CHARACTERIZATION AND BIOLOGICAL

ACTIVITIES OF ESSENTIAL OILS OF

Cade (Juniperus oxycedrus)

By

Muhammad Muddassar Afzal

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

MASTER OF PHILOSPHY

IN

CHEMISTRY

DEPARTMENT OF CHEMISTRY

FACULTY OF SCIENCES

UNIVERSITY OF SURGHODA,

LYALLPUR CAMPUS, FAISALABAD

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CHAPTER 2
Review of Literature

2.1 Essential Oils

The fragrant mixture of liquids, obtained through distillation of aromatic plant
materials, is known as an essential oil (Burt, 2004). Essential oils are mixtures of fragrant
substances or mixtures of fragrant and odorless substances. A fragrant substance is a
chemically pure compound, which is volatile under normal conditions and which owing to
its odour can be useful for the society (Gunther, 1952).

2.2 Cade (Juniperus Oxycedrus) Essential Oils

In the present study, evaluation of the essential oil (EO) of the aerial parts (leaves, ripe
berries and unripe ber-ries) of Juniperus phoenicea L. collected from Eastern Morocco, and
comparison of their chemical composition and their antibacterial and antifungal activities
were carried out. The average yields of EOs obtained was varied; the unripe berries sample
was the highest. The EOs components were analyzed and identified chromatographi-cally
by using (GC and GC/MS). Forty one compounds were identified in the leaves oil, while
34 and 28 com-pounds were identified in unripe and ripe berries, respectively. J. phoenicea
is dominated by the presence of the major compound -pinene only in leaves and unripe
berries with 34.36% and 33.7%, respectively, while the major compound in the ripe berries
was -pinene oxide (18.17%). The antibacterial and antifungal activities of the EOs of
J.phoenicea were evaluated against four ATTC types of bacterial strains, (Bacillus subtilis,
Es-cherichia coli, Staphylococcus aureus and Micrococcus luteus) and seven ATCC types
of fungal strains in which three are molds (Asper gillusniger, Penicillium digitatum and
Penicillium expansum), the others are fun-gal species (Gloeophyllum trabeum,
Coniophoraputeana, Poria placenta and Coriolus versicolor). The mini-mum inhibitory
concentration was determined and the results obtained led to a significant inhibitory effect
against most of studied microorganisms. The results showed that, EOs inhibited the growth
of all bacterial strains at highest concentration (1/100 v/v) from all samples, and the most
effective EO was obtained from the ripe berries. Additionally, the four wood rot fungi were
sensitive to the EO from all samples at highest concen-tration (1/100 v/v), and only EO
from ripe berries has antifungal activity even at low concentration (1/1000 v/v). The
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sensitivity was appeared also in all molds in case of ripe berries and leaves EOs at high
concentration (1/100 v/v), while unripe berries EO inhibited the growth of Penicillium
expansum only, the other molds were resistant.
The chemical composition of the essential oil obtained from the leaves of Juniperus
phoenicea L. (Fam. Cupressaceae) was investigated. Samples were collected from Al -
Jabel Al-Akhdar (Green Mountain) in the northeastern region of Libya. Hydro-distillation
was performed to obtain the essential oils of the leaves while gas chromatography coupled
with mass spectrometry system (GC/MS) was used for the analysis of their chemical
composition. Thirty four (34) compounds were identified in essential oil representing
88.29% of the total oil composition. The major compound was identified as -pinene
(20.85%) followed by germacrene D (16.49%).
Antimicrobial activity and chemical composition of the essential oil of Juniperus
communis (L.), originated from east part of Kosova, was investigated. The essential oil
from berries of J. communis (L.), obtained by hydro-distillation was analyzed by GC and
GC-MS. Antimicrobial properties of the essential oil of J. communis (L.) are investigated
and results are submitted for their activities against Staphylococcus aureus,

Escherichia coli, Hafnia alvei and Pseudomonas aeruginosa. Applying the agar disc
diffusion technique, we measured diameters of the inhibition zone around discs, which are
previously wetted with DMF solution of the essential oil with three different
concentrations, 1, 3 and 5 mg mL-1. Analysis of the oil resulted in the identification of 41
peaks, representing 96% of the oil. Berry essential oil composed mainly of monoterpenoids
which amounted to 83%, of which 69.4% was monoterpene hydrocarbons. The main
monoterpene hydrocarbons were -pinene (36.2%) and -myrcene (21.1%). The
sesquiterpene accounted for about 13.4% of the total oil composition. Germacrene D
(2.2%), -cadinol (1.6%), -humulene (1.5%), spathulenol (1.4%), epi--bisabolol (1.3%)
and germacrene B (1.1%) were the main constituents of the sesquiterpenes. The inhibition
zone depends from concentrations and also from sort of bacteria. The inhibition zones
differ from 0-39 mm. The present work presents the chemical composition of the
hydrodistilled oil of J. communis (L.) from East part of Kosova and the results are
compared to those reported in the literature. This study demonstrates the occurrence of -
pinene chemotype of J. communis (L.) from east part of Kosova. The essential oil of J.
communis (L.) growing wild in Kosova, showed moderate to high activities against

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Staphylococcus aureus, Escherichia coli, Hafnia alvei. Pseudomonas aeruginosa is resistant
to the essential oil of J. communis (L.) growing wild in east part of Kosova.
Chemical composition and antimicrobial activity of essential oil isolated from berries
from 2 different samples of Juniperus oxycedrus L. (Cupressaceae), growing wild in
Republic of Macedonia was investigated. Performing GC/FID/MS analysis, one hundred
components were identified, representing 96.0-98.95% of the oil. The major components
were -pinene (22.54- 27.12%), myrcene (11.26- 15.13%) and limonene (2.78-18.06%).
Antimicrobial screening of the J. oxycedrus essential oils was made by disc diffusion and
broth dilution method against 16 bacterial isolates of Gram positive and Gram negative
bacteria and one strain of Candida albicans. The most sensitive bacteria was Haemophilus
influenzae (MIC = 125 ml/ml). The essential oils showed moderate antimicrobial activity
against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae,
Streptococcus pyogenes, Corynebacterium spp., Escherichia coli and Campilobacter jejuni
(MIC > 500 ml/ml) and no activity against Candida albicans, Staphylococcus epidermidis,
Acinetobacter spp.Salmonella enteritidis, Shigella flexnery, Klebsiella pneumonia,
Pseudomonas aeruginosa, Enterococcus and Proteus mirabilis.

Chemical composition and antimicrobial activity of essential oil isolated from berries
from 2 different samples of Juniperus oxycedrus L. (Cupressaceae), growing wild in
Republic of Macedonia was investigated. Performing GC/FID/MS analysis, one hundred
components were identified, representing 96.0-98.95% of the oil. The major components
were -pinene (22.54- 27.12%), myrcene (11.26- 15.13%) and limonene (2.78-18.06%).
Antimicrobial screening of the J. oxycedrus essential oils was made by disc diffusion and
broth dilution method against 16 bacterial isolates of Gram positive and Gram negative
bacteria and one strain of Candida albicans. The most sensitive bacteria was Haemophilus
influenzae (MIC = 125 ml/ml). The essential oils showed moderate antimicrobial activity
against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae,
Streptococcus pyogenes, Corynebacterium spp., Escherichia coli and Campilobacter jejuni
(MIC > 500 ml/ml) and no activity against Candida albicans, Staphylococcus epidermidis,
Acinetobacter spp.
It was found for the first time that needles and berries of the same junipers growing wild
in Lithuania produced essential oils of different chemotypes. The Juniperus communis L.
var. communis with leaves (needles) producing sabinene-chemotype essential oils were
found only in three localities from the 34 investigated habitats, beside the plants with
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nee-dles biosynthesizing -pinene-chemotype oils. The dominant compounds in four from
49 (including earlier results) investigated needle essential oils were sabinene (34.1
40.8%), -pinene (11.727.8%) and terpinen-4-ol (6.99.3%), while in the rest needle oils
the main component was -pinene (41.266.5%). The first major constituent of all berry
essential oils under study was -pinene (21.067.4 %). The second and third main
constituents in unripe berry oils from bushes with needle oils of sabinene chemotype were
sabinene (6.319.6%), myrcene (4.312.8%) and terpinen-4-ol (13.1%) and in the ripe
berry oils myrcene (7.818.7%) and terpinen-4-ol (3.29.6%). The content of sabinene in
all ripe berry oils was only 0.42.9%.
In this study, the chemical compositions of the leaves, berries and twigs essential oils of J.
oxycedrus L. subsp. oxycedrus, collected in Turkey, were determined. The oils were
analyzed by GC and GC-MS. 15-21 volatile compounds were identified of the leaves,
berries and twigs essential oils representing 82.4-98.0% of the total oils. The essential oils
were obtained from leaves, berries and twigs by yielding 0.02%, 2.12% and 0.01%, resp.
The major compounds were determined manoyl oxide (32.8%) and caryophyllene oxide
(11.9%) in leaf oil, myrcene (44.6%), -pinene (19.9%) and germacrene D (15.5%) in berry
oil, manoyl oxide (35.4%) and caryophyllene oxide (16.8%) in twig oil.
This study was designed to examine the phytochemistry of the essential oil obtained from
aerial parts of Juniperus oxycedrus collected in Atlas median region from Morocco. The
essential oil was extracted by hydro-distillation and analysed by gas chromatography
equipped with flame

ionisation detector (GC-FID) and gas chromatography coupled to mass spectrometry

system (GC/MS). 48 Constituents were identified in leaves oil representing 84.05% of the
total oil and the yield was 1.66%. The Juniperus oxycedrus leaves oil was characterised by
high contents of -pinene
(31.25%) followed by sabinene (5.21%), limonene (5.02%), pinene (4.58%),
caryophyllene oxide (4.12%), myrcene (3.56%), -cymene (3.21%),
phellandrene (3.01%), terpinene (2.19%), terpinen-4-ol (2.01%), germacrene-D
(1.57%), (E)-caryophyllene (1.25%) and -ocimene (1.09%).
The present study evaluates the chemical composition and antimicrobial activity of
essential oils (EOs) of Juniperus Phoenicea of five localities from eastern Algeria. The
analysis and identification of the components of the Eos was performed using the (GC-

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MS). The average yield of essential oil of the samples is 0.82%. The chemical composition
of the EOs of J. Phoenicea is dominated by the presence of a major product, -pinene (36.3-
55.9%). Three components are represented with large concentrations, terpinolene (0-13%),
3-carene (0-12.4%) and the -phellandrene (0-7.3%). Our investigation allows us to
support the species Juniperus phoenicea of eastern Algeria has several variability
quantitative and qualitative. The antimicrobial activity of the essential oils of J. phoenicea
was evaluated against nine bacteria. The results showed a variable degree of antibacterial
activity being the population Elhadjaz most effective.
The essential oil constituents from the needles, twigs and berries of Juniperus chinensis
from Korea were investigated by gas chromatography-mass spectrometry (GC-MS). The
essential oils from the different plant parts were obtained by steam distillation and the
yields were 0.34, 0.11 and 0.12% (v/w), respectively. The GC-MS analysis revealed the
identification of 36 different components from needles, twigs and berries, which were
mostly monoterpene hydrocarbons (42.05-48.15%) followed by oxygenated monoterpenes
(28.53-39.92%).Among the 36 components, 17 components were identified in all the three
essential oils. The components such as bornyl acetate (2.85 20.70%), sabinene (10.23
18.13%), -pinene (5.80 16.26%), terpinen-4-ol (5.98 31.10%), limonene (3.98
6.96%), -pinene (3.05 4.39%), -terpinene (2.24 8.36%), -elemol (1.74 4.77%) and
-cadinol (2.49 3.39%) were detected as the major components in the essential oils from
the three different parts of J. chinensis. The main differences between the three essential
oils can be referred to terpinen-4-ol and bornyl acetate. The essential oil of the berries
contained the highest level of terpinen-4-ol (31.10%) than needles (7.51%) and twigs
(5.98%). On the other hand, bornyl acetate content was very less in berries (2.85%) when
compared with twigs (20.70%) and needles (16.43%).
In this work, the chemical composition and antimicrobial activity of essential oils obtained
from Juniperus phoenicea were determined. Juniperus species from the Cupressaceae
family are widely distributed in Morocco. In this study, the essential oils of J. phoenicea
collected from Atlas median in the region of Boulmane (Morocco) were obtained by hydro-
distillation of the aerial parts and analysed by gas chromatography equipped with flame
ionisation detector (GC-FID) and gas chromatography coupled to a mass spectrometry
system (GC/MS) for their chemical composition. Their antibacterial activity was studied
in vitro on seven bacterial strains: Escherichia coli, Staphylococcus aureus, Staph.
intermedius, Klebsiella pneumonia, Pseudomonas aeruginosa, Bacillus subtilis and

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Streptococcus mutans. Twenty tree compounds were identified in leaves oil representing
81.87% of the total oil composition. The yield of essential oil of J. phoenicea was 1.62%
and the major compound in aerial parts was -pinene (49.15%) followed by -phyllandrene
(7.39%), mycene (5.24%), -pinene (3.58%), linalool (2.54%), piperitone (1.56%), -
terpinene (1.28%), Trans-pinocarveole (1.23%) -cymene (1.10%), terpineol (1.02%)
and -cardinene (1.01%). The bacterial strains tested were found to be sensitive to essential
oils studied and showed a very effective bactericidal activity with minimum inhibitory
concentrations (MIC) ranging from 0.02 to 0.40 mg per mL. 2010 Friends Science
Publishers
Essential oils from Juniperus oxycedrus L. have been used since antiquity for fragrance,
flavoring, medicinal, antimicrobial, insecticidal, and cosmetic purposes. Several works
studied the chemical composition of the essential oils of Juniperus oxycedrus leaves. The
aim of this study is to investigate the chemotaxonomic relationships and antibacterial
activity of two Tunisian subspecies:

Juniperus oxycedrus ssp. oxycedrus (L. K.) Deb. and Juniperus oxycedrus ssp. macrocarpa
(S. & m.) Ball. In addition, and for the first time, we reported the antibacterial activities of
Tunisian J. oxycedrus ssp. macrocarpa and J. oxycedrus ssp. oxycedrus against four
bacteria. Essential oils obtained by hydrodistillation were analysed by GC and GC/MS.
Fifty-five constituents were identified. Thirty four major compounds were retained for the
study of the chemical variability, and -pinene, sylvestrene, p-cymene, and 13-epi-manoyl
oxide were the main ones. The chemical principal components analysis (PCA) identified
three chemotypes. The study of the antibacterial activity showed that Escherichia coli was
found to be extremely resistant (zone diameter 0 mm) to all the oils tested, while
Staphylococcus aureus was the most sensitive strain (zone diameter 13.5 mm and MIC
ranged from 600 to 650 g/mL).
Nowadays, the overdose use of antibiotics and the increase of microbial resistances
have made researchers to carry out more studies about the new drugs with new chemical
composition. In order to produce these new drugs, different species of special plants can
be useful by scientists and researchers. Base on this, the leaves and fruits of Juniperus
excelsa and Juniperus horizontalis were collected from Iranian National Botanic Garden.
Essential oils were isolated by hydro-distillation method. The essential oil components
were analyzed by gas chromatographymass spectrometry (GC-MS). The yields of the

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leaves and fruits of J. excelsa and J. horizontalis essential oils were 0/79 to 4/15% and
1/083 to 2/7%, respectively. There were 15 and 27 components in the essential oil of J.
excelsa and J. horizontalis, respectively. The major compound in the essential oils obtained
from the leaves and fruits of J. excelsa were -pinene (79/95 and 89/49%, respectively).
The main compound in the essential oils obtained from the leaves and fruits of J.
horizontalis were sabinene (30/21 and 38%, respectively). In addition, bornyl acetate
(10/66%) and delta-cadinene (3/79%) were identified as major components in the essential
oil of the leaves obtained from J. horizontalis. Juniper essential oils were evaluated for the
antibacterial activity against thirteen bacterial species by disk diffusion and micro dilution
method. Juniper essential oils showed more antibacterial activities against Gram-positive
as compared to Gram-negative bacteria species. The antibacterial activity of essential oils
may be related to presence of - pinene, limonene, and sabinene which are known to have
antibacterial properties.
The yield and composition of the essential oil of common juniper (Juniperus
communis L.) branches from Estonia were analyzed. The yields and composition of the oil
isolated by hydrodistillation from dried juniper branches, gathered from one single juniper
plant every month in 2006 and in 2007 (12 samples), were compared. The oil yields ranged
between 0.05 0.70%. A total sixty seven compounds were identified, representing over
96% of the total oil yield. The essential oil from Estonian juniper branches showed a high
content of a-pinene (40.4 62.0%); the other predominant constituents were limonene (4.2
10.0%), a-cadinol (1.9 6.3%), d-cadinene (2.1 4.8%), g-muurolene and germacrene
D (1.6 4.4%), b-myrcene (2.6 3.1%), a- and b-selinene (0.9 3.1%), germacren D-4-ol
(0.8 3.0%) and ?-pinene (1.4 2.2%). The yield of essential oil and amounts of mono-
and sesquiterpenes depend on the month of collecting the plant material. The best time for
harvesting juniper branches seems to be from January to April. The yield and composition
of essential oils in branches are rather similar, but did not completely correspond to the
standards of European Pharmacopoeia stated to juniper berries.
Vaiiulyt V., Loien K., 2013: Variation of chemical and morphological characters
of leaves and unripe cones in Juniperus communis [Paprastojo kadagio (Juniperus
communis) lap bei neprinokusi kankorli chemini ir morfologini parametr
vairavimas]. Bot. Lith., 19(1): 3747.

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 The variation of content of essential oil and pinene isomers and morphological
parameters of leaves and unripe cones in Juniperus communis L. was studied. Leaves and
cones were collected separate from 110 J. communis individuals in 11 different habitats
throughout Lithuania. Essential oils were analysed by capillary gas chroma-tography (GC).
The study showed more intensive essential oil accumulation and higher intraspecific
variation of essential oil amount in unripe cones than in leaves. Significant differences
between - and - pinene amounts were observed in J. communis. -Pinene was detected
in much higher quantities than -pinene. Positive cor-relation was detected between pinene
isomers in cones and leaves (r = 0.44, p < 0.05 and r = 0.50, p < 0.05, respectively) of the
same tree showing pinene isomers common biosynthetic pathway. Significant differences
between populations were characteristic of J. communis according to leaf length, unripe
cones mass and essen-tial oil yield in leaves, however, significant correlation was
established between essential oil yield of leaves and habitat illumination (r = -0.67, p <
0.05) and soil acidity (r = -0.80, p < 0.05) only.
The composition of the essential oil from ripe and unripe berries and leaves of Juniperus
oxycedrus L. ssp. oxycedrus, Juniperus phoenicea ssp. turbinata and Juniperus communis
ssp. communis was analyzed by GC-MS, and microbiological assays were carried out.
Samples were collected in different localities (Sardinia, Italy) and hydro distilled. The
yields ranged between 2.54% ( 0.21 (v\w dried weight) and 0.04% ( 0.00. A total of 36
components were identified. The major compounds in the essential oils were R-pinene, -
pinene, -3-carene, sabinene, myrcene, -phellandrene, limonene, and D-germacrene. Both
qualitative and quantitative differences between species and between different parts of the
plant were observed. The essential oils and their major compounds were tested against
Candida albicans, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa,
and the minimum inhibitory concentration and minimum bactericidal concentration were
determined. The results obtained led to a nonsignificant inhibitory effect, although all the
essential oils from

Juniperus phoenicea ssp. turbinata and the essential oil from leaves of Juniperus oxycedrus
ssp. oxycedrus exhibited rather good or weak activity against Candida albicans and
Staphylococcus aureus.
The qualitative and quantitative composition of the essential oils obtained from male and
female leaves of Juniperus thurifera L., (growing in Algeria) has been investigated for the

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first time. The essential oils were obtained by hydrodistillation (0.45% from female trees
and 0.53% from male trees, v/w dried material) and analyzed by gas choromatography
(GC) and gas choromatography-mass spectrometery (GC-MS). Seventy-seven compounds
were identified, representing more than 97% of the oils. The major components were
Sabinene, -pinene and terpinene-4-ol. The concentrations of the oil constituents: -
thujene, -pinene, -phellandrene, p-cymene, linalyl acetate, -amorphene, germacrene D-
4-ol, and 4-epi-abietal were greater in the oil of the female tree than in the oil of the male
tree. Conversely, the concentrations of -terpinene, -terpinene, terpinene-4-ol, elemol, -
epi-cadinol and -eudesmol were greater in the oil of the male tree than in the oil of the
female tree. However, the concentration gradient trends for both female and male trees
were similar for sabinene, myrcene, linalool, -pinene, limonene, cis-sabinene hydrate
terpinolene, -terpineol. The antimicrobial activity of male and female J. thurifera essential
oils was evaluated against 14 bacteria. The results showed a variable degree of antibacterial
activity depending from the type of the oil (extracted from male or female trees). Essential
oils of female trees were most effective.
The essential oil of common juniper (Juniperus communis L., from the south-ern part of
Serbia) and its fractions of different composition, as well as commercial anti-biotics were
used for testing their antimicrobial activity against bacteria, yeast and fungi. The essential
oil was produced by hydro-distillation in a pilot plant (130 dm3) and then fractionated by
distillation over a column, with 36 theoretical stages, under vacuum (2666 mbar). The
essential oil was also fractionated using pure CO2 or CO2 and metha-nol as co-solvent
under supercritical conditions. The native oil showed weak anti-microbial activity, while
the fractions with a high content of -pinene, and mixture of

-pinene and sabinene showed the highest antimicrobial activity, especially against fungi.
In comparison to the commercial antibiotics, the oil fractions showed more exten-sive
spectra of antimicrobial activity, as well as wider inhibition zones.
Essential oils are complex mixtures isolated from aromatic plants which may possess
antioxidant and anti-inflammatory activities of interest in thye food and cosmetic industries
as well as in the human health field. In this work, a review was done on the most recent
publications concerning their antioxidant and anti-inflammatory activities. At the same
time a survey of the methods generally used for the evaluation of antioxidant activity and

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some of the mechanisms involved in the anti-inflammatory activities of essential oils are
also reported.
Salmonella enteritidis, Shigella flexnery, Klebsiella pneumonia, Pseudomonas
aeruginosa, Enterococcus and Proteus mirabilis.
In this study, the chemical compositions of the leaves, berries and twigs essential oils
of J. oxycedrus L. subsp. oxycedrus, collected in Turkey, were determined. The oils were
analyzed by GC and GC-MS. 15-21 volatile compounds were identified of the leaves,
berries and twigs essential oils representing 82.4-98.0% of the total oils. The essential oils
were obtained from leaves, berries and twigs by yielding 0.02%, 2.12% and 0.01%, resp.
The major compounds were determined manoyl oxide (32.8%) and caryophyllene oxide
(11.9%) in leaf oil, myrcene (44.6%), -pinene (19.9%) and germacrene D (15.5%) in berry
oil, manoyl oxide (35.4%) and caryophyllene oxide (16.8%) in twig oil.

2.3 Methods of Isolation of Essential Oils

Methods to isolate essential oils may be categorized into enfleurage, steam

distillation, solvent extraction, hydrodistilation, and supercritical fluid extraction.
Hydrodistillation or steam distillation is the most widely utilized physical method for
isolating essential oils from the botanical material (Whish, 1996; Masango, 2004).
Although steam distillation is much popular for the isolation of essential oils on
commercial scale and 93% of the oils are produced by this process, but it is not a preferred
method in research laboratories (Masango, 2004). This is probably due to unavailability of
steam generators and suitable distillation vessels. Most studies which focus on the essential
oil of herbs have made use of hydrodistillation in Clevenger-type apparatus (Kulisic et al.,
2004; Sokovic and Griensven, 2006; Hussain et al., 2008).
In hydrodistillation procedure, the material is immersed in water, which is heated
to boiling point using an external heat source. In both hydro-,and steam- distillation
techniques, the vapours are allowed to condense and the oil is then separated from the
aqueous phase (Houghton and Raman, 1998). Care must be taken to ensure efficient
condensation of steam, thereby preventing the loss of the more volatile oil components.
There are reports in literature on the significance of both hydro-, and steam-
distillation processes. In hydro-distillation the plant material and water are combined in the
still and the whole things is then brought to a boil. The hot water draw out the oils, just as

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steam does, and it is carried to the condenser and cooled into hydrosol and essential oil.
This method produces a finer, more complete product, as hot water is collar than steam
distillation and shocks the plant material less (Ackerman, 2001). However, it has been
suggested by Charles and Simon (1990) that steam distillation is more efficient than
hydrodistillation in removing oil from plant material.
They found that steam distillation consistently gave higher yields of oil than
hydrodistillation, although the same compounds were obtained. They also approved the
hydrodistillation, a simpler and more rapid method for oil isolation. In contrast, Whish
(1996) found no difference between oil yields, when oils from Melaleuca alternifolia (tea
tree) were produced via steam distillation and hydrodistillation, on both macro-, and
semimicro-scales.
while, Khanavi et al., (2004) reported better essential oils yield of Stachys persica
and S. byzantine from hydrodistillation than steam distillation. Sefidkon et al. (2007)
isolated the essential oils from the aerial parts of Satureja rechingeri by steam, hydro- and
water-steam-distillation. The highest oil yield was obtained with hydro-distillation and the
lowest with steam-distillation (Sefidkon et al. 2007).

Terblanche et al. (1998) used both hydrodistillation and microwave distillation

methods for the isolation of the essential oil from Lippa scaberrima. Only slight differences
were observed between the compositions of the oils obtained from the two methods,
although the yields obtained through microwave distillation were generally lower.
This is probably due to unavailability of steam generators and suitable distillation vessels.
Most studies which focus on the essential oil of herbs have made use of hydrodistillation
in Clevenger-type apparatus (Kulisic et al., 2004; Sokovic and Griensven, 2006; Hussain
et al., 2008).

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Several found that hydro/steam-distilled oil contained higher percentages of terpene
hydrocarbons. In contrast, the super critical extracted oil contained a higher percentage of
oxygened compounds (Reverchon, 1997; Donelian et al., 2009).
Khajeh et al. (2004) reported variation in the chemical composition of Carum
copticum essential oil isolated by hydrodistillation and supercritical fluid extraction
menthods. Silva et al. (2004) reported that essential oils from leaves of Ocimum
gratissimum, Ocimum micranthum and Ocimum selloi obtained by steam distillation (SD),
microwave oven distillation (MO) and supercritical (SC) extraction with CO2 showed
different composition by GC/MS analysis. The authors reported that main components in
the three essential oils were the same but in different relative amounts.

The duration of the distillation process is also an important parameter that influence
on the yield and composition of essential oils (Koedam, 1982; Masango, 2004). The long
distillation cycles should be avoided, as only a small increase in the oil yield is obtained
towards the end of the cycle, which is counteracted by the greater loss of polar compounds
to the increased aqueous fraction (Masango, 2004). Commercially, use of shorter
distillation cycles results in a reduction in production costs. Koedam (1982) has shown in
hydrodistillation experiments that alonger the length of distillation process, the acidity of
the distillation water also influenced the composition of the oil obtained. Throughout
distillation process, oxygenated compounds are released earlier from intact plant material,
than lower boiling point, non-oxygenated compounds (Baker et al., 2000). This might be
due to that boiling water (steam) which penetrates the oil gland and dissolves part of the
oil present in the gland. Once released from the glands, the oil components are immediately
vapourised. Polar oxygenated compounds are more water-soluble than non-oxugentaed
compounds and thus diffuse faster and distilled first (Koedam, 1982; Baket et al., 2000).
Koedam (1982) found that initial distillate fractions, obtained during hydrodistillation of
dill seed, contained predominantly carvone (b.p. 230 C), and only small amounts of
limonene (b.p. 176 C), in spite of the latter having a lower boiling point. However, the
limonene quantities gradually increased throughout the distillation, with a simultaneous
decrease in carvone. Baker et al. (2000) explained that the affinity of some volatile
components for lipids, prevent their release from the plants.

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 Sesquiterpenes, probably because of their large molecular size, tend to distill later
than oxygenated monoterpenes. Sabinene, cis-sabinene hydrate and trans-sabinene hydrate
are known to undergo thermal transformation to terpinen-4-ol, -terpinene, -terpinene and
terpinolene, during hydroand steam distillation (Koedam, 1982; Baker et al., 2000).
MacTavish and Harris (2002) reported that a distillate temperature between 105 and 110
C is critical for the optimum distillation of peppermint (Mentha spicata).

2.4 Chemistry of Essential Oils

Essential oils are made up of three elements almost exclusively carbon, hydrogen,
and oxygen. By far the most common component class is the terpenes. Terpenes are made
from combinations of several 5-carbon- base (C5) units called isoprene (Gunther, 1952).
Terpenes can form building blocks by joining together in a "head-to-tail" configuration to
form monoterpene, sesquiterpenes, diterpene and larger sequences (Pinder, 1960). The
chief terpenes are the monoterpenes (C10) and sesquiterpenes (C15) and in some cases
hemiterpenes (C5), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40) also exist.
A terpene containing oxygen is called a terpenoid.
The monoterpenes are generally formed by the combination of two isoprene units.
They are the mainly representative molecules constituting 80-90% of the essential oils and
allow a vast variety of structures. They also contain several functional groups like carbures
(ocimene, myrcene, terpinenes, phellandrenes, pinenes, etc.), aldehydes (geranial,
citronellal, etc.), ketone (menthones, pulegone, carvone, fenchone, pinocarvone, etc.),
alcohols (geraniol, citronellol, nerol, menthol, carveol, etc.), esters (linalyl acetate,
citronellyl acetate, isobornyl acetate, etc.), ethers (1,8-cineole, menthofurane, etc.) (Burt,
2004).
There is plenty of literature on the characterization of essential oils. Capillary gas
chromatography (GC) with flame ionisation detection (FID), are, in most cases, the method
of choice for quantitative determinations. Many researchers make use of mass
spectrometers (MS), coupled with GC, to determine the identities of components (Salzer,

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1977; Wilkins and Madsen, 1991; Daferera et al., 2000; Juliano et al., 2000; Jerkovic et
al., 2001; Delaquis et al., 2002; Hussain et al., 2008; Anwar et al., 2009a; Anwat et al.,
2009b). Alternatively, Kovats indices, determined by co-injection of the oil with a
homologous series of n-alkanes, are also widely used to identify compounds, where
authentic standards are not available (Juliani et al., 2002). Hernandez-Arteseros et al.
(2003) identified 150 constituents, using Kovats retention indices, in the essential oil of
Lippia chiapanensis, from South America. Time-of-flight mass spectrometric (TOF-MS)
detection has been increasingly used as a qualitative tool, for the detection of volatile
components (Adahchour et al., 2003). Capillary columns selected, in most cases, are HP-
5ms, DB-5 (cross-linked 5% diphenyl/95% dimethyl siloxane) or DB-1, also known as SE-
30, (polydimethyl siloxane) stationary phases. These more non-polar stationary phases are
often complimented by the use of a more polar stationary phase, such as polyethylene
glycol (Cavaleiro et al., 2004).
Essential oils are very complicated mixtures of natural compounds at quite different
concentrations (Burt, 2004; Bakkali, 2008). They are characterized by two or three major
components at fairly high concentrations (2070%) compared to others components
present in trace amounts (Bauer et al., 2001; Burt, 2004). For example, carvacrol (30%)
and thymol (27%) are the major components of the Origanum compactum essential oil,
linalol (68%) of the Coriandrum sativum essential oil, - and -thujone (57%) and
camphor (24%) of the Artemisia herba-alba essential oil, 1,8-cineole (50%) of the
Cinnamomum camphora essential oil, -phellandrene (36%) and limonene (31%) of leaf
and carvone (58%) and limonene (37%) of seed Anethum graveolens essential oil, menthol
(59%) and menthone (19%) of Mentha piperita essential oil. Generally, these major
components determine the biological properties of the essential oils. The components
include two groups of distinct biosynthetical origin (Croteau et al., 2000; Betts, 2001;
Bowles, 2003; Pichersky et al., 2006).
The main components of M. arvensis essential oils from China town and Hamaliyan
regions of India were reported to be menthol, p-mentone and iso-menthone (Singh et al.,
2005; Pandey et al., 2003). Yadegarinia et al. (2006) reported that essential oil of M.
piperita from Iran contained following major components: -terpinene, piperittinone oxide
and trans-carveol. Gulluce et al. (2007) reported the cis-piperitone

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epoxide, pulegone and menthone as the major components of M. longifolia essential oil
native to Turkey. The essential oilc of M. spicata from Edhessa Greece were characterized
by the presence of carvone and dihydrocarveol, cis-dihydrocarvone, respectively
(Sivropoulou et al., 1995).
Literature revealed that the chemical composition of the O. sanctum, O.
gratissimum and O. basilicum essential oils varied depending upon the origins and cultivars
(Silva et al., 2004; Hussain et al., 2008; Zheljazkov et al., 2008). Within the O. basilicum,
there is a clear variation in phenotype and chemotype in terms of oil content and oil
composition (Zheljazkov et al., 2008). Linalool and methylchavicol were the main
components of common and European basils, whereas methyl-chavicol was present at high
concentration in the Reunion basil. The tropical chemotype of basil is known to have
methyl cinnamate as the major component of its essential oil. Another basil
chemotype,frequently grown in North Africa, Russia, Eastern Europe, and parts of Asia
was high in eugenol (Marotti et al., 1996).
Moraes et al. (2002) reported the variation in the yield of Ocimum selloi Benth
essential oils of the inflorescences, containing flowers and seeds. Telci et al. (2006)
investigated the chemical composition of eighteen Turkish basil essential oils by GC and
GC-MS. On the basis of chemical composition, Telci et al. (2006) classified the basil into
seven chemotypes: (1) linalool, (2) methyl cinnamate/linalool, (3) methyl cinnamate, (4)
methyl eugenol, (5) methyl chavicol (estragol), (6) citral, and (7) methyl chavicol/citral.
Methyl chavicol with high citral contents can be considered as a new chemotype in the
Turkish basils.
The genus Thymus has numerous species and varieties and their essential oil
composition have been studied (Sotomayor, 1998; Sotomayor et al., 2004). Nickavar et al.
(2005) characterized the essential oils of Thymus daenensis and Thymus kotschyanus by
using GC and GC-MS. Both the essential oils were found to be rich in thymol and
carvacrol. Jordan et al. (2006) reported the variations in the chemical compositions among
plants of of Thymus hyemalis L. and Spanish Thymus vulgaris L. shrubs. From Thymus
vulgaris grown in France and in other countries (Piccaglia et al., 1993), six chemotypes
have been reported, geraniol, linalool, -terpineol, carvacrol, thymol and trans-thujan-4-
ol/terpinen-4-ol. Studies carried out by Morales (1986) on the essential oil

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variability of Thymus zygis growing wild in South Eastern Spain, demonstrated that the
most common chemotype of this thyme was thymol. The chemical variability of the
essential oil from wild T. hyemalis of the south eastern Iberian Peninsula has been reported
(Adzet et al., 1976; Sotomayor, 1998). These researchers stated that thymol, carvacrol,
borneol and linalool were the chemotypes most abundant in this area.
There are numerous reports on the chemical composition and diverse biological
activities of Origanum species (Muller-Riebau et al., 1995; Sokovic et al., 2002; Bouchra
et al., 2003; Yildirim et al., 2005; Kizil and Uyar, 2006; Caglar et al., 2007; Esen et al.,
2007; Lee et al., 2007; Soylu et al., 2006; Souza et al., 2007). Origanum acutidens is an
endemic species growing in northeastern Turkey. The essential oil composition of this
species has been previously reported (Baser et al., 1997; Baser, 2002; Figueredo et al.,
2006). D-Antuone et al. (2000) analyzed the native populations of Origanum vulgare L.
from the Liguria and Emilia regions of northern Italy for essential oil content and
composition. Baydar et al. (2004) characterized essential oils from the wild oregano
(Origanum minutiflorum) (endemic in Turkey), oregano (Origanum onites), black thyme
(Thymbra spicata) and wild savory (Satureja cuneifolia) by GC-MS. The major constituent
of the oils was cavracrol (86.9% in O. onites, 84.6% in O. minutiflorum, 75.5% in T. spicata
and 53.3% in S. cuneifolia). Kokkini et al. (1997) analyzed the composition of six wild
Greek populations of Origanum vulgare ssp. hirtum and four of the Turkish oregano (O.
onites) essential oils by means of GC and GC/MS. Analysis showed that Turkish oregano
oils had higher amounts of sabinene, myrcene, -terpinene, borneol and carvacrol, and
Greek oregano oils had higher p-cymene content.
Bernotiene et al. (2002) collected the inflorescences, the leaves with stems and the
unseparated aerial parts of plants of Origanum vulgare L. ssp vulgare in 9 habitats in
eastern Lithuania in 1995-2000. The essential oils of the inflorescences and the leaves with
stems contained different quantities of major constituents. Gaspar et al. (2004) appraised
the yield and composition of the essential oils from different size bracts of Origanum
vulgare L. ssp. virens. Jelali et al. (2007) investigated the composition ofOriganum
majorana fresh leaves essential oils, extracted by hydrodistillation, by means of capillary
gas chromatography (CGC). The results showed that Origanum majorana leaf essential oil
mainly contained terpinen-4-ol, -terpinene. -terpinene and

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-terpineol. Tabanca et al. (2004) investigated two chemotypes of Origanum majorana L.
(Lamiaceae) in Turkey.
Atti-Santos et al. (2005) extracted the essential oils from the nineteen samples of
Rosmarinus officinalis by steam distillation using a pilot plant and evaluated them in terms
of chemical compositions and physico-chemical characteristics. Delamare et al. (2007)
analyzed the essential oils of Salvia officinalis and Salvia triloba cultivated in South Brazil
by GCMS. Flaminiet al. (2005) studied the compositions of the essential oils of three
Jordanian Salvia species, S. lanigera and S. spinosa from a desert climate, and S. syriaca
from a Mediterranean habitat. Skovia and Griensven (2006) characterized the essential oils
of M. spicata, Mentha piperita, Ocimum basilicum, Thymus vulgaris, Origanum vulgare,
Salvia officinalis, Lavandula angustifolia, Citrus limon and C. Aurantium. Angioni et al.
(2006) isolated essential oils from the stems/leaves (L) and flowers (F) of Lavandula
stoechas L. ssp. Stoechas growing wild in southern Sardinia (Italy) by hydrodistillation
and analyzed by gas chromatography coupled with flame ionization detector and ion trap
mass spectrometry.

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