Chemical composition of the essential oil from the leaves of Anaxagorea brevipes (Annonaceae) and evaluation of its bioactivity

Abstract The essential oil obtained by hydrodistillation from leaves of Anaxagorea brevipes was analysed by gas chromatography fitted with a flame ionisation detector (GC–FID) and coupled to mass spectrometry (GC–MS). Thirty one components were identified, representing around 75.7% of total oil. The major components were β-eudesmol (13.16%), α-eudesmol (13.05%), γ-eudesmol (7.54%), guaiol (5.12%), caryophyllene oxide (4.18%) and β-bisabolene (4.10%). The essential oil showed antimicrobial activity against Gram-positive bacteria and yeast with the MIC values between 25.0 and 100 μg/mL. The highest antiproliferative activity was observed for the oil against MCF-7 (breast, TGI = 12.8 μg/mL), NCI-H460 (lung, TGI = 13.0 μg/mL) and PC-3 (prostate, TGI = 9.6 μg/mL) cell lines, while against no cancer cell line HaCat (keratinocyte) the TGI was 38.8 μg/mL. The oil exhibited a small antioxidant activity assessed through ORAC-FL assay (517 μmol TE/g). This is the first report regarding the chemical composition and bioactivity of A. brevipes essential oil.


Introduction
Annonaceae family is widely distributed around the world and is the largest family in Magnoliales with approximately 130 genera and 2500 species. Represented by trees, shrubs, and woody climbers, they are valuable for their large pulpy fruits and others are used in traditional medicine (Richardson et al. 2004). Essential oil of several Annonaceae plants has been demonstrated promissory biological effects such as: insecticidal (Babarinde et al. 2015); spasmolytic (Correia et al. 2015); antitumour and trypanocidal activities . The genus Anaxagorea A. St. Hil includes 26 species with pantropical distribution across the USA and Asia and in Brazil with occurrence in Amazon forest (Lobão et al. 2005). Of the total species of the Anaxagorea, only five were phytochemically investigated, from which lignoids (Puentes De Díaz 1997), flavonoids (Gonda et al. 2002), polyprenoides (Sasak & Chojnacki 1973, xanthones (Gonda et al. 2002), steroids (Puentes De Díaz 1997 and alkaloids were isolated (Hocquemiller et al. 1981). The essential oil of Anaxagorea dolichocarpa showed terpenoid β-elemene in the leaves and the δ-cadinol, caryophyllene oxide, δ-cadineno, α-copaene and the γ-muurolene in the fruits as the main components (Fournier et al. 1994). Investigation of fragrances in the flowers of Anaxagoreaa brevipes and A. dolichocarpa showed esters of aliphatic acids, including ethyl 2-methylbutanoate in A. brevipes and ethyl 3-methylbutanoate in A. dolichocarpa as the major components (Jürgens et al. 2000).
The A. brevipes is commonly known as the 'envira preta' in Colombia, Venezuela, Suriname and in much of the Amazon. To our knowledge, this is the first report on the analysis of the chemical composition and evaluation of antimicrobial, antiproliferative and antioxidant activities of their essential oil and for the bioactivity of this plant.

Essential oils analysis
The hydrodistillation of A. brevipes leaves produced a yellow crude essential oil, with a yield of 0.52%, in relation to the dry weight of the plant material. In total, 31 compounds were identified in the essential oil. The sesquiterpenes were the most abundant constituents with 72.34% of the crude essential oil (Table S1). The major compounds identified in the essential oil of A. brevipes were β-eudesmol (13.16%), α-eudesmol (13.05%), γ-eudesmol (7.54%), guaiol (5.12%), caryophyllene oxide (4.18%) and β-bisabolene (4.10%), and have been described for the first time in this plant. However, some compounds identified in the essential oil of A. brevipes have been described in other species of Anaxagorea (Fournier et al. 1994;Jürgens et al. 2000), indicating that this species is a typical member of the Anaxagorea genus. In addition, some of these compounds are also reported as constituents in the essential oil of other genera of Annonaceae family (Costa et al. 2008;Britto et al. 2012;Thang et al. 2013Thang et al. , 2014. The essential oil of A. dolichocarpa showed as main component the terpenoid β-elemene in the leaves and the δ-cadinol, caryophyllene oxide, δ-cadineno, α-copaene and the γ-muurolene in the fruits (Fournier et al. 1994). The chemical presence of the eudesmols (α, β and γ) as constituents in the essential oil of A. brevipes is interesting from the point of view about the chemistry of the essential oil of Annonaceae species. These compounds α-, β-and γ-eudesmols together are the major compounds as observed only in Guatteria friesiana (W.A. Rodrigues) Erkens & Maas, indicating some chemotaxonomic relationship (Costa et al. 2008;Britto et al. 2012). Further investigations are necessary to confirm this hypothesis.

Biological activity
The essential oil of A. brevipes showed antibacterial and antifungal inhibitory effect, early against Kocuria rhizophila (ATCC 9341), Staphylococcus aureus penicillinase-negative (strain, 8-), Candida albicans (ATCC 10231) and Candida parapsilosis (ATCC 22019) with MIC values between 25.0 and 100 μg/mL (Table S2). This result is very similar to the recent studies performed with Annonaceae plants. These works have demonstrated that essential oils with high sesquiterpene contents, mainly germacrene D, caryophyllene and β-eudesmol, possess antimicrobial properties (Costa et al. 2008;Ho et al. 2010).
The antiproliferative activity of the essential oil from the leaves of A. brevipes was investigated on a series of cancer cell lines and highest activity was observed (Table S3). The bioactivity was particularly found against MCF-7 (breast, TGI (total growth inhibition) = 12.8 μg/mL), NCI-H460 (lung, TGI = 13.0 μg/mL) and PC-3 (prostate, TGI = 9.6 μg/mL) cell lines, while against no cancer cell line Hacat (keratinocyte, TGI = 38.8 μg/mL). Antitumor properties of the essential oil of Annonaceae species were described in vitro and in vivo and germacrene B, germacrene D and caryophyllene were found as the main compounds (Ferraz et al. 2013;Quintans et al. 2013). Also, in vitro and in vivo antitumour effects were documented for the essential oil from the leaves of G. friesiana and these effects could be assigned to its components α-, β-and γ-eudesmol. Therefore, the antiproliferative activity found here is also attributed to the sesquiterpenes constitution of the essential oil of A. brevipes, mainly the presence of α-, β-and γ-eudesmol (Britto et al. 2012). The same result was observed for the antimicrobial activity of the oil of A. brevipes (Costa et al. 2008).
Regarding antioxidant activity, the oil exhibited a small antioxidant activity assessed through TLC autographic assay for DPPH Radical-Scavenging (qualitative method) and by ORAC FL assay (quantitative method). In ORAC FL assay, the antioxidant capacity of the essential oil was 517.04 (15.20) μmol TE/g and in this assay is considered to have good antioxidant capacity with values >800 μmol TE/g (Costa et al. 2011). Similar results were verified for essential oil of other Annonaceae species (Costa et al. 2011;Siqueira et al. 2015).

Conclusion
In this study the chemical composition of the essential oil from the leaves of A. brevipes, and 31 components were identified, representing around 75.7% of total oil. The major components were β-bisabolene, caryophyllene oxide, guaiol and α-, β-and γ-eudesmol. The chemical composition of this oil is in agreement with the chemistry of the Annonaceae family. The essential oil showed antimicrobial and antiproliferative activity. This is the first report regarding the chemical composition and bioactivity of A. brevipes essential oil and the results confirm that the Annonaceae species are natural source of biologically active compounds. Further investigations (in vitro and in vivo) are necessary to confirm the potential of the A. brevipes essential oil as active and to study their toxicity and efficacy toward a clinical employment.

Supplementary material
Experimental details relating to this article are available online, alongside with Tables S1-S3.