Determination of Essential Oils and Heavy Metals Accumulation in Salvia officinalis Cultivated in three Intra-raw Spacing in Ash-Shoubak, Jordan

Abstract

Essential oil yields and heavy metals content were determined in Salvia officinalis, cultivated in three intra-raw spacing (15, 30 & 45 cm) in the research farm of Ash-Shoubak University College. Specimens were harvested at the vegetative, beginning of blooming, full-blooming and fruit maturation stages. Essential oil concentration and content of heavy metals in the plant specimens were determined by using hydrodistillation and atomic absorption spectrometry methods, respectively. The yields of essential oil and heavy metal contents were affected by intra-raw spacing and phenological stage. The maximum oil yield was obtained in plant cultivated in 15 cm planting space (2.00) and harvested during vegetative stage, while the minimum oil content was detected in the plants cultivated in the same raw planting space and harvested at maturation stage. Heavy metals contents were variable depending on both intra-raw spacing and phenological stage. Co, Cd, and Pb were not detected. Contents of Ni, Zn, Fe and Cu were increased during the vegetative stage of the plant but still below their toxic level. Results indicated that S. officinalis cultivated in 15 cm planting space and harvested at the vegetative stage in Ash-Shoubak is rich in essential oils and free from hazardous heavy metals.

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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
ISSN Print: 1560–8530; ISSN Online: 1814–9596
11–264/AWB/2011/13–6–981–985
http://www.fspublishers.org
Full Length Article
To cite this paper: Abu-Darwish, M.S., A.H. Al-Fraihat, S.Y.A. Al-Dalain, F.U. Afifi and J.A. Al-Tabbal, 2011. Determination of essential oils and heavy
metals accumulation in Salvia officinalis cultivated in three intra-raw spacing in ash-shoubak, Jordan. Int. J. Agric. Biol., 13: 981–985
Determination of Essential Oils and Heavy Metals Accumulation
in Salvia officinalis Cultivated in three Intra-raw Spacing in
Ash-Shoubak, Jordan
MOHAMMAD S. ABU-DARWISH1, AHMAD H. AL-FRAIHAT1†, SATI Y.A. AL-DALAIN, FAT MA U. AFIFI‡ AND JALA
A. AL-TABBAL¶
Ash-Shoubak University College, Al-Balqa’ Applied University, Al-Salt 19117, Jordan
†Department of Applied Science, Ajloun University College, Al-Balqa` Applied University, Ajloun, Jordan
‡Faculty of Pharmacy, University of Jordan, Amman, Jordan
¶Al-Huson University College, Al-Balqa’ Applied University, Al-Salt 19117, Jordan
1Corresponding author’s e-mails: maa973@yahoo.com; drfrehat@yahoo.com
ABSTRACT
Essential oil yields and heavy metals content were determined in Salvia officinalis, cultivated in three intra-raw spacing (15,
30 & 45 cm) in the research farm of Ash-Shoubak University College. Specimens were harvested at the vegetative, beginning
of blooming, full-blooming and fruit maturation stages. Essential oil concentration and content of heavy metals in the plant
specimens were determined by using hydrodistillation and atomic absorption spectrometry methods, respectively. The yields
of essential oil and heavy metal contents were affected by intra-raw spacing and phenological stage. The maximum oil yield
was obtained in plant cultivated in 15 cm planting space (2.00) and harvested during vegetative stage, while the minimum oil
content was detected in the plants cultivated in the same raw planting space and harvested at maturation stage. Heavy metals
contents were variable depending on both intra-raw spacing and phenological stage. Co, Cd, and Pb were not detected.
Contents of Ni, Zn, Fe and Cu were increased during the vegetative stage of the plant but still below their toxic level. Results
indicated that S. officinalis cultivated in 15 cm planting space and harvested at the vegetative stage in Ash-Shoubak is rich in
essential oils and free from hazardous heavy metals. © 2011 Friends Science Publishers
Key Words: Aromatic; Pollution; Sage; Soil; Trace elements; Volatile oil
INTRODUCTION
Sage (Salvia officinalis L.) is a well-known medicinal
and culinary herb that has been used for centuries in the
Mediterranean region. The widespread genus Salvia has 500
and 900 species (Hedge, 1992). The most popular species of
the genus is sage, which is used as spice and flavoring agent
in food industry, perfumery and cosmetics. In Jordan, 19
species of the genus Salvia are recorded (Al-Eisawi, 1982).
Amr and Đorđević (2000) reported that the sage originating
from Jordan complied with the standard requirements for
the plant species, and can be used as a high quality raw
material for the production of phytopreparations. The leaves
of S. officinalis are used to relive headache, flatulence,
toothache, abdominal pain, and common cold or as a
sedative agent, wound healing and antidiabetic preparation
(Abu-Irmaileh & Afifi, 2003; Otoom et al., 2006; Al-
Qura’n, 2009). Several studies have been published
indicating the antioxidant and antimicrobial properties of the
essential oils extracted from S. officinalis (Velickovic et al.,
2003a; Velickovic et al., 2003b). Moreover, S. officinalis
infusions are used as haemostatic, estrogenic, anti-
perspiration, anti-neuralgic, spasmolytic, antiseptic,
astringent and as hypoglycemic agent (Istudor, 2001).
Heavy metals are detected in many medicinal plants
(Başgel & Erdemoğlu, 2005; Razic et al., 2005; Sumontha
et al., 2006; El-Rjoob et al., 2008; Abu-Darwish et al.,
2009; Massadeh et al., 2009). They can affect the
production and yield of certain biological compounds with
different roles in living tissues of animals and plants
(Malencic et al., 2003; Oktem, 2005; Aziz & Gad, 2007).
The content of heavy metals in aromatic medicinal plants
may be affected by geochemical characteristics of soil, or
location in which the plant is cultivated (Chan, 2003; Abu-
Darwish et al., 2009a; Abu-Darwish et al., 2009b).
Moreover, rain, atmospheric dust, plant protective agents,
and fertilizers could be additive factors of plant
contamination with heavy metals (Malencic et al., 2003).
On the other hand, the distribution and accumulation of
heavy metals among plant species and their organs is found
to be selective and depends on the plant species and the
individual ability of their parts to accumulate metals

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(Szentmihályi & Csedó, 2002; Prasad & Freitas, 2003;
Angelova et al., 2005; Rio-Celestino et al., 2006; El-Rjoob
et al., 2008).
The objectives of this study were to determine the
essential oil yield and lead (Pb), cadmium (Cd), chromium
(Cr), cobald (Co), nickel (Ni), zinc (Zn), iron (Fe), cupper
(Cu) and manganese (Mn) contents in S. officinalis
cultivated in 15, 30 and 45-cm intra-raw spacing in Ash-
Shoubak, south of Jordan.
MATERIALS AND METHODS
Location: This study was performed in the research farm of
Ash-Shoubak University College, Ash-Shoubak, Jordan
(Latitude 30 31N, Longitude 32 35 E). Ash-Shoubak is
1365 m above the sea level. Ambient temperature and
seasonal means of rainfall during the studying period in
2007 were (4.11 - 19.9C) and 294.2 mm, respectively. Soil
texture in the first 30 cm of the cultivation area can be
defined as clay loam, with a pH 7, 6 (Table I).
Plant cultivation: S. officinalis seedlings with a well-
developed root system were transplanted and cultivated in
the experimental area in May of 2007 in 50 cm-rows with
15, 30 and 45 cm intra-row spacing. A drip irrigation system
and other field practices had been done without addition of
fertilizers. Five plants were harvested from each intra-row
spacing at the following phenological stages, vegetative
(VEG), beginning of blooming (BB), full-blooming (FB)
and fruit maturation (FM) during June, July, August,
September 2007, respectively. All the plant samples were
dried at about 20C.
Essential oil extraction: A 20 g of the dried and coarsely
powdered specimens including leaves, stems, flowers, and
fruits (depending on the phenological stage) of S. officinalis
were steam distilled in triplicate for 2 h using a Clevenger-
type distillation apparatus (European Pharmacopoeia, 2005).
Yield percentage was calculated as volume of essential oil
per 100 g of plant dry matter.
Determination of heavy metals: Heavy metals content
were analyzed in selected samples with highest and lowest
essential oil contents in 15, 30 and 45 cm intra-raw planting
spaces by Atomic Absorption Flame Emission
Spectrophotometer, (Model AA-6200 Shimadzu Japan) (Al-
Alawi et al., 2007). The plant samples were oven dried at 70
C for 24 h until the dry weight was constant. The dried
samples were then ground and passed through a 0.2 mm
plastic sieve. Then, 0.5 gm of plant sample was wet digested
with an ultra-pure nitric acid (HNO3) (15 mL) in a
polyethylene test tube using a heating blocks digestion unit
at 120 C. The final solution was filtered into a 25 or 50 mL
volumetric flask through a 45 µm filter paper and diluted to
the mark with ultra−pure water. Ultra-pure water was used
for all dilutions and sample preparation. All reagents used
were of analytical grade (Sigma-Aldrich, Switzerland).
Soil samples: Three soil samples, collected in a depth of
approximately 0-15 cm from the location of S. officinalis
growth area, were sieved through 2-mm stainless steel sieve.
Samples were dried at 110oC for 24 h to achieve a constant
weight. After cooling, the sample was passed through 2 mm
sieve and stored in polyethylene bottles for chemical
treatment using acid digestion. Acid digestion was
performed by placing 0.5 g of soil sample in a beaker and
digested with 8 mL mixture of concentrated HCl and HNO3
with a ratio of (6:2 v/v) for 6 h at 90ºC and 2 mL of
concentrated HCl was added. The residue was filtered and
diluted to 25 mL with deionized water. The solution was
stored in a refrigerator at 4ºC for analysis (El-Rjoob et al.,
2008).
RESULTS AND DISCUSSION
The essential oil contents of S. officinalis samples
varied between 0.80% and 2.00% (Table II). Maximum
yield was recorded during the VEG stage in inter-raw
distance of 15-cm (2.0%) and the minimum was recorded at
45 cm during the FM stage (0.87%). The detected heavy
metal contents varied depending upon inter-raw spacing and
phenological stage. The contents of Cd, Pb, Cr and Co were
not detected in any specimen at all inter-raw planting
spaces. Other metal concentrations varied at different inter-
raw planting spaces during the VEG and FM stages. Mean
levels of Ni ranged from 0.42% to 4.17% at 15 cm inter-raw
planting space. Average Fe concentration varied from
524.67% at 45 cm to 935.40% at 15 cm during the VEG and
FM stages, respectively. The mean concentrations of Fe, Cu,
Zn, Mn, Cd and Pb in all investigated soil samples were
lower than the permissible limits (Table I). pH values for
soil samples were moderately basic (Table I).
Essential oils: Essential oils extracted from S. officinalis
were influenced by both, growth stage and inter-raw
planting space (Table II). The oil content extracted during
VEG and BM stages planted in 15, 30, and 45 cm intra-raw
spacing were found to satisfy the requirements of European
Pharmacopoeia (EP), while samples collected at FB and FM
stages from all studied intra-raw spacing plants were lower
than EP. The EP requires an oil yield of 1.0% v/w or above
(Anonymous, 2005). The results showed a clear effect of
both, intra–raw planting space and phenological stage on the
oil yields. The maximum oil yields were 2.00, 1.80 and
1.73% during the VEG stage of S. officinalis planted in 15,
30 and 45 cm intra-raw spaces, respectively, which
decreased to 0.80, 0.90 and 0.87% during the FM and BB
stages, respectively although earlier the highest oil content
was detected in four years old S. officinalis samples from
central Jordan, collected during the blooming phase
(2.13%). Nevertheless, the obtained results in both locations
were higher than those recorded in other parts of the world
(Dob et al., 2007; Raal et al., 2007; Khalil et al., 2008).
In the present study, essential oils extracted during the
life cycle stages of all studied inter-raw planting spaces of S.
officinalis decreased in the order: VEG>BB>FB>FM.
Similar trend was observed with an increase in the second

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term crop (Mirjalili et al., 2006; Zawislak & Dyduch, 2006).
Oil concentration is influenced by environmental factors,
methods of cultivation, seasonal variations and harvesting
time (Qiu et al., 2005; Maric et al., 2006; Bernotienė et al.,
2007). Also Qiu et al. (2005) reported qualitative and
quantitative differences in the oil of S. officinalis collected
in Shanghai, China at various seasonal periods. The highest
yield was observed in three-years-old samples. The
increasing effect on essential oil contents during the
vegetative stage of plant cycle could also be influenced by
light level (Al-Ramamneh, 2009). Light stimulated the
production of peltate glanular trichomes, the formation of
which is a prerequisite for the accumulation of essential oils
in thyme plants. On the other hand, the decreased amount of
essential oil from sage samples collected at FB and FM
stages may be due to drought caused by high temperature
during these periods. A significant reduction in the essential
oil content in chamomile when exposed to drought
conditions was observed (Razmjoo et al., 2008).
Soil pH and electrical conductivity (EC): The pH values
of all examined soil samples were moderately basic and
were 7.8 in soil samples planted at 15, 30 cm and 7.9 in soil
samples planted at 45 cm intra-row spacing. Similar values
were recorded earlier for the study area. Nevertheless, they
were higher than those reported in Thailand (5.8 to 6.67)
(Parkpian, 2003; El-Zuraiqi et al., 2004; El-Rjoob et al.,
2008). EC values ranged from 1.40 to 1.81 in all
investigated soil samples which indicated a relative water-
soluble salt content in the soil (El-Rjoob et al., 2008). The
soil in Ash-Shoubak region is similar to those studied in
other parts of Jordan and is characterized by its poor fertility
due to the fact that the soil is under cultivation for centuries.
Heavy metals in soil: The mean average concentrations of
Fe, Cu, Zn, Mn, Cd, and Pb in all soil samples were 6.00,
2.58, 3.78, 23.17, 0.11 and < 0.01, respectively. They were
lower than the permissible limits of rare elements in
agricultural soils in Canada, USA, France, Germany and
United Kingdom. This indicated that the soil growth area of
Ash-Shoubak region is clean and not polluted. In a study in
Irbid, Jordan, El-Rjoob et al. (2008) detected high
concentrations of heavy metals in soil samples indicating
clearly the effect of condensed traffic on the soil
characteristics.
Heavy metals in plant samples: The results of trace
element contents are shown in (Table III). Pb was not
detected during the VEG and FM pheneological stages of S.
officinalis planted at 15, 30 and 45 cm inter-raw spaces. The
absence of Pb might be mainly due to the cultivation
location, distanced from the main road and motor vehicles,
the leading factors of plant contamination with Pb (Łozak et
al., 2002; Malencic et al., 2003; Prasad & Freitas, 2003;
Angelova et al., 2005; El-Rjoob et al., 2008; Massadeh et
al., 2009). On the contrary, S. officinalis cultivated in two
other locations in the central parts of Jordan has been found
to be contaminated with Pb. Also S. officinalis cultivated in
other regions of the world were found to be contaminated
with Pb, and exceeded the toxic level (Malencic et al., 2003;
Angelova et al., 2005).
Cd is toxic for plants in high concentrations due to its
high affinity to –SH groups of enzymes and proteins. The
average normal and toxic Cd contents in plant leaves are
0.05-0.20 mg/kg and 3-30 mg/kg, respectively. Cd was not
detected in any of the examined specimens of S. officinalis
collected from Ash-Shoubak region in all inter-raw planting
spaces, which was similar to that found in the central
Table I: Means of heavy metals contents (mg/kg) and pH values in 15, 30, and 45 planting space soils
Intra-raw Spacing Extract mg/kg %
Ph EC Fe Cu Zn Mn Cd Pb Clay Silky Sandy Texture
15 cm 7.8 1.81 6.84 2.85 5.07 25.38 0.12 <0.01 28.0 39.0 33.1 Clay loam
30 cm 7.8 1.40 6.03 2.49 3.43 26.05 0.11 <0.01 27.3 38.1 34.5 Clay loam
45 cm 7.9 1.49 5.13 2.40 2.83 18.09 0.10 <0.01 27.2 38.4 34.4 Clay loam
Table II: The % of oil content in Saliva officinalis L. cultivated at 15, 30, and 45 cm planting spaces in Ash-
Shoubak region
Inter-raw planting space Phenological Stage
VEG BB FB FM
15 cm 2.00±0.1155 1.8±0.2517 0.84±0.0058 0.80±0.00
30 cm 1.80±0.0058 1.70±0.1155 0.93±0.0058 0.90±0.001
45 cm 1.73±0.1155 1.50±0.1155 0.93±0.002 0.87±0.001
Table III: Concentration of heavy metals (mg/kg) in Saliva officials L. cultivated in Ash-Shoubak region depending
on phenological stages and intra-raw spacing
Plant Stage Pb Cd Cr Co Ni Zn Fe Cu Mn
VEG /15 cm Ph nd nd nd 0.42±0.25 116.91±0.0.36 736.17±6.94 7.32±0.81 45.0±0.46
VEG/30 cm nd nd nd nd 2.78±1.08 95.81±1.19 768.97±5.41 7.02±0.50 51.35±0.42
VEG/45 cm nd nd nd nd 4.17±1.38 108.85±0.52 524.67±3.35 13.07±0.70 44.63±0.72
FM/15 cm nd nd nd nd 4.94±1.09 125.71±0.29 935.40±8.84 12.14±0.93 45.00±0.64
nd: not detected

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regions of the country (Amr & Đorđević, 2000). On the
other hand, Cd was detected below the toxic level (3-30
mg/kg) in S. officinalis cultivated in other regions of the
world. Its value was higher in samples collected near the
main roads exposed to high (Malencic et al., 2003;
Angelova et al., 2005; El-Rjoob et al., 2008). Likewise, Cr
was not detected. As previously reported for central regions
of Jordan. High concentrations of Cr were recorded in some
other countries such as Turkey and Hungary which differed
significantly of the plant species and location of cultivation
area (Başgel & Erdemoğlu, 2005).
Co was not detected in the samples of the present
study and in the samples from the central parts of the
country. This confirms the assumption that Co distribution
in plants is entirely species-dependent. The uptake of Co is
controlled by different mechanisms in different species.
Physical conditions like salinity, temperature, pH of the
medium, and presence of other metals in the soil influence
the Co uptake and accumulation in medicinal plants.
Low concentrations of Ni are beneficial for plant
growth and development, respiration intensity and
photosynthesis, as well as for the activity of antioxidant
enzymes. The average content of Ni in plant is 0.10 – 5.0
mg/kg, the toxic level ranges from 10 to 100 mg/kg. The
lowest content of Ni recorded 0.42 mg/kg at 15 cm inter-
raw planting space and harvested during the VEG stage,
while the highest was 4.94 mg/kg, during the FM stage at
the same inter-raw planting space. Ni content during the
VEG stage at 30 and 45 cm inter-raw planting spaces were
2.78 and 4.17 mg/kg, respectively. These results showed
that the content of Ni was increased by increasing the inter-
raw planting space, indicating to the activity of the root, Ni
absorbing system and/or the metabolic activity of the tissue-
metal accumulating (Sengar et al., 2008). Low level of Ni
might be due to the geographical effect of Ash-Shoubak
location, which is windy and wide area, whereas the wind
direction affects its concentration in the plant (El-Rjoob et
al., 2008).
Zn has an important role in the biosynthesis of
enzymes and some proteins. The concentration of Zn in
plant may vary between 30-150 mg/kg, but usually it is
between 20-50 mg/kg (Malencic et al., 2003). The lowest
concentration of Zn (95.81 mg/kg) was found in S.
officinalis during the VEG stage within 30 cm intra-raw
planting space, while the highest was 125.71 mg/kg in
plants collected during FM stage in 15 cm inter-raw
planting space. On the other hand, Zn content was increased
during the life cycle of S. officinalis cultivated at 15 cm
intra-raw space from 116.91 mg/kg in VEG stage to 125.71
mg/kg during FM stage indicating the Zn accumulation in
the leaf tissue of S. officinalis (Angelova et al., 2005; Rio-
Celestino et al., 2006). Lower concentrations were recorded
in S. officinalis cultivated in the central region of Jordan
(Amr & Đorđević, 2000).
Fe concentration in dry plant material reaches 1000
mg/kg or higher (Malencic et al., 2003). In the present study
the concentration of Fe reached its maximum (935.40
mg/kg) at 15 cm inter-raw planting space and harvested
during FM stage, while the minimum was 524.67 mg/kg in
the samples cultivated within 45 cm inter-raw planting
space and harvested during VEG stage. The content of Fe
increased proportionately to progressive growth stage of
samples planted at 15 cm inter-raw space, where it was
increased from 736.17 mg/kg at the VEG stage, to a
maximum of 935.40 mg/kg at FM stage. The average Fe
values in S. officinalis, cultivated in the central parts of the
country, ranged between 122 mg/kg to 148 mg/kg in
samples collected at BB stage while the maximum level was
recorded in samples collected during blooming and FM i.e.,
184 and 182 mg/kg, respectively. The high content of Fe in
S. officinalis could be attributed to the absence of Co, since
high levels of Co induce Fe deficiency in plants and
suppress uptake of Cd by roots. These results seem to
indicate that both soils and plants in Ash-Shoubak south
region are well supplied with this essential microelement
compared to the central regions of Jordan and other
geographical locations reported in the literature (Malencic et
al., 2003; Başgel & Erdemoğlu, 2005).
Cu is an essential microelement for plants. The
average content of Cu in dry plant material is reported to be
2.0-20 mg/kg (Malencic et al., 2003). It affects respiration,
metabolism of carbohydrates, lipids and proteins. In the
present study the concentrations of Cu in all samples of S.
officinalis were within the normal range and lower than
those recorded in the central region of Jordan and ranged
between 7.02 and 13.07 mg/kg during the VEG stage of S.
officinalis planted at 30 and 45 cm inter-raw planting
spaces, respectively. On the other hand, the contents of Cu
in S. officinalis grown in Serbia, Yugoslavia, Turkey and
Bulgaria were 25.1, 26, 5.5 and 35.8, respectively
(Malencic et al., 2003; Angelova et al., 2005; El-Rjoob
et al., 2008). These findings could be due to the effect
of anthropogenic activities and heavy traffic observed in
these regions that may accumulate Cu in the soil (Razic
et al., 2005; El-Rjoob et al., 2008). Cu availability for
plant uptake is dependent upon number of plant factors
such as root intrusion, water and ion fluxes and their
relationship to the kinetics of metal solubilization in soils;
biological parameters, including kinetic of membrane
transport, ion interactions and metabolic fate of absorbed
ions.
The contents of Mn in all samples of S. officinalis
were similar. The highest content was 51.35 mg/kg in
samples cultivated at 30 cm inter-raw spacing and harvested
during the VEG stage, while the lowest was 44.63 mg/kg at
45 cm inter-raw space. All findings were lower than those
detected in the central regions of Jordan, which was ranged
from 92 to 108 mg/kg. The recorded data were higher than
those recorded in Serbia and Turkey, where the content of
Mn was 39.25 and 32.6 mg/kg, respectively (Malencic et
al., 2003; Başgel & Erdemoğlu, 2005). These variations
could be due to the differences in plants uptake, which can

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985
be explained by differences in the ability of plants to bring
about the dissolution of oxidized manganese.
CONCLUSION
The yields of essential oil and of heavy metals content
in S. officinalis are affected by phenological stages and
intra-raw planting spaces. S. officinalis L. cultivated in 15
cm planting space and harvested during the VEG stage in
Ash-Shoubak is rich in essential oils content and free from
hazardous heavy metals.
Acknowledgement: The authors express their great thanks
to the Deanship of Scientific Research and Al-Balqa`
Applied University for their financial support.
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(Received 20 April 2011; Accepted 13 September 2011)