PHYTOTHERAPY RESEARCH Phytother. Res. 19, 75–77 (2005) Published online in Wiley InterScience DOI: 10.1002/ptr.1613 BIOACTIVE(www.interscience.wiley.com). TRITERPENOIDS FROM VOCHYSIA PACIFICA
75
SHORT COMMUNICATION
Bioactive Triterpenoids from Vochysia pacifica Interact with Cyclic Nucleotide Phosphodiesterase Isozyme PDE4 Bernard Weniger1, Annelise Lobstein1, Byung-Hun Um1, Catherine Vonthron-Sénéchau1, Robert Anton1, Nora Jiménez Usuga2, Hélène Basaran3 and Claire Lugnier3* 1
UMR/CNRS 7081, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg, France Corporación de Patologías Tropicales, Grupo de Investigación en sustancias Bioactivas, Universidad de Antioquia, Medellín, Colombia 3 UMR/CNRS 7034, Faculté de Pharmacie Université Louis Pasteur, Strasbourg, France 2
Several species of the genus Vochysia (Vochysiaceae) are used by traditional communities in South America to relieve ailments linked to inflammation, such as skin sores, asthma and pulmonary congestion. As the cAMP phosphodiesterase 4 isozyme (PDE4) is currently considered as an intracellular target for new antiinflammatory drugs, several constituents of Vochysia pacifica Cuatrec., an endemic tree from the Western coast of Colombia, were tested for their ability to inhibit PDE4. Purification of the methanol extract of the stem bark of this species led to the isolation of seven known triterpene derivatives: betulinic acid, sericic acid, 24-hydroxytormentic acid, trachelosperogenin B, 24-hydroxytormentic acid glucosyl ester, quadranoside I and niga-ichigoside F1. One triterpene glycoside, quadranoside I, and two triterpenes, betulinic and sericic acids, exhibited mild inhibitory activity on the isolated PDE4 isozyme. Copyright © 2005 John Wiley & Sons, Ltd. Keywords: Vochysia; quadranoside I; betulinic acid; sericic acid; PDE4 inhibition.
INTRODUCTION The approximately 100 species of Vochysia (Vochysiaceae) are large trees and shrubs occurring throughout tropical America from Mexico to Peru. Several of these species are used by traditional Amerindian communities in South America for a variety of therapeutic uses linked to inflammation, such as to treat skin sores and to relieve respiratory ailments, such as asthma and pulmonary congestion (Schultes and Raffauf, 1990). Previous phytochemical work on related Vochysia species has led to the isolation of polyphenols and triterpenes (Zucaro et al., 2000). It is well known that the cyclic nucleotide phosphodiesterase isozyme PDE4 constitutes an intracellular target for antiinflammatory agents and that PDE4 inhibitors can be considered as potential therapeutic agents for the treatment of asthma and chronic obstructive pulmonary disease (Huang et al., 2001). In the course of our investigation on bioactive constituents from Latin American plants, seven known triterpene derivatives were evaluated: betulinic acid, sericic acid, 24-hydroxytormentic acid, trachelosperogenin B, 24hydroxytormentic acid glucosyl ester, quadranoside I and niga-ichigoside F1, isolated from Vochysia pacifica Cuatrec., an endemic tree from the Pacific coast of Colombia, for their possible inhibitory activity on this * Correspondence to: Dr C. Lugnier, UMR/CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur, BP 60024, 67401 Illkirch cedex, France. E-mail:
[email protected] Contract/grant sponsor: ECOS-Nord/ICFES/COLCIENCIAS/ICETEX. Contract/grant sponsor: COLCIENCIAS; Contract/grant number: 111505-353-96. Copyright © 2005 John Wiley & Sons, Ltd. Copyright © 2005 John Wiley & Sons, Ltd.
isozyme, using Rolipram, a selective PDE4 inhibitor, as a positive control.
MATERIALS AND METHODS Plant material. The stem bark of Vochysia pacifica (Vochysiaceae) was collected near Buenaventura, Colombia in March 1999. A voucher specimen (BW136) was determined by Lic. R. T. González and deposited at the Herbarium of the University del Valle (CUVC). Authority to access plant samples used for research was obtained through COLCIENCIAS project No. 1115-05-353-96. Extraction, isolation and structural determination of test material. The defatted powdered stem bark (180 g) was extracted successively with CH2Cl2, EtOAc and n-BuOH at room temperature to give a CH2Cl2-soluble extract (9 g), an EtOAc-soluble extract (2.7 g) and a n-BuOHsoluble extract (14 g). The CH2Cl2 extract was chromatographed on silica gel 60 (230–400 mesh) eluting with CH2Cl2 containing increasing amounts of MeOH to yield five fractions. Betulinic acid (12.5 mg) was obtained from the 10% MeOH eluate (Fr.3), after purification using preparative TLC with CH2Cl2–MeOH (98:2) as eluant. The EtOAc-soluble extract was separated over a silica gel column (230–400 mesh), eluted with a gradient of MeOH in CH2Cl2, affording six fractions. Fraction 4 obtained upon elution with CH2Cl2– MeOH (8:2) was further purified using gel permeation CC (Sephadex LH-20; MeOH) yielding sericic acid (7.5 mg). The same procedure was used with Fr.5, Received 2004 Phytother. Res. 11 19,February 75–77 (2005) Accepted 13 November 2004
76
B. WENIGER ET AL.
obtained upon elution with CH2Cl2–MeOH (7:3), yielding 24-hydroxytormentic acid (11 mg). Part of the concentrated n-BuOH-soluble fraction (100 mg) was fractionated by preparative HPLC [WaterSymetry R Prep C18, 19 × 300 mm, 7 mm column, eluted with a gradient of CH3CN–H2O (0.07% TFA) (10%–30% in 30 min), flow rate = 12 mL/min, detection at 205 nm] to give five fractions (Fr.1–Fr.5). This procedure was repeated five times and the identical fractions were combined. The combined Fr.4 were purified again by preparative HPLC [CH3CN–H2O (26%–30% in 30 min), flow rate = 12 mL/min, detection at 205 nm] to give nigaichigoside F1 (3.5 mg) and quadranoside I (4.5 mg), respectively. Purification of the combined Fr.5 using the same chromatographic conditions gave 24hydroxytormentic acid glucopyranosyl ester (48 mg). NMR spectra were carried out on a NMR Bruker DRX-300 spectrometer at 300 MHz using standard Bruker microprograms. FABMS and HRFABMS were taken on a JEOL-NS700 instrument. Optical rotations
were taken with a Perkin Elmer 241 MC automatic polarimeter. All triterpene derivatives were identified on the basis of physical and spectroscopic data ([α]D20, 1 H NMR, 13C-NMR, MS) compared with literature values. Copies of the original spectra are obtainable from the author. Chemicals. All chemicals were of analytical grade and were purchased from Sigma. Rolipram was a kind gift of Schering (Berlin). PDE4 inhibition method. Cytosolic PDE4 was purified by anion exchange chromatography from the media layer of bovine aorta by a modification of a previously described method (Komas et al., 1991). Its activity was measured by radioenzymatic assay (Keravis et al., 1980) at a substrate concentration of 1 µM cAMP in the presence of 15 000 cpm of [3H]-cAMP (Amersham) as a tracer. The buffer solution contained the following components: 50 µM Tris-HCl pH 7.5, 2 mM magnesium
Figure 1. Structure of betulinic acid, sericic acid and quadranoside I. Copyright © 2005 John Wiley & Sons, Ltd.
Phytother. Res. 19, 75–77 (2005)
77
BIOACTIVE TRITERPENOIDS FROM VOCHYSIA PACIFICA
acetate, 50 µg/mL bovine serum albumin and 1 mM EGTA. To prevent the interaction of contaminating PDE3 in the assay of isolated PDE4, studies with the use of [3H]-cAMP as a substrate were always carried out in the presence of 100 µmol/L cGMP. Rolipram and the isolated compounds were dissolved in DMSO, with a final concentration (1%) which did not affect PDE activity. The inhibition study on PDE4 activity included six concentrations of the drug. The results are expressed as the percentage inhibition of substrate hydrolysis. The IC50 value was calculated by non-linear regression (Prism software) and represented the mean value of three determinations.
Table 1. Inhibitory effects of triterpenoids from Vochysia pacifica and reference compound Rolipram on the cyclic nucleotide phosphodiesterase isoenzyme PDE4 Compound
IC50 (µM)
Betulinic acid Sericic acid 24-Hydroxytormentic acid Trachelosperogenin B 24-Hydroxytormentic acid glycosyl ester Quadranoside I Nigaichigoside F1 Rolipram
125.0 ± 6.9 178.7 ± 8.4 >300 >300 >300 114.7 ± 6.5 >300 1.1 ± 0.2
IC50 values are mean ± SD (n = 3), p < 0.05.
RESULTS AND DISCUSSION Three of the isolated compounds, betulinic acid, sericic acid and quadranoside I (Fig. 1) showed mild in vitro inhibition of PDE4 with IC50 values in the 100 µmolar range (Table 1). Betulinic acid has been isolated previously from many plant sources and has demonstrated several biological activities, and in particular in vitro and in vivo antiinflammatory activity (Safayhi and Sailer, 1997). Antifungal activity has been reported for sericic acid and antihepatotoxic activity for quadranoside I (Hess et al., 1995; Adnyana et al., 2000). The observed inhibitory activities are markedly weaker than those obtained with the selective PDE4 inhibitor Rolipram, but are similar to that reported for visnagin (Duarte et al., 1999) and better than those reported on this isozyme for the natural non-selective reference PDE inhibitor
theophylline (Lugnier et al., 1992). The other four isolated compounds showed IC50 values above 300 µM. These results, in addition with the known antiinflammatory activity reported before for betulinic acid, might support the traditional use of Vochysia spp. as an antiinflammatory remedy. Furthermore, this is the first report showing that betulinic acid, a lupane-type triterpene with selective cytotoxicity on different tumour cell lines (Zuco et al., 2002) is able to inhibit PDE4, a potential target for tumour growth inhibition (Huang et al., 2001). Acknowledgements The authors wish to thank ECOS-Nord/ICFES/COLCIENCIAS/ ICETEX program and COLCIENCIAS (project No. 1115-05-35396) for financial support.
REFERENCES Adnyana IK, Tezuka Y, Banskota AH, et al. 2000. Quadranosides I–V, new triterpene glucosides from the seeds of Combretum quadrangulare. J Nat Prod 63: 496–500. Duarte J, Lugnier C, Torres AI, et al. 1999. Effects of visnagin on cyclic nucleotide phosphodiesterases and their role in its inhibitory effects on vascular smooth muscle contraction. Gen Pharmacol 32: 71–74. Hess SC, Brum RL, Honda NK, et al. 1995. Antibacterial activity and phytochemical analysis of Vochysia divergens (Vochysiaceae). J Ethnopharmacol 47: 97–100. Huang Z, Ducharme Y, Macdonald D, Robichaud A. 2001. The next generation of PDE4 inhibitors. Curr Opin Chem Biol 5: 432–438. Keravis TM, Wells JN, Hardman JG. 1980. Cyclic nucleotide phosphodiesterase activities from pig coronary arteries. Lack of interconvertibility of major forms. Biochim Biophys Acta 613: 116–129. Komas N, Lugnier C, Stoclet JC. 1991. Endothelium-dependent
Copyright © 2005 John Wiley & Sons, Ltd.
and independent relaxation of the rat aorta by cyclic nucleotide phosphodiesterase inhibitors. Br J Pharmacol 104: 495–503. Lugnier C, Gauthier C, Le Bec A, Soustre H. 1992. Cyclic nucleotide phosphodiesterases from frog atrial fibers: isolation and drug sensitivities. Am J Physiol 262: H664– H660. Safayhi H, Sailer ER. 1997. Anti-inflammatory actions of pentacyclic triterpenes. Plants Med 63: 487–493. Schultes RE, Raffauf RF. 1990 The Healing Forest, Medicinal and Toxic Plants of the Northwest Amazonia. Dioscorides Press: Portland, Oregon, 484. Zucaro YL, Compagnonea RS, Hess SC. 2000. Hydromaslinic acid, a triterpene from Vochysia ferruginea. J Braz Chem Soc 11: 241–244. Zuco V, Supino R, Righetti SC, et al. 2002. Selective cytotoxicity of betulinic acid on tumor cell lines, but not normal cells. Cancer Lett 175: 17–25.
Phytother. Res. 19, 75–77 (2005)
