Abstract
Several Lippia species have been used in folk medicine mainly for gastrointestinal and respiratory diseases. Their biological properties have been partially associated to the terpenoids found in their essential oils. According to the World Health Organization, cancer is the leading cause of death worldwide and is described as a complex group of diseases with several hallmarks. One of its acceptable defining features is the cell proliferation beyond their boundaries forming the tumors. Importantly, some drugs currently available were discovered by the investigation of plant secondary metabolites. Thus, this study aimed to evaluate in vitro cytotoxic effect of the essential oils extracted from five Lippia species against tumor cell lines. The results indicated that mouse colon carcinoma CT26.WT cell line viability was significantly reduced showing an IC50 of 19.05, 30.20 and 36.30 µg/ml when treated with the essential oils of L. sidoides, L. salviifolia and L. rotundifolia, respectively. Human lung carcinoma A549 cell line also had a compromised viability to the action of L. alba carvone chemotype essential oil. The tested essential oils did not compromise viability of the normal cell line CHO. These finds suggest that the studied Lippia essential oils might be good candidates for further in-depth studies.
Cytotoxicity; Essential oil; Lippia; Terpenes
Introduction
It has been reported that incidence and mortality rates for most cancers are decreasing in the United States and many other western countries, however they are increasing in several less developed and economically transitioning countries. The proportion of new cancer cases diagnosed in less developed countries is projected to increase from about 56% of the total world in 2008 to more than 60% in 2030 (Jemal et al., 2010). Worldwide, lung cancer is the most common in term of cases and deaths. Colorectal cancer is the third most common cancer in men and the second in women, and breast and prostate cancer are the leading cause of cancer death in women and men worldwide, respectively (Ferlay et al., 2010).
Several strategies, including surgery, radio and chemotherapy have been developed to treat different types of cancer. Chemotherapy has proven efficient in many cases, however the development of resistance and toxicity continues to be problems during the treatment. Therefore, it is still essential the identification of new agents demonstrating chemotherapeutic and chemopreventive activities. In this sense the monoterpenes have been suggested as good candidates for the study of antitumor agents. Shoff et al. (1991) demonstrated that the monoterpene geraniol increase the population doubling time of leukemia and melanoma cells. Several authors demonstrated that synthetic geraniol compromise growth of in vitro tumor cell lines and the wild variety of in vivo tumors types, including hepatoma, pancreatic and colon which is highly resistant to chemotherapy (Yu et al., 1995; Burke et al., 1997; Duncan et al., 2004; Carnesecchi et al., 2001; 2002; Ong et al., 2006; Wiseman et al., 2007). In the same way, the monoterpene limonene exerts therapeutic activity against breast, skin, liver, lung and stomach tumors in rodents (Elegbede et al., 1986; Wattenberg and Coccia, 1991; Crowell and Gould, 1994; Kawamori et al., 1996; Crowell, 1999). Additionally, tumor-suppressive activity has been reported to monoterpenes like carvone, carveol, mentol and perillyl alcohol (He et al., 1997).
Brazil has one of the richest biodiversity worldwide with nearly 19% of the world flora (MMA, 1998). Monoterpenes are found in several plant species, including species of the Lippia genus. Furthermore, Brazil has one of the main diversity centers of the genus Lippia, which is located at the Espinhaço Mountain Range, in the State of Minas Gerais, Brazil (Salimena-Pires 1991; Viccini et al., 2004). The genus Lippia belongs to the Verbenaceae family and comprises approximately 200 species of herbs, shrubs and small trees (Sanders, 2001). Several species of the genus Lippia are used for treatment of respiratory disorders, being employed as remedy for colds, grippe, bronchitis, coughs and asthma (Caceres et al., 1991; Forestieri et al., 1996; Pascual et al., 2001; Jean et al., 2012). They are also commonly utilized as gastrointestinal remedies and as seasoning for food preparation (Hennebelle et al., 2008; Oliveira et al., 2007, Pascual et al., 2001). Additionally, some Lippia species are used to treat hepatic diseases, burns, wounds, fever, syphilis, gonorrhea, diarrhea, dysentery, malaria, among others. (Pascual et al., 2001). Importantly for the two best studied species of this genus, L. alba and L. sidoides, previous studies reported antioxidant activity, indicating that these plants might be a potential source of antitumor biomolecules (Ramos et al., 2003; Monteiro et al., 2007). Yet, other specie is which antioxidant activity was reported was L. salviifolia (Silva, 2008). The pharmacological properties of the Lippia species have been related to the components of their secondary metabolism, specifically to their essential oils (Pascual et al., 2001). The main constituents of Lippia essential oil are the monoterpenes, and according to the major monoterpene present in their oil they can be classified in different chemotypes (Pascual et al., 2001). Although several pharmacological studies have been performed for many Lippia species, very few studies were performed for the ones native to Brazil, such as L. lacunosa and L. rotundifolia (Jardim Botânico do Rio de Janeiro, 2013).
The aim of the present study was to chemically characterize the essential oils extracted from L. alba, L. sidoides, L. salviifolia, L. rotundifolia and L. lacunosa and to evaluate their effect on viability of tumor cell lines.
Materials and methods
Plant material
Fresh leaves were collected for each one of the Lippia species, Verbenaceae, L. alba (Mill.) N.E. Brown geraniol and carvone chemotypes, L. sidoides Cham., L. salviifolliaCham., L. rotundifolia Cham. and L. lacunosa Mart. and Schauer at the Experimental Station located on the campus of the Federal University of Juiz de Fora, Juiz de Fora, Brazil (21º46'48.4"S 43º22'24.4" W). Each one of the Lippia species was collected in the early morning or late afternoon, without rain, from October 2009 to February 2010. The voucher specimens of the Lippia species evaluated in this study are deposited at the CESJ Herbarium of Federal University of Juiz de Fora and the voucher specimens numbers are: L. alba geraniol chemotype: 48374, L. albacarvone chemotype: 48463, L. sidoides: 49007, L. salviifolia: 47444, L. rotundifolia: 31376 and L. lacunosa: 51920.
Essential oil extraction
The essential oils from fresh leaves of the Lippia species were obtained separately by hydrodistillation in a Clevenger-type apparatus for 4 h. The oils were measured and aliquots of 5 mg of each one of them were stored at -80ºC in sealed vials covered with aluminum foil until use. For each one of the assays described below one aliquot was defrost and dissolved in 4% dimethyl sulfoxide, DMSO (Sigma, St. Louis, MO, USA) and purified water, making up a stock solution of 1 mg/ml.
Gas chromatography/mass spectrometry analysis
The chemical composition of the essential oil of each Lippia specie was determined by gas chromatography coupled to mass spectrometry performed on a Shimadzu QP5050A GC/ MS instrument, equipped with a PTE-5 Supelco column (30 m × 0,25 mm × 0,25 μm). The carrier gas was helium (1 ml/ml). The column temperature ranged from 30º to 250ºC. The split rate was 1:20 and the injector and interface temperature were 250ºC. The total analysis time was 39.17 min with a flux of 19.8 ml/min. Retention indexes (RI) were calculated from retention times generated from the analysis of each oil in comparison with the standard n-alkanes solution, C8-C20, analyzed, and used to determine the components of each one of the essential oils, according to Adams (1995). The amount of compounds was determined by peaks area integration of the spectrograms.
Cell line and culture condition
Mouse colon carcinoma CT26.WT cells were provided by Dra. Lucíola Bastos from the Department of Physiology and Biophysics, Biological Sciences Institute, Federal University of Minas Gerais, Brazil; human lung carcinoma A549 cells was obtained by cell bank of Rio de Janeiro, Brazil; human mammary gland adenocarcinoma MDA MB-231 cells was provided by Dr. Ricardo R. Brentani from Ludwig Institute for Cancer Research, São Paulo, Brazil; human colon adenocarcinoma CACO-2 cells was obtained by cell bank of Rio de Janeiro, Brazil and the hamster normal ovary CHO cells was obtained by Pan American Foot-and-Mouth Disease Center, Rio de Janeiro, Brazil. Each one of the cancer cell lines were cultured in appropriate medium at 37ºC with 5% CO2. CT26.WT, MDA MB-231 and CHO were grown in RPMI 1640 medium, pH 7.4 (Cultilab, Campinas, SP, Brazil) supplemented with 10% fetal bovine serum (FBS), 0.1 mg/ml ampicillin, 0.1 mg/ml kanamycin, 0.005 mg/ml amphotericin, 0.2% NaHCO3 and 0.2% HEPES (Sigma, St. Louis, MO, USA). A549 and CACO-2 were grown in DMEM medium (Cultilab, Campinas, SP, Brazil) supplemented with 10% FBS, 0.1 mg/ml kanamycin, 0.005 mg/ml amphotericin and 0.37% NaHCO3 (Sigma, St. Louis, MO, USA).
Cytotoxicity assay
The cytotoxicity of all six essential oils were measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetratozolium bromide] (Sigma, St. Louis, MO, USA) colorimetric assay (Denizot and Lang, 1986). Cells from each cell line were seeded into 96-well plates at a density of 2 × 103 cells/well in 100 µl of appropriated medium supplemented with 10% FBS. After 48 h of incubation at 37ºC, cells were treated with medium supplemented with 10% FBS containing different concentrations of each one of the six essential oils, separately. The stock solutions of each essential oil were diluted in four concentrations, 0.1, 1.0, 10 and 100 µg/ ml. The negative control samples contained 0.4% DMSO, which is equivalent to the percentage found in the highest concentration evaluated. The positive control samples contained 5-fluorouracil (5-FU), an analogue of the pyrimidine uracil and one of the oldest anticancer agents (Casale et al., 2004), at four different concentrations, 0.1, 1.0, 10 and 100 µM. The blank control contained only RPMI medium supplemented with 10% FBS. After 72 h of incubation at 37ºC, medium was discarded and 10 µl tetrazolium dye (MTT) solution (5 mg/ ml in PBS) was added to each well. Cells were incubated at 37ºC for 4 h, then the solution was discarded and 100 µl of dimethyl sulfoxide (DMSO) was added to dissolve the formed formazan crystals. The microplates were read in a TP-reader (Thermoplate) at 540 nm.
Statistical analysis
At each experimental plate, all the samples were repeated six time and all MTT assays were performed in different independent experiments. The results were analyzed using the software GraphPad Prisma 5.0 (GraphPad Software, Inc.). Each treated group and the control was compared by Bonferroni Test and when p < 0.05 the differences were considered significant. Cell viability was calculated using the following equation: A/C × 100, where A is the arithmetic average of the OD of each sample and C is the arithmetic average of the OD observed in the negative control. The cell viabilities values were used to calculate the concentration that corresponds to 50% of cell lethality (IC50), through linear regression using the software Sigma Plot 10.0 (Systat Software, Inc.).
Results
Composition of the essential oils obtained from five Lippia species
Table 1 presents the compounds identified by gas-chromatography followed by mass spectrometry in the essential oils of L. alba geraniol and carvone chemotypes, L. sidoides, L. salviifolia, L.rotundifolia and L. lacunosa. Approximately fourteen main compounds were detected, but to the most of them the contents were very low. For L. alba geraniol chemotype the compounds with the highest percentages were geranial and citral and to carvone chemotype were carvone and limonene. In the oil of L. sidoides the main compounds were thymol and o-cymene. The major constituents of L. salviifolia essential oil were nerolidol and germacrene D and of L. rotundifolia were β-myrcene and (E,Z)-, (E,E)-, and (Z,Z)-farnesol. Finally, the most abundant components for the essential oil of L .lacunosa were myrcenone and β-myrcene.
Percentage chemical composition of the compounds of the essential oils extracted from leaves of L. alba geraniol and carvone chemotypes, L. sidoides, L. salviifolia, L. rotundifolia and L. lacunosa, as determined by gas-chromatography followed by mass spectrometry. The high content compounds were highlighted.
Effect of the essential oils of Lippia species on tumor cell lines viability
Table 2 shows the IC50 values (which are the concentrations that corresponds to 50% cell lethality) of cells treated with each one of the essential oils extracted from the Lippia species. It was determined that IC50 values were obtained only in the presence of essential oils of L. sidoides (19.05 µg/ ml), L. salviifolia (30.20 µg/ml) and L. rotundifolia (36.30 µg/ ml) on CT26.WT cell line and L. alba carvone chemotype essential oil (47.80 µg/ml) on A549 cell line. In the Fig. 1 we observe the representative curve concentration-response as a measurement of the OD values determined by the MTT assay. It illustrates the reduction of CT26.WT cell viability after treatment with the essential oils of L. sidoides, L. salviifolia and L. rotundifolia and the reduction of A549 cell line viability with the essential oil from L. alba carvone chemotype. On the other hand, MDA MB-231 and CACO-2 cell lines did not have their viability significantly compromised by the Lippia essential oils evaluated in this study (Table 2). Except for CACO-2 cell line, the positive control 5-FU reached IC50 levels were 7.94 µg/mlto CT26.WT, 8.30 µg/ml to A549 and 7.60 µg/ml to MDA MB-231 cell lines. The essential oils of Lippia also did not compromise cell viability of normal cell line CHO (Table 2).
IC50 values (μg/ml) of cell lines treated for 72 h with the four different concentrations, 0.1, 1, 10 and 100 μg/ml of each one of the essential oils extracted from the Lippia species. The negative control samples contained 0.4% DMSO, which is equivalent to the percentage found in the highest concentration evaluated. The positive control samples contained 5-FU at four different concentrations, 0.1, 1, 10 and 100 μM.
The cytotoxic effect, as a measurement of the OD values determined by the MTT assay, of the different concentrations (0.1-100 μg/ml) of L. sidoides, L. salviifolia and L. rotundifolia essential oils on CT26.WT cell line (A) and L. alba carvone chemotype essential oil on A549 cell line (B). The control samples contained 0.4% DMSO, which is equivalent to the percentage found in the highest concentration evaluated.
* p < 0.05.
** p< 0.01.
*** p < 0.001 are significantly different from the control value.
Discussion
Historically, numerous drugs have been developed from compounds originally isolated from medicinal plants (Lee, 1999). Since 1961 plant-derived compounds have been approved as anticancer drugs to, such as vinblastine (Velban), vincristine (Oncovin), etoposide (VP-16), teniposide (VM-26), paclitaxel (Taxol), navelbine (Vinorelbine), taxotere (Docetaxel), topotecan (Hycamitin) and irinotecan (Camptosar) (Dholwani et al., 2008). Recently, several studies have suggested that monoterpenes could represent a new class of agents to be used as anticancer drugs, which is especially important for tumors highly resistant to chemotherapy and to minimize the side effects of the current treatments (Shoff et al., 1991; Yu et al., 1995; Burke et al., 1997; He et al., 1997; Crowell, 1999; Duncan et al., 2004; Wiseman et al., 2007; Chaouki et al., 2009). Several of these monoterpenes are found in Lippia species and Brazil is one of the largest centers of diversity of this genus, comprising 70-75% of the known species (Viccini et al., 2006). In this study, it was initially identified the chemical compounds of the essential oils extracted from each one of the five Lippia species. GC-MS was performed and the results are shown in Table 1. For Lippia albageraniol chemotype, geranial and citral were the major compounds, comprising approximately 63%. Tavares et al. (2005) obtained the same major compounds. For the chemotype carvone, carvone and limonene were the main constituents, comprising around 70% of the essential oil. Zoghbi et al. (1998) obtained these same components among the majority ones. For L. sidoides, the major compound was thymol (63.20%), which is in agreement with Costa et al. (2005). L. salviifolia had nerolidol (49.22%) being the major compound of its essential oil, as Singulani et al. (2012). For L. rotundifolia and L. lacunosa the major compounds were β-myrcene (18.48%) and myrcenone (58.57%) respectively. Leitão et al. (2008) obtained the same ones for L. lacunosa. Therefore, these results showed that the major components identified in each one of the five species are in agreement with previous studies, and confirmed the identity of the Lippia chemotypes used in this study.
In order to evaluate effect of the essential oil extracted from Lippiaspecies on tumor cell viability, the MTT assay was undertaken. According to the National Cancer Institute (USA), vegetables crude extracts are cytotoxic considered when their IC50 values are less than 30 µg/ml (Hennebelle et al., 2008; Mesa-Arango et al., 2009). This assay showed that 50% of cell inhibition was obtained with concentrations under or near to 30 µg/ml for CT26. WT with the essential oils of L. sidoides (19.05 µg/ml) and L. salviifolia(30.20 µg/ml) (Fig. 1 and Table 2). The growth suppression effect of thymol, the major compound of L. sidoides essential oil, has been reported through IC50 value on the B16F10 mouse melanoma cell line (He et al., 1997). Nerolidol, the major compound of L. salviifolia essential oil, had been previously also demonstrated antiproliferative action on human leukemia HL-60. (Tatman and Mo, 2002). The IC50 values obtained for A549 cells treated with L. alba carvone chemotype essential oil (47.80 µg/ml) and CT26. WT cell treated with L. rotundifolia essential oil (36.30 µg/ml) (Fig. 1 and Table 2) did not classify them as cytotoxic crude extracts by the National Cancer Institute. Besides the selectivity on colon tumor cell line, it was not observed toxic effect in normal cell (CHO) exposed to each one of the essential oil tested.
In conclusion the present results showed a significant cytotoxic effect of L. sidoides and L. salviifolia essential oils on CT26.WT colon tumor cells which might be attributed to their major compounds. Also importantly it showed the absence of this effect on the normal cell line. Therefore, these finds stimulates further studies on their mechanisms of action in order to be considered for in vivo studies.
Acknowledgment
The authors thank the Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil, that fully supported the research.
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Publication Dates
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Publication in this collection
Nov-Dec 2013
History
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Received
10 Sept 2013 -
Accepted
28 Nov 2013
