to their geological features were selected. These ones, with similar climatic

conditions, are some of the most representative habitats concerning soil composition (calcicolous and

siliceous) in Valencia (Spain) natural environment.


Plant material

Three samples each one coming from ten individuals randomly selected were collected within a 10 ha land

(approximately) in two different locations distant from each other by 90 km, approximately. Both sampling

zones (Segart and Xeraco in Valencia, Spain) were Southeast-oriented and their climatic and geological

features are detailed in Table 2.

Table 2. Climatic and geological features of sampling areas

Location Altitude

(m a.s.l.)

Ave

rage

ye

arly

pre

cip

itat

ion

a

Ave

rage

ye

arly

tem

pe

ratu

rea

The

rmic

ity

Ind

exb

Coordinates

(centre of sampling zone)

Geological featuresc

Segart 300 m 469 mm 15,8 ºC 341 39º 40’ 57’’ N; 0º 22´38 ‘’ W Silicious

(argillites, sandstone)

Xeraco 250 m 485 mm 18,0 ºC 405 39º 01’ 18’’ N; 0º 15’ 26’’ W Calcicolous

(limestone lagoon,

limestone with dolomite

and orbitoline) aMeteorological data from the nearest climatic station, bThermicity index calculated as described by Martinez (1983) cSource: terrasit.gva.es/


21

The leaves and fruits were harvested all around the shrub. A voucher specimen was placed in the

Herbarium of Mediterranean Agroforestry Institute at the Universitat Politècnica de València (Spain) (VALA

9570-9571). After removing damaged and yellowish material, each whole sample was dried in a dry and

shady place at room temperature for 20 days just before essential oil extraction.

Essential oil extraction

After homogenizing each sample, 50 g were subjected to hydrodistillation for 2.5 h in a Clevenger type

apparatus (fruits and leaves were crushed with a domestic grinder immediately before extraction). The

essential oil was swept away with 2 mL of dichloromethane (DCM). After removing aqueous phase, the

essential oil extract was dried over anhydrous sodium sulphate. The solvent was removed by N2 stream. A

solution containing 10 L EO/1 mL of DCM was kept in the dark at -18ºC in sealed vials until analysis.

GC analysis

The analysis of samples was carried out by gas chromatography with flame ionization detector (GC-FID) in a

Clarus 500 GC (Perkin-Elmer Inc.) chromatograph equipped with a capillary column ZB-5 (30 m x 0.25 mm

i.d. x 0.25 mm film thickness; Phenomenex Inc.).The injection volume was 1 µL. The GC oven temperature

was programmed from 50ºC to 250°C at a rate of 3°C min-1. Helium was the carrier gas (1.2 mL min-1).

Detector temperature was set at 250ºC. The percentage composition of the essential oil was computed

from GC peak areas without correction factors by means of the software Total Chrom 6.2 (Perkin-Elmer

Inc.).

Analysis by GC-MS was carried out using a Clarus 500 GC-MS (Perkin-Elmer Inc.) equipped with the same

column mentioned above. Ionization source temperature was set at 200˚C and 70 eV electron impact mode

was employed. MS spectra were obtained by means of total ion scan (TIC) mode (mass range m/z 45-500

uma). The total ion chromatograms and mass spectra were processed with the software Turbomass 5.4

(Perkin-Elmer Inc.).

Retention indices were determined by injection of C8–C25 n-alkanes standard (Supelco®) under the same

GC conditions. The essential oil components were identified by comparison of their mass spectra with

those of computer library NIST MS Search 2.0 and available data in the literature. The identification of the

following compounds was confirmed by comparison of their experimental RI with those of authentic

reference standards (Sigma-Aldrich®): -pinene, -pinene, camphene, myrcene, camphor, terpinolene,

borneol, terpinen-4-ol, bornyl acetate and linalool.

Statistical analysis

The statistical analyses were carried out by means of one-way ANOVA using Statgraphics 5.1. Software. It

was applied to the main compounds, as well as to the terpene classes. As the raw data were expressed as %

peak areas, they were subjected to arcsin [square root (%/100)] transformation and previous

homocedasticity test and the means were compared by Fisher’s least significant difference (LSD).


Oil yields (calculated as oil V/wt. of air dried leaves) were 0.13 ± 0.03 % and 0.12 ± 0.02 % for samples from

Xeraco and Segart, respectively. The average of triplicate values (± standard deviation) of EO composition

are displayed in Table 3. 77 compounds, which represented 94.4-98.7 % were identified. Two main EO

profiles could be distinguished according the rate of monoterpene/sesquiterpene composition in leaf


22

samples. A predominance of monoterpenoid over sesquiterpenoid fraction was noted in samples collected

from Xeraco (calcicolous soil) (64.2 %-33.8 %) whereas the opposite tendency was found in Segart (siliceous

soil) (31.6 %-55.1 %). These different profiles agree with those referred in the literature. Indeed, data from

other Spanish locations (Boelens & Jiménez, 1991; Fernández et al., 2000), Morocco (Amhamdi et al., 2009;

Hafsé et al., 2013; Haloui et al., 2015) and Tunisia (Ismail et al., 2012) are quite similar to those that were

found in calcicolous location. On the contrary, profiles as those reported by Dob et al. (2006) in Algiers

(Algeria) and Lo Presti et al. (2008) in Sicily show a clear predominance of sesquiterpenes over

monoterpenes, being closer to siliceous location. It is worth to mention the data reported by Mecherara-

Idjeri et al. (2008) coming from Algeria, in which very wide range of variation concerning both terpene

classes and main components was found.

Significant differences were found within these fractions. Monoterpene hydrocarbons rate was higher for

calcicolous soil (57.5 %), as against 20.4 % in siliceous one. On the contrary, oxygenated monoterpenes and

sesquiterpenes were also significantly higher for siliceous soil (11.2 – 6.7 % and 15.2 - 1.6 %, respectively).

No significant differences were observed for sesquiterpene hydrocarbons fraction.

With regard to main compounds, EO from leaves showed significant differences concerning the main

monoterpene hydrocarbons. -pinene (19.0 %) and limonene (8.5 %), together with -3-carene, -pinene

and sabinene reach the higher levels in calcicolous soil. In the same way, samples rich in -pinene,

limonene and some of the aforementioned compounds are also reported by Fernández et al. (2000), Ben

Douissa et al. (2005), Dob et al. (2006), Barra et al. (2007) in samples from Alghero and Gardeli et al. (2008).

However, myrcene is the predominant monoterpene hydrocarbon in the siliceous location. This profile is

also reported by Boelens & Jiménez (1991), Amhamdi et al. (2009) and Djenane et al. (2011). Other data

from literature show more equilibrate rates between myrcene and the rest of monoterpene hydrocarbons

(Zrira et al., 2003, in Oulmes (Morocco) and Barra et al. 2007).

In agreement with previous studies, terpinen-4-ol is the main oxygenated monoterpene accounting for 5.1-

6.7 %, without significant difference between locations. It is worth mentioning that this compound reaches

high rates in P. lentiscus EO from several origins being reported as major component by Zrira et al. (2003) in

samples from Mehdia (Morocco), Kivçak et al. (2004) and Barra et al. (2007) –samples from Alghero-.

In respect of sesquiterpene components, germacrene-D and -caryophyllene are the major components as

referred to by the majority of previous researches. Nevertheless, noticeable amounts of other

sesquiterpene hydrocarbons such as longifolene (Dob et al., 2006), -cadinene (Lo Presti et al., 2008;

Mecherara-Idjeri et al. 2008, in two locations) or -gurjunene (Amhamdi et al., 2009) are also reported.

Within the sesquiterpene fraction, oxygenated sesquiterpenes are very significant higher in siliceous

location, which agrees with results from Turkey (Kivçak et al., 2004) and Sicily (Lo Presti et al., 2008).

Moreover, this agreement also concerns to major compounds as epi- -cadinol or epi--muurolol. It is

worth to mention the occurrence of diterpenic fraction in siliceous location (5.9 %). This type of compounds

are not reported in P. lentiscus aerial parts in the majority of previous studies except by Boelens & Jiménez

(1991) and Lo Presti et al. (2008).


23

Table 3. Constituents (% peak areas) of EO of Pistacia lentiscus leaves from each sampling location.

% peaks areaa

RI Identification Segart Xeraco

tricycleneb 925c MS, RId 0.1 ± 0.1 0.3 ± 0.1

α-thujene 929 MS, RI, St. tre 0.2 ± 0.1

α-pinene 937 MS, RI 2.4 ± 1.3a 19.0 ± 5.7b

camphene 953 MS, RI, St. 0.5 ± 0.4 1.5 ± 0.4

sabinene 975 MS, RI 0.9 ± 0.2a 6.1 ± 1.3b

β-pinene 979 MS, RI, St. 0.7 ± 0.5a 6.4 ± 1.1b

myrcene 996 MS, RI, St. 11.0 ± 8.3a 1.1 ± 0.0b

-3-carene 1010 MS, RI 1.4 ± 1.1a 7.2 ± 0.4b

α-terpinene 1019 MS, RI, St. 0.4 ± 0.7 1.7 ± 0.5

p-cymene 1027 MS, RI, St. 0.2 ± 0.4 1.1 ± 0.4

limonene 1032 MS, RI, St. 1.3 ± 1.6a 8.5 ± 0.4b

(Z)-β-ocimene 1040 MS, RI -f tr

(E)-β-ocimene 1052 MS, RI 0.2 ± 0.3 0.3 ± 0.0

γ-terpinene 1061 MS, RI, St. 1.0 ± 1.2 3.0 ± 0.7

terpinolene 1086 MS, RI, St. 0.4 ± 0.6 0.9 ± 0.2

p-cimenene 1090 MS, RI tr tr

monoterpenes 20.4 ± 16.6a 57.5 ± 11.3b

linalool 1097 MS, RI, St. tr -

menhta-2-en-1-ol 1125 MS, RI tr 0.1 ± 0.0

3-terpinen-1-ol 1134 MS, RI tr tr

menta-2-en-1-ol 1141 MS, RI - tr

camphor 1148 MS, RI, St. 0.1 ± 0.1 0.0 ± 0.0

(Z)-β-terpineol 1152 MS, RI tr -

borneol 1173 MS, RI, St. 0.1 ± 0.2 -

terpinen-4-ol 1181 MS, RI, St. 6.7 ± 1.6a 5.1 ± 0.5a

α-terpineol 1194 MS, RI 2.8 ± 0.6 1.0 ± 0.3

γ-terpineol 1199 MS, RI tr -

β-cyclocitral 1223 MS, RI 0.0 ± 0.1 -

bornyl acetate 1283 MS, RI, St. 1.4 ± 1.1 0.5 ± 0.1

verbenone 1204 MS, RI, St. tr -

(E)-piperitol 1205 MS, RI - tr

carvacrol 1298 MS, RI, St. tr tr

Oxygenated monoterpenes 11.2 ± 3.8a 6.7 ± 0.9b

δ-elemene 1338 MS, RI - 0.1 ± 0.0

α-copaene 1373 MS, RI - 0.3 ± 0.1

Ylangene 1375 MS, RI tr tr

β-elemene 1386 MS, RI 0.4 ± 0.1 -

β-bourbonene 1380 MS, RI - 0.8 ± 0.2


24

β-caryophyllene 1415 MS, RI 12.8 ± 2.6a 11.1 ± 2.1a

β-copaene 1432 MS, RI - tr

(Z)-muurola-3,5-diene 1450 MS, RI tr tr

α-humulene 1451 MS, RI 1.5 ± 0.2 1.3 ± 0.3

aromadendrene 1456 MS, RI - 0.2 ± 0.0

γ-gurgujene 1472 MS, RI - 0.3 ± 0.0

germacrene-D 1485 MS, RI 17.5 ± 5.4a 10.8 ± 3.1a

β-selinene 1488 MS, RI - -

bicyclogermacrene 1490 MS, RI 1.6 ± 0.5 0.9 ± 0.3

(E)-muurola-4(14),5-diene 1494 MS, RI 0.1 ± 0.1 0.4 ± 0.1

α-muurolene 1500 MS, RI 0.9 ± 0.2 0.9 ± 0.2

β-cadinene 1501 MS, RI - 0.1 ± 0.0

β-bisabolene 1504 MS, RI - 0.1 ± 0.1

(E,E)-α-farnesene 1506 MS, RI 0.4 ± 0.4 0.2 ± 0.1

γ-cadinene 1508 MS, RI 0.6 ± 0.2 0.5 ± 0.0

δ-cadinene 1514 MS, RI 4.0 ± 1.4 3.6 ± 0.8

(E)-cadina-1,4-diene 1528 MS, RI - 0.1 ± 0.0

zonarene 1530 MS, RI - 0.3 ± 0.1

(E)-γ-bisabolene 1531 MS, RI 0.1 ± 0.2 0.1 ± 0.1

α-cadinene 1533 MS, RI - 0.1 ± 0.0

Sesquiterpenes 39.9 ± 11.5a 32.2 ± 7.6a

elemol 1550 MS, RI tr 0.1 ± 0.1

(E)-nerolidol 1563 MS, RI - 0.1 ± 0.0

caryophyllene oxide 1576 MS, RI 0.9 ± 0.3 0.1 ± 0.1

globulol 1585 MS, RI 1.1 ± 0.4 0.1 ± 0.1

viridiflorol 1593 MS, RI 0.8 ± 0.4 0.1 ± 0.1

rosifoliol 1599 MS, RI 0.1 ± 0.1 -

junenol 1605 MS, RI tr -

cubenol 1622 MS, RI 0.1 ± 0.2 tr

γ-eudesmol 1632 MS, RI 0.5 ± 0.2 0.2 ± 0.1

cadinol-epi-α 1638 MS, RI 4.9 ± 3.4a 0.6 ± 0.2b

muurolol-epi-α 1640 MS, RI 9.1 ± 4.8a 0.8 ± 0.5b

α-muurolol 1642 MS, RI 0.5 ± 0.5 -

Oxygenated sesquiterpenes 15.2 ± 9.2a 1.6 ± 0.9b

unidentified diterpene (C20H32) g 1949 MS, RI 4.8 ± 2.2 -

manoyl oxide 1991 MS, RI 1.1 ± 0.8 -

diterpenoids 5.9 ± 3.0 0

3-hexen-1-ol 854 MS, RI tr tr

2-hexenal 855 MS, RI - tr

nonanal 1105 MS, RI 0.2 ± 0.3 tr

2-nonanone 1092 MS, RI tr tr

isopentyl isovalerate 1107 MS, RI 0.1 ± 0.1 0.1 ± 0.0

2-methyl butyl hexanoate 1253 MS, RI 0.1 ± 0.1 0.1 ± 0.0


25

2-undecanone 1292 MS, RI 1.2 ± 1.5 0.3 ± 0.2

(Z)-3-hexenyl n-hexanoate 1379 MS, RI - 0.1 ± 0.0

3-methyl-1-butyl benzoate 1441 MS, RI 0.3 ± 0.2 0.2 ± 0.1

Other 1.8 ± 2.2 0.7 ± 0.4

Total identified 94,4 98,7

a Values within a row for each compound having different letters are significantly different from each other according Fisher’s least significant difference (LSD) test (P


26

Hafsé, M.; Benbrahim, K. F.; Saidi, A. & Farah, A. (2013). Volatile Components and Antibacterial Profile of

Essential Oils Extracted from Leaves and Twigs of Lentiscus L. British Microbiology Research Journal, 3(4),

602-611.

Haloui, T., Farah, A., Balouiri, M., Chraibi, M., Fadil, M., Benbrahim, K. F., & Alaoui, A. B. (2015).

Bacteriostatic and Bactericidal Profile of Leaves and Twigs Essential oils of Moroccan Pistacia lentiscus

L. Journal of Applied Pharmaceutical Science, 5(06), 050-053.

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Nat. Volatiles & Essent. Oils, 2015; 2(4): 27-39 Kaczmarczyk et al.

27

RESEARCH ARTICLE

Selected Essential Oils in Cosmetic Emulsions: Process Oriented

Stability Studies and Antimicrobial Activity

Daria Kaczmarczyk1, Daniel J. Strub1, Anna Polowy2, Kazimiera A. Wilk2 and Stanisław Lochyński1,3*

1 Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology,

WybrzeżeWyspiańskiego 27, 50-370 Wrocław, Poland. 2 Organic and Pharmaceutical Technology Group, Faculty of Chemistry, Wrocław University of Technology,

WybrzeżeWyspiańskiego 27, 50-370 Wrocław, Poland. 3 Institute of Cosmetology, Wroclaw College of Physiotherapy, Kościuszki 4, 50-038 Wrocław, Poland.

*Corresponding author: [email protected]

Abstract

Essential oils show self-preserving properties, which gives an opportunity for the cosmetic industry to reduce or even eliminate the

application of synthetic preservatives like parabens, which are thought to be endocrine-disrupting compounds. The present study

was focused on advancing of our understanding on antibacterial and antifungal properties of essential oils in cosmetic products.

Especially, towards the development of practical and applicable aspects of abovementioned natural products. Presented procedures

and results can be easily up-scaled and transferred to cosmetic industry. Results comprising procedure for preparation of a stable

cosmetic emulsion using essential oils as natural preservatives, the evaluation of its stability by the turbidimetric method and

estimation of preserving capabilities of essential oils including comparison with commonly used synthetic preservatives are presented

in this present study. The stability evaluation of cosmetic emulsions revealed that the most stable emulsion is one comprising citrus

oil (C= 2%) and vitamin E (C= 1.5%). The most unstable compositions are ones with rosemary oil (C= 2%), vitamin E (C= 1.5%) and

citrus oil (C= 1%). Microbiological tests showed that the prepared emulsions consist of less than 10 CFU/g, which proves that the

compositions are microbial clean and thus high quality.

These results confirm our hypotheses that essential oils can be effective natural preservatives in cosmetic formulations with

concurrent stability and other features oriented on the possible commercial application.

Keywords: Essential oils, cosmeceuticals, emulsions, natural preservatives

Introduction

Essential oils show self-preserving properties, which gives an opportunity for the cosmetic industry to reduce

or even eliminate the application of synthetic preservatives. Mutations of microorganisms leading to

resistance towards conventional preservatives triggered research for finding alternative preserving systems

in various branches of industry, including cosmetic industry (Sheldon, 2005). In addition, common

preservatives – parabens (p-hydroxybenzoate esters) – possess estrogenic activity in various in-vitro and in-

vivo tests (Table 1); are thought to act as endocrine disruptive compounds (Darbre and Harvey, 2008; Byford

et al., 2008; Routledge et al., 1998) and induce proliferation of cancer cells (Byford et al., 2008; Darbre et al.,

2003; Darbre et al., 2002).

Antimicrobial activity enables application of essential oils as food preservatives (Burt, 2004), crop cultivation

growth stimulants (Calsamiglia et al., 2007) and as natural pesticides in ecological agriculture (ElShafei et al,

2010). Literature references point out their high activity against gram-positive bacteria and slightly lower

activity against gram-negative (Burt 2004; Skočibušić et al., 2006). In addition to antimicrobial properties,

essential oils exhibit therapeutic properties over a wide range of targets. Oils isolated from thyme, rosemary

and lavender counteract alopecia areata. Chamomile and lavender oils show anti-inflammatory and anti-acne


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activities. In addition, rosemary and lemon oils exhibit anti-wrinkle properties (Chaturvedi et al, 2007). It is

worth to notice that essential oils may induce allergic reactions, despite their natural origin (Maddocks-

Jennings, 2004). To minimize the risk of their occurrence, the concentration of essential oils in cosmetic

formulations is limited to 1-2%. The selection of essential oils for purposes of our study was made based on

an analysis of their specific activity and direct influence on the skin.

Table 1. Estrogenic activity of parabens.

Paraben Estrogen receptor recombined in yeasts

Human receptor MCF-7

Rat ovarian estrogen receptor

Cells of ovarian cancer HeLa

Methylparaben + + - n/a

Ethylparaben + + + +

Propylparaben + + + +

Butylparaben + + + +

Isopropylparaben + + n/a n/a

Isobutylparaben + + n/a n/a

Benzylparaben + + + n/a

Nowadays, the nano-encapsulation of active substances is very important in cosmetic formulations. Some

research has been made regarding the use of essential oils as preservatives in cosmetics (Varvaresou et al.,

2009). Lemon and lavender oils were previously investigated as potential natural preservatives in cosmetics.

Previous studies confirmed growth inhibitory properties of lemon oil towards Pseudomonas aeruginosa,

Staphylococcus aureus and Candida albicans (Kunicka-Styczyńska et al., 2011). Another studies focused on

antimicrobial and antifungal properties of sage, eucalyptus and laurel tree oils (Maccioni and Anchisi, 2002).

Synergistic effects of combination of essential oils and low doses of synthetic preservative were observed.

Our results also confirm this statement. All previously conducted studies focused on basic research that only

proves potential of essential oils as constituents of cosmetics.

Presented study was focused on two directions in natural products research. The first was to advance our

understanding on antibacterial and antifungal properties of essential oils in cosmetic products. Nevertheless,

the main purpose of our study was directed towards practical and applicable aspects of abovementioned

natural products. Presented procedures and results can be easily up-scaled and transferred to cosmetic

industry. This study is also a continuation of our interest in preparation of new semisynthetic therapeutic

agents (Siemieniuk et al., 1992a; Siemieniuk et al., 1992b) and novel nano-carriers and encapsulation

processes (Jarzycka et al., 2013; Bazylińska et al., 2012a; Bazylińska et al., 2012b; Bazylińska et al., 2011; Wilk

et al., 2009). We have taken an advantage of new technologies and methods for process-oriented

preparation of stable cosmetic emulsions using essential oils as natural preservatives, the evaluation of their

stability by the turbidimetric method and estimation of preserving capabilities of essential oils including

comparison with commonly used synthetic preservatives. Results of this study bring answer to the question

if essential oils can be effective natural preservatives in cosmetic formulations with concurrent stability and

other features facilitating the possibility of commercial application.


Material

Glycerine was purchased from PPH Stanlab; potassium hydroxide from POCH S.A.; stearin from cosmetic

warehouse “ZrobSobieKrem”; tocopheryl acetate (vitamin E) and L-Ascorbic acid (vitamin C) from cosmetic

warehouse e-Naturalne. Rosemary, lemon, lavender and eucalyptus oils were purchased from Avicenna-oil


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and Phenonip (mixture of phenoxyethanol, methylparaben, ethylparaben, butylparaben, propylparaben and

isobutylparaben) from cosmetic warehouse “mazidla.pl”

Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 6538 and Candida albicans ATCC 10231

were used for microbial quality control studies.

Sample preparation

Emulsions were prepared using Homogenizer Ultra Turrax T25 Basic, IKA. All cosmetic emulsions were

prepared using hot-hot cold method. Choice of the method was based on different physical states of water

and oil phases and temperature sensitivity of essential oils and vitamin E. Components of water and oil

phases (Table 2) were weighed separately. Both phases were stirred vigorously and heated to 70-80˚C. When

both phases reached the same temperature, oil phase was transferred steadily into water phase. In the next

step mixture was homogenized at 19000 rpm for 10 minutes and subsequently at 11000 rpm for 20 minutes.

Emulsion was cooled down to room temperature, temperature-sensitive components were added and final

mixture was homogenized at 19000 rpm for 10 minutes. E

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