The Diterpenoid ent-16-Kauren-19-oic Acid Acts as an Uncoupler of the Bacterial Respiratory Chain

Abstract

ent-16-Kauren-19-oic acid is a bacteriolytic diterpenoid present in the resin of the medicinal plant Pseudognaphalium vira vira. The compound and its methyl ester showed strong activity against gram-positive bacteria, whereas the derivative 3β-hydroxy-ent-kauren-19-oic acid was inactive against all assayed bacteria at the maximal concentration used (250 µg/mL). The bacteriolytic effect of ent-16-kauren-19-oic acid (5 µg/mL) was confirmed with cultures of Bacillus cereus and Staphylococcus aureus, whereas the methyl derivative (12 µg/mL) showed only a bacteriostatic effect. Both compounds stimulated oxygen consumption and proton conduction of whole cells, as reflected by an abrupt increase in the extracellular pH. These results indicate that ent-16-kauren-19-oic acid acts as a respiratory chain uncoupler, and that this function is strongly affected by minor structural substitutions, suggesting a tight activity-structure relationship. The ultimate effect of the uncoupling mechanism demonstrated by ent-16-kauren-19-oic acid is bacterial lysis. The disruption of the bacterial membrane integrity caused by the diterpenoid compound was determined using SYTOX Green stain and visualized by fluorescence microscopy.

Key words

ent‐16‐kauren‐19‐oic acid - Pseudognaphalium vira vira - Asteraceae - diterpenoid - antibacterial activity - respiratory chain uncoupler

References

• 1 Rahman A, Nasreen A, Akhtar F, Shekhani M S, Clardy J, Parvez M, Choudhary M I. Antifungal diterpenoid alkaloids from Delphinium denudatum.  J Nat Prod. 1997;  60 472-474
• 2 Cotoras M, Folch C, Mendoza L. Characterization of the antifungal activity on Botrytis cinerea of the natural diterpenoids kaurenoic acid and 3β-hydroxy-kaurenoic acid.  J Agric Food Chem. 2004;  52 2821-2826
• 3 Tada M, Okuno K, Chiba K, Ohnishi E, Yoshii T. Antiviral diterpenes from Salvia officinalis.  Phytochemistry. 1994;  35 539-541
• 4 Ghisalberti E. The biological activity of naturally occurring kaurane diterpenes.  Fitoterapia. 1997;  68 303-324
• 5 Chinou I. Labdanes of natural origin – biological activities (1981–2004).  Curr Med Chem. 2005;  12 1295-1317
• 6 Rezende M C, Urzúa A, Bortoluzzi A J, Vásquez L. Variation of antimicrobial activity of Pseudognaphalium vira vira (Asteraceae): isolation and X‐ray structure of ent-3β-hydroxy-16-kauren-19-oic acid.  J Ethnopharmacol. 2000;  72 459-464
• 7 Tapia L, Torres J, Mendoza L, Urzúa A, Ferreira J, Pavani M, Wilkens M. Effect of 13-epi-sclareol on the bacterial respiratory chain.  Planta Med. 2004;  70 1058-1063
• 8 Micol V, Mateo C R, Shapiro S, Aranda F J, Villalain J. Effects of (+)-totarol, a diterpenoid antibacterial agent, on phospholipid model membranes.  Biochim Biophys Acta. 2001;  1511 281-290
• 9 Mateo C R, Prieto M, Micol V, Shapiro S, Villalain J. A fluorescence study of the interaction and location of (+)-totarol, a diterpenoid bioactive molecule, in model membranes.  Biochim Biophys Acta. 2000;  1509 167-175
• 10 Haraguchi H, Oike S, Muroi H, Kubo I. Mode of antibacterial action of totarol, a diterpene from Podocarpus nagi.  Planta Med. 1996;  62 122-125
• 11 Mendoza L, Wilkens M, Urzúa A. Antimicrobial study of the resinous exudates and of diterpenoids and flavonoids isolated from some Chilean Pseudognaphalium (Asteraceae).  J Ethnopharmacol. 1997;  58 85-88
• 12 Wilkens M, Alarcón C, Urzúa A, Mendoza L. Characterization of the bactericidal activity of the natural diterpene kaurenoic acid.  Planta Med. 2002;  68 452-454
• 13 Haraguchi H, Tanimoto K, Tamura Y, Mizutani K, Kinoshita T. Mode of antibacterial action of retrochalcones from Glycyrrhiza inflata.  Phytochemistry. 1998;  48 125-129
• 14 Marumo H, Kitaura K, Morimoto M, Tanaka H, Omura S. The mode of action of nanaomycin A in gram-positive bacteria.  J Antibiot (Tokyo). 1980;  33 885-890
• 15 Lebaron P, Catala P, Parthuisot N. Effectiveness of SYTOX Green stain for bacterial viability assessment.  Appl Environ Microbiol. 1998;  64 2697-2700
• 16 Gil F, De la Iglesia R, Mendoza L, González B, Wilkens M. Soil bacteria are differentially affected by the resin of the medicinal plant Pseudognaphalium vira vira and its main component kaurenoic acid.  Microbial Ecol. 2006;  52 10-18
• 17 Tojo E, Rial M E, Urzúa A, Mendoza L. Clerodane diterpenes from Haplopappus deserticola.  Phytochemistry. 1999;  52 1531-1533
• 18 Urzúa A, Caroli M, Vásquez L, Mendoza L, Wilkens M, Tojo E. Antimicrobial study of the resinous exudate and of diterpenoids isolated from Eupatorium salvia (Asteraceae).  J Ethnopharmacol. 1998;  62 251-254
• 19 Urzúa A, Torres R, Mendoza L, Delle Monache F. Antibacterial new clerodane diterpenes from the surface of Haplopappus foliosus.  Planta Med. 2003;  69 675-677
• 20 Urzúa A, Jara F, Tojo E, Wilkens M, Mendoza L, Rezende M C. A new antibacterial clerodane diterpenoid from the resinous exudates of Haplopappus uncinatus.  J Ethnopharmacol. 2006;  103 297-301