**5.1 Medium**

74 Fungicides for Plant and Animal Diseases

(Schulz, 1996), and the long chain alkanols enter in part into the lipid bilayers (Franks and Lieb, 1986). The amount of alkanols entering into the cytosol or lipid bilayer is dependent on the length of the alkyl chain. Nonetheless, alkanols are chemically stable compounds and may not react with any biologically important substances in the cytosol or lipid bilayer. Hence, the primary antifungal action of alkanols comes largely from their ability to function as nonionic surfactants (physical disruption of the membrane). In the case of 2*E*-alkenals, their α,β-unsaturated aldehyde group should not be overlooked because this group is chemically highly reactive and readily reacts with biologically important nucleophilic groups, such as sulfhydryl, amino, or hydroxyl (Schauenstein et al., 1977). For example, the yeast plasma-membrane H+-ATPase was reported to contain nine cysteines. 2*E*-Alkenals may bind directly to the plasma membrane H+-ATPase probably with sulfhydryl groups of the three cysteines in the presumed transmembrane segments (C148, C312, C867). However, Petrov and Slayman (1995) reported that no single cysteine is required for activity based on their site-directed mutagenesis study. This previous result does not exclude the possibility to assume that 2*E*-alkenals first break the hydrogen bond as nonionic surfactants and then react with the freed sulfhydryl group of the H+-ATPase as well as other plasma membrane proteins. This can be supported by the previous report that covalent modification of the conserved C148 in the transmembrane segment 2 may be important for inhibition of H+- ATPase activity and cell growth (Monk et al., 1995). However, the observation that alkanals and 2*E*-alkenals exhibit similar antifungal activity against *S. cerevisiae* as shown in Table 2 and also inhibit glucose-induced acidification, may not support the above assumption because the conjugated double bond is not essential to elicit the activity. The possibility of

this concerted function of 2*E*-alkenals is unlikely but cannot be excluded.

concept.

The leakage of carboxyfluorescein (CF) in liposomes of phosphatidylcholine (PC) following exposure to 2*E*-alkenals was previously reported (Trombetta et al., 2002), similar to those described for alkyl gallates (Fujita and Kubo, 2002). Interestingly, 2*E*-alkenals tested caused rapid CF leakage from PC liposomes and the effectiveness order correlated well with the alkyl chain length. Thus, 2*E*-nonenal was more effective in inducing CF leakage from PC liposomes than that of 2*E*-hexenal (Trombetta et al., 2002). This also supports the surfactant

In general terms, aldehydes may enter the cell by passive diffusion across the membrane. Once inside the cells, the following reactions are known for the 2*E*-alkenals. Reactions with the sulfhydryl group, for which primary addition to the ,-olefinic group occurs exclusively. Reactions with amino groups, where the formations of Schiff bases and 1,4 addition products is possible. It is generally true, however, that the reaction with the sulfhydryl groups takes place much faster, in fact by several orders of magnitude. These reactions may lead to the deactivation of enzymes – in particular of sulfhydryl enzymes. The main problem of general significance remains the certain and unequivocal experimental proof that 2*E*-alkenals recognized as highly reactive compounds is in fact essential bioregulators of metabolism. For example, sulfhydryl groups in proteins and lower molecular weight compounds such as glutathione are known to play an important role in the living cell. Bacteria protect themselves against hydrogen peroxide in various ways (Brul and Coote, 1999), and some of the most ubiquitous systems include glutathione. 2*E*-Alkenals causes depletion of cytoplasmic and mitochondrial glutathione, which functions in eliminating reactive oxygen species, similar to found for polygodial (Machida et al., 1999). *Saccharomyces cerevisiae* was maintained at -80 °C in yeast nutrient broth (YNB; Difco Laboratories, Detroit, MI) containing 25% glycerol and subcultured at 30 °C in Sabouraud's dextrose agar (SDA) medium (Bactopeptone 1%, dextrose 4%, Bacto-agar 1.8%). A fresh culture was preincubated with shaking for 16 h at 30 °C in 2.5% malt extract (ME) broth (BBL) medium.

#### **5.2 Acidification measurement**

The glucose-induced medium acidification of *S. cerevisiae* was measured with a modified procedure (Haworth et al., 1993). The test strain was cultured with shaking in YPD (Glucose 2%, Bactopeptone 2%, Yeast extract 1%) broth overnight at 30 °C and washed twice with cold distilled water. The cells were diluted to 5 X 107 colony forming units (CFU) per mL with cold distilled water and kept on ice. The reaction mixture contained 2.7 mL of cells and 30 L of the inhibitor in DMSO, and was preincubated at 30 °C for 5 min. A 20% glucose solution of 0.3 mL was added (final 2%) to induce acidification. After 10 min incubation, the pH of external medium was checked (Orion 8175 Ross semimicro electrode).

#### **5.3 Antifungal assay**

The test compounds were first dissolved in DMF and the concentration of DMF in each medium was always 1%. The highest concentration tested was 1600 µg/mL, unless otherwise specified. The maximum extent and rate of activity is known to vary with the seed culture mediums, the physiological age of the culture, and the type of culture medium. For example, the minimum inhibitory concentration (MIC) of anethole significantly varied with the inoculum size. All antifungal susceptibility tests in this study were performed under a standard condition using fresh inoculum from a 5 h shaking culture in ME medium, final inoculum size of 105 CFU/mL, and 48 h stationary incubation in ME medium, unless otherwise specified.

Broth macrodilution minimum inhibitory concentrations (MICs) were determined as previously described (Kubo and Himejima, 1992). Briefly, serial 2-fold dilutions of the test compounds were made in DMF and 30 µL of 100 X conc. solution was added to 3 mL of ME

Naturally Occurring Antifungal Agents and Their Modes of Action 77

Fig. 13. Antifungal action involves multifunction. The amphipathic medium-chain aldehydes and alcohols are nonionic surfactants and disrupt the hydrogen bonding in the lipid-protein interface of integral proteins, such as ion channels and/or transport proteins, denaturing their functional conformation. 2*E*-Alkenals react with biologically important nucleophilic groups such as sulfhydryl, amino, or hydroxyl. For example, sulfhydryl groups in proteins and lower molecular weight compounds such as glutathione are known to play an important role in the living cell and 2*E*-alkenal mediated depletion of intercellular glutathione can be explained by a direct interaction between the enal moiety and the

alcohol dehydrogenase competitively but not phosphofructokinase or pyruvate

decarboxylase.

**8. References** 

**7. Acknowledgements** 

support during his study at UC Berkeley.

*europea* L. *FEMS Microbiol. Lett*., *198*, 9-13.

biological membranes. *Lipids*, *9*, 645-650.

resistance mechanisms. *Int. J. Food Microbiol*., *50*, 1-17.

sulfhydryl group of glutathione by a Michael-type addition. Aldehydes are known to inhibit

The authors are indebted to Dr. S. H. Lee, Dr. M. Himejima and Dr. C. S. Lunde for performing antimicrobial assay at earlier stage of the work, and Dr. H. Haraguchi and Dr. D. G. Hammond for performing the respiratory inhibition assay. K. F. thanks for financial

Bisignano, G.; Lagana, M. G.; Trombetta, D.; Arena, S.; Nostro, A.; Uccella, N.; Mazzanti, G.;

Brockerhoff, H. (2001) Model of interaction of polar lipids, cholesterol, and proteins in

Brul, S.; Coote, P. (1999) Preservative agents in foods, mode of action and microbial

Saija, A. (2001) In vitro antibacterial activity of some aliphatic aldehydes from *Olea* 

media. These were inoculated with 30 µL of seed culture to give the final inoculum of 105 CFU/mL. The assay tubes were incubated without shaking at 30 °C for 48 h. The MIC is the lowest concentration of test compound that demonstrated no visible growth. The minimum fungicidal concentrations (MFCs) were examined as follows. After the MIC had been determined, a 30 µL of aliquot was taken from each clear tube and added into 3 mL of drug free fresh medium. After 48 h incubation, the MFC was determined as the lowest concentration of the test compounds in which no recovery of microorganism was observed.

Time kill studies were performed to examine the effects of combinations of compounds in more detail. The culture tubes were prepared as described above and incubated at 30 °C for 16 h. A 30 µL aliquot of the culture was inoculated into 3 mL of ME broth containing appropriate concentrations of the test compounds. The initial population size for *S. cerevisiae* was 5.8 X 105 CFU/mL. Samples were taken at selected times during 48 h of exposure, and serial dilutions were made in sterile saline before the samples were plated onto YPD agar plates. The plates were incubated at 30 °C for 48 h before the number of CFU was determined.
