- Email: [email protected]
In vitro antifungal and anti-elastase activity of some aliphatic aldehydes from Olea europaea L. fruit
ARTICLE IN PRESS
Phytomedicine 13 (2006) 558–563 www.elsevier.de/phymed
In vitro antifungal and anti-elastase activity of some aliphatic aldehydes from Olea europaea L. fruit L. Battinellia, C. Danielea, M. Cristianib, G. Bisignanob, A. Saijab, G. Mazzantia, a
Department of Human Physiology and Pharmacology, University ‘‘La Sapienza’’, P.le Aldo Moro 5, 00185 Rome, Italy Dipartimento Farmaco-Biologico, University of Messina, Contrada Annunziata, 98168 Messina, Italy
b
Received 1 March 2005; accepted 25 July 2005
Abstract Olea europaea preparations are traditionally employed in a variety of troubles, including skin infections. Olive extracts and some of their pure compounds have shown antimicrobial activity in vitro. The present study deals with the antifungal activity of some aliphatic aldehydes from olive fruit [hexanal, nonanal, (E)-2-hexenal, (E)-2-heptenal, (E)-2octenal, (E)-2-nonenal] against Tricophyton mentagrophytes (6 strains), Microsporum canis (1 strains) and Candida spp. (7 strains). The capability of these substances to inhibit elastase, a virulence factor essential for the dermatophytes colonization, and their cytotoxicity on cultures of reconstructed human epidermis, are also described. Aldehydes tested, inhibited the growth of T. mentagrophytes and M. canis in the range of concentration between o1.9 and 125 mg/ml; the unsaturated aldehydes showed the most broad spectrum of activity in that inhibited all strains tested. None of the aldehydes exhibited activity against Candida spp. strains. (E)-2-octenal and (E)-2-nonenal inhibited the elastase activity in a concentration-dependent manner; the anti-elastase activity suggests an additional target of the antimicrobial activity of these compounds. Aldehydes were devoid of cytotoxicity on cultures of human reconstructed epidermis. The antifungal activity of the aldehydes from olive fruit here reported, substantiates the use of olive and olive oil in skin diseases and suggests that these natural compounds could be useful agents in the topical treatment of fungal cutaneous infections. r 2005 Elsevier GmbH. All rights reserved. Keywords: Olea europaea; Antifungal activity; Dermatophytes; Candida spp.; Aliphatic aldehydes; Olive fruit
Introduction Olea europaea L. (Oleaceae) preparations have been used widely in folk medicine in European Mediterranean area, Arabia peninsula, India and other tropical and subtropical regions, as diuretic, hypotensive, Corresponding author. Tel.: +39 0649912903; fax: +39 0649912480. E-mail address: [email protected] (G. Mazzanti).
0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2005.09.009
emollient and for urinary and bladder infections (Samova et al., 2003); they are also employed in the treatment of skin diseases (Elkhalifa, 2002). In a recent open pilot study olive oil mixed with honey and beeswax showed to be effective, after topical application, in the treatment of skin fungal infections; clinical response was obtained in 86% of patients with Pityriasis versicolor, 78% of patients with Tinea cruris and in 75% of patients with Tinea corporis (Al-Waili, 2004). The same preparation resulted also effective in
ARTICLE IN PRESS L. Battinelli et al. / Phytomedicine 13 (2006) 558–563
reducing the symptoms of diaper dermatitis and eradicated Candida albicans from 50% of culturepositive patients during a 7-day trial (Al-Waili, 2005). Several investigations deal with the ability of O. europaea extracts or their pure components to inhibit or delay the growth of microorganisms. An olive leaf water extract was tested against bacteria and fungi: the extract killed almost all bacteria while dermatophytes were inhibited following a 3-day exposure and C. albicans was killed following a 24 h (Markin et al., 2003). Oleuropein and hydroxytyrosol, secoiridoides contained in olive and olive oil, showed antimicrobial activity on ATCC and clinical isolated bacteria responsible for intestinal or respiratory tract infections in man. In particular, oleuropein inhibited the growth of Salmonella spp., Vibrio spp. and Staphylococcus aureus with minimum inhibitory concentration (MIC) between 62.5 and 125 mg/ml for ATCC strains and between 31.25 and 250 mg/ml for clinical isolates. Hydroxytyrosol, derived from oleuropein by enzymatic hydrolysis, showed a more broad spectrum and a higher potency in that inhibited also Haemophilus influenzae and Moraxella catharralis; its MIC values were between 0.24 and 7.85 mg/ml for ATCC strains and between 0.97 and 31.25 mg/ml for clinical isolates (Bisignano et al., 1999). Furthermore, oleuropein showed activity against several species of Mycoplasma (Furneri et al., 2002). Noteworthy is the activity of some aldehydes, volatile flavor components of olive fruit and oil, against different fungal and bacterial strains. Kubo et al. (1995) described the antimicrobial activity of long chain saturated and unsaturated aldehydes from olive fruit against a broad spectrum of food-borne microfungal and bacteria strains; among the microorganisms tested, fungi were the most sensitive. This activity is of particular interest since most of plant secondary metabolites show in general more potent activity against Gram-positive bacteria than against fungi. More recently, Bisignano et al. (2001) described the activity of some of these compounds against a number of standard bacteria strains that may be causal agents of human infections. The results obtained pointed out that unsaturated aldehydes have a broad antimicrobial spectrum and show similar activity against Grampositive and Gram-negative bacteria. It has been hypothesized that these phytochemicals act both on the plasmatic membrane, by perturbating its lipidic fraction, and on intracellular targets (Trombetta et al., 2002; Kubo et al., 2003). Finally, a, b-unsaturated aldehydes for their antimicrobial properties are considered to be involved in the resistance of olive to microbe and insect attack (Kubo and Hanke, 1985). The current study was focused on the antifungal activity of some aliphatic aldehydes from olive fruit against Tricophyton mentagrophytes and Microsporum
559
canis, dermatophytes responsible for infection of keratinized tissue, and Candida spp. All microorganisms used, were also examined for their capability of producing elastase, a serine proteinase enzyme that hydrolytically degrades elastin. Elastase together with other proteolytic enzymes, enables microorganisms to invade and subsequently disseminate through the Stratum corneum (Muhsin et al., 1997); for this reason the aliphatic aldehydes were also tested for their ability to inhibit elastase activity. Finally, the cytotoxicity of tested substances on cultures of reconstructed human epidermis (RHE) was evaluated to assess their tolerability after topical application.
Materials and methods Substances Hexanal (98%), nonanal (95%), (E)-2-hexenal (X98%), (E)-2-heptenal (97%), (E)-2-octenal (94%) and (E)-2-nonenal (97%) were obtained from Aldrich (Milan, Italy). Aldehydes were dissolved in dimethyl sulfoxide (DMSO). Sodium dodecyl sulfate (SDS), Phosphate buffer solution (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), MCDB 153 medium, Elastin-Congo red, Elastin and Pancreatic Porcin Elastase type II were obtained from Sigma (St. Louis, MO, USA), phenylmethylsulfonyl fluoride (PMFS), 98% pure, was obtained from Aldrich. All culture media were purchased from Oxoid (Milan, Italy) and Miconazole from ICN Biomedicals, INC. (Irvine, CA).
Microorganisms Experiments were performed using 6 T. mentagrophytes strains, 1 M. canis strain and 7 Candida strains (4 C. albicans, 2 C. glabrata, 1 C. krusei). All microorganisms used, were clinically isolated. The yeasts were maintained in Saboraud dextrose agar slants at room temperature and subcultured routinely. The dermatophytes were grown in Dermasel agar plus 0.5% yeast extract.
Susceptibility test The antimicrobial activity of the aldehydes from olive fruits was determined by a broth dilution assay. The experiments were carried out in Mueller-Hinton Broth using 96-well microtiter trays for yeasts; the tests for dermatophytes were performed in Triptone Soy Broth plus 5% of peptone, using tubes in order to avoid contamination. Several two-fold dilutions of aldehydes were carried out starting from the concentration of
ARTICLE IN PRESS 560
L. Battinelli et al. / Phytomedicine 13 (2006) 558–563
250 mg/ml (0.3% of DMSO). The wells were inoculated with suspensions of Candida spp. strains (103 cells/ml) and incubated for 48 h at 37 1C; the tubes were inoculated with dermatophytes (4 104 spores/ml) and incubated for 7 days at 37 1C; then the MIC was determined. To establish minimum fungicidal concentration (MFC) the cultures that did not present growth were used to inoculate plates of Sabouraud dextrose agar and incubated at 37 1C for 48 h or 7 days, respectively, for yeasts and dermatophytes. Miconazole, used as reference substance, was dissolved in DMSO then diluted in culture media to obtain concentrations ranging from 16 to 0.031 mg/ml. To test the effect of the solvent and the broth sterility, proper blanks were assayed simultaneously; the solvent, at the higher concentration used did not interfere with the growth of microorganisms. Miconazole was used as positive control. All samples were tested in triplicate.
Evaluation of elastase production Elastase production was examined by a qualitative assay using screening plates for elastase activity (Williams et al., 1988). Microorganism cultures were plated onto agar plates prepared with a nutrient agar base (8 g/l of Nutrient Broth and 5 g/l of Agar Noble in water) and an insoluble overlay of agar containing elastin (1.5 g of Agar Noble, 250 mg of elastin, 600 mg of Nutrient Broth in 75 ml of water). The plates were incubated at 37 1C for 48 h for yeasts and at 35 1C for 6 days for dermatophytes. The overlay contained visible particulate elastin; the elastinolitic activity was indicated by zones of elastin clearing.
Elastase activity assay Elastase activity was detected by a procedure described by Naughton and Sanger (1961) with slight modifications. Briefly, 12.5 mg of Elastin-Congo red were put in 10 ml tubes containing 5 ml of PBS. After equilibration of the reaction temperature at 37 1C the solution containing elastase was added, then the tubes were closed and incubated horizontally at 37 1C under rapid shaking. After 2 h, the tubes were placed in an ice water bath. After centrifugation at 4000 rpm the nonhydrolyzed substrate was removed by membrane filtration using 0.45 mm filter, then the absorbance of the samples was determined at 495 nm. To evaluate the inhibitory effect of the aldehydes, the compounds were added before the enzyme solution. PMFS was used as reference protease inhibitor (positive control).
Cytotoxicity on reconstructed human epidermis Cultures of RHE, purchased from SkinEthic Laboratories (Nice, France), were used to evaluate the
cytotoxicity of the aldehydes under study following application on the epidermis. RHE is obtained by culturing normal human keratinocytes in a modified and chemically defined MCDB 153 medium on Millipore polycarbonate culture inserts lifted to the air–liquid interface for 16 days (Rosdy et al., 1993; Gazel et al., 2003). This in vitro 3-D human skin model has been shown capable of identifying toxic potential of both soluble and insoluble compounds; moreover it allows the application of test ingredients and/or formulations to the air-exposed surface, thus mimicking the in vivo situation. The cultures (0.63 cm2) of RHE were shipped to the laboratory in 24-well plates on sponges containing the nutrient medium. At their arrival, the sponges were removed and replaced by 0.5 ml of MCDB 153 medium. The RHE cultures were incubated for 24 h at 37 1C (5% CO2) before testing, according to the supplier’s specifications. The aldehydes were diluted in water containing 0.06% of ethanol at the concentration of 250 and 500 mg/ml. A volume of 0.5 ml of fresh medium was placed under each insert and 100 ml of the diluted aldehydes were applied directly on the Stratum corneum of the RHE. Cultures were also treated with 100 ml of SDS 20% w/v (positive control) or of the medium alone (negative control). After an incubation for 24/48 h at 37 1C (5% CO2), the RHE cultures were washed twice with 1 ml of PBS at pH 7.4 and tested for cytotoxicity by MTT assay (Mosmann, 1983). For this purpose the cultures were transferred to 24-well plates containing 0.3 ml of MTT solution (0.5 mg/ml dissolved in culture medium) per well and incubated for 3 h at 37 1C (5% CO2). Cultures were then washed with 1 ml of PBS at pH 7.4 and the formazan was extracted by shaking for 2 h with 2 ml of isopropanol at room temperature. Aliquots (200 ml) were transferred from each well to corresponding well of a 96-well plate than the optical density (OD) at 570 nm was determined spectrophotometrically using a Biorad microplate reader (Biorad, Hercules, California). The mean OD570 of the untreated control tissues exposed to culture medium was set to represent 100% of viability and the results were expressed as percentage of the controls.
Results Antifungal activity In our experiments the aldehydes investigated showed a different spectrum of antimicrobial activity (Table 1); in particular hexanal and nonanal inhibited the growth of T. mentagrophytes 7 (MIC 3.9 mg/ml and 15.6 mg/ml respectively) and T. mentagrophytes 61 (MIC 125 mg/ml and o1.9 mg/ml, respectively). Nonanal was also active against T. mentagrophytes 13 and M. canis 70 at the
ARTICLE IN PRESS L. Battinelli et al. / Phytomedicine 13 (2006) 558–563
Table 1.
561
Antifungal activity of saturated and unsaturated aliphatic aldehydes from olive fruit: MICa (mg/ml)
Microorganism
Hexanal
Nonanal
E-2-hexenal
E-2-heptenal
E-2-octenal
E-2-nonenal
Miconazole
Tmb 7 Tm 13 Tm 46 Tm 52 Tm 61 Tm 62 Mcc 70
3.9 — — — 125 — —
15.6 o1.9 — — o1.9 — 3.9
15.6 62.5 62.5 62.5 31.2 62.5 62.5
o1.9 o1.9 125 125 o1.9 125 125
15.6 15.6 125 125 o1.9 62.5 62.5
62.5 o1.9 31.2 62.5 250 62.5 15.6
0.5 4 0.5 1 1 0.5 0.5
— No effect at 250 mg/ml. a MIC, minimum inhibitory concentration. b Tm, T. mentagrophytes. c Mc, M. canis.
Elastase production and elastase activity The qualitative assay for detection of elastase activity on plate showed that all dermatophytes displayed elastinolytic activity; on the contrary Candida spp. strains were not able to do so. The colorimetric quantitative assay for elastase activity, performed using an enzyme of porcine origin, revealed that the unsaturated aldehydes (E)-2-octenal and (E)-2-nonenal inhibited the elastase activity in a concentration-dependent manner (Fig. 1), while all the other aldehydes were inactive. The positive control PMFS, at the concentration of 3 10 6 M, gave an inhibition of the enzyme activity higher than 90%.
Cytotoxicity on reconstructed human epidermis The evaluation of the cytotoxicity of the aldehydes under study, following topical application on the epidermis, showed that at the maximum concentration tested of 500 mg/ml, the average percentage of surviving cells was higher than 95%. In the same condition SDS 20% w/v, used as positive control, gave a percentage of surviving cells of 2% whereas with the medium alone the percentage was 98%.
Discussion The present study was aimed to assess the antifungal activity of some aldehydes from O. europaea, namely
100
80
Inhibition (%)
concentrations of o1.9 and of 3.9 mg/ml, respectively. The other aldehydes inhibited all the dermatophytes tested with MIC between o1.9 and 125 mg/ml. The antimicrobial activity observed was always cytocidal and the MFC values were the same of the MIC ones. None of the aldehydes under study exhibited activity against Candida spp. strains.
60
40 (E) -2- OCTENAL (E) -2-NONENAL 20
0 0.00
0.01
0.02
0.03
0.04
0.05
Concentration (M)
Fig. 1. Inhibition of pancreatic porcine elastase activity by aliphatic aldehydes from olive fruits. The elastase activity was evaluated by the capability of the enzyme to hydrolyze the Elastine-Congo red and was determined by measuring the absorbance (495 nm) of the sample containing the hydrolized substrate. Values are means7SE of 3 replicates.
hexanal and nonanal (saturated), (E)-2-hexenal, (E)-2heptenal, (E)-2-octenal and (E)-2-nonenal (unsaturated). The aldehydes from olive exhibited a significant antifungal activity against Tricophyton mentagrophytes and Microsporum canis strains. The unsaturated ones showed to be active against all dermatophytes tested with MIC starting from o1.9 mg/ml; particularly (E)-2hexenal and (E)-2-nonenal showed a more broad spectrum than the corresponding saturated compound hexanal and nonanal. These data confirm previous results by Bisignano et al. (1999) who asserted that the presence of a double bond in the molecule is important for the antimicrobial activity of olive aldehydes. The substances under study showed to inhibit also the elastase activity. In general, the organisms characterized by the ability to induce marked inflammatory skin
ARTICLE IN PRESS 562
L. Battinelli et al. / Phytomedicine 13 (2006) 558–563
infections are those producing elastase (Rippon and Varadi, 1968). Elastase is a virulence factor for dermatophytes and other microorganisms; it permits skin colonization by hydrolizing elastin then allowing a better tissue penetration and spreading of infection. It is noteworthy that all dermatophytes tested in this work produce elastase. The inhibition of the enzyme seems to depend both on the presence of a double bond and on the length of the alkylic chain; in fact only the unsaturated long chain aldehydes (E)-2-octenal and (E)-2-nonenal inhibited the enzyme. On the whole, present findings allow us to speculate that olive aldehydes tested could act by different mechanisms: by inhibiting the fungal replication and/ or by inhibiting elastase, thus reducing the dermatophytes colonization. The data on RHE also suggest that a low cutaneous toxicity may be elicited by topical application of these biomolecules. The antifungal activity of aldehydes may also contribute to explain the antimicrobial properties of olive preparations; aldehydes infact represent the major flavor components in olive leaves, fruit and oil: their content in olives is between 50% and 75% (Montedoro et al., 1978) and in leaves it varies from 40% to 60% (Campeol et al., 2003). As regards the single aldehydes, the content of (E)-2-hexenal for instance, can reach 438 mg/kg in the olive fruit (Ranalli and Ferante, 1996) and 10574 mg/kg in olive oil (Guth and Grosch, 1993): this concentration in olive oil (about 10 mg/ml) is similar to the MIC values (15.6–62.5 mg/ml) obtained in our experiments with this substance. A direct determination of the aldehydes concentration in olive leaf aqueous extracts has not been performed at our knowledge, but Flamini et al., (2003), who analyzed comparatively the composition of volatiles obtained from O. europaea leaves, fruits and virgin oil, found in leaves a percentage of (E)-2-hexenal about four times higher than in the oil. Moreover, besides their particular antimicrobial activity, aldehydes when mixed all together, as happens in olive extracts and olive oil, may act synergistically producing an increase in the antimicrobial activity of the whole product. In this connection our previous studies showed that when (E)-2-heptenal, (E)-2-nonenal, (E)-2decenal and (E,E)-2,4-decadienal were tested together (ratio 1:1:1:1) against ATCC and clinically isolated microbial strains, the MIC values were considerably lower than those obtained when the same aldehydes were tested alone (Bisignano et al., 2001). On the basis of these observations a synergistic effect between the aldehydes is probable; similarly a possible synergistic effect between the aldehydes and the secoiridoides, oleuropein and hydroxytyrosol, has to be taken in account. In conclusion, the antifungal activity here reported, along with the antibacterial activity of aldehydes and secoiridoides from olive fruit and oil previously de-
scribed, give a scientific support to the traditional use of O. europaea preparations in skin diseases. Only few substances are known to inhibit human pathogenic fungi, which are often completely resistant to antibiotics, and most of them are relatively toxic. The increased incidence of therapeutic failure in the treatment of fungal infections and the prevalence of opportunistic infections has renewed interest in the search for new antifungal agents, including those obtained from higher plants. Present results allow to suppose that these natural compounds could be useful agents in the topical treatment of fungal infections.
Acknowledgments Dr. Lucia Battinelli was supported by the ‘‘Enrico and Enrica Sovena’’ Foundation (Italy).
References Al-Waili, N.S., 2004. An alternative treatment for pityriasis versicolor, tinea cruris, tinea corporis and tinea faciei with topical application of honey, olive oil and beeswax mixture: an open pilot study. Complement. Ther. Med. 12 (1), 45–47. Al-Waili, N.S., 2005. Clinical and mycological benefits of topical application of honey, olive oil and beeswax in diaper dermatitis. Clin. Microbiol. Infect. 11 (2), 160–163. Bisignano, G., Tomaino, A., Lo Cascio, R., Crisafi, G., Uccella, N., Saija, A., 1999. On the in vitro antimicrobial activity of oleuropein and hydroxytyrosol. J. Pharm. Pharmacol. 51 (8), 971–974. Bisignano, G., Lagana`, M.G., Trombetta, D., Arena, S., Nostro, A., Uccella, N., Mazzanti, G., Saija, A., 2001. In vitro antibacterial activity of some aliphatic aldehydes from Olea europaea L. FEMS Microbiol. Lett. 198, 9–13. Campeol, E., Flamini, G., Cioni, P.L., Morelli, I., 2003. Volatile fractions from three cultivars of Olea europea L. collected in two different seasons. J. Agric. Food Chem. 51, 1994–1999. Elkhalifa, K.F., 2002. Treatment of skin diseases in the Sudan using tree materials. Arab. Gulf J. Sci. Res. 20 (4), 232–235. Flamini, G., Cioni, P.L., Morelli, I., 2003. Volatile from leaves, fruits, and virgin oil from Olea europea cv. Olivastra Seggianese from Italy. J. Agric. Food Chem. 51, 1382–1386. Furneri, P.M., Marino, A., Saija, A., Uccella, N., Bisignano, G., 2002. In vitro antimycoplasmal activity of oleuropein. Int. J. Antimicrob. Agents 20 (4), 293–296. Gazel, A., Ramphal, P., Rosdy, M., De Wever, B., Tornier, C., Hosein, N., Lee, B., Tomic-Canic, M., Blumenberg, M., 2003. Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays. J. Invest. Dermatol. 121 (6), 1459–1468. Guth, H., Grosch, W., 1993. Quantitation of potent odorants of virgin olive oil by stable-isotope dilution assays. J. Am. Oil Chem. Soc. 70, 513–518.
ARTICLE IN PRESS L. Battinelli et al. / Phytomedicine 13 (2006) 558–563
Kubo, I., Hanke, F.J., 1985. Multifaceted chemically based resistance in plants. In: Conn, E. (Ed.), Recent Advances in Phytochemistry, Chemically Mediated Interactions between Plants and Other Organisms, Vol. 19. Plenum, New York, pp. 171–194. Kubo, A., Lunde, S., Kubo, I., 1995. Antimicrobial activity of the Olive oil flavor compounds. J. Agric. Food Chem. 43, 1629–1633. Kubo, I., Fujita, K., Kubo, A., Nihei, K., Lunde, C.S., 2003. Modes of antifungal action of (2E)-alkenals against Saccharomyces cerevisiae. J. Agric. Food Chem. 51 (14), 3951–3957. Markin, D., Duek, L., Berdicevsky, I., 2003. In vitro antimicrobial activity of olive leaves. Mycoses 46, 132–136. Montedoro, G., Bertuccioli, M., Anichini, F., 1978. Aroma analysis of virgin olive oil by head space volatile extraction techniques. In: Charalampous, G., Inglet, G. (Eds.), Flavor of Foods and Beverages. Academic Press, New York, pp. 247–281. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Muhsin, T.M., Aubaid, A.H., Al-Duboon, A.H., 1997. Extracellular enzyme activity of dermatophytes and yeasts on solid media. Mycoses 40, 465–469.
563
Naughton, M.A., Sanger, F., 1961. Purification and specificity of pancreatic elastase. Biochem. J. 78, 156–163. Ranalli, A., Ferante, M.L., 1996. Physico-chemical and analytical characteristics of extra virgin olive oils extracted by using a pectolytic enzymatic processing aid. Olivae 60, 27–32. Rippon, J.W., Varadi, D.P., 1968. The elastases of pathogenic fungi and actinomycetes. J. Invest. Dermatol. 50, 54–57. Rosdy, M., Pisani, A., Ortonne, J.P., 1993. Production of basement membrane components by a reconstructed epidermis cultured in the absence of serum and dermal factors. Br. J. Dermatol. 129 (3), 227–234. Samova, L.I., Shode, F.O., Ramnanan, P., Nadar, A., 2003. Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies africana leaves. J. Ethnopharmacol. 84, 299–305. Trombetta, D., Saija, A., Bisignano, G., Arena, S., Caruso, S., Mazzanti, G., Uccella, N., Castelli, F., 2002. Study on the mechanisms of the antibacterial action of some plant a,bunsaturated aldehydes. Lett. Appl. Microbiol. 35 (4), 285–290. Williams, K., Phillips, K.D., Willis, A.T., 1988. A simple and sensitive method for detecting bacterial elastase production. Lett. Appl. Microbiol. 7, 173–176.
Phytomedicine 13 (2006) 558–563 www.elsevier.de/phymed
In vitro antifungal and anti-elastase activity of some aliphatic aldehydes from Olea europaea L. fruit L. Battinellia, C. Danielea, M. Cristianib, G. Bisignanob, A. Saijab, G. Mazzantia, a
Department of Human Physiology and Pharmacology, University ‘‘La Sapienza’’, P.le Aldo Moro 5, 00185 Rome, Italy Dipartimento Farmaco-Biologico, University of Messina, Contrada Annunziata, 98168 Messina, Italy
b
Received 1 March 2005; accepted 25 July 2005
Abstract Olea europaea preparations are traditionally employed in a variety of troubles, including skin infections. Olive extracts and some of their pure compounds have shown antimicrobial activity in vitro. The present study deals with the antifungal activity of some aliphatic aldehydes from olive fruit [hexanal, nonanal, (E)-2-hexenal, (E)-2-heptenal, (E)-2octenal, (E)-2-nonenal] against Tricophyton mentagrophytes (6 strains), Microsporum canis (1 strains) and Candida spp. (7 strains). The capability of these substances to inhibit elastase, a virulence factor essential for the dermatophytes colonization, and their cytotoxicity on cultures of reconstructed human epidermis, are also described. Aldehydes tested, inhibited the growth of T. mentagrophytes and M. canis in the range of concentration between o1.9 and 125 mg/ml; the unsaturated aldehydes showed the most broad spectrum of activity in that inhibited all strains tested. None of the aldehydes exhibited activity against Candida spp. strains. (E)-2-octenal and (E)-2-nonenal inhibited the elastase activity in a concentration-dependent manner; the anti-elastase activity suggests an additional target of the antimicrobial activity of these compounds. Aldehydes were devoid of cytotoxicity on cultures of human reconstructed epidermis. The antifungal activity of the aldehydes from olive fruit here reported, substantiates the use of olive and olive oil in skin diseases and suggests that these natural compounds could be useful agents in the topical treatment of fungal cutaneous infections. r 2005 Elsevier GmbH. All rights reserved. Keywords: Olea europaea; Antifungal activity; Dermatophytes; Candida spp.; Aliphatic aldehydes; Olive fruit
Introduction Olea europaea L. (Oleaceae) preparations have been used widely in folk medicine in European Mediterranean area, Arabia peninsula, India and other tropical and subtropical regions, as diuretic, hypotensive, Corresponding author. Tel.: +39 0649912903; fax: +39 0649912480. E-mail address: [email protected] (G. Mazzanti).
0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2005.09.009
emollient and for urinary and bladder infections (Samova et al., 2003); they are also employed in the treatment of skin diseases (Elkhalifa, 2002). In a recent open pilot study olive oil mixed with honey and beeswax showed to be effective, after topical application, in the treatment of skin fungal infections; clinical response was obtained in 86% of patients with Pityriasis versicolor, 78% of patients with Tinea cruris and in 75% of patients with Tinea corporis (Al-Waili, 2004). The same preparation resulted also effective in
ARTICLE IN PRESS L. Battinelli et al. / Phytomedicine 13 (2006) 558–563
reducing the symptoms of diaper dermatitis and eradicated Candida albicans from 50% of culturepositive patients during a 7-day trial (Al-Waili, 2005). Several investigations deal with the ability of O. europaea extracts or their pure components to inhibit or delay the growth of microorganisms. An olive leaf water extract was tested against bacteria and fungi: the extract killed almost all bacteria while dermatophytes were inhibited following a 3-day exposure and C. albicans was killed following a 24 h (Markin et al., 2003). Oleuropein and hydroxytyrosol, secoiridoides contained in olive and olive oil, showed antimicrobial activity on ATCC and clinical isolated bacteria responsible for intestinal or respiratory tract infections in man. In particular, oleuropein inhibited the growth of Salmonella spp., Vibrio spp. and Staphylococcus aureus with minimum inhibitory concentration (MIC) between 62.5 and 125 mg/ml for ATCC strains and between 31.25 and 250 mg/ml for clinical isolates. Hydroxytyrosol, derived from oleuropein by enzymatic hydrolysis, showed a more broad spectrum and a higher potency in that inhibited also Haemophilus influenzae and Moraxella catharralis; its MIC values were between 0.24 and 7.85 mg/ml for ATCC strains and between 0.97 and 31.25 mg/ml for clinical isolates (Bisignano et al., 1999). Furthermore, oleuropein showed activity against several species of Mycoplasma (Furneri et al., 2002). Noteworthy is the activity of some aldehydes, volatile flavor components of olive fruit and oil, against different fungal and bacterial strains. Kubo et al. (1995) described the antimicrobial activity of long chain saturated and unsaturated aldehydes from olive fruit against a broad spectrum of food-borne microfungal and bacteria strains; among the microorganisms tested, fungi were the most sensitive. This activity is of particular interest since most of plant secondary metabolites show in general more potent activity against Gram-positive bacteria than against fungi. More recently, Bisignano et al. (2001) described the activity of some of these compounds against a number of standard bacteria strains that may be causal agents of human infections. The results obtained pointed out that unsaturated aldehydes have a broad antimicrobial spectrum and show similar activity against Grampositive and Gram-negative bacteria. It has been hypothesized that these phytochemicals act both on the plasmatic membrane, by perturbating its lipidic fraction, and on intracellular targets (Trombetta et al., 2002; Kubo et al., 2003). Finally, a, b-unsaturated aldehydes for their antimicrobial properties are considered to be involved in the resistance of olive to microbe and insect attack (Kubo and Hanke, 1985). The current study was focused on the antifungal activity of some aliphatic aldehydes from olive fruit against Tricophyton mentagrophytes and Microsporum
559
canis, dermatophytes responsible for infection of keratinized tissue, and Candida spp. All microorganisms used, were also examined for their capability of producing elastase, a serine proteinase enzyme that hydrolytically degrades elastin. Elastase together with other proteolytic enzymes, enables microorganisms to invade and subsequently disseminate through the Stratum corneum (Muhsin et al., 1997); for this reason the aliphatic aldehydes were also tested for their ability to inhibit elastase activity. Finally, the cytotoxicity of tested substances on cultures of reconstructed human epidermis (RHE) was evaluated to assess their tolerability after topical application.
Materials and methods Substances Hexanal (98%), nonanal (95%), (E)-2-hexenal (X98%), (E)-2-heptenal (97%), (E)-2-octenal (94%) and (E)-2-nonenal (97%) were obtained from Aldrich (Milan, Italy). Aldehydes were dissolved in dimethyl sulfoxide (DMSO). Sodium dodecyl sulfate (SDS), Phosphate buffer solution (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), MCDB 153 medium, Elastin-Congo red, Elastin and Pancreatic Porcin Elastase type II were obtained from Sigma (St. Louis, MO, USA), phenylmethylsulfonyl fluoride (PMFS), 98% pure, was obtained from Aldrich. All culture media were purchased from Oxoid (Milan, Italy) and Miconazole from ICN Biomedicals, INC. (Irvine, CA).
Microorganisms Experiments were performed using 6 T. mentagrophytes strains, 1 M. canis strain and 7 Candida strains (4 C. albicans, 2 C. glabrata, 1 C. krusei). All microorganisms used, were clinically isolated. The yeasts were maintained in Saboraud dextrose agar slants at room temperature and subcultured routinely. The dermatophytes were grown in Dermasel agar plus 0.5% yeast extract.
Susceptibility test The antimicrobial activity of the aldehydes from olive fruits was determined by a broth dilution assay. The experiments were carried out in Mueller-Hinton Broth using 96-well microtiter trays for yeasts; the tests for dermatophytes were performed in Triptone Soy Broth plus 5% of peptone, using tubes in order to avoid contamination. Several two-fold dilutions of aldehydes were carried out starting from the concentration of
ARTICLE IN PRESS 560
L. Battinelli et al. / Phytomedicine 13 (2006) 558–563
250 mg/ml (0.3% of DMSO). The wells were inoculated with suspensions of Candida spp. strains (103 cells/ml) and incubated for 48 h at 37 1C; the tubes were inoculated with dermatophytes (4 104 spores/ml) and incubated for 7 days at 37 1C; then the MIC was determined. To establish minimum fungicidal concentration (MFC) the cultures that did not present growth were used to inoculate plates of Sabouraud dextrose agar and incubated at 37 1C for 48 h or 7 days, respectively, for yeasts and dermatophytes. Miconazole, used as reference substance, was dissolved in DMSO then diluted in culture media to obtain concentrations ranging from 16 to 0.031 mg/ml. To test the effect of the solvent and the broth sterility, proper blanks were assayed simultaneously; the solvent, at the higher concentration used did not interfere with the growth of microorganisms. Miconazole was used as positive control. All samples were tested in triplicate.
Evaluation of elastase production Elastase production was examined by a qualitative assay using screening plates for elastase activity (Williams et al., 1988). Microorganism cultures were plated onto agar plates prepared with a nutrient agar base (8 g/l of Nutrient Broth and 5 g/l of Agar Noble in water) and an insoluble overlay of agar containing elastin (1.5 g of Agar Noble, 250 mg of elastin, 600 mg of Nutrient Broth in 75 ml of water). The plates were incubated at 37 1C for 48 h for yeasts and at 35 1C for 6 days for dermatophytes. The overlay contained visible particulate elastin; the elastinolitic activity was indicated by zones of elastin clearing.
Elastase activity assay Elastase activity was detected by a procedure described by Naughton and Sanger (1961) with slight modifications. Briefly, 12.5 mg of Elastin-Congo red were put in 10 ml tubes containing 5 ml of PBS. After equilibration of the reaction temperature at 37 1C the solution containing elastase was added, then the tubes were closed and incubated horizontally at 37 1C under rapid shaking. After 2 h, the tubes were placed in an ice water bath. After centrifugation at 4000 rpm the nonhydrolyzed substrate was removed by membrane filtration using 0.45 mm filter, then the absorbance of the samples was determined at 495 nm. To evaluate the inhibitory effect of the aldehydes, the compounds were added before the enzyme solution. PMFS was used as reference protease inhibitor (positive control).
Cytotoxicity on reconstructed human epidermis Cultures of RHE, purchased from SkinEthic Laboratories (Nice, France), were used to evaluate the
cytotoxicity of the aldehydes under study following application on the epidermis. RHE is obtained by culturing normal human keratinocytes in a modified and chemically defined MCDB 153 medium on Millipore polycarbonate culture inserts lifted to the air–liquid interface for 16 days (Rosdy et al., 1993; Gazel et al., 2003). This in vitro 3-D human skin model has been shown capable of identifying toxic potential of both soluble and insoluble compounds; moreover it allows the application of test ingredients and/or formulations to the air-exposed surface, thus mimicking the in vivo situation. The cultures (0.63 cm2) of RHE were shipped to the laboratory in 24-well plates on sponges containing the nutrient medium. At their arrival, the sponges were removed and replaced by 0.5 ml of MCDB 153 medium. The RHE cultures were incubated for 24 h at 37 1C (5% CO2) before testing, according to the supplier’s specifications. The aldehydes were diluted in water containing 0.06% of ethanol at the concentration of 250 and 500 mg/ml. A volume of 0.5 ml of fresh medium was placed under each insert and 100 ml of the diluted aldehydes were applied directly on the Stratum corneum of the RHE. Cultures were also treated with 100 ml of SDS 20% w/v (positive control) or of the medium alone (negative control). After an incubation for 24/48 h at 37 1C (5% CO2), the RHE cultures were washed twice with 1 ml of PBS at pH 7.4 and tested for cytotoxicity by MTT assay (Mosmann, 1983). For this purpose the cultures were transferred to 24-well plates containing 0.3 ml of MTT solution (0.5 mg/ml dissolved in culture medium) per well and incubated for 3 h at 37 1C (5% CO2). Cultures were then washed with 1 ml of PBS at pH 7.4 and the formazan was extracted by shaking for 2 h with 2 ml of isopropanol at room temperature. Aliquots (200 ml) were transferred from each well to corresponding well of a 96-well plate than the optical density (OD) at 570 nm was determined spectrophotometrically using a Biorad microplate reader (Biorad, Hercules, California). The mean OD570 of the untreated control tissues exposed to culture medium was set to represent 100% of viability and the results were expressed as percentage of the controls.
Results Antifungal activity In our experiments the aldehydes investigated showed a different spectrum of antimicrobial activity (Table 1); in particular hexanal and nonanal inhibited the growth of T. mentagrophytes 7 (MIC 3.9 mg/ml and 15.6 mg/ml respectively) and T. mentagrophytes 61 (MIC 125 mg/ml and o1.9 mg/ml, respectively). Nonanal was also active against T. mentagrophytes 13 and M. canis 70 at the
ARTICLE IN PRESS L. Battinelli et al. / Phytomedicine 13 (2006) 558–563
Table 1.
561
Antifungal activity of saturated and unsaturated aliphatic aldehydes from olive fruit: MICa (mg/ml)
Microorganism
Hexanal
Nonanal
E-2-hexenal
E-2-heptenal
E-2-octenal
E-2-nonenal
Miconazole
Tmb 7 Tm 13 Tm 46 Tm 52 Tm 61 Tm 62 Mcc 70
3.9 — — — 125 — —
15.6 o1.9 — — o1.9 — 3.9
15.6 62.5 62.5 62.5 31.2 62.5 62.5
o1.9 o1.9 125 125 o1.9 125 125
15.6 15.6 125 125 o1.9 62.5 62.5
62.5 o1.9 31.2 62.5 250 62.5 15.6
0.5 4 0.5 1 1 0.5 0.5
— No effect at 250 mg/ml. a MIC, minimum inhibitory concentration. b Tm, T. mentagrophytes. c Mc, M. canis.
Elastase production and elastase activity The qualitative assay for detection of elastase activity on plate showed that all dermatophytes displayed elastinolytic activity; on the contrary Candida spp. strains were not able to do so. The colorimetric quantitative assay for elastase activity, performed using an enzyme of porcine origin, revealed that the unsaturated aldehydes (E)-2-octenal and (E)-2-nonenal inhibited the elastase activity in a concentration-dependent manner (Fig. 1), while all the other aldehydes were inactive. The positive control PMFS, at the concentration of 3 10 6 M, gave an inhibition of the enzyme activity higher than 90%.
Cytotoxicity on reconstructed human epidermis The evaluation of the cytotoxicity of the aldehydes under study, following topical application on the epidermis, showed that at the maximum concentration tested of 500 mg/ml, the average percentage of surviving cells was higher than 95%. In the same condition SDS 20% w/v, used as positive control, gave a percentage of surviving cells of 2% whereas with the medium alone the percentage was 98%.
Discussion The present study was aimed to assess the antifungal activity of some aldehydes from O. europaea, namely
100
80
Inhibition (%)
concentrations of o1.9 and of 3.9 mg/ml, respectively. The other aldehydes inhibited all the dermatophytes tested with MIC between o1.9 and 125 mg/ml. The antimicrobial activity observed was always cytocidal and the MFC values were the same of the MIC ones. None of the aldehydes under study exhibited activity against Candida spp. strains.
60
40 (E) -2- OCTENAL (E) -2-NONENAL 20
0 0.00
0.01
0.02
0.03
0.04
0.05
Concentration (M)
Fig. 1. Inhibition of pancreatic porcine elastase activity by aliphatic aldehydes from olive fruits. The elastase activity was evaluated by the capability of the enzyme to hydrolyze the Elastine-Congo red and was determined by measuring the absorbance (495 nm) of the sample containing the hydrolized substrate. Values are means7SE of 3 replicates.
hexanal and nonanal (saturated), (E)-2-hexenal, (E)-2heptenal, (E)-2-octenal and (E)-2-nonenal (unsaturated). The aldehydes from olive exhibited a significant antifungal activity against Tricophyton mentagrophytes and Microsporum canis strains. The unsaturated ones showed to be active against all dermatophytes tested with MIC starting from o1.9 mg/ml; particularly (E)-2hexenal and (E)-2-nonenal showed a more broad spectrum than the corresponding saturated compound hexanal and nonanal. These data confirm previous results by Bisignano et al. (1999) who asserted that the presence of a double bond in the molecule is important for the antimicrobial activity of olive aldehydes. The substances under study showed to inhibit also the elastase activity. In general, the organisms characterized by the ability to induce marked inflammatory skin
ARTICLE IN PRESS 562
L. Battinelli et al. / Phytomedicine 13 (2006) 558–563
infections are those producing elastase (Rippon and Varadi, 1968). Elastase is a virulence factor for dermatophytes and other microorganisms; it permits skin colonization by hydrolizing elastin then allowing a better tissue penetration and spreading of infection. It is noteworthy that all dermatophytes tested in this work produce elastase. The inhibition of the enzyme seems to depend both on the presence of a double bond and on the length of the alkylic chain; in fact only the unsaturated long chain aldehydes (E)-2-octenal and (E)-2-nonenal inhibited the enzyme. On the whole, present findings allow us to speculate that olive aldehydes tested could act by different mechanisms: by inhibiting the fungal replication and/ or by inhibiting elastase, thus reducing the dermatophytes colonization. The data on RHE also suggest that a low cutaneous toxicity may be elicited by topical application of these biomolecules. The antifungal activity of aldehydes may also contribute to explain the antimicrobial properties of olive preparations; aldehydes infact represent the major flavor components in olive leaves, fruit and oil: their content in olives is between 50% and 75% (Montedoro et al., 1978) and in leaves it varies from 40% to 60% (Campeol et al., 2003). As regards the single aldehydes, the content of (E)-2-hexenal for instance, can reach 438 mg/kg in the olive fruit (Ranalli and Ferante, 1996) and 10574 mg/kg in olive oil (Guth and Grosch, 1993): this concentration in olive oil (about 10 mg/ml) is similar to the MIC values (15.6–62.5 mg/ml) obtained in our experiments with this substance. A direct determination of the aldehydes concentration in olive leaf aqueous extracts has not been performed at our knowledge, but Flamini et al., (2003), who analyzed comparatively the composition of volatiles obtained from O. europaea leaves, fruits and virgin oil, found in leaves a percentage of (E)-2-hexenal about four times higher than in the oil. Moreover, besides their particular antimicrobial activity, aldehydes when mixed all together, as happens in olive extracts and olive oil, may act synergistically producing an increase in the antimicrobial activity of the whole product. In this connection our previous studies showed that when (E)-2-heptenal, (E)-2-nonenal, (E)-2decenal and (E,E)-2,4-decadienal were tested together (ratio 1:1:1:1) against ATCC and clinically isolated microbial strains, the MIC values were considerably lower than those obtained when the same aldehydes were tested alone (Bisignano et al., 2001). On the basis of these observations a synergistic effect between the aldehydes is probable; similarly a possible synergistic effect between the aldehydes and the secoiridoides, oleuropein and hydroxytyrosol, has to be taken in account. In conclusion, the antifungal activity here reported, along with the antibacterial activity of aldehydes and secoiridoides from olive fruit and oil previously de-
scribed, give a scientific support to the traditional use of O. europaea preparations in skin diseases. Only few substances are known to inhibit human pathogenic fungi, which are often completely resistant to antibiotics, and most of them are relatively toxic. The increased incidence of therapeutic failure in the treatment of fungal infections and the prevalence of opportunistic infections has renewed interest in the search for new antifungal agents, including those obtained from higher plants. Present results allow to suppose that these natural compounds could be useful agents in the topical treatment of fungal infections.
Acknowledgments Dr. Lucia Battinelli was supported by the ‘‘Enrico and Enrica Sovena’’ Foundation (Italy).
References Al-Waili, N.S., 2004. An alternative treatment for pityriasis versicolor, tinea cruris, tinea corporis and tinea faciei with topical application of honey, olive oil and beeswax mixture: an open pilot study. Complement. Ther. Med. 12 (1), 45–47. Al-Waili, N.S., 2005. Clinical and mycological benefits of topical application of honey, olive oil and beeswax in diaper dermatitis. Clin. Microbiol. Infect. 11 (2), 160–163. Bisignano, G., Tomaino, A., Lo Cascio, R., Crisafi, G., Uccella, N., Saija, A., 1999. On the in vitro antimicrobial activity of oleuropein and hydroxytyrosol. J. Pharm. Pharmacol. 51 (8), 971–974. Bisignano, G., Lagana`, M.G., Trombetta, D., Arena, S., Nostro, A., Uccella, N., Mazzanti, G., Saija, A., 2001. In vitro antibacterial activity of some aliphatic aldehydes from Olea europaea L. FEMS Microbiol. Lett. 198, 9–13. Campeol, E., Flamini, G., Cioni, P.L., Morelli, I., 2003. Volatile fractions from three cultivars of Olea europea L. collected in two different seasons. J. Agric. Food Chem. 51, 1994–1999. Elkhalifa, K.F., 2002. Treatment of skin diseases in the Sudan using tree materials. Arab. Gulf J. Sci. Res. 20 (4), 232–235. Flamini, G., Cioni, P.L., Morelli, I., 2003. Volatile from leaves, fruits, and virgin oil from Olea europea cv. Olivastra Seggianese from Italy. J. Agric. Food Chem. 51, 1382–1386. Furneri, P.M., Marino, A., Saija, A., Uccella, N., Bisignano, G., 2002. In vitro antimycoplasmal activity of oleuropein. Int. J. Antimicrob. Agents 20 (4), 293–296. Gazel, A., Ramphal, P., Rosdy, M., De Wever, B., Tornier, C., Hosein, N., Lee, B., Tomic-Canic, M., Blumenberg, M., 2003. Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays. J. Invest. Dermatol. 121 (6), 1459–1468. Guth, H., Grosch, W., 1993. Quantitation of potent odorants of virgin olive oil by stable-isotope dilution assays. J. Am. Oil Chem. Soc. 70, 513–518.
ARTICLE IN PRESS L. Battinelli et al. / Phytomedicine 13 (2006) 558–563
Kubo, I., Hanke, F.J., 1985. Multifaceted chemically based resistance in plants. In: Conn, E. (Ed.), Recent Advances in Phytochemistry, Chemically Mediated Interactions between Plants and Other Organisms, Vol. 19. Plenum, New York, pp. 171–194. Kubo, A., Lunde, S., Kubo, I., 1995. Antimicrobial activity of the Olive oil flavor compounds. J. Agric. Food Chem. 43, 1629–1633. Kubo, I., Fujita, K., Kubo, A., Nihei, K., Lunde, C.S., 2003. Modes of antifungal action of (2E)-alkenals against Saccharomyces cerevisiae. J. Agric. Food Chem. 51 (14), 3951–3957. Markin, D., Duek, L., Berdicevsky, I., 2003. In vitro antimicrobial activity of olive leaves. Mycoses 46, 132–136. Montedoro, G., Bertuccioli, M., Anichini, F., 1978. Aroma analysis of virgin olive oil by head space volatile extraction techniques. In: Charalampous, G., Inglet, G. (Eds.), Flavor of Foods and Beverages. Academic Press, New York, pp. 247–281. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Muhsin, T.M., Aubaid, A.H., Al-Duboon, A.H., 1997. Extracellular enzyme activity of dermatophytes and yeasts on solid media. Mycoses 40, 465–469.
563
Naughton, M.A., Sanger, F., 1961. Purification and specificity of pancreatic elastase. Biochem. J. 78, 156–163. Ranalli, A., Ferante, M.L., 1996. Physico-chemical and analytical characteristics of extra virgin olive oils extracted by using a pectolytic enzymatic processing aid. Olivae 60, 27–32. Rippon, J.W., Varadi, D.P., 1968. The elastases of pathogenic fungi and actinomycetes. J. Invest. Dermatol. 50, 54–57. Rosdy, M., Pisani, A., Ortonne, J.P., 1993. Production of basement membrane components by a reconstructed epidermis cultured in the absence of serum and dermal factors. Br. J. Dermatol. 129 (3), 227–234. Samova, L.I., Shode, F.O., Ramnanan, P., Nadar, A., 2003. Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies africana leaves. J. Ethnopharmacol. 84, 299–305. Trombetta, D., Saija, A., Bisignano, G., Arena, S., Caruso, S., Mazzanti, G., Uccella, N., Castelli, F., 2002. Study on the mechanisms of the antibacterial action of some plant a,bunsaturated aldehydes. Lett. Appl. Microbiol. 35 (4), 285–290. Williams, K., Phillips, K.D., Willis, A.T., 1988. A simple and sensitive method for detecting bacterial elastase production. Lett. Appl. Microbiol. 7, 173–176.