Leu T.*1, Soulet S.1, Herbette G.2, Faure R.2, Bianchini J.-P.1, Meijer L.3 and Raharivelomanana P1. *E-mail to: Tinihauarii.Leu@upf.pf

1 Laboratoire Biotem EA 4239, Université de la Polynésie Française, BP 6570, 98702 Faa’a, Tahiti, French Polynesia2 Spectropole de Marseille, Université Paul Cézanne, 13397 Marseille Cedex 20, France3 Station Biologique de Roscoff, CNRS/USR 3151, Place G. Tessier, BP 74, 29682, Roscoff, Cedex, France

GA 2009

Elastase inhibitors and cancer preventive potential agents from Calophyllum

inophyllum (L.) grown in French Polynesia

Introduction:

Experimental. Resin of Tamanu was extracted from seed oil with 96° EtOH and neutral fraction was separated from the acidic one (fig. 1). Products of NRT and ART were isolated by chromatographic methods and identified by spectroscopic analyses (UV, IR, HRMS, 1D and 2D NMR). In vitro enzymatic inhibition assay was carried out at different steps of purification. Bioguided fractionation led to the isolation of new elastase inhibitory compounds.

Tamanu oil(TO)

Resin of Tamanu(RT)

Neutral Resin of Tamanu (NRT)

Acidic Resin of Tamanu (ART)

Deresined Tamanu oil(DTO)

Tamanu oil(TO)

Tamanu oil(TO)

Resin of Tamanu(RT)

Resin of Tamanu(RT)

Neutral Resin of Tamanu (NRT)

Neutral Resin of Tamanu (NRT)

Acidic Resin of Tamanu (ART)Acidic Resin of Tamanu (ART)

Deresined Tamanu oil(DTO)

Deresined Tamanu oil(DTO)

Fig 1: Separation of resins from Tamanu oil

References:1. Barnes, P.J., Hansel, T.T. (2004) The Lancet

364: 985-996.2. Akli, S., Keyomarsi, K. (2004) Br Cancer Res 6:

188-191.3. Pétard, P. (1986) Plantes utiles de Polynésie –

Raau Tahiti, Editions Haere Po No Tahiti, Papeete, Tahiti.

4. Laure, F. et al. (2008) Anal Chim Act 624: 147-153.

Acknowledgement: The Ministère chargé de la Recherche en Polynésie française is greatly acknowledge for financial support (Convention n°5.0314/PR du 30/11/05, avenant n°6.0360/PR du 24/08/06). The authors are also grateful to Dr. J.-Y. Meyer and Dr. P. Frogier for helpful commentaries.

Conclusions. 16 coumarin derivatives were identified from the resin of Tamanu including the new tamanolides and the firstly occuring in C. inophyllum calanolides. Among those, 5 compounds led to significant inhibition of elastase, the most active compound being calophyllolide. Phenyl C-4 susbtituent and opened D-ring are structural requirements for elastase inhibition by coumarin derivatives.

Elastase is a serin endo-proteinase responsible for extracellular matrix proteins degradation.Overexpression was linked to COPD, Cystic Fibrosis1 and associated to increased low-molecular weight form Cyclin E correlating with poor diagnosis and morbidity increase in breast cancer2.

Calophyllum inophyllum, L.

Division: Magnoliophyta Class: Magnoliopsida Order: Theales Family: Clusiaceae Genus: Calophyllum Species: C. inophyllum

Tamanu (C. inophyllum) is a pantropical species widely used in Polynesian folk medicine for skin treatments, wound healing and as pain killer3.Previous studies have shown the presence of bioactive coumarin derivatives4.

Elastase.

Enzymatic screening assay revealed elastase inhibiting properties of fractions of Tamanu oil. Bioguided phytochemical investigations led

to the identification of elastase inhibiting new pyranocoumarin compounds.

Calophyllolide Inophyllum C Inophyllum E Inophyllum PInophyllum D

Calanolide « No name » Calanolide A Calanolide B Calanolide D

Inocalophyllin A Inocalophyllin B Tamanolide Tamanolide D Tamanolide P

Coumarin derivatives identified in Tamanu oil.Inophyllum

serie

Calanolide serie

Inocalophyllin serie

Tamanolide serie

Epicalanolide C12-oxocalanolide A

Tamanolide is a new serie of coumarin derivatives

Compounds IC50 (µM)Calophyllolide 8.4Inophyllum C -Inophyllum E -Inophyllum D -Inophyllum P -

Calanolide "No name" 26.3Calanolide A -

12- oxocalanolide A -Calanolide B -

Epicalanolide C -Calanolide D -

Inocalophyllin A -Inocalophyllin B 9.3

Tamanolide 17.7Tamanolide D 26.1Tamanolide P -

Elastase inhibiting activity.

Structure-activity relationship.

Calophyllolide is the most active compound (IC50 = 8.4 µM)

Phenyl C-4 substituent, methoxyl C-8b and tigloyl C-12a groups appears to be structural requirements for elastase inhibition by coumarin derivatives