Cytotoxicity screening of essential oils in cancer cell lines

Abstract

This study evaluated the cytotoxicity activity of the essential oils of Tagetes erecta L., Asteraceae (TE-OE), Tetradenia riparia (Hochst.) Codd, Lamiaceae (TR-OE), Bidens sulphurea (Cav.) Sch. Bip., Asteraceae (BS-OE), and Foeniculum vulgare Mill., Apiaceae (FV-OE), traditionally used in folk medicine, against the tumor cell lines murine melanoma (B16F10), human colon carcinoma (HT29), human breast adenocarcinoma (MCF-7), human cervical adenocarcinoma (HeLa), human hepatocellular liver carcinoma (HepG2), and human glioblastoma (MO59J, U343, and U251). Normal hamster lung fibroblasts (V79 cells) were included as control. The cells were treated with essential oil concentrations ranging from 3.12 to 400 µg/ml for 24 h. The cytotoxic activity was evaluated using the XTT assay; results were expressed as IC₅₀, and the selectivity index was calculated. The results were compared with those achieved for classic chemotherapeutic agents. TE-OE was the most promising among the evaluated oils: it afforded the lowest IC₅₀ values for B16F10 cells (7.47 ± 1.08 µg/ml) and HT29 cells (6.93 ± 0.77 µg/ml), as well as selectivity indices of 2.61 and 2.81, respectively. The major BS-EO, FV-EO and TE-EO chemical constituents were identified by gas chromatography mass spectrometry as being (E)-caryophyllene (10.5%), germacrene D (35.0%) and 2,6-di-tert-butyl-4-methylphenol (43.0%) (BS-EO); limonene (21.3%) and (E)-anethole (70.2%) (FV-EO); limonene (10.4%), dihydrotagetone (11.8%), α-terpinolene (18.1%) and (E)-ocimenone (13.0%) (TE-EO); and fenchone (6.1%), dronabinol (11.0%), aromadendrene oxide (14.7%) and (E,E)–farnesol (15.0%) (TR-EO). 2,6-di-tert-butyl-4-methylphenol (43.0%), (E)-anethole (70.2%) and α-terpinolene (18.1%), respectively. These results suggest that TE-OE may be used to treat cancer without affecting normal cells.

Keywords:
Tagetes erecta ; Tetradenia riparia ; Bidens sulphurea ; Foeniculum vulgare ; Cytotoxicity

Introduction

The search for new drugs that display activity against several types of cancer has become one of the most interesting subjects in the field of natural products research. In this area, plants have played a dominant role in the development of sophisticated traditional medicine systems, especially those with a long history in the treatment of cancer (De Mesquita et al., 2007). Reports on the use of herbs are as old as humanity and have demonstrated that plant-derived essential oils exert better therapeutic activity than their isolated major compounds. In addition, the essential oils are the products of extraction of a plant species, so they are more concentrated and may exhibit higher toxicity than the original plant (Bisset, 1994).

Tagetes erecta L., Asteraceae, an ornamental plant known as marigold, is commonly used to treat bronchitis, rheumatic pain, cold, and respiratory diseases, and which can also be employed as stimulant and muscle relaxant (Neher, 1968). The essential oil from T. erecta leaves displays schistosomicidal properties and is utilized as antihelminthic in the Amazonia region (Stasi and Hiruma-Lima, 2002; Tonuci et al., 2012). The monoterpenes α-terpinolene, (E)-ocimenone, limonene, (Z)-β-ocimene, linalool, dihydrotagetone, piperitone, piperitenone and (E)-tagetone are the main chemical constituents of this essential oil (Baslas and Singh, 1981; Krishna et al., 2004; Ogunwande and Olawore, 2006; Sefidkon et al., 2004; Sharma et al., 1961; Singh et al., 2003; Tonuci et al., 2012).

Tetradenia riparia (Hochst.) Codd., Lamiaceae, possesses a variety of medicinal properties in cases of cough, dropsy, diarrhea, fever, headaches, malaria, and toothache (Campbell et al., 1997). The essential oil from T. riparia leaves displays repellent (Omolo et al., 2004), insecticidal (Dunkel et al., 1990), ascaricidal (Peter and Deogracious, 2006), antimalarial (Campbell et al., 1997), antimicrobial and antinociceptive actions (Gazim et al., 2010). Its oil presents a complex mixture of monoterpenes, sesquiterpenes and diterpenes. The oxygenated diterpenes calyculone, 9β,13β-epoxy-7-abietene and 6,7-dehydroroyleanone; the oxygenated sesquiterpenes 14-hydroxy-9-epi-caryophyllene, cis-muurolol-5-en-4-α-ol, α-cadinol and ledol and the oxygenated monoterpene fenchone, perillyl alcohol, α-terpineol and β-fenchyl alcohol have been reported as the main chemical constituents of the essential oil of T. riparia (Fernandez et al., 2014; Gazim et al., 2010, 2014; Omolo et al., 2004).

Bidens sulphurea (Cav.) Sch. Bip., Asteraceae, many times referred to Cosmos sulphureus Cav., a synonymous of B. sulphurea in the literature, has anti-icteric and hepatoprotective effects and is traditionally used to treat malaria in Brazil (Botsaris, 2007). The essential oil extracted from the flowers of B. sulphurea displays schistosomicidal properties and exhibited significant antibacterial activity that support folkloric use in the treatment of some diseases as broad spectrum antibacterial agents (Aguiar et al., 2013; Ram et al., 2013). 2,6-di-tert-butyl-4-methylphenol and the sesquiterpenes β-caryophyllene and germacrene D are reported as the major constituents of the essential oil from B. sulphurea flowers (Aguiar et al., 2013).

Foeniculum vulgare Mill., Apiaceae, commonly known as "fennel", is a medicinal and aromatic plant used as carminative, digestive, lactogogue, and diuretic agent, and which can also help to treat respiratory and gastrointestinal disorders (Agarwal et al., 2008). The essential oil of fennel is used as additive in the food, pharmaceutical, cosmetic, and perfume industries (Tinoco et al., 2007), besides having important medicinal properties, such as diuretic, anti-inflammatory, analgesic, antioxidant (Gross et al., 2002), antiseptic, sedative, carminative, stimulant, and vermifugal activities (He and Huang, 2011; Tinoco et al., 2007). In the literature, (E)-anethole and the monoterpenes limonene and fenchone are often reported as the main constituents of this essential oil of fennel (Akgul and Bayrak, 1988; Anwar et al., 2009; Cosge et al., 2008; Garcia-Jimenez et al., 2000).

In the present study, we screened the cytotoxicity of essential oils extracted from T. erecta, T. riparia, B. sulphurea and F. vulgare against different cell lines. Despite the reports on the biological activities of these essential oils, data on their cytotoxicity are still scarce in the literature (Fabio et al., 2007; Gazim et al., 2014; Villarini et al., 2014).

Materials and methods

Plant material and essential oil extraction

Specimens of Tagetes erecta L., Asteraceae, Tetradenia riparia (Hochst.) Codd., Lamiaceae, Bidens sulphurea (Cav.) Sch. Bip., Asteraceae, and Foeniculum vulgare Mill., Apiaceae, were collected at "Sítio 13 de maio" (20°26' S 47°27' W 977 m) near Franca, State of São Paulo, Brazil on May 10, 2012 and identified by Prof. Dr Milton Groppo (Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo). Voucher specimens (SPFR 10014, 12421, 12020 and 12024, respectively) were deposited at the Herbarium of this institution (Herbarium SPFR).

Fresh leaves (450 g) of F. vulgare (FV-EO), T. erecta (TE-EO) and T. riparia (TR-EO) and fresh flowers (300 g) of B. sulphurea (BS-EO) were submitted to hydrodistillation in a Clevenger-type apparatus for 3 h. After manual collection of the essential oils (EO), anhydrous sodium sulfate was used to remove traces of water, which was followed by filtration. The EO were stored in an amber bottle and kept in the refrigerator at 4 °C until further analysis. The essential oil yields were calculated from the weight of fresh leaves and expressed as the average of triplicate analysis.

GC-FID and GC–MS analyses

BS-EO, FV-EO, TE-EO and TR-EO were analyzed by gas chromatography (GC) on a Hewlett-Packard G1530A 6890 gas chromatograph fitted with FID and data-handling processor. An HP-5 (Hewlett-Packard, Palo Alto, CA, USA) fused-silica capillary column (30 m × 0.25 mm i.d.; 0.33 µm film thickness) was employed. The operation conditions were as follows: the column temperature was programmed to rise from 60 to 240 °C at 3 °C/min and then held at 240 °C for 5 min; carrier gas = H₂, at a flow rate of 1.0 ml/min; injection mode; injection volume = 0.1 µl (split ratio of 1:10); injector and detector temperatures = 240 and 280 °C, respectively. The components relative concentrations were obtained by peak area normalization (%). The relative areas were the average of triplicate GC-FID analyses.

GC–MS analyses were carried out on a Shimadzu QP2010 Plus (Shimadzu Corporation, Kyoto, Japan) system equipped with an AOC-20i autosampler. The column consisted of Rtx-5MS (Restek Co., Bellefonte, PA, USA) fused silica capillary (30-m length × 0.25-mm i.d. × 0.25-µm film thickness). The electron ionization mode was used at 70 eV. Helium (99.999%) was employed as the carrier gas at a constant flow of 1.0 ml/min. The injection volume was 0.1 µl (split ratio of 1:10). The injector and the ion-source temperatures were set at 240 and 280 °C, respectively. The oven temperature program was the same as the one used for GC. Mass spectra were taken with a scan interval of 0.5 s, in the mass range from 40 to 600 Da. BS-EO, FV-EO, TE-EO and TR-EO components identification was based on their retention indices on an Rtx-5MS capillary column under the same operating conditions as in the case of GC relative to a homologous series of n-alkanes (C₈–C₂₄); structures were computer-matched with the Wiley 7, NIST 08, and FFNSC 1.2 spectra libraries, and their fragmentation patterns were compared with literature data (Adams, 2005). Standard compounds available in our laboratory were also co-eluted with the essential oils to confirm the identity of some of their components.

Cell lines

Eight different tumor cell lines were used during the experiments: murine melanoma (B16F10), courtesy by Departamento de Bioquímica da Faculdade de Medicina da Universidade de São Paulo, Campus de Ribeirão Preto, São Paulo; colon adenocarcinoma (HT29), human glioblastoma (MO59J, U343, and U251) and human cervical adenocarcinoma (HeLa), obtained from the Cell Bank of Universidade Federal do Rio de Janeiro; human breast adenocarcinoma (MCF-7) and human hepatocellular carcinoma (HepG2), courtesy of Laboratório de Mutagênese do Departamento de Ciências Biológicas da Universidade Estadual Paulista, Campus de Araraquara, São Paulo. In order to compare the cytotoxic effects and the selectivity obtained on tumor cells after the treatment with the essential oils, we also included treatments in a normal cell line (Chinese hamster lung fibroblasts; V79), courtesy of Laboratório de Mutagênese da Universidade Estadual de Londrina, Paraná. The different cell lines were maintained as monolayers in plastic culture flasks (25 cm²) in culture medium (HAM-F10 + DMEM, 1:1, Sigma–Aldrich or only DMEM) supplemented with 10% fetal bovine serum (Nutricell), antibiotics (0.01 mg/ml streptomycin and 0.005 mg/ml penicillin; Sigma–Aldrich), and 2.38 mg/ml Hepes (Sigma–Aldrich), at 36.5 °C, with 5% CO₂ or in a BOD-type chamber. The cells were used from the 4th passage.

Cytotoxic activity of the essential oils

The cytotoxic activity of the essential oils against different cell lines was screened using the Colorimetric Assay In Vitro Toxicology – XTT Kit (Roche Diagnostics). For the experiments, 1 × 10⁴ cells were seeded into microplates with 100 µl of culture medium (1:1 HAM F10/DMEM or DMEM alone) supplemented with 10% fetal bovine serum containing concentrations of the essential oils that ranged from 3.12 to 400 µg/ml. Negative (no treatment), solvent (0.02% DMSO, dimethylsulfoxide, Sigma-Aldrich), and positive (25% DMSO) controls were included. The classic chemotherapeutic agents doxorubicin (DXR, Pharmacia Brasil Ltda., 98% purity), (S)-(+)-camptothecin (CPT, Sigma–Aldrich, ≥90% purity), and etoposide (VP16, Sigma–Aldrich, ≥98% purity) were also tested. After incubation at 36.5 °C for 24 h, the culture medium was removed. The cells were washed with 100 µl of PBS (phosphate buffered saline) to remove the treatments and exposed to 100 µl of culture medium HAM -F10 without phenol red. Then, 25 µl of XTT was added, and the cells were incubated at 36.5 °C for 17 h. The absorbance of the samples was determined using a multi-plate reader (ELISA – Tecan – SW Magellan vs 5.03 STD 2P) at a wavelength of 450 nm and reference length of 620 nm.

The experiments were performed in triplicate; 50% inhibition of cell growth (IC₅₀) was used as the analysis parameter calculated by Prism Graphpad (version 5.0) software. The One-Way ANOVA analysis was used to compare the mean values (p < 0.05).

Results and discussion

Table 1 shows the chemical composition of the essential oils investigated in this study. Most of the compounds identified in these EO are monoterpenes, sesquiterpenes and phenylpropanoids. Diterpenes were detected only in the essential oil of T. riparia (TE-EO).

Table 1
Chemical composition and yields (w/w) of the essential oils of B. sulphurea (BS-EO), F. vulgare (FV-EO), T. erecta (TE-EO) and T. riparia (TR-EO).

Compound	RIexp	RIlit	% RA				Identification
			BS-EO	FV-EO	TE-EO	TR-EO
α-pinene	938	939		1.5	1.3		RL, MS, Co
camphene	954	953			0.2	0.6	RL, MS
sabinene	978	976		0.8	0.8	1.0	RL, MS
β-pinene	983	980			tr	0.5	RL, MS, Co
myrcene	988	991		1.7			RL, MS
α-phellandrene	1007	1005			0.5	tr	RL, MS
limonene	1033	1031		21.3	10.4	1.2	RL, MS, Co
(Z)-β-ocimene	1040	1043	0.5		4.2	0.5	RL, MS
(E)-β-ocimene	1043	1050		0.9	0.7		RL, MS
dihydrotagetone	1049	1054			11.8		RL, MS
α-terpinolene	1084	1088			18.1		RL, MS
fenchone	1095	1094		1.2		6.1	RL, MS, Co
(E)-tagetone	1151	1146			6.9		RL, MS
camphor	1151	1143				2.0	RL, MS, Co
borneol	1173	1165				0.8	RL, MS, Co
terpinen-4-ol	1184	1177				0.7	RL, MS
α-terpineol	1197	1189				1.0	RL, MS
estragol	1198	1195		1.8			RL, MS
verbenone	1218	1230			9.7		RL, MS
(E)-ocimenone	1238	1239			13.0		RL, MS
piperitone	1252	1252			8.8		RL, MS
(E)-anethole	1287	1293		70.2			RL, MS, Co
piperitenone	1335	1342			9.7		RL, MS
α-copaene	1399	1376				0.8	RL, MS
β-elemene	1416	1391	2.1			1.5	RL, MS
α-gurjunene	1426	1409				4.5	RL, MS
(E)-caryophyllene	1441	1428	10.5		1.2	1.6	RL, MS, Co
α-trans-bergamotene	1460	1436				0.4	RL, MS
precocene I	1458	1467			1.4		RL, MS
α-humulene	1467	1467	0.5			0.7	RL, MS, Co
germacrene D	1488	1480	35.0		0.9	tr	RL, MS
aromadendrene	1490	1491				0.5	RL, MS
viridiflorene	1500	1493				1.3	RL, MS
bicyclogermacrene	1507	1494	5.8			0.6	RL, MS
E,E-α-farnesene	1503	1508	0.4			3.1	RL, MS
2,6-di-tert-butyl-4-methylphenol	1523	1519	43.0			0.3	RL, MS, Co
Δ-cadinene	1527	1524	0.4			2.7	RL, MS
cis-nerolidol	1531	1539				1.5	RL, MS
germacrene-D-4-ol	1572	1574			tr	5.4	RL, MS
spathulenol	1575	1576	0.5			0.3	RL, MS
caryophyllene oxide	1577	1581	1.0		tr	tr	RL, MS
viridiflorol	1590	1590				2.4	RL, MS
α-cadinol	1650	1653				3.0	RL, MS
α-muurolol	1655	1657				0.5	RL, MS
t-cadinol	1664	1660				5.6	RL, MS
aromadendrene oxide	1672	1668				14.7	RL, MS
E,E-farnesol	1702	1706				15.0	RL, MS
13-epimanoyl oxide	1996	2002				7.2	RL, MS
dronabinol	2190	2202				11.0	RL, MS
Total			99.7	99.4	99.8	99.2
Monoterpene hydrocarbons	0.5	26.2	18.1	4.1
Oxygenated monoterpenes		1.2	78.0	10.6
Sesquiterpene hydrocarbons	54.7		2.1	17.9
Oxygenated sesquiterpenes	1.5		0.2	55.6
Others	43.0	72.0	1.4	11.0
Yield (w/w)	0.24%	1.60%	0.53%	1.26%

• RIexp: Retention index determined relative to n-alkanes (C8–C20) on the Rtx-5MS column. RIlit: Retention index from the literature (Adams, 2005). Compound identification: RL, comparison of the RI with those of the literature (Adams, 2005); RA: relative area (peak area relative to the total peak area in the GC-FID chromatogram), average of three replicates; MS, comparison of the mass spectra with those of the Wiley 7, NIST 08, and FFNSC 1.2 spectral libraries as well as with those of literature (Adams, 2005); Co: co-elution with standard compounds available in our laboratory. tr: relative area lower than 0.1%. Yields were expressed as the average of three replicates (3×150g for FV-EO, TE-EO and TR-EO and 3×100g for BS-EO).

(E)-caryophyllene (10.5%), germacrene D (35.0%) and 2,6-di-tert-butyl-4-methylphenol (43.0%) were identified as the major constituents in the essential oil of B. sulphurea (BS-EO). These compounds were also the main chemical constituents of a sample of the essential oil from a specimen of B. sulphurea from Southeast Brazil (Aguiar et al., 2013).

Limonene (21.3%) and (E)-anethole (70.2%) were the major compounds in the essential oil of F. vulgare (FV-EO). These compounds have also been found to be the major constituents in essential oils of specimens of F. vulgare from different countries (Cosge et al., 2008; Garcia-Jimenez et al., 2000; Wakabayashi et al., 2015). On the other hand, methyl-chavicol and α-phellandrene, which are often detected in the essential oil of F. vulgare and were reported as its major compounds in some studies (Chung et al., 2011; Ozcan and Chalchat, 2006), were not detected in FV-EO.

Limonene (10.4%), dihydrotagetone (11.8%), α-terpinolene (18.1%) and (E)-ocimenone (13.0%) were found to be the major compounds in the essential oil of T. erecta (TE-EO). These monoterpenes have been reported among the main constituents of the essential oil of other T. erecta specimens (Baslas and Singh, 1981; Krishna et al., 2004; Ogunwande and Olawore, 2006; Sefidkon et al., 2004; Sharma et al., 1961; Singh et al., 2003). On the other hand, the oxygenated monoterpenes (Z)-ocimenone, (Z)-tagetone, linallyl acetate and linalool, which were found in the essential oil of other T. erecta specimens (Baslas and Singh, 1981; Sharma et al., 1961; Singh et al., 2003), were not detected in TE-EO.

Finally, the major constituents in the essential oil of T. riparia (TR-EO) were identified as being fenchone (6.1%), dronabinol (11.0%), aromadendrene oxide (14.7%) and (E,E)–farnesol (15.0%). The chemical composition of TR-EO differs from most of the previously investigated T. riparia essential oils in the low content of diterpenes. 13-Epimanoyl oxide (7.2%) was the only diterpene identified in TR-EO. The oxygenated diterpenes calyculone, 14-hydroxy-9-epi-caryophyllene, cis-muurolol-5-en-4-ol, which were identified as major constituents in the essential oil of other T. riparia specimens (Fernandez et al., 2014; Gazim et al., 2010, 2014) were not detected in TR-EO.

Table 2 shows the IC₅₀ values for the normal and tumor cell lines treated with TE-OE, TR-OE, BS-OE, and FV-OE. Our results showed that the IC₅₀ values for tumor cell lines treated ranged from 6.93 ± 0.77 to 161.60 ± 1.41 µg/ml for TE-OE; from 77.46 ± 1.75 to 272.37 ± 18.45 µg/ml for TR-OE; from 229.23 ± 10.40 to 334.17 ± 15.50 µg/ml for BS-OE, and from 112.78 ± 13.74 to 406.00 ± 1.57 µg/ml for FV-OE. Regarding the normal cell line V79, the IC₅₀ values were 19.50 ± 5.96, 76.33 ± 3.44, 96.50 ± 1.19 and 448.00 ± 19.52 µg/ml for TE-OE, TR-OE, BS-OE, and FV-OE, respectively.

Table 2
IC50 values found for the different cell lines after treatment with the essential oils of T. erecta (TE-EO), T. riparia (TR-EO), B. sulphurea (BS-EO), F. vulgare (FV-EO) and the positive control: etoposide (VP-16), (S)-(−)camptothecin (CPT) and doxorrubicin (DXR).

Treatments	IC50 (μg/ml)a
	V79	B16F10	HT29	MCF-7	HeLa	HepG2	MO59J	U343	U251
TE	19.50 ± 5.96	7.47 ± 1.08	6.93 ± 0.77	63.42 ± 2.42	26.02 ± 5.52	161.60 ± 1.41	38.69 ± 5.51	83.56 ± 10.81	75.56 ± 7.11
TR	76.33 ± 3.44	272.37 ± 18.45	77.46 ± 1.75	129.57 ± 10.68	155.70 ± 19.09	140.97 ± 11.29	217.97 ± 13.55	221.30 ± 9.80	109.90 ± 2.83
BS	96.50 ± 1.19	230.00 ± 19.50	268.70 ± 8.23	253.75 ± 15.91	334.17 ± 15.50	241.07 ± 9.49	229.23 ± 10.40	243.53 ± 0.87	236.30 ± 2.33
FV	448.00 ± 19.52	112.78 ± 13.74	NE	NE	NE	NE	406.00 ± 1.57	NE	NE
VP-16	1.28 ± 0.05	48.91 ± 8.53	325.40 ± 6.79	82.67 ± 15.62	225.50 ± 31.82	235.37 ± 6.47	58.94 ± 3.10	2.18 ± 0.99	42.97 ± 0.40
CPT	3.27 ± 0.14	20.17 ± 1.98	7.34 ± 1.47	36.09 ± 12.46	19.38 ± 0.76	11.87 ± 1.96	15.55 ± 0.65	5.71 ± 1.05	11.14 ± 2.79
DXR	0.57 ± 0.27	3.81 ± 0.18	101.62 ± 24.15	5.39 ± 1.35	21.90 ± 9.09	62.13 ± 2.04	6.98 ± 2.13	0.70 ± 0.35	16.28 ± 2.51

• ^a
  The values are mean ± standard deviation. IC50 (concentration inhibiting 50% growth). V79 (normal hamster lung fibroblasts); B16F10 (murine melanoma); HT29 (human colon carcinoma); MCF-7 (human breast adenocarcinoma); HeLa (human cervical adenocarcinoma); HepG2 (human hepatocellular carcinoma); and MO59J, U343 and U251 (human glioblastoma). NE – not effective.

TE-OE exerted the most pronounced antiproliferative effect against the tumor cell lines. The lowest IC₅₀ values were 7.47 ± 1.08 and 6.93 ± 0.77 µg/ml for the B16F10 and HT29 cell lines, respectively, which were significantly lower than the IC₅₀ value obtained for the normal cell line (19.50 ± 5.96 µg/ml). On the other hand, recent studies using the MTT assay reported that Tagetes erecta extracts in EtOH and EtOAc did not significantly affect the cell viability of H460 (pleural carcinoma) and Caco-2 (colon carcinoma) cells as compared with the control groups (Vallisuta et al., 2014).

FV-EO was more cytotoxic to B16F10 tumor cells (IC₅₀ = 112.78 ± 13.74 µg/ml) than to normal cells (IC₅₀ = 448.00 ± 19.52 µg/ml); the selectivity index was 3.97 (Tables 2 and 3). However, according to American National Center Institute, only extracts with IC₅₀ values lower than 30 µg/ml against experimental tumor cell lines constitute promising anticancer agents for drug development (Suffness and Pezzuto, 1990). In this sense, TR-EO and BS-EO showed IC₅₀ values greater than 30 µg/ml for all cell lines tested, and were more cytotoxic to normal line to which the tumor cell lines.

Table 3
Selectivity of the cytotoxicity of T. erecta (TE-EO), T. riparia (TR-EO), B. sulphurea (BS-EO) and F. vulgare (FV-EO) essential oils to tumor cells as compared with V79 cells.

Treatments	Selectivity indexa
	B16F10	HT29	MCF-7	HeLa	HepG2	MO59J	U343	U251
TE	2.61	2.81	0.30	0.74	0.12	0.50	0.23	0.25
TR	0.28	0.98	0.58	0.49	0.54	0.35	0.34	0.34
BS	0.41	0.35	0.38	0.28	0.39	0.42	0.39	0.40
FV	3.97	NE	NE	NE	NE	1.10	NE	NE
VP16	0.00	0.00	0.00	0.00	0.00	0.00	0.60	0.00
CPT	0.10	0.40	0.40	0.00	0.10	0.20	0.60	0.30
DXR	0.10	0.00	0.00	0.00	0.00	0.00	0.70	0.00

• ^a
  The selectivity index is the ratio of the IC50 values of the treatments on V79 cells to those in the cancer cell lines. B16F10 (murine melanoma); HT29 (human colon carcinoma); MCF-7 (human breast adenocarcinoma); HeLa (human cervical adenocarcinoma); HepG2 (human hepatocellular carcinoma); and MO59J, U343, and U251 (human glioblastoma). NE–not effective

Table 3 shows the selectivity indices of the essential oils tested against the tumor cell lines and the non-tumor cell line. For all natural products tested, the selectivity indexes were greater than those observed for the chemotherapeutic agents, especially VP16. Literature papers have considered that a value greater than or equal to 2.0 is an interesting selectivity index (Suffness and Pezzuto, 1990). This value means that the compound is more than twice more cytotoxic to the tumor cell line as compared with the normal cell line. According to Bézivin et al. (2003), the selectivity index is interesting in the case of values greater than three.

Treatments with TE-EO and FV-EO afforded the highest selectivity indices, which ranged from 0.23 to 2.81 for TE-EO and from 1.10 to 3.97 for FV-EO (Table 3). These findings demonstrated that TE-EO constitutes a promising essential oil for the development of anticancer drugs, because it provided indices greater than 2. For FV-EO, although the selectivity indices suggested promising antitumor activity, the IC₅₀ values were greater than that recommended by Suffness and Pezzuto (1990). In addition, it was not possible to calculate the selectivity index for the majority of the cancer cell lines treated with FV-EO, since it was more cytotoxic to the normal cell line.

The cytotoxicity of the essential oils used in present study can be attributed to the major chemical constituents in the essential oils tested. (E)-anethole, the major compound found in FV-EO, caused a concentration and time-dependent cell death in freshly isolated rat hepatocytes (Nakagawa and Suzuki, 2003). Limonene, one the major compounds found on the BS-EO and TE-EO, combined with docetaxel significantly enhanced the cytotoxicity to normal prostate epithelial cells (DU-145) (Rabi and Bishayee, 2009).

Our results demonstrated that TE-EO exerted a selective and cytotoxic activity against tumor cell lines. Therefore, this essential oil should be considered a promising source to develop specific antitumor drugs.

Acknowledgments

The authors thank the FAPESP Brazil, for a PhD scholarship (P.F.O; grant #2009/21310-2) and the financial support (grant #2007/54241-8). The authors are also grateful to CNPq for fellowships.

References

• Adams, R., 2005. Identification of Essential Oils Components by Gas Chromatography/Mass Spectrometry, 4th ed. Allured Publishing, Carol Stream, IL.
• Agarwal, R., Gupta, S.K., Agarwal, S.S., Srivastava, S., Saxena, R., 2008. Oculohypotensive effects of Foeniculum vulgare in experimental models of glaucoma. Indian J. Physiol. Pharmacol. 52, 77–83.
• Aguiar, G.P., Melo, N.I., Wakabayashi, K.A.L., Lopes, M.H.S., Mantovani, A.L.L., Dias, H.J., Fukui, M.J., Keles, L.C., Rodrigues, V., Groppo, M., Silva-Filho, A.A., Cunha, W.R., Magalhães, L.G., Crotti, A.E.M., 2013. Chemical composition and in vitro schistosomicidal activity of the essential oil from the flowers of Bidens sulphurea (Asteraceae). Nat. Prod. Res. 27, 920–924.
• Akgul, A., Bayrak, A., 1988. Comparative volatile oil composition of various parts from Turkish bitter fennel (Foeniculum vulgare var. vulgare). Food Chem. 30, 319–323.
• Anwar, F., Ali, M., Hussain, A.I., Shahid, M., 2009. Antioxidant and antimicrobial activities of essential oil and extracts of fennel (Foeniculum vulgare Mill.) seeds from Pakistan. Flavour Frag. J. 24, 170–176.
• Baslas, R.K., Singh, A.K., 1981. Chemical examination of essential oil from the leaves of Tagetes erecta Linn. J. Indian Chem. Soc. 58, 104.
• Bézivin, C., Tomasi, F., Lohézie-Le, D., Boustie, J., 2003. Cytotoxic activity of some lichen extracts on murine and human cancer cell lines. Phytomedicine 10, 499–503.
• Bisset, N.G., 1994. Herbal Drugs and Phytopharmaceuticals – A Handbook for Practice on a Scientific Bases. London/Stuttgart, Medpharm/CRC Press.
• Botsaris, A.S., 2007. Plants used tradicionally to treat malaria in Brazil: the archives of flora medicinal. J. Ethnobiol. Ethnomed. 3, 2–8.
• Campbell, W.E., Gammon, D.W., Smith, P., Abrahams, M., Purves, T., 1997. Composition and antimalarial activity in vitro of the essential oil of Tetradenia riparia Planta Med. 63, 270–272.
• Chung, I.-M., Ro, H.-M., Moon, H.-I., 2011. Major essential oils composition and immunotoxicity activity from leaves of Foeniculum vulgare against Aedes aegypti L. Immunopharmacol. Immunotoxicol. 33, 450–453.
• Cosge, B., Gurbuz, B., Kendir, H., Ipek, A., 2008. Composition of essential oil in sweet fennel (Foeniculum vulgare mill. var. dulce) lines originated from Turkey. Asian J. Chem. 20, 1137–1142.
• De Mesquita, M.L., de Paula, J.E., Pessoa, C., De Moraes, M.O., Costa-Lotufo, L.V., Grougnet, R., Michel, S., Tillequin, F., Espindola, L.S., 2007. Cytotoxic activity of Brazilian Cerrado plants used in traditional medicine against cancer cell lines. J. Ethnopharmacol. 123, 439–445.
• Dunkel, F., Weaver, D., Van Puyvelde, L., Cusker, J.L., Serugend, A., 1990. Population suppression effects of Rwandan medicinal plant, Tetradenia riparia (Hochst.) Codd (Lamiaceae) on stored grain and bean insects. In: Proc. 5th Int. Wkg. Conf. Stored Prod. Prot, pp. 1609–1617.
• Fabio, A., Carmelli, C., Fabio, G., Nicoletti, P.I., Quaglio, P., 2007. Screening of the antibacterial effects of a variety of essential oils on microorganisms responsible for respiratory infections. Phytother. Res. 21, 374–377.
• Fernandez, C.M.M., Barba, E.L., Fernandez, A.C.M., Cardoso, B.K., Borges, I.B., Takemura, O.S., Martins, L.D., Cortez, L.E.R., Cortez, D.A.G., Gazim, Z.C., 2014. Larvicidal activity of essential oil from Tetradenia riparia to control of Aedes aegypti larvae in function of season variation. J. Essent. Oil Bear. Pl. 17, 813–823.
• Garcia-Jimenez, N., Perez-Alonso, M.J., Velasco-Negueruela, A., 2000. Chemical composition of fennel oil, Foeniculum vulgare Miller, from Spain. J. Essent. Oil Res. 12, 159–162.
• Gazim, Z.C., Amorim, A.C.L., Hovell, A.M.C., Rezende, C.M., Nascimento, I.A., Ferreira, G.A., Cortez, D.A.G., 2010. Seasonal variation, chemical composition, and analgesic and antimicrobial activities of the essential oil from leaves of Tetradenia riparia (Hochst.) Codd in Southern Brazil. Molecules 15, 5509–5524.
• Gazim, Z.C., Rodrigues, F.F.G., Amorin, A.C.L., de Rezende, C.M., Sokovic, M., Tesevic, V., Vuckovic, I., Krstic, G., Cortez, L.E.R., Colauto, N.B., Linde, G.A., Cortez, D.A.G., 2014. New natural diterpene-type abietane from Tetradenia riparia essential oil with cytotoxic and antioxidant activities. Molecules 19, 514–524.
• Gross, M., Friedman, J., Dudai, N., Larkov, O., Cohen, Y., Bar, E., Ravid, U., Putievsky, E., Lewinsohn, E., 2002. Biosynthesis of estragole and trans-anethole in bitter fennel (Foeniculum vulgare Mill. var. vulgare) chemotypes. Changes in SAM:phenylpropene O-methyltransferase activities during development. Plant Sci. 162, 1047–1053.
• He, W.P., Huang, B.K., 2011. A review of chemistry and bioactivities of a medicinal spice: Foeniculum vulgare J. Med. Plants Res. 5, 3595–3600.
• Krishna, A., Kumar, S., Mallavarapu, G.R., Ramesh, S., 2004. Composition of the essential oils of the leaves and flowers of Tagetes erecta L. J. Essent. Oil Res. 16, 520–522.
• Nakagawa, Y., Suzuki, T., 2003. Cytotoxic and xenoestrogenic effects via biotransformation of trans-anethole on isolated rat hepatocytes and cultured MCF-7 human breast cancer cells. Biochem. Pharmacol. 66, 63–73.
• Neher, R.T., 1968. The ethnobotany of Tagetes Econ. Bot. 22, 317–325.
• Ogunwande, I.A., Olawore, N.O., 2006. The essential oil from the leaves and flowers of African Marigold, Tagetes erecta L. J. Essent. Oil Res. 18, 366–368.
• Omolo, M.O., Okinyo, D., Ndiege, I.O., Lwande, W., Hassanali, A., 2004. Repellency of essential oils of some Kenyan plants against Anopheles gambiae Phytochemistry 65, 2797–2802.
• Ozcan, M.M., Chalchat, J.C., 2006. Effect of collection time on chemical composition of the essential oil of Foeniculum vulgare subsp piperitum growing wild in Turkey. Eur. Food Res. Technol. 224, 279–281.
• Peter, W., Deogracious, O., 2006. The in vitro ascaricidal activity of selected indigenous medicinal plants used in ethnoveterinary practices in Uganda. Afr. J. Tradit. Complem. 3, 94–103.
• Rabi, T., Bishayee, A., 2009. d-Limonene sensitizes docetaxel-induced cytotoxicity in human prostate cancer cells: Generation of reactive oxygen species and induction of apoptosis. J. Carcinog. 8, 9.
• Ram, B., Mandlik, M., Kumar, K., 2013. Antimicrobial activity of Cosmos sulphureus flowers around Pune. Int. J. Pharm. Res. Dev. 5, 27–31.
• Sefidkon, F., Salehyar, S., Mirza, M., Dabiri, M., 2004. The essential oil of Tagetes erecta L. occurring in Iran. Flavour Frag. J. 19, 579–581.
• Sharma, M.L., Nigam, M.C., Handa, K.L., Chopra, I.C., 1961. Essential oil of Tagetes erecta Perfum. Essential Oil Rec. 4, 561–562.
• Singh, G., Singh, O.P., De Lampasona, M.P., Catalan, C., 2003. Studies on essential oils. Part 35: Chemical and biocidal investigations on Tagetes erecta leaf volatile oil. Flavour Fragr. J. 18, 62–65.
• Stasi, L.C., Hiruma-Lima, C.A., 2002. Plantas medicinais na Amazônia e na Mata Atlântica, 2nd ed. UNESP, São Paulo.
• Suffness, M., Pezzuto, J.M., 1990. Assays related to cancer drug discovery. In: Hostettmann, K. (Ed.), Methods in Plant Biochemistry: Assays for Bioactivity. Academic Press, London, pp. 71–133.
• Tinoco, M.T., Martins, M.R., Cruz-Morais, J., 2007. Antimicrobial activity of Foeniculum vulgare Miller essential oil. Rev. Ciências Agrárias 30, 448–454.
• Tonuci, L.R.S., Melo, N.I., Dias, H.J., Wakabayashi, K.A.L., Aguiar, G.P., Aguiar, D.P., Mantovani, A.L.L., Ramos, R.C., Groppo, M., Rodrigues, V., Veneziani, R.C.S., Cunha, W.R., Silva-Filho, A.A., Magalhães, L.G., Crotti, A.E.M., 2012. In vitro schistosomicidal effects of the essential oil of Tagetes erecta Rev. Bras. Farmacogn 22, 88–93.
• Vallisuta, O., Nukoolkarn, V., Mitrevi, A., Sarisuta, N., Leelapornpisid, P., Phrutivorapongkul, A., Sinchaipanid, N., 2014. In vitro studies on the cytotoxicity and elastase and tyrosinase inhibitory activities of marigold (Tagetes erecta L.) flower extracts. Exp. Ther. Med. 7, 246–250.
• Villarini, M., Pagiotti, R., Dominici, L., Fatigoni, C., Vanini, S., Levorato, S., Moretti, M., 2014. Investigation of the cytotoxic, genotoxic, and apoptosis-inducing effectsof estragole isolated from the fennel (Foeniculum vulgare). J. Nat. Prod. 77, 773–778.
• Wakabayashi, K.A.L., Melo, N.I., Aguiar, D.P., Oliveira, P.F., Groppo, M., Silva-Filho, A.A., Rodrigues, V., Cunha, W.R., Tavares, D.C., Magalhães, L.G., Crotti, A.E.M., 2015. Anthelmintic effects of the essential oil of fennel (Foeniculum vulgare Mill., Apiaceae) against Schistosoma mansoni Chem. Biodiv. 12 (Epub ahead of print).

Publication Dates

• Publication in this collection
  Mar-Apr 2015

History

• Received
  2 Dec 2014
• Accepted
  14 Feb 2015