essential oil composition and antimicrobial activity of three ocimum ...

Research Article
ESSENTIAL OIL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF THREE OCIMUM SPECIES
FROM UTTARAKHAND (INDIA)
ANKUR KUMAR ANAND 1, MANINDRA MOHAN 2, S. ZAFAR HAIDER 2*, AKASH SHARMA 3
1Planet Herbs Life Sciences, Industrial Estate, Selaqui­248197, Dehradun (Uttarakhand) India, 2Centre for Aromatic Plants (CAP),
Industrial Estate, Selaqui­248197, Dehradun (Uttarakhand) India, 3Clinical Microbiology Division, Indian Institute of Integrative
Medicine, Canal Road, Jammu (J&K), India. Email: zafarhaider.1@rediffmail.com
Received: 25 March 2011, Revised and Accepted: 23 April 2011
ABSTRACT
The essential oils obtained from aerial parts of three Ocimum species from India were investigated by GC and GC/MS. The volatile oil of Ocimum
basilicum afforded methyl chevicol (70.04%) as a major constituent followed by linalyl acetate (22.54%). Camphor (56.07%), DL‐limonene
(13.56%) and camphene (7.32%) were obtained in a high concentration in Ocimum kilimandscharicum, whereas in Ocimum gratissimum eugenol
(53.89%) was the most abundant component followed by cis‐ocimene (23.97%) and germacrene‐D (10.36%). Antimicrobial activities of the three
oils were found against Gram +ve bacteria (Staphylococcus aureus, Enterococcus faecalis) and Gram ‐ve bacteria (Escherichia coli, Pseudomonas
aeruginosa) as well as yeast Candida albicans, and these results were discussed with the compositions of each sample.
Keywords: Ocimum basilicum, Ocimum kilimandscharicum, Ocimum gratissimum, Essential oil compositions, Methyl chevicol, Camphor, Eugenol,
Antimicrobial activity.
INTRODUCTION
The genus Ocimum L. (Lamiaceae) is an important group of aromatic
and medicinal flora which yield many essential oils and aroma
chemicals and find diverse uses in the perfumery and cosmetic
industries as well as in indigenous systems of medicine. The genus is
distributed in tropical and warm temperate regions of the world and
represented by nine species in India1. Among the various Ocimum species, O. basilicum L. (sweet basil) is
commercially and extensively cultivated for essential oil production
in India and abroad. O. kilimandscharicum Guerke (Camphor basil), a
native of Kenya is cultivated in some parts of India and attracted
attention as a source of camphor1, whereas O. gratissimum L. (wild
basil), found almost throughout India is often cultivated.
Earlier studies reveal that the essential oils of these species have
been the subject of several studies with varying chemical
composition and antimicrobial activities2‐13. The essential oil
composition of these three Ocimum species were assigned some
highly valuable compounds as methyl chevicol, linalool, eugenol,
thymol, 1,8‐cineole, methyl eugenol, camphor, p‐cymene, γ‐
terpinene, myrcene and α‐thujene in abundance as reported in the
literature2‐5,7,8,11,12. On continuation of our research on essential oil bearing plants from
India14, we investigated the essential oil composition and the
antimicrobial activity from aerial parts of O. basilicum, O.
kilimandscharicum and O. gratissimum.
MATERIALS AND METHODS
Plant material
The aerial parts of O. basilicum, O. kilimandscharicum and O.
gratissimum were collected in flowering stage from Central Nursery,
Forest Research Institute, Dehradun (Uttarakhand). Voucher
specimens were deposited at Centre for Aromatic Plants, Selaqui,
Dehradun.
Essential oil extraction
For the extraction of essential oils, shade dried aerial parts of three
Ocimum species were subjected to hydro‐distillation using a
Clevenger‐type apparatus for 3.5 h. The maximum oil yield obtained
in O. kilimandscharicum (1.92%) followed by O. basilicum (0.53%)
and O. gratissimum (0.28%). The essential oils collected were dried
over anhydrous sodium sulphate and stored at 4 °C until the analysis
was carried out.
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 3 Suppl 3, 2011
GC and GC/MS analysis
GC analyses were performed by an Agilent Technology 6890 N gas
Chromatograph data handling system equipped with a split‐
splitless injector and fitted with a FID using N2 as the carrier gas.
The column was HP‐5 capillary column (30m x 0.32mm, 0.25µm
film thickness) and temperature program was used as follows:
initial temperature of 600 C (hold: 2 min) programmed at a rate of
30C /min to a final temperature of 2200C (hold: 5 min).
Temperatures of the injector and FID were maintained at 2100C and 2500C, respectively.
The GC/MS analyses were carried out on a Perkin Elmer Clarus
500 gas chromatograph equipped with a split‐splitless injector
(split ratio 50:1) data handling system. The column was an Rtx®‐5
capillary columns (60 m x 0.32mm, 0.25µm film thickness). Helium
(He) was the carrier gas at a flow rate 1.0 ml/min. The GC was
interfaced with (Perkin Elmer clarus 500) mass detector operating
in the EI + mode. The mass spectra were generally recorded over
40‐500 amu that revealed the total ion current (TIC)
chromatograms. Temperature program was used as the same as
described above for GC analyses. The temperatures of the injector,
transfer line and ion source were maintained at 2100C, 2100C and
2000C, respectively.
Identification of the individual components was made by matching
their recorded mass spectra with the library (NIST/ Pfleger
/Wiley) provided by the instrument software, and by comparing
their calculated retention indices with GC alkane standard solution
(C8‐C20) as well as literature value15. Relative area percentages of
the individual components were obtained from GC‐FID analyses.
Antimicrobial screening
The antimicrobial tests were carried out at Clinical Microbiology
Division, Indian Institute of Integrative Medicine, Canal Road,
Jammu (J&K) India using the following microorganisms:
Staphylococcus aureus ATCC 29213, methicillin‐resistant
Staphylococcus aureus (MRSA ATCC 15187), Vancomycin Resistant
Enterococcus faecalis (VRE, clinical isolate), Escherichia coli ATCC
25922, Pseudomonas aeruginosa ATCC 29212 and Candida albicans
ATCC 90028.
MIC determination of microorganisms: MIC of bacterial pathogens
was determined by Broth Dilution method using 96‐well micro‐
titre plates. All cultures were preserved in 50% glycerol at ‐700C. The bacterial and fungal pathogens were inoculated in MHB broth
and RPMI media for the preparation of inoculum, respectively.

Inoculum was prepared from overnight grown culture on
TSA/MHB agar plates by suspending well‐isolated colonies of
culture in 2ml sterile saline and the turbidity of suspension was
adjusted to 0.5 McFarland (1.5 x 108 cfu/mL). The cultures were
further diluted in medium to get 2 times the final inoculum (5 x
105 cfu/ml), blank media used as negative control. Stock solution
of the oils was prepared in dimethyl sulfoxide (DMSO), 50% DMSO
or water, depending upon the solubility. 2 fold concentrations of
the solution were prepared in micro‐centrifuge tube and then
transferred to micro‐titre plates. Each microorganism suspension
was then added into the wells. The last two columns containing
100 μL and 200 μL of medium, without drug served as growth and
medium control, respectively. After incubation at 370C for
overnight the first well showing no turbidity of growth was
determined as minimal inhibitory concentration (MIC).
Ciprofloxacin and amphotericin‐B were used as standard
antibacterial and antifungal agents, respectively.
RESULTS AND DISCUSSION
The compositions of the oils of O. basilicum (sample A), O.
kilimandscharicum (sample B) and O. gratissimum (sample C) are
listed in table 1. Total 30 compounds were identified in all the oils,
representing 95.41%, 94.19% and 94.03% in the oil samples A, B
and C, respectively. In the oil of O. basilicum methyl chevicol
(70.04%) detected as the major component, followed by linalyl
acetate (22.54%). There are several chemotype of sweet basil oil
Haider et al.
Int J Pharm Pharm Sci, Vol 3, Suppl 3, 2011, 223­225
such as methyl chevicol, linalool, methyl eugenol and methyl
cinnamate rich types are reported in different regions16. Maheshwari17 also reported the presence of high amount of methyl
chevicol, but our result is different due to the presence linalyl
acetate. O. kilimandscharicum oil was characterized with camphor
(56.07%) as the most abundant constituent. Other compounds
that were present in appreciable amounts in the oil were DL‐
limonene (13.56%), camphene (7.32%) and terpinen‐4‐ol (3.50%),
whereas in O. gratissimum eugenol (53.89%) was found to be
major constituent followed by cis‐ocimene (23.17%) and
germacrene‐D (10.36%). Earlier investigations also revealed the
presence of eugenol in abundance9,12. All the oils afforded
maximum amount of monoterpenoids (monoterpene
hydrocarbons and oxygenated monoterpenes), except sample A,
which showed very lesser amount of monoterpenes.
Sesquiterpene hydrocarbons were found negligible in oils A and B,
whereas more than 12% were in O. gratissimum due to the
presence of germacrene D.
The essential oil of Ocimum species were tested against standard
bacterial strains S. aureus, E. faecalis, E. coli, P. aeruginosa and the
yeast Candida albicans (Table 2). Among the three oil samples, O.
gratissimum oil was found to be more active against all tested
micro‐organisms, especially against C. albicans and Gram +ve
bacteria. The oil of O. basilicum showed best MIC against C.
albicans, whereas oil of O. kilimandscharicum did not show any
remarkable activity against all tested micro‐organisms.
Table 1: Percentage composition of the essential oils of three Ocimum species
Components RI A B C
α‐pinene 939 0.06 1.23 0.83
camphene 953 0.01 7.32 0.73
β‐pinene 980 0.12 ‐ ‐
β‐myrcene 993 ‐ 1.58 0.20
α‐phellandrene 1008 ‐ 0.26 0.46
α‐terpinene 1019 ‐ 0.33 ‐
p‐cymene 1023 ‐ 0.62 0.06
DL‐limonene 1031 0.11 13.56 0.05
1,8‐cineole 1033 0.09 0.85 0.09
cis‐ocimene 1042 0.17 ‐ 23.97
β‐ocimene 1054 0.03 2.00 0.97
γ‐terpinene 1058 ‐ 0.88 ‐
cis‐sabinene hydrate 1068 ‐ 0.47 ‐
α‐terpinolene 1088 ‐ 1.33 ‐
trans‐sabinene hydrate 1097 ‐ 0.49 ‐
linalool 1098 0.20 1.70 0.23
camphor 1142 ‐ 56.07 ‐
terpinen‐4‐ol 1166 ‐ 3.50 0.33
methyl chevicol 1193 70.04 ‐ ‐
myrtenol 1194 ‐ 1.24 ‐
(Z)‐citral 1253 0.46 ‐ ‐
linalyl acetate 1261 22.54 ‐ ‐
(E)‐citral 1267 0.72 ‐ ‐
trans‐caryophyllene 1298 0.24 0.33 1.86
eugenol 1357 ‐ ‐ 53.89
α‐humulene 1450 0.10 ‐ ‐
β‐farnesene 1454 0.19 ‐ ‐
β‐selinene 1479 0.26 ‐ ‐
germacrene D 1481 ‐ 0.43 10.36
β‐bisabolene 1509 0.07 ‐ ‐
Total identified 95.41 94.19 94.03
monoterpene hydrocarbons 0.5 29.11 27.27
oxygenated monoterpenes 94.05 64.32 54.54
sesquiterpene hydrocarbons 0.86 0.76 12.22
A: O. basilicum; B: O. kilimandscharicum; C: O. gratissimum
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Haider et al.
Int J Pharm Pharm Sci, Vol 3, Suppl 3, 2011, 223­225
Microorganisms
Table 2: The antimicrobial activity results of the oils (mg/mL) of Ocimum species
MIC
MIC
(mg/mL)
(μg/mL)
A B C S
Bacterial strains
S. aureus ATCC 29213 12.5 12.5 3.125 0.25
MRSA 15187 6.25 25 3.125 16
VRE (Clinical isolate) 6.25 25 3.125 16
E. coli ATCC 25922 6.25 25 6.25 0.03
P. aeruginosa ATCC 29212
Fungal strain
12.5 50 12.5 0.06
C. albicans ATCC 90028 1.56 6.25 1.56 0.5*
A: O. basilicum; B: O. kilimandscharicum; C: O. gratissimum; S: Standard antimicrobial agents ‐ Ciprofloxacin, *Amphotericin‐B
ACKNOWLEDGEMENT
The authors are thankful to Dr. I.A. Khan, Clinical Microbiology
Division, Indian Institute of Integrative Medicine, Jammu (J&K) India
for his valuable contribution in carrying out microbial testing.
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