Chemical investigations of male and female leaf extracts from Schinus molle L.

Abstract The pepper-tree Schinus molle is an evergreen ornamental plant with various and diversified list of medical uses. In this article we analysed the chemical composition of male and female leaves of this plant during the off-flowering and flowering seasons. The leaf extracts were obtained by using a sequential extraction with solvents of different polarities and the chemical composition was investigated by GC-MS. The results showed a total of twenty-three components, in which elemol is the most abundant constituent followed by bicyclogermacrene, γ-eudesmol, α-eudesmol, β-eudesmol and isocalamendiol. The petroleum ether and diethyl ether extracts from male and female flowering and off-flowering leaves consisted of sesquiterpene hydrocarbons as a major constituent followed by monoterpene hydrocarbons, while the acetone extracts showed a different composition. The obtained results show differences in the chemical composition between male and female and flowering and not flowering.


Introduction
Schinus molle L. is an evergreen plant belonging to the Anacardiaceae family. Commonly called 'pink' or 'false pepper' , S. molle is a dioecious plant with compounds, imparipinnate and lanceolate leaves having a peppery smell when crushed. It has pendulous branches with yellowish-white flowers arranged in clusters; the fruits are coral-red in the size of peppercorns (Kasimala and Kasimala 2012).
Essential oils purified from S. molle are mainly constituted by monoterpenoids and sesquiterpenoids, among which the major components were α-phellandrene and sylvestrene (Bendaoud et al. 2010).
Different studies carried out on essential oils have shown that the sexual differences in dioecious plant (Sharma et al. 2015), the vegetative cycle or physiological variations (Figueiredo et al. 2001), the seasonal variations (Choudhry et al. 2014) and the geographical and environmental factors (Borges et al. 2017) can affect the chemical variability concerning plant composition and/or secondary metabolites productions (Figueiredo et al. 2008).
Based on these evidences, we aimed to investigate the chemical composition of leaf extracts from male and female S. molle plants collected during the off-flowering season and the flowering season. The samples were obtained by using a sequential extraction with solvents of different polarities (petroleum ether, diethyl ether and acetone), with the purpose to achieve a selectivity for the extracted phytochemicals polarity. The extracts were then chemically analyzed by using GC-MS.

Results and discussion
The composition of the male and female S. molle petroleum ether extract from leaves in the flowering and off-flowering periods reveals that the main component of the considered stages was elemol (53.68 and 65.83%) for the male and (69.53 and 54.82%) for female, respectively (Table S1). The second most abundant component was bicyclogermacrene (9.73 and 11.59%) for the male and germacrene D (15.58 and 15.23%) was for the female. Elixene (1.21 and 1.71%) and spathulenol (0.64 and 2.22%) were present only in the male leaves while α-thujene (0.72% for male and 0.99% for female), germacrene D-4-ol (5.20% for male and 8.31% for female) and n-hexadecanoic acid (0.24% for male and 0.32% for female) were present only in the off-flowering season's leaves. Viridiflorol (4.85%) was present only in the female leaves of off-flowering season. The composition of the male and female S. molle diethyl ether extract from leaves in the flowering and off-flowering periods are reported in Table S2. As for petroleum ether extracts, elemol (57.57 and 72.58%) for the male and (71.37 and 60.80%) for the female was the main component and bicyclogermacrene (9.56 and 7.76%) was the second most abundant component for the male while the germacrene D (11.39 and 10.86%) was for the female. Elixene (1.46 and 1.11%) and spathulenol (3.22 and 0.65%) were present only in the male leaves. α-thujene (0.67% for male and 0.72 for female), germacrene D-4-ol (1.65% for male and 3.83% for female) and n-hexadecanoic acid (0.89% for male and 10.41% for female) were present only in the leaves of off-flowering season. In acetone extract the number of identified components was lower than the other two extracts. 2-pentanone, 4-hydroxy-4-methyl (57.47% for the male and 59.92% for the female) was the most abundant component of extract obtained from off-flowering leaves (Table S3). N-hexadecanoic acid (49.77% for the male), was the most abundant component of extract obtained from flowering male leaves and absent in the female flowering leaves. The percentage (9.98 and 17.42%) of elemol from the male was lower if compared to the extract in diethyl ether and in petroleum ether and remained high for the female (51.89 and 25.72%). In the male flowering season leaves β-eudesmol (24.68%) was the most abundant component. Sabinene (0.75 and 0.39%), humulene (1.07 and 0.30%), δ-cadinene (0.96 and 0.84%) and 2-propanone, 1-hydroxy (1.04 and 0.32%) were present only in the female leaves.
The GC/MS analysis of the extracts showed elemol as the most abundant component in the petroleum ether and diethyl ether extracts while its percentage was lower in the acetone extract. A total of twenty-three components were identified by GC-MS. The petroleum ether and diethyl ether extracts from male and female flowering and off-flowering leaves consisted of sesquiterpene hydrocarbons as a major constituent followed by monoterpene hydrocarbons. The acetone extracts showed different composition and the extracts from the male flowering and off-flowering samples were characterized by the presence of n-hexadecanoic acid (49.77%) and 2-pentanone, 4-hydroxy-4-methyl (57.47%), respectively. On the other hand, the extracts from the female flowering and off-flowering leaves were mainly characterized by the presence of 2-pentanone, 4-hydroxy-4-methyl (17.86 and 59.92%).
Taken together, the obtained results suggest that the use of solvents having different polarity grade is very important for investigations of the extracts from S. molle and clearly show differences in the chemical composition between male and female flowering and off-flowering leaves. Further investigations will need to point out if such chemical differences could be related to the different role of male and female plants in the fertilization process.
There are many studies related to the extracts composition of S. molle leaves, berries and aerial parts from different regions of the world (Bendaoud et al. 2010;Salem et al. 2016). As reported by dos Santos Cavalcanti et al. (2015), the different chemical composition can be related to various factors. The main factor that determines the concentration of each compound is its genotype (Gomes et al. 2013). However, variations in the extract contents from plant tissues should be also dependent from different factors, some of them intrinsic and controlled by the plant's genetic variability and others dependent on the extraction method applied. Furthermore, quantitative profiles are susceptible to climatic conditions, such as seasonality, water availability and soil nutrients (dos Santos Cavalcanti et al. 2015).

Experimental
Supplied as supplementary materials.

Conclusions
Our investigations confirm that the extracts of S. molle are an important source of chemical constituents and in this view further studies need to address such compounds for possible uses in aromatherapy and pharmacy.

Disclosure statement
No potential conflict of interest was reported by the authors.