**5. Structure requirement of eugenol derivatives for antifungal activity**

It was proposed that the inhibition of ergosterol synthesis leads to the damage of cell membrane functionality and integrity [42]. However, the effect of eugenol is not reversed by osmotic support, indicating that its effect does not affect the cell wall synthesis and assembly. Furthermore, eugenol does not bind ergosterol, the main sterol of fungal membrane [43].

Eugenol is suggested to block aromatic and branched chain amino acid synthesis across the cytoplasmic membrane. Eugenol inhibits growth of yeast strain carrying a mutation in gene encoding an enzyme, a tryptophan, phenylalanine, tyrosine, and isoleucine biosynthesis

There are two approaches to obtain antifusarium from clove. Firstly, the secondary metabolites from clove leaves or buds can be extracted using nonpolar solvent such as hexane, petroleum ether, gasoline, or kerosene. Subsequently, the solvent is removed through evaporation leaving the concentrated extract containing antifusarium and antiphytophthora compounds. Hexane and petroleum ether have relatively low boiling point; therefore, it is easy to evaporate, and while the boiling point of gasoline and kerosene is higher than 100°C, higher temperature or lower pressure is needed to evaporate. By using extraction combined with distillation to recover the solvent, more efficient production system can be developed. Secondly, since eugenol is a component of volatile oil, the oil of clove leaves can be obtained through steam distillation by which the oil will evaporate together with steam, and upon condensation the oil will separate from water and the oil can be collected. To obtain pure eugenol, further separation processes will be needed, such as liquid–liquid extraction, vacuum fraction distillation, and chromatographic

There are some other plant metabolites having antifungal activity, and the effect is stronger than eugenol. Thymol and other components of volatile oil had been compared, and the results are as shown in **Table 3** [45]. Thymol is the component of volatile oil from *Thymus vulgaris*, herbal medicine commonly used in cough mixture. Most of these compounds demonstrate minimal inhibition concentration (MIC) above 50 ppm. The natural antifungal that demonstrates antifungal activity similar to that of commercially distributed is xanthorrhizol with MIC lower than 10 ppm [17]. Xanthorrhizol is the major component of volatile oil iso-

*Botrytis cinerea Fusarium* 

*oxysporum*

*Alternaria solani*

pathway, in a medium supplemented with the related amino acid [44].

72 Fusarium - Plant Diseases, Pathogen Diversity, Genetic Diversity, Resistance and Molecular Markers

techniques.

lated from the rhizome of *C. xanthorrhiza*.

*Sclerotium rolfsii Rhizoctonia* 

**Table 3.** Relative antifungal activity of plant component compared to thymol.

*solani*

Thymol 100 100 100 100 100 Eugenol 42.2 62.02 17.23 38.14 37.77 Methyl cinnamate 49.65 57.68 16.43 28.16 81.31 Linalool 11.8 11.33 4.45 6.03 0.85 1,8-Cineol 0.0008 0.468 — — —

**Compound Toxicity index**

Eugenol derivatives had been synthesized and their antifungal activities evaluated [43]. Some structures and their antifungal activities are shown in **Figure 2**. It seems that the aromatic, ortho-oxygenation, and the double bond at the terminal of side chain are essential for

[G] MIC=125ppm [H] MIC > 250 ppm

**Figure 2.** Derivatives of eugenol and their antifungal activities.

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**Figure 3.** The important sites for antifungal activity of eugenol derivatives.

the antifungal activity. The presence of substituents on the hydroxy phenolic reduces the activity. Compound F by which the orthodioxy is connected by a methine bridge becomes inactive (MIC > 250 ppm). The absence of double bond in the side chain eliminates the antifungal activity; this is shown by compound D with MIC >250 ppm and considered to be inactive. If the position of double bond of the side chain is moved to the middle, the antifungal activity also disappears. This is demonstrated by compound H that is inactive. The presence of nitro substituent attached to the aromatic increases antifungal activity, and the nitro at ortho-position to the hydroxy group gives higher activity than at meta-position (compounds B and C).

Base on the above data, the structure requirement for eugenol derivatives to be active as a fungicide is shown in **Figure 3**.
