*2.1. Alantolactone*

The eudesmanolide alantolactone (**1**) (C15H20O2) (Figure 2) was first isolated from *Inula helenium* L. roots, and later was also found in other *Inula* species [24], as well as non-*Inula* spp. such as *Saussurea lappa* C.B. Clarke [25] and *Aucklandia lappa* DC. [26].

**Figure 2.** Structure of alantolactone (**1**).

Alantolactone (**1**) is obtained mainly by extraction and purification from natural sources, although its total synthesis is possible [27,28]. Modern laboratory techniques have resulted in a significant increase in its extraction yield [29–31]. For example, ~3% pure alantolactone (**1**) yields were obtained from *Inula helenium* roots by Zhao et al. [30], which meant a significant improvement over the ~1% yields from the roots of *Inula magnifica* Lipsky [32].

Although alantolactone is associated with allergic contact dermatitis triggered by *Inula* species [33–35], this compound was first described as anti-helminthic [36], with subsequent studies demonstrating its tremendous potential, mainly as antitumor [37–40], anti-inflammatory [26,41], and antioxidant [42] agent.

Regarding anticancer activity, the effect of alantolactone (**1**) on leukemia is well documented and was recently reviewed by Da Silva Castro et al. [43]. Xu et al. [44] reported a positive and significant effect (**1**) on B-cell acute lymphoblastic leukemia. In this study, 100 mg/kg (b.w.) doses were intravenously administered every two days in leukemia xenografted NOC/SCID mice. Results showed that treated mice lived an average of eight days more than non-treated mice (31.5 days after xenografting in treated compared to 23.5 days in non-treated mice), without significant weight loss. Before this study, Chun et al. [45] had also reported an in vivo antitumor effect on triple negative breast cancer in mice. The researchers used only a 2.5 mg/kg (b.w.) dose every two days to treat athymic nude mice xenografted with MDA-MB-231 cells. In this assay, alantolactone (**1**) reduced tumor size by over half, and significantly reduced tumor weight by about half after 24 days. Alantolactone (**1**) also exhibits in vivo activity against gastric cancer. Human gastric cancer cells (SGC-7901) were xenografted onto nude athymic mice, which were treated with 15 mg/kg (b.w.) of alantolactone (**1**) injected every two days [46]. Alantolactone (**1**) caused significant tumor growth inhibition and reduced Ki-67 and Bcl-2 expression (tumor associated proteins), without significant liver and kidney toxicity or impact on mouse weight [46].

Alantolactone (**1**) has also proven to be an especially potent sensitizing agent. In fact, it exhibits a synergistic effect with known chemotherapy drugs, such as oxaliplatin. Cao et al. [47] showed that a 10:2 mg/kg (b.w.) dose of alantolactone: oxaliplatin reduced tumor volume and weight by more than 50% in athymic mice xenografted with colorectal tumor cells (HCT116). The anticancer effect increased substantially when both compounds were used together, as opposed to alone. This result is in line with that obtained by He et al. [48], according to which, using a small weekly injected dose of 3 mg/kg (b.w.), tumor volume significantly decreased by around 75% in xenografted pancreatic cancer cells (PANC-1), while increasing cancer cell chemosensitivity to oxaliplatin, revealing a synergistic action.

Alantolactone's anticancer mechanism was also the main focus of several studies, some of them mentioned in recent reviews [49,50] The authors attribute its activity to the multiple pathways it activates. Alantolactone (**1**) acts as an alkylating agen<sup>t</sup> leading to inhibition of key enzymes and proteins, and as an apoptosis inducer in cancer cells at mitochondrial level by interacting with cytochrome c. It promotes overproduction of reactive oxygen species (ROS) due to specific caspase activation, by inhibiting autophagic deregulation, among other processes.

Beyond its anticancer activity, compound **1** also exhibits interesting anti-inflammatory activity. Ren et al. [51] used the DSS-induced colitis mouse model to test alantolactone (**1**) anti-inflammatory activity. A 50 mg/kg dose reversed colitis symptoms (bloody diarrhea, colon shortening and weight loss), besides significantly reducing pro-inflammatory cytokine TNF-α expression (to about half that of positive control) and IL-6 (by over 2.5 times compared to positive control) [51]. Wang et al. [41], showed that a 10 mg/kg (b.w.) dose of alantolactone (**1**) significantly improved neurological function and reduced cerebral edema in a traumatic brain injury mouse model. This neuroprotective effect was attributed to alantolactone's capacity to inhibit the NF-κB inflammatory pathway and the cytochrome c/caspase-mediated apoptosis pathways [41]. To the best of our knowledge, no other authors have elaborated on this interesting double action: the fact that alantolactone (**1**) can simultaneously activate apoptotic pathways in cancer cells and inhibit these pathways in a cerebral edema model.

Seo et al. [52] described the neuroprotective effect of alantolactone (**1**) using amyloid β25–35- induced ex vivo neuronal cell death and scopolamine-induced amnesia in mouse models meant to emulate conditions common to neurodegenerative conditions like Alzheimer's disease. A 1 μM alantolactone (**1**) treatment increased cortical neuron viability to almost baseline control readings, and 1 mg/kg (b.w.) significantly decreased scopolamine-induced cognitive impairments. It is a interesting to note that this particular neuroprotective effect is attributed to a drop in ROS levels. High ROS concentrations are associated with the neurodegenerative damage of Alzheimer's disease. Again, alantolactone (**1**) exhibits a double action: it raises ROS levels to induce apoptosis in murine models of neurodegenerative damage.

The interesting advances with alantolactone (**1**) derivatives are also worth mentioning. Kumar et al. [53] assayed three of the 17 thiol derivatives synthesized, at 10 mg/kg (b.w.) doses, in mice showing that they have in vivo anti-inflammatory activity comparable to alantolactone (**1**). The novel compounds shared alantolactone's anti-inflammatory mechanisms. Another noteworthy study involving alantolactone (**1**) derivatives was published in 2018 by Li et al. [54], testing 44 derivatives for their ability to inhibit induced pulmonary fibrosis in mice. The results showed that 2 of these compounds are particularly active at 100 mg/kg (b.w.), reducing the fibrotic area by more than 60%. This is achieved by inhibition of the TGF-β1 pathway of myofibroblast differentiation. It should be noted that no toxic effects were observed for either compound in the chronic toxicity test (seven days), using a dose of 2 g/kg (b.w.) administered orally. Finally, a patent involving an alantolactone (**1**) spiro-isoxazoline derivative was filed in June 2019 (US patent N◦ 20190185487) for the development and production of these compounds that exhibit significant anti-inflammatory activity.

One factor which contributes to making alantolactone (**1**) very interesting as a future medicine is its low toxicity. In work by Khan et al. [55], Kunming mice treated with 100 mg/kg (b.w.) alantolactone (**1**) showed no significant signs of hepatotoxicity or nephrotoxicity, in line with the previously cited work by He et al. [38]. This is especially important for alantolactone (**1**) as an anticancer drug, because it is well known that the liver and kidneys are particularly susceptible to negative side effects from chemotherapy approaches [56,57].

Another engaging finding obtained by Khan et al. [55] is alantolactone's ability to cross the blood-brain barrier. It may thus become useful in the treatment of brain tumors or other conditions involving the central nervous system (CNS), since the blood-brain barrier is the greatest obstacle for drug delivery to those areas [58].

The metabolism and pharmacokinetics of alantolactone (**1**) have also been studied in vivo, with future pre-clinical testing in mind. Research shows that alantolactone (**1**) exhibits low absorption and is rapidly eliminated after intravenous and oral administration. Its metabolism involves conjugation with thiol, and α, β-unsaturated carbonyl is the preferential structural metabolic site. The low aqueous solubility of alantolactone (**1**) causes low oral bioavailability [59–61].

All these recent reports show there is grea<sup>t</sup> interest in alantolactone (**1**), and that it has potent proven in vivo activities, mainly several different types of anticancer activity. This broad-spectrum activity, combined with its synergistic action with known cancer therapy agents, shows alantolactone (**1**) has grea<sup>t</sup> potential for future drug development. However, further research is necessary, especially clinical trials, to identify its intracellular action sites and secondary targets and thereby elucidate its mode of action.
