ln a preliminary screening of 34 plants extracts, the roots of Vernonia gigantea (Walt.) Trelease (Fam. Compositae) showed good antimicrobial activity in vitro against the Gram positive bacterium Bacillus subtilis, the acid-fast bacterium Mycobacterium smegmatis and the dermatophytic fungi Microsporum gypseum and Trichophyton mentagrophytes.
Bioassay-guided fractionation of the ethanolic extracts of V. gigantea roots afforded the sesquiterpene lactone zaluzanin C (35) as the active antifungal principle. Phytochemical investigation of V. gigantea roots afforded also the zaluzanin C related compounds : glucozaluzanin C (36), 6'-O-caffeoyl-glucozaluzanin C (37), 8α-hydroxy-11β,13-dihydrozaluzanin C (38) and 8α-hydroxy-11β,13-dihydrozaluzanin C-3-O-β-glucopyranoside (39); and the caffeoylquinic acid derivatives : 3,5-di-O-caffeoylquinic acid (42), methyl 3,5-di-O-caffeoyl quinate (43), 1,5-di-O-caffeoylquinic acid (44), methyl 3,4,5-tri-O-caffeoyl quinate (45) and ethyl 3,4,5-tri-O-caffeoyl quinate (46). All these compounds were isolated for the first time from V. gigantea; while 37, 39 and 46 are novel compounds.
The zaluzanin C related compounds 36-39 have weak or no antifungal activity at all. Comparison of the structures of these compounds suggested that the α-methylene-γ-lactone group and the nature of the substituent at C-3 are key factors for the presence of antifungal activity.
Microbial transformation studies on zaluzanin C were performed in order to obtain other closely related derivatives with increased or decreased antifungal activity that could yield structure-activity relationships.
The bacteria Streptomyces sp. ATCC 15077, Streptomyces griseus B ATCC 8090; and the fungi Aspergillus ochraceous ATCC 1080 and Aspergillus flavipes ATCC 11013 were able to biotransform zaluzanin C into five metabolites : 4β,15,11β,13-tetrahydrozaluzanin C (57), 4β,15,11β,13-tetrahydro-epi-zaluzanin C (58), 4β ,15,11β ,13-tetrahydro-3-dehydrozaluzanin C (59), 11β,13-dihydro-10α-epoxyzaluzanin C (60) and 4β,15,11β,13-tetrahydro-10α-epoxyzaluzanin C (61). All these metabolites have no antimicrobial activity. The microorganisms used in the biotransformation studies regioselectively reduced the C-11 to C-13 double bond (which is required for activity) suggesting that the microorganisms performed this type of reaction as a defense mechanism.
Even though glucozaluzanin C (36) and compound 37 still conserve intact the C-11 to C-13 double bond, they are devoid of activity. The difference between these two compounds and zaluzanin C is the substituent at C-3.
Based on these facts, two chemical modifications to the structure of zaluzanin C were performed in order to enhance its antifungal activity. The C-3-OH group was acetylated to yield zaluzanin D (62); and oxidized with chromium oxide to yield dehydrozaluzanin C (63).
Zaluzanin D is less polar than zaluzanin C and possess better antifungal activity. DehydrozaIuzanin C has the same polarity as zaluzanin C, but it has an extra α,β-unsaturated ketone group, which is responsible for a remarkably enhancement of antifungal activity. MIC (Minimum lnhibitory Concentration) values for zaluzanin C, zaluzanin D and dehydrozaluzanin C against T. mentagrophytes were 25, 12.5 and 0.78 μg/ml, respectively; compared to 1.56 μg/ml of miconazole nitrate, a reference antifungal compound in current clinical use.
A bioassay for ergosterol-sensitive antifungal activity demonstrated that the mechanism of action of dehydrozaluzanin C is different from that of amphotericin B.
Zaluzanin C (35) was isolated as a minor compound of the V. gigantea roots. Although the respective glucoside, glucozaluzanin C (36), is inactive; the yield of its isolation was 250 times higher than its aglycone (35). It appears that glucozaluzanin C is acting as a "post-inhibitin" (an inactive compound normally present in the plant that can rapidly be converted into the active form by a simple biochemical reaction).