Fixed and volatile constituents of Croton heliotropiifolius Kunth from Bahia-Brazil

Essential oils from Croton heliotropiifolius Kunth roots and stems, popularly known in Bahia (Brazil) as velame or cassutinga, were obtained by hydro distillation. Fixed oil from the roots of this species was obtained by maceration in hexane. The oils were analyzed by nuclear magnetic resonance (NMR) and gas chromatography coupled with mass spectrometry (GCMS). 46 compounds were identified in the stems and 35 substances were found in the roots. The principal components oils were camphor, β-pinene, and α-pinene in the stems, and camphor, borneol, valencene, and viridiflorol in the roots. The fixed oil contained C8 to C18 fatty acids. It is the first study that reports the presence of fixed oil from the roots of this genus.


INTRODUCTION
Croton is distinguished by its morphological diversity and interintraspecific and it is the second largest genus of Euphorbiaceae, comprising around 1,200 species distributed in tropical and subtropical regions, particularly in America.Brazil is a country that congregates a large number of species, about 350 species (Lima and Pirani, 2008).
Several Croton species have been used in traditional medicine for different purposes (Randau et al., 2004;Vunda et al., 2012).The genus contains various substances with biological activities such as against cancerditerpenoids (clerodane, furoclerodane and acyclic diterpenes), alkaloids (taspine), and essential oils (Salatino et al., 2007).Essential oils have also been evaluated for their larvicidal efficacy against Aedes aegypti, amoebicidal, antifungal, and antimicrobial activities (Salatino et al., 2007;Souza et al., 2006;Rodrigues et al., 2009;Motta et al., 2013).These oils present variable components depending on the climate and soil conditions in which the specimens are found (Maia and Ming 1998).
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License velame or cassutinga, is an aromatic plant, endemic species in the Northeast of Brazil, used in folk medicine as tea for gastrointestinal diseases and to alleviate fever (Randau et al., 2004).Depending on its location, C. heliotropiifolius shows great variation in size and shape of the leaves, fuzz color and length of inflorescences.It is a shrubby species that ranges from 0.7 to 2.5 m in height; it may have colorless latex or orange when oxidized.The branches are cylindrical and gray-green.Alternating leaves sub-opposite the apex of the branches and may be the entire or serrated margin.It can be distinguished from other species mainly by tripartite columella of fruit at the apex after dehiscence of the fruit (Silva et al, 2010).
Some previous studies presented variations in the composition of oils from C. heliotropiifolius stems due to the collection period and the region where the specimen was collected (Araújo-Neves and Camara, 2012;Souza et al. 2010).Araújo-Neves and Camara (2012) identified the volatile constituents of this species present in the stems collected in the region of Pernambuco, Brazil, identified guaiol (18.38%), β-elemene (17.28%) and valerianol (10.62%) as significant components of the oil.Souza et al. (2010) identified as major components of oil from stems as the α-pinene, β-pinene, camphor and germacrene D and found a significant seasonal variation in major constituents, especially the decrease in concentration in the dry season (winter).
Considering the above, this study aimed to analyze the chemical composition of volatile and fixed oils extracted from the stems and roots of C. heliotropiifolius Kunth and to check the alterations of the constituents according to region where the specimen was collected.Moreover, this is the first phytochemical investigation of constituents of fixed and volatile oils presents in the roots of Croton.

Plant material
Roots and stems were collected in the Morro do Chapéu City located at 1011 m above sea level with highland tropical climate in the Chapada Diamantina, Bahia, Brazil.The Croton heliotropiifolius was identified and a voucher specimen is deposited in the Alexandre Leal Costa Herbarium of the Institute o f Biology, at the Federal University of Bahia, Brazil under number 106168.

Oil isolation
Around 100 g of material was triturated and then subjected to hydro distillation for three hours in a modified Clevenger-type apparatus.The oil was collected from a condenser, dried over anhydrous sodium sulfate, and stored at 4°C till it was analyzed.The other portion of dried roots (784 g) of C. heliotropiifolius was macerated with 500 mL of n-hexane at room temperature.The oil was filtered and 6 mL were obtained.The fixed oil was purified by column chromatography in silica gel eluted with 20 mL of n-hexane and dichloromethane (8:2).

Chemical analysis of the oils
The oils were analyzed by gas chromatography coupled with mass spectrometry (GC-MS), the analyses were performed on a Shimadzu QP 2010 model mass spectrophotometer with ionization source of 70 eV and with a split/splitless injector, and a Shimadzu AOC-20i auto injector (Shimadzu, Kyoto, Japan).The volume of 1.0 μL of pure oil for each sample was injected at 220°C in a DBWAX column (polyethylene glycol) 30 m × 0.25 mm × 0.25 μm film thickness (Agilent J&W DB-WAX, Santa Clara, California).The analysis occurred with 1-minute sample time in the split mode, a column flow of 1.3 mL/min, a linear velocity of 41.4 cm/s, and scan between m/z 40 and 500.T h e oven temperature was initially set at 50°C.It was increased by 20°C/min until 240°C was reached, maintaining this temperature for 5 min.The temperature was then once again increased this time by 5°C/min until a temperature of 280°C was reached, maintaining this temperature for 30 min.Inlet pressure: 37.1 kPa.Carrier gas: He, linear velocity (ū): 32.4cm/sec.Injection mode: split (10:1).For identification of compounds, the peaks were compared with some standards, always consulting the libraries WILEY version nº.7 and NIST version nºs. 12 and 62.

Identification of constituents of oils
The chromatograms are shown in Figure 1 and their compositions are presented in Tables 1 to 3. The constituent's fragmentation patterns in the mass spectra were compared with those from the libraries WILEY version nº 7 and NIST version nº 12 and 62.

Essential oil composition
Sesquiterpenes were the major components in all the root and stem oils studied.These results suggest that r e g a r d i n g the distribution and accumulation of monoterpenes and sesquiterpenes of the root and stem oil from C. heliotropiifolius, the chemical composition of the oils is similar to that obtained by Araújo-Neves and Camara (2012) and Souza et al. (2010).
The fatty acids present in the fixed oil were oleic, stearic, pelargonic, and palmitic acids.This was first observed in the family of the latter substance.Prior to this research, fixed oil was only obtained from the stems of C. cajucara Benth.(Souza et al., 2006).

Chemotaxonomic analysis
The variability in the composition of oils shows a characteristic chemical profile of the genus Croton.
In the stems, the main components found were the monoterpenes camphor, β-pinene, and α-pinene respectively.The α-pinene is a terpene with anti-inflammatory, antifungal, and antioxidant activity and was seen in others species of Croton.β-pinene, which has antifungal efficacy, was found in a smaller number of species such as C. argyrophylloides, C. zenhtneri, and C. nepetaefolius (Morais et al., 2006).Camphor is an antiseptic found in the species C. crassifolius Geisel (Yang and Deng, 2009), C. regelianus Muell.Arg.(Torres et al., 2008), C. cajucara Benth (Souza et al., 2006) and C. hieronymi Griseb and the In the roots, the monoterpenes camphor (antiseptic)and borneol (gastroprotective), the sesquiterpenes valencene (flavor and mild oxidant) and viridiflorol (antiseptic, antibacterial, and anti-inflammatory) are the main constituents.Valencene was found in the leaves of C. matourensis and flowers of C. micans Swarts and viridiflorol has not been found in others species of Croton (Compagnone et al., 2010).Borneol is reported principally in barks of Croton urucurana (Simionatto et al., 2007).

Conclusion
This paper describes for the first time the chemical composition of the fixed oil present in C. heliotropifolius's roots, as well as the volatile oils found in the roots and stems of this species with some differences from previous studies, which may be related to the region wherein the plant was collected and the stress level in the specimen used was submitted.Besides contributing to chemosystematics of the genus, this study has the perspectives to investigate the biological activities of the extracts; the phytochemical study of the polar extracts and other parts of the plant, as well as research for the future use of oil as cosmetics.It emphasizes the importance of the study of C. heliotropifolius found in the semi-arid of Bahia in order to contribute to the knowledge of the caatinga biome, unique in the world and has huge variety of plant species, but is threatened by human action.This knowledge can help promote their better preservation, creating jobs and reducing regional inequalities through sustainable exploitation of this and other plant species.

Figure 1 .
Figure 1.Cromatogram of fixed oil from roots from C. heliotropiifolius.

Table 1 .
Major constituents in the essential oil stems (OS) from C. heliotropiifolius

Table 2 .
Major constituents in the essential oil roots (OR) from C. heliotropiifolius

Table 3 .
Major constituents in the fixed oil roots from C. heliotropiifolius.