Essential Oil Composition of Croton palanostigma Klotzsch from North Brazil

Os óleos essenciais das folhas, ramos finos, galhos, cascas do caule e frutos de Croton palanostigma foram analisados por CG e CG-EM. Os componentes principais determinados no óleo das folhas foram linalol (25,4%), (E)-cariofileno (21,0%), metileugenol (17,2%) e b-elemeno (6,0%); no óleo dos ramos finos foram a-pineno (41,4%), limoneno (29,0%), sabineno (11,5%) e b-pineno (5,7%); no óleo dos galhos foram metileugenol (24,1%), (E)-metilisoeugenol (15,3%), a-pineno (11,2%) e (E)-cariofileno (8,5%); no óleo das cascas do caule foram a-pineno (31,6%), metileugenol (25,6%) e (E)-metilisoeugenol (23,7%); e no óleo dos frutos foram linalol (42,7%), metileugenol (16,3%) e b-elemeno (6,4%). Análise estatística mostrou que as folhas e os frutos apresentam significante similaridade entre si, assim como os galhos e as cascas do caule. Adicionalmente, o óleo obtido das cascas do caule possui elevada atividade larvicida sobre Artemia salina (CL 50 , 3,71 ± 0,01 mg mL).


Introduction
Croton is a genus of Euphorbiaceae comprising about 1300 species widespread in Africa, Asia and South America.Many species are used in the traditional medicine of these continents, especially to treat cancer, diabetes, hypercholesterolemia, malaria and ulcers, among other diseases. 1Croton palanostigma Klotzsch (syn.C. benthamianuns Müll.Arg.) 2 is a medium-sized tree which is native to the Amazon region, known as "marmeleiro" (Brazil) 3 and "sangre de drago" or "sangre de grado" (Peru, Colombia, Venezuela, Guyanas and Bolivia). 4hytochemical studies with the trunk bark of C. palanostigma furnished aparisthman and cordatin, two furan diterpenes with a clerodane skeleton that show anti-ulcer activity similar to cimetidine, a drug used for the treatment of peptic ulcers. 5Previously, these results were reported for the species Aparisthmium cordatum Baill., now identified as C. palanostigma. 6The species C. palanostigma produces a red viscous sap that was reported to have gastroprotective and gastrointestinal anticancer activities. 7The chemical studies from another red sap of Croton spp led to the isolation of the alkaloid taspine, 8 the dihydrobenzofuran lignans 3',4-O-dimethylcedrusin 9 and 4-O-methylcedrusin 10 and proanthocyanidins. 11ecently, Salatino and coworkers 1 have reported the study of the essential oils of about thirty species of Croton.The results indicate that some of these oils are rich in terpenoids and phenylpropanoids and others are rich only in terpenoids. 1 In the work reported here, the essential oils of the leaves, twigs, branches, trunk bark and fruits of C. palanostigma were analyzed by GC-FID and GC-MS.Statistical analysis was performed to determine the similarities of chemical composition of the various plant parts.In addition, a brine shrimp lethality bioassay was carried out to investigate the toxicity of the trunk bark oil.

Plant processing
The specimen C. palanostigma was collected in the locality of Terra Alta, Municipality of Castanhal, Pará, Brazil, in March 2006.The plant was identified by Dr. Ricardo Secco, a specialist on Euphorbiaceae of the Museu Paraense Emílio Goeldi, Belém, Brazil.A voucher of C. palanostigma (MG 182.822) was deposited in the herbarium of Museu Paraense Emílio Goeldi.The moisture contents of leaves, twigs (diameter of approximately 1.5 cm), branches (diameter of approximately 3.5 cm), trunk bark and fruits were calculated after phase separation in a Dean-Stark trap (2 h, 5 g) using toluene.All parts of the plant from C. palanostigma were dried separately at room temperature (5-7 days) and submitted to hydrodistillation (3 h, 100 g each) using a Clevenger-type apparatus.The oils were dried over anhydrous sodium sulfate and their percentage contents were calculated on basis of the plant dry weight.

Oil composition analysis
Qualitative analysis of the volatile compounds was performed on a Thermo DSQII GC-MS instrument, with the following conditions: WCOT DB-5ms (30 m × 0.25 mm; 0.25 μm film thickness) fused silica capillary column; temperature programmed from 60 to 240 °C (3 °C min -1 ); injector temperature, 250 °C; carrier gas, helium, adjusted to a linear velocity of 32 cm s -1 (measured at 100 °C); injection type, splitless (0.1 µL of a 2:1000 hexane solution); the split flow was adjusted to give a 20:1 ratio; septum sweep was a constant 10 mL min -1 ; EIMS, electron energy, 70 eV; ion source temperature and connection parts, 200 °C.The quantitative data of oils were obtained by peak area normalization using a Focus GC-FID operated under the same conditions, except that the carrier gas that was nitrogen.The retention index was calculated for all volatile constituents using a homologous series of n-alkanes.

Brine shrimp bioassay
The brine shrimp (Artemia salina Leach) lethality bioassay was carried out to investigate the toxicity of the essential oils of the trunk bark.Brine shrimp eggs were hatched in artificial salt water and used after 48 h using the method of Parra et al. 12 Experiments were conducted along with control and different concentrations (1, 5 and 10 µg mL -1 ) in a set of three tubes per dose.The percentage lethality was determined by comparing the mean surviving larvae of the test and control tubes.Lethal concentration (LC 50 ) values were obtained from the best-fit line plotting concentration versus percentage lethality. 13

Hierarchical Cluster Analysis (HCA)
The oils were submitted to the HCA technique taking into account their chemical composition and the major components.HCA examines the distances between the samples in a data set and the information is then represented in a two-dimensional plot (dendrogram).The most similar points were grouped forming the clusters and the process was repeated until all the points are inserted into a unique group. 14,15
The essential oils of Croton palanostigma are rich in terpenoids and phenylpropanoids.Linalool, a-pinene, limonene, methyleugenol and (E)-methylisoeugenol were the main compounds.According to Salatino et al, 1 the essential oils of Croton species are rich in terpenoids and phenylpropanoids, or only in terpenoids. 1 Concerning Hierarchical Cluster Analysis (HCA), the resulting dendogram is shown in the Figure 1.It can be observed that the volatile compositions from different parts of C. palanostigma are separated into three groups.The first group comprises the samples of leaves and fruits, the second group is represented by the sample of twigs, and the third group is composed of the samples of branches and trunk bark.Based on this classification we can say that the volatile composition of leaves and fruits, as well as, the branches and trunk bark, are similar to each other.Linalool and methyleugenol characterize the first group, while limonene characterizes the second one, and, finally, a-pinene and methyleugenol the last group.
According to Meyer et al., 12 crude extracts and pure substances are toxic when LC 50 value < 1000 µg mL -1 , that is, the lower the value of LC 50 , the higher the biological activity.So the trunk bark oil of C. palanostigma can be considered highly toxic.

Conclusions
The essential oils of Croton palanostigma furnished volatiles belonging to the classes of phenylpropanoids and terpenoids.The HCA analysis showed that the oils from different parts of the plant are dominated by linalool and methyleugenol in the first group (leaves and fruits), limonene in a second group (twigs) and a-pinene and methyleugenol in the third group (branches and trunk bark).The trunk bark oil of C. palanostigma showed high brine shrimp larvicidal activity.

Figure 1 .
Figure 1.Dendrogram (HCA analysis) for the main compounds identified in the essential oils of C. palanostigma.