Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Potential use of Tunisian Pituranthos chloranthus essential oils as a natural disinfectant T. Yangui*, M. Bouaziz*, A. Dhouib and S. Sayadi Laboratoire des Bioproce´de´s, Poˆle d’Excellence Re´gionale AUF, (PER-LBP) Centre de Biotechnologie de Sfax, Sfax, Tunisie
Keywords antimicrobial, antioxidant, essential oil, natural disinfectant, Pituranthos chloranthus. Correspondence Abdelhafidh Dhouib, Laboratoire des Bioproce´de´s, Poˆle d’Excellence Re´gionale AUF, (PER-LBP), Centre de Biotechnologie de Sfax, BP: 1177, 3018 Sfax, Tunisie. E-mail:
[email protected]
*The first two authors contributed equally to this work. 2008 ⁄ 0609: received 9 April 2008, revised 6 September 2008 and accepted 22 September 2008 doi:10.1111/j.1472-765X.2008.02499.x
Abstract Aims: To highlight the bactericidal and fungicidal activities of Tunisian Pituranthos chloranthus essential oils and to study their potential use as powerful and natural disinfectant. Methods and Results: The essential oils were obtained by hydro-distillation of the aerial part of P. chloranthus. The bactericidal and fungicidal properties of essential oils were investigated by using the NCCLS broth dilution method and the EN 1275 and EN 1276 European standard methods. High bactericidal and fungicidal effects of 1Æ87–3Æ75 and 7Æ5 mg l)1 were obtained, respectively. Essential oils concentrations of 0Æ5% and 1% (w ⁄ v) allowed reductions in viability higher than 5 and 4 log units per ml for standard bacteria and fungi, respectively, within a contact time of 5 min under dirty conditions. Conclusions: Our results support the traditional uses of P. chloranthus as a natural disinfectant and insecticide. It could be used to manage life-threatening pathogens as well as food preservative. Significance and Impact of the Study: This natural disinfectant could play a vital role in alleviating the spread of pathogenic micro-organisms and environmental problems associated with the indiscriminate use of synthetic chemicals.
Introduction Plants, naturally, produce essential oils to protect themselves from pathogen micro-organisms. These essential oils have been used in the folk medicine since thousands of years as antimicrobial (Fisher and Phillips 2008). Essential oils were frequently referred to as the natural and environmentally friendly cleaning solutions. They are used as a substitute to chemicals to disinfect and spread a pleasant scent in the air (Segvic Klaric et al. 2007). They are also used to control human diseases of microbial origin and to cure such diseases as atherosclerosis and cancer (Warnke et al. 2006). Insecticidal properties of essential oils have been extensively studied against various insect species (Kordali et al. 2007). Furthermore, the use of antioxidants having natural origin has become more popular as a means to increase the shelf-life of food products, to improve the stability of fats and oils and to slow down the ageing process. 112
Pituranthos chloranthus Benth. and Hook. is a NorthAfrica endemic plant, locally named Guezzah (Touil et al. 2006). Stems of P. chloranthus have been, traditionally, used as straw for farmers to dry figs and grapes. This plant has a double advantage; first, it has been used for its aroma and distinctive taste that adhere to the dry fruits. Second, it has an insecticidal effect. In the Tunisian South, a tuft of P. chloranthus was, traditionally, suspended to the surface of the water to disinfect the underground cisterns of the rain water storage used for the drink. Furthermore, Pituranthos species are used in traditional medicine for the treatment of asthma, rheumatism, postpartum care, spasms, pains, fevers, diabetes, lice (head and pubis), hepatitis, digestive difficulties, urinary infections and scorpions stings (Ve´rite´ et al. 2004). The aim of this study was to highlight this nonexploited endemic plant as a new material in the production of essential oils, which were worthily evaluated thanks to their antimicrobial activities and possible exploitation as a natural disinfectant and food preservative.
ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009) 112–117
T. Yangui et al.
Materials and methods Plant material Three samples of about 1 kg each one, of the aerial part of P. chloranthus, were collected on May 2007 in three different random localities (11 km north, 8 km west and 17 km south) of Sfax, Tunisia. Botanical identification of P. chloranthus was carried out by Professor Makki Boukhris, Faculty of Sciences of Sfax, Tunisia. Essential oil extraction The fresh plant material (800 g of each sample in the three localities), was subjected to hydro-distillation during approx. 4 h in a Clevenger-type apparatus. The distilled essential oils of each sample were separately collected. They were dried over anhydrous sodium sulfate and stored in tightly closed dark vials at 4C until use. Gas chromatography-mass spectrometry The analysis of the essential oils was performed on a HP model 5975B inert MSD, equipped with a capillary DB5MS column (30 m length, 0Æ25 mm i.d. and 0Æ25 mm film thickness; Agilent Technologies, J&W Scientific Products, Palo Alto, CA, USA). The carrier gas was He and was used at 1 ml min)1 flow rate. The oven temperature programme was as follows: 1 min at 100C ramped from 100C to 260C at 4C min)1 and 10 min at 260C. The chromatograph was equipped with a split ⁄ splitless injector used in the split mode. The split ratio was 1 : 100. Identification of components was assigned by comparison of the retention indices (RI) relative to C9–C26 n-alkanes and MS corresponding database (Wiley and NIST library) and with mass spectral literature. Determination of antioxidant activity Antioxidant activity using the system b-carotene-linoleate was determined according to the method described by Hidalgo et al. (1994). The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging effect was evaluated according to the method employed by Gulluce et al. (2007). Antimicrobial assay The bacteria were cultivated in tryptic soy broth (TSB) or agar (Sigma) at the appropriated temperature (30C or 37C) of the strain. Fungi and yeasts were cultured on malt extract broth (MEB) or agar (Fluka, Madrid, Spain) at 28C. Inocula were prepared by adjusting the
Pituranthos chloranthus essential oil as disinfectant
turbidity of each bacterial and yeast cultures to reach an optical comparison to that of a 0Æ5 McFarland standard, corresponding to approx. 1–5 · 108 CFU ml)1. The concentration of spore suspensions was determined by using a haematocytometer (Thoma cell) and adjusted to 1– 5 · 107 spore per ml. Minimal inhibitory concentration, minimal bactericidal concentration and minimal fungicidal concentration Minimal inhibitory concentrations (MICs), minimal bactericidal concentrations (MBCs) and minimal fungicidal concentration (MFCs) were determined by NCCLS (2000) broth dilution method. To obtain stable diffusion, stock solutions of essential oils and eugenol were prepared in 0Æ1% ethanol. Further dilutions were, then, performed using twofold dilution in TSB for bacteria and MEB for fungi. Ethanol at 0Æ1% had no inhibition effect. Disinfectant properties To evaluate P. chloranthus essential oils as a disinfectant, the European standard EN 1276 (1997) and EN 1275 (1997) methods were used employing the standard bacterial and fungal strains under dirty conditions that are representative of surfaces which are known to or may contain organic and inorganic materials (3 g l)1 bovine albumin, 300 mg kg)1 CaCO3). The method of dilution-neutralization was employed, using (3% Tween80, 3% Saponin, 0Æ1% Histidine and 0Æ1% Cysteine) autoclaved solution as a neutralizer. Sterile hard water (300 mg kg)1 CaCO3) was used as a diluent during the test. The product test concentrations were 0Æ5%, 0Æ75% and 1% (v ⁄ v) for bactericidal activity and 0Æ5%, 1% and 2% (v ⁄ v) for fungicidal activity. The contact time and test temperature were t = 5 min ± 10 s and h = 20 ± 1C, respectively. Pituranthos chloranthus essential oils were compared with the chemical eugenol (Sigma-Aldrich, France) and the orange essential oils (Sigma-Aldrich). Results Chemical composition The average percentage composition of individual components of essential oils from the three samples of P. chloranthus are cited in Table 1. A total of 40 compounds representing 78Æ10% of the oils were identified by gas chromatography-mass spectrometry (GC-MS) spectra and compared with those of the reference substances. Quantitative data were based on peak area normalization without using a correction factor. The average yield in essential oils was 0Æ07% (w ⁄ w). Monoterpenes were the
ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009) 112–117
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Pituranthos chloranthus essential oil as disinfectant
T. Yangui et al.
Table 1 Composition of the essential oils of the aerial part of Pituranthos chloranthus analysed by gas chromatograph-mass spectrometer Peak number*
Compound
IR
TIC (%)
1 2 3 4 5 6 7 12 13 15 16 18 20 21 22 24 27 28 32 36 38 41 42 44 45 46 48 49 50 51 53 54 56 57 61 64 68 84 85 89
Heptanal a-Thujene a-Pinene Benzaldehyde Sabinene b-Myrcene Hexanoic acid a- Terpinene o-Cymene a-Limonene 1, 8-Cineole c-Terpinene 1-Octanol Linalool oxide a-Terpinolene Linalool Thujone D-3-Carene p-Menth-2-en-1-ol Sabina ketone Pinocarvone Thujen-2-one Terpinen-4-ol 8-Hydroxy-p-cymene a-Terpineol Myrtenol Phellandrene epoxide c-Terpinene l-Verbenone trans-Carveol b-Citronellol Cis-carveol Cuminic aldehyde Carvone Carvenone Decan-1-ol Cuminyl alcohol Vanillin Methyl eugenol BHT (Ional) % Identification Yield (w ⁄ f.w.)
966 1004 1009 1027 1038 1040 1053 1072 1079 1083 1086 1116 1132 1135 1157 1174 1197 1204 1230 1256 1263 1280 1290 1298 1306 1314 1322 1331 1334 1348 1363 1365 1379 1386 1406 1431 1459 1633 1647 1835
0Æ05 ± 0Æ19 ± 0Æ38 ± 0Æ07 ± 0Æ18 ± 0Æ13 ± 0Æ74 ± 0Æ34 ± 0Æ52 ± 1Æ56 ± 0Æ07 ± 1Æ12 ± 0Æ12 ± 0Æ43 ± 2Æ30 ± 2Æ14 ± 0Æ21 ± 2Æ82 ± 3Æ97 ± 1Æ25 ± 0Æ36 ± 0Æ22 ± 30Æ34 ± 4Æ23 ± 3Æ50 ± 4Æ12 ± 1Æ32 ± 1Æ11 ± 0Æ94 ± 3Æ15 ± 0Æ88 ± 0Æ56 ± 0Æ20 ± 1Æ73 ± 0Æ2 ± 1Æ82 ± 0Æ61 ± 0Æ07 ± 1Æ47 ± 2Æ33 ± 78Æ10 0Æ070 ±
0Æ00 0Æ01 0Æ03 0Æ01 0Æ02 0Æ01 0Æ01 0Æ02 0Æ00 0Æ12 0Æ01 0Æ08 0Æ01 0Æ00 0Æ01 0Æ07 0Æ01 0Æ15 0Æ09 0Æ01 0Æ03 0Æ03 2Æ43 0Æ51 0Æ39 0Æ21 0Æ04 0Æ09 0Æ06 0Æ36 0Æ07 0Æ03 0Æ02 0Æ12 0Æ01 0Æ15 0Æ05 0Æ00 0Æ16 0Æ27 0Æ009
Values are mean ± SD of three different experiments. RI, retention index relative to n-alkanes (C9–C26) on a polar HP-5 column. *Numbers correspond to the peaks observed in the GC-MS chromatogram.
most abundant compound group of the oil (71Æ05%). The major constituents of the oil were terpinen-4-ol (30Æ34%), 8-hydroxy-p-cymene (4Æ23%), myrtenol (4Æ12%), p-menth-2-en-1-ol (3Æ97%) and a-terpineol (3Æ50%). 114
Table 2 Antioxidant activity of the essential oils of the aerial part of Pituranthos chloranthus, orange essential oils, eugenol and BHT
P. chloranthus essential oils Orange essential oils BHT Eugenol
DPPH bleaching method IC50 (mg l)1)
b-Carotene activity (%)
59Æ98 10245 8Æ13 5Æ6
73Æ24 41Æ37 93Æ60 94Æ11
± ± ± ±
1Æ12 41Æ0 1Æ07 1Æ58
± ± ± ±
2Æ11 2Æ11 1Æ29 1Æ35
Values are mean ± sd of three repetitions.
Antioxidant capacity Table 2 shows the free radical-scavenging capacities and the percentage of inhibition of linoleic acid oxidation as exhibited by the essential oils of P. chloranthus compared with synthetic antioxidant BHT (2,6-di-tat-butyl-4-hydroxytoluene), eugenol and orange essential oils. Pituranthos chloranthus essential oils exhibit an intermediate antioxidant activity that is lower than BHT and eugenol but higher than orange essential oils. Antimicrobial activity According to the results of the dilution method (Table 3), the growth of all the tested bacteria and fungi was inhibited by the essential oils of P. chloranthus in the range of Table 3 Minimum inhibitory concentration and minimum bactericidal concentration or minimal fungicidal concentration of Pituranthos chloranthus and orange essential oils and eugenol
Organisms
MBC or MFC MIC (mg l)1) (mg l)1)
P. chloranthus essential oils Pseudomonas aeruginosa ATCC 15442 3Æ75 Escherichia coli ATCC 10536 3Æ75 Staphylococcus aureus ATCC 9144 1Æ875 Enterococcus hirae ATCC 10541 1Æ875 Candida albicans ATCC 10231 7Æ5 Aspergillus niger ATCC 16404 7Æ5 Orange essential oils Ps. aeruginosa ATCC 15442 1Æ875 E. coli ATCC 10536 1Æ875 Staph. aureus ATCC 9144 0Æ938 Ent. hirae ATCC 10541 0Æ938 C. albicans ATCC 10231 3Æ75 A. niger ATCC 16404 3Æ75 Eugenol Ps. aeruginosa ATCC 15442 1Æ875 E. coli ATCC 10536 1Æ875 Staph. aureus ATCC 9144 0Æ938 Ent. hirae ATCC 10541 0Æ938 C. albicans ATCC 10231 3Æ75 A. niger ATCC 16404 3Æ75
± ± ± ± ± ±
1Æ25 3Æ75 1Æ25 3Æ75 0Æ625 1Æ875 0Æ625 1Æ875 2Æ5 7Æ5 2Æ5 7Æ5
± ± ± ± ± ±
1Æ25 1Æ25 0Æ625 0Æ625 2Æ5 2Æ5
± ± ± ± ± ±
0Æ625 0Æ625 0Æ313 0Æ313 1Æ25 1Æ25
30 30 15 15 60 60
± ± ± ± ± ±
10 10 5 5 20 20
± ± ± ± ± ±
0Æ625 1Æ875 0Æ625 1Æ875 0Æ313 0Æ938 0Æ313 0Æ938 1Æ25 3Æ75 1Æ25 3Æ75
± ± ± ± ± ±
0Æ625 0Æ625 0Æ313 0Æ313 1Æ25 1Æ25
Values are mean ± SD of three repetitions.
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Pituranthos chloranthus essential oil as disinfectant
MICs from 1Æ875 to 7Æ5 mg l)1. As the values of MIC are low and equal to the values of MBC and MFC, essential oil effect is considered as a strong bactericidal and fungicidal. Compared with eugenol, P. chloranthus essential oil was slightly less effective. Comparing the means of MBC and MFC values of all the tested strains, P. chloranthus essential oils exhibited approximately two times lower and eight times stronger bactericidal and fungicidal effects than eugenol and essential oil of orange, respectively. Disinfecting property At 0Æ5% and 1% (w ⁄ v) concentrations, in dirty conditions and after 5 min of contact-time, P. chloranthus essential oils show a powerful disinfectant effect with a reduction in viability >5 and >4 log units of standard bacteria and fungi, respectively. Compared with the chemical eugenol and orange essential oils, P. chloranthus essential oils exhibit similar and much higher bactericidal effect than eugenol and orange, respectively. However, the disinfectant effect of the three compounds on fungi is practically identical and requires a higher concentration of 1% to reach the 4 log units of reduction recommended by the European norm. Discussion To the best of our knowledge, the antimicrobial activities and disinfectant properties of the Tunisian P. chloranthus essential oils have never been reported. Therefore, this work is the first report on the biological characteristics of this herb of arid-zone. Table 1 indicates that, P. chloran-
thus essential oils contain a high proportion of oxygenated monoterpenes. Among identified components in P. chloranthus essential oils, terpinen-4-ol is the major one (30Æ34%). This terpene is widely known as the major component of tea tree Melaleuca alternifolia Cheel (42Æ35%) and Alpinia zerumbet (28Æ09%) essential oils and mandarin juices (Lahlou et al. 2003; Pe´rez-Lo´pez et al. 2006). The antioxidant and DPPH radical-scavenging activities of P. chloranthus essential oils (Table 2) can be attributed to the presence of some components that have an antioxidant activity: terpinen-4-ol (Ruberto and Baratta 2000), myrtenol (Lee and Shibamoto 2001), p-menth-2-en-1-ol (Tuzun and Yegen 2000), 8-hydroxy-p-cymene (Bedoukian and Weldon 2007). Previous researches demonstrated that essential oils and especially oxygenated monoterpenes such as borneol, terpinene-4-ol, a-terpineol and menthol have an antimicrobial broad spectrum (Loughlin et al. 2008). In fact, the results of MIC, MBC and MFC determinations (Table 3) clearly showed that, P. chloranthus essential oils exert slightly lower inhibition effect than eugenol and orange essential oils against both standard bacteria and fungi. However, P. chloranthus essential oils and eugenol exhibited bactericidal and fungicidal effects as the (MBC or MFC) ⁄ MIC ratio is equal to one. As it was expected, MIC values were higher for fungi than for bacteria and Gram-positive are more sensitive than Gram-negative bacteria (Wilkinson et al. 2003). The reduction in viability results (Tables 4 and 5) showed a powerful antimicrobial effect of P. chloranthus essential oils against standard bacteria and fungi, suggesting
Table 4 Reduction in viability of standard bacteria after 5 min contact with Pituranthos chloranthus and orange essential oils and eugenol determined by EN 1276: 1997 standard method
Test procedure at concentration % (w ⁄ v)
Organism test P. chloranthus essential oils Pseudomonas aeruginosa ATCC 15442 Escherichia coli ATCC 10536 Staphylococcus aureus ATCC 6538 Enterococcus hirae ATCC 10541 Orange essential oils Ps. aeruginosa ATCC 15442 E. coli ATCC 10536 Staph. aureus ATCC 6538 Ent. hirae ATCC 10541 Eugenol Ps. aeruginosa ATCC 15442 E. coli ATCC 10536 Staph. aureus ATCC 6538 Ent. hirae ATCC 10541
N : bacterial test suspensions (CFU ml)1)
N N N N
: : : :
3Æ1 2Æ2 2Æ9 2Æ4
· · · ·
108 108 108 108
N N N N
: : : :
3Æ1 2Æ2 2Æ9 2Æ4
· · · ·
108 108 108 108
N N N N
: : : :
3Æ1 2Æ2 2Æ9 2Æ4
· · · ·
108 108 108 108
ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009) 112–117
Reduction in viability (CFU ml)1) 0Æ5
3Æ1 8Æ1 2Æ9 2Æ4
0Æ75
· · · ·
108 105 108 108
3Æ1 2Æ2 2Æ9 2Æ4
