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Rev. latinoam. quím vol.38 n.3 Naucalpan de Juárez Dec. 2010
Antifungal activity of extracts and terpene constituents of aerial parts of Juniperus Lucayana
Yarelis Ortiz Núñezª*, Iraida Spengler Salabarriab, Isidro G. Colladoc, Rosario HernándezGalánc
ª Instituto de Investigaciones Fundamentales en Agricultura Tropical Alejandro de Humboldt (INIFAT), Calle: 2, Esq.1, Santiago de las Vegas, CP 17200, Cuba. *To whom correspondence should be addressed: Tel.: +53 7 57 9010; Fax: + (537) 57 9014; email: firstname.lastname@example.org
b Centro de Estudios de Productos Naturales, Universidad de La Habana, Zapata y G. Vedado, Cuba.
c Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain.
Received July 2010.
Accepted December 2010.
Ethanolic and hexane extracts from the aerial parts of Juniperus lucayana were assayed against the phytopathogenic fungus Botrytis cinérea by the poisoned food technique. The hexane extract showed to have a higher antifungal activity than ethanolic extract. Fractionation of hexane extract by silica gel open column chromatography and HPLC afforded seven known compounds sandaracopimaric acid (1), 4epidehydroabietic acid (2), oplopanone (3), oplodiol (4), nephtediol (5), 7αhydroxycallitrisic acid (6), and 7oxocallitrisic acid (7). Compound 1 showed a significative fungal growth inhibition during the assay. The effect of compounds 27 on spore germination fungus was also studied.
Keywords: Juniperus lucayana, terpenoids, antifungal activity, Botrytis cinerea, growth inhibition, spore germination.
Los extractos etanólicos y nhexánico obtenidos a partir de las partes aéreas de Juniperus lucayana fueron evaluados sobre el hongo fitopatógeno Botrytis cinerea por el método de envenenamiento del medio. El extracto nhexánico mostró una mayor actividad antifúngica que el etanólico. El fraccionamiento del extracto nhexánico mediante cromatografía de columna abierta sobre gel de silice y CLAE permitió el aislamiento de siete compuestos conocidos, el ácido sandaracopimárico (1 ), el ácido 4epidehidroabiético(2), la oplopanona (3), el oplodiol (4), el neftediol (5), el ácido 7αhidroxicallitrisico (6), y el ácido 7oxocallitrisico (7). El compuesto 1 presentó una inhibición significativa del crecimiento del hongo durante el experimento. Adicionalmente se estudió el efecto de los compuestos 27 sobre la germinación de esporas del hongo.
Palabras clave: Juniperus lucayana, terpenoides, actividad antifúngica, Botrytis cinerea, inhibición del crecimiento, germinación de esporas.
In recent years, public pressure to reduce the use of synthetic fungicides in agriculture has increased (Shafique et al., 2007). Concerns have been raised about both the environmental impact and the potential health risk related to the use of these compounds. In contrast, natural productbased fungicides have the ability to decompose rapidly, reducing risk to the environment with the added advantage in that they have both unique modes of action and low mammalian toxicity (Duke et al, 2003; Ortiz et al., 2008). During our search for antifungal substances from Cuban plants, we found that the ethanolic extracts from the wood and aerial parts of Juniperus lucayana Britton (Cupressaceae) exhibited antifungal activity against Botrytis cinerea, with IC50 values ranging from 125 to 250 μg/mL (Ortiz et al., 2004). This fungus is a serious pathogen that attacks economically important crops such as lettuce, carrots, tobacco, strawberry and grapes (Aleu et al., 2001; Daoubi et al., 2005). Juniperus species have been extensively investigated as a source of natural products with potential antimicrobial, acaricidal and insecticidal activities (Karaman et al., 2003; Schmidt, 2004; Barrero et al., 2005). To our knowledge, no fungicidal studies had been carried out on this species. In previous works we reported the bioassayguided fractionation of ethanolic extract from the wood of J. lucayana which afforded three new sesquiterpenes along with six known sesquiterpenes and two flavonoids (Ortiz et al., 2007). In this work, we reported the antifungal activity of the nhexane and ethanolic extracts from the aerial parts of J. lucayana, as well as fractionation of the nhexane extract by open column chromatography and HPLC to afford the isolation of seven compounds, first time reports for this species.
MATERIAL AND METHODS
General experimental procedures
The melting points (m.p.) were determined on a ReichertThermovar apparatus. The optical rotations were measured in CHCl3 solution on a PerkinElmer 341 polarimeter. IR spectra were recorded on a Mattson Genesis spectrophotometer, series FTIR. 1H and 13C NMR measurements were obtained on Varian Inova 400 and 600 MHz NMR spectrometers, using SiMe4 as the internal reference. HPLC was performed with a Hitachi/Merck L6270 apparatus equipped with an UV/Vis detector (L 4250) and a differential refractometer detector (RI71). TLC was performed on Merck Kiesegel 60 F254 layers, 0.2 mm thick. Sephadex LH20 and Sigel (Merck) were used for column chromatography. Semipreparative HPLC purification was conducted using a Sigel column Lichrospher Si60 column (10 μm, 1 cm wide, 25 cm long).
J. lucayana was collected at the Institute of Fundamental Research on Tropical Agriculture ''Alexander von Humboldt'' in Santiago de las Vegas, Cuba, in March, 2005 and identified by Dr. Pedro Sánchez. A specimen of this plant is deposited in the herbarium of the ''Instituto de Ecología y Sistemática de las plantas en Cuba'' with the number HAC42498.
Extraction and isolation
The airdried powdered aerial parts (460 g) of J. lucayana were extracted using successively nhexane and ethanol in a Soxhlet apparatus, for 4 h. The solvent was evaporated under reduced pressure to yield 31.7 g of an ethanol crude extract ( JFetd). The nhexane extract was cooled at 10 0C for 24 h, yielding an insoluble fraction. The defatted hexane extract (JFhex, 15.0 g) was fractionated by Sephadex LH20 column chromatography using a mixture of nhexane, CHCl3, and MeOH (2:1:1) yielding six fractions (F16). TLC revealed that fractions F3 and F4 contained the same products. These fractions were purificated by means of column chromatography on Sigel with CHCl3, and MeOH and CHCl3 mixtures containing increasing percentages of MeOH to give the compounds 1 (91.2 mg) and 2 (19.0 mg). Fraction F1 was subjected to Sephadex LH20 column yielding three fractions (F79), Fraction F8 was purificated by column chromatography on Sigel eluting with MeOH and CHCl3 mixtures containing increasing percentages of MeOH to give 15 fractions (F1024). Finally, fractions F11, F19, and F20 were thus purificated by means of semipreparative HPLC column [mixture of acetone and nhexane (20:80), 2.8 mL. min1] to afford compounds 3 (8.4 mg), 4 (4.3 mg), 5 (11.2 mg), 6 (1.9 mg), and 7 (4.4 mg).
B. cinerea culture used in this work, Bc 2100, was obtained from the ''Colección Española de Cultivos Tipos (CECT)'', Universidad de Valencia, Facultad de Biología, Spain, where a culture of this strain is on deposit.
Poison food technique
Extracts and fractions (F16) were dissolved in ethanol to give final of 500 mg/ L for fractions and concentrations ranging from 125 to 500 mg/L for extracts. Compound 1 was evaluated at 150 mg/L. Antifungal assays were then carried out in accordance with the poison food technique (Soundharrajan et al, 2003). The solutions were added to a glucosemaltpeptoneagar [61 g/L of glucose (20 g)malt (20 g)peptone (1 g)agar (20 g), pH 6.57.0]. The final ethanol concentration was identical in both control and treated cultures. The medium was poured into sterile plastic Petri dishes measuring 9 cm in diameter and 1.0cm diameter mycelial discs of fungus cut from an actively growing culture were placed at the center of the agar plates. Inhibition of radial growth was measured during 6 days. Growth inhibition was calculated as the percentage of inhibition of radial growth relative to the negative control. Two independent assays were conducted, each in triplicate. The results are shown as mean values of colony diameters; (±SD).
Compounds 27 were dissolved in ethanolwater (1:1) at 100 and 200 mg/L. B. cinerea conidia used for this experiment were collected from cultures of fungi growing in the PDA media. The conidia were collected from the plates with 1 mL sterile distilled water, passed through a glass wool filter to remove hyphae, diluted, counted, and immediately used in the bioassay. Spore germination assays were carried out on multiwell microscope slides containing 10 wells. Each well was filled with 3 μL of the compound solutions at 100 and 200 mg/L, sterilized by filtration, together with conidia (2 μL, to a final concentration of 1 x 105 conidias/mL) of the fungus to be tested. Assays with sterile distilled water and waterethanol, were used as controls. The plates were incubated at 25 °C and after 8 h, numbers of germinated and nongerminated conidias were counted in a light microscope and the spore germination percentage was calculated. Three wells per compounds concentration were prepared, and three areas per well were recorded (each containing 50 conidia). The experiment was performed by duplicate.
RESULTS AND DISCUSSION
Ethanolic and hexane extracts from the aerial parts of Juniperus lucayana were assayed against the phytopathogenic fungus B. cinerea, using the poisoned food technique (Soundharrajan et al., 2003). As observed in Figure 1 , the hexane extract (JFhex) proved to be more active than ethanolic extract (JFetd), at different concentrations evaluated. The hexane extract exhibited a significant inhibition in fungal growth at concentrations of 250 and 500 mg/L (50.1 and 61.5%, respectively), while the ethanolic extract was less effective. In both extracts, in concentrations lower than 250 mg/L, a delay in fungal growth was observed.
Although the doses recorded in our experiments are high (125500 mg/L) these results are highly relevant, since a fungicidal effect is not previous observed in products derived from aerial parts of J. lucayana.
As mentioned before, the hexane extract was more effective on B. cinérea than ethanolic extract. Therefore, we decided as a first step to isolate the active principles from hexane extract. The hexane extract was chromatographed over a Sephadex LH20 column (nhexane/CHCl3/MeOH, 2:1:1). All fractions obtained, F16, were also tested against B. cinérea using the same assay. Fractions F1, F3, and F4 exhibited a 59.2, 71.5 and 70% inhibition in the growth of this fungus, respectively, at concentrations
of 100 mg/L (see Figure 2). On the basis of their antifungal activity and their thinlayer chromatographic profiles, fractions F1, F3 and F4 were purified by a combination of Sigel column and semipreparative HPLC analysis affording seven known compounds. The structures of the compounds were identified by physical and spectroscopic data measurement (mp, , IR, 1H NMR, 13C NMR and 2D NMR) and by comparing the data obtained with published values, such as sandaracopimaric acid (()pimara8(14), 15dien19oic acid) (1) (Edwards et al., 1960; Dang et al., 2005), 4epidehydroabietic acid (2) (San Feliciano et al., 1992), oplopanone (2amethyl5aisopropil7αoxomethylbiciclo[4.3.0]nonan2pol) (3) (Su et al., 1995; Kuo et al., 2002), oplodiol (7eudesmene1P,4Pdiol) (4) (Minato and Ishikawa, 1967; Tchuendem et al., 1999), nephtediol ((1S, 4R, 7S)germacra5E, 10(15)diene1,4diol) (5) (Kitagawa et al., 1987), 7ahydroxycallitrisic acid (7ahydroxyabieta8,11,13trien19oic acid) (6) (De Pascual et al., 1983) and 7oxocallitrisic acid (7oxoabieta8,11,13trien19oic acid) (7) (Prinz et al., 2002) (see Figure 3).
Compound 5 is isolated for the first time from a Juniperus species, meanwhile compounds 1, 2, 3, 4, 6, and 7 have been previously obtained from the genus Juniperus (Kuo et al., 1994; San Feliciano et al., 1995; Fang et al., 1996). The isolation of all this compounds from J. lucayana has not before been reported to the best of our knowledge.
The antifungal properties of the compounds 17 was evaluated for first time against B. cinerea, as described in the part experimental. The sandaracopimaric acid (1), evaluated by poison food technique, reduced the radial fungus growth (70.8% growth inhibition) in the first day of assay. However, it showed a weak activity after five days of experiment (52.7% growth inhibition) (see Figure 4). Compounds 27 did not inhibited the spore germination of B. cinerea at concentrations tested (Table 1).
The ethanolic and hexane extracts from J. lucayana showed an important antifungal effect against B. cinerea. The presence of sandaracopimaric acid (1) contributes to this effect. Further studies on the isolated of additional compounds responsible of the activity of the extract are required.
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