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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
doi: 10.1111/1750-3841.12457
Further reproduction without permission is prohibited
MTCC 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.
M: Food Microbiology
& Safety
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.
M: Food Microbiology
& Safety
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
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)
M: Food Microbiology
& Safety
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.
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
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
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
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
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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M: Food Microbiology
& Safety