**5.4 Adsorption test**

The test strain was cultured with shaking in YPD broth overnight at 30 ºC and washed twice with 50 mM MOPS buffer (pH 6.0). After each 2*E*-alkenal was mixed with or without *S. cerevisiae* cells (108 cells/mL) in the above buffer at 30 ºC, the suspension was vortexed for 5 seconds. Absorbance of the supernatants obtained by centrifugation for 5 min was measured at 255 nm.

### **6. Summary**

The antifungal activity of alkanols comes from their ability to act as surfactants and the maximum activity can be designed by selecting a alkyl chain length to give the appropriate partition coefficient (log P) as a standard. Similarly, 2*E*-alkenals also act as surfactants, but their ,-unsaturated aldehyde moiety needs to be taken into account. 2*E*-Alkenals may not act by a single defined process but have multiple functions (Figure 13). The surfactant concept, disrupting and disorganizing the lipid bilayer-protein interface nonspecifically, can be extended to answer many other problems related to membrane-bound enzymes and receptors, and the fluidity of the membrane lipids. For example, the anesthesia cutoff phenomenon among alkanols is well known and a long standing problem. Anesthesia involves many membrane-bound proteins such as synaptosomal ATPases and acetylcholine receptor (Edelfors and Ravn-Jonsen, 1990; Elliott and Haydon, 1989). The same surfactant concept, disrupting and disorganizing the lipid bilayer-protein interface, seems applicable to explain the anesthesia cutoff phenomena of alkanols. The knowledge obtained may provide insights into fungicidal action of aldehydes and alkanols on a molecular basis, and a more rational and scientific approach to use or design efficient and safe antifungal agents. Based on the data obtained, the hydrophilic head portion can be replaced by any hydrophilic groups as long as the "head and tail" structure is balanced. Hence, various additional biological activities can be introduced mainly by selecting appropriate head portions. For example, each series of alkyl gallates (Kubo et al., 2001b; Fujita and Kubo, 2001 and 2002) and alkyl protocatechuates (Nihei et al., 2003) were synthesized as antioxidation antifungal agents.

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

The test strain was cultured with shaking in YPD broth overnight at 30 ºC and washed twice with 50 mM MOPS buffer (pH 6.0). After each 2*E*-alkenal was mixed with or without *S. cerevisiae* cells (108 cells/mL) in the above buffer at 30 ºC, the suspension was vortexed for 5 seconds. Absorbance of the supernatants obtained by centrifugation for 5 min was measured

The antifungal activity of alkanols comes from their ability to act as surfactants and the maximum activity can be designed by selecting a alkyl chain length to give the appropriate partition coefficient (log P) as a standard. Similarly, 2*E*-alkenals also act as surfactants, but their ,-unsaturated aldehyde moiety needs to be taken into account. 2*E*-Alkenals may not act by a single defined process but have multiple functions (Figure 13). The surfactant concept, disrupting and disorganizing the lipid bilayer-protein interface nonspecifically, can be extended to answer many other problems related to membrane-bound enzymes and receptors, and the fluidity of the membrane lipids. For example, the anesthesia cutoff phenomenon among alkanols is well known and a long standing problem. Anesthesia involves many membrane-bound proteins such as synaptosomal ATPases and acetylcholine receptor (Edelfors and Ravn-Jonsen, 1990; Elliott and Haydon, 1989). The same surfactant concept, disrupting and disorganizing the lipid bilayer-protein interface, seems applicable to explain the anesthesia cutoff phenomena of alkanols. The knowledge obtained may provide insights into fungicidal action of aldehydes and alkanols on a molecular basis, and a more rational and scientific approach to use or design efficient and safe antifungal agents. Based on the data obtained, the hydrophilic head portion can be replaced by any hydrophilic groups as long as the "head and tail" structure is balanced. Hence, various additional biological activities can be introduced mainly by selecting appropriate head portions. For example, each series of alkyl gallates (Kubo et al., 2001b; Fujita and Kubo, 2001 and 2002) and alkyl protocatechuates (Nihei et al., 2003) were synthesized as antioxidation

determined.

at 255 nm.

**6. Summary** 

antifungal agents.

**5.4 Adsorption test** 

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 sulfhydryl group of glutathione by a Michael-type addition. Aldehydes are known to inhibit alcohol dehydrogenase competitively but not phosphofructokinase or pyruvate decarboxylase.

#### **7. Acknowledgements**

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 support during his study at UC Berkeley.

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**4** 

*México* 

**Antifungal Properties of** 

*1Universidad Autónoma Agraria Antonio Narro* 

*2Universidad Autónoma de Coahuila* 

**Bioactive Compounds from Plants** 

F. Castillo1, D. Hernández1, G. Gallegos1, R. Rodríguez2 and C. N. Aguilar2

Currently, the consequences derived from application of fungicides in traditional agricultural production systems for control of crop diseases have impacted negatively this activity. Fungicides application, where the indiscriminate use and application frequency high has led to problems and constraints in the control of these diseases by loss in efficiency, increased resistance to active ingredients, ecological damage and a serious negative impact on the human health. For this reason, it is had carryed out research to develop new products, methods and strategies for diseases control. The investigation and development of bio-based products is of great interest to subtract the negative effects generated by traditional agricultural production systems. The use and application of bioactive phytochemicals with antifungal properties represent an attractive and efficient alternative to

These bioactive compounds are naturally produced in the plants how secondary metabolites, the principal groups with antifungal activity were terpenes, tannins, flavonoids, essential oil, alkaloids, lecithin and polypeptides. These groups of compounds are important for the physiology of plants contributing properties confer resistance against microorganisms, other organisms and help preserve the integrity of the plant with continuous exposure to environmental stressors, such as ultraviolet radiation, high

Plants have developed natural defense mechanisms to protect themselves long before the man played an active role in protecting them. It is known that plants synthesize a variety of groups of bioactive compounds in plant tissues as secondary metabolites that have antifungal activity to stop or inhibit the development of mycelia growth, inhibition of germination or reduce sporulation of fungal pathogens, each these groups presented variable mechanisms of action, for example, the toxicity of polyphenols in microorganisms is attributed to enzyme inhibition by oxidation of compounds. For essential oils is

**1. Introduction** 

inhibit the growth of several fungal pathogens.

**2. Bioactive antifungal activity groups** 

temperatures or dehydration.

**2.1 General** 

