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Antibacterial Activity of Essential Oils of Edible

Spices, Ocimum canum and Xylopia aethiopica


N. A. Vyry Wouatsa, Laxminarain Misra, and R. Venkatesh Kumar

The essential oils of 2 Cameroonian spices, namely, Xylopia aethiopica and Ocimum canum, were chemically
investigated and screened for their antibacterial activity. The essential oils were analyzed by means of GC, GC/MS,
and NMR. X. aethiopica oil contained myrtenol (12%), a monoterpenoid in highest concentration. The essential oil of
O. canum belonged to the known linalool (44%) rich chemotype. The results of the antibacterial screening against the
food spoiling bacteria revealed a significant and broad spectrum of activity for these essential oils. The present material
of X. aethiopica, which is having myrtenol in relatively higher concentration, has shown moderate antibacterial activity.
The bioassay-guided fractionation of Ocimum canum oil through flash chromatography showed that minor compounds,
namely, -terpineol, chavicol, chavibetol, and trans-p-mentha-2,8-dien-ol, significantly contributed for the overall activity
observed. Hence, these results evidenced the possible potential of the essential oil of O. canum as a suitable antibacterial
for controlling food-borne pathogens whereas the X. aethiopica oil has moderate possibility.
Abstract:

Keywords: antibacterial, chemical composition, essential oil, Ocimum canum Sims., Xylopia aethiopica (Dun.) A. Rich

There is a strong global demand for the microbe-free, safe, and healthy foods. In this study, we
showed that the essential oil of O. canum (wild basil) can be used as antibacterial for food items. Also, we showed that
a value addition in the antibacterial potential of O. canum oil can be done by processing the essential oil through flash
chromatographic separations.

Practical Application:

M: Food Microbiology
& Safety

Introduction
Many bacterial and fungal infections occur through the ingestion of spoiled foods and water. These infections, caused mainly
by bacterial agents such as Staphylococcus aureus, Escherichia coli,
Salmonella spp., Streptococcus spp. etc, still remain a leading cause
of health hazard with high morbidity and mortality, especially in
the developing world. This is mostly due to the limited access
of lower economy population to integrated health care, prevention tools, and medications. It is further important due to the
emergence of bacterial resistance to current antibiotics. To disentangle this problem of emergence and spread of antimicrobial
resistant microorganisms, there is an urgent need to search and develop new, natural, and cheap leads as antimicrobial agents which
could be used as potent alternatives to the current synthetic drugs
and which will represent safe alternative for controlling food- and
water-borne pathogens.
Used mostly in perfurmery, cosmetic, and pharmaceutical industries, essential oils are mainly exploited for their fragrance,
flavor, and biological properties. Several biological activities have
been attributed to essential oils, namely, antimicrobial, antifungal,
antiinflammatory, antiplasmodial, antioxidant, and so on. (Burt
2004; Zeng and others 2012; Kavoosi and others 2013). Owing
to their potency and safety, many essential oil components have
been recognized as GRAS (generally recognized as safe) by the
Food Drug Administration. Therefore, the use of essential oils as
MS 20131899 Submitted 12/20/2013, Accepted 3/10/2014. Authors Wouatsa
and Misra are with CSIR-Central Inst. of Medicinal and Aromatic Plants,
Lucknow, 226015, India. Author Kumar is with Babasaheb Bhimrao Ambedkar Univ, Lucknow, 226025, India. Author Wouatsa is also with Babasaheb
Bhimrao Ambedkar Univ, Lucknow, 226025, India. Direct inquiries to author Misra
(E-mail: laxmisra@hotmail.com).

M972

Journal of Food Science r Vol. 79, Nr. 5, 2014

food additives to enhance the shelf life of many food products


is primed. Moreover, owing to their complex composition, the
development of bacterial resistance to essential oils is less likely
to occur as compared to synthetic drugs (Burt 2004; Brenes and
Roura 2010). Also, there is a safe demand by consumers for safe
and healthy foods devoid of chemicals. Hence, in view of this, the
essential oils of 2 Cameroonian spices, namely, Ocimum canum and
Xylopia aethiopica, which are common edible spices with antimicrobial properties, were examined for their chemical composition
and their antibacterial activity for possible use as food preservatives.
Ocimum canum Sims. (Lamiaceae), commonly known as wild
basil, is called koti in Cameroon and its leaves are used locally
as spice in an ethnodietary soup known as Mbongo tchobi.
Traditional practitioners in Cameroon employ this plant for abdominal pain, malaria, diarrhea, and stomach-related disorders.
It has been demonstrated that its leaf essential oil shows antimicrobial properties when tested through agar diffusion assay (Bassole and others 2005; Nascimento and others 2011). The fruits
of Xylopia aethiopica (Dun.) A. Rich. (Annonaceae) are used as
antitussive, analgesic, and painkiller. Other ethnomedicinal applications involve the treatment of skin infections, dysentery, and
bronchitis. The fruits of X. aethiopica also constitute the key ingredients of Nah poh and Nkui, the 2 ethnodietary soups from
West region of Cameroon. Previous studies on the essential oil
of X. aethiopica have shown its biological properties, such as antioxidant, antiplasmodial, cytotoxic, and insecticidal (Adegoke and
others 2003; Boyom and others 2003; Kouninki and others 2007;
Tatsadjieu and others 2007). Antifungal and antibacterial properties have also been reported in this oil but on limited number
of food-borne bacteria (Tatsadjieu and others 2003; Asekun and
Adeniyi 2004; Fleischer and others 2008). Moreover, the antibacterial activity of the essential oil of Cameroonian O. canum is now
R

C 2014 Institute of Food Technologists

doi: 10.1111/1750-3841.12457
Further reproduction without permission is prohibited

Antibacterial essential oils of spices . . .


Table 1Bacterial strains used.
Strain name
Bacillus subtilis
Escherichia coli
Klebsiella pneumoniae
Micrococcus luteus
Pseudomonas aeruginosa
Raoultella planticola
Salmonella typhimurium
Staphylococcus aureus
Staphylococcus aureus
Streptococcus mutans

MTCC code

ATCC equivalent code

Affection(s) caused

121
739
109
2470
741
530
1251
96
2940
890

6051
10536
15380
NA
25668
15050
NA
9144
NA
NA

Food contamination
Food poisoning, diarrhea
Pneumonia, urinary tract infections
Opportunistic pathogen
Nosocomial infection, urinary and gastrointestinal tracts infections, pneumonia
Pancreatic complication
Gastrointestinal complication
Food poisoning and toxic shock syndrome
Food poisoning and toxic shock syndrome
Tooth decay

NA, not applicable; ATCC, American type culture collection; MTCC, microbial type culture collection.

Flash chromatography of the essential oil of Ocimum


canum
The essential oil of O. canum (16 mL) was further fractionated
for bioassay-guided studies by flash chromatography (124 g, 230
to 400 mesh, 36 230 mm glass column C-690 Sepacore Buchi)
with n-Hexane as solvent A and EtOAc as solvent B. The flow rate
was set up to 5 mL/ min. A total of 50 fractions (approximately
50 mL each) were collected and pooled according to their TLC
Material and Methods
pattern using Toluene-EtOAc (93:7) as solvent system into 16
General experimental procedures
fractions. Each fraction was further tested for antibacterial activity
Optical rotations were taken with a Horiba SEPA-300 po- and the active fractions were then subjected to GC, GC/MS, or
larimeter (Horiba, Kyoto, Japan). Refractive indexes were ob- NMR measurements to identify the bioactive components.
tained on ATAGO refractometer (Atago Co., Ltd, Tokyo, Japan)
while densities were measured with density/specific gravity meter Bacterial strains
DA-500 KEM (KEM Co., Ltd, Tokyo, Japan).1 H and 13 C NMR
Ten food pathogenic strains (Table 1) were used for the screening
spectra were obtained with an FT-NMR 300 MHz (Bruker, Bil- of the antibacterial activity of the essential oils of O. canum and X.
lerica, Mass., U.S.A.) equipped with a 5-mm 1 H and 13 C (ATP) aethiopica.
probe operating at 300 and 75 MHz, respectively, with TMS (Tetra
methyl silane) as internal standard. Precoated aluminum sheets sil- Antibacterial activity
ica gel 60 F254 TLC plates (Merck) were used to check the purity
The antibacterial activity of the essential oils was screened by
of compounds. Flash chromatography was performed with a Buchi agar disc diffusion and dilution assays following the methodology
Pump manager C-615 flash model (Buchi Labortechnik, Flawil, previously described (Misra and others 2013). The fractions of O.
Switzerland) operating with 2 pump modules C-605. Spots were canum essential oil were prepared in ethanol (99.9% purity, Merck
viewed under UV lamps (254 and 365 nm) and sprayed with 1% KGaA, Darmstadt, Germany) at the concentration of 20 L/mL
ethanolic vanillin solution mixed with 5% ethanolic sulfuric acid. for all the fractions except fractions F-6 and F-7 which were
All reagents used were of analytical grades.
prepared at 4 L/mL. Serial 2-fold dilutions in Mueller Hinton
broth were made in a series of concentration with 1 L/mL being
the highest concentration tested. Kanamycin diluted prior in water
Plant materials
The aerial parts of Ocimum canum were collected at Douala, was used as reference antibiotic. Negative control was made with
Littoral Region of Cameroon in August 2009 while the dried fruits ethanol. All tests were made in triplicate.
of Xylopia aethiopica were purchased from the general spice market
of Douala in December 2009. The plants were identified at the Statistical analyses
Statistical analyses were performed using Statgraphics Centurion
National Herbarium of Cameroon against samples deposited under
the voucher specimen nr 26804SFR/Cam and 16419SFR/Cam XVI. Multifactor ANOVA at 95% confidence level was used.
Duncan test was employed for multiple comparison tests.
for O. canum and X. aethiopica, respectively.
Essential oils extraction
X. aethiopica fruits (900 g) gave 5.5 mL and O. canum aerial
part (7.87 kg) afforded 23.61 mL essential oils by hydrodistillation
for 6 h using a Clevenger type apparatus and dried over anhydrous sodium sulfate following the procedure described previously
(Misra and others 2013).

Results and Discussion

Essential oils composition


The yellow colored oils of O. canum and X. aethiopica showed
the following physical properties: yield (% w/w) 0.276, 0.564;
refractive index at 20 C 1.480, 1.488; optical rotation at 20 C,
() 5.723, (+) 6.133; density at 20 C 0.9150, 0.9239, respectively. The qualitative and quantitative analysis of their chemical
GC, GC/MS, and NMR analyses
composition were studied by GC, GC/MS, Kovats index, and
The essential oils and their components were analyzed using the by comparison with mass spectral data of the library and the
instruments and conditions already reported by Misra and others literature (Adams 2001). For the essential oil of O. canum, 43
components (Table 2) were identified accounting for 96.05% of
(2013).
Vol. 79, Nr. 5, 2014 r Journal of Food Science M973

M: Food Microbiology
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being reported for the 1st time. In continuation of our interest on biologically active phytochemicals (Misra and others 2013;
Wouatsa and others 2013a, 2013b; Joshi and others 2014; Siddique
and others 2014), we report a detailed study on the chemical composition of the essential oils of O. canum and X. aethiopica and their
activity against food-borne microbes.

Antibacterial essential oils of spices . . .

M: Food Microbiology
& Safety

Table 2Composition of the essential oils of O. canum and Table 2Continued.


X. aethiopica (%).
Compounds
KI
O. canum
X. aethiopica
Compounds
KI
O. canum
X. aethiopica
-Muurolene
1497

0.11
(Z)-3-hexen-1-ol
902
0.50

-Bulnesene
1500

0.15
-Bisabolene
1509
0.52

-Thujene
936
0.11
0.15
-Cadinene
1516
0.28
0.27
-Pinene
939
0.56
3.42
Camphene
955

0.18
Cadina-1,4-diene
1533
0.62

Sabinene
977
0.13
0.54
Sesquisabinene hydrate cis
1547

0.27
E-Nerolidol
1565

0.21
-Pinene
984
0.94
9.00
Spathulenol
1577

0.12
-Myrcene
990
0.67

-Phellandrene
1003
0.07
0.13
Caryophyllene oxide
1580

0.38
Delta-3-carene
1011

0.90
Globulol
1585

0.15
Viridiflorol
1590

0.24
-Terpinene
1018
0.31
0.36
Humulene epoxide
1608

0.53
p-Cymene
1026
0.27
2.32
Limonene
1030
2.38
6.00
-Eudesmol
1631
0.98
0.85
1,8-Cineole
1033
10.19
2.00
-Cadinol
1656
0.07
0.09
Bulnesol
1666

0.11
(Z)--Ocimene
1044
0.17

(E)--Ocimene
1052
1.66

Epi--bisabolol
1692

0.12
-Terpinene
1069
0.69
0.21
Caryophyllene acetate
1700

0.26
p-Cresol
1076

0.33
Class composition
Monoterpene hydrocarbons
7.96
23.21
Fenchone
1083
2.25

Terpinolene
1089

0.59
Oxygenated monoterpenes
64.18
34.78
Linalool
1100
44.13
1.83
Phenyl derivatives
17.68
16.53
Sesquiterpene hydrocarbons
6.81
2.24
cis-Thujone
1103

0.21
Oxygenated sesquiterpenes
1.05
3.33
Fenchol endo
1114

0.15
Allo ocimene
1128

0.74
Linear compounds
0.50
0.0
Limonene oxide
1136

0.26
Total
98.18
80.09
Sabinol trans
1140
0.21
7.00
Camphor
1143
0.12

Isopulegol
1147

2.55
Citronellal
1154

0.31
the oil mainly composed of oxygenated monoterpenes (61.95%).
Borneol
1164

0.33
Linalool (Figure 1B, 44.13%), eugenol (17.68%), and 1,8-cineole
Pinocampheol
1171
0.25
0.26
(10.19%) were the major components followed by terpinen-4-ol
Terpinen-4-ol
1178
4.83
0.34
-Terpineol
1189
1.65
0.21
(4.83%) and -trans-bergamotene (2.94%). Previous investigations
Myrtenol
1193

12.00
on the chemistry of the essential oil of O. canum have shown the
Methylchavicol
1200

4.45
presence of several chemotypes, namely, limonene-rich (NgasD-Verbenone
1201

4.43
soum and others 2004; Ngassoum and others 2007), 1,8-cineole
cis Carveol
1203

0.24
Cuminic aldehyde
1212

2.85
rich (Bassole and others 2005), camphor-rich (Upadhyay and othNerol
1229

0.64
ers 1991; Chagonda and others 2000), linalool-rich (Ngassoum
Isobornyl formate
1233

0.13
and others 2004), -terpineol-rich, chavicol- and -terpineolPhellandral
1240

2.96
rich (Chalchat and others 1999), and methyl trans-cinnamate-rich
ascaridole cis
1242

chemotypes (Martins and others 1999). The present chemotype,


Isogeijerene C
1250

0.32
Geraniol
1257
0.57
0.75
described in this study, has a similar composition with those of
Geranial
1272

0.27
the North Cameroon type reported by Ngassoum and others
Safrole
1284

0.24
(2004) wherein the oxygenated monoterpenoids were the major
Pregeijerene
1287

0.22
components (91.9%) with linalool at 44.9%, as one of the main
Thymol
1292

2.98
Carvacrol
1300

0.54
constituents.
Terpinylacetate
1317

0.45
trans-dihydro-alpha
Eugenol
1357
17.68
0.25
Neryl acetate
1366

0.12
10 OH
-Ylangene
1373
0.10

9
-Copaene
1379

0.34
OH
-Cubebene
1389
0.43

1
6
-Elemene
1391

0.09
-Gurjunene
1415
0.16

5
2
6
-Cis Bergamoptene
1417
0.07

7
-Gurjunene
1427

0.10
5
-Elemene
1432
0.14

7
9
-Trans Bergamoptene
1435
2.94

3
4
8
Aromadendrene
1438
0.07

-Humulene
1442
0.06
0.66
3
8
4
Z--Farnesene
1447

0.13
epi--Santalene
1448
0.19
0.39
-Humulene
1456
0.10

2
Alloaromadendrene
1465
0.11

A
1
10
-Muurolene
1479
0.42

Germacrene D
1482
0.21
0.11
B
-Selinene
1489
0.28
0.15
-Selinene
1496
0.21
0.29
Figure 1Structures of myrtenol (A) and linalool (B).
(Continued)

M974 Journal of Food Science r Vol. 79, Nr. 5, 2014

Antibacterial essential oils of spices . . .


agents as compared to Gram-positive bacteria. This may be due to
their extra membranous layer made of lipopolysaccharide, which
has been shown to act as barrier against external agents especially
hydrophobic agents (Hancock 1997; Helander and others 1998).
The present study reveals that the essential oil of O. canum has successfully inhibited various Gram-negative bacteria among which
E. coli, S. typhimurium, and K. pneumoniae are responsible for food
poisoning, gastrointestinal, and nosocomial infections, respectively,
at concentrations 2 L/mL (Nissen and others 2010).
The relationship studies between chemical composition and antimicrobial activity revealed that the activity of the essential oils
of O. canum and X. aethiopica could be attributed to the higher
content in oxygenated monoterpenes (50%) which are well recognized for their antimicrobial potential (Devi and others 2010;
Nissen and others 2010). The essential oil of O. canum having
pronounced activity contained more oxygenated monoterpenes
(61.95%) than X. aethiopica (48.83%). Moreover, O. canum contained linalool at 44.13% and 1,8-cineole at 10.19% in contrast
to the essential oil of X. aethiopica in which the same compounds
were present in less than 2% concentration. Linalool and eugenol
have shown a broad spectrum of antibacterial activity indicating
that they may be responsible for the activity of O. canum essential
Antimicrobial activity
oil. Eugenol, a well-known aromatic compound with remarkable
An inhibition diameter greater or equal to 20 mm was consid- antimicrobial properties and recognized as safe by FDA, was found
ered as significant cutoff point relevant for a strong antimicrobial in good concentration in the essential oil of O. canum at 17.68%.
activity. Likewise, a moderate activity was attributed to an inhibition zone between 10 and 20 mm and no activity was coined Bioassay-guided fractionation of O. canum oil
for zones lower than 10 mm. Based on these criteria, the results
In an attempt to identify the bioactive compounds, the bioassay(Table 3) showed that the essential oils of O. canum and X. aethiopica guided fractionation was performed on the essential oil of
demonstrated significant activity (P < 0.001, F = 21.06) with in- O. canum. The essential oil of O. canum was subjected to flash
hibition diameters ranging between <10 and 14.33 mm. The chromatography and afforded 16 fractions, which were further
inhibition of bacterial growth was significantly (P < 0.001) de- tested for their antibacterial properties (Experimental). The results
pendent upon the oils tested and the microorganism used. In this showed that all the tested fractions displayed moderate to low acregard, O. canum essential oil was found to be more active (P < tivity against S. typhimurium (Table 6). Fractions F-9, F-11, and
0.05) than the essential oil of X. aethiopica and the most suscep- F-14 were found active against at least 2 bacteria whereas fraction
tible organisms (P < 0.05) were P. aeruginosa MTCC741 and S. F-11 showed MIC below 1 L/mL vis-`a-vis the 2 susceptible
typhimurium MTCC1251.
bacteria K. pneumoniae and S. typhimurium.
For quantitative determination of the antimicrobial activity,
GC-MS of the bioactive fractions of the essential oil of O. canum
minimal inhibitory concentration (MIC) (Table 4) was calculated. revealed the presence of oxygenated monoterpenes (Table 6). The
A significant difference (P < 0.001, F = 48.42) among the most active but small in quantity, fraction (F-11) was composed of
antimicrobial potential of the essential oils of O. canum and X. -terpineol, chavicol, chavibetol, and trans p-mentha-2,8-dien-ol
aethiopica was observed. The essential oil of O. canum showed better (Table 6). Previous studies demonstrated the antibacterial activity
(P < 0.05) antimicrobial activity, which was higher than that of of -terpineol against a wide range of microorganisms (Cosentino
X. aethiopicas oil as inferred by its low MIC values (0.43 2.08 and others 1999). Hence, this compound may also be responsible
L/mL) against 7 out of 10 bacteria tested. In contrast, the essen- for the broad range of activity displayed by the essential oil of O.
tial oil of X. aethiopica failed to show any interesting activity with canum. NMR studies on the isolated compounds from fractions
MIC 4.16 L/mL. Nonetheless, a low MIC (0.69 L/mL) F-9 and F-10 confirmed that they contain linalool as the major
was obtained for this oil against the Gram-negative bacterium compound of the essential oil of O. canum and showed poor activity
S. typhimurium. With regard to the susceptibility of the microor- than fraction F-11, which happened to be the mixture of several
ganism to the action of the essential oils tested, S. typhimurium minor components. The 1 H NMR spectrum of linalool (Figure
was found to be the most sensitive (P < 0.05) microorganism 1B) showed typical double doublet (J = 16.0, 12.0 Hz) at 5.92
while P. aeruginosa was the most resistant (P < 0.05).
and 2 doublets (J = 16.0 and 2.0 Hz, 14.0, and 2.0 Hz) at 5.0
To assess the bactericidal or bacteriostatic potential of the es- and 5.2 for H-7 and H-8, respectively. An overlapping triplet at
sential oils, minimal bactericidal concentration (MBC) was eval- 5.15 for H-3 and 2 singlets (3H each) at 1.60 and 1.65 for
uated and the results are presented in Table 5. The essential oils H-1 and H-10 along with another singlet (3H) at 1.30 for Hof O. canum and X. aethiopica were both more bactericidal than 9 clearly indicated the presence of linalool in the oil as one of
bacteriostatic. The higher bactericidal and bacteriostatic proper- the major compounds. 13 C NMR of pure linalool was recorded
ties were observed with O. canum, which had lower MBC values showing the signals that matched well with the standard linalool
(2 L/mL) compared to X. aethiopica.
( 146.2, 133.0, 125.8, 112.5, 72.5, 42.2, 28.0, 25,8, 23.2, 17.8).
Overall, the essential oil of O. canum displayed a broad spectrum These characteristic signals were used to monitor the presence of
of antimicrobial activity. Both Gram-negative and Gram-positive linalool in the chromatographic fractions. This clearly evidenced
bacteria were susceptible to its action. Interestingly, Gram-negative that the antibacterial activity of the essential oil of O. canum is due
bacteria are well recognized to be less sensitive to antibacterial to combined effects of both its major and minor constituents.
Vol. 79, Nr. 5, 2014 r Journal of Food Science M975

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In the essential oil of X. aethiopica, 68 constituents (Table 2) constituting 80.28% of the total detected components were identified.
The main constituents were oxygenated monoterpenes (48.83%)
followed by monoterpene hydrocarbons (25.08%). Sesquiterpenes
constituted less than 10% of the oil. -Pinene has earlier been
reported as the main component of this oil (13.78% to 23.60%)
(Tatsadjieu and others 2003; Karioti and others 2004; Jirovetz and
others 2005; Olonisakin and others 2007; Koba and others 2008),
but in our material it constituted to 9.0% only. Now, the myrtenol
(Figure 1A) has been found as the 1st major constituent (12%),
whereas earlier, myrtenol was reported as the minor component
(2.4%) of the essential oil of X. aethiopica (Jirovetz and others 2005).
Hence, the present study for the 1st time describes the occurrence
of myrtenol (Figure 1A) as one of the main constituents of X.
aethiopica. Other constituents identified in the essential oil of X.
aethiopica below 10% concentration included mainly trans sabinol (7%), limonene (6%), methyl chavicol (4.45%), d-verbenone
(4.43%), -pinene (3.42%), 1,8-cineole (2.0%), and cuminic aldehyde (2.85%). The presence of myrtenol (Figure 1A) in good concentration in this oil could be attributed to the location of plant
material collected by us.

Antibacterial essential oils of spices . . .


Table 3Inhibition diameters in mm of the essential oils of O. canum and X. aethiopica.
Gram-positive bacteria
E. oils

BS

ML

SA96

Gram-negative bacteria

SA2940

SM

EC

KP

PA

RP

STM

O. canum
12 1 13.33 0.58 11.66 0.58
10 0
10.33 0.58 7.67 0.58 10.33 0.58 14.33 0.58 11.33 0.58 13.66 0.58
X. aethiopica
8
9 1
12
8.67 1.15 10.67 0.58 10.33 0.57 8.33 0.57
11
10. 33 0.57
11 1
E. oils, essential oils; BS, Bacillus subtilis MTCC121; ML, Micrococcus luteus MTCC2470; SA96, Staphylococcus aureus MTCC96; SA2940, Staphylococcus aureus MTCC2940; SM,
Streptococcus mutans MTCC890; EC, Escherichia coli MTCC739; KP, Klebsiella pneumoniae MTCC109; PA, Pseudomonas aeruginosa MTCC741; RP, Raoultella planticola MTCC530;
STM, Salmonella typhimurium MTCC1251.

Table 4Minimal inhibitory concentrations (MIC ) of the essential oils of O. canum and X. aethiopica.
Gram-positive bacteria
E. oils
O. canum
X. aethiopica
Kanamycin

Gram-negative bacteria

BS

ML

SA96

SA2940

SM

EC

KP

PA

RP

STM

2.08
nt
0.52 0.45

2.08
4.16
4.16

3.47 1.20
>8.33
6.94 2.40

4.16
>8.33
nt

1.39 0.60
4.16
0.86 0.30

2.08
8.33
8.33

1.73 0.60
nt
4.85 3.19

>8.33
>8.33
62.5

1.04
4.16
0.35 0.15

0.43 0.15
0.69 0.30
0.52

nt, not tested.

MIC is expressed in L/mL for the essential oils and in g/mL for kanamycin the standard antibiotic.
E. oils, essential oils; BS, Bacillus subtilis MTCC121; ML, Micrococcus luteus MTCC2470; SA96, Staphylococcus aureus MTCC96; SA2940, Staphylococcus aureus MTCC2940; SM,
Streptococcus mutans MTCC890; EC, Escherichia coli MTCC739; KP, Klebsiella pneumoniae MTCC109; PA, Pseudomonas aeruginosa MTCC741; RP, Raoultella planticola MTCC530;
STM, Salmonella typhimurium MTCC1251.

Table 5Minimal bactericidal concentration (MBC ) of the essential oils of O. canum and X. aethiopica.
Gram-positive bacteria

Gram-negative bacteria

M: Food Microbiology
& Safety

E. oils

BS

ML

SA96

SA2940

SM

EC

KP

PA

RP

STM

O. canum
X. aethiopica

2.08
nt

>33.33
>33.33

16.66
33.33

8.33
33.33

>33.33
nt

2.08
8.33

2.08
nt

nt
nt

1.39 0.60
6.94 2.41

0.69 0.30
1.04

nt, not tested.

MBC is expressed in L/mL for the essential oils. To determine the MBC, 100 L of Mueller Hinton Broth (MHB) from the wells which showed no visible bacterial growth
were transferred onto Mueller Hinton Agar (MHA) plates and the bacterial revival was assessed after 24-h incubation. Hence, MBC was the lowest concentration at which bacteria
failed to grow in MHB and in the subsequent transfer to MHA.
E. oils, essential oils; BS, Bacillus subtilis MTCC121; ML, Micrococcus luteus MTCC2470; SA96, Staphylococcus aureus MTCC96; SA2940, Staphylococcus aureus MTCC2940; SM,
Streptococcus mutans MTCC890; EC, Escherichia coli MTCC739; KP, Klebsiella pneumoniae MTCC109; PA, Pseudomonas aeruginosa MTCC741; RP, Raoultella planticola MTCC530;
STM, Salmonella typhimurium MTCC1251.

Table 6Minimal inhibitory concentration (MIC) and composition of the fractions of the essential oil of O. canum.
MIC in L/mL (vol/vol)
Fractions
F-2
F-3
F-6
F-7
F-8
F-9
F-10
F-11
F-12
F-13
F-14
O. canum oil

KP

PA

STM

Main constituents

>1
>1
>0.2
>0.2
>1
1
>1
0.5
>1
>1
1
1.73 0.60

>1
>1
>0.2
>0.2
>1
>1
>1
>1
>1
>1
>1
>8

0.6 0.57
1
>0.2
>0.2
1
0.5
0.5
0.5
0.35 0.21
>1
0.25
0.43 0.15

- and -Pinene, sabinene


Eucalyptol, aristolene epoxide
Trans--bergamoptene
Eugenol
Fenchone, 4-terpinylacetate, chavibetol (m-eugenol), methyl eugenol
Linalool
Linalool
-Terpineol, chavicol, trans-p-mentha-2,8-dienol
-Elemene, dehydroaromadendrene
Elemene, dehydroaromadendrene
-Eudesmol
Linalool, eugenol, and 1,8- cineole

Conclusion
The present collection of X. aethiopica from Cameroon contained myrtenol as one of the prominent constituents of its essential oil whereas the essential oil of O. canum belonged to one of the
existing chemotype earlier reported from North Cameroon. The
results of the antibacterial screening demonstrated that the essential
oil of the aerial parts of O. canum can be used as a food additive for
controlling food-borne pathogens. Furthermore, bioassay-guided
fractionation of this oil through flash chromatography revealed that
minor compounds contribute significantly for the overall activity
observed. Flash chromatography enabled the purification and isolation of several constituents among which linalool and eugenol
were the major ones. Moreover, the fractionation of the essential

M976 Journal of Food Science r Vol. 79, Nr. 5, 2014

oil through flash chromatography allowed the separation and precise identification of eugenol derivatives, namely, meta eugenol
and methyl eugenol.

Acknowledgments
N.A. Vyry Wouatsa is grateful to TWAS, Italy and CSIR, India
for the award of a doctoral fellowship. Director CSIR-CIMAP,
Lucknow is also acknowledged for providing necessary research
facilities. Mr. M.P. Darokar is thanked for permitting us to test the
antibacterial activity in his laboratory. Thanks are also due to Mrs.
Anju Yadav and Namita Gupta for recording GC, GC/MS, and
NMR spectra, respectively.

Author Contributions
N.A. Vyry Wouatsa and Laxminarain Misra designed the study.
N.A. Vyry Wouatsa collected the plants, performed the extraction of the essential oil, the antibacterial assays and isolated the
compounds. She also drafted the manuscript. Laxminarain Misra
supervised the work, identified the isolated compounds, and revised the manuscript. R. Venkatesh Kumar supervised the work
and edited the manuscript.

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Vol. 79, Nr. 5, 2014 r Journal of Food Science M977

M: Food Microbiology
& Safety

Antibacterial essential oils of spices . . .