INTRODUCTION
Sesquiterpene lactones are a group of secondary metabolites found across the plant kingdom, comprising a large group of over 5000 known compounds from different families, such as Cactaceae, Solanaceae, Araceae, and Euphorbiaceae. However they are mostly prevalent in the Asteraceae, where they can be found almost ubiquitously. The most representing natural element from Asteraceae is Arnica Montana (Figure1-2): known commonly as leopard's bane or wolf's bane, it is a European flowering plant charaterized by tall stems, 20–60 centimetres high, supporting usually a single yellow flower head, approximately 5 cm in diameter, that appear from May to August.
Fig. 1 
Fig. 2
Arnica Montana grows in nutrient-poor siliceous meadows up to nearly 3,000 metres. It is endemic to Europe, from southern Iberia to southern Scandinavia and the Carpathians, but is becoming rarer, particularly in the north of its distribution, because of the increasing intensive agriculture.
At times where extraction and synthetic chemistry were not yet invented, hematomas, contusions, sprains, rheumatic diseases, and superficial inflammations of the skin were treated all over the world with preparations from medicinal plants. The plethora of bioactive compounds, generally called phytochemicals, found in Asteraceae, such as terpenoids, alkaloids, phenolics and polyacetylenes make it a highly significant genus both biochemically and pharmacologically. Sesquiterpene lactones are one of the most prevalent and biologically significant classes of secondary metabolite present, so that they have been subject in plenty of studies which have been trying to explain the role of endogenous sesquiterpene lactones in the plants that produce them, and explore mechanisms by which they interact in animal and human consumers of these plants.
Several mechanisms are proposed for the reduction of inflammation and tumorigenesis at potentially achievable levels in humans. Plants can be classified by their specific array of produced sesquiterpene lactones, showing high levels of translational control. In addition, folk medicines implicate sesquiterpene lactones as the active ingredient in many treatments for other ailments such as diarrhea, burns, influenza, and neurodegradation. Finally recent studies explain how sesquiterpene lactones have been found to sensitize tumor cells to conventional drug treatments. (Sesquiterpenoids Lactones: Benefits to Plants and People, International Journal of Medical Science, 2013)
As regards the results in A. Montana, one of the most investigated moleculas it is called Helenalin.
BIOCHEMICAL FEATURES
Sesquiterpenes are colorless lipophilic compounds. Biosynthesis in plants is from three isoprene units, and occurs via farnesyl pyrophosphate (FPP), in the endoplasmic reticulum. Sesquiterpenes consist of a 15 carbon backbone, and whilst diverse in their structure, the majority, and the most functional forms are cyclic, and consequently the focus of this review will rest upon these compounds. Sesquiterpene lactones can be divided into several main classes including germacranolides, heliangolides, guaianolides, pseudoguaianolides, hypocretenolides, and eudesmanolides.
Fig.3
Biosynthesis of sesquiterpene lactones is highly characterized, with detailed reports available among others. Germacranolides, guaianolides, pseudoguianolides and eudesmanolides are the most representative classes. Germacranolides (Figure 3A) is the most significant with regards to their function in humans: they have a 10 membered ring; Guaianolides (Figure 3C-D) have a 7-membered and a 5-membered ring, and a methyl group at C-4; Pseudoguaianolides (Figure 3E) have a 7-membered and a 5-membered ring and a methyl group at C-5. Eudesmanolides (Figure 3G) have two fused 6 membered rings All contain a fused 5-membered lactone group (γ lactone) with a carbonyl moiety at the alpha position.
Fig.4
Helenalin (Figure 4) in particular is a sesquiterpene lactones of the 10a-methylpseudoguaianolide type, same as its derivate 11a,13-dihydrohelenalin: these moleculas possess two alkylant structure elements which may be responsible for their remarkably high activity. However, other factors, such as lipophilicity, molecular geometry, and the chemical environment of the target sulfhydryl may also influence the activity of sesquiterpene lactones. (The Anti-inflammatory Sesquiterpene Lactone Helenalin Inhibits the Transcription Factor NF-kB by Directly Targeting p65. The Journal of Biological Chemistry, 1998)
HELENALIN ANTI-INFLAMMATORY ACTIVITY
Sesquiterpene lactones have been shown to modulate many processes that influence inflammatory reactions, for example, oxidative phosphorylation, platelet aggregation, histamine and serotonin release.
These activities are mediated chemically by a,b-unsaturated carbonyl structures such as an a-methylene-g-lactone (αMγL), an oxygen-containing ring structure with a carbonyl function: this group is the most responsible for biological effects, because its alkylation power (especially of thiol groups) acts on transcription factors and enzymes in the human body, causing steric and chemical changes. These structure elements react with nucleophiles, especially cysteine sulfhydryl groups, by a Michael type addition.
About this, Siedle et al. conducted a comprehensive study (Quantitative Structure-Activity Relationship of Sesquiterpene Lactones as Inhibitors of the Transcription Factor NF-KB, Journal of Medicinal Chemistry, 2004) based upon analysis of over 100 sesquiterpene lactones for six families and they found that presence of an α,β-unsaturated carbonyl group was more important to cytotoxicity than αMγL groups. A consequence of their capacity for Michael addition with cysteine containing proteins and enzymes may be that two cysteine sulfhydryl groups (*Cys 38* and Cys 120) in the p65 subunits become the targets for inhibitory action on NF-κB (a protein that mediates immune response in humans by controlling effectors such as cytokines, inflammatory molecules and cell adhesion molecules) which is comprised of a p50 and p65 subunit, and acts as a transcription factor when released from the IκB subunit.
Fig.5
Many studies show that the primary mode of this action for this is by preventing its release from the IκB complex. On the other hand both Lyss and Siedle suggested that helenalin may use a different molecular mechanism of inhibition of p65, not preventing the IkB degradation, but by direct alkylation of the molecule, stating that IκB inhibition plays only a minor, secondary role in most sesquiterpene lactones. Lipophilicity was also considered to be a major factor aspect of sesquiterpene lactone activity, potentially due to greater membrane permeability allowing the sesquiterpenes into cells and nuclei where they exert their main effects.
HELENALIN AS ANTICANCER AGENT
Recently, a group of scientists from Singapore deepened Helenalin’s multi modal action on cellular proliferation and apoptosis because the mechanisms underlying its action was still largely unexplained. In this study, to deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers.
Results showed that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death (NF-κB p65 repression by the sesquiterpene lactone, Helenalin, contributes to the induction of autophagy cell death. BMC Complementary and alternative medicine, 2012).
In the same year as well, another group in South Korea followed this trend, and published their work about whether helenalin could induce apoptosis in human renal carcinoma Caki cells. In order to investigate the effect of helenalin on Caki cells, series of experiments were conducted. Caki cells were incubated for 24 h at various concentrations: helenalin induced morphological features of apoptosis in Caki cells, in a dose dependent manner. Furthermore, a flow cytometric analysis was performed to calculate the haplodiploid cell population. The results suggested that:
1. Helenalin-treated Caki cells remarkably increased the Sub G1 population in a dose dependent manner.
2. Helenalin reduced cell viability and induced cytoplasmic histones associated with DNA fragmentation and chromatin condensation in the nuclei, indicating the key characteristics of apoptosis.
3. Exposure to helenalin led to increase cleaved form of PARP.
After exploring the anti-neoplastic effect of helenalin on renal carcinoma Caki cells, further investigation was performed on effect of helenalin on other human carcinoma cells. For this purpose, carcinoma cell lines of different origins were used including human renal carcinoma and human colon carcinoma. Expectedly, helenalin induced similar apoptotic effects in these cell lines. The mechanisms investigated to clearify how these processes could operate, were essentialy two: 
Fig.6
1. Oxidative stress: Helenalin increased intracellular ROS levels, and pretreatment with ROS scavengers, N-acetylcystine (NAC) and glutathione ethyl ester (GEE), blocked helenalin-induced ROS production. (Figure 6)
2. Helenalin induced morphological change and apoptosis in Bcl-2 overexpressed cells, even though increased Bcl-2 levels is one of mechanism to increase a resistance in cancer cells. (Helenalin-induced apoptosis is dependent on production of reactive oxygen species and independent of induction of endoplasmic reticulum stress in renal cell carcinoma, Toxicology in vitro, 2012)
CONCLUSIONS
As described until here, there are many Asiatic studies that support benefits of using extracts from Arnica Montana containing Sequiterpene Lactones and furthermore in Europe this kind of preparations have been largely promoted especially as antinflammatory products for low grade pathologies.
On the other hand, some studies conducted on atopic and allergic patients warn us about risks they can occure using these treatments, even though as regards reactions to Arnica may be partly due to other allergens than SLs, e.g. polyacetylenes, as showed in their study (Quantification of Sesquiterpene Lactones in Asteraceae Plant Extracts: Evaluation of their Allergenic Potential. Scientia Pharmaceutica, 2013), comparing the SL contents of the single plant extracts (results of spectrophotometric analyses) with the results of a clinically conducted patch test with patients who reacted positively to at least one the Compositae extracts. (Figure 7)
Fig.7
Bibliography
Chadwick M, T. H. (2013). Sesquiterpenoids Lactones: Benefits to Plants and People. Int. J. Mol. Sci.14, 12780-12805;.
Jang JH, T. I.-j. (2012). Helenalin-induced apoptosis is dependent on production of reactive oxygen species and independent of induction of endoplasmic reticulum stress in renal cell carcinoma. Tox In Vit.
Lim CB, P. Y. (2012). NF-κB p65 repression by the sesquiterpene lactone, Helenalin, contributes to the induction of autophagy cell death. BMC Complementary and alternative medicine.
Lyss G, K. S. (1998). The Anti-inflammatory Sesquiterpene Lactone Helenalin Inhibits the Transcription Factor NF-kB by Directly Targeting p65. The Journal of Biological Chemistry.
Siedle B, P. M. (2004). Quantitative Structure-Activity Relationship of Sesquiterpene Lactones as Inhibitors of the Transcription Factor NF-KB. J Med Chem.
Salapovic H, J. G. (2013). Quantification of Sesquiterpene Lactones in Asteraceae Plant Extracts: Evaluation of their Allergenic Potential. Scientia Pharmaceutica
