Essential oil composition of Croton palanostigma Klotzsch from north Brazil

SHORT REPORT

Essential oil composition of Croton palanostigma Klotzsch from north Brazil

Davi do Socorro B. Brasil^{I, III}; Adolfo Henrique Muller^{I, II}; Giselle Maria S. P. Guilhon^I, Cláudio Nahum Alves^I; Eloísa Helena A. Andrade^{I, III}; Joyce Kelly R. da Silva^I; José G. S. Maia^{I, III,*}

^IPrograma de Pós-Graduação em Química, Instituto de Ciências Exatas e Naturais, Universidade Federal do Pará, 66075-900 Belém-PA, Brazil

^IICentro Universitário do Estado do Pará, 66035-170 Belém-PA, Brazil

^IIIFaculdade de Engenharia Química, Universidade Federal do Pará, 66075-900 Belém-PA, Brazil

ABSTRACT

The essential oils of leaves, twigs, branches, trunk bark and fruits of Croton palanostigma were analyzed by GC and GC-MS. The main compounds found in the oil of the leaves were linalool (25.4%), (E)-caryophyllene (21.0%), methyleugenol (17.2%) and β-elemene (6.0%); in the oil of the twigs were α-pinene (41.4%), limonene (29.0%), sabinene (11.5%) and β-pinene (5.7%); in the oil of the branches were methyleugenol (24.1%), (E)-methylisoeugenol (15.3%), α-pinene (11.2%) and (E)-caryophyllene (8.5%); in the oil of the trunk bark were a-pinene (31.6%), methyleugenol (25.6%) and (E)-methylisoeugenol (23.7%); and in the oil of the fruits were linalool (42.7%), methyleugenol (16.3%) and β-elemene (6.4%). Statistical analysis showed that the leaves and fruit, and the branches and trunk bark, have significant similarities between them. In addition, the trunk bark oil has high brine shrimp larvicidal activity (LC₅₀, 3.71 ± 0.01 mg mL^-1).

Keywords: Croton palanostigma, Euphorbiaceae, essential oil composition, α-pinene, linalool, methyleugenol, limonene, brine shrimp larvicidal activity.

RESUMO

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 β-elemeno (6,0%); no óleo dos ramos finos foram α-pineno (41,4%), limoneno (29,0%), sabineno (11,5%) e β-pineno (5,7%); no óleo dos galhos foram metileugenol (24,1%), (E)-metilisoeugenol (15,3%), α-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 β-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 (CL₅₀, 3,71 ± 0,01 mg mL^-1).

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).⁴

Phytochemical 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.⁵ Previously, these results were reported for the species Aparisthmium cordatum Baill., now identified as C. palanostigma.⁶ The species C. palanostigma produces a red viscous sap that was reported to have gastroprotective and gastrointestinal anticancer activities.⁷ The chemical studies from another red sap of Croton spp led to the isolation of the alkaloid taspine,⁸ the dihydrobenzofuran lignans 3',4-O-dimethylcedrusin⁹ and 4-O-methylcedrusin¹⁰ and proanthocyanidins.¹¹

Recently, Salatino and coworkers¹ 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.

Experimental

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 ºC (3 ºC min^-1); injector temperature, 250 ºC; carrier gas, helium, adjusted to a linear velocity of 32 cm s^-1 (measured at 100 ºC); 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 ºC. 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 (LC₅₀) 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

Results and Discussion

The leaves, twigs, branches, trunk bark and fruits of C. palanostigma provided oil yields of 0.7, 0.6, 0.3, 2.2 and 0.5%, respectively and their volatile constituents were analyzed by GC-FID and GC-MS. The individual components of the oils were identified by comparison of both their mass spectra and their GC retention data with those of authentic compounds previously analyzed and stored in the data system. Other identifications were made by comparison of mass spectra with those existing in data system libraries or cited in the literature.^16,17 The main compounds found in the oil of leaves were linalool (25.4%), (E)-caryophyllene (21.0%), methyleugenol (17.2%) and β-elemene (6.0%). The oil of twigs was dominated by α-pinene (41.4%), limonene (29.0%), sabinene (11.5%) and β-pinene (5.7%). The major constituents identified in the oil of branches were methyleugenol (24.1%), (E)-methylisoeugenol (15.3%), α-pinene (11.2%) and (E)-caryophyllene (8.5%). The oil of trunk bark was dominated by the volatiles α-pinene (31.6%), methyleugenol (25.6%) and (E)-methylisoeugenol (23.7%). The principal components found in the oil of fruits were linalool (42.7%), methyleugenol (16.3%) and β-elemene (6.4%). The seventy-one constituents identified in the oils of C. palanostigma are listed in Table 1.

The essential oils of Croton palanostigma are rich in terpenoids and phenylpropanoids. Linalool, α-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, α-pinene and methyleugenol the last group.

The trunk bark oil of C. palanostigma showed a high brine shrimp larvicidal activity (LC₅₀, 3.71 ± 0.01 µg.mL^-1). According to Meyer et al.,¹² crude extracts and pure substances are toxic when LC₅₀ value < 1000 µg mL^-1, that is, the lower the value of LC₅₀, 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.

Acknowledgments

We are grateful for the financial support of FAPESPA/SEDECT, MCT/FINEP, MCT/CNPq and MCT/PPBio, and to Dr Ricardo Secco, from Emílio Goeldi Museum, for the plant identification.

References

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Received: April 16, 2009

Web Release Date: June 26, 2009

Publication Dates

• Publication in this collection
  04 Aug 2009
• Date of issue
  2009

History

• Accepted
  26 June 2009
• Received
  16 Apr 2009