Variations in essential oil compositions of Lavandula
pubescens (Lamiaceae) aerial parts growing wild in Yemen
Rowaida N. Al-Badani*a), Joyce Kelly R. Da Silvab)c), William N. Setzer*b), Nasser A. Awadh
Alid)e), Bushra A. Muharame), and Ahmed J. A. Al-Fahadf)
a
) Department of chemistry and chemistry of natural product, Faculty of Pharmacy, University
of Science and Technology, Sana’a, Yemen. (e-mail: [email protected])
b
) Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899,
USA (phone: 1-256-824-6519; fax: 1-256-824-6519; e-mail: [email protected])
) Programa de Pós-Graduação em Biotecnologia, Universidade Federal do Pará, Belém, PA
66075-900, Brazil (e-mail: [email protected])
c
d
) Pharmacognosy Department, Faculty of Clinical Pharmacy, Albaha
University, Al Baha, KSA (e-mail: [email protected])
) Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Sana’a
University, Sana’a, Yemen (e-mail: [email protected])
e
f
) Department of Chemistry, Faculty of Science, Albaha University, Al
Baha, KSA (e-mail: [email protected])
Abstract:
Lavandula pubescens Decne. is one of five Lavandula species growing wild in Yemen. The
plant is used in Yemeni traditional medicine, and the essential oil tends to be rich in carvacrol.
In this work, L. pubescens was collected from eight different locations in Yemen, the essential
oils obtained by hydrodistillation, and the oils analyzed by gas chromatography – mass
spectrometry (GC-MS). Principal component analysis (PCA) and hierarchical cluster analysis
(HCA) were used to differentiate between the L. pubescens samples. The essential oils were
rich in carvacrol (60.9-77-5%), with lesser concentrations of carvacrol methyl ether (4.011.4%), caryophyllene oxide (2.1-6.9%), and terpinolene (0.6-9.2%).
The essential oil
compositions in this study showed very high similarity, but it was possible to discern two
separate groups based on minor components, in particular the concentrations of terpinolene,
carvacrol methyl ether, m-cymen-8-ol, and caryophyllene oxide.
Keywords: gas chromatography – mass spectrometry; cluster analysis; principal component
analysis; carvacrol
1
Introduction
The genus Lavandula (Lavender) in the Lamiaceae comprises about 39 species, five of which
are wild-growing in Yemen, namely L. atriplicifolia Benth., L. citriodora A.G. Mill., L.
coronopifolia Poir., L. dentata L., and L. pubescens Decne. All species have their own distinct
smell and show a strong preference for volcanic soil [1]. A large part of the aroma and flavor
of the Lavandula genus is due to the presence of essential oils, some constituents of which
have also shown biological activity and could be responsible for the plants’ use in folk
medicine [2]. Some Lavandula species, and in particular L. dentata and L. pubescens, have
been used in Yemeni folk medicine. L. pubescens is used as an antiseptic and as a remedy for
broncho-pulmonary infections [3][4], as a carminative, insect repellent, and antiseptic [5]. L.
pubescens essential oils from Asir, Saudi Arabia [6] and Taiz, Yemen [7], were both rich in
carvacrol (85.3% and 20.6%, respectively). L. pubescens essential oil has shown antibacterial
activity against Salmonella enterica and Staphylococcus aureus, antifungal activity against
Aspergillus fumigatus and Candida albicans [8], and larvicidal activity against Culex pipiens
[6]. The high carvacrol concentration in L. pubescens likely accounts for the observed
bioactivity and its use in folk medicine.
L. pubescens is one representative of natural vegetation growing in Yemen. It occurs
naturally in mountainous regions including Taiz, Ibb, Yarim, Alkafer, Dhamar, Sana’a,
Amran, Huth, Khamir, Wadi Dhahr, Manakhaa, Shahara, Almahwit, Almahabishah and
Hajjah town.
The plant grows mainly in the slopes and wadis (streams), which are
characterized by steep ground with shallow, rocky soil [9].
There are several reports on the essential oil analyses of Lavandula species and the
results revealed that the essential oil content varies in different species growing in different
parts of the world. The percentage of the major chemical constituents (mainly oxygenated
2
monoterpenoids, monoterpene hydrocarbons and sesquiterpenoids) invariably differ from
species to species [10][11]. It was found that the variation in the essential oil composition is
influenced mainly by individual genetic variability, variation among different plant parts and
their different stages of development, and modifications due to the environment, e.g., weather,
light, latitude, altitude, soil, stress, etc. These factors, in particular the genetic makeup,
influence the plant’s biosynthetic pathways and, consequently, the relative proportion of the
main constituents [12][13]. Along these lines, several different chemotypes, based on
essential oil compositions, have been defined for Lavandula spp. [14][15]. For example, L.
pedunculata Cav., which has at least 10 subtaxa [16], has two recognized chemotypes from
Portugal [17][18]. Similarly, L. dentata has nine different subtaxa [16], and two different
chemotypes have been found in Tunisia [19]. L. stoechas L. has 32 recognized subtaxa [16],
and an analysis of eleven different populations of L. stoechas from Algeria revealed three
clusters [20].
Because of the chemical diversity observed in Lavandula essential oils, we
hypothesized that L. pubescens from Yemen may also show similar diversity in its essential
oils. The purpose of this study, therefore, was to determine chemical compositions of essential
oils from eight samples of L. pubescens plants collected from various localities in Yemen in
order to examine possible chemodiversity based on geographical location, and to define the
potential chemotypes within this species growing wild in Yemen.
Results and Discussion
L. pubescens samples were collected from eight different sites in Yemen (Table 1). A total of
56 compounds were identified in the L. pubescens essential oils from Yemen, accounting for
97.7-99.8% of the oil compositions (Table 2).
The major components in L. pubescens
essential oils were carvacrol (60.9-77-5%), carvacrol methyl ether (4.0-11.4%), caryophyllene
3
oxide (2.1-6.9%), and terpinolene (0.6-9.2%).
The compositions in this study are
qualitatively similar to those previously reported from Saudi Arabia [6] and Yemen [7].
Table 1. Collection data and essential oil yields of the samples of Lavandula pubescens
collected in Yemen.
Collection date
Altitude
Oil Yield
Latitude
Longitude
(time)
(m)
(%) ± SDa
26/10/2015
Taiz
13º 20′ 8.8″ N
44º 8′ 31.7″ E
1755.3
0.7± 0.028
I
(9 am)
17/10/2015
Sana’a
15º 26′ 29.5″ N
44º 6′ 39.3″ E
2419.1
0.4 ± 0.028
II
(7-9 am)
15/10/2015
Amran
15º 7′ 44.1″ N
44º 6′ 51.8″ E
2733.8
0.5 ± 0
III
(8-10 am)
3/10/2015
Ibb
14º 16′ 34.6″ N
44º 10′ 56.6″ E
1556.3
0.6 ± 0.057
IV
(8 am)
15/10/2015
Hajjah city
15º 49′ 59.9″ N
43º 24′ 59.9″ E
1446.9
0.5 ± 0
V
(8 am)
25/10/2015
Almahabeshah
15º 57′ 0.1″ N
43º 26′ 24.0″ E
1510.0
0.5 ± 0
VI
(9-11am)
22/10/2015
Almahweet
15º 28′ 0.6″ N
43º 28′ 47.6″ E
1335.0
0.4 ± 0.028
VII
(8-9 am)
3/11/2015
Dhamar
14º 32′ 23.4″ N
44º 18′ 0.7″ E
2451.0
0.4 ± 0
VIII
(7 am)
a
The yields are averages of four replicates collected from several different plants at each collection site.
Sample
Collect site
The occurrence of carvacrol as the major compound is common for essential oils from
Lavandula species. The main components of L. multifida oil were carvacrol (65.1%) and βbisabolene (24.7%), while high proportions of (E)-β-ocimene (26.9%), carvacrol (18.5%) and
β-bisabolene (13.1%), characterized L. coronopifolia oil [21]. The oil of L. coronopifolia
from Morocco was rich in carvacrol (48.9%), β-caryophyllene (10.8%) and caryophyllene
oxide (7.7%) [22].
In order to differentiate between the analyzed L. pubescens samples, a hierarchical
cluster analysis (HCA) using the chemical profile has been carried out and the resulting
dendrogram is shown in Fig. 1. The samples (LpI – LpVIII) showed a high similarity level
(98.96%).
4
Table 2. Chemical compositions of essential oils of Lavandula pubescens from Yemen.
Component
β-Pinene
Myrcene
α-Phellandrene
δ-3-Carene
α-Terpinene
p-Cymene
Limonene
1,8-Cineole
(Z)-β-Ocimene
(E)-β-Ocimene
Terpinolene
Linalool
endo-Fenchol
allo-Ocimene
neo-allo-Ocimene
Cyclononanone
trans-3-Caren-2-ol
(E)-Epoxy-ocimene
Citral
Terpinen-4-ol
m-Cymen-8-ol
p-Cymen-8-ol
α-Terpineol
β-Cyclocitral
p-Menth-1-en-9-al
Carvacrol methyl ether
Carvotanacetone
Carvacrol
trans-Pinocarvyl acetate
Eugenol
α-Duprezianene
RIcalca
985
987
997
1003
1011
1020
1023
1027
1035
1046
1088
1100
1112
1131
1134
1134
1134
1147
1175
1175
1183
1186
1198
1200
1216
1235
1245
1291
1302
1357
1388
RIlitb
979
990
1002
1011
1017
1024
1029
1031
1037
1050
1088
1096
1116
1132
1140
Lp-I
2.60
0.42
0.14
0.60
0.61
0.76
2.01
0.12
0.27
1244
1247
1299
1298
1356
1388
Lp-III
0.98
0.26
0.27
Lp-IV
1.39
1.57
0.32
0.29
0.26
0.31
0.58
0.72
0.14
1.14
7.53
0.06
0.15
0.06
Lp-V
1.07
0.10
0.38
0.29
1.18
Lp-VI
0.80
0.09
0.21
0.12
0.23
0.26
3.86
0.13
Lp-VII
1.15
Lp-VIII
1.08
0.99
0.07
0.11
0.27
0.30
0.44
0.72
2.20
0.08
2.22
0.84
0.83
0.17
0.27
0.18
0.17
1136
1142
1174
1174
1179
1182
1186
1217
Lp-II
0.97
1.03
0.28
0.44
0.44
0.37
0.39
0.39
0.30
0.05
9.16
0.07
0.09
0.13
0.31
0.16
0.05
0.10
0.09
2.03
2.16
0.57
1.28
0.53
5.90
0.33
4.32
0.30
67.83
0.63
0.23
0.86
0.76
0.69
0.18
0.45
0.16
3.30
0.33
2.78
0.16
7.31
0.09
0.48
1.75
0.09
0.13
1.94
0.10
2.88
6.95
0.17
11.39
3.45
6.74
3.96
72.72
60.87
9.52
2.27
61.79
73.28
75.77
0.11
0.10
0.12
77.46
0.09
0.16
0.33
69.82
0.09
0.14
0.05
0.12
0.05
5
β-Caryophyllene
1418
1419
2.08
3.74
1.96
2.47
3.38
3.57
1.95
1432
1436
0.10
-Elemene
α-Humulene
1451
1454
0.12
0.19
0.13
0.15
0.19
0.18
0.12
(E)-β-Ionone
1484
1487
0.06
0.05
Aciphyllene
1498
1501
0.05
β-Bisabolene
1509
1505
2.46
1.65
2.42
2.41
2.08
2.10
2.04
cis-Dihydroagarofuran
1519
1520
0.11
Silphiperfol-5-en-3-one B
1546
1551
0.10
Germacrene B
1554
1561
0.10
Silphiperfol-5-en-3-one A
1570
1575
0.17
Caryophyllene oxide
1581
1583
2.13
3.74
2.65
5.22
5.91
6.92
5.00
Guaiol
1596
1600
0.08
0.09
0.07
0.07
0.11
0.09
0.08
Rosifoliol
1605
1600
0.21
Humulene epoxide II
1606
1608
0.36
0.34
0.30
0.41
0.26
0.29
1,10-di-epi-Cubenol
1612
1619
0.09
0.08
Caryophylla-4(12),8(13)dien-5-α-ol
1633
1640
0.13
0.09
0.12
0.11
0.20
0.09
0.12
-Eudesmol
1636
1632
0.30
0.08
0.07
epi-α-Cadinol
1638
1640
0.07
β-Eudesmol
1647
1649
0.44
0.45
0.34
0.55
0.36
0.32
Pogostol
1651
1653
0.08
0.11
0.08
0.08
0.11
0.09
α-Eudesmol
1654
1652
0.14
0.18
0.16
0.14
0.34
0.25
0.20
Bulnesol
1664
1670
0.10
14-hydroxy-9-epi-(E)Caryophyllene
1667
1669
0.13
0.09
0.18
0.11
0.19
0.07
0.17
Germacra-4(15),5,10(14)trien-1-α-ol
1690
1685
0.05
α-Bisabolol
1691
1685
0.05
Monoterpene hydrocarbons
7.41
13.43
3.56
12.57
3.02
5.57
7.12
Oxygenated monoterpenoids
81.00
75.42
83.99
76.51
82.0
83.59
82.09
Sesquiterpene hydrocarbons
4.66
5.83
4.51
5.03
5.65
5.90
4.16
Phenylpropanoids⁄Others
0.26
0.36
0.34
0.10
0.12
0.21
Total
99.63
99.16
99.76
99.23
99.66
99.22
99.64
a
RIcalc = Calculated retention indices determined with reference to a homologous series of n-alkanes on an HP-5ms column.
b
RIlit = Retention indices published in Adams (2007) [23].
6
0.08
2.54
5.52
0.06
0.26
0.08
0.09
0.22
0.17
0.13
5.25
83.16
2.62
0.12
97.68
Figure 1. Dendrogram obtained by hierarchical cluster analysis (HCA), based on the
composition of essentials oils from Lavandula pubescens.
Despite the similarities between the essential oils, we were able classify the
samples into two separate groups (Fig. 1). Group 1 is composed of samples Lp-I, LpIII, Lp-V, Lp-VII and Lp-VIII and shows a similarity level of 99.5%. In the oils Lp-I,
Lp-III, Lp-VII and Lp-VIII, the main compounds were carvacrol, carvacrol methyl
ether and caryophyllene oxide with average concentrations of 72.9, 5.9, and 5.4%,
respectively. However, sample Lp-V showed a high content of carvacrol (61.8%),
carvacrol methyl ether (9.5%) and m-cymen-8-ol (7.3%). In addition, higher
concentrations of oxygenated sesquiterpenoids (6.3 to 8.8%) were detected in this
group.
Group 2 is composed of samples Lp-II, Lp-VI and Lp-IV and shows a
similarity level of 99.3%. The samples Lp II and Lp-VI showed the highest
concentrations of monoterpene hydrocarbons (13.4 and 12.6%) and lower
concentrations of oxygenated monoterpenoids (75.4 and 76.5%) in comparison with
other samples. The oils Lp-II and Lp-VI showed as the main compounds carvacrol
7
(69.8 and 77.5%) and terpinolene (9.2 and 3.6%). Furthermore, in these samples were
detected lower concentrations of carvacrol methyl ether (4.3 and 3.5%) and
caryophyllene oxide (2.1 and 2.7%). The main compounds of Lp-IV oil were
carvacrol (60.9%), carvacrol methyl ether (11.4%) and terpinolene (7.53%).
Recently, principal component analysis (PCA) has been shown to be an
important multivariate statistical method that can be applied to differentiation of
chemical composition of essential oils among individuals from different populations
[24][25]. PCA analysis of the L. pubescens essential oils in this work shows that the
components PC1 and PC2 have explained 100% of phytochemical variation among all
samples, which were classified in three groups with higher similarity (Fig. 2). The
first PC (PC1) explained 99.3%. Generally, the major variation of the data can be
represented by the first component [26]. PC1 had positive correlations with
terpinolene and carvacrol methyl ether and negative correlations with carvacrol. The
more positive loadings were observed in samples Lp-II, Lp-IV and Lp-V, which
displayed the lower concentrations of carvacrol with values of 67.83, 60.87 and
61.79%, respectively. PC2, with only 0.99% of variance showed high positive
contributions from minor compounds such as p-cymen-8-ol, eugenol and βcaryophyllene, and negatively correlated with m-cymen-8-ol, carvacrol methyl ether,
caryophyllene oxide and β-pinene.
Group I is characterized by samples Lp-II and Lp-VI (see Fig. 2), which
displayed more positive loadings in PC2 (0.573 and 0.385, respectively). The oil Lp-II
showed a higher concentration of terpinolene (9.16%). However, there is similarity
between Lp-II and Lp-VI due to the concentrations of carvacrol methyl ether (4.32
and 3.45%, respectively), β-caryophyllene (3.74 and 3.57%, respectively) and other
minor components. Group II includes the samples Lp-I, Lp-III, Lp-IV, Lp-VII and
8
Lp-VIII, which the main constituents were carvacrol (60.87-75.77%), carvacrol
methyl ether (3.96-11.39%), caryophyllene oxide (3.74-6.92%) and terpinolene (1.147.53%). Group III was composed of the individual sample Lp-V, which the main
compounds were carvacrol (61.79%) and carvacrol methyl ether (9.52%). In addition,
this sample showed higher concentrations of m-cymen-8-ol (7.31%) and
caryophyllene oxide (6.92%).
Figure 2. Bidimensional plot of first two components obtained by PCA analysis of
Lavandula pubescens based on chemical composition of the essential oils.
There is a clear relationship between mean annual rainfall and topography in
Yemen. Rainfall ranges from of 400-800 mm in the southern, central, and western
highlands and decreases steadily to below 300 mm in northern and eastern highlands.
Average temperatures are dominantly controlled by altitude [27]. Climate data for the
collection sites in this work are summarized in Table 3. Soil surveys have shown that
most Yemeni soils are calcareous, tend to be alkaline (pH ~ 7.0-7.8), with low levels
of organic matter. Available nitrogen is generally low to very low, with medium to
9
very low levels of phosphates, and high levels of potassium (Table 3) [28][29]. In
order to determine any correlation between the principle components of the essential
oils and the geographical location, elevation and climatic conditions, Pearson’s
correlation coefficient was carried out. As shown in Table 4, Pearson correlation (p <
0.05) reveals that β-pinene concentration negatively correlates with latitude, while
myrcene concentrations positively correlates with rainfall. The other major
components showed no statistically significant correlations to climatic or location
data; the surprising situation is the improbable similarity of the essential oils, despite
the differences in collection sites.
Table 3. Climate data and soil properties for the Lavandula pubescens collection
sites in Yemen.
Soil Propertiesd
Collection site
a
RF (mm)
HT (ºC)
LT (ºC)
b
c
pH
OM
N
P
K
Taiz
400
25
9
7.4
L
L
L
H
Sana’a
350
25
9
7.8
VL
VL
L
H
Amran
300
25
9
7.9
VL
VL
VL
H
Ibb
700-800
18
11
7.2
M
F
M
H
Hajjah city
300-400
22
8
7.5
L
L
L
H
Almahabeshah
300-350
22
8
7.4
M
L
L
H
Almahweet
350-400
25
9
7.7
L
L
L
H
Dhamar
400
20
6
7.8
VL
VL
VL
H
Average annual rainfall (mm) [28][29]. Average High Temperature (October/November), ºC [30]. c
Average Low Temperature (October/November), ºC [30]. d Soil properties taken from [28][29], OM =
organic matter, N = nitrogen, P = phosphates, K = potassium, L = low, VL = very low, M = medium, F
= fair, H = high.
a
b
Analogous to the results in this study, the chemical composition of floral
essential oils of L. stoechas collected from three areas of Sicily (Italy) did not display
10
significant variation in the main component, the oxygenated monoterpenoid fenchone
(52.8-71.1%). However, the essential oils revealed a high variability in minor
compounds as (0.1-12.8%) and camphor (6.6-12.1%) [31]. On the other hand, the EOs
of twelve wild Tunisian populations of L. multifida showed significant chemical
variation between their main compounds: carvacrol (10.73 – 42.99%), β-bisabolene
(12.23 – 21.00%), acrylic acid dodecyl ester (2.31 – 25.9%) and β-phellandrene (3.13
– 17.21%). These data were analyzed by principal component analysis (PCA) and
hierarchical cluster analysis (HCA) and divided into three groups, which were defined
by variations in the minor components, such as caryophyllene oxide (trace – 42.99%).
Additionally, the chemical variation of L. multifida specimens displayed little
correlation with their bioclimatic or geographic location [13].
Table 4. Pearson’s correlation (p < 0.05) between environmental variables and
Lavandula pubescens essential oil major component concentrations.
Compounds
Latitude
Longitude
Altitude
Rainfall
High Temp
Low Temp
Carvacrol
0.142
-0.063
0.275
-0.585
0.312
-0.580
Carvacrol methyl ether
-0.147
-0.041
-0.383
0.662
-0.396
0.639
Caryophyllene oxide
-0.144
-0.081
0.001
-0.175
0.031
-0.291
Terpinolene
0.045
0.213
-0.012
0.464
-0.142
0.588
m-Cymen-8-ol
0.112
-0.224
-0.150
0.015
-0.229
-0.137
β-Caryophyllene
0.506
-0.510
-0.348
-0.101
0.214
0.431
β-Bisabolene
-0.658
0.453
0.139
0.320
-0.402
-0.116
β-Pinene
-0.862a
0.342
-0.181
0.237
0.171
0.256
p-Cymen-8-ol
-0.234
-0.001
-0.095
-0.245
0.366
0.022
(Z)-β-Ocimene
-0.107
-0.072
-0.067
-0.171
0.435
-0.053
Myrcene
-0.281
0.621
0.219
0.738a
-0.644
0.239
a
Correlation coefficient statistically significant at p < 0.05.
11
Conclusions
Principal component analysis (PCA) and cluster analysis have allowed separation of
the L. pubescens populations into two groups that are defined by minor components
rather than major compounds, and therefore L. pubescens samples in this study
represent a single chemotype. There are, however, no apparent correlations between
the groups and their bioclimatic or geographical locations.
Material and Methods
Plant Material
Eight specimens (aerial parts) of Lavandula pubescens Decne. were collected from
different localities in Yemen during the period of October and November 2015. Each
sample was collected from several plants at each collection site. The plant samples
were taxonomically identified by the Botanist Dr. Hassan M. Ibrahim at the Botany
Department, Faculty of Sciences, Sana’a University. A voucher specimen (YMPLam-4) has been deposited at the Pharmacognosy Department, Faculty of Pharmacy,
University of Science and Technology, Yemen. Plant materials were air-dried in the
shade for 10 days before hydrodistillation. The collection data are summarized in
Table 1.
Essential Oil Distillation
Dried, crushed aerial parts (50 g each, four replicates) from eight samples of L.
pubescens were hydrodistilled for 3 h in a Clevenger type apparatus according to the
European Pharmacopoeia method [32]. The obtained oils were subsequently dried
over anhydrous Na2SO4 and kept at 4°C until analysis.
12
Oil Composition Analysis
The essential oils of L. pubescens were analyzed by GC-MS using an Agilent 6890
GC with Agilent 5973 mass selective detector (MSD) [operated in the EI mode
(electron energy = 70 eV), scan range = 40 – 400 amu, and scan rate = 3.99
scans/sec], and an Agilent ChemStation data system. The GC column was an HP-5ms
fused silica capillary with a (5% phenyl)-polymethylsiloxane stationary phase, film
thickness of 0.25 μm, length of 30 m, and internal diameter of 0.25 mm. The carrier
gas was helium with a column head pressure of 48.7 kPa and a flow rate of 1.0
mL/min. Injector temperature was 200ºC and detector temperature was 280ºC. The
GC oven temperature program was used as follows: 40ºC initial temperature held for
10 min; increased at 3ºC/min to 200ºC; increased at 2ºC/min to 220 °C. A 0.2% w/v
solution of the sample in CH2Cl2 was prepared and 1 μL was injected using a splitless
injection technique.
Identification of the oil components was based on their retention indices
determined by reference to a homologous series of n-alkanes, and by comparison of
their mass spectral fragmentation patterns with those reported in the literature [23]
and
stored
on
the
MS
library
[NIST
database
(G1036A,
D.01.00)/ChemStation data system (G1701CA, version C.00.01.080].
revision
The
percentages of each component are reported as raw percentages based on total ion
current without standardization.
Statistical Analysis
Cluster analysis was used to classify and group the essential oils according to their
main volatile constituents. Complete linkage and absolute correlation coefficient
distance was selected as a measure of similarity. For the grouping of the oil samples
the agglomerative and hierarchical method was applied. Principal component analysis
13
was performed on a correlation matrix for the visual comparison of the chemical
compositions of the different L. pubescens samples. Pearson correlation coefficient
was used to determine any correlation between the principle components of the
essential oils and the geographical and climate data. All data were statistically
analyzed using the MINITAB 14.0 software.
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