Antimycoplasmic activity and seasonal variation of essential oil of Eugenia hiemalis Cambess. (Myrtaceae)

Abstract The purpose of this work was to study the chemical composition and antimycoplasmic and anticholinesterase activities of the essential oil of Eugenia hiemalis leaves collected throughout the year. A total of 42 compounds were identified by CG, and are present in almost every seasons. Sesquiterpenes were dominant (86.01–91.48%), and non-functionalised sesquiterpenes comprised the major fraction, which increased in the summer; monoterpenes were not identified. The major components were spathulenol (5.36–16.06%), δ-cadinene (7.50–15.93%), bicyclogermacrene (5.70–14.24%) and β-caryophyllene (4.80–9.43%). The highest oil yield was obtained in summer and autumn. Essential oils presented activity against three evaluated Mycoplasma strains, but no activity was observed in the anticholinesterase assay. Graphical abstract

Due to their medicinal properties, EOs have important applications as antimicrobial, analgesic, sedative, anti-inflammatory, spasmolytic and local anaesthesic remedies (Bakkali et al. 2008). These properties are related to its chemical composition, which can be affected by environmental and geographic factors, and seasonal and circadian variations (Keszei et al. 2010).
Eugenia genus is characterised by considerable chemical diversity (Frohne & Jensen 1992;Stefanello et al. 2011;Faqueti et al. 2013). Eugenia hiemalis Cambess. (known as 'guamirim') is a tree that is native to Brazil, Argentina, Uruguay and Paraguay (Kawasaki 1989). There are few studies about chemical constitution and biological activities of the extract of the aerial parts (Falkenberg 1996;Monks et al. 2002;Bokesch et al. 2008). The EO of leaves collected in the state of Rio Grande do Sul (Brazil) has already been analysed; however, previous studied did not consider the effects of seasonal variations and climatic conditions (Apel et al. 2004).
Therefore, the present study describes the chemical composition of EO of fresh leaves of E. hiemalis collected in Blumenau (Santa Catarina, Brazil) in all seasons of the year. The antimycoplasmic and anticholinesterase activities of these EOs were also evaluated.

Seasonal chemical composition
The results of the analyses of the chemical composition of EOs collected in all seasons of the year (between 2013 and 2014) and climatic conditions of this period are presented in Table S1 and S2 (Supplementary material). The highest oil yield was obtained in the summer (0.12% v/w), followed by the autumn (0.09% v/w), winter (0.06% v/w) and spring (0.04% v/w). Thus, higher oil content was obtained during the periods with higher temperatures and rainfall. It is known that temperature is an important factor for the enzymatic activity, and some enzymes increase their activity at higher temperatures, i.e. in summer (Barros et al. 2009). Furthermore, since the flowering of this species occurs from March to May (Sobral 2003), the higher production of EOs in this period could be explained for this atypical characteristic in comparison to other Myrtaceae (the name epithet 'hyemalis' is used for winter-flowering species and in this case was graphed by Cambèssedes as 'hiemalis').
An interesting variation between the functionalised and non-functionalised compounds was observed, with an increase in non-functionalised compounds during the summer months, especially α-copaene, α-gurjunene, β-caryophyllene, aromadendrene, germacrene D and bicyclogermacrene. That is contrary to what was generally described for Eugenia EOs in the literature ( Lima et al. 2008;Siebert et al. 2015).
The main components were spathulenol (5.36-16.06%), δ-cadinene (7.50-15.93%), bicyclogermacrene (5.70-14.24%) and β-caryophyllene (4.80-9.43%). They were amongst the four main compounds in at least three of the evaluated seasons. Spathulenol and δ-cadinene were the major compounds in winter and autumn, respectively, while bicyclogermacrene was the major compound in spring and summer; δ-cadinene presented high levels in the other seasons too. Higher contents of β-caryophyllene were found in summer as well as in spring. The calculated coefficients of variation of all compounds in EOs range from 7% to 81%.

Antimycoplasmic activity
The assay was performed against Mycoplasma strains. There have been few studies evaluating the antibacterial activity of natural products against mollicutes. The demand for specific media, the long incubation time, and the fact that the culture medium is easily contaminated may explain why the cultivation of these micro-organisms is considered a difficult challenge (Razin 2006).
The results (Table S3 -Supplementary material) indicated good activity for the analysed EOs, with MIC < 500 μg mL −1 against Mycoplasma capricolum subsp. capricolum, M. hominis and M. pneumoniae FH. The oil obtained from plant material collected in spring seems to be slightly more active than the other oils for M. capricolum subsp. capricolum and M. pneumoniae FH, but the observed differences amongst MIC values for the different seasons are not statistically significant.
These results were more promising than those of previous reports in the literature with EOs from the leaves of E. brasiliensis (Siebert et al. 2015) and Thyme vulgaris (Arjoon et al. 2012).

Anticholinesterase activity
The EOs showed no activity in the acetylcholinesterase assay (IC 50 > 1000 μg mL −1 ), despite the fact that some other Eugenia species presented interesting results, like EOs of E. riedelina (Souza 2009) and E. sulcata (Lima et al. 2012), presenting valerianol and monoterpenes, respectively, as major components.

Conclusions
The chemical composition of the essential oil of leaves of E. hiemalis varies quantitatively throughout the year; sesquiterpenes were predominant, and non-functionalised ones represented the major fraction in the all seasons. Furthermore, all the EOs presented antimycoplasmic activity, but no anticholinesterase activity. Future studies are encouraged, to find the components that are responsible for the antibacterial activity as well as to test EOs against cell wall bacteria.