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Antibacterial Activity of Essential Oils of EdibleSpices, Ocimum canum and Xylopia aethiopicaN. A. Vyry Wouatsa, Laxminarain Misra, and R. Venkatesh Kumar

Abstract: The essential oils of 2 Cameroonian spices, namely, Xylopia aethiopica and Ocimum canum, were chemicallyinvestigated 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 ofO. canum belonged to the known linalool (44%) rich chemotype. The results of the antibacterial screening against thefood spoiling bacteria revealed a significant and broad spectrum of activity for these essential oils. The present materialof 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 activityobserved. Hence, these results evidenced the possible potential of the essential oil of O. canum as a suitable antibacterialfor controlling food-borne pathogens whereas the X. aethiopica oil has moderate possibility.

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

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

IntroductionMany bacterial and fungal infections occur through the inges-

tion of spoiled foods and water. These infections, caused mainlyby bacterial agents such as Staphylococcus aureus, Escherichia coli,Salmonella spp., Streptococcus spp. etc, still remain a leading causeof health hazard with high morbidity and mortality, especially inthe developing world. This is mostly due to the limited accessof lower economy population to integrated health care, preven-tion tools, and medications. It is further important due to theemergence of bacterial resistance to current antibiotics. To dis-entangle this problem of emergence and spread of antimicrobialresistant microorganisms, there is an urgent need to search and de-velop new, natural, and cheap leads as antimicrobial agents whichcould be used as potent alternatives to the current synthetic drugsand which will represent safe alternative for controlling food- andwater-borne pathogens.Used mostly in perfurmery, cosmetic, and pharmaceutical in-

dustries, essential oils are mainly exploited for their fragrance,flavor, and biological properties. Several biological activities havebeen attributed to essential oils, namely, antimicrobial, antifungal,antiinflammatory, antiplasmodial, antioxidant, and so on. (Burt2004; Zeng and others 2012; Kavoosi and others 2013). Owingto their potency and safety, many essential oil components havebeen recognized as GRAS (generally recognized as safe) by theFood Drug Administration. Therefore, the use of essential oils as

MS 20131899 Submitted 12/20/2013, Accepted 3/10/2014. Authors Wouatsaand Misra are with CSIR-Central Inst. of Medicinal and Aromatic Plants,Lucknow, 226015, India. Author Kumar is with Babasaheb Bhimrao Ambed-kar Univ, Lucknow, 226025, India. Author Wouatsa is also with BabasahebBhimrao Ambedkar Univ, Lucknow, 226025, India. Direct inquiries to author Misra(E-mail: laxmisra@hotmail.com).

food additives to enhance the shelf life of many food productsis primed. Moreover, owing to their complex composition, thedevelopment of bacterial resistance to essential oils is less likelyto occur as compared to synthetic drugs (Burt 2004; Brenes andRoura 2010). Also, there is a safe demand by consumers for safeand healthy foods devoid of chemicals. Hence, in view of this, theessential oils of 2 Cameroonian spices, namely, Ocimum canum andXylopia aethiopica, which are common edible spices with antimi-crobial properties, were examined for their chemical compositionand their antibacterial activity for possible use as food preservatives.

Ocimum canum Sims. (Lamiaceae), commonly known as wildbasil, is called koti in Cameroon and its leaves are used locallyas spice in an ethnodietary soup known as Mbongo tchobi.Traditional practitioners in Cameroon employ this plant for ab-dominal pain, malaria, diarrhea, and stomach-related disorders.It has been demonstrated that its leaf essential oil shows antimi-crobial properties when tested through agar diffusion assay (Bas-sole and others 2005; Nascimento and others 2011). The fruitsof Xylopia aethiopica (Dun.) A. Rich. (Annonaceae) are used asantitussive, analgesic, and painkiller. Other ethnomedicinal appli-cations involve the treatment of skin infections, dysentery, andbronchitis. The fruits of X. aethiopica also constitute the key ingre-dients of Nah poh and Nkui, the 2 ethnodietary soups fromWest region of Cameroon. Previous studies on the essential oilof X. aethiopica have shown its biological properties, such as an-tioxidant, antiplasmodial, cytotoxic, and insecticidal (Adegoke andothers 2003; Boyom and others 2003; Kouninki and others 2007;Tatsadjieu and others 2007). Antifungal and antibacterial prop-erties have also been reported in this oil but on limited numberof food-borne bacteria (Tatsadjieu and others 2003; Asekun andAdeniyi 2004; Fleischer and others 2008). Moreover, the antibac-terial activity of the essential oil of Cameroonian O. canum is now

C 2014 Institute of Food Technologists RM972 Journal of Food Science Vol. 79, Nr. 5, 2014 doi: 10.1111/1750-3841.12457

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Table 1Bacterial strains used.

Strain name MTCC code ATCC equivalent code Affection(s) caused

Bacillus subtilis 121 6051 Food contaminationEscherichia coli 739 10536 Food poisoning, diarrheaKlebsiella pneumoniae 109 15380 Pneumonia, urinary tract infectionsMicrococcus luteus 2470 NA Opportunistic pathogenPseudomonas aeruginosa 741 25668 Nosocomial infection, urinary and gastrointestinal tracts infections, pneumoniaRaoultella planticola 530 15050 Pancreatic complicationSalmonella typhimurium 1251 NA Gastrointestinal complicationStaphylococcus aureus 96 9144 Food poisoning and toxic shock syndromeStaphylococcus aureus 2940 NA Food poisoning and toxic shock syndromeStreptococcus mutans 890 NA Tooth decay

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

being reported for the 1st time. In continuation of our inter-est on biologically active phytochemicals (Misra and others 2013;Wouatsa and others 2013a, 2013b; Joshi and others 2014; Siddiqueand others 2014), we report a detailed study on the chemical com-position of the essential oils of O. canum and X. aethiopica and theiractivity against food-borne microbes.

Material and Methods

General experimental proceduresOptical rotations were taken with a Horiba SEPA-300 po-

larimeter (Horiba, Kyoto, Japan). Refractive indexes were ob-tained on ATAGO refractometer (Atago Co., Ltd, Tokyo, Japan)while densities were measured with density/specific gravity meterDA-500 KEM (KEM Co., Ltd, Tokyo, Japan).1H and 13C NMRspectra were obtained with an FT-NMR 300 MHz (Bruker, Bil-lerica, Mass., U.S.A.) equipped with a 5-mm 1H and 13C (ATP)probe operating at 300 and 75MHz, respectively, with TMS (Tetramethyl silane) as internal standard. Precoated aluminum sheets sil-ica gel 60 F254 TLC plates (Merck) were used to check the purityof compounds. Flash chromatography was performed with a BuchiPump manager C-615 flash model (Buchi Labortechnik, Flawil,Switzerland) operating with 2 pump modules C-605. Spots wereviewed under UV lamps (254 and 365 nm) and sprayed with 1%ethanolic vanillin solution mixed with 5% ethanolic sulfuric acid.All reagents used were of analytical grades.

Plant materialsThe aerial parts of Ocimum canum were collected at Douala,

Littoral Region of Cameroon in August 2009while the dried fruitsof Xylopia aethiopica were purchased from the general spice marketof Douala in December 2009. The plants were identified at theNational Herbarium of Cameroon against samples deposited underthe voucher specimen nr 26804SFR/Cam and 16419SFR/Camfor O. canum and X. aethiopica, respectively.

Essential oils extractionX. aethiopica fruits (900 g) gave 5.5 mL and O. canum aerial

part (7.87 kg) afforded 23.61 mL essential oils by hydrodistillationfor 6 h using a Clevenger type apparatus and dried over anhy-drous sodium sulfate following the procedure described previously(Misra and others 2013).

GC, GC/MS, and NMR analysesThe essential oils and their components were analyzed using the

instruments and conditions already reported by Misra and others(2013).

Flash chromatography of the essential oil of OcimumcanumThe essential oil of O. canum (16 mL) was further fractionated

for bioassay-guided studies by flash chromatography (124 g, 230to 400 mesh, 36 230 mm glass column C-690 Sepacore Buchi)with n-Hexane as solvent A and EtOAc as solvent B. The flow ratewas set up to 5 mL/ min. A total of 50 fractions (approximately50 mL each) were collected and pooled according to their TLCpattern using Toluene-EtOAc (93:7) as solvent system into 16fractions. Each fraction was further tested for antibacterial activityand the active fractions were then subjected to GC, GC/MS, orNMR measurements to identify the bioactive components.

Bacterial strainsTen food pathogenic strains (Table 1) were used for the screening

of the antibacterial activity of the essential oils of O. canum and X.aethiopica.

Antibacterial activityThe antibacterial activity of the essential oils was screened by

agar disc diffusion and dilution assays following the methodologypreviously described (Misra and others 2013). The fractions of O.canum essential oil were prepared in ethanol (99.9% purity, MerckKGaA, Darmstadt, Germany) at the concentration of 20 L/mLfor all the fractions except fractions F-6 and F-7 which wereprepared at 4 L/mL. Serial 2-fold dilutions in Mueller Hintonbroth were made in a series of concentration with 1 L/mL beingthe highest concentration tested. Kanamycin diluted prior in waterwas used as reference antibiotic. Negative control was made withethanol. All tests were made in triplicate.

Statistical analysesStatistical analyses were performed using Statgraphics Centurion

XVI. Multifactor ANOVA at 95% confidence level was used.Duncan test was employed for multiple comparison tests.

Results and Discussion

Essential oils compositionThe 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, respec-tively. The qualitative and quantitative analysis of their chemicalcomposition were studied by GC, GC/MS, Kovats index, andby comparison with mass spectral data of the library and theliterature (Adams 2001). For the essential oil of O. canum, 43components (Table 2) were identified accounting for 96.05% of

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Table 2Composition of the essential oils of O. canum andX. aethiopica (%).

Compounds KI O. canum X. aethiopica

(Z)-3-hexen-1-ol 902 0.50 -Thujene 936 0.11 0.15-Pinene 939 0.56 3.42Camphene 955 0.18Sabinene 977 0.13 0.54-Pinene 984 0.94 9.00-Myrcene 990 0.67 -Phellandrene 1003 0.07 0.13Delta-3-carene 1011 0.90-Terpinene 1018 0.31 0.36p-Cymene 1026 0.27 2.32Limonene 1030 2.38 6.001,8-Cineole 1033 10.19 2.00(Z)--Ocimene 1044 0.17 (E)--Ocimene 1052 1.66 -Terpinene 1069 0.69 0.21p-Cresol 1076 0.33Fenchone 1083 2.25 Terpinolene 1089 0.59Linalool 1100 44.13 1.83cis-Thujone 1103 0.21Fenchol endo 1114 0.15Allo ocimene 1128 0.74Limonene oxide 1136 0.26Sabinol trans 1140 0.21 7.00Camphor 1143 0.12 Isopulegol 1147 2.55Citronellal 1154 0.31Borneol 1164 0.33Pinocampheol 1171 0.25 0.26Terpinen-4-ol 1178 4.83 0.34-Terpineol 1189 1.65 0.21Myrtenol 1193 12.00Methylchavicol 1200 4.45D-Verbenone 1201 4.43cis Carveol 1203 0.24Cuminic aldehyde 1212 2.85Nerol 1229 0.64Isobornyl formate 1233 0.13Phellandral 1240 2.96ascaridole cis 1242 Isogeijerene C 1250 0.32Geraniol 1257 0.57 0.75Geranial 1272 0.27Safrole 1284 0.24Pregeijerene 1287 0.22Thymol 1292 2.98Carvacrol 1300 0.54Terpinylacetate 1317 0.45

trans-dihydro-alphaEugenol 1357 17.68 0.25Neryl acetate 1366 0.12-Ylangene 1373 0.10 -Copaene 1379 0.34-Cubebene 1389 0.43 -Elemene 1391 0.09-Gurjunene 1415 0.16 -Cis Bergamoptene 1417 0.07 -Gurjunene 1427 0.10 -Elemene 1432 0.14 -Trans Bergamoptene 1435 2.94 Aromadendrene 1438 0.07 -Humulene 1442 0.06 0.66Z--Farnesene 1447 0.13epi--Santalene 1448 0.19 0.39-Humulene 1456 0.10 Alloaromadendrene 1465 0.11 -Muurolene 1479 0.42 Germacrene D 1482 0.21 0.11-Selinene 1489 0.28 0.15-Selinene 1496 0.21 0.29

(Continued)

Table 2Continued.

Compounds KI O. canum X. aethiopica

-Muurolene 1497 0.11-Bulnesene 1500 0.15-Bisabolene 1509 0.52 -Cadinene 1516 0.28 0.27Cadina-1,4-diene 1533 0.62 Sesquisabinene hydrate cis 1547 0.27E-Nerolidol 1565 0.21Spathulenol 1577 0.12Caryophyllene oxide 1580 0.38Globulol 1585 0.15Viridiflorol 1590 0.24Humulene epoxide 1608 0.53 -Eudesmol 1631 0.98 0.85-Cadinol 1656 0.07 0.09Bulnesol 1666 0.11Epi--bisabolol 1692 0.12Caryophyllene acetate 1700 0.26Class compositionMonoterpene hydrocarbons 7.96 23.21Oxygenated monoterpenes 64.18 34.78Phenyl derivatives 17.68 16.53Sesquiterpene hydrocarbons 6.81 2.24Oxygenated sesquiterpenes 1.05 3.33Linear compounds 0.50 0.0Total 98.18 80.09

the oil mainly composed of oxygenated monoterpenes (61.95%).Linalool (Figure 1B, 44.13%), eugenol (17.68%), and 1,8-cineole(10.19%) were the major components followed by terpinen-4-ol(4.83%) and -trans-bergamotene (2.94%). Previous investigationson the chemistry of the essential oil of O. canum have shown thepresence of several chemotypes, namely, limonene-rich (Ngas-soum and others 2004; Ngassoum and others 2007), 1,8-cineolerich (Bassole and others 2005), camphor-rich (Upadhyay and oth-ers 1991; Chagonda and others 2000), linalool-rich (Ngassoumand others 2004), -terpineol-rich, chavicol- and -terpineol-rich (Chalchat and others 1999), and methyl trans-cinnamate-richchemotypes (Martins and others 1999). The present chemotype,described in this study, has a similar composition with those ofthe North Cameroon type reported by Ngassoum and others(2004) wherein the oxygenated monoterpenoids were the majorcomponents (91.9%) with linalool at 44.9%, as one of the mainconstituents.

OH

OH

1

2

3

4

56

7

8

9

10

1

2

3

4

7

65

8

9

10

AB

Figure 1Structures of myrtenol (A) and linalool (B).

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In the essential oil of X. aethiopica, 68 constituents (Table 2) con-stituting 80.28% of the total detected components were identified.The main constituents were oxygenated monoterpenes (48.83%)followed by monoterpene hydrocarbons (25.08%). Sesquiterpenesconstituted less than 10% of the oil. -Pinene has earlier beenreported as the main component of this oil (13.78% to 23.60%)(Tatsadjieu and others 2003; Karioti and others 2004; Jirovetz andothers 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 ofX. aethiopica (Jirovetz and others 2005).Hence, the present study for the 1st time describes the occurrenceof 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 sabi-nol (7%), limonene (6%), methyl chavicol (4.45%), d-verbenone(4.43%), -pinene (3.42%), 1,8-cineole (2.0%), and cuminic alde-hyde (2.85%). The presence of myrtenol (Figure 1A) in good con-centration in this oil could be attributed to the location of plantmaterial collected by us.

Antimicrobial activityAn inhibition diameter greater or equal to 20 mm was consid-

ered as significant cutoff point relevant for a strong antimicrobialactivity. Likewise, a moderate activity was attributed to an inhi-bition zone between 10 and 20 mm and no activity was coinedfor zones lower than 10 mm. Based on these criteria, the results(Table 3) showed that the essential oils ofO. canum andX. aethiopicademonstrated significant activity (P < 0.001, F = 21.06) with in-hibition diameters ranging between

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Table 3Inhibition diameters in mm of the essential oils of O. canum and X. aethiopica.

Gram-positive bacteria Gram-negative bacteria

E. oils BS ML SA96 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 0.58X. 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 1E. 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 Gram-negative bacteria

E. oils BS ML SA96 SA2940 SM EC KP PA RP STM

O. canum 2.08 2.08 3.47 1.20 4.16 1.39 0.60 2.08 1.73 0.60 >8.33 1.04 0.43 0.15X. aethiopica nt 4.16 >8.33 >8.33 4.16 8.33 nt >8.33 4.16 0.69 0.30Kanamycin 0.52 0.45 4.16 6.94 2.40 nt 0.86 0.30 8.33 4.85 3.19 62.5 0.35 0.15 0.52nt, 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

E. oils BS ML SA96 SA2940 SM EC KP PA RP STM

O. canum 2.08 >33.33 16.66 8.33 >33.33 2.08 2.08 nt 1.39 0.60 0.69 0.30X. aethiopica nt >33.33 33.33 33.33 nt 8.33 nt nt 6.94 2.41 1.04nt, 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 growthwere 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 bacteriafailed 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 KP PA STM Main constituents

F-2 >1 >1 0.6 0.57 - and -Pinene, sabineneF-3 >1 >1 1 Eucalyptol, aristolene epoxideF-6 >0.2 >0.2 >0.2 Trans--bergamopteneF-7 >0.2 >0.2 >0.2 EugenolF-8 >1 >1 1 Fenchone, 4-terpinylacetate, chavibetol (m-eugenol), methyl eugenolF-9 1 >1 0.5 LinaloolF-10 >1 >1 0.5 LinaloolF-11 0.5 >1 0.5 -Terpineol, chavicol, trans-p-mentha-2,8-dienolF-12 >1 >1 0.35 0.21 -Elemene, dehydroaromadendreneF-13 >1 >1 >1 Elemene, dehydroaromadendreneF-14 1 >1 0.25 -EudesmolO. canum oil 1.73 0.60 >8 0.43 0.15 Linalool, eugenol, and 1,8- cineole

ConclusionThe present collection of X. aethiopica from Cameroon con-

tained myrtenol as one of the prominent constituents of its essen-tial oil whereas the essential oil ofO. canum belonged to one of theexisting chemotype earlier reported from North Cameroon. Theresults of the antibacterial screening demonstrated that the essentialoil of the aerial parts of O. canum can be used as a food additive forcontrolling food-borne pathogens. Furthermore, bioassay-guidedfractionation of this oil through flash chromatography revealed thatminor compounds contribute significantly for the overall activityobserved. Flash chromatography enabled the purification and iso-lation of several constituents among which linalool and eugenolwere the major ones. Moreover, the fractionation of the essential

oil through flash chromatography allowed the separation and pre-cise identification of eugenol derivatives, namely, meta eugenoland methyl eugenol.

AcknowledgmentsN.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 researchfacilities. Mr. M.P. Darokar is thanked for permitting us to test theantibacterial activity in his laboratory. Thanks are also due to Mrs.Anju Yadav and Namita Gupta for recording GC, GC/MS, andNMR spectra, respectively.

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Author ContributionsN.A. Vyry Wouatsa and Laxminarain Misra designed the study.

N.A. Vyry Wouatsa collected the plants, performed the extrac-tion of the essential oil, the antibacterial assays and isolated thecompounds. She also drafted the manuscript. Laxminarain Misrasupervised the work, identified the isolated compounds, and re-vised the manuscript. R. Venkatesh Kumar supervised the workand edited the manuscript.

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