Gc ms analysis and antimicrobial activity of essential oil of nepeta coerulescens

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ISSN Print: 2278 – 2648 IJRPP |Vol 3 | Issue 1 | Jan – Mar -2014
ISSN Online: 2278-2656 Journal Home page: www.ijrpp.com
Research article Open Access
GC-MS Analysis and antimicrobial activity of essential oil
of Nepeta coerulescens
Navadha Joshi*1
, G.C.Sah2
1
Department of Chemistry, S. S.J. campus, Kumaon University, Almora-263601,
Uttrakhand, India.
2
Department of Chemistry, S. S.J. campus, Kumaon University, Almora-263601,
Uttrakhand, India.
*Corresponding author: Navadha Joshi.
Email address : joshinavadha@gmail.com
ABSTRACT
The chemical composition of the essential oil obtained from the leaves of Nepeta coerulescens collected from the
Zanskar sub district of Kashmir, was analyzed by GC-MS. The major constituent terpene hydrocarbon was found
out to be caryophyllene oxide (25.146%). The antimicrobial activity of essential oil of N.coerulescens was tested
against five bacteria and two fungi, by disc diffusion method. Results showed that the oil exhibited mild
antibacterial activity and good antifungal activity.
Keywords: Nepeta coerulescens, Antibacterial, Antifungal, Caryophyllene oxide, Lamiaceae, Essential oil.
INTRODUCTION
Nepeta L. with approximately 300 species, most of
which are herbaceous perennials, is one of the largest
genera in the family Lamiaceae, subfamily
Nepetoideae, tribe Mentheae. Its species are
distributed throughout Southwest and Central Asia,
Europe, North Africa, North and Central America,
Japan, Korea, China, and the Canary Islands [1-3].
Most of the species are found in South-western Asia,
especially Iran, Central Asia and Trans-Caucasia.
Nepeta is the second largest genus of the Indian
labiates, with 41 species in all, 37 of which occur in
the Western Himalaya [4]. N. campestris and N.
eriostachya were observed to be endemic to India [5].
In the Kashmir Himalaya, about 27 species have been
reported [6], but some of them are now considered as
synonyms to other species. For the first time the
exact number of species of Nepeta was recorded [7]
through extensive survey of Kashmir Himalaya and
consultation of literature as well as specimens
preserved in different herbaria. N. elata, N. royleana,
N. pamirensis and N. vakanica recorded previously
from the study area are actually the synonyms of N.
laevigata, N.salviaefolia, N.kokanica and N.floccosa
respectively.
International Journal of Research in
Pharmacology & Pharmacotherapeutics

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The study therefore, records only twenty two species
of Nepeta from the entire Kashmir Himalayan region.
Several Nepeta species are used in folk medicine as
diuretic, diaphoretic, antitussive, antispasmodic, anti-
asthmatic, febrifuge, emmenagogue, and sedative
agents, and for antiseptic and astringent properties as
a topical remedy in children with cutaneous
eruptions, and for snake and scorpion bites. Some
species are used as medicinal herbs in Iran, for
example, N.ispahanica, N.binaloudensis, N.bracteata,
N. pogonosperma and N.pungens, while N.crispa is
used as culinary herb.[8].
MATERIALS AND METHODS
Plant Material
The leaves of Nepeta coerulescens, were collected
from Zanskar subdistrict , of Kargil district, which
lies in the eastern half of the Jammu and Kashmir,
India. The leaves of the plant were botanically
authenticated by Professor P.C.Pandey, Department
of Botany, Kumaun University, S.S.J. campus,
Almora-263601, Uttarakhand.
Methodology
About 8 kg sample of dried leaves of Nepeta
coerulescens were subjected to hydrodistillation for 8
hours using a Clevenger-type apparatus. The oil was
extracted over ether and dried over anhydrous
Na2SO4. The yield was 0.06% (v/w).
GC and GC/MS: GC/MS analyses were performed
with a Perkin Elmer Clarus 500 gas chromatograph
equipped with a split/splitless injector (split ratio
50:1) data handling system. The column was Rtx-5
capillary columns (60 m x 0.32 mm, 0.25 µm 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
follows: initial temperature of 60o
C (hold: 2 min)
programmed at a rate of 3o
C /min to a final
temperature of 220o
C (hold: 5 min). The temperatures
of the injector, transfer line and ion source were
maintained at 210o
C, and 200o
C, respectively.
Identification of Compounds
The components of the oils were identified by
comparison of their mass spectra with those of
computer library (NIST/ Pfleger /Wiley) or with
authentic compounds and confirmed by comparison
of their retention indices either with those of
authentic compounds or with data published in
literature [9-10]. Isolation of the compounds: The
fractionation of the oil was carried over silica gel
(230-400 mesh, Loba) by column chromatography
using n-hexane (Qualigens) and varying percentages
of diethylether (Qualigens) in n-hexane as mobile
phase [11]. Monitoring was done on pre coated silica
gel TLC plates using iodine as visualizing agent.
Repeated column chromatography of the column
fractions gave one compound coded as C.
Microorganisms
Three gram negative bacteria viz. Pasteurella
multicoda (MTCC 1148), Escherichia coli (MTCC
443), and Salmonella enterica (MTCC 3223), and
two gram positive bacteria viz. Staphylococcus
aureus (MTCC 737) and Bacillus subtilis (MTCC
441) were used for the study of antibacterial activity.
Fungi used were Candida albicans (MTCC 854) and
Aspergillus flavus (MTCC 871). Standard pure
cultures of these bacteria were procured from the
Institute of Microbial Technology (IMTECH),
Chandigarh, India as Microbial Type Culture
Collection (MTCC) and maintained in the laboratory
by regular sub culturing on to nutrient agar.
Antimicrobial activity
Antimicrobial screening of the oil was done by Disc
diffusion method as reported in literature, with slight
modification [12]. Bacterial and fungal suspensions
of 0.25 ml (4 times diluted) was added to the
previously prepared nutrient agar plate and the strain
was thoroughly spread on the surface of agar media,
using a bent rod. The sterilized Whatmann filter
paper No.1 disc (5mm in diameter) was thoroughly
soaked with essential oil and placed in the inoculated
plates. Gentamycin and Nystatin were used as a
reference drugs. Fine pointed forceps were used to
place the disc on the previously inoculated plates
with the maximum possible aseptic precautions. The
discs were firmly pressed against the nutrient agar
medium so that they come in complete contact with

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the agar surface. The discs were placed at equal
distances from each other on the seeded plates and
the plates were incubated at 370
C overnight, to
observe the zone of inhibition around the disc. They
were then compared with the zone of inhibition using
standard antibiotic after overnight incubation on the
nutrient agar plates.
Result and discussion
The essential oil obtained from the leaves of Nepeta
coerulescens were analyzed by GC, GC-MS and
NMR (Table 1) and found to contain Caryophyllene
oxide (25.146%) as the major constituent, 4-(1,5-
dimethylhex-4 enyl)cyclohex-2-enone (10.376%)
was the second major constituent and 1-Butanone,1-
(2-furanyl)- (8.204%) the third major constituent in
the oil. A total of 19 components were listed,
constituting 76.59% of the oil.
The antimicrobial activity of essential oils measured
by disc diffusion method is given in Table 2. The oil
showed mild activity against the bacterial strains and
good activity against fungal strains at a dilution of
1/4 of essential oil. Possibly the antifungal activity is
attributed to the presence of caryophyllene oxide at a
higher concentration in the oil, since it is reported in
a paper as, an oxygenated terpenoid, well known as
preservative in food, drugs and cosmetics, has been
tested in vitro as an antifungal against dermatophytes
[13].
Table 1. Chemical constituents.
S.No. Chemical constituents R.time Leaf oil (%MS)
1 Benzene,(1,3,3-trimethyl nonyl) 13.412 1.628
2 D-limonene 14.851 1.675
3 1-butanone,1-(2-furanyl)- 16.915 8.204
4 Decane,6 ethyl 2 methyl 17.538 1.046
5 Isomenthol 21.505 1.324
6 4-2(-methylcyclo hex-1-enyl)-but-2-enal 22.603 2.807
7 1,4-Benzenediol,2,6-dimethyl 27.263 1.853
8 Trans-4a-methyl decahydro nephthalene 28.008 1.218
9 Caryophyllene 30.022 4.209
10 Bicyclo[3.1.1]hept-2-ene,2,6 dimethyl-6-(4-methyl-3-pentenyl) 32.830 3.582
11 6-(p-tolyl)-2 methyl-2 heptenol 33.555 1.067
12 2-Methyl bicyclo[3.2.1] octane 33.837 1.722
13 Trans-a-bergamotene 34.250 2.229
14 Caryophyllene oxide 39.052 25.146
15 4-(1,5-dimethylhex-4 enyl) cyclohex-2-enone 42.857 10.376
16 2-buten-1-one-1 phenyl 43.632 1.416
17
Bicyclo[3.3.1] nonan-9-one,1,2,4-trimethyl 3-nitro,(2-endo,3-
exo,4-exo)
50.880 1.404
18 N-hexadecanoic acid 51.685 1.355

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Table 2. Antimicrobial activity of essential oils
Microorganism Leaves oil Gentamycin Nystatin (μg)
E.coli 9 40 -
Pasturella multocida 10 32 -
Salmonella enterica 7 22 -
Staphylococus aureus 18 29 -
Bacillus subtillis 17 20 -
Candida albicans 14 - 11
Aspergillus flavus 14 - 14
CONCLUSION
GC-MS analysis of the essential oil of leaves of
N.coerulescens reported Caryophyllene oxide as the
major constituent. Caryophyllene oxide constituted
25.146 % of the total essential oil, followed by
10.376 % of 4-(1,5-dimethylhex-4 enyl) cyclohex-2-
enone. The oil showed mild antibacterial activity and
good antifungal activity, possibly due to the presence
of caryophyllene oxide in moderate concentration.
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