Journal of Medicinal Plants Research Vol. 6(3), pp. 455-460, 23 January, 2012 Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1166 ISSN 1996-0875 ©2011 Academic Journals
Full Length Research Paper
Phytochemical and antimicrobial activities of Lavandula
officinalis leaves and stems against some pathogenic
Ali Shafaghat*, Farshid Salimi and Vahid Amani-Hooshyar
Department of Chemistry, Ardabil Branch, Islamic Azad University, Ardabil, Iran.
Accepted 4 October, 2011
The leaves and stem-bark of Lavandula officinalis were investigated for activity against some
pathogenic organisms. Phytochemical screening revealed the presence of coumarins, tannins,
flavonoids, volatile oil and fatty acids. Twenty six and twenty were characterized representing 84.5%
(leaf oil) and 91.4% (stem oil) of the lot of components detected. The leaf oil of the major constituents
was identified as borneol (23.6%), 1, 8-cineol (17.6%), camphor (12.6%). In the stem oil of plant 1, 8-
cineol (20.8%), borneol (19.2%), α-cadinol (11.3%), caryophyllene oxide (10.4%) and camphor (7.4%)
were the predominating compounds. The hexane extracts of leaf and stem of plant were obtained by
soxhlet apparatus. The fatty acids were derived to methyl esters and determined by gas chromatograph
and mass spectrometer (GC/MS) systems. The main components of the leaf and stem (hexanic extracts)
were ω-3 (43.2 and 21.0%), ω-6 (3.4 and 14.5%), palmitic acid (7.4 and 12.4%) and Bis (2-ethylhexyl)
phthalate (12.8 and 16.7%), respectively. Antimicrobial activities of the crude methanol, hexane extracts
and essential oil from leaf and stem were evaluated using agar diffusion method. Results, suggest
potential antimicrobial activity of the essential oil and extracts of L. officinalis, which may find those
application in future research for the food and pharmaceutical industry.
Key words: Lavandula officinalis, Lamiaceae, fatty acid, essential oil phytochemical, crude extract,
The genus Lavandula (lavender, Lamiaceae) is
represented in the flora of Iran by two species: Lavandula
stricta Del. and Lavandula sublepidota Rech. f. of which
L. sublepidota is endemic plant (Mozaffarian, 2007). This
genus is distributed from the Canary and Cape Verde
Islands and Madeira, across the Mediterranean Basin,
North Africa, South, West Asia, the Arabian Peninsula
and tropical NE Africa with a disjunction to India. The
*Corresponding author. E-mail: firstname.lastname@example.org.
Abbreviations: GC-MS, Gas chromatograph and mass
spectrometer; IOOC, international olive oil council; TLC, thin
layer chromatography; DDM, disc diffusion method; PUFAs,
polyunsaturated fatty acids; ALA, α-linolenic acid; LA, linoleic
acid; EFAs, essential fatty acids; HCL, hydrogen chloride;
GC/FID, gas chromatography - flame ionization detector; UFA,
unsaturated fatty acid.
English lavender is widely cultivated in gardens and is
also an important essential oil crop from which many
cultivars have been selected (Imelouane et al., 2009).
This herbal drug is popular in folk medicine as
spasmolytic, carminative, stomachic and diuretic.
Nowadays, it is applied as a mild sedative and
cholagogue in various phytopharmaceuticals as well
(Wichtl, 1994). In food manufacturing, lavender essential
oil is employed in flavoring beverages, ice-cream, candy,
baked goods and chewing gum (Kim and Lee, 2002).
Leaf and flower of Lavandula has the highest amount of
essential oils. Lavender oil, which is the essential oil
obtained by the aerial part of L. officinalis, is
predominantly used in aromatherapy as a relaxant and
sedative agent (Cavanagh and Wilkinson, 2002; Sanz et
al., 2004). Also, it was traditionally used as an antiseptic
agent in swabbing of wounds, for burns and insect bites
and in veterinary practice to kill lice and other animal
parasites. There are various reports about combination of
456 J. Med. Plants Res.
Lavandula component in different regions of the world
and most of them showed that phenol compounds are the
main components (Meftahizade et al., 2011).
An ample literature exists on the essential oils in
Lavandula, reviewed by Boelens (1995). For taxonomists,
use of essential oils as characters for the classification is
limited by inherent problems of natural variability,
although at lower taxonomic levels this can be used to
help recognize cultivars (Grayer et al., 1996). The
chemical composition and ratios of the individual
components making up the oils are also known to change
in response to environmental conditions, such as water
and nutrient stress or time of year (Ross and Sombrero,
1991). Lavender essential oil is one of the most
appreciated in the perfume and soap industry. Generally,
it is produced by short steam distillation of fresh flowers.
The quality of the essential oil, besides other parameters,
strongly depends on the total amount of ester
compounds, which has to be 35 to 55%, calculated as
linalyl acetate. Because of the great usage of both, the
flowers and the essential oil, lavender is cultivated in
many countries worldwide (Argentina, Brazil, Bulgaria,
Cyprus, Greece, Croatia, Hungary, Iran, Italy, Russia,
Spain, Turkey, Japan and United Kingdom) (Trease and
Evans, 1978). Several plants of this genus have been
studied from the chemical, biological and
pharmacological point of view (Pascual et al., 1983;
Gamez et al., 1987; Kokkalou, 1988; Ghelardini et al.,
1999; Lis-Balchin and Hart, 1999; An et al., 2001;
Cavanagh and Wilkinson, 2002; Nogueira and Romano,
2002). In this study, antimicrobial activity of the crude
methanol and hexane extracts and essential oil from leaf
and stem of L. officinalis cultivated in Ardabil (northwest
Iran) were examined using different bacterial species. In
addition, phytochemical active constituents and
composition of volatile compounds from leaf and stem
were also determined.
MATERIALS AND METHODS
Leaf and stem of L. officinalis were collected separately in the
botanical farm of agricultural research center (belong to I.A.
University Ardabil Branch) at an altitude of 1550 m in August 2010.
A voucher specimen (L-112) is kept at the Herbarium of Agriculture
Research in Ardabil Center, Iran. The samples were air-dried in the
laboratory at ambient temperature (30±2°C) for 14 days; pulverized
using a mechanical grinder and the obtained powders was stored
until further use.
Essential oil isolation
Air dried leaf (110 g) and stem (250 g) of L. officinalis were
submitted to Hydro distillation for 4 h using Clevenger type
apparatus. Briefly, the samples were immersed in water and heated
to boiling, after which the essential oil was evaporated together with
water vapour and finally collected in a condenser. The distillates
were isolated and dried over anhydrous sodium sulfate. The oils
were stored at 2°C until analysis by GC–MS.
Preparation of methanol extract
100 g of dried and powdered of each plant part (leaf and stem)
were packed in a Soxhlet extractor and extracted with methanol
(MeOH). The methanol extracts were evaporated to dryness using
a rotary evaporator to obtain 21.3 g (leaf) and 19.7 g (stem) of
crude extracts. The various crude extracts were later subjected to
Preparation of hexane extract
100 g of dried and powdered of each material (leaf and stem) were
extracted with hexane using a Soxhlet apparatus (70°C, 4h) to
obtain the fatty acids and the other apolar components. During
extraction procedures, Merck hexane (95%) was used. The hexane
extracts were concentrated by rotary evaporator under vacuum at
45°C to obtain 13.1g (leaf) and 12.4 g (stem) of extracts.
Methylation of hexane extract
After removing hexane using rotary evaporator, the oily mixtures
were derived to their methyl esters by the International Olive Oil
Council (IOOC, 2001) reports by trans-esterification process. In this
process, dried hexane extracts were dissolved in hexane and then
extracted with 2 M methanolic KOH at room temperature for 30 s.
The upper phases were analyzed by gas chromatography- flame
ionization detector (GC/FID) and GC/MS systems.
GC/MS analysis of hexane extract and essential oil
GC analysis: Gas chromatograph (GC) analysis was performed on
a Shimadzu 15 A GC equipped with a split/splitless injector (250°C)
and a flame ionization detector (250°C). N
was used as carrier gas
(1 ml/min) and the capillary column used was DB-5 (50 m × 0.2
mm, film thickness 0.32 µm). The column temperature was kept at
60°C for 3 min and then heated to 220°C with a 5°C/ min rate and
kept constant at 220°C for 5 min. The relative percentages of the
characterized components are given in Table 1 (essential oil) and
Table 2 (hexane extract).
GC/MS analysis: GC/MS analysis was performed using a Hewlett
Packard 5973 with an HP-5MS column (30 m × 0.25 mm, film
thickness 0.25 µm). The column temperature was kept at 60°C for 3
min and programmed to 220°C at a rate of 5°C/min and kept
constant at 220°C for 5 min. The flow rate of helium as carrier gas
was 1 ml/min. mass spectrometer (MS) were taken at 70 eV. The
fatty acids were identified by comparing their retention times and
mass peaks with those of standard methyl ester mixtures and by
NIST-Wiley library data search. Relative percentage amounts were
calculated from peak area using a Shimadzu C-R4A chromatopac
without the use of correction factors.
Flavonoids: The alcoholic extract (0.5 g) was treated with a few
drops of concentrated Hydrogen chloride (HCl) and magnesium
turnings (100 mg). The presence of flavonoids was indicative if pink
or magenta-red color developed within 3 min. A pink or red color
Table 1. Chemical composition (%) of the essential oil from leaf
(l) and stem (s) of L. officinalis.
Compound * KI l (%) S (%)
Tricyclene 919 0.2 -
α-Pinene 938 3.2 1.0
Camphene 953 1.9 0.4
β-Pinene 981 3.8 1.0
β-Myrcene 991 1.6 -
δ-3-Carene 1011 2.5 0.9
1,8-Cineol 1032 17.6 20.8
γ-Terpinene 1061 0.9 -
α-Terpinolene 1088 1.4 -
Camphor 1142 12.6 7.4
Borneol 1168 23.6 19.2
α-Thujenale 1183 0.3 -
Trans-Carveol 1218 2.9 -
Phellandral 1248 1.0 -
Piperitone 1252 1.1 -
Bornyl acetate 1289 0.5 2.3
β-Cubebene 1391 - 1.9
Aromadendrene 1438 0.1 -
Trans-β-Farnesene 1456 - 3.9
γ-Gurjunene 1477 0.2 1.3
α-Amorphene 1484 0.3 2.0
Valencene 1495 - 0.7
Germacrene A 1503 0.1 -
γ-Cadinene 1514 0.1 -
δ-Cadinene 1524 - 0.6
Ldol 1566 0.2 -
Caryophyllene oxide 1581 1.4 10.4
β-Bisabolenal 1770 4.6 -
Eicosane 1999 - 0.6
2,13-Octadecadienol 2065 0.1 0.4
Total - 84.6% 91.4%
*The composition of the essential oils was determined by
comparison of the mass spectrum of each component with Wiley
GC/MS library data and also from its Kovats retention indices (KI).
developed which could be extracted with amyl alcohol. The
presence of flavonoids was numbered, according to color intensity
(Salehi- Surmaghi et al., 1992).
Tannins: 5 mg of the powdered extracts was stirred with 10 ml of
hot distilled water, filtered and ferric chloride was added to the
filtrate and observed for blue-black, blue-green or green precipitate
Coumarins: The methanolic extract was analyzed for coumarins by
running Thin Layer Chromatography (TLC) of the extract on Silica
gel 60F 254 precoated sheets with 10% AcOH (Acetic acid) as
mobile phase and NH
/KOH as detecting reagent to observed deep
blue color (Horborne, 2005).
Shafaghat et al. 457
The in vitro antibacterial and antifungal activities of the extracts and
essential oil were evaluated by the disc diffusion method (DDM)
using Mueller-Hinton agar for bacteria and Sabouraud Dextrose
agar for fungi (Baron and Finegold, 1990). Discs containing 30 µL
of the methanol and hexanic extracts of leaf and stem were used
and growth inhibition zones were measured after 24 and 48 h of
incubation at 37 and 24°C for bacteria and fungi, resp ectively.
Gentamicin and tetracycline for bacteria and nystatin for fungi were
used as positive controls. The microorganisms used were: Bacillus
subtilis ATCC 9372, Staphylococcus epidermidis ATCC 12228,
Enterococcus faecalis ATCC 15753, Staphylococcus aureus ATCC
25923, Klebsiella pneumoniae ATCC 3583, Pseudomonas
aeruginosa ATCC 27852, Escherichia coli ATCC 25922, Aspergillus
niger ATCC 16404, Candida albicans ATCC 5027 and
Saccharomyces cerevisiae ATCC 9763.
RESULTS AND DISCUSSION
The results obtained in the analyses of the essential oil
and hexane extract of leaf and stem from L. officinalis are
listed in Tables 1 and 2, respectively, in which the
percentage and retention indices of components are
given. As can be seen in Table 1, about 84.6% (27
components) of the oil of L. officinalis leaf and 91.2% ( 19
components) of the oil of stem oil were identified. The oil
of leaf was characterized by the presence of borneol
(23.6%), 1, 8- cineole (17.6%), camphor (12.6%), and β-
bisabolenal (4.6%). The volatile oil of stem, contained five
main compounds were characterized by 1, 8- cineole
(20.8%), borneol (19.2%), α- cadinol (11.3%),
caryophyllene oxide (10.4%) and camphor (7.4%). The
dominant compounds in the leaf and stem oils are
oxygenated monoterpenes (58.3 and 49.7%,
respectively). In investigation on the oil of the aerial parts
of L. officinalis collected in mountain Kozjak, Republic of
Macedonia, the main constituents were linalool (25.7%),
linalyl acetate (23.2%) and lavandulyl acetate (12.4%)
(Kulevanova et al., 2000). The chemical constituents of
the essential oil from flowers of L. officinalis growing in
Iran has been reported, the main constituents were
linalool (34.1%), 1,8- cineole (18.5%), borneol (14.5%),
camphor (10.2%), terpinen-4-ol (4.5%), linalyl acetate
(3.7%), α-bisabolol (3%), α-terpineol (2.2%) and (Z)-β-
farnesene (2.2%) (Afsharypuor and Azarbayejany, 2006).
According to the results, the hexane extract yields of
the studied different part of L. officinalis were found 3.1%
(leaf extract) and 2.4%( stem extract) on the basis of dry
weight of the plant materials. The highest total
percentage was detected in leaf. The total fatty acid
contents of hexane extracts varied from 95.6 to 96.2%
(Table 2). The major saturated and unsaturated
components including linoleic (ω-6) and palmitic acids
are shown in the Table. The major polyunsaturated fatty
acids (PUFAs) were α-linolenic and linoleic acids. As can
be seen in Table 2, about 96.2% (27 components) of the
extract from leaf, and 95.6% (25 components) from stem
extract were identified. There were some differences in
458 J. Med. Plants Res.
Table 2. Chemical composition (%) of the hexanic extract from leaf (l) and stem (s) of L. officinalis.
Compound * (related fatty acid) Rt (min) l (%) s (%)
Dodecanoic acid, methyl ester (lauric acid) 7.8 1.3 0.6
Hexadecane 8.5 - 0.1
Tetradecanoic acid, methyl ester (myristic acid) 9.7 3.4 1.7
Pentadecanoic acid, methyl ester (Pentadecanoic acid) 10.6 0.2 0.3
2-Pentadecanone, 6, 10, 14-trimethyl. 10.7 0.2 -
1-Hexadecanol 11.0 0.1 -
Nonadecane 11.2 0.1 -
Hexadecanoic acid, methyl ester (palmitic acid) 11.4 7.4 12.4
Heptadecanoic acid, methyl ester (margaric acid) 12.2 0.2 0.3
9,12-Octadecadienoic acid, methyl ester (linoleic acid) or ω- 6 12.7 3.4 14.5
9,12,15-Octadecatrienoic acid, methyl ester (linolenic acid) or ω- 3 12.8 43.2 21.0
Phytol 12.9 1.3 1.6
Octadecanoic acid, methyl ester(stearic acid)
Nonadecanoic acid, methyl ester (Nonadecanoic acid) 13.7 - 0.1
9-Tricosene 14.0 - 0.3
Tricosane 14.2 0.7 -
Eicosanoic acid, methyl ester (arachidic acid) 14.4 1.6 1.9
Tetracosane 14.9 0.4 -
Heneicosanoic acid, methyl ester ( Heneicosanoic acid) 15.1 - 0.2
Pentacosane 15.5 2.1 1.3
Docosanoic acid, methyl ester (behenic acid) 15.7 1.4 1.7
Bis(2-ethylhexyl) phthalate 15.9 12.8 16.7
Oxirane, heptadecyl 16.4 1.8 7.8
Tetracosanoic acid, methyl ester (lignoceric acid) 16.9 1.6 1.3
2,6,10,14,18,22-Tetracosahexaene 17.5 0.5 -
Squalene 17.51 - 0.6
Tetracosanal 17.6 2.2 -
Nonacosane 17.9 3.4 13.7
Hexacosanoic acid, methyl ester (cerotic acid) 18.1 1.3 0.7
1-Dotriacontanol 20.5 0.3 0.9
γ-Sitosterol 20.65 - 3.2
Stigmasterol, 22,23-dihydro 20.7 1.2 0.9
Total - 96.2 95.6
*The composition of the extracts was determined by comparison of the mass spectrum of each component with Wiley
GC/MS library data and also from its retention times (Rt).
the fatty acid profiles of the different part of this plant. The
unsaturated fatty acid contents were higher than
saturated ones, whereas some of the fatty acids were not
observed in all parts of this plant. In fact, both fractions
mainly include unsaturated fatty acids, with a clear
predominance of α-linolenic acid (ALA or ω-3), linoleic
acid (LA) and Bis (2-ethylhexyl) phthalate. One of the
essential fatty acids (EFAs), ω-3 (ALA) was a
predominant component in leaf of L. officinalis. The
hexanic extract of leaf had a higher proportion of
unsaturated fatty acid (UFA) compared to stem part.
Some hydrocarbon compounds were found in leaf and
stem (Table 2). The essential oils and extracts of leaf
and stem from L. officinalis were tested against four
Gram-positive and three Gram-negative bacteria, as well
as three fungi. The results, presented in Table 3, show
that the both essential oils and methanolic extracts
exhibited strong biological activities against all tested
fungi and bacteria, versus, the hexane extracts exhibited
moderate biological activity against all tested fungi and
bacteria except for two resistant Gram-negative bacteria,
K. pneumoniae and P. aeruginosa, as well as a fungi, A.
The most sensitive microorganisms were S.
epidermidis and S. aureus, with inhibition zones of (8.9 to
25.6) mm. Other microorganisms were found to be less
Shafaghat et al. 459
Table 3. Antimicrobial activities of the essential oils, MeOH and hexane extracts of leaf (l) and stem (s) of L. officinalis.
Zone of inhibition (mm) *
Essential oil MeOH ext Hexane ext Antibiotics
Microorganism l s l s l s G** N** T**
18.7 16.3 22.1 21.3 11.7 12.3 NT
S. epidermidis 25.6 23.7 24.9 22.6 9.7 8.9 NT NT 34.1
E. faecalis 16.3 16.7 22.3 19.9 10.3 9.2 NT NT 9.7
S. aureus 21.5 20.9 21.1 16.7 7.2 9.1 NT NT 21.6
P. aeruginosa 10.3 10.6 9.9 10.8 8.4 NA 11.7 NT NT
E. coli 18.0 17.9 19.4 20.3 7.5 8.0 24.5 NT NT
A. niger 14.7 15.6 12.7 13.1 8.2 NA NT 16.7 NT
C. albicans 15.5 14.2 14.7 16.1 11.4 9.9 NT 18.9 NT
S. cerevisiae 13.9 14.7 16.9 16.3 10.0 9.5 NT 18.4 NT
*Inhibition zone diameter (mm), including diameter of sterile disk 6 mm. **
G: gentamicin; N: nystatin; T: tetracycline;
NT: not tested.
Table 4. Phytochemical analyses of the methanolic extract of leaf
and stem from L. officinalis.
Phytochemical* Leaf Stem
Flavonoids + +
Coumarins + -
Tannins + +
*: (+), present; (-), absent.
sensitive to the hexane extracts with inhibition zones
ranged from 8 to 12.3 mm. It is conceivable that the
antimicrobial property of the essential oils from L.
officinalis might be ascribed to its high content of
oxygenated terpenes and of the methanolic extracts
might be ascribed to its high content of phenolic
compounds. The phytochemical screening of the leaf and
stem revealed the presence of flavonoids, tannins and
coumarins in methanolic extracts (Table 4) and fatty
acids in hexanic extracts. The presence of flavonoids,
tannins, coumarins, fatty acids and volatile compounds in
the plant’s part is an indication that the plant is of
pharmacological importance (Hostettmann and Marston,
1995). The essential oil, hexane and methanolic extracts
evaluated in this work have a great variety of
phytochemicals that could be considered as responsible
for a larger or smaller part of the antimicrobial activities.
Although they usually occur as complex mixtures, their
activity can generally be accounted for in terms of their
major oxygenated monoterpenoid and natural
compounds. Research into the antimicrobial actions of
monoterpenes suggests that they diffuse into and
damage cell membrane structures (Sikkema et al., 1995).
Antimicrobial activities of essential oils are difficult to
correlate to a specific compound due to their complexity
The author wish to thank Islamic Azad University of
Ardabil Branch for financial support for this investigation.
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