Chemical Composition and Antimicrobial Activity of The Essential Oils of Four Ocimum Species Growing in Tanzania

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Food Chemistry 119 (2010) 311–316

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Food Chemistry j o u r n a l h o m e p a g e :  w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m

Chemical composition and antimicrobial activity of the essential oils of four Ocimum  species growing in Tanzania D. Runyoro a, O. Ngassapa a, K. Vagionas b, N. Aligiannis b, K. Graikou b, I. Chinou b,

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a b

Department of Pharmacognosy, School of Pharmacy, Muhimbili University of Health and Allied Sciences (MUHAS), P.O. Box 65013, Dar es Salaam, Tanzania Division of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, University Campus of Zografou, 157 71 Athens, Greece

a r t i c l e

i n f o

 Article history: Received 29 January 2009 Received in revised form 19 May 2009 Accepted 16 June 2009

Keywords: Ocimum basilicum O. kilimandscharicum O. lamiifolium O. suave Antimicrobial activity Essential oils

a b s t r a c t

As part of ongoing research on Tanzanian plants used as edibles or spices, six samples of essential oils from four  Ocimum  species (O. basilicum, O. kilimandscharicum,  O. lamiifoliu lamiifolium, m, O. suave) were analyzed by GC and GC–MS. Eighty-one compounds, compounds, corresponding to 81.1–98.2% of the chemical components of the oils, were were identifie identified. d. Major Major comp compound oundss were were eith either er phen phenyl yl prop propane ane derivatives derivatives or terpenoi terpenoids, ds, including methyl eugenol, 1,8-cineole, camphor, bornyl acetate, germacrene-D, germacrene-D, E-myroxide, germacrene-B, caryophylene oxide and  p -cymene. The oils were also evaluated for antimicrobial activity against eight bacterial strains and three fungi. The oil of  O. suave  (B) showed the strongest antibacterial activity; O. suave  (A), O.  kilimandscharicum  and,  O. lamiifolium  were moderately active, while  O. basilicum  oil was weakly active. However, none of the oils was active against the fungi species. The study has shown that, Ocimum oils could potentially be used as anti-infective agents.   2009 Elsevier Ltd. All rights reserved.

1. Introduction In the third world and developing countries, and even in developed nations, food-borne diseases are a major dilemma. The consumptio sum ption n of food foodss conta contamin minated ated with som some e mic microo roorgan rganism ismss represents a serious health risk to humans. The subsistence and gr grow owth th of mi micro croor organ ganism ismss in foods foods ma may y lead lead to spoil spoilage age,, form formati ation on of toxins and quality deterioration of food products (Celiktas ( Celiktas et al., 2007). 2007 ). Since ancient times, herbs and spices have been added to food to improve the flavour and organoleptic properties, but also as preservatives. In recent years, the essential oils and the herbal extracts from various species of edible and medicinal plants have attracted a great deal of scientific interest due to their potential as a source of natural agents to increase the safety and shelf life of food foodss and of na natur tural al bio biolo logic gicall ally y act active ive com compo pound undss (Bozin, Mimica-Dukic, Simin, & Anackov, 2006). 2006). Especially, the antimicrobial activity of essential oils have formed the basis of many applica cati tion ons, s, incl includ udin ing g fr fres esh h and and pr proc oces esse sed d food food pr pres eser erva vati tion on,, pharma pha rmaceuti ceuticals cals,, alte alternat rnative ive medicine medicine and natural natural therapie therapiess (Celiktas et al., 2007 2007). ). Guided by ethnobotanical literature and availability from natural sources, our main objective is to validate the use of selected African aromatic plants for their antimicrobial properties and to   Correspond Corresponding ing author. author. Address: Address: University University of Athens, Athens, School of Pharmacy, Pharmacy, Division of Pharmacognosy and Chemistry of Natural Products, PanepistimiopolisZografou, 157 71 Athens, Greece. Fax: +30 210 7274115.  [email protected]  (I. Chinou). E-mail address:  [email protected] (I.

emphasise the need to promote their natural botanical resources in Africa as well well as their uses worl worldwi dwide. de. In the frame framewor work k of  our research on odoriferous Tanzanian plants and their biological activities, we report herein the analysis of essential oils from four Ocimum   species. The genus  Ocimum   (Lamiaceae) consists of about 50–150 species (Simon, (Simon, Quinn, & Murray, 1990) 1990) with a large number of varieties containing both terpene and non-terpene constituents in their People le of th the e Haya Haya tribe tribe of N Nor orth th We West st essential oils (Evans, (Evans, 1995). 1995). Peop Ta Tanz nzani ania a refer refer to plants plants of thi thiss genus, genus, alm almost ost wi witho thout ut excep exceptio tion, n, as ‘‘ Akashwagara  Akashwagara”. Members of the genus find a number of uses in Afr African ican trad traditio itional nal medici medicine ne (Chogo Chogo & Cra Crank, nk, 19 1981 81;; Git Githin hinji ji & Kokwar Kok waro, o, 199 1993; 3; Janss Janssen, en, Sche Scheffer ffer,, Ntez Ntezurub urubanz anza, a, & Baer Baerheim heim-Svendsen, 1989). 1989). The Haya use the decoctions prepared from the leaves of the plants for relief of stomach upsets. Leaves from the plants in the genus are either, rubbed between the palms and inhaled, or are boiled and the hot vapour inhaled for treatment of  1993). ). TanzaTanzablocked blocked nostril nostrilss and bronchial bronchial cata catarrh rrh (Kokwaro, Kokwaro, 1993 nians, nian s, especia especially lly those living living along along the Indian Ocea Ocean n coastal coastal regions, gions, use the plants plants to repe repell mo mosqu squito itoes es and as flavo flavouri uring ng agents. agen ts. Pl Plants ants of the genu genuss Ocimum are also also rep repor orted ted for many many bio bio-logical logi cal activ activities ities,, such as mosqui mosquito to repe repellen llentt and antimicro antimicrobial bial Chogo go & Cra Crank, nk, 19 1981 81;; Git Githin hinji ji & Ko Kokw kwar aro, o, 199 1993; 3; Ko Kokw kwar aro, o, act activi ivity ty (Cho 1993), 1993 ), insect insectici icida dall act activi ivity ty agains againstt cro crop p pest pest insect insectss (Bekele Bekele &

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0308-8146/$ - see front matter    2009 Elsevier Ltd. All rights reserved. 10.1016/j.foodchem.2009.06.028 doi: doi:10.1016/j.foodchem.2009.06.028

Hassanali, 2001), 2001), antipyretic (Makonnen, (Makonnen, Debella, Zerihun, Abebe, & Te Teka, ka, 20 2003 03)) and and ant antio ioxid xidant ant act activi ivity ty ( Javanmar  Javanmardi, di, Stush Stushnoff noff,, Locke, & Vivanco, 2003). 2003).

 

 

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D. Runyoro et al. / Food Chemistry 119 (2010) 311–316 

As part of ongoing chemical and biological studies of essential oils from odoriferous Tanzanian plants, which are used as spices, six oil sam sampl ples es from from aer aerial ial pa parts rts of fou fourr   Ocimum   species were investigated. The plants included  O. basil basilicum icum  Linn. (2 samples samples), ), O. kilimandschar kilimandscharicum icum   Baker Baker ex Gurk Gurke, e, O. lamii lamiifoliu folium m   Hochst Hochst ex Benth. and  O. suave  Willd (2 samples). Ocimum basilicum   is a stout, bushy aromatic herb with white flowers in loose racemes (Hutchinson (Hutchinson & Dalziel, 1963 1963). ). It is mostly Graye ayerr et al. al.,, 199 1996 6) and it is cultivate culti vated d for culi culinary nary purposes purposes (Gr known as ‘‘Lutatambwa lunywamu” by the Nyakyusa of the Mbeya region (Tanzania), where it is used as a tick repellent in chickens. The plant is found in European and African countries as a major essential oil crop, producing 42.5 tonnes of oil worldwide (Grayer (Grayer et al., 1996). 1996). The essential oil from the plant, which is known as ‘‘sweet basil”, is widely employed in flavouring and is one of the most studied oils of the genus. Ocimum kilimandscharic kilimandscharicum um   is an aro aromat matic ic per perenni ennial al woo woody dy shr shrub ub up to 2 m tall. tall. In Rw Rwan anda da the pl plant ant is used used in traditi tradition onal al medicine to cure eye infections (Ntezurubanza, (Ntezurubanza, Scheffer, Looman, & Baerheim Baerheim-Svendse -Svendsen, n, 1984) 1984) and and in Ke Keny nya a it is us used ed as a gr grai ain n pro ro- Jembere bere,, Obe Obeng-O ng-Ofori fori,, Hass Hassana anali, li, & tectant tecta nt agai against nst insect pests pests ( Jem Nyamasyo, Nyama syo, 1995 1995). ). Ocimum lamiifolium  is an erect, hairy perennial, several feet in height; flowers are white in long lax racemes. The Nyakyusa refer to the plant as ‘‘Lufisu lunyambala”. In Ethiopia fresh leaves of  O. lamii lamiifoliu folium m   ar are e sq sque ueez ezed ed an and d snif sniffe fed d to tr trea eatt cold coldss an and d coughs cou ghs;; and as an eye rinse for for ey eye e infec infectio tions, ns, while while cru crushe shed d Asfaw, w, leaves leaves ar are e used used to arr arrest est no nose se bleed bleeding ing (Demissew Demissew & Asfa 1994). 1994 ). O.   suave   is a br branc anche hed d er erect ect,, pu pube besce scent, nt, ar arom omati atic c shr shrub ub,, reaching reac hing one metre in heig height, ht, with dense dense spikes spikes of sma small ll greengreenish white flowers. flowers. In Afr Africa ica it is used as a hemorrh hemorrhoid oidss rem remedy edy and to pe perfu rfume me chewi chewing ng tobacc tobacco o and snu snuffs ffs.. Sm Smok oke e from from the burning burn ing plant is used as a mo mosqui squito to repellen repellent, t, and Maasai girls and warriors use the leaves and flowers as perfume. An infusion of leaves is used as a disinfectant and an insecticide and in Kenya, leaves are used as an insect repellent repellent and grain protectant protectant 1993). ). In the literature on Tanzaagainst insect pests (Kokwaro, (Kokwaro, 1993 nian  O. suave   (Arusha, (Arusha, Northeastern Tanzania), there is only one study by   Chogo and Cra Crank nk (198 (1981) 1)   that rep reporte orted d eugenol eugenol as the major maj or volatile volatile cons constitue tituent nt of its esse essential ntial oil. To our knowledge, there are no previous studies on  O. basilicum,   O. kilimandscharicum kilimandscharicum   and  O. lamiifoliu lamiifolium m   growing growing in TanzaTanzania, while several several studies studies have been published published on the volatiles volatiles basilicum icum   (Gith Githinji inji & Kok Kokwar waro, o, 199 1993; 3; Lach Lachowi owicz cz et al., from   O. basil 1998; 199 8; Lew Lewinso inson n et al., 2000; Wan Wan,, Wil Wilcock cock,, & Cove Coventry ntry,, 1998 1998), ), Kokwar waro, o, 1993 1993;; Jemb Jembere ere et al., O. kilimandscharic kilimandscharicum um   (Githinji Githinji & Kok lami miif ifol oliu ium m 1995; Nte tez zur uru uban anz za et al. l.,, 1984) and   O. la mbou ougn gnang ang et al. al.,, 200 2006 6) from from dif diffe feren rentt geogra geographi phical cal (Tchou Tchoumb origins.  report herein the chemical composition and antimicrobial We report We activity activ ity of esse essential ntial oils from O. basil basilicum, icum, O. kilimand kilimandschar scharicum icum, O. lamiifolium   and   O. suave   growing in the Mbeya region, in the southwestern part of Tanzania.

2. Materials and methods  2.1. Plant material Leaves and flowering tops of various  Ocimum  species were collected from the wild, in the Mbeya region, Tanz Tanzania. ania. Locations Locations and Table le 1. The plants plants we were re da dates tes of co colle llecti ction on are are as detai detailed led in   Tab authenticated by comparison with herbarium specimens, by the staff of the Departmen Departmentt of Bota Botany, ny, University University of Dar es Salaam. Salaam. Voucher specimens are deposited in the herbarium of the Department of Pharmacognosy, School of Pharmacy, Muhimbili University of Health Health and Alli Allied ed Sciences. Sciences. The materia materials ls were were airair-dri dried ed indoors prior to the isolation of essential oils.

 2.2. Isolation procedure The plant materials of   O. basilicum  sam  sampl ple e A (42 (420 0 g), O. basilicum  sam  sample ple B (205 g), O. kilimandscharicum  (73  (735 5 g), O. lamiifolium (107 (10 7 g), O. suave  sam  sample ple A (940 (940 g) and  O. suave   sample sample B (670 g), were wer e subjected subjected to hydr hydro-d o-distil istillati lation on for 3 h, in a modifie modified d Clevenger-type apparatus, with a water-cooled oil receiver to reduce formatio form ation n of arti artifacts facts due to over overheat heating ing duri during ng hyd hydro-d ro-distill istillaation. The essential oils were collected over water, separated and dr dried ied ov over er anhyd anhydro rous us so sodiu dium m sul sulfat fate. e. The They y we were re sto stored red at 4– 6 C prior to chemical analysis and antimicrobial studies. The colours of six samples of essential oils from  Ocimum   species varied from bright yellow (O. basilicum,  O. lamiifolium) to almost colourless (O. kilimandscharicum). It is noteworthy noteworthy that a consider considerable able kilimandscharicum um   underwent am amou ount nt of ess essent ential ial oil oil from from   O. kilimandscharic crystallization, crystallizati on, forming colourless fragrant crystals. The yields of  oils ranged from about 0.5% (O. basilicum  B) to about 4% (O. basilicum   A); those of   O. basilicum   A, O. lamiifolium   and   O. kilimandscharicum   were were re reas aso onabl nably y hi high gh (4.0 (4.05% 5%,, 3. 3.3% 3% and and 3. 3.13 13%, %, respectively).   Table Table 1   shows shows the yiel yield d of the vari various ous oils under investigation.

 2.3. Gas chromatography GC analyses were carried out on a Perkin–Elmer 8500 gas chromatograph with FID, fitted with a Supelcowax-10 fused silica capillary illar y column column (30 m  0.32 mm i.d., i.d., 0.25 lm film thickness). The column temperature was programmed from 75 C to 200 C at a rate of 2.5 C/m C/min. in. The injec injector tor and dete detector ctor tem temper peratur atures es were were programmed at 230 C and 300 C, respectively. Helium was used as carrier carrier gas, at a flow rat rate e of 1 ml/ ml/min min..

 2.4. Gas chromatography– chromatography–mass mass spectrometry The GC–MS analyses were carried out using a Hewlett–Packard 5973-6890 GC–MS system operating on EI mode (equipped with a HP 5M 5MS S 30 m  0.25 0.25 mm  0.25 lm film thickness capillary column). Helium Helium (2 ml/min) ml/min) was used as carrie carrierr gas. The tempe temperature rature gradient was programmed from 60 C to 280 C, at 3 C/min. Helium was was used, at a flow rate of 1 ml/ ml/min min.. Split ratio, 1:10 1:10..

 Table 1

Oil yield of investigated  Ocimum  species. Plant species I   II   III   IV   V   VI  

O. O. O. O. O. O.

basilicum   A basilicum   B kilimandscharicum   lamiifolium suave   A suave   B

 

Voucher specimen number

Local name

Collection location (District)

Date of collection

Oil yield (% v/w /w))

EO-015 EO-063 EO-049 EO-071 EO-025 EO-045

Lutatambwa lunywamu Lutatambwa lunywamu – Lufisu lunyambala – –

Lema (Rungwe) Utengule (Mbeya) Kawetere (Mbeya) Mwakaleli (Rungwe) Lwangwa-Manow (Rungwe) Uyole (Mbeya)

March, 2000 March, 2000 July, 2000 July, 2000 March, 2000 March, 2000

4.05 0.54 3.13 3.3 1.15 1.01

 

D. Runyoro et al. / Food Chemistry 119 (2010 (2010)) 311–316 

 

313

 Table 2

The chemical composition (%) of essential oils of various  Ocimum   species. Compoundsa

No

1 2 3 4 5. 6. 7 8 9 10 11 12 13 14 15 16 17 18 19 20. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 42 43 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73

   

 

       

   

     

 

 

    

 

 

 

   

             

         

     

    

Essential oilsb I

II

III

IV

V

VI

RI

Identificationc

Tricyclene a-Thujene a-Pinene Camphene Sabinene b-Pinene 1-Octen-3-ol

– 0.33 4.39 – – 8.15 –

– – 1.39 0.54 – – 1.00

0.2 – 1.07 5.36 tr 0.87 –

0.3 0.46 5.7 5.91 0.5 4.2 0.2

– 0.34 2.17 – 0.18 – 0.14

– – tr tr – tr tr

927 930 939 954 975 979 979

1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3

Myrcene 3-Octanol a-Phellandrene a-Terpinene p-Cymene Limonene b-Phellandrene 1,8-Cineole cis-ocimene trans-b-ocimene c-Terpinene cis-sabinene hydrate trans-linalool oxide cis-linalool oxide a-Terpinolene Rosefuran Linalool trans-sabinene hydrate 1-Octen-3-yl acetate a-Camphonelal

1.06 – – – – 2.30 – 54.3 – – – 1.91 – – – – – tr – –

– tr – – 1.24 1.86 – 3.10 – – – – – – – – – – – –

1.09 – 0.41 0.49 – 7.13 – 14.3 0.15 1.39 0.87 0.24 – – 2.23 – 1.6 – – 0.35

0.2 – – – 11.4 – 2.94 – – – – – 0.10 0.15 – – 2.99 – 1.35 –

– 0.09 – – 0.76 0.41 – 0.19 0.24 0.11 0.35 – – – 0.15 – – – – –

– – – – tr tr – tr – – – – 0.17 0.15 – 0.18 1.21 – – –

991 – 1003 1017 1025 1029 1030 1031 1037 1050 1060 1070 1073 1087 1089 – 1097 1098 1113 1126

1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 3 1,2,3 1,2,3 1,2,3 1,2,3

cis Viem rboenneonle oxide cis--L E-myroxide Camphor Borneol Rosefuran epoxide Terpinen-4-ol p-Cymen-8-ol Cryptone a-Terpineol Myrtenol Mytenal Verbenone trans-carveol Cuminal Carvone Bornyl acetate p-Cymen-7-ol d-Elemene a-Cubebene Eugenol

0 –.16 – – – – 1.56 – – 6.6 – – – 0.36 – 0.24 – – – – –

– – 19.6 – – 6.03 – – – – – – – – – – – – 0.82 –

– – – 52.4 0.67 – 3.24 tr – 1.01 0.66 – – – – – – – – – –

– – – 0.53 3.68 – 0.71 – 1.67 0.81 – 0.16 – – 0.39 – 30.3 0.72 – – –

–.23 0 – – – – 0.14 0.16 – 0.18 – – 0.1 0.08 – – – – 0.70 0.08 0.12

– – – – – – – – – – – – – – – – – – – 0.08 0.43

11 14 31 7 1 1145 1146 1169 1177 1177 1183 1186 1189 1196 1196 1205 1217 1242 1243 1289 1291 1338 1351 1359

1,,2 2,,3 3 1 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1 1,,2,3 1,2,3 1,2,3

a-Copaene b-Bourbonene b-Cubebene b-Elemene Methyl eugenol b-Caryophyllene b-Gurjunene c-Elemene a-Humulene trans-b-farnesene cis-muurola-4(14)5-diene Germacrene-D b-Selinene a-Selinene Bicyclogermacrene a-Muurolene b-Bisabolene Germacrene-A c-Cadinene 7-Epi-a-selinene

3.01 0.23 – – – 1.34 0.26 – 3.55 – – 0.21 0.24 – – – – – – –

7.50 – 1.79 – – 2.53 – – 6.28 – – – 1.71 3.57 – – – – – –

– – – – – 1.12 – – – 0.82 – 0.46 – – – – – – – –

– 0.65 – – – 0.52 – – – – – – – – – – – – – –

0.58 0.36 0.15 0.49 – 5.13 – 0.51 2.62 – 0.09 29.2 2.76 1.44 1.60 1.2 – 0.22 1.81 1.01

0.85 0.46 – – 82.7 – – – – – – – – – – – 0.64 – – –

1377 1388 1388 1391 1404 1419 1434 1437 1455 1457 1467 1485 1490 1498 1500 1500 1506 1509 1514 1522

1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3

d a--CCaaddiinneennee Germacrene-B Palustrol Germacrene-D-4-ol

0 –.81 – 0.26 –

0 –.61 – – –

– – – 1.16

– – – – –

3 06 4 0..1 14.0 – 1.28

0.09 – – – –

15 1 52 33 9 1561 – 1576

1 1,,2 2,,3 3 1,2,3 3 1,2,3 (continued on next page)

 

 

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D. Runyoro et al. / Food Chemistry 119 (2010) 311–316 

 Table 2  ( continued)

Compoundsa

No

74 75 76 77 78 79 80 81

a b c

     

 

Essential oilsb I

II

Spathulenol Caryophyllene oxide Humulene epoxide II 10-Epi-c-eudesmol c-Eudesmol s-Cadinol b-Eudesmol a-Ca -Cadin dinol ol + a-eudesmol

– 0.76 – – – – – –

– 11.4 11.0 – – – – –

Total

91.99

82.11

IV

V

VI

RI

Identificationc

– – – –

4.08 1.87 0.65 – – – – –

0.50 1.43 – 0.39 2.91 0.1 3.11 8.11

– 4.13 0.37 – – – – –

1578 1583 1608 1624 1632 1640 1651 1654

1,2,3 1,2,3 1,2,3 1 1,,2,3 1,2,3 1 1,,2,3 1,2,3 1,2,3

98.2

83.14

92.01

91.41

III 0.13 –

Compounds listed in order of elution. I = O. basilicum  A; II II = O. basilicum  B; III III = O. kilimandscharicum; IV IV = O. lamiifolium; V = O. suave  A; VI VI = O. suave  B. 1 = Retent Retention ion time time;; 2 = Kovat’ Kovat’ss retent retention ion indices indices;; 3 = mass mass spe spectr ctra. a.

 2.5. Identification of components

The chemical chemical compo compositio sition n analysis analysis of all studied studied samples samples is shown in Table in  Table 2. 2. In total, 81 components were identified, representin sen ting g 82 82–9 –98% 8% of all compo componen nents ts in the oils. oils. The com compo pone nents nts var var-ied between the oils and only  a -pinene and caryophyllene oxide were were ident identifie ified d in va varie ried d am amou ounts nts in all the studi studied ed sampl samples es of Ocimum  oils. Twenty-three and nineteen compounds were identified in oils of the two samples of   O. basilicum, corresponding to 91.99% and 82.11% of the chemical components for samples A and B, respectively. tivel y. The maj major or compone components nts for sam sample ple A were, were, 1,81,8-cineo cineole le

However,, the two oils differed However differed in that some of the compo component nentss present in one of the oils in appreciable amounts were absent in the other oil. For examp example le E-myr E-myroxide, oxide, a major com component ponent of sample B, was absent in sample A while  b -pinene and and a -terpineol, present in sample A, in appreciable amounts, were absent in sample B. In additio addition n hum humulen ulene e epo epoxide xide II, rosefura rosefuran n epoxide epoxide,, and sel seline inene ne,, wh which ich we were re identi identifie fied d in sampl sample e B in appr appreci eciabl able e am amou ount nts, s, we were re abse absent nt in sa sam mpl ple e A. Th The e two two sa sam mpl ples es of  O.  O. basilicum could actually be two different chemical races. This is in line with previous studies which have described various chemotypes of   O. basilicum   and othe otherr  Ocimum  specie  species, s, such as methyl methyl cinn cinnama amate, te, linalool, methyl chavicol and methyl eugenol chemotypes (Simon ( Simon et al., 1990; Vina & Murillo, 2003). 2003). Phenyl propane derivatives previously reported in the essential oils of  O. basil basilicum icum, inclu including ding methy methyll chavicol chavicol ((Lach Lachow owicz icz et al., 1998; Wan et al., 1998), 1998), methyl eugenol (Lachowicz (Lachowicz et al., 1998; Lewinso Lew inson n et al., 2000) 2000) methy methyll cinnam cinnamate ate (Simon Simon et al. al.,, 19 1990 90), ), and eugenol (Lachowicz (Lachowicz et al., 1998; Lewinson et al., 2000), 2000), were absent in both oil samples of  O. basilicum. Linalool, a monoterpene alcohol, alco hol, prev previous iously ly reported reported in O. basilicum oils oils (Gr Gray ayer er et al. al.,, 19 1996; 96; Keita, Vincent, Schmit, & Belanger, 2000; Simon et al., 1990; Wan et al., 1998), 1998), was not detected in either oil sample. The compound 1996), ), was the ma1,8-cineole, previously reported (Grayer (Grayer et al., 1996  jor compon component ent of sample sample A and was fou found nd in app appreci reciable able amo amounts, unts, in sample B. Another compound, limonene, also previously identified in the oil (Grayer (Grayer et al., 1996), 1996), was present to the extent of  2.30% and 1.86% in samples A and B, respectively. Twenty-eight compounds, corresponding to 98.2% of the chemical components in the oil from  O. kilimandscharicum, were identified. The major components of the oil were mainly mono mo noter terpe peno noids ids and includ included ed ca camp mpho horr (52.4% (52.4%), ), 1,8-c 1,8-cine ineol ole e (14.3%) (14. 3%),, lim limone onene ne (7.1 (7.13%) 3%) and cam camphen phene e (5.3 (5.36%) 6%).. Cam Campho phorr and eugenol euge nol chem chemotyp otypes es have been des describ cribed ed for this plant plant spec species ies Ntezurub urubanz anza a et al., 1984 1984). ). Studies Studies on exp experim erimenta entall plants plants in (Ntez the USA showed that the fresh flowering herb yielded 0.5–1% of  essential esse ntial oil, inclu including ding campho camphor, r, that could could be separat separated ed easi easily ly fromtheoil fr omtheoil (Nte Ntezu zurub ruban anza za et al. al.,, 198 1984 4). As has has alre alread ady y been been no note ted, d, in the Section 2.2, characteristic colourless crystals were observed in the oil of  O. kilimandscharicum. These crystals have been sub jected  jecte d to GC–M GC–MS S anal analysis ysis and wer were e dete determi rmined ned to be pure cam cam-phor,, whi phor which ch has been the major constituent constituent (52.4%) (52.4%) of the oil. According to this finding, the oil could be classified as a camphor chemo che motyp type, e, as it has been been descr describe ibed d by   Nte Ntezuru zurubanz banza a et al. (1984).. The oil also contained 1,8-cineole in appreciable amounts, (1984) a compound, which was previously identified in  O. kilimandschari-

(54.3%), -pi -pinene neneB(8.15%) (8.1 5%) and   a-terpineo -terpineol l (6.6%). Major comp compoonents for  bsample were E-myroxide (19.6%), caryophyllene oxide (11.4%), humulene epoxide II (11.0%),  a -copaene (7.5%), v-humulene (6.28%), (6.28%), and ros rosefur efuran an epo epoxide xide (6.03 (6.03%). %). Compon Components ents ide identifie ntified d in appreciable amounts in both samples included 1,8-cineole,   apinene, limonene, limonene,   a-copaene,   b-caryop -caryophyllene hyllene and   a-humulene.

in Rwanda (this 1984in Ntezurubanza etreported al., 1984). ). Limonene, cum  growing third major compound in(Ntezurubanza oil, was also appreciablea amounts in the oil from Rwanda. In the case of   O. lamiifolium, twenty-ei twenty-eight ght compo compounds unds correspond spo nding ing to 83 83.1% .1% of all the co comp mpone onents nts,, we were re ident identifie ified d and we were re,, mainly monoterpenoids. monoterpenoids. The major compound compoundss included bornyl

The compounds were identified by comparison of their retention tion ind indice icess (KI (KI)) (Van Van de den n Do Dool ol & Kr Kratz atz,, 19 1963 63), ), rete retentio ntion n times times (RT) and mas masss spectra spectra with those of authe authentic ntic samples samples and/or and/or the NIST/NBS, Wiley libraries spectra and the literature (Adams, (Adams, 1995;; Mas 1995 Massada sada,, 1976 1976). ). Th The e pe perce rcenta ntage ge comp compos ositi ition on of the ess essent ential ial oils is base based d on com compute puter-ca r-calcul lculated ated peak peak areas areas with without out corr correctio ection n for FID response factor.

 2.6. Antimicrobial activity Antimicrobial activity of the essential oils against bacteria and  Jans-fungi was determined by using the agar dilution technique ( Jans sen, Scheffer, & Baerheim Svedsen, 1987). 1987). The microorganisms included four Gram-positive Gram-positive bacteria:   Staphylococcus aureus   (ATCC 25923),   Staphylococcus epidermidi epidermidiss   (ATCC 12228), 12228),   Streptococcus mutans  (clinica  (clinicall isolates) isolates) and   Streptococcus viridans   (clinical isolates); the last two are oral pathogens. Also, four Gram-negative bacteria: Escherichia coli  (ATCC 25922),  Enterobacter cloacae  (ATCC 13047),   Klebsiella pneumoniae   (ATCC (ATCC 138 13883 83)) and   Pseudomonas aeruginosa (ATCC 227853) and three species of  Candida:  C. albicans (ATCC 10231),   C. tropi tropicalis calis   (ATCC 13801) and   C. glabrata glabrata   (ATCC 28838). 2883 8). Stan Standar dard d antib antibiotic ioticss (neti (netilmi lmicin, cin, and amo amoxicil xicillin lin wit with h clavclavulanic acid) were used as positive controls for bacteria, 5-flucytocine cine an and d am amph phote oteric ricin in B as a po posit sitive ive control control for for   Candida   and sanguinarine, an alkaloid, as a positive control for oral pathogens. Technica Tech nicall data have bee been n describe described d previou previously sly (Vag Vagiona ionass et al., 2007). 2007 ). Min Minimu imum m inhibitor inhibitory y concentr concentratio ations ns (MIC (MICs) s) wer were e dete deterrmi mine ned d for all the sampl samples es and the stand standard ard pu pure re compo compound unds, s, und under er identical conditions, for comparison purposes.

3. Results and discussion

 

D. Runyoro et al. / Food Chemistry 119 (2010 (2010)) 311–316 

 

315

acetate acet ate (30.3%), (30.3%),   p-cym -cymene, ene, (11.4 (11.4%), %), cam camphe phene ne (5.91%) (5.91%) and   apinene (5.7%). A previous study by Tchoumbougnang by  Tchoumbougnang et al. (2006) reported sabinene as the major compound identified in the essential oils of  O.   O. lamiifolium  growing in tropical Africa. In O. suave, forty eight and twenty compounds were identified, corresponding to 92% and 91.4% of the total chemical components in sam sampl ples es A and B, res respe pecti ctivel vely. y. Sa Samp mple le A consis consisted ted ma mainl inly y of ses ses-quiterp quit erpenoi enoids, ds, which which included included germ germacre acrene-D ne-D (29.2 (29.2%), %), germ germacre acreneneB (14.0%),  a -cadinol and  a -eudesmol (8.11%) and  b -caryophyllene (5.13%). The only monoterpenoid found in sample A, in an appre-

tococcus viridans   and   S. mutans. Between Between the two samp samples les of   O. basilicum, sample B was relatively more active than was sample A. Th The e esse essent ntia iall oil oil of O. basilicum wh which ich is commo commonly nly kn know own n as basil oil, has been reported previously to have antimicrobial activity on a number number of GramGram-nega negative tive and posi positive tive bacteri bacteria a and fungi, some of which are food spoilage microorganisms (Koga, (Koga, Hirota, & Takumi, 1999; Lachowicz et al., 1998; Wan et al., 1998; Wannissorn,, Jarik orn Jarikasem asem,, Siri Siriwan wangcha gchai, i, & Thub Thubthim thimthed thed,, 2005 2005). ). However, However, the two samples of  O. basilicum  used in this study were the least active. Ocimum basilicum  exist  existss in a numb number er of chem chemotyp otypes es includinclud-

ciable ciab le amount, amount, was a-pine -pinene ne (2. (2.17% 17%). ). Th The e oil of sam sampl ple e B was was ma made de up ma mainl inly y of a ph pheny enyll pr prop opan ane e compo compound und,, me methy thyll eu eugen genol ol (82.7% (82.7%). ). This compound has also been reported as the main component in oils of other Lamiaceae species, such as  Melaleuca ericifolia  and  O. sanctum   (Farag et al., 2004 2004). ). Other components in sample B included caryophylene oxide (4.13%) and linalool (1.21%). The two oil samples from O. suave  were similar in that both contained very little amounts of monoterpenoids. Eugenol, which was previously reported as the major component of the oil from  O. suave  growing in Arusha (Chogo (Chogo & Crank, 1981), 1981), was not identified in the present samples sam ples,, while, while, instead instead of eugenol, eugenol, methyl euge eugenol nol was dete deterrmined as the major component of sample B in our study. Generally, the observed differences in chemical composition of  the various oils, when compared with those reported in previous studies, could be due to a number of factors. Such factors may include clud e diff differe erences nces in climatic climatic condition conditions, s, geograp geographica hicall loca location tions, s, season at the time of collection, stage of development, processing of plant materials before extraction of the oils and occurrence of  chemotypes. The oils were also evaluated for antimicrobial activity against four Gram-positive Gram-positive bacteria (S. aureus aureus,   S. epid epidermi ermidis dis,   S. mutan mutanss and S. viridans), four Gram-negative bacteria (P. aeruginosa,  E. cloacae,  K. pneumoniae  and  E. coli) and three species of the yeast  Candida   (C. albicans, C. tropicalis, and  C. glabrata). Oils from the two samples of  O.   O. suave  were the most active and those of  O. basilicum samples showed weaker activities (Table (Table 3). 3). However, none of the oils exhibited activity against the tested human pathogenic fungi. Oils from from   O. basil basilicum icum   showe showed d a weak weak act activi ivity ty (MIC (MIC 3.14– 3.14– 12.5 mg/ml) mg/ml) a again gainst st six tes tested ted bact bacteria eria,, but w were ere inac inactive tive on Strep-

ing linalool, methylchavicol, geraniol, methyl eugenol and eugenol 1996)) and these particular oils did not belong to any (Grayer et al., 1996 of these chemotypes, and this may be the reason for the observed weak activity. The major compound in the oil from  O. basilicum  A, 1,8-cineole, also exhibited a weak activity on the test microorgan2.0–9.5 mg/ml). mg/ml). isms   (MIC 2.0–9.5 isms The oil of  O.   O. kilimandscharicum  showed a moderate activity on lamiifoliu folium m six teste tested d bacteria bacteria (MIC 1.55 1.55–3.3 –3.35 5 mg/ml) mg/ml),, whi while le   O. lamii showed a moderate activity against  S. aureus, S. epidermidis,   and P. aeruginosa  (MIC 1.75–2.7 1.75–2.75 5 mg/ml) mg/ml) and a wea weak k activity activity against E. cloacae, K. pneumoniae  and  E. coli. The level of activity of  O.   O. lamiifolium oil was comparable with that of its major constituent, bornyl acetate. Hence, the observed activity could mainly be due to this component. The oil of   O. suave   (B) show showed ed activity on all tested bacteri bacteria a (MIC 0.05–1.45 mg/ml); mg/ml); this could be attributed to its major comcomponent, methyl eugenol (82.7%). A previous study by Farag by  Farag et al. (2004)   on essential essential oils of four   Melaleuca   species species reve revealed aled that the oil from  M. ericifolia, which had methyl eugenol as the major compon com ponent ent (96.8%) (96.8%) exhi exhibite bited d the highest highest antimicro antimicrobial bial activity activity when whe n com compar pared ed with other other  Melaleuca   species investigated. This suggests that the antimicrobial effect is most likely due to methyl eugenol.  Ocimum suave  (A), though devoid of methyl eugenol, was found found to be mo mode dera ratel tely y act active ive on all the tested tested bacte bacteria ria (MI (MIC C 1.1 1.19– 9– 3.10 mg/ml mg/ml). O. suave  samp  sample le B wa wass sev sever eral al ttim imes es mo more re act active ive on S. aureus wh when en compar compared ed to the Ar Arush usha a sampl sample e pr previ eviou ously sly stu studi died ed by Chogo and Crank (1981) (1981).. However, the oil from sample B was less active on  E. coli  when compared to the oil from Arusha plants. A previous study also showed that, the essential oil from Rwandese

 Table 3

Antimicrobial activity (MIC mg/ml) of the studied  Ocimum  species essential oils and their main components. Tested s am amples

O. basilicum   (A) O. basilicum   (B) O. kilimandscharicum   O. lamiifolium O. suave   (A) O. suave   (B) Borneol Bornyl acetate Camphor Caryophyllene oxide 1,8-Cineole Limonene Linalool a-Pinene b-Pinene Spathulenol trans -caryophyllene Amoxycillin Amoxycill in + clavu clavulanic lanic acid Amphotericin B 5-Flucytocine Netilmicin Sanguinarine

Micr o oo organisms

 

S. aureus

S. epidermidis

P. aeruginosa

E. cloacae

K.  pneumoniae

E. coli

S. mutans

S. viridans

C. albicans

C. tropicalis

C.  glabrata

12.5 10.7 2.85 2. 1.95 1.35 0.05 1.25 1.95 2.70 0.073 9.5 >20 0.25 7.50 12.00 1.35 >20

1 11 1.5 9. 9.45 3 3..35 1.75 1.28 0.90 1.57 1.75 1.95 0 0..90 9.5 >20 0. 0.25 9. 9.50 16.00 1.50 >20

6.84 5.37 2.50 2.75 2.37 1.20 2.50 2.30 2.80 0.87 2.75 >25 >20 6.00 >20 >20 >20

6.85 4.21 2.97 4.10 3.10 1.37 4.20 3.75 2.75 2.43 3.00 >25 1.75 8.00 >20 >20 >20

5.30 4.79 2.70 3.50 2.75 1.18 3 3..75 3.25 3.24 1.23 2.35 >25 >20 15.0 >20 >20 >20

4.25 3.14 1.55 4.90 1.95 1.45 4.50 4.88 1.33 >6.40 2.00 >20 1.25 2.00 9.75 8.50 >20

– – – – 1.65 0.19 – – – 0.25 – – 0 0..37 – – – –

– – – – 1.19 0.92 – – – 0.75 – – 0.45 – – – –

– – – – – – – – – – – – – 4.00 – – –

– – – – – – – – – – – – – 4.00 – – –

– – – – – – – – – – – – – 2.00 – – –











– – – 0.015

– – – 0.015

1X10 3 0.1X10 – –



3X10 – – 4X10 –





3

3X10

3

– – 4X10 –





3

3.1X10

3

– – 8.8X10 –





3

4.2X10

3

– – 8X10 –



3

3



4.8X10 – – 8X10 –



3

3



5X10

3

– – 10X10 –



3







3

0.5X10 1X10 3 – – 

3



0.4X10 10X10 – –



3

3

 

316

 

D. Runyoro et al. / Food Chemistry 119 (2010) 311–316 

plants, eval plants, evaluate uated d by bioautog bioautograp raphy hy agar overlay overlay,, possess possessed ed anti antimimi Janssen en et al., 1989). 1989). crobial activity ( Janss

4. Conclusions This study has shown that essential oils from all four studied Ocimum  specie  speciess could could be used as pote potentia ntiall antim antimicro icrobia biall agents, agents, as well as accordingly, as food preservatives against food spoilage microorganisms. basilicum icum   (Sample Besid Bes ides, es, as the yields yields of the oils of   O. basil (Sample A), O. kilimandscharicum   and   O. lamiifolium  are reasonably high, the plants have the potential for a further large-scale cultivation and possible source of income for farmers, especially in a developing African country, like Tanzania.

 Acknowledgments Th This is study study was was pa parti rtiall ally y sup suppo porte rted d by a gra grant nt from from the Direc Director tor-ate of Rese Research arch and Publ Publicati ications ons (Sida/SA (Sida/SAREC REC funds), funds), Muh Muhimb imbili ili Unive Universi rsity ty of Healt Health h and Al Allie lied d Sci Scien ences ces,, which which is gra gratef tefull ully y acknowledged. The assistance of Mr. and Mrs. Rev. Moses Mwakyendel kyen delwa wa of Lutengan Lutengano, o, Tuk Tukuyu, uyu, Mbeya, in the collectio collection n and hydrodistillation of plant materials, is highly appreciated.

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