On-line version ISSN 1678-7064
Quím. Nova vol.32 no.2 São Paulo 2009
José F. CiccióI,*; Carlos ChaverriI; Cecilia DíazII
ICentro de Investigaciones en Productos Naturales and Escuela de Química, Universidad de Costa Rica, 2060 San José, Costa Rica
IIInstituto Clodomiro Picado, Facultad de Microbiología and Departamento de Bioquímica, Escuela de Medicina, Universidad de Costa Rica, 2060 San José, Costa Rica
The chemical composition of the volatiles of Nectandra salicina growing wild in Costa Rica was determined by capillary GC/FID and GC/MS. Thirty-seven and forty-two compounds were identified in the leaf and branch oils respectively corresponding to about 92.6 and 86.2% of the total amount of the oils. The major components of the leaf oil were: atractylone (14.6%), viridiflorene (10.1%), α-pinene (9.4%), β-caryophyllene (7.2%), α-humulene (7.0%), δ-cadinene (6.1%), β-pinene (6.0%) and germacrene D (5.8%). The major components of the branch oil were: atractylone (21.1%), germacrene D (10.7%), viridiflorene (7.9%) and 7-epi-α-selinene (5.0%). When the oils were tested on different cell lines, all the LD50 values were higher than 150 µg/mL, with values very similar for the leaf and branch oils. Low toxicity could be explained by antagonistic effects among the main compounds present in the oils.
Keywords: Nectandra salicina; volatiles; cytotoxicity.
Nectandra is a New World genus constituted by approximately 100 to 150 species, ranging from Florida to Argentina, with the majority of species present in South America.1 In Costa Rica, this genus is represented by about 20 species. It belongs to the Lauraceae family, which is present with abundance and diversity of species in the Cloud Forests of Costa Rica, together with plants of the Leguminosae (Fabaceae) family.2 The majority of the species produce relatively small fruits which are of great ecological importance for the sustenance of several mammals such as monkeys and kinkajous, and birds such as quetzals and toucans.2,3 The Lauraceae family is recognized by the simple, alternate, stiff and aromatic elliptic to obovate leaves, and by the fruits often borne in a cup. Worldwide, this family has a considerable economic value because it is used as a source of timber for construction and furniture, as food (Persea americana Mill., Avocado), to obtain flavors for food industry, drinks and perfumery and medicines [Cinnamomum camphora (L.) J. Presl., Camphor Laurel].
Nectandra salicina C. K. Allen is a tree (5-10 m tall) from evergreen forests, which is found in both the Caribbean and Pacific slopes of Costa Rica, from about 600 to 1,700 m of elevation. It is commonly known as ira, canelo and aguacatillo4 and is recognized by its small lustrous narrowly elliptic and acuminated leaves, with the tertiary veins, usually prominent on both surfaces. Inflorescences are small, few-flowered with pink red rachises and puberulent little flowers. Fruits are ellipsoid to globose and borne in shallow cups.1
Many members of the Nectandra genus have been chemically investigated and they are mainly characterized by the occurrence of alkaloids,5-10 lignans,11-13 neolignans,14-17 tetrahydrofuranoid lignans,18 norlignans,19 dehydrodieugenols20 and γ-lactones.21,22
The composition of several essential oils from N. angustifolia (syn. N. falcifolia),23-25 N. coriacea,26 N. elaiophora27 and N. rigida28 has been published. However, to the best of our knowledge, only one previous report on the composition of the leaf oil from N. salicina appears in the scientific literature.29
Leaves and branches of Nectandra salicina C. K. Allen, Lauraceae, growing wild in Costa Rica were collected in June 2001, in Fraijanes, Miramar, Province of Puntarenas, Costa Rica. A voucher specimen was deposited at the Herbarium of the University of Costa Rica (USJ 76991).
Fresh leaves (1.0 kg) and chipped fresh branches (1.5 kg) were subjected to hydrodistillation for 3 h using a modified Clevenger-type apparatus. The distilled light yellow oils were collected and dried over anhydrous sodium sulfate and stored in a freezer (0-10 ºC). Leaf and branch essential oil yields were 0.1% (v/w) and 0.2% (v/w), respectively.
General analytical procedures
The oils of N. salicina were analyzed by GC/FID using a Shimadzu GC-17 gas chromatograph. The data were obtained on a 5% phenyl- 95% methylpolysiloxane fused silica capillary column (30 m x 0.25 mm; film thickness 0.25 µm), Heliflex (Alltech) AT-5, with a Shimadzu Class-VP, version 4.3 software. Operating conditions were: carrier gas N2, flow 1.0 mL/min; oven temperature program: 60-220 ºC at 3 ºC/min, 220 ºC (10 min); sample injection port temperature 250 ºC; detector temperature 275 ºC; split 1:50.
The analyses by GC/MS were performed using a Shimadzu GC-17A gas chromatograph coupled with GCMS-QP5050 apparatus and CLASS 5000 software with Wiley 139 and NIST computer databases. The data were obtained on a 5% phenyl- 95% methylpolysiloxane fused silica capillary column (30 m x 0.25 mm; film thickness 0.25 µm). Operating conditions were: carrier gas He, flow 1.0 mL/min; oven temperature program: 60-220 ºC at 3 ºC/min; sample injection port temperature 250 ºC; detector temperature 260 ºC; ionization voltage: 70 eV; ionization current 60 µA; scanning speed 0.5 s over 38-400 amu range; split 1:70.
Identification of the oils components was performed using the arithmetic retention indices (RI) on DB-5 type column,30 and by comparison of their mass spectra with those published in the literature31 or those of our own database. Integration of the total chromatogram, expressed as area percent, has been used to obtain quantitative compositional data.
Mouse macrophage J774, human hepatoma HepG2, human leukemic K562 and mouse myoblastic C2C12 cell lines were obtained from American Type Culture Collection (ATCC). Cells were maintained in Dulbecco essential medium supplemented with 10% fetal bovine serum, 2 mmol/L of glutamine, 100 IU/mL of penicillin and amphotericin B in a 37 ºC humidified incubator under an atmosphere of 7% CO2 in air. For the experiments, adherent cells were cultured in 96-well plates to confluence (15,000 cells/well) and allowed to adhere overnight. Non-adherent cells were also plated at 15,000/well.
Various concentrations of essential oils, previously dissolved in 95% ethanol, were added to the plates in 100 µL of fresh medium and incubated for 48 h. After that, 10 µL of [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) (0.5 mg/mL) was added to the culture and, after 2 h at 37 ºC, medium was carefully removed and 95% ethanol was added to the wells with the purpose of dissolving formazan crystals. Absorbances were read at 570 nm and results were expressed as viability percentages, using samples incubated with 95% ethanol dissolved in culture medium as 100% viability values. LD50 values were calculated with SlideWrite® Plus 6.1 (Advanced Graphics Software, Inc., Carlsbad, CA).
RESULTS AND DISCUSSION
N. salicina leaf and branch oil constituents are listed in Table 1. As it can be observed, 37 components were identified from the leaf, representing 92.6% of the essential oil. Monoterpene hydrocarbons (17.4%), sesquiterpene hydrocarbons (51.1%) and oxygenated sesquiterpenes (22.4%) were the main constituents of the oil, and contained the monoterpenes α-pinene (9.4%), β-pinene (6.0%); the sesquiterpenes viridiflorene (10.1%), β-caryophyllene (7.2%), α-humulene (7.0%), δ-cadinene (6.1%) and germacrene D (5.8%), and the oxygenated sesquiterpene atractylone (14.6%), as a main constituents. From the branch, 42 compounds were identified, representing 86.2% of the oil. This oil is constituted mainly of sesquiterpene hydrocarbons (40.7%) and oxygenated sesquiterpenes (36.2%). The major components of the branch oil were atractylone (21.1%), germacrene D (10.7%), viridiflorene (7.9%) and 7-epi-α-selinene (5.0%).
N. salicina from Costa Rica produced oils which are terpenoid in nature, as well as the oil from leaves of N. rigida from Brazil, which contains α- and β-phellandrenes (72.8%)28 and the oil from the leaf of N. angustifolia from Argentina, which contains p-menth-1(7),8-diene (25.2%) and terpinolene (20.9%), as main constituents.25 The major component of both leaf and branch oils of N. salicina from Costa Rica was the oxygenated sesquiterpene atractylone (14.6 and 21.1% respectively). In the leaf oil, lesser amounts of the monoterpenes α-pinene (9.4%) and β-pinene (6.0%); and the sesquiterpenes: viridiflorene (10.1%), β-caryophyllene (7.2%), α-humulene (7.0%), δ-cadinene (6.1%) and germacrene D (5.8%) are also present. In the branch oil, together with atractylone, we also found sesquiterpenes germacrene D (10.7%), viridiflorene (7.9%) and 7-epi-α-selinene (5.0%).
A previous report on the leaf volatile composition of N. salicina29 (from the Species Collection at South Coast Research and Experiment Station, UC, Riverside, USA), had indicated that the main constituents were indene (30.1%) and the furanocoumarin methoxsalen (24.5%). In our study, which characterizes the leaf oil of the plants growing in Costa Rica, the composition differs both qualitative and quantitatively from that previous report. We were unable to find any evidence of the presence of indene or methoxsalen in the leaf or branch volatiles of this wild tree. On the other hand, these results could be identifying a different chemotype of N. salicina, rich in atractylone, or could be reflecting edaphic and climate factors (ecotypes), since plants were not grown in the same environments.
When both volatiles were tested on four different cell lines: leukemic and hepatoma cells and two non-tumor cells (macrophages and myoblasts), low toxicity was observed. There was a little variation in the LD50 values on tumor cells, which were around 175 µg/mL for the branch oil whereas for the leaf oil, values were over 230 µg/mL (Figure 1). Both volatiles gave almost identical LD50 values on non-tumor cells C2C12 and J774 (from 125 to 200 µg/mL) (Figure 2).
Almost no cytotoxic effect was previously observed at the concentration of 100 µg/mL with Nectandra membranacea on the hepatoma cell line HepG2,32 the same cell line tested in our study. Some studies showed that leaf oils from Ocotea veraguensis, O. whitei and Persea americana, other members of the Lauraceae family, have no cytotoxic effect on mammary ductal carcinoma (MDA-MB-435) and ovarian adenocarcinoma (OVCAR-5), but they show toxicity at concentrations of 100 µg/mL in other mammary adenocarcinoma (MCF7, MDA-MB-468, MDA-MB-231) and malignant melanoma UACC-257.33
In an interesting study, Wright et al.34 previously showed that essential oil components such as β-caryophyllene and α-humulene present antagonistic effects when combined with α-pinene. Since these are some of the main compounds present in N. salicina volatiles, it could partially explain the low cytotoxic activities observed in this study.
Also, even though it has been shown that atractylone has antiproliferative activity on leukemia cell lines (HL-60 and P-388) and normal peripheral blood mononuclear cells and is able to trigger apoptosis,35 its presence seems insufficient to induce high toxicity on the cells tested here. However, different atractylone concentrations present in N. salicina leaf and branch volatiles could partially explain differences in LD50 values observed between both samples.
This is the first report in the literature that shows the composition and cytotoxic characterization of the essential oils obtained from leaves and branches of N. salicina. It seems clear that, as other members of the Lauraceae family, these oils have low toxicity, possibly due to antagonistic effects induced by their main compounds, since some of these compounds (α- and β-pinene, β-caryophyllene, α-humulene), when tested individually, are able to induce high toxicity on tumor cells.34
The authors are grateful to Vicerrectoría de Investigación (UCR) (Project 809-A4-006) for financial support, to L. J. Poveda (Escuela de Ciencias Ambientales, Universidad Nacional) for the botanical identification and to L. Hernandez (CIPRONA) for her technical assistance. Partial funding was also obtained from FEES through Project No. 809-A8-518.
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Recebido em 1/8/08; aceito em 8/9/08; publicado na web em 5/2/09