Chemical Composition and Cytotoxicity Evaluation of Essential Oil from Leaves of Casearia Sylvestris, Its Main Compound α-Zingiberene and Derivatives

Casearia sylvestris (Salicaceae), popularly known as “guaçatonga”, is a plant widely used in folk medicine to treat various diseases, including cancer. The present work deals with the chemical composition as well as the cytotoxic evaluation of its essential oil, its main constituent and derivatives. Thus, the crude essential oil from leaves of C. sylvestris was obtained using a Clevenger type apparatus and analyzed by GC/MS. This analysis afforded the identification of 23 substances, 13 of which corresponded to 98.73% of the total oil composition, with sesquiterpene α-zingiberene accounting for 50% of the oil. The essential oil was evaluated for cytotoxic activity against several tumor cell lines, giving IC50 values ranging from 12 to 153 μg/mL. Pure α-zingiberene, isolated from essential oil, was also evaluated against the tumor cell lines showing activity for HeLa, U-87, Siha and HL60 cell lines, but with IC50 values higher than those determined for the crude essential oil. Aiming to evaluate the effect of the double bonds of α-zingiberene on the cytotoxic activity, partially hydrogenated α-zingiberene (PHZ) and fully hydrogenated α-zingiberene (THZ) derivatives were obtained. For the partially hydrogenated derivative only cytotoxic activity to the B16F10-Nex2 cell line (IC50 65μg/mL) was detected, while totally hydrogenated derivative showed cytotoxic activity for almost all cell lines, with B16F10-Nex2 and MCF-7 as exceptions and with IC50 values ranging from 34 to 65 μg/mL. These results indicate that cytotoxic activity is related with the state of oxidation of compound.


Results and Discussion
Drug discovery from plants still provides important new drugs, especially for cancer. Using this approach, several plants with metabolites that exhibit antitumor activity have been described [27]. Aiming at the discovery of new anti-tumoral compounds from Brazilian flora, fresh leaves of C. sylvestris were extracted by steam distillation for 5 h, giving 2.92 g of a pale yellow oil (corresponding to a yield of 0.23% w/w). The analysis of its chemical components was performed by gas chromatography associated with mass spectrometry using a mass spectra library (LIB NIST107) for identification and comparison with the mass spectra available in the literature [28], strictly considering fragmentation patterns, and Kovats indexes of each constituent. This analysis allowed the identification of 23 substances (Table 1), corresponding to 98.73% of the total oil composition. The oil was shown to be composed mainly of α-zingiberene (48.31%) followed of the E-caryophyllene (14.27%), γ-muurolene (5.16%), viridiflorene (5.07%), and acoradiene (4.11%), contrary to previously analyzed essential oil, in which bicyclogermacrene, with about 41%, followed by β-caryophyllene (18%) and spathulenol (16%) were described as the major components [22,29]. Other constituents that were also observed as major components of the essential oil of C. sylvestris were β-elemene (31.7%) [30] and germacrene D [21]. These differences in chemical composition can be attributed, at least in part, to several factors such as the presence of certain micronutrients in the soil, temperature, environmental factors and chemotypes [31]. Considering that α-zingiberene represents about 50% of crude oil from C. sylvestris, and in order to evaluate the biological activities of the pure component, this material was fractionated over a SiO 2 /AgNO 3 column to afford 200 mg of α-zingiberene, which was identified by 1 H-, 13 C-NMR and LREIMS.
In the 1 H-NMR spectrum of α-zingiberene signals assigned to the olefinic protons were observed at δ 5.38 (br s, H-2), δ 5.57 (dd, J = 9.8 and 2.6 Hz, H-4), δ 6.70 (dd, J = 9.8 and 2.0 Hz, H-5) and δ 5.02 (br t, J = 7.0 Hz, H-10). Hydrogens of the methyl groups linked to the sp 2 carbons of double bonds C-12, C-13 and C-15 showed peaks at δ 1.61 (s), δ 1.64 (s) and δ 1.68 (d, J = 2.0 Hz), respectively, while methyl hydrogens of the C-14 was detected at δ 0.83 (d, J = 6.2 Hz). Additional peaks ranging from δ 1.16 to δ 2.14 correspond to remaining hydrogens (H-1, H-6, H-7, H-8 and H-9). The 13 C-NMR spectrum showed fifteen signals, being six at the region of double bonds (δ 120-135), referring to carbons C-2, C-3, C-4 C-5, C10 and C-11, besides signals at δ 26.2, 17.9, 17.8 and 21.3 corresponding to methyl groups in C-12, C-13, C14 and C-15 respectively, thus confirming its structure as α-zingiberene ( Figure 1) [32,33]. α-Zingiberene was further subjected to analysis by LREIMS and its mass spectrum showed the molecular ion [M + ] at m/z 204, confirming the formula C 15 H 24 , which characterizes the compound as α-zingiberene [28]. The initial fragmentation with the loss of hexenyl radical (C 6 H 11 ) with m/z 83 generates the fragment at m/z 121. The additional loss of a molecule of H 2 with simultaneous rearrangement generates the observed base peak at m/z 119. The peak at m/z 91 indicates the formation of a benzene ring with alkyl side chain, the cation tropilium. Despite the occurrence of α-zingiberene as the most important compound of the oil of ginger (Zingiber officinale) [32], this is the first reported occurrence of this sesquiterpene as the main constituent in the essential oil from leaves of C. sylvestris. Aiming at establishing the relationships between the structure/activity of α-zingiberene, this was subjected to a double bond reduction reaction by catalytic hydrogenation. Initially α-zingiberene was mixed with H 2 and Raney-Ni catalyst. After purification the product was subjected LREIMS analysis to confirm the hydrogenation. The mass spectrum revealed a molecular ion at m/z 208, i.e., four units of mass higher than the α-zingiberene, indicating that only two double bonds were hydrogenated. The mass spectrum indicated the loss of a hexyl fragment radical (m/z 85) to give a monounsaturated [C 9 H 15 ] + cation peak at m/z 123. A further fragmentation of this peak with loss of ethylene produced the base peak of spectrum m/z 95 (Scheme 1). This pattern indicated the reduction of the exocyclic double bond and one of the ring alkenes, suggesting that the partially hydrogenated product corresponds to the PHZ structure shown in Figure 1. α-Zingiberene was also further subjected to hydrogenation under more drastic conditions. LREIMS analysis of the purified product showed the molecular ion at m/z 210, confirming that α-zingiberene was fully hydrogenated, as shown in Figure 1 (THZ).
The crude oil as well as purified α-zingiberene were evaluated for its antitumor activity in vitro, initially against murine melanoma cell line B16F10-Nex2. The obtained results indicated that the essential oil was cytotoxic against this cell line with an IC 50 value of 61.5 μg/mL. Further, the cytotoxic potential of the crude oil, α-zingiberene and their hydrogenated derivatives were evaluated against different human tumor cell lines (Table 2). Positive control assay was carried out with cisplatin (standard drug), with IC 50 of 176.0 μg/mL on B16F10-Nex2 cells. The crude oil displayed activity against all cell lines tested with IC 50 values ranging from 12 to 42 μg/mL, being HL60 the most sensitive cell line to the crude oil, with an IC 50 of 12.0 μg/mL. Otherwise, the purified α-zingiberene from the essential oil showed a cytotoxic activity against HeLa, U-87, Siha and HL60 cell lines, but the IC 50 values were higher than those obtained for the crude oil, indicating that there may be a synergism or additive activity between α-zingiberene and other substances present in the crude oil for the observed activity against these cell lines. However, when evaluated against B16F10-Nex2 cell line, α-zingiberene cytotoxicity was intensified, with an IC 50 value of 27.0 μg/mL, compared to the crude oil (61.5 μg/mL), suggesting that this sesquiterpene may be primarily responsible for the cytotoxic activity against this particular cell line.
Additionally, it was observed that at the tested concentrations PHZ was only active against the B16F10-Nex2 cell line, with an IC 50 of 65.2 μg/mL, and IC 50 values greater than 200 μg/mL for the other tumor cell lines, indicating that PHZ has low cytotoxic activity against these tumor cells.
The totally hydrogenated zingiberene (THZ) showed cytotoxic activity against almost cell lines, except for B16F10-Nex2 and MCF-7, with IC 50 values ranging from 34-65 μg/mL. The higher lipophilicity of totally hydrogenated zingiberene, compared to other compounds, could be responsible for enhanced cell penetration through the plasma membrane.
Although there are no studies concernng the cytotoxic activity of sesquiterpenes isolated from oil of C. sylvestris, Salvador et al. [34] showed that essential oil from C. lasiophylla were cytotoxic against tumor cell lines U251, UACC-62, MCF-7, NC1-ADR/RES, NCI-H460, PC03, OVCAR-3, HT-29 K562. Silva et al. [22], also showed the cytotoxic activity of the crude essential oil of leaves of C. sylvestris collected in Campinas against several tumor cell lines (A-549, HT-29 and HeLa). However, these oils were shown to be composed by bicyclogermacrene and β-caryophyllene as main compounds, a different profile of that detected in the present work.

Plant Material
Leaves of Casearia sylvestris with no deformation and degradation by exogenous factors were selected and collected with the aid of sterile pliers from a single tree at the Atlantic Forest area in São Paulo city, SP, Brazil (coordinates 23 53'08.86''S, 46 40'10.45''O), in October, 2012.

Essential Oil Extraction and Analysis
Fresh leaves (1,278 g) of C. sylvestris were extracted over 5 h by steam distillation in a Clevenger type apparatus to afford 2.9 g of crude essential oil. The oil was analyzed by GC-FID and GC-LR-EI-MS using a RtX-5 capillary column. The identification of the individual compounds was performed by comparison of retention indexes (determined relatively to the retention times of a series of n-alkanes) and comparison of recorded mass spectra with those available in the system library [28]. GC chromatograms were obtained on a Shimadzu GC-2010 gas chromatograph equipped with an FID-detector and an automatic injector (Shimadzu AOC-20i) using a RtX-5 (5% phenyl, 95% polydimethylsiloxane (Restek, Bellefonte, PA, USA, 30 m × 0.32 mm × 0.25 μm film thickness) capillary column. These analyses were performed by injecting 1.0 μL of a 1.0 mg/mL solution of volatile oil in CH 2 Cl 2 in a split mode (1:10) employing helium as the carrier gas (1 mL/min) under the following conditions: injector and detector temperatures of 220 °C and 250 °C, respectively; oven programmed temperature from 40-240 °C at 3 °C/min, holding 5 min at 240 °C. GC-FID was performed in quantitative analysis. The percentage compositions of the oil samples were computed by internal normalization from the GC peak areas without using correction for response factors. GC-LR-EI-MS analysis was conducted on a Shimadzu GC-17A chromatograph interfaced with a MS-QP-5050A mass spectrometer. Helium was used as the carrier gas. The LR-EI-MS operating conditions were an ionization voltage of 70 eV and an ion source temperature of 230 °C with the same conditions described above.

Hydrogenation of α-Zingiberene
In a high-pressure reactor (stainless steel), pure α-zingiberene (90 mg, 0.44 mmol), Ni-Raney catalyst (10%, 9 mg; Aldrich; pore size: 50 μm, superficial area: 80-100 m 2 /g) and hexane (5 mL) were added. After addition of H 2 (8 atm), the mixture were stirred for 3 h at 90 °C. Then, the reactor was cooled and the product was filtered [35]. After solvent evaporation, 30 mg (33%) of a colorless oil was obtained, which was characterized as PHZ.  (3H, H-15). This data suggests that the remaining double bond is located as shown in Figure 2, but there are some inconsistencies of these multiplicities with the theoretical spectrum and we did not have sufficient sample for more extensive NMR studies that would have allowed us to confirm this structure unambiguously. This product was fully hydrogenated, under more drastic conditions (26 atm H 2 , Ni-Raney 20%, 30 mg, 120 °C, 3 h) to obtain 60 mg (65%) of the fully saturated product THZ.

Cell Lines
The murine melanoma cell line B16F10 was originally obtained from the Ludwig Institute for Cancer Research (São Paulo, Brazil). The melanotic B16F10-Nex2 subline, characterized at the Experimental Oncology Unit (UNIFESP-Federal University of São Paulo), is characterized by low immunogenicity and moderate virulence. Human melanoma (A2058), glioblastome (U87), leukemia (HL-60), uterus carcinoma (Siha) and breast cancer (MCF-7) cell lines were obtained from the Ludwig Institute for Cancer Research. Human cervical carcinoma (HeLa) was acquired from Dr. Hugo Pequeno Monteiro (UNIFESP).

In Vitro Cytotoxic Activity
The essential oils extracted from leaves of C. sylvestris, as well as pure α-zingiberene, zingiberene partially hydrogenated and zingiberane, were resuspended in dimethyl sulfoxide (DMSO) at a final concentration of 10 mg/mL, diluted in RPMI medium containing 10% fetal calf serum ranging from 100 to 0 µg/mL and incubated with 1 × 10 4 cells in a 96-well plate. After 18 h of incubation, cell viability was measured using the Cell Proliferation Kit I (MTT) (Sigma, St. Louis, MO, USA), an MTT-based colorimetric assay [36]. Readings were made in a plate reader at 570 nm. All experiments were performed in triplicate.

Conclusions
Essential oil from leaves of C. sylvestris analyzed in this work showed a different profile in comparison to other previously analyzed oils, being composed by approximately 50% of α-zingiberene. The cytotoxic evaluation displayed that the crude essential oil was active against all cell lines tested, while purified α-zingiberene showed less activity, indicating that there may be synergism of zingiberene with other substances present in the crude oil for activity. For PHZ, the cytotoxic activity decreased, while THZ showed cytotoxic activity against almost all the tested cell lines. These findings suggests that oil from C. sylvestris may be an interesting agent for cancer treatment; however additional studies must be done including isolation of active compound, mechanism of action on tumor cells as well as experimental assays.