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Chemical Diversity and Antioxidant Capacity of

Essential Oils of Marjoram in Northwest Egypt
ab
Hosam O. Elansary
a
Biodiversity Institute of Ontario, University of Guelph, ON N1G 2W1, Canada
b
Floriculture, Ornamental Horticulture and Garden Design Department, Faculty of
Agriculture (El-Shatby), Alexandria University, Alexandria, Egypt
Published online: 04 Sep 2015.

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To cite this article: Hosam O. Elansary (2015) Chemical Diversity and Antioxidant Capacity of Essential Oils of Marjoram in
Northwest Egypt, Journal of Essential Oil Bearing Plants, 18:4, 917-924, DOI: 10.1080/0972060X.2014.958561

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 TEOP 18 (4) 2015 pp 917 - 924 917
ISSN Print: 0972-060X
ISSN Online: 0976-5026

Chemical Diversity and Antioxidant Capacity

of Essential Oils of Marjoram in Northwest Egypt
1,2
Hosam O. Elansary *

1
Biodiversity Institute of Ontario, University of Guelph, ON N1G 2W1, Canada
2
Downloaded by [University of Guelph Library] at 05:58 04 September 2015

Floriculture, Ornamental Horticulture and Garden Design Department,

Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, Egypt
Received 14 April 2014; accepted in revised form 24 October 2014

Abstract: Origanum majorana “Marjoram” cultivation in Egypt dates back to 3000BC with current
global distribution and economic importance in medicinal and herbal products. Plant natural diversity and
scattered cultivated populations may harbor chemical diversity useful in agricultural and pharmaceutical
industries. To investigate this assumption in Northwest Egypt, leaves essential oil (EO) composition of local
accessions of Origanum majoranaL. are examined and in comparison in this study. Antioxidant activities (AA)
using both of 2,2’-diphenypicrylhydrazyl (DPPH) and β-carotene-linoleic acid assay were examined for EOs.
Main EOs constituent were cis-sabinene hydrate 34.3 %, α-terpinen 18 %, 4-terpineol 15.20 %, terpinolene 11.8
%, and sabinene 7.4 %; α-terpinen 29.2 %, cis-sabinene hydrate 15.40 %, terpinolene 19.50 %, and 4-terpineol
14.40 %; 4-terpineol 35 %, cis-sabinene hydrate 20 %, terpinolene 10.33 %, α-terpinen 8.96 %, and sabinene 8.4
% in the accessions OM550, OM555 and OM560; respectively. Examined accessions were classified into two
chemotypes easily using their Eos harboring either sabinene or terpinens as major constituents. High antioxidant
capacities were related to high sabinene and terpinens contents. It was concluded that Egyptian marjoram
harbor good quality essential oils could be utilized as food additives and for improving human health.

Key words: Marjoram; GC-MS; antioxidant; Origanum majorana.

Introduction
The genus Origanum (family Lamiaceae) is
characterized by large morphological and chemical
diversity resulting in the distinction of 49 taxa and
42 species 1. One of the main species of the genus
Origanum is O. majorana L. which is a tender
perennial herb native to North Africa, some parts
of Asia and southern Europe and was formerly
classified as Majorana hortensis Moench 2.
Marjoram is widely cultivated medicinal plant in
Egypt, Greece, France and other Mediterranean
countries in addition to USA, India and Canada 3,
furthermore, it was cultivated in Egypt over 3000
years ago and used medicinally by ancient
Greeks 4. The plants are usually harvested when
*Corresponding author (Hosam O. Elansary)
E-mail: < hosammail2003@yahoo.com; hoelansa@uoguelph.ca >
full blooming occurs, and are used either fresh or
dried as food spices either in the Arabian or
western kitchen. Leaves, flowers and tender
stems flavor syrups, stews, dressing liqueurs,
sausages, meats and seafoods and contain
antioxidants in their extracts or in essential oils
3,5,6
. The plant has been used in folk medicine for
the treatment of asthma, indigestion, headaches,
rheumatism, toothache, muscular pain, relief of
childbirth pain, flatulence, and ear problems 7,8.
Marjoram volatile oils were used in the treatment
of flatulence, colic, rheuma-tism, toothaches and
to promote perspiration in people suffering measles
3,9
. Recently, marjoram oils are used for scenting
soaps, detergents, creams, lotions and perfumes
© 2015, Har Krishan Bhalla & Sons
 Hosam O. Elansary / TEOP 18 (4) 2015 917 - 924
with a maximum level of 0.60 % in perfumes, below
0.004 % in fresh foods and less than 1 % in
processed foods 10.
There are a wide range of ecotypes and chemo-
types of marjoram and there are confusions
among cultivated species especially with the pot
marjoram or the Turkish oregano (Origanum
onites) and wild marjoram (Thymus mastichina
L.). Pot marjoram is native to Syria, Turkey and
southern Europe whereas wild marjoram is native
to Spain and North Africa and all of them are
named or used as oregano 2. The complexity
increases, knowing that marjoram was already a
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hybrid and hybridized during cultivation with
Origanum vulgare to form O. x applii which is
grown in west and central Europe 3.
Marjoram oil is yellowish with a spicy aroma,
reminiscent of lavender and accurate species and
subspecies identification is directly related to its
economic value and commercial uses. Several
methods of species identification in Origanum had
been proposed using molecular markers such as
RAPD 11, AFLP 12 and rDNA sequencing 13or
morphological and chemotyping of essential oils
of accessions 14,15. Within species variation had
not been studies intensively in Egypt.
In Egypt, Origanum is used as meat spices and
for culinary purposes also. Egypt is one of the
world producers of dried plants and fresh oil and
Figure 1. Showing the locations of samples in Northwest Egypt
918
one of the main suppliers of Western Europe
countries such as Germany. Egyptian flora
includes some Oreganos pecies such as O.
syriacum and O. majorana 16,17 in addition to a
large variation of unstudied accessions scattered
across the country. The need for accurate identi-
fication tools using either genotyping or chemo-
typing is rising in the local and international
community.
The objective of this work was to test available
chemotypes in Northern west Egypt and especially
Alexandria area using essential oil analyses.
Furthermore, to define the best way to identify
local accessions accurately and evaluated the
antioxidant capacities of the essential oil.
Materials and methods
Plant material
Leaf samples were collected in April 2012 from
three accessions (OM550, OM555 and OM560)
of (marjoram) Origanum majorana L. from
three different geographic locations in Northwest
Egypt (Figure 1) and were grown in similar
growing conditions at latitude (30.925579), longi-
tude (29.775524) located at Alexandria-Cairo
desert road, Alexandria Egypt. All samples (5 of
each case) obtained vouchers at Egypt Barcode
of Life project (www.egyptbol.org), faculty of
Agriculture, Alexandria University (voucher No.
 Hosam O. Elansary / TEOP 18 (4) 2015 917 - 924
Hosam00550-Hosam00565; respectively).
Chemicals
Methanol, Tween 40, linoleic acid, β-carotene
and 2,2’-diphenyl-1-picrylhydrazyl (DPPH) were
bought from Sigma Aldrich Egypt.
Isolation of essential oils
200 Grams of fresh leaves were cut into small
pieces and hydro-distilled for 2 h, using a
Clevenger-type apparatus. The distilled essential
oils were dried over anhydrous sodium sulphate,
filtered and stored in sealed vials at 4oC.
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Gas chromatography/mass spectrometry
(GC-MS)
Essential oils of O. majorana accessions were
analyzed with Thermo Scientific gas chromato-
graph (Trace GC Ultra) coupled with Thermo
Scientific Mass spectrometer (ISQ) instrument
available at the Institute of Graduate Studies and
Research, Alexandria University. Separation was
accomplished with TG-1MS narrow bore column
(length 30 m × 0.32 mm ID, 0.25 μm film thick-
ness). Helium was used as carrier gas. The oven
temperature was programmed to increase from
an initial temperature of 45°C to 165°C at 4°C
min-1with holding time of 2min at 45°C, then
15°Cmin-1 to 280°C, with a final holding time of
15 min. Samples (2 μL) were injected at 250°C
on splitless mode flow (1 ml min-1), and splitless
time (3 min) then split flow (10 ml min-1).
The GC-FID analysis was conducted on the
same column and temperature program.
Identification of compounds based on retention
time and retention indices related to homologous
series of n-alkanes (C10-C36) analyzed on the
same conditions of sample analysis on GC-FID
and computer matching with the NIST mass spec-
tral search programVer. 2.0 and WILEY libraries
in addition to references from literature 18.
Antioxidant capacity
Free radical scavenging activity of the samples
was determined using the 2,2’-diphenypicryl-
hydrazyl (DPPH) method 19 with some modi-
fications. The reaction mixture was mixed for 10s
and left to stand in fibber box at room temperature
919
in the dark for 30 min. The absorbance was
measured at 517 nm, using a UV scanning spectro-
photometer (Unico® 1200). Total antioxidant
activity (TAA) was expressed as the percentage
inhibition of the DPPH radical and was determined
by the following equation:
Abs control - Abs
% TAA =
sample
x 100
Abs control
Where TAA is the total antioxidant activity and
Abs is the absorbance, Abs control: The absorb-
ance of control reaction, and Abs sample: The
absorbance of sample.
Tests were carried out in triplicate. The β-
Carotene-linoleic acid assay was made using the
method described by Tepe, et. al.19 with some
modifications by Ferreira, et. al., 20. The absorb-
ance was measured at 470 nm.
Results
Chemotyping using essential oils analyses
Essential oils yields were 2.3, 2.62 and 2.5 %
for OM550, OM555 and OM560; respectively.
The main oil constituents in OM550 were cis-
sabinene hydrate 34.3 %, β-terpinen 18 %, 4-
terpineol 15.20 %, terpinolene 11.8 % and sabinene
7.40 %; in OM555 were α-terpinen 29.20 %, cis-
sabinene hydrate 15.40 %, terpinolene 19.50 %,
and 4-terpineol 14.40 %; and in OM560 were 4-
terpineol 35 %, cis-sabinene hydrate 20 %,
terpinolene 10.33 %, α-terpinen 8.96 %, and
sabinene 8.40 % (table 1).
Two chemotypes were found, one of them was
rich in cis-sabinene which is OM550 and the
second chemotype was rich in either terpinen or
terpineol, both of OM555 and OM560 showed
lower content of cis-sabinene hydrate whereas
sabinene was found in the three accessions with
an almost similar value ranging from 7.40 to 8.40
%. α-Terpinen was the highest in OM555whereas
much lower in OM550 and OM560. Accession
OM550 could be easily distinguished by the high
content of cis-sabinene hydrate. Accession
OM555 could be distinguished with the high value
of α-terpinen reaching approximately 30 %,
whereas accession OM560 is characterized by
high percentage of 4-terpineol (35 %).
 Hosam O. Elansary / TEOP 18 (4) 2015 917 - 924 920
Table 1. Chemical constituents of the essential oils from Origanum
majorana L. accessions leaves collected from Northwest Egypt

RI Compounds OM550 OM555 OM560

946 α-Pinene 0.30 0.10 0.22

974 Sabinene 7.40 7.30 8.40
978 β-Pinene 0.60 0.70 0.90
985 Myrcene 0.23 0.11 0.12
1015 α-Terpinene 18.00 29.20 8.96
1035 Terpinolene 11.80 19.50 10.33
1037 Limonene 0.20 0.00 0.11
1040 Cineole 0.13 0.12 0.15
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1067 γ-Terpinen 0.50 0.43 0.44

1080 cis-Sabinene hydrate 34.30 15.40 20.00
1117 Linalool 0.24 0.25 0.20
1142 cis-p-Menth-en-1-ol 0.11 0.09 0.06
1160 trans-p-Menth-en-1-ol 0.40 1.57 2.70
1188 Borneol 0.12 0.20 0.10
1190 Myrtenol 0.06 0.10 0.12
1192 Terpinen-4-ol/4-Terpineol 15.20 14.40 35.00
1198 Terpineol 2.40 3.60 3.20
1233 Geraniol 0.30 0.50 0.12
1322 Methyl (E)-cinnamate 0.12 0.21 0.13
1336 Myrtenyl acetate 0.23 0.10 0.30
1366 Copaene 0.15 0.14 0.10
1371 Methyl eugenol 0.23 0.22 0.15
1373 Anisole 0.31 0.30 0.15
1379 β-Elemene 0.12 0.12 0.23
1384 β-Cubebene 0.10 0.35 0.25
1386 α-Guaiene 0.15 0.20 0.20
1434 α-Bergamotene 0.11 0.12 0.11
1436 α-Caryophyllene 0.12 0.22 0.23
1438 Germacrene D 0.11 0.10 0.31
1442 β-Farnesene 0.14 0.15 0.24
1443 Caryophyllene 2.30 1.60 1.80
1466 γ-Elemene 0.12 0.08 0.04
1470 γ-Gurjunene 0.23 0.08 0.13
1552 epi-Globulol 0.20 0.21 0.24
1571 Caryophyllene oxide 0.60 0.31 1.60
1584 Spathulenol 0.30 0.12 0.36
1605 Cubenol 0.40 0.12 0.11
1620 Methyl jasmonate 0.22 0.23 0.21
1624 α-Cadinol 0.12 0.09 0.22
1643 β-Eudesmol 0.26 0.23 0.16
1699 α-Bisabolol 0.19 0.22 0.40
Unidentified compounds 1.11 0.91 1.20
Total 100.00 100.00 100.00
 Hosam O. Elansary / TEOP 18 (4) 2015 917 - 924
Caryophyllenes were presented ranging from
1.80 to 2.30 % in the three accessions and were
the highest in accession OM550. Pinenes, cineole,
linalool, geraniol, myrcene and cis-p-Menth-en-
1-ol didn’t exceed 1 % in all examined accessions.
Antioxidant capacity
Free radicals such as super oxide and hydroxyl
induce damage in the human body and could cause
aging and may cause diseases such as cancer and
heart diseases 21. Researchers around the world
are in active mining for new natural resources of
antioxidants which eliminates much of the damage
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of free radicals and does not have hazardous
effects on the human health. Essential oils as
natural products represent an important resources
of antioxidants especially the family Lamiaceae
members such as Ocimum 23 and Oregano 24.
Antioxidant capacity could be measured by
reducing capacity, free radical scavenging and
other methods. In this study, two methods were
used for the evaluation of antioxidant activity of
the essential oils of O. majorana which are
DPPH and β-carotene-linoleic acid assay. Free
radical scavenging activity using DPPH results
presented as TAA (Total Antioxidant Activity) %
were 80.10, 73.70 and 62.40 % for OM550,
OM555 and OM560; respectively. The β-carotene
assay results were 83, 78.60 and 66.50 % for O.
majorana A, B and C; respectively.Accession
OM550, was much higher than both accession of
OM555 and OM560 in both methods in addition
that both methods had similar trends.
Discussion
In this study, 43 compounds were identified in
O. majorana essential oils, many of them worked
as identifiers for each accession. The results
agrees with Baatour et al. 25, they reported that
large percent of the total volatiles quantified
originated from cis-sabinene hydrate, 4-terpineol
(terpinen-4-ol), and sabinene whereas no thymol
was detected in the three accessions examined in
this study. Contradicting one report by Solimanet
al. 16 on one accession in northeast Egypt, although
they didn’t detect thymol in most examined cases
except one case. The O. syriacum L. is either
reach in thymol or in carvacrol 26-29. In general,
921
O. majorana is characterized for its high content
of each of cis and trans-sabinene hydrate 30-32.
Exception for this pattern had been described in
Turkey. In Turkey, three O. majorana chemo-
types were described one of them contains cis-
sabinene hydrate, 4-terpineol (terpinen-4-ol) and
linalool and the second contains mainly carvacrol
33-35
, furthermore the second chemotype produce
high amounts of oils ranging from 5-8 %, whereas
the third chemotype contained linalool mainly 33.
It was suggested that the O. majorana section
vary not only at the sectional level, but also within
the species 36. Furthermore the genus Oregano
was classified according to chemotype into three
groups, the first contains mainly p-cymyl
compounds in their essential oils such as thymol,
carvacrol, p-cymene and others in species such
as in O. syriacum and O. rotundifolia, the
second group contains mainly sabinene and cis or
trans-sabinene hydrate such as in O. majorana ,
and the third group contains minor compound but
in high ratios such as linalool, geraniol, and linalyl
acetate in addition to other compounds. In addition
to these groups there are hybrids such as the
hybrid between O. majorana and O. vulgare
resulting in cymol compounds reaching to 65 %
simulating O. vulgare as reported by Bosabalidis
and Skoula 37 and Skoula et al.38.
The presence of high ratios of carvacrol in
Oregano (white oregano) and cis-sabinene
(sweet oregano) in O. majorana are the major
factors of high quality of their respective essential
oils in addition to the high production of essential
oils and their green oil color 39. In the Egyptian
accessions of O. majorana examined in this
study, we found high ratios of sabinene which may
qualify for economic production and exporting due
to international standards of their essential oils
qualities. Chemotypes, environmental factors and
growing conditions had significant effect on the
oil composition of marjoram oils 17, indicating that
commercial production requires determining
specific chemotype and stabilizing growing con-
ditions.
All of the examined accessions showed high
antioxidant capacities which might be explained
by the similar chemical composition of their volatile
oil especially the content of terpineols and sabi-
 Hosam O. Elansary / TEOP 18 (4) 2015 917 - 924
nenes. Terpenoids found in this study (terpineol,
4-terpineol & terpinolen) played a major rule in
antioxidant activity as found before in other genera
40
. Nejla and Moncef 41 reported that Tunisian O.
majorana had high antioxidant activity and was
useful in the suppression of the oxidation process
of vegetable oils and animal fats such as lard.
Tunisian marjoram shows similar chemical pattern
to the examined ones in this study which indicates
the use of these accessions in food processing
and inhibition of oxidative reactions in oils.
Furthermore, Alizadeh et al. 24 reported essential
oils of O. majorana from Iran with similar
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922
the high amount of cis-sabinene hydrate in
accession OM550 and also available in lesser
ratios in accessions OM555 and OM560 which is
similar case to that found in Iran and Tunisia.
Conclusion
High diversity of O. majorana was found in
the Egyptian accessions by means of their essen-
tial oils composition “Chemotyping”. Furthermore,
Egyptian marjoram has promising future in the field
of industrial applications of essential oils especially
as food additive and for human health improve-
ment. Indeed further investigations could be

composition of cis and trans-sabinene hydrate and conducted on the genetic background of taxa
terpeniols and they had high antioxidant capacities. diversity of Origanum comparing larger sampling
The highest antioxidant capacity of O. majorana areas including southern parts of Egypt which are
accessions examined in this study may be due to known for the production of medicinal herbs.

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