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Open Access Highly Accessed Preliminary communication

Antiproliferative activity of hexane extract from Tunisian Cistus libanotis, Cistus monspeliensis and Cistus villosus

Mariem Ben Jemia1, Mohamed Elyes Kchouk1, Felice Senatore2, Giuseppina Autore3, Stefania Marzocco3, Vincenzo De Feo3* and Maurizio Bruno4

Author Affiliations

1 Laboratoire des Plantes Extremophiles - Biotechnologic Center Borj-Cedria Technopark, B.P. 901, 2050, Hammam-Lif, Tunisie

2 Department of Pharmacy, University of Naples “Federico II”, Via D. Montesano, 49-80131, Naples, Italy

3 Department of Pharmacy, University of Salerno, via Ponte Don Melillo, 84084, Fisciano (Salerno), Italy

4 Department STEMBIO, Sect. of Organic Chemistry, University of Palermo, Viale delle Scienze, Parco d’Orleans II, 90128, Palermo, Italy

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Chemistry Central Journal 2013, 7:47  doi:10.1186/1752-153X-7-47


The electronic version of this article is the complete one and can be found online at: http://journal.chemistrycentral.com/content/7/1/47


Received:4 November 2012
Accepted:4 February 2013
Published:5 March 2013

© 2013 Ben Jemia et al; licensee Chemistry Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

As a part of our investigation on Tunisian medicinal plants, we have carried out a phytochemical investigation of the hexane extracts from leaves of Cistus libanotis, C. villosus and C. monspeliensis, evualuating also their possible antiproliferative activity in vitro.

Results

The major compounds of hexane extracts were identified and quantified by GC-MS. The composition of the three species, although belonging to the same genus, is completely different. The antiproliferative activity was evaluated against murine monocyte/macrophages (J774.A1), human melanoma cells (A-375), and human breast cancer cells (MCF-7), showing major activity against the human melanoma cell line A-375.

Conclusions

The chemical composition of the hexane extracts from the three Cistus species can be useful in the chemosystematics of this complex genus. The preliminary antiproliferative activity against human melanoma cell line A-375 deserve further investigations in order to determine the compounds, or their combinations, which are the main responsible for the antiproliferative activity and its possible mechanism(s) of action.

Background

Cistaceae is a Mediterranean native family of almost 200 species of shrubs. Most members of this family are very fragrant and sweet smelling, being much appreciated in the perfume industry and for ornamental purposes. Also, Cistaceae plants adapt easily to wildfires that destroy large forest areas, their seeds resisting and repopulating rapidly in the following season [1]. This family is formed by different genera, including Helianthemum, Halimium and Cistus. This latter contains between 16 and 28 different species, depending on the source [2]. Some of the Cistus species are endemic and others are widespread in the Iberian Peninsula, Canary Islands, Northwestern Africa, Italy, Greece and Turkey [3]. The species are disseminated over different areas of the Mediterranean area, but not all the species are distributed following the same pattern. Thereby, each area is colonised by different Cistus species depending on climatological and soil conditions.

Traditional folk medicine has used Cistus species as antiinflammatory, antiulcerogenic, wound healing, antimicrobial, cytotoxic and vasodilator remedies. Recent studies highlighted some information on the possible candidate compounds for these effects, and new activities are being discovered and attributed to Cistus extracts. These include antimicrobial, antioxidant, antiproliferative, antinociceptive and analgesic effects [4-6].

A comprehensive study on the qualitative composition of the hexane extract of C. monspeliensis L. leaves has been reported [7] as well as the catechin related compounds in aqueous extracts of the same species [8]. The composition of aqueous extracts from Cistus libanotis L. has also been reported [9]. No previous reports on the composition of C. villosus L. are available.

Here we present a comparative qualitative and quantitative study of the composition of hexane extracts from the aerial parts of three Cistus species grown in Tunisia. Hexane extracts from Cistus monspeliensis, C. libanotis and C. villosus leaves were analyzed by GC-MS. Moreover, the antiproliferative activity against a panel of cancer cell lines has been evaluated.

Results and discussion

Chemical composition of hexane extracts

As a part of our investigation on Tunisian medicinal plants, we have conducted a phytochemical investigation of the hexane extracts of Cistus libanotis, Cistus villosus and Cistus monspeliensis, evaluating also their possible antiproliferative activity against murine monocyte/macrophages (J774.A1), human melanoma cells (A-375), and human breast cancer cells (MCF-7). The composition of the three hexane extracts was achieved by GC-MS. Although the three species belong to the same genus, the composition of their hexane extract is completely different (Table 1). A total of 47 constituents, representing 90.1% of the total extract, have been identified from the hexane extract from the leaves of C. libanotis. In Table  1, the retention indices, retention times and percentage composition are given; the components, grouped in class of substances, are listed in order of elution on a HP 5MS column. By far flavonoids (30.2%) were the main fraction of the extract, with quercetagetin 3',4',6,7-tetramethyl ether (24.6%) as the principal compound. The main component of fatty acids fraction (24.4%) was (Z,Z,Z)-9,12-15-octadecatrienoic acid (23.5%). Monoterpene hydrocarbons were also present in good amount (10.5%), being camphene (3.6%) and β-pinene (3.0%) the principal compounds. Hydrocarbons and oxygenated monoterpenes represented 9.3% and 7.1%, respectively. Diterpenes (2.2%), sesquiterpenes (1.3%) and oxygenated sesquiterpenes (0.7%) were present in small amount. In C. monspeliensis (36 compounds) extract the principal class was represented by fatty acids (43.3%) among which the most abundant were (Z,Z,Z)-9,12-15-octadecatrienoic acid (14.7%) and (Z,Z)-9,12-octadecadienoic acid (6.6%). Hydrocarbons (22.8%) are also present in good amount with nonacosane (7.1%) and heptacosane (6.1%) as principal ones. Vitamin E was present (11.5%) in higher amount with respect to C. libanotis (3.3%). Monoterpenes, oxygenated monoterpenes, diterpenes and triterpenes were present in quite low amount. The peculiar characteristic of the composition of the extract of C. villosus is the high quantity of hydrocarbons (37.3%), being nonacosane (18.3%) and hentriacontane (9.2%) the main compounds. Fatty acids (10.6%) and diterpenes (4.8%) were also present in good amount. It is noteworthy the good quantity of vitamin E (22.7%), the most abundant products among the 31 compounds of the extract of C. villosus.

Table 1. Percentage composition of the hexane extract from aerial parts of three Cistus spp

Few reports are available in literature about the chemical constituents of Cistus species. The analysis of the composition of a hexane extract of leaves of C. monspeliensis collected in the island of Crete [7] indicated the presence of 13-epi-manoyl oxide, completely absent in our sample, which contains on the contrary a good quantity of manoyloxide. The available literature reports also chemical studies on the composition of extracts of Cistus species, obtained by different solvents. Catechin related compounds were also identified in the aqueous extracts of Cistus monspeliensis[8]. Some studies have reported the existence of monomeric and polymeric flavanols, gallic acid, rutin and diterpenes in several parts of Cistus incanus[10-12]. Previous studies have shown the presence of oligomeric proanthocyanidins in Cistus albidus[13]. Polyphenols in water extracts of C. libanotis and C. monspeliensis collected in Spain, have also been reported [9]. The concentration and the presence of different compounds in plants are not only species specific but they also depend on soil fertility and pH, light intensity, plant age or temperature stress [14].

The presence of flavonoids in Cistus has been well documented. In fact, previous reports showed the occurrence of apigenin, quercetin and kaempferol derivatives in exudates of C. ladanifer leaves and in soil where these plants grew [15,16], and this has been related to its allelopathic potential. In the extract of C. libanotis we detected as main compounds quercetin 3,7,3',4'-tetramethyl ether (retusin) (24.6%) and 5.6% of apigenin dimethyl ether (genkwanin 4'-methyl ether).

Cytotoxic activity of the extracts

The cytotoxic activity of three Cistus extracts against three cancer cell lines, including murine monocyte/macrophages, J774.A1, human melanoma cells, A-375, and human breast cancer cells MCF7, was determined, through the MTT conversion assay [17]. In Table  2 we showed the IC50 values, that represent the concentration expressed as mg of dry extract/ml of the different hexane extracts of C. libanotis, C. villosus and C. monspeliensis that affords a 50% reduction in cell growth after 72 h incubation time. Both extracts obtained from C. libanotis and C. villosus were inactive against all tested cell lines. The 50% cytotoxic concentration (IC50) could not be estimated. A pronounced growth inhibition was showed by C. monspeliensis hexane extract against A-375 cell line, with a IC50 value of 82.42 ± 2.92 mg/ml at 24 h and 52.44 ± 3.69 mg/ml at 72 h. Our results indicated higher activity of C. monspeliensis extract if compared to 6-mercaptopurine (means IC50 = 142,36 mg/ml at 72h) used as reference drug (Table 2).

Table 2. In vitro antiproliferative activity of C. libanotis, C. monspeliensis and C. villosus hexane extracts against J774.A1 macrophages, A-375 human melanoma cells and MCF-7 breast cancer cells at 72 h

Natural extracts have been previously reported as a potential source of antiproliferative compounds [18-20]. In this sense, it is accepted that the chemopreventive and tumor-inhibitory effect associated to some dietary antioxidant polyphenols could be due to their capability to inhibit oxygen reactive species (ROS) or free radicals [21]. More recently, a large body of studies is evidencing the ability of these compounds to modulate uncontrolled proliferation pathways or protooncogen expression [22]. Therefore, it is certainly plausible that the antiproliferative activity against A-375 cell line of the hexane extract of Cistus monspeliensis compounds could be related to their radical scavenging activity too.

Phenolic compounds have been traditionally associated to biological activities such as antioxidant, antimicrobial or cytotoxic. A recent study on the anticancer activity of several tea extracts with high polyphenolic content has reported IC50 values within the range 0.1–0.5 mg/ml for several cancer cell lines [23].

Therefore, the concentration ranges of C. monspeliensis extract displaying cytotoxicity against A-375 might be significant to support further studies since hexane extract of C. monspeliensis is enriched in vitamin E (11.5%) which possesses well known antioxidant activity. Vitamin E acts as a peroxyl radical scavenger, preventing the propagation of free radicals in tissues, by reacting with them to form a tocopheryl radical which will then be oxidized by a hydrogen donor (such as Vitamin C) and thus return to its reduced state [24]. As it is fat soluble, it is incorporated into cell membranes and protects them from oxidative damage. The cancer preventive properties of vitamin E were firstly suspected when some studies showed that people in the Mediterranean area who consume diets enriched in vitamin E displayed a lower risk of colon cancer than people in Northern Europe and the U.S. [25,26]. More recently, the Melbourne Colorectal Cancer Study showed that dietary vitamins E and C were protective for both colon and rectal cancer, and that for both vitamins there was a dose–response effect of increasing protection [27]. Another clinical study supported a preventive effect of vitamin E in the development of prostate cancer. This study included over 29,000 elderly male smokers and showed that those taking vitamin E for six years had 32% fewer diagnoses of prostate cancer and 41% fewer prostate cancer deaths than men who did not take vitamin E [28]. More recently it has been demonstrated that Vitamin E also protects lipids and prevents the oxidation of polyunsaturated fatty acids [29].

Experimental studies also suggested detrimental effects of omega-6 polyunsaturated fatty acids (PUFA), and beneficial effects of omega-3 PUFAs on mammary carcinogenesis, possibly due to the interaction with antioxidants. Significant interactions were also found between omega-6 and long-chain omega-3 PUFAs, with breast cancer risk inversely related to long-chain omega-3 PUFAs [30]. In this light, it is interesting to note that the hexane extract of C. monspeliensis was represented by fatty acids (43.3%) among which the most abundant was the polyunsaturated fatty acid (Z,Z,Z)-9,12-15-octadecatrienoic acid (14.7%) or linolenic acid.

In this sense, C. monspeliensis extract could be capable to exert its antiproliferative activity by the presence of the large amounts of fatty acids and vitamin E, in the light of the available literature that reports that dietary antioxidant polyphenols are capable to inhibit reactive oxygen species or free radicals [21] and/or to modulate uncontrolled proliferation [22-24].

Experimental

Plant material

Leaves of C. libanotis, C. monspeliensis and C. villosus were collected on March 2012 from plants growing in the National Park of Boukornine (Tunisie).

Extraction

The plant material was dried under shade and gross powdered prior to extraction. The powdered leaf (30 g) was extracted three times with 300 mL of hexane for 3 days, than the extracts were filtered through a filter paper, after that the extracts were concentrated by rotatory evaporation, and kept at 4°C until use.

Gas chromatography

Analytical gas chromatography was carried out on a Perkin-Elmer Sigma 115 gas chromatograph fitted with a HP-5 MS capillary column (30 m × 0.25 mm i.d.; 0.25 μm film thickness). Column temperature was initially kept at 45°C for 8 min, then gradually increased to 280°C at 2.5°C min-1, held for 15 min and finally raised to 295°C at 10°C min-1. Diluted samples (1/100 v/v, in n-pentane) of 1 μL were injected manually at 250°C, and in the splitless mode with a 1 minute purge-off due to the small amount of oil partially utilized for biological tests. Flame ionization detection (FID) was performed at 280°C. Helium was the carrier gas (1 mL min-1).

Gas chromatography - mass spectrometry

GC-MS analysis was performed on an Agilent 6850 Ser. II apparatus, fitted with a fused silica HP-1 capillary column (30 m × 0.25 mm i.d.; 0.33 μm film thickness), coupled to an Agilent Mass Selective Detector MSD 5973; ionization energy voltage 70 eV; electron multiplier voltage energy 2000 V. Mass spectra were scanned in the range 35–450 amu, scan time 5 scans/s. Gas chromatographic conditions were as reported above; transfer line temperature, 295°C.

Identification of components

Most constituents were identified by gas chromatography by comparison of their retention indices (LRI) with either those of the literature [31,32] or with those of authentic compounds available in our laboratories. The retention indices were determined by GC-FID mode in relation to a homologous series of n-alkanes (C8-C28) under the same operating conditions. Further identification was made by comparison of their mass spectra on both columns with either those stored in NIST 02 and Wiley 275 libraries or with mass spectra from the literature [32,33] and our home made library. Component relative concentrations were calculated based on GC-FID peak areas without using correction factors.

Cell lines

J774.A1 murine monocyte/macrophage, A-375 human melanoma cell line and MCF-7 human breast cancer cell line were purchased from ATCC and used to evaluate the antiproliferative activity of the hexane extracts of Cistus spp.. All the media and sera were purchased from Hy-Clone (Euroclone, Paignton, Devon, UK); MTT [3 (4,5-dimethylthiazol-2-yl)-2,5-phenyl-2H-tetrazolium bromide] and 6-mercaptopurine (6-MP) were from Sigma Chemicals (Milan, Italy). Cell culture was maintained at 37°C in a Hera Cell humidified CO2 incubator (Kandro Laboratory, Germany) with 5% CO2.

MTT antiproliferative assay

Cells (J774.Al, A-375, and MCF-7 ) were harvested and suspended in complete culture media. Approximately 100 μl of the cell suspension with a concentration of 2.0×104, 3.0×103, 5.0×103 cells, respectively were plated on 96-well microtiter plates and allowed to adhere at 37°C in 5% CO2 and 95% air for 24 h. Thereafter, the medium was replaced with 90 μL of fresh medium, and a 10 μL aliquot of serial dilution of each extract to test was added and the cells were incubated for tested time. Incubation was carried out for 72 h in the dark and at the end of the period, 20 μl of MTT at a concentration of 5 mg/ml was added to each well. After 3 hours of incubation, culture media in each well was aspirated and 100 μl of DMSO was added to dissolve the formazan products formed prior to recording the optical density (O.D.) at 570 nm with respect to the reference wavelength at 620 nm. In some experiments, serial dilutions of 6-MP, as reference drug, were added. The cell viability was assessed through an MTT conversion assay (Bianco et al., 2012). The optical density (OD) of each well was measured with a microplate spectrophotometer (Titertek Multiskan MCC/340) equipped with a 620 nm filter. The test concentration which inhibits 50% of the cell population (IC50) was obtained by Probit Analysis (SPSS Version 12.0.1, Chicago, IL, USA). All the experiments were carried out in triplicates and two independent experiments were performed for each test sample. The viability of each cell line in response to treatment with Cistus extracts was calculated as % dead cells: 100 - (OD treated/OD control) × 100.

Conclusion

The chemical composition of the hexane extracts from the three Cistus species can be useful in the chemosystematics of this complex genus. The antiproliferative activity of the Cistaceae hexane extracts observed for the first time in this study against human melanoma cell line A-375 deserve further investigations in order to determine the compounds, or their combinations, which are the main responsible for antiproliferative activity and its potential mechanism.

Findings and description of additional material

Plant material and extracts of the plants are available (MB). The GC-MS data are also available (FS).

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

MBJ Collected and identified the plant material, prepared the extracts and drafted the manuscript. MEK Collected and identified the plant material and drafted the manuscript. FS performed the GC-MS analysis, identified the components and drafted the manuscript. GA performed the MTT antiproliferative assay and drafted the manuscript. SM performed the MTT antiproliferative assay and drafted the manuscript. VDF identified the components and drafted the manuscript. MB prepared the extracts, identified the components and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgments

This research was supported by Italian Government fund MIUR PRIN 2009 “Composti naturali da piante mediterranee e loro derivati sintetici con attivita’ antitumorale”. The GC-MS spectra were performed at the "C.S.I.A.S." of the University "Federico II" of Napoli. The assistance of the staff is gratefully appreciated.

References

  1. Ferrandis P, Herrantz JM, Martínez-Sánchez JJ: Effect of fire on hardcoated Cistaceae seed banks and its influence on techniques for quantifying seed banks.

    Plant Ecol 1999, 144:103-114. Publisher Full Text OpenURL

  2. Guzmán B, Vargas P: Systematics, character evolution, and biogeography of Cistus L. (Cistaceae) based on ITS, trnL-trnF, and matK sequences.

    Mol Phylogenet Evol 2005, 37:644-660. PubMed Abstract | Publisher Full Text OpenURL

  3. Andrade D, Gil C, Breitenfeld L, Domingues F, Duarte AP: Bioactive extracts from Cistus ladanifer and Arbutus unedo L.

    Ind Crop Prod 2009, 30:165-167. Publisher Full Text OpenURL

  4. De Andres AI, Gomez-Serranillos MP, Iglesias I, Villar AM: Effects of extract of Cistus populifolius L. on the central nervous system.

    Phytother Res 1999, 13:575-579. PubMed Abstract | Publisher Full Text OpenURL

  5. Küpeli E, Yesilada E: Flavonoids with anti-inflammatory and antinociceptive activity from Cistus laurifolius L. leaves through bioassayguided procedures.

    J Ethnopharmacol 2007, 112:524-530. PubMed Abstract | Publisher Full Text OpenURL

  6. Barrajon-Catalan E, Fernandez-Arroyo S, Guillén E, Segura-Carretero A, Fernandez-Gutierrez A, Micol V: Cistaceae aqueous extracts containing ellagitannins show antioxidant and antimicrobial capacity, and cytotoxic activity against human cancer cells.

    Food Chem Toxicol 2010, 48:2273-2282. PubMed Abstract | Publisher Full Text OpenURL

  7. Angelopoulou D, Costas Demetzos L, Dimas C, Perdetzoglou1 D, Loukis A: Essential oils and hexane extracts from Leaves and fruits of Cistus monspeliensis. Cytotoxic activity of ent-13-epi-Manoyl Oxide and its isomers.

    Planta Med 2001, 67:168-171. PubMed Abstract | Publisher Full Text OpenURL

  8. Pomponio R, Gotti R, Santagati NA, Cavrini V: Analysis of catechins in extracts of Cistus species by microemulsion electrokinetic chromatography.

    J Chromatogr A 2003, 990:215-223. PubMed Abstract | Publisher Full Text OpenURL

  9. Barrajon-Catalan E, Fernandez-Arroyo S, Roldan C, Guillen E, Saura D, Segura-Carretero A, Micol V: A systematic study of the polyphenolic composition of aqueous extracts deriving from several Cistus Genus species. Evolutionary Relationship.

    Phytochem Anal 2011, 22:303-312. PubMed Abstract | Publisher Full Text OpenURL

  10. Chinou I, Demetzos C, Harvala C, Roussakis C, Verbist JF: Cytotoxic and antibacterial labdane-type diterpenes from the aerial parts of Cistus incanus subsp. creticus.

    Planta Med 1994, 60:34-36. PubMed Abstract | Publisher Full Text OpenURL

  11. Kreimeyer J, Petereit F, Nahrstedt A: Separations of flavan-3-ols and dimeric proanthocyanidins by capillary electrophoresis.

    Planta Med 1998, 64:63-67. PubMed Abstract | Publisher Full Text OpenURL

  12. Santagati NA, Salerno L, Attaguile G, Savoca F, Ronsisvalle G: Simultaneous determination of catechins, rutin, and gallic acid in cistus species extracts by HPLC with diode array detection.

    J Chromatogr Sci 2008, 46:150-156. PubMed Abstract | Publisher Full Text OpenURL

  13. Qa’dan F, Petereit F, Nahrstedt A: Prodelphinidin trimers and characterization of a proanthocyanidin oligomer from Cistus albidus.

    Pharmazie 2003, 58:416-419. PubMed Abstract OpenURL

  14. Adamczyk B, Kitunen V, Smolander A: Protein precipitation by tannins in soil organic horizon and vegetation in relation to tree species.

    Biol Fert Soils 2008, 45:55-64. Publisher Full Text OpenURL

  15. Chaves N, Escudero JC, Gutierrez-Merino C: Quantitative variation of flavonoids among individuals of a Cistus ladanifer population.

    Biochem Syst Ecol 1997, 25:429-435. Publisher Full Text OpenURL

  16. Chaves N, Sosa T, Escudero JC: Plant growth inhibiting flavonoids in exudate of Cistus ladanifer and in associated soils.

    J Chem Ecol 2001, 27:623-631. PubMed Abstract | Publisher Full Text OpenURL

  17. Bianco G, Russo R, Marzocco S, Velotto S, Autore G, Severino L: Modulation of macrophage activity by aflatoxins B1 and B2 and their metabolites aflatoxins M1 and M2.

    Toxicon 2012, 59:644-50. PubMed Abstract | Publisher Full Text OpenURL

  18. Kaefer CM, Milner JA: The role of herbs and spices in cancer prevention.

    J Nutr Biochem 2008, 19:347-361. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  19. Perchellet JP, Gali HU, Perchellet EM, Klish DS, Armbrust AD: Antitumor-promoting activities of tannic acid, ellagic acid, and several gallic acid derivatives in mouse skin.

    Basic Life Sci 1992, 59:783-801. PubMed Abstract OpenURL

  20. Shoemaker M, Hamilton B, Dairkee SH, Cohen I, Campbell MJ: In vitro anticancer activity of twelve Chinese medicinal herbs.

    Phytother Res 2005, 19:649-651. PubMed Abstract | Publisher Full Text OpenURL

  21. Halliwell B: Antioxidants in human health and disease.

    Ann Rev Nut 1996, 16:33-50. Publisher Full Text OpenURL

  22. Menendez JA, Vazquez-Martin A, Colomer R, Brunet J, Carrasco-Pancorbo A, Garcia-Villalba R, Fernandez-Gutierrez A, Segura-Carretero A: Olive oil’s bitter principle reverses acquired autoresistance to trastuzumab (Herceptin) in HER2-overexpressing breast cancer cells.

    BMC Cancer 2007, 7:80. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  23. Friedman M, Mackey BE, Kim HJ, Lee IS, Lee KR, Lee SU, Kozukue E, Kozukue N: Structure-activity relationships of tea compounds against human cancer cells.

    J Agric Food Chem 2007, 55:243-253. PubMed Abstract | Publisher Full Text OpenURL

  24. Traber Maret G, Stevens JF: Vitamins C and E: beneficial effects from a mechanistic perspective.

    Free Radic Biol Med 2001, 51:1000-1013. OpenURL

  25. Berrino F, Muti P: Mediterranean diet and cancer.

    Eur J Clin Nutr 1989, 43(Suppl 2):49-55. PubMed Abstract OpenURL

  26. Khlat M: Cancer in Mediterranean migrants–based on studies in France and Australia.

    Cancer Causes Contr 1995, 6:525-31. Publisher Full Text OpenURL

  27. Kune G, Watson L: Colorectal cancer protective effects and the dietary micronutrients folate, methionine, vitamins B6, B12, C, E, selenium, and lycopene.

    Nutr Cancer 2006, 56:11-21. PubMed Abstract | Publisher Full Text OpenURL

  28. Heinonen OP, Albanes D, Virtamo J, Taylor PR, Huttenen JK, Hartman AM: Prostate cancer and supplementation with α- tocopherol and β- carotene: incidence and mortality in a controlled trial.

    J Natl Cancer Inst 1998, 90:440-446. PubMed Abstract | Publisher Full Text OpenURL

  29. Ellie W, Rady Rolfes S: Understanding Nutrition. 12th edition. Edited by editionEdited by Wadsworth WP. California: Cengage Learning; 2011.

  30. Thiébaut AC, Chajès V, Gerber M, Boutron-Ruault MC, Joulin V, Lenoir G, Berrino F, Riboli E, Bénichou J, Clavel-Chapelon F: Dietary intakes of omega-6 and omega-3 polyunsaturated fatty acids and the risk of breast cancer.

    Int J Cancer 2009, 124:924-931. PubMed Abstract | Publisher Full Text OpenURL

  31. Davies NW: Gas chromatographic retention indexes of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases.

    J Chromatogr A 1990, 503:1-24. OpenURL

  32. Jennings W, Shibamoto T: Qualitative Analysis of Flavour and Fragrance volatiles by Glass Capillary Gas Chromatography. New York: Academic Press; 1980. OpenURL

  33. Adams RP: Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. 4th edition. Carol Stream, Illinois: Allured Publishing Co; 2007. OpenURL