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1 Introduction

Plant secondary metabolites have amazing structural diversity and biological activities, including anticancer proprieties [1, 2]. Among these, essential oils are odoriferous substances traditionally used in the perfumery, food, and pharmaceutical industries. Essential oils may be found in different plant parts generally in flowers (e.g. Acacia spp., Dianthus caryophyllus, Jasminum spp., Lavandula spp., Rosa spp., Rosmarinus officinalis, Syzygium aromaticum, etc.), fruits (Citrus spp., Juniperus communis, etc.), leaves (e.g. Cymbopogon spp., Mentha spp., Ocimum spp., etc.), leaves and stems (e.g. Aloysia citriodora, Cinnamomum spp., Pelargonium spp., Pogostemon cablin, etc.). Moreover, bark (e.g. Betula pubescens, Cinnamomum cassia), rhizomes (e.g. Acorus calamus, Curcuma longa, Zingiber officinale, etc.), roots (e.g. Angelica archangelica, Vetiveria zizanioides, etc.), seeds (e.g. Coriandrum sativum, Foeniculum vulgare, Myristica spp., etc.), and wood (e.g. Aniba rosaeodora, Eremanthus erythropappus, Pinus sylvestris, Santalum album, etc.) are also natural sources of commercially important essential oils [3, 4].

The most volatile fraction (constituting 90–95 % of total oil) comprises, in most cases, complex mixtures that may contain hundreds of compounds which are composed mainly of terpenoids (mono-, sesqui-, and even diterpenes). Besides, it may contain benzenoids, phenylpropanoids, aliphatic aldehydes, alcohols, and esters. Terpenoids (mono- and sesquitepenes) are the primary constituents of the essential oils of many types of herbs; many of them are commercially important, and are widely used as flavoring agents, perfumes, insecticides, antimicrobial agents, and raw material for important chemicals [5].

In this chapter, a total of 20 essential oil constituents, which presented positive results on cytotoxic drug screening, were selected (Fig. 19.1). In addition, we highlighted the recent advances in the knowledge of the chemical and anticancer properties of these compounds, establishing new goals for future research.

Fig. 19.1
figure 1

Chemical structures of selected essential oil components

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2 Chemical and Botanical Data

A rigid scheme for classifying secondary metabolites is not applicable due to their immense structural diversity; however, three main classes are often used: terpenoids and steroids; fatty-acid derivatives and polyketides; and alkaloids. Terpenoids constitute the largest and one of the most diverse classes of secondary metabolites and they are classified according to the number of containing five-carbon units coupled through biosynthetic pathways [6, 7]. In volatile fraction, we found terpenoids that are classified as hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), and even some diterpenes (C20).

Despite their diversity, all plant terpenoids derive from the common building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) that are synthesized via two parallel pathways known as mevalonate (MVA) and methylerythritol 4-phosphate (MEP) [6]. In general, MVA pathway leads to the synthesis of some sesquiterpenes and triterpenes (sterols) in the cytoplasm, while the MEP pathway is responsible for the synthesis of monoterpenes, diterpenes, tetraterpenes (carotenoids) and polyterpenes [7, 8].

In the most volatile fraction of essential oils, terpenoids are generally unsaturated compounds which easily decomposable by light, heat or oxygen to produce undesirable compounds. So, the steps of isolation, concentration and purification in methods for extraction of essential oil become critical. The most commonly used technique is the so-called traditional methods [9], i.e., those based on mechanical pressing (e.g. citrus oils) and by hydro- or steam-distillation. Steam distillation is the worldwide procedure for extraction of essential oils from plant material, which is usually made by Clevenger-type apparatus in laboratory scale (hydro-distillation). The main drawback of this technique is related to the decomposition of labile compounds and the possibility of formation of non-natural compounds (artifacts). Therefore, it is desirable to employ mild conditions to avoid oxidation, thermal deterioration or other chemical changes. Modern methods of sampling using small volume or no organic solvent have emerged in recent years. Techniques such as supercritical fluid extraction with CO2 (SFE-CO2), microwave assisted extraction (MAE) and pressure liquid extraction (PLE) have gained in interest as a green approach in volatile fraction extractions which can be used in small and large scale. These techniques have advantages and limitations, which have been recently reviewed [10–12].

Gas chromatograph (GC) coupled with a mass spectrometer (MS) has been the main analytical technique for the chemical characterization of essential oils [10] and together forms a powerful tool for high quality quantitative and qualitative analysis. However, GC/MS analysis of this complex mixture often is a time-consuming task due to limitations in techniques and instrumentation. Advances in this field have led to increased sensitivity, reproducibility and shorter times for analysis of volatile components. For example, fast GC often yields faster analysis times than conventional GC while maintaining resolution and allowing more samples to be analyzed per shift. Nevertheless, the use of fast GC for essential oils analysis is still exploring [13, 14].

For identification of essential oil constituents, computerized matching of acquired mass spectrum with those stored in the mass spectral libraries (NIST and Wiley) and with that of an authentic standard, comparison of retention indices [15] determined at least on two columns with distinct polarities together are currently the most widely used for structural identification [7]. Quadrupole mass spectra obtained by electron ionization (EI) at 70 eV has been preferred to the ion-trap-derived mass spectra due to resulting in fragmentation characteristics of each compound [16], especially for the identification of unknown compounds. Tandem mass spectrometry (MS–MS), Fourier transform infrared spectroscopy (FT-IR) and time-of-flight mass spectrometry (TOF–MS) are alternative techniques for detection employed to identify closely related isomers or overlapping compounds. The latter has been the detector of choice for GC × GC analysis [10, 17].

In addition, enantiomer separation and determination of enantiomeric ratio or enantiomeric excess (e.e.) remains with increasing interest mainly due to possible difference in enantiomer biological properties [18]. Two-dimensional (2D) chromatographic approach has been required for chiral recognition of components in complex samples. The most used techniques is heart-cutting multidimensional GC (MDGC) and comprehensive 2D GC (GC × GC). Advantages and limits of both techniques are well-known, although recently mass spectrometry as a second dimension in detection has gained further interest because of the role it can play in speeding up enantioselective GC analysis (es-GC) [18, 19]. Derivatized cyclodextrins (CDs) based columns are the most popular chiral stationary phase for es-GC [20] and has been successfully applied in the 2D GC analysis of chiral components from essential oils [21].

Recently there has been growing interest in terpenoids compounds due to the biological activity shown by some of them, especially as anticancer agents. In addition, terpenoid enantiomers are particularly useful chiral building blocks for the chemical and biotechnological syntheses [22]. Thus, essential oils are a rich source of highly value compounds and their commercialization has increasing because they are also used widely in prevent and therapy several diseases [23].

Despite the importance of the issue, to our knowledge, little attention was paid on the investigation of chiral components of essential oils and their anticancer activity [24, 25].

It is widely known that many substances produced by living organisms are chiral and can occur alone (only one enantiomer) or can be present in varying amounts or even as racemic mixtures, exerting the same or distinct activities. For example, (4S)-(+)-carvone smell like caraway and (4R)-(−)-carvone has a distinct sweet spearmint odor with both enantiomers presenting different effects on the central nervous system [26]. Moreover, the isomers of the monoterpene alcohol (3R)-(−)- and (3S)-(+)-linalool have distinctive odors [27], but show the same antifungal activity [28].

The biosynthetic pathways of the essential oil components can produce one form only of an optically active chemical. Sometimes one form of an enantiomer may be produced in larger amounts, but often the relative abundances of both enantiomeric forms are very specific to the species and geographical origin of an essential oil [29].

Except for α-humulene, γ-terpinene, eugenol, and thymol exists chirality (stereogenic centers) in the other molecules mentioned in Fig. 19.1. Moreover, enantiomeric distribution of constituents from essential oils is still scarce, especially for sesquiterpenes. Nevertheless, some papers have been published mainly on the enantiomeric variation of monoterpenes such as (±)-limonene, (±)-linalool, (±)-α-pinene, and (±)-terpinen-4-ol [27, 28, 30, 31].

The enantiomers of the linalool are found in variable distribution from different plant species [21, 28]. In general, the optically active (3S)-(+)- and (3R)-(−)-linalool were isolated from lavender oil and coriander oil respectively, but both forms can be found in variable proportion in the wood and leaves essential oils of Brazilian rosewood (Aniba rosaeodora Ducke) [32]. It is noteworthy that the chiral stability may be influenced by the developmental stage of plant material, pH value and sampling techniques. In comparison, increasing amounts of the (3S)-(+) linalool was detected in oils produced by hydro-distillation of longer than 1 h duration [33]. In the same way, ascaridole, a monoterpene endoperoxide found as major component (up to 92 % of total oil) of essential oil from Chenopodium ambrosioides from various origins, is a heat-sensitive compound which rearranges to isoascaridole [34].

The enantiomeric composition of the monoterpene limonene is different for the various plant parts and changes during the development of the umbels as well as antimicrobial efficacy of the individual enantiomers and the racemic mixture showed variation [35]. For example, (4R)-(+)-limonene, the major component orange and other citrus peel oils, is one of the most investigated monoterpenes regarding the prevention of chemically induced tumors [36]. From a biosynthetic point of view, (4S)-(−)-limonene then serves as a precursor to other oxygenated monocyclic monoterpenes such as (−)-perillyl alcohol and (−)-perillaldehyde which are minor components in many aromatic plants including Perilla frutescens (Lamiaceae) and also have been linked to anticancer activity [23, 24, 36].

The sesquiterpene α-(−)-bisabolol was first isolated from the blossoms of Matricaria chamomilla (Asteraceae) which may exist in three others possible stereoisomers: (+)-α-bisabolol and (+)- and (−)-epi-α-bisabolol. Besides, chamomile, another source of α-(−)-bisabolol is sage (Salvia runcinata) that contain up to 90 % and candeia (Eremanthus erythropappus) which may contain up to 85 % from the wood [37].

(−)-trans-β-elemene is a sesquiterpene that has attracted attention due to recent developments on their use in the antitumor therapy of many kinds of cancer. A comprehensive review that includes its natural occurrence, biogenesis, anticancer activity, and synthesis and chemical characterization was recently published [38].

Caryophyllene is probably the most widely distributed sesquiterpene in nature. The pure form of the (−)-(Ε)-caryophyllene is frequently present in the essential oil from Humulus lupulus, Piper nigrum, Syzygium aromaticum leaf, and Copaifera officinalis, etc. However, (+)-(Ε)-caryophyllene is rare and has been found in essential oils of liverworts [39].

Phenylpropanoid volatile compounds are found in essential oil composition of many plant species presenting significant biological activities. One such compound is eugenol which has been used by humans since antiquity for food preservation and flavoring and for medicinal purpose. Essential oil from clove (Syzigium aromaticum) is the main natural source of eugenol (up to 90 % of total oil) [40].

3 Anticancer Proprieties

As already mentioned, we selected a total of 20 compounds that presented positive results on cytotoxic drug screening; among them, ascaridole, α-bisabolol, (E)-caryophyllene, β-elemene, β-eudesmol, d-limonene, terpinen-4-ol, and thymol have been extensively studied with promissory results. Therefore, a special attention was given to these compounds. Moreover, the relevant compounds were summarized and included in a condensed form in Table 19.1.

Table 19.1 Summary of the anticancer proprieties of essential oil constituents
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Ascaridole exhibits cytotoxic activity against leukemia, melanoma, brain, and colon cancer cell lines, as well as, multiple drug resistance cancer cell lines [41, 42]. It also presented in vivo antitumor in a sarcoma murine model with no toxic side effects [42]. More recently, cell cycle and DNA damage analyses revealed a remarkable NER (nucleotide excision repair) specificity of ascaridol. Ascaridole decreased the G1 phase in three cells lines, but it caused G2/M phase arrest only in NER-deficient cells. It also induced an increase in the subG1 peak which was considerably higher in NER-deficient cells than in proficient cells. Moreover, DNA damage induction was substantially higher in NER-deficient cells. In addition, ascaridole led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites [43].

α-Bisabolol showed cytotoxic effect on several human and rat malignant cell lines. The action of α-bisabolol seems to be selective as its effect in normal mouse astroglial cells was not cytotoxic. It also exhibited apoptotic activity by induction of the liberation of cytochrome c and via Fas receptor [44, 45]. In addition, Gomes-Carneiro et al. [46] showed that α-bisabolol is non-mutagenic in the Salmonella microsomal test, and it can even neutralize the effect of various mutagenic substances. Moreover, α-bisabolol also showed an antigenotoxic effect against the hydrogen peroxide effect [45].

(E)-Caryophyllene has been reported to have cytotoxic activity over a wide range of tumor cell lines, but not against normal cells [47–49]. In addition, it caused an induction of apoptosis accompanied by DNA ladder and caspase-3 catalytic activity in tumor cell lines [49].

β-Elemene exhibits in vitro and in vivo antitumor activity on human and murine tumor cells. Many studies showed that the cell proliferation inhibited by β-elemene is correlated to G2/M phase arrest and induction of apoptotic cell death by reduction of Bcl-2 protein expression. β-Elemene also enhances caspase-3 activity, and inhibits protein expression of eukaryotic initiation factors eIFs (4E, 4G), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) [50–57].

Furthermore, β-elemene markedly enhanced taxanes or cisplatin-induced cytotoxicity [57–59]. The combination treatments induced increased cytochrome c release from mitochondria, significant caspase-8 and -3 cleavage, and downregulation of Bcl-2 and Bcl-XL expression. The suppression of specific ‘survival’ gene expression appears to be the key action leading to the synergistic effect of combination treatments with β-elemene and taxanes [58, 59]. In vivo, the growth of laryngeal cancer cell-transplanted tumors in nude mice was inhibited by intraperitoneal injection of elemene. Compared with control groups, elemene significantly inhibited the protein expression of eIFs (4E and 4G), bFGF, and VEGF and decreased the microvessel density (MVD) [57]. Moreover, some clinical trials indicated that the possible side effects of β-elemene given intravenously include slight fever (usually lower than 38 °C), gastro-intestinal reactions, allergic reactions, local pain, and phlebitis. No bone marrow, liver, cardiac, or renal toxicities were found to be related to clinical treatment with β-elemene [50, 60, 61].

β-Eudesmol produced inhibitory effect on the growth of various tumor cells lines, but it had no effect on the proliferation of the rat aortic smooth muscle cells and astrocytes [62–64]. In addition, β-eudesmol induced apoptosis accompanied by cleavage of caspase-3, caspase-9, and poly (ADP-ribose) polymerase; downregulation of Bcl-2 expression; release of cytochrome c from mitochondria; and decrease in mitochondrial membrane potential (MMP). Activation of c-Jun N-terminal kinases (JNK) mitogen-activated protein kinases was observed in β-eudesmol-treated cells, and the inhibitor of JNK blocked the β-eudesmol-induced apoptosis, downregulation of Bcl-2, and the loss of MMP, suggesting that β-eudesmol induces apoptosis by mitochondrial apoptotic pathway, which is controlled through JNK signaling [63].

β-Eudesmol also inhibited angiogenesis by blocking extracellular regulated protein kinases (ERK) MAPK signaling [62]. Moreover, it inhibited the growth of mouse H22 and S180 tumor and the formation of new blood vessels in tumor tissues in vivo [65].

d-limonene is a known anticarcinogenic compound that it was proved to have antitumor activity [66–70]. In recent studies, d-limonene showed able to inhibit the growth of human gastric cancer cell in vitro through a mechanism of inducing the apoptosis of tumor cells [70].

In xenograft model, d-limonene alone or combined with 5-FU decreased drastically, the metastasis to liver, peritoneum and the occurrence of ascites were inhibited significantly compared with the control group. In addition, a notably decreased expression of MVD and VEGF in d-limonene and combined group were observed, suggesting the anti-angiogenic mechanism of d-limonene via down-regulation of VEGF [71].

Many studies have indicated that terpinen-4-ol exerts cytotoxic effects against several tumor cell lines without affecting normal cells. It is also able to induce cell-cycle arrest and cell death through apoptosis or necrosis pathways [72, 73]. In addition, Wu et al. [74] demonstrated that caspase-dependent mitochondrial dysfunction is the mechanism of terpinen-4-ol-induced apoptosis. Downregulation of Bcl-2, XIAP and survivin suggests that terpinen-4-ol increases the susceptibility of cancer cells to apoptosis induction. Notably, the ability of terpinen-4-ol to induce apoptosis in tumor cells was p53-dependent. Furthermore, the growth of s.c. xenograft tumors was remarkably inhibited by intratumoral injection of terpinen-4-ol, indicating that the agent also has potential for clinical anticancer activity.

The cytoprotective and antimutagenic effects of thymol has been extensively reported [75–78]. Anyway, some studies also have indicated the antitumor activity of thymol [79–81]. Thymol caused activation of caspase-9, -8 and -3 and concomitant PARP cleavage and it induced disruption of mitochondrial membrane potential, which is associated with caspase-dependent apoptosis. The disruption of mitochondrial membrane and activation of apoptosis appears to be dependent on reactive oxygen species. The translocation of AIF from mitochondria to cytosol and then to nucleus indicates thymols’ ability to induce apoptosis through caspase independent pathway as well [79]. In addition, Hsu et al. [80] demonstrated that thymol induces a [Ca2+] concentration rise by inducing phospholipase C- and protein kinase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via non store-operated Ca2+ channels. Thymol induced cell death that may involve apoptosis.

4 Conclusions and Perspectives

A great amount of essential oil constituents with anticancer potential are found. The most of them presented cytotoxic activity only high range of IC50 values; therefore, they present weak clinical potential use. On the other hand, some of them have been extensively studied with promissory results. In short, 20 compounds were identified for their activities in the experimental models used for cytotoxic drug screening; among them, ascaridole, α-bisabolol, (E)-caryophyllene, β-elemene, β-eudesmol, d-limonene, terpinen-4-ol, and thymol have been shown promissory results. Anyway, further investigations are necessary to validate these compounds as novel clinically useful cancer chemotherapeutic agents.