Pentacyclic triterpenoids with antimicrobial activity from the leaves of Vernonanthura patens ( Asteraceae )

Vernonanthura patens (Kunth) H. Rob, is native to Mexico and South America and found in dry or wet forests or pine forests. They are reaching in height from 0 to 1865 meters above sea level (masl). They are distributed throughout South America, in Central America: Mexico, Belize, Costa Rica, El Salvador, Guatemala, Honduras, Nicaragua, Panama, and northern South America: Venezuela, western South America: Bolivia, Colombia, Ecuador and Peru. Three pentacyclic triterpenoids from the leaves were isolated by chromatographic (Colum Chromatography (CC) and Thin Layer Chromatography (TLC)) techniques. The structures of these isolated compounds were identified by 1H NMR and 13C NMR spectroscopic and MS analysis as lupeol, epilupeol and acetyl lupeol. Lupeol and epilupeol showed antifungal activity against Fusarium oxysporum and Penicillium notatum, whereas acetyl lupeol was inactive.


Introduction
Vernonanthura patens (Kunth) H. Rob (Asteraceae) is a wild plant broadly distributed throughout America.It grows from 0 to 2200 meters above sea level in the Ecuadorian region.Its leaves are cooked and used against malaria, for postpartum treatment and for healing infected wounds of animals as part of a plant mixture (Blair, 2005;Kvist, 2006).It is also used against headaches, treatment of leishmaniasis (Gacheta, 2010), preparation of antivenos (Tene, 2007), and to combat athlete's foot (Valadeau, 2009).Its usefulness for treating certain types of cancer has also been referred to by indigenous healers.However, there are scarce biological and chemical studies on this species.The only results published so far refer to the antimalarial activity against Plasmodium falciparum, Itg2 strain (Blair, 2005), anti-Leishmania activity (Valadeau, 2009) of the leaves and lack of antiprotozoal activity against different strains of Leishmania (Fournet, 1994).On the chemical composition of the species, there are reports of sesquiterpene lactones and sesquiterpenes present in the aerial parts (Mabry, 1975: Jakupovic, 1986).There are some references on Vernonanthura species that shows the presence of diterpene compounds (Portillo, 2005;Valadeau, 2009), flavonoids (Borkosky, 2009;Mendonça, 2009), triterpenes (Tolstikova, 2006;Gallo, 2009), saponins (Borkosky, 2009) and sesquiterpene lactones.In addition, different biological activities have been described assuming that certain chemical groups could be responsible for the therapeutic properties attributed to species of this genus (Pollora, 2003(Pollora, , 2004;;Portillo, 2005;Bardó n, 2007).In this work, we performed the extraction and fractionation of leaves of the species in the vegetative state using column chromatography increasingly polar solvent and thin layer chromatography.Through these procedures, three triterpenoids isolated, which were identified using Nuclear Magnetic Resonance Spectroscopy of 1 H and 13 C.For these compounds, antimicrobial activity was determined against Fusarium oxysporum and Penicillium notatum.

Plant material
Mature leaves of Vernonanthura patens (laritaco) were collected at the vegetative state in the Marcabelí town, province of El Oro, Ecuador, with the following coordinates S 3°47' 06,14" W 79°54' 32.66", to 316,58 m.Leaves were collected in the early morning in February 2011.A voucher specimen is on deposit in the National Herbarium of Ecuador (QCNE) and a duplicate sample (CIBE37) is kept as a herbal witness in the laboratory of the CIBE-ESPOL Bioproducts.

Extraction and isolation
Coarsely ground leaves (67 g) were extracted by maceration, with two liters of methanol (HPLC grade) in a closed container and in the absence of light and with agitation in a New Brunswick Scientific shaker model C-10.The extraction time was 8 days and was conducted until total depletion of plant material.The crude extract was evaporated to dryness on a rotary evaporator Heidolph brand Laborota 4001 model with reduced pressure at 50°C.
Mass spectra were performed on an Agilent 7890A gas chromatograph, with Agilent 5975 detector (Avondale, PA.USA).
NMR analyses [ 1 H NMR (500 MHz) and 13 C NMR (125 MHz)] were recorded at room temperature with a Bruker DRX500 spectrometer with an inverse multinuclear 5 mm probe head equipped with a shielded gradient coil.The spectra were recorded in CDCl 3 , and the solvent signals (7.26 and 77.0 ppm, respectively) were used as reference.

Bioassays
The diffusion method with potato dextrose agar (PDA) was used to determine the antifungal activity of three isolated pure compounds from V. patens leaves at 100 and 200 μg/mL (Avello, 2009).Dilutions were made with 10% dimethylsulfoxide (DMSO).Strains of Fusarium oxysporum and Penicillium notatum were isolated from infected Pinus radiata and Citrus sinense fruits and maintained in the Collection of Fungi at the University of Concepcion-Chile.
Holes of 5 mm diameter were made in the agar with a sterile cork borer and filled with 20 μL of each concentration of pure compound.DMSO 10% was used as a negative control in each plate.A disc (5 mm diameter) of already grown fungus was placed in the center of Petri dishes and incubated at 22°C.Radial growth assessment was made during 2 weeks.
Experimental design was completely randomized and each assay was performed in triplicate.Descriptive statistics of the experimental data were made in order to represent and point out its most important features.
In Tables 1 and 2, the spectroscopic data for lupeol and epilupeol are presented for the identified compounds compared with those reported in the literature.
The most relevant antifungal activity was observed in the pure compounds 1 and 3 at both concentrations tested.The pure compounds showed selective inhibition properties and a certain concentration dependence on its antifungal activity.Compound 1 showed a rate of inhibition of 50 and 90% (100 and 200 μg/mL respectively) against Penicillium notatum, while compound 3 was capable to inhibit 80 and 100% of Fusarium oxysporum growth for each assayed concentrations.
Compound 2 showed no antifungal activity against the microorganisms tested.Statistical differences (P ≤ 0.05) with negative controls indicated that DMSO did not influence the results of biological evaluation (Figure 1).

Figure 1 .
Figure 1.Percent Inhibition of isolated compounds V. patents against F. oxysporum and P. notatum at different concentrations.

Table 1 .
1 HNMR shifts values of compounds 1 and 3 and reference compounds used as ( H ).