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  • br The cluster analysis of chemical data by HCA permitted

    2022-04-26


    The cluster analysis of chemical data by HCA permitted to group chemically the samples in three classes according to the type of SPME fibers and GC-injection types, in order to express their similarities and differences. The dendrograms of Fig. 2 showed that the DVB/CAR/ PDMS and PDMS protocols presented a similarity that exceeds 97% for fruits EO and 92% for leaves EO (Class I). This means that the chemical composition obtained by the two protocols was similar. The analysis of the EO after using the PA fibers (Class II) showed 6% dissimilarity for the fruit oils and 36% dissimilarity for leaves EOs when compared with PDMS and DVB/CAR/PDMS fibers. The data provided by direct splitless injection were different from those obtained by SPME methods and have been grouped in Class III. The profiles of each class (Fig. 3) showed that α-pinene and β-myrcene were detected in high quantities in fruits oils, after the microextraction by all fibers. This confirmed that these are the major molecules found in fruits oils. Concerning leaves oils, it was found that the PDMS and DVB/CAR/PDMS protocols re-vealed a high level of α-pinene, β-myrcene, β-pinene, D-limonene and 3-carene, while PA fiber microextraction detected large amounts of α-pinene, β-myrcene and Cyclofenchene. r> 3.3. Effects of J. Oxycedrus L. essential oils, α-pinene and β-myrcene in cell viability
    A major concern about bioactive compounds from plants is that some can be toxic to non-cancer cells. Thus, taking into account that safety is an essential MIK665 criterion for the discovery of novel drugs (Morobe et al., 2012), the cytotoxic effects of EOs of fruits and leaves from J. oxycedrus L., at concentrations ranging from 0.0195 to 0.625 μl/mL, were investigated through an MTT-assay on a non-cancer human fore-skin fibroblastic cell line (HFF-1). Having into account the results ob-tained with the non-cancer MIK665 (data not shown), the non-toxic con-centrations (0.0195–0.078 to μl/mL) for HFF-1 cells, were further tested in MCF-7aro breast cancer cells.
    The results obtained for the extracts on MCF-7aro cells are pre-sented in Fig. 4. The EO of fruits was the most efficient (p < 0.05,
    p < 0.001) in decreasing cell viability (Fig. 4b). For EO of leaves, only the highest concentration decreased MCF-7aro cell viability (Fig. 4a). Comparing both extracts, the highest concentration of EO fruits induced a reduction of 24.2% in viability of MCF-7aro cells (p < 0.001), while EO of leaves only caused a decrease of 16.8% (p < 0.001). To de-termine if the reduction in cell viability was caused by a loss of cell membrane integrity, the LDH assay was carried out. Results demon-strated that the incubation of MCF-7aro cells with both EOs did not induce LDH release to the medium (Fig. 4f), suggesting that these concentrations did not cause rupture of cell membrane.
    Considering the chemical composition of EOs, it was noted that α-
    Name EO of Leaves
    EO of Fruits
    DVB/CAR/PDMS fiber PDMS fiber Polyacrylate fiber Direct injection DVB/CAR/PDMS fiber PDMS fiber Polyacrylate fiber Direct injection
    The chemical analysis was carried out by HS-SPME-GC–MS using DVB/CAR/PDMS, PDMS and polyacrylate fibers and by GC–MS with direct injection. The empty boxes indicate that the compound was not detected by the used method. The analysis was carried out in three independent experiments and the results were expressed as mean ± SEM.
    Fig. 1. Principal Components Analysis (PCA) of the oil constituents obtained from fruits (a) and leaves (b) of Juniperus oxycedrus L., by HS-SMPE-GC–MS analysis using three SPME fibers (PDMS, DVB/CAR/PDMS and Polyacrylate) and by GC–MS with direct injection.
    Fig. 2. Hierarchical Cluster Analysis (HCA) of the oil constituents obtained from fruits (a) and leaves (b) of Juniperus oxycedrus L. by SMPE-GC–MS analysis using three SPME fibers (DVB/CAR/PDMS, PDMS and PA) and by GC–MS with direct injection.
    pinene and β-myrcene were the most abundant compounds found in both EOs extracts. Therefore, the effects of α-pinene and β-myrcene, isolated or in combination, were also evaluated on MCF-7aro cell via-bility, in the proportions that were found in EOs from fruits and leaves. The concentrations of β-myrcene and α-pinene that were present in the final concentration of 0.078 μl/mL of EO of fruits were 7.04 μM and 8.01 μM, respectively, while in the case of EO of leaves were 2.3 μM of α-pinene and 3.37 μM of β-myrcene. Results showed that these pure compounds reduced, in a dose-dependent manner, the viability of MCF-7aro cells (Fig. 4c and 4d). Curiously, in our conditions and in the concentrations tested, the combination of β-myrcene and α-pinene were less effective than the isolated compounds (Fig. 4e).