Natural matrices are important sources of new antitumor and antimicrobial compounds. Species such as
Natural matrices, like
Nowadays, there is a worldwide concern about the use of synthetic chemical compounds as antitumor agents due to their potential negative health effects, opening ways to use plants as sources of natural compounds with similar activity [
There are some reports on the antitumor potential of
Despite the previous findings, and as far as we know, this is the first study exploring
Cultivated
Fetal bovine serum (FBS), L-glutamine, Hank’s balanced salt solution (HBSS), trypsin-EDTA (ethylenediaminetetraacetic acid), nonessential amino acids solution (2 mM), penicillin/streptomycin solution (100 U/mL and 100 mg/mL, resp.), RPMI-1640, and DMEM media were from HyClone (Logan, UT, USA). Acetic acid, ellipticine, sulforhodamine B (SRB), trypan blue, trichloroacetic acid (TCA), and Tris were from Sigma Chemical Co. (Saint Louis, USA). Mueller-Hinton agar (MH) and malt agar (MA) were obtained from the Institute of Immunology and Virology, Torlak (Belgrade, Serbia). Dimethyl sulfoxide (DMSO) (Merck KGaA, Germany) was used as a solvent. Phosphate buffered saline (PBS) was obtained from Sigma Chemical Co. (St. Louis, USA). Methanol and all other chemicals and solvents were of analytical grade and purchased from common sources. Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, USA).
Methanolic extracts were obtained from cultivated and wild plant material. Each sample (
For aqueous extracts, plant material (
Methanolic and aqueous extracts were redissolved in water (8 mg/mL) or 5% DMSO (10 mg/mL) for antitumor and antimicrobial activity evaluation, respectively. The final solutions were further diluted to different concentrations for bioactivity evaluation.
Five human tumor cell lines were tested: MCF7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), HCT15 (colon carcinoma), HeLa (cervical carcinoma), and HepG2 (hepatocellular carcinoma). Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium containing 10% heat-inactivated FBS and 2 mM glutamine (MCF7, NCI-H460, and HCT15) or in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (HeLa and HepG2 cells), at 37°C, in a humidified air incubator containing 5% CO2. Each cell line was plated at an appropriate density (7.5 × 103 cells/well for MCF-7, NCI-H460, and HCT15 or 1.0 × 104 cells/well for HeLa and HepG2) in 96-well plates. Sulforhodamine B assay was performed according to a procedure previously described by the authors [
For hepatotoxicity evaluation, a cell culture (PLP2) was prepared from a freshly harvested porcine liver obtained from a local slaughter house, according to an established procedure [
The following gram-positive bacteria:
The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were determined by the microdilution method. Briefly, fresh overnight culture of bacteria was adjusted by the spectrophotometer to a concentration of 1 × 105 CFU/mL. The requested CFU/mL corresponded to a bacterial suspension determined in a spectrophotometer at 625 nm (OD625). Dilutions of inocula were cultured on a solid medium to verify the absence of contamination and check the validity of the inoculum. Different solvent dilutions of methanolic extract/fractions were placed in the wells containing 100
For the antifungal bioassays, the following microfungi were used:
The fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v/v). The spore suspension was adjusted with sterile saline (
For wild and cultivated plant material, three samples were used and all the assays were carried out in triplicate. Data were expressed as means ± standard deviations, maintaining the decimal places allowed by the magnitude of standard deviation.
An analysis of variance (ANOVA) with type III sums of squares was performed using the GLM (general linear model) procedure of the SPSS software. The dependent variables were analyzed using 2-way ANOVA with the factors “extract” (E) and “origin” (O). When a statistically significant interaction (E×O) was detected, the two factors were evaluated simultaneously by the estimated marginal means plots for the two levels of each factor. Alternatively, if no statistical significant interaction was verified, means were compared using results obtained for EB and GI that were classified using a simple
Principal components analysis (PCA) was applied as pattern recognition unsupervised classification method. The number of dimensions to keep for data analysis was assessed by the respective eigenvalues (which should be greater than one), by Cronbach’s alpha parameter (that must be positive), and also by the total percentage of variance (that should be as high as possible) explained by the number of components selected. The number of plotted dimensions was chosen in order to allow meaningful interpretations.
All statistical tests were performed at a 5% significance level using the SPSS software, version 20.0 (SPSS Inc.).
The interactions among
Table
Phenolic compounds (mg/g) of different extracts of
Total flavan-3-ols | Total flavones | Total flavonols | Total phenolic | |
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Methanolic |
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Aqueous |
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O×E | ||||
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The detailed phenolic profile of all laurel samples was previously described by Dias et al. [
The interaction among factors was again significant in all cases, except MCF7 line (Table
Antitumor activity and hepatotoxicity (GI50,
MCF7 | NCI-H460 | HCT15 | HeLa | HepG2 | PLP2-hepatotoxicity | |
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Origin (O) | ||||||
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Extract (E) | ||||||
Methanolic |
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Aqueous |
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O×E | ||||||
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Ellipticine |
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Extract type and origin had a significant interaction in the antibacterial activity against all species, except
Antibacterial activity (MIC and MBC, mg/mL) of different extracts of
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MIC | ||||||||
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Wild |
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Extract (E) | ||||||||
Methanolic |
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Aqueous |
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O×E | ||||||||
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Ampicillin |
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Streptomycin |
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MBC | ||||||||
Origin (O) | ||||||||
Cultivated |
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Wild |
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Extract (E) | ||||||||
Methanolic |
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Aqueous |
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O×E | ||||||||
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Ampicillin |
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Streptomycin |
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All presented MICs were much better than those obtained by Al-Hussaini and Mahasneh [
The interaction among factors was once more significant in almost all cases, except MIC values for
Antifungal activity (MIC and MFC, mg/mL) of different extracts of
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MIC | ||||||||
Origin (O) | ||||||||
Cultivated |
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Wild |
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Extract (E) | ||||||||
Methanolic |
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Aqueous |
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O×E | ||||||||
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Bifonazole |
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Ketoconazole |
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MFC | ||||||||
Origin (O) | ||||||||
Cultivated |
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Wild |
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Extract (E) | ||||||||
Methanolic |
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Aqueous |
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O×E | ||||||||
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Bifonazole |
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Ketoconazole |
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Concerning fungicidal activity, MFCs varied from 0.03 to 0.6 mg/mL and from 0.03 to 0.5 mg/mL for cultivated and wild samples, respectively. Both origins had the same effect on
For both origins and extracts,
After analysing individually each bioactivity indicator and the contents in phenolic compounds, PCA was applied to understand the true effect of either origin or extract type in a global manner. That is, instead of evaluating individual changes caused in each bioactivity indicator or phenolic compounds group, it was intended to obtain an integrated output dealing with all the effects at once. The plot of component loadings forextract type was designed with the first two dimensions (first: Cronbach’s
Biplot of objects (extraction solvents) and component loadings (evaluated parameters).
Group corresponding to cultivated samples extracted with methanol (solid grey line ellipse) was characterized by the high amounts of bioactive compounds, specifically flavonols, flavones, and total phenolics, and its high bioactivity against bacteria (
The most distinctive features in cultivated samples extracted with water (solid black line ellipse) were the low content in flavan-3-ols, despite having high antifungal activity against
A third group (dashed grey line ellipse), corresponding to wild samples extracted with methanol, was characterized as having high antibacterial (
The high bioactivity of wild methanolic extracts might be related to their high content in flavan-3-ols, mainly epicatechin and procyanidin trimer with an A-linkage [
Similarly, wild samples extracted with water (dashed black line ellipse) had the reverse behavior in comparison to cultivated samples extracted with methanol. These aqueous extracts were mostly active against
The extract type induced the most marked changes in bioactivity of laurel samples. Furthermore, each of the assayed factors (origin and extract type) acts in a differentiated manner; that is, the same evaluated parameter gave sometimes statistically significant differences regarding laurel origin, but no effect at all from extract type or vice versa. From the PCA biplot, it became clear that wild laurel samples were more effective to inhibit tumor cell lines growth, especially HeLa, MCF7, NCI-H460, and HCT15. HepG2, as previously highlighted, had the same reaction to laurel from wild and cultivated origin. It was also observed that methanolic extracts tended to have higher antimicrobial activity, except
The most interesting finding in this work was the bioactive specificity of each laurel extract, considering its wild or cultivated origin. In fact, from the obtained results it is possible to choose the combination extract type/origin with potentially highest effect against determined bacteria, fungi, or tumor cell line. These findings should, however, be analysed within the geographical area of study, considering eventual specific features of the used samples.
The authors declare that they have no conflict of interests regarding the publication of this paper.
The authors are grateful to Fundacão para a Ciência e a Tecnologia (FCT, Portugal) for the financial support to CIMO (strategic project PEst-OE/AGR/UI0690/2011) and REQUIMTE (PEst-C/EQB/LA0006/2011). M. I. Dias, R. Calhelha, and J. C. M. Barreira also thank FCT, POPHQREN, and FSE for their Grants (SFRH/BD/84485/2012, SFRH/BPD/68344/2010, and BPD/72802/2010). The authors also thank to Serbian Ministry of Education and Science for financial support (grant number 173032).