Antibacterial activity of some Lamiaceae essential oils

LWT - Food Science and Technology 44 (2011) 1199e1206

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Antibacterial activity of some Lamiaceae essential oils using resazurin as an indicator of cell growth Abdullah I. Hussain a, c, Farooq Anwar b, **, Poonam S. Nigam c, *, Satyajit D. Sarker d, John E. Moore c, e, Juluri R. Rao c, f, Anisha Mazumdar c a

Department of Chemistry, GC University, Faisalabad-38000, Pakistan Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan Centre of Molecular Biosciences, Institute of Biomedical Sciences Research, University of Ulster, Coleraine, BT52 1SA, UK d Department of Pharmacy, School of Applied Sciences, University of Wolverhampton, Wolverhampton WV1 1SB, England, UK e Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast, BT9 7AD, UK f Applied Plant Science Division, Agri-Food & Bioscience Institute (AFBI), Belfast BT9 5HX, UK b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 June 2009 Received in revised form 1 October 2010 Accepted 7 October 2010

The aim of this work was to investigate the antibacterial activity of six Lamiaceae essential oils, against pathogenic and food spoilage bacteria. The chemical profiles of essential oils were evaluated by the means of GC and GCeMS. The major constituents of the oils were 1,8-cineol (29.2%), camphor (17.2%), a-pinene (11.5%) in Rosmarinus officinalis, citronellal (20.5%), b- geraniol (17.0%), b-citronellol (11.5%) in Melissa officinalis, 1,8-cineol (27.4%), a-thujone (16.3%), b-thujone (11.2%), borneol (10.4%), camphor (7.98%) in Salvia officinalis, linalool (25.1%), linalyl acetate (22.5%) in Lavandula angustifolia, thymol (52.4%), p-cymene (17.9%) in Thymus vulgaris and Patcholene alcohol (22.7%), a-bulnesene (17.1%), a-guaine (13.8%) in Pogostemon cablin. On quantitative basis, the amounts of 1,8-cineol, citronellal, 1,8cineol, linalool, thymol and patchouli alcohol, calculated using calibrated curve with pure standard compounds, in the respective essential oils were found to be 28.4, 19.0, 26.7, 23.3, 51.1 and 21.1 g/100 g of oil, respectively. The modified resazurin microtitre-plate assay was used to evaluate the antibacterial activity of the essential oils and their principal components. All the essential oils analyzed presented inhibitory effects on most of the strains tested. Thymus vulgaris essential oil showed the highest inhibition. It was concluded that modified resazurin assay could be effectively used for reliable assessment of antibacterial activity of the tested essential oils against several Gram positive and negative bacterial taxa. The present results also demonstrated that Lamiaceae essential oils exhibiting higher antibacterial activity were generally rich in oxygenated monoterpens. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Thymus vulgaris Posostemon cablin Resazurin MIC MBC Gram positive Thymol Patchouli alcohol

1. Introduction The foods contaminated with pathogens are often indentified as primary sources of food-borne diseases in humans. The survival and growth of microorganisms in food products may lead to their spoilage and quality deterioration (Celiktas et al., 2007). Moreover, the emergence of multi-drug resistant pathogens now presents an increasing global challenge to both human and veterinary medicine. Therefore, there is a continuous need to develop novel

* Corresponding author. Tel.: þ44 287 032 4053. ** Corresponding author. Tel.: þ92419200161 69x3309; fax: þ92419200764. E-mail addresses: [email protected] (A.I. Hussain), [email protected] (F. Anwar), [email protected] (P.S. Nigam), [email protected] (S.D. Sarker), [email protected] (J.E. Moore), [email protected] (J.R. Rao), [email protected] (A. Mazumdar). 0023-6438/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.10.005

antimicrobial agents to minimize the threat of further antimicrobial resistance. Plant essential oils and extracts containing physiologically active phytochemicals have immense potential for producing new drugs of great benefit to mankind. In this context, a systematic screening of secondary metabolites of folk herbs and medicinal plants may result in the discovery of novel and effective antimicrobial compounds (Janovská, Kubíková, & Kokoska, 2003). Essential oils are considered to be one of the potential sources for the screening of anticancer, antimicrobial, antioxidant, and free radical scavenging agents (Celiktas et al., 2007; Hussain, Anwar, Sherazi, & Przybylski, 2008). In particular, antimicrobial activities of essential oils have formed the basis of many applications, including raw and processed food preservation, pharmaceuticals, alternative medicine and natural therapies (Bozin, Mimica-Dukic, Simin, & Anackov, 2006). Plant essential oils and extracts have broadespectrum activity against both Gram-negative and Gram-

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A.I. Hussain et al. / LWT - Food Science and Technology 44 (2011) 1199e1206

Table 1 Yields and chemical composition of the essential oils of six Lamiaceae species. Componentsh

RIi

% Compositiong Rosmarinus officinalis

Monoterpene hydrocarbons tricyclene a-thujene a-pinene camphene b-pinene b-myrcene a-phellandrene 3-carene o-cymene para-cymene limonene (Z)-b-ocimene (E)-b-ocimene g-terpinene Oxygenated monoterpenes 1-octen-3-ol 3-octanone 1,8-cineol cis-sabinenehydrate cis-linaloloxide terpinolene linalool a-thujone b-thujone cis2menthenol (þ)-camphor isopulegol citronellal Isoborneol lavandulol borneol menthenol terpinen-4-ol a-terpineol estragol verbinone nerol carvone b-citronellol geraniol linalyl acetate methyl citronelate geranial dihydrolinalool acetate bornyl acetate isobornyl acetate cuminyl alcohol lavandulyl acetate thymol carvacrol neryl acetate geranyl acetate methyl eugenol Sesquiterpene hydrocarbons d-elemene a-copaene b-patchoulene b-elemene cis-a-bergamotene a-cedrene b-caryophyllene a-bergamotene a-guaiene g-patchoulene a-amorphene a-humulene a-patchoulene b-farnesene allo aromadendrene g-gurjunene g-muurolene

0.10

927 930 939 954 979 991 1003 1011 1021 1025 1029 1037 1049 1060

1.83 e 11.5 5.99 6.46 0.73 0.21 e e e 4.51 e e 0.90

980 984 1031 1070 1088 1089 1097 1116 1119 1121 1144 1146 1153 1156 1165 1169 1172 1177 1189 1195 1207 1222 1224 1228 1255 1257 1260 1271 1275 1285 1286 1287 1289 1290 1299 1365 1380 1404

e 1.61 29.2 e e e 3.35 e e e 17.2 e e 0.25 e 6.33 e e e e 3.82 e e e e e e e e e e e e e e e e e

1327 1375 1380 1391 1403 1409 1421 1439 1440 1441 1442 1454 1456 1458 1460 1472 1480

e e e e e e 3.15 0.15e e e e e e e 0.33 0.04a e e 0.33 0.01a

0.3f 0.26e 0.24d 0.07c 0.02b

0.29d

0.10c

0.10b 0.99c

0.19d

1.0c

0.02b 0.12c

0.28

Mode of Identificationj

Melissa officinalis

Salvia officinalis

Lavandula angustifolia

Thymus vulgaris

Pogostemon cablin

e e 0.09 0.02 0.06 e e e e e 2.25 0.05 0.15 0.05

e 0.06 2.95 2.35 4.56 0.30 0.10 e e 0.23 e 0.96 0.29 0.98

e e 0.28 0.21 0.58 0.15 0.11 0.97 0.17 0.62 0.13 2.18 1.64 0.14

e e 0.65 0.50 0.50 e e e e 17.9 1.00 e e 1.70

e e 0.04 0.00a e 0.04 0.01a e e e e e e e e e

RI, MS RI, MS RT, RI, MS RI, MS RT, RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RT, RI, MS RI, MS RI, MS RI, MS

e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e

RI, MS RI, MS RT, RI, MS RI, MS RI, MS RI, MS RT, RI, MS RI, MS RI, MS RI, MS RT, RI, MS RI, MS RI, MS RI, MS RI, MS RT, RI, MS RI, MS RT, RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RT, RI, MS RT, RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RT, RI, MS RT, RI, MS RI, MS RI, MS RI, MS

e e e e e 0.08 1.30 e e e e 0.19 20.5 e e e 0.15 e 0.23 0.18 e e e 11.5 17.0 e 1.37 5.77 e e e e e e e 2.13 6.79 0.14 e 0.11 e 2.65 e 0.11 0.78 0.35 e e 1.45 0.60 e e e 0.33 0.71

0.01b 0.01a 0.02a

0.06c 0.02a 0.01a 0.01a

0.01a 0.14b

0.02 0.6

0.01 0.01b 0.01

0.9 0.6 0.08 0.20

0.02b 0.42b 0.01

0.03b 0.19b 0.01b 0.10a 0.03a

0.09b 0.09b

0.03 0.04b

e e 27.4 e e e 0.10 16.3 11.2

0.02 0.01e 0.07d 0.10c 0.03b 0.02a

0.02a 0.05b 0.02b 0.06c

c

1.1

0.02a 0.5 0.3

7.98 0.31b e e e e 10.4 0.4d e e 0.10 0.01a e e e e e e e e e e e e e e e e e e e e e e 3.23 0.16c e e 2.75 0.08d e e e 0.30 0.02a e e e e e

0.77 0.10 3.11 0.13 0.18 0.35 25.1 e e 0.23 0.58 e e 0.03 1.20 1.45 e 4.73 2.76 e e 0.04 0.66 e e 22.5 e e 0.18 0.23 0.07 0.22 2.52 0.23 e 0.77 1.23 e e 0.06 e e 0.18 0.06 1.16 0.88 e e 1.15 e e 3.29 5.27 e e

0.01c 0.02b 0.04b 0.01a 0.02a 0.06 0.01 0.04b 0.01a 0.11c 0.06c 0.01b

0.03 0.02a 0.15a 0.02a 0.01 0.01b 0.9e

0.02 0.04a

0.01a 0.06 0.03a 0.18 0.19c

0.02 0.05a

0.6

0.02 0.01 0.01 0.02 0.10 0.01a

0.04a 0.08a

0.01a

0.02 0.02a 0.03c 0.04b

0.07a

0.15b 0.11

e e 5.65 0.86 e e 2.39 e e e e e e e e 2.32 e e 2.98 e e e 0.89 e e e e e e e e e e 52.4 4.21 e e e

0.04d 0.05c 0.05b

0.4c 0.04b

0.05d

b

0.31 0.05b

0.09c

0.19b

0.16c

0.10b

2.3b 0.16

e e e e e e 1.00 0.03b e e e e e e e e e e

0.29 e 7.45 1.89 e e 3.62 e 13.8 9.47 e 1.42 2.32 e e e e

0.03 0.34 0.11a

0.19f 0.36 0.24 0.06c 0.06

RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RT, RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS


1201

Table 1 (continued ) Componentsh

germacrene D

RIi

1485

b-cadinene eremophilene a-muurolene bicyclogermacrene a-bulnesene b-bisabolene g-cadinene b-sesquiphellandrene calamenene virdiflorene calarene Oxygenated sesquiterpenes a-elemol caryophyllene oxide t-b-elemenone g-eudesmol virdiflorol b-eudesmol a-cadinol patchouli alcohol farnesol aromadendrene oxide nonadecane d-patchoulene Total Oil Yield (g/kg)

1486 1500 1503 1505 1509 1515 1519 1520 1531 1639 1550 1583 1601 1632 1634 1651 1654 1659 1713 1749 1902

% Compositiong Rosmarinus officinalis


e e e e e e e e e e e e

2.06 3.44 e 1.15 e e 0.61 e e e e 2.15

e 0.20 0.03a e e e e e e e e e e 97.9 10.5 0.8

4.84 0.21 1.34 0.06d e 0.10 0.02 e 0.14 0.01 3.39 0.09b e 0.27 0.01a 0.03 0.01a 0.07 0.03a e 96.7 2.5 0.1

0.17c 0.15b 0.07b

0.09b

0.15

Salvia officinalis


0.05 0.02a e e e e e e e e e 1.88 0.11b e

1.51 0.31 e 0.11 0.05 0.10 0.15 0.20 0.08 0.15 e e

e e e e 2.96 0.14 e 0.05 0.01a e e e e e 97.5 4.6 0.2

e 1.95 0.10e e e e e e e 1.11 0.06b e e e 94.3 5.8 0.3

0.05b 0.03a

0.02a 0.01 0.02a 0.01a 0.03 0.01 0.01

Thymus vulgaris

Pogostemon cablin

e e e e e e e e e e e e

e e 3.93 e e 17.1 2.32 e e e 0.19 e

e 0.45 0.02b e e e e e e e e e e 95.2 2.9 0.3

e 0.67 2.08 e e e e 22.7 2.15 1.20 0.71 0.25 93.7 19.8

0.09

0.65b 0.13c

0.02a

0.05c 0.16

0.5 0.17c 0.08b 0.08b 0.02

Mode of Identificationj

RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,

MS MS MS MS MS MS MS MS MS MS MS MS

RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS MS

0.9

aef

Mean followed by different superscript letters (aef) in the same row represent significant difference (p < 0.05). g Values are mean standard deviation of three different samples of each Lamiaceae species, analyzed individually in triplicate. h Compound listed in order of elution from an HP-5MS column. i Retention indices relative to C9eC24 n-alkanes on the HP-5MS column. j RT, identification based on comparison of retention time with standard compounds; RI, Identification based on retention index; MS, identification based on comparison of mass spectra.

positive food-borne microbes (Celiktas et al., 2007; Hussain, Anwar, Shahid, Ashraf, & Przybylski, 2010; Hussain et al., 2008; Kotzekidou, Giannakidis, & Boulamatsis, 2008). Antimicrobial susceptibility testing can be carried out by different techniques (Hammer, Carson, & Riley, 1999; Sarker, Nahar, & Kumarasamy, 2007). The selection of correct assay to assess the antimicrobial potential of plant essential oils and extracts is important for generating high-quality data with greater accuracy, speed and efficiency. In most of the antibacterial assays, the bacterial concentration decreased serially during the serial dilution of the test materials and thus cannot be a ‘true’ indicator of the minimum inhibitory concentration (Sarker et al., 2007). Moreover, the bacterial concentration is only approximate as they are compared to the MacFarland standards. The method in which turbidity is evaluated by comparing with MacFarland standards generally is not able to provide standardized number of colony formation unit (CFU) for all strains, because this is operator dependent and subjective. It is also not easy to compare diverse bacterial species because they might have differing optical densities. In order to ensure that a standardized number of bacteria are always used, a set of graphs of killing/ viability curves for each bacterial strain have to be employed (MacGowan, Wootton, & Holt, 1999; Sarker et al., 2007). Sarker et al. (2007) modified the resazurin microtitre-plate assay and applied it successfully for the assessment of antibacterial activity of the plant extracts and standard drugs. The newly modified resazurin assay was found to be accurate, reliable, sensitive and user-friendly because it allowed the detection of microbial growth in extremely small volumes of solution in microtitre-plates without the use of spectrophotometer. In the present study we employed the modified resazurin microtitre-plate assay, developed previously by Sarker et al. (2007), to assess the antibacterial activity of six essential oils of Lamiaceae species native to Pakistan, against

a diverse range of Gram positive and Gram-negative bacteria. The main purpose of this study was to evaluate this method against the essential/volatile oils and to create a directly comparable and quantitative antibacterial data for oils for which little/no information exists. Moreover, the selected essential oils were also characterized quantitatively by GC and GCeMS. 2. Material and methods 2.1. Collection of plant materials The leaves of Rosmarinus officinalis L. (R. officinalis) and Salvia officinalis L. (S. officinalis) were collected in May and March 2008, respectively while those of Melissa officinalis L. (M. officinalis), Lavandula angustifolia Miller (L. angustifolia), Thymus vulgaris L. (T. vulgaris) and Pogostemon cablin (Blanco) Benth (P. cablin) were collected in August 2008 from the cultivated fields of the Botanical Gardens, University of Agriculture, Faisalabad, Pakistan. The plant specimens were further identified and authenticated by Dr. Mansoor Hameed, Taxonomist of the Department of Botany, University of Agriculture, Faisalabad, Pakistan. 2.2. Chemicals Ciprofloxacin, homologous series of C9eC24 n-alkanes and various reference chemicals used in this study were obtained from SigmaeAldrich (St Louis, MO, USA). Sterile resazurin tablets were obtained from BDH Laboratory Supplies (Poole, UK). All other chemicals (analytical grade) i.e. sodium chloride, dimethyl sulfoxide used in this study were purchased from Merck (Darmstadt, Germany), unless stated otherwise. Sterile isosensitest broth (pH buffered medium) was purchased from Oxoid Ltd., (Hampshire, UK)

1202


Fig. 1. Typical GCeMS chromatogram of Lavandula angustifolia essential oil showing the separation of chemical components.

and sterile isosensitest agar was obtained from Southern Group Laboratory, SGL (Northamptonshire, UK).

2.3. Isolation of the essential oil The air-dried and finely ground (80 mesh) leaves samples of Rosmarinus officinali, S. officinalis, M. officinalis, L. angustifolia, T. vulgaris and P. cablin were subjected to hydro-distillation for 3 h using a Clevenger-type apparatus. Distillates of essential oils were dried over anhydrous sodium sulfate, filtered and stored at 4 C until analyzed.

2.4.2. Gas chromatography/mass spectrometry analysis GCeMS analysis of the essential oils was performed using an Agilent-Technologies (Little Falls, California, USA) 6890 N Network gas chromatographic (GC) system, equipped with an Agilent-Technologies 5975 inert XL Mass selective detector and Agilent-Technologies 7683B series auto injector. Compounds were separated on HP-5 MS capillary column (30 m 0.25 mm, film thickness 0.25 mm; Little Falls, CA, USA). A sample of 1.0 mL was injected in the split mode with split ratio 1:100. For GCeMS detection, an electron ionization system, with ionization energy of 70 eV, was used. Column oven temperature program was the same as in GC analysis. Helium was used as a carrier gas at a flow rate of 1.5 mL min1. Mass scanning range was 50e550 m/z while injector and MS transfer line temperatures were set at 220 and 290 C, respectively.

2.4. Analysis of the essential oil 2.4.1. Gas chromatography The essential oils were analyzed using a PerkineElmer gas chromatograph model 8700, equipped with flame ionization detector (FID) and HP-5MS capillary column (30 m 0.25 mm, film thickness 0.25 mm). Injector and detector temperatures were set at 220 and 290 C, respectively. Column oven temperature was programmed from 80 to 220 C at the rate of 4 C min1, initial and final temperatures were held for 3 and 10 min, respectively. Helium was used as carrier gas with flow of 1.5 mL min1. A sample of 1.0 mL was injected, using split mode (split ratio, 1:100). All quantifications were done by a built-in data-handling program provided by the manufacturer of the gas chromatograph (PerkineElmer, Norwalk, CT, USA). The composition of the essential oil component was reported as a relative percentage of the total peak area. Furthermore, the major components of the essential oils, 1,8-cineol, citronella, linalool, thymol and patchouli alcohol were quantified by constructing standard calibration curve using the internal standard addition method as described by Kowalski (2008).

2.4.3. Compounds identification The identification of the oil constituents was based on computer matching with the NIST02.L and WILEY7n.L mass spectral libraries as well as by comparison of the mass spectra and retention indices with those of published data or with our own data (MS for pure reference compounds) (Adams, 2007; Anwar, Ali, Hussain, & Shahid, 2009; Mimica-Dukic, Bozin, Sokovic, Mihajlovic, & Matavulj, 2003; Vagionas, Graikou, Ngassapa, Runyoro, & Chinou, 2007). Where possible, compounds were also identified by comparison of their retention indices, retention times and mass spectra relative to n-alkanes (C9eC24) with those of authentic compounds.

2.5. Antibacterial activity 2.5.1. Microbial strains The Lamiaceae essential oils were individually tested against a panel of pathogenic and clinically isolated microorganisms, including: Staphylococcus aureus (S. aureus) NCTC 6571, S. aureus


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Fig. 2. Typical GCeMS chromatogram of Pogostemon cablin essential oil showing the separation of chemical components.

(NCTC 1803), Bacillus cereus (B. cereus) ATCC 11778, B. cereus NCTC 7464, Basilius subtilis NCTC 10400, Bacillus pumilis (wild type), Pseudomonas aeruginosa (P. aeruginosa) NCTC 1662, Salmonella poona (S. poona) NCTC 4840, Escherichia coli (E. coli) ATCC 8739 and Ampicillin resistant E. coli NCTC 10418. The bacterial strains were obtained from Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast and Microbiology Laboratory, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK. 2.5.2. Disc diffusion method The antibacterial activity of the essential oils was determined by disc diffusion method (Kelen & Tepe, 2008). Briefly, 100 mL of suspension containing approx 5 105 colony-forming units (CFU)/ mL of bacteria cells on nutrient agar. The sterile filter discs (6 mm in diameter) were separately impregnated with 10 mL of essential oils and placed on the inoculated agar which had previously been

inoculated with the tested microorganism. Ciprofloxacin (25 mg/ disc) was used as positive reference, while discs without oil were used as a negative control. The plates were incubated at 37 C for 24 h for bacteria and at 30 C for 48 h for fungal strains. Antibacterial activity was assessed by measuring the diameter of the inhibition zone in millimeters, including disc diameter (6 mm) for the test organisms, compared to the controls. 2.5.3. Resazurin microtitre-plate assay For the minimum inhibitory concentration (MIC) of the essential oils and main components, a modified resazurin microtitre-plate assay was used as reported by Sarker et al. (2007). Briefly, a volume of 100 mL essential oil solutions (2.5 mg/mL, w/v in DMSO:water (10 mL:90 mL) solution), pure components (2.0 mg/mL in DMSO solution) and standard antibiotic (1.0 mg/mL in DMSO solution) was pipetted into the first row of the 96 well plates. To all other wells 50 mL of nutrient broth was added. Two fold serial dilutions

Table 2 Composition of major constituents in six Lamiaceae essential oils. Compounds

Content (g/100 g)a Rosmarinus officinalis


Salvia officinalis


Thymus vulgaris

Pogostemon cablin

1,8-cineol citronellal linalool thymol patchouli alcohol

28.4 0.8 e e e e

e 19.0 0.4 e e e

26.7 0.9 e e e e

e e 23.3 0.7 e e

e e e 51.1 1.9 e

e e e e 21.1 0.9

a

Values are mean standard deviation of three different samples of each Lamiaceae essential oils, analyzed individually in triplicate.

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Table 3 Antibacterial activity (inhibition zone measured in mm)f of six Lamiaceae essential oils against selected strains of bacteria. Microorganisms

Essential oils R. officinalis

Staphylococcus aureus (NCTC 1803) S. aureus (NCTC 6571) Bacillus cereus (ATCC 11778) B. cereus (NCTC 7464) Bacillus subtilis (NCTC 10400) Bacillus pumilis (wild type) Pseudomonas aeruginosa (NCTC1662) Salmonella poona (NCTC 4840) Escherichia coli (ATCC 8739) E. coli (NCTC 10418)g

24.4 24.3 26.2 22.0 26.2 16.0 19.3 18.2 16.4 14.0

1.0c 1.1b 1.2a 1.2a 0.9b 0.8b 0.9d 0.7c 0.7a 1.1d

Ciprofloxacin M. officinalis 22.0 24.3 28.1 24.2 28.2 19.7 13.7 16.9 18.3 17.7

0.6b 0.7b 0.6b 1.0a 0.9c 0.5c 0.3b 0.3b 0.8b 0.9b

S. officinalis

L. angustifolia

22.0 1.0b 24.3 0.8b 26.2 1.3a 26.1 0.6b 22.4 0.6a 12.4 0.4a 9.0 0.3a 16.6 0.4b 19.7 0.9b 20.9 0.8c

26.2 24.1 26.1 24.3 22.2 20.0 21.0 15.4 16.3 13.1

1.0c 1.4b 1.3a 1.1a 1.2a 0.8c 1.1d 0.6a 0.3a 1.0a

T. vulgaris 27.9 27.6 28.2 27.1 28.0 24.3 17.1 27.5 23.2 25.3

P. cablin

0.6d 0.6c 0.7b 1.0b 0.8c 0.9d 0.8c 1.0e 1.2c 2.0d

16.4 20.3 29.9 30.3 28.1 25.4 12.4 14.5 21.9 23.0

0.3a 0.5a 0.5c 1.2c 0.7c 1.1d 1.1b 0.3a 0.6c 1.0d

32.5 27.3 34.2 34.0 32.1 22.6 30.1 24.7 31.0 33.0

1.1e 1.0c 1.4d 1.6d 1.0d 1.0d 1.8e 1.1d 1.7d 1.5e

aee

Mean followed by different superscript letters (aee) in the same row represent significant difference (p < 0.05). Diameter of inhibition zone (mm) including disc diameter of 6 mm. Values are mean standard deviation of three different samples of each Lamiaceae essential oils, analyzed individually in triplicate. g Amphicilin resistant strain. f

were performed using a multichannel pipette such that each well had 50 mL of the test material in serially descending concentrations. 30 mL of 3.3 strength isosensitised broth and 10 mL of resazurin indicator solution (prepared by dissolving 270 mg tablet in 40 mL of sterile distilled water) were added in each well. Finally, 10 mL of bacterial suspension was added to each well to achieve a concentration of approx 5 105 cfu/mL. Each plate was wrapped loosely with cling film to ensure that bacteria did not become dehydrated. Each plate had a set of controls: a column with a ciprofloxacin as positive control; a column with all solutions with the exception of the test compound; a column with all solutions with the exception of the bacterial solution adding 10 mL of nutrient broth instead and a column with DMSO solution as a negative control. The plates were prepared in triplicate, and incubated at 37 C for 24 h. The color change was then assessed visually. The growth was indicated by color changes from purple to pink (or colorless). The lowest concentration at which color change occurred was taken as the MIC value. 2.5.4. Determination of minimum bactericidal concentration (MBC) of essential oils The wells showing complete absence of growth in the MIC assay were identified and 10 mL of each well were transferred to nutrient agar plates (Celiktas et al., 2007). The agar plates were incubated with timeetemperature profiles identical to that in disc diffusion assay. The well showing the complete absence of growth on agar plates was considered as the minimum bactericidal concentration (MBC).

2.6. Statistical analysis All the experiments were conducted in triplicate and the data are presented as mean values (standard deviation) of triplicate

determinations. Statistical analysis of the data was performed by Analysis of Variance (ANOVA) using STATISTICA 5.5 (Stat Soft Inc, Tulsa, OK, USA) software and a probability value of p 0.05 was considered to denote a statistical significance difference.

3. Results and discussion 3.1. Yield and chemical composition of the essential oils The essential oil yields ranged from 2.5 to 19.8 g/kg (w/w) based on the dry weight of the plant material (Table 1). The maximum and minimum oil contents were found in P. cablin and M. officinalis, respectively. The variations in the content of essential oils with respect to Lamiaceae species were significant (p < 0.05). The results obtained by GC and GCeMS analysis of the Lamiaceae essential oils are presented in Table 1. Nineteen, 43, 24, 58, 16 and 21 compounds were identified in the essential oils of Rosmarinus officinalis, M. officinalis,S. officinalis, L. angustifolia, T. vulgaris and P. cablin, representing 97.9, 96.7 97.5, 94.3, 95.2 and 93.7% of the total oil, respectively. The main components of essential oils were 1,8-cineol (29.2%), camphor (17.2%), a-pinene (11.5%) in R. officinalis, citronellal (20.5%), b-geraniol (17.0%), b-citronellol (11.5%) in M. officinalis, 1,8-cineol (27.4%), a-thujone (16.3%), b-thujone (11.2%), borneol (10.4%), camphor (7.98%) in S. officinalis, linalool (25.1%), linalyl acetate (22.5%) in L. angustifolia, thymol (52.4%), p-cymene (17.9%) (Fig. 1) in T. vulgaris and Patcholene alcohol (22.7%), a-bulnesene (17.1%), a-guaine (13.8%) in P. cablin (Fig. 2). Except, P. cablin, all other analyzed essential oils mainly rich in oxygenated monoterpenes. R. officinalis, M. officinalis, S. officinalis, L. angustifolia and T. vulgaris contained 56.3, 67.1. 73.5, 68.1 and 71.7% oxygenated monoterpenes, respectively. P. cablin essential oils contained 63.5% sesquiterpene hydrocarbons (Table 1). The variations in the

Table 4 Minimum inhibitory concentration (MIC; mg mL1) of six Lamiaceae essential oils against selected strains of bacteria.f Microorganisms

R. officinalis

M. officinalis

S. officinalis

L. angustifolia

T. vulgaris

P. cablin

Staphylococcus aureus (NCTC 1803) S. aureus (NCTC 6571) Bacillus cereus (ATCC 11778) B. cereus (NCTC 7464) Bacillus subtilis (NCTC 10400) Bacillus pumilis (wild type) Pseudomonas aeruginosa (NCTC1662) Salmonella Poona (NCTC 4840) Escherichia coli (ATCC 8739) E. coli (NCTC 10418)g

310.4 12.4b 305.3 6.0b 203.3 4.9d 91.3 2.9b 97.3 3.3c 403.7 9.3b 1113.3 36.7b 310.3 6.9b 733.7 17.3d 810.7 7.0d

330.3 12.3b 300.6 11.8b 80.1 2.9a 94.2 2.0b 72.0 2.5a 320.4 6.4a 1000.3 33.2a 847.0 22.7d 442.3 10.8c 567.4 17.2b

324.3 8.5b 302.4 6.8b 147.4 3.2c 136.2 3.9d 95.2 3.6c 594.0 9.2d 1250.3 36.0c 404.3 10.6c 475.0 9.7a 548.0 8.7a

320.4 12.0b 310.0 9.7b 210.4 10.4d 80.4 3.3a 70.3 2.5a 470.0 20.0c 1040.0 36.2b 830.3 25.3d 730.1 18.7d 722.2 30.3c

210.0 4.9a 160.5 3.7a 100.3 2.3b 100.2 3.1c 70.4 2.2a 310.2 7.0a 1250.3 24.7c 160.3 4.0a 430.4 12.6bc 360.6 9.4a

520.0 10.1c 395.2 9.7c 100.7 4.5b 80.9 2.4a 80.0 3.0b 310.3 8.0a 1200.0 28.3c 950.4 45.2e 410.7 12.3b 530.2 20.0b

aee f g

Mean followed by different superscript letters (aee) in the same row represent significant difference (p < 0.05). Values are mean standard deviation of three different samples of each Lamiaceae essential oils, analyzed individually in triplicate. Amphicilin resistant strain.


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Table 5 Minimum inhibitory concentration (mg mL1) of pure compounds.g Tested Microorganisms

1,8-cineol

Staphylococcus aureus (NCTC 1803) S. aureus (NCTC 6571) Bacillus cereus (ATCC 11778) B. cereus (NCTC 7464) Bacillus subtilis (NCTC 10400) Bacillus pumilis (wild type) Pseudomonas aeruginosa (NCTC1662) Salmonella Poona (NCTC 4840) Escherichia coli (ATCC 8739) E. coli (NCTC 10418)h

930.0 830.3 930.3 910.7 600.0 980.2 > 1000 > 1000 > 1000 > 1000

27.0f 20.2a 26.6f 28.1e 20.3f 33.3f

citronellal 590.9 490.0 398.5 375.0 385.6 823.2 > 1000 > 1000 > 1000 > 1000

16.7e 14.3b 9.9e 12.3d 11.2e 24.9e

linalool 380.0 360.3 326.2 300.5 350.0 790.0 > 1000 > 1000 980.0 > 1000

thymol 9.5b 9.9b 9.8d 8.5c 11.2d 21.0d

30.0d

550.5 540.5 310.4 290.6 250.1 720.5 > 1000 900.4 910.3 890.0

Patchouli alcohol 13.5d 16.2c 9.4c 9.3c 7.3c 19.4c 27.4b 22.5c 28.5c

490.3 470.9 290.5 200.3 185.7 340.0 > 1000 > 1000 600.1 635.5

13.9c 13.5c 7.9b 7.8b 4.4b 9.1b

19.7b 19.0b

Ciprofloxacin 6.23 0.17a 15.60 0.19a 6.66 0.34a 8.00 0.47a 4.47 0.10a 62.25 1.70a 30.20 1.70a 2.53 0.13a 4.48 0.22a 5.50 0.21a

aef

Means followed by different superscript alphabets (aef) in the same rows present significant difference (p < 0.05). Values are mean standard deviation of three different samples of each Lamiaceae essential oils, analyzed individually in triplicate. h Amphicilin resistant strain. g

concentration of essential oils with respect to Lamiaceae species were significant (p < 0.05). The amounts of the main components i.e. 1,8-cineol, citronellal, 1,8-cineol, linalool, thymol and patchouli alcohol, calculated using calibrated curves with pure standards compounds, in the R. officinalis, M. officinalis, S. officinalis, L. angustifolia, T. vulgaris and P. cablin, essential oils were found to be 28.4, 19.0, 26.7, 23.3, 51.1 and 21.1 g/100 g of oil, respectively (Table 2). There are no previous data available in the literature on the quantitative analysis of Lamiaceae essential oil’s components with which to compare our present results. However, there are some reports on the chemical composition of the oils isolated from these plants of Lamiaceae family grown in diverse climate of different countries (Celiktas et al., 2007; Hayouni et al., 2008; Shellie, Mondello, Marriott, & Dugo, 2002; Maksimovic et al., 2007; Mimica-Dukic, Bozin, Sokovic, & Simin, 2004; Wang, Wu, Zu, & Fu, 2008). 3.2. Antibacterial activity of essential oils The antibacterial effectiveness of essential oils from the six species of Lamiaceae was qualitatively and quantitatively evaluated by the development of inhibition zones (IZ), measurement of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. The essential oils were inhibitory to the growth of all the bacteria under test, and these findings are summarized in Tables 3 and 4. Generally, Gram-positive bacteria are more susceptible than Gram-negative bacteria. Basilius subtilis and B. cereus were the most sensitive, while P. aeruginosa was the most resistant strain tested (against all essential oils). All the tested essential oils showed the antibacterial activity. The best antibacterial activity was observed with the essential oils of T. vulgaris with larger IZ, 17.1e28.2 mm and smallest MIC, 70.4e1250.3 mg mL1 values against selected strains of bacteria. Melissa officinalis, R. officinalis, S. officinalis, L. angustifolia and P. cablin also exhibited antibacterial activities with IZ and MIC values ranging from 13.7 to 28.2, 16.0e26.2, 9.0e26.4, 13.1e26.2 and 12.4e30.3 mm and 72.0e1000.3, 91.3e1113.3, 95.2e1250.3, 70.3e1040.0 and 80.0e1200.0 mg mL1, against all the tested strains, respectively. Among all the essential oils evaluated, S. officinalis exhibited comparatively weaker antibacterial activity. It is generally accepted that essential oils having higher contents of oxygenated terpenes exhibit potent antibacterial potential (Hussain et al., 2008). All the tested essential oils are bactericidal and showed MBC values (data not shown) almost equal to MIC values. Table 5 showed the antibacterial activity of 1,8-cineol, the major constituents of R. officinalis and S. officinalis, citronellal, linalool, thymol and patchouli, the main components of M. officinalis, L. angustifolia, T. vulgaris and P. cablin essential oils, respectively. Patchouli alcohol and thymol exhibited highest antibacterial

activity against B. subtilis with MIC 185.7 and 250.1 mg mL1, respectively. Citronellal and linalool also showed appreciable but 1,8-cineol exhibited least antibacterial activity against the selected microorganisms. The higher antibacterial activity of T. vulgaris essential oil could be attributed to the presence of high content of thymol. Similarly, the antibacterial activity of P. cablin essential oil might be in due part to the high concentration of patchouli alcohol. Literature revealed that the thymol and patchouli alcohol are potent antimicrobial agents and the antibacterial activity of essential oils might be ascribed to the presence of high contents of these compounds (Bozin et al., 2006; Yang et al., 1996). Ciprofloxacin revealed stronger antibacterial activity with large IZ and small MIC values (Tables 3 and 5). In the present study, we used the modified resazurin microtitreplate assay to evaluate the antimicrobial activity of essential oils for the first time. As reported earlier,8 we found that this method is also effective, sensitive and quick against essential/volatile oils. This provided the reproducible and accurate results and allowed direct comparison of the antibacterial activity of the tested essential oils. Essential oils rich in phenolic compounds are widely reported to possess high level of antimicrobial activity (Sivropoulou, Kokkini, Lanaras, & Arsenakis, 1995), which has been confirmed and extended in the present studies. It is believed that the phenolic components of essential oils show strongest antimicrobial activity, followed by aldehyde, ketones and alcohols (Tepe et al., 2006). Our results are in good agreement with the finding of Cantore, Iacobellis, Marco, Capasso, and Senatore (2004) who reported that Gram-positive bacteria are more sensitive to plant essential oils than Gram-negative bacteria (especially E. coli). There is evidence in the literature that the essential oils of some Lamiaceae plants possess considerable antibacterial activities (Celiktas et al., 2007; Delmare, Moschen-Pistorello, Artico, Atti-Serafini, & Exheverrigaray, 2007; Gören et al., 2002; Kelen & Tepe, 2008; Mimica-Dukic et al., 2004; Rota, Herrera, Martinez, Sotomayor, & Jordan, 2008). 4. Conclusion The modified resazurin assay was found to be applicable for reliable assessment of antibacterial activity of the tested essential oils against several Gram positive and negative bacterial taxa. These results indicate that the all the analyzed essential oils presented inhibitory effects on most of the strains tested and may therefore be used as safe antimicrobial and antiseptic supplements in preventing food deteriorations. The present results also demonstrated that Lamiaceae essential oils which exhibited higher antibacterial activity were generally rich in oxygenated monoterpens. A further study under in vivo condition is recommended to elaborate the antimicrobial mechanism of the tested essential oils

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for various applications. The investigated essential oils may be used for preservation of processed foods, as well as pharmaceutical and natural therapies for the treatment of infectious diseases in human and plants. Ethical approval Not required. Competing interests None declared. Acknowledgement We would like to extend our special gratitude to Professor M.I. Bhanger, Director, National Center of Excellence in Analytical Chemistry (NCEAC), University of Sindh, Jamshoro, Pakistan for allowing us to use GCeMS instrumental facility. Thanks are due to the Higher Education Commision for the award of scholarship to A.I. Hussain under the scheme of International Research Support Initiative Program (IRSIP) to conduct the part of project work in University of Ulster, Coleraine, UK. References Adams, R. P. (2007). Identification of essential oil components by gas chromatography/ quadrupole mass spectroscopy. Carol Stream, IL: Allured. Anwar, F., Ali, M., Hussain, A. I., & Shahid, M. (2009). Antioxidant and antimicrobial activities of essential oil and extracts of fennel (Foeniculum vulgare Mill.) seeds from Pakistan. Flavour and Fragrance Journal, 24, 170e176. Bozin, B., Mimica-Dukic, N., Simin, N., & Anackov, G. (2006). Characterization of the volatile composition of essential oils of some lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. Journal of Agricultural and Food Chemistry, 54, 1822e1828. Cantore, P. L., Iacobellis, N. S., Marco, A. D., Capasso, F., & Senatore, F. (2004). Antioxidant activity of Coriandrum sativum L. and Foeniculum vulgare Miller Var. vulgare (Miller) essential oils. Journal of Agricultural and Food Chemistry, 52, 7862e7866. Celiktas, O. Y., Kocabas, E. E. H., Bedir, E., Sukan, F. V., Ozek, T., & Baser, K. H. C. (2007). Antimicrobial activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and seasonal variations. Food Chemistry, 100, 553e559. Delmare, A. P. L., Moschen-Pistorello, I. T., Artico, L., Atti-Serafini, L., & Exheverrigaray, S. (2007). Antibacterial activity of the essential oils of Salvia officinalis L. and Salvia triloba L. cultivated in South Brazil. Food Chemistry, 100, 603e608. mus, Z., & Pezzuto, J. M. (2002). The Gören, A. C., Topçu, G., Bilsel, G., Bilsel, M., Aydog chemical constituents and biological activity of essential oil of Lavandula stoechas ssp. stoechas. Zeitschrift für Naturforschung, 57, 797e800. Hammer, K. A., Carson, C. F., & Riley, T. V. (1999). Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology, 86, 985e990. Hayouni, E. A., Chraief, I., Abedrabba, M., Bouix, M., Leveau, J.-Y., Mohammed, H., et al. (2008). Tunisian Salvia officinalis L. and Schinus molle L. essential oils: their chemical compositions and their preservative effects against Salmonella inoculated in minced beef meat. International Journal of Food Microbiology, 125, 242e251.

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