*2.3. Costunolide*

Costunolide (**3**) (Figure 4), has the same chemical formula as alantolactone (**1**) (C15H20O2). It is a member of the germacranolide subclass and was first isolated from *Saussurea costus* (Falc.) Lipsch. roots in 1960 [82]. It is present in many Asteraceae genera such as *Inula* [24], *Lactuca* and *Helianthus* [83], but also those from other families like Magnoliaceae [84].

**Figure 4.** Structure of costunolide (**3**).

Biosynthesis of costunolide (**3**) is well documented and occurs through the mevalonate pathway [85]. Briefly, the process starts with the cyclization of farnesyl pyrophosphate, forming germacrene A. Next, the isoprenyl side chain of germacrene A undergoes hydroxylation by germacrene A hydroxylase, followed by oxidation to germacrene acid. It is finally synthesized after oxidation and cyclization of germacrene acid [85]. Some species are rich sources of costunolide (**3**), such as essential oil of *Saussurea lappa* roots (yield ~3%), of which 52% was identified as costunolide (**3**) [86]. The total synthesis of costunolide (**3**) has been described using different methods/strategies [87–89].

In a recent review [90], Kim and Choi detail the activities of custonolide (**3**) and discuss its therapeutic potential. Like alantolactone (**1**), compound **3** also exhibits anticancer bioactivity against different cancer cells via various routes. In fact, it acts as an apoptosis inducer, cell cycle regulator, angiogenesis and metastasis inhibitor and can also reverse the drug resistance mechanism [91].

There are several recent publications describing in vivo costunolide (**3**) studies, such as the work by Jin et al. [92] on costunolide's anticancer effect on osteosarcoma xenografted mice. Results showed a daily 20 mg/kg (b.w.) dose was enough to significantly reduce tumor weight by about half, as well as reduce the number of lung metastases to around one third of that in the control group. Western blot analysis of the tissues revealed this effect could be attributed to costunolide (**3**) inhibition of STAT3 transcription, a factor that is widely known to be linked to oncogenesis and cancer proliferation [93]. It is interesting to note the similarity between previously mentioned STAT3 inhibitory activity by alantolactone (**1**) [45]. To highlight another similarity, costunolide (**3**) also exhibits activity against gastric adenocarcinoma in xenografted mice [94]. Results from this study showed costunolide (**3**) induced caspase-mediated mitochondrial apoptosis in cancer cells and a 50 mg/kg (b.w.) dose on alternate days significantly reduced tumor size to about half. It achieved very similar results to the positive control cisplatin, a chemotherapeutic agen<sup>t</sup> used in clinical treatment [94]. Interesting to note is costunolide's anticancer e ffect through telomerase reverse transcriptase inhibition [95], a mechanism not known in alantolactone (**1**). As reported by the researchers, a 5 mg/kg (b.w.) dose injected on alternate days in glioma xenografted mice significantly reduced tumor size by over 50%. This inhibition was associated with reduced telomerase activity, which leads to ROS-associated apoptosis [95]. A subsequent work by the same research group also showed the same dose a ffects lipid metabolism in glioma xenograft tumors, by lowering expression of FASN, SREBP-1, and PGC-1 α [96], which are key genes targeted for cancer treatment [97–99].

Beyond costunolide's anticancer e ffect, it has also proven to be a potent anti-inflammatory agen<sup>t</sup> [90], as well as an antidiabetic, antihelminth, antimicrobial, antiulcer and antioxidant [91]. Its anti-inflammatory action has been well documented in vitro [84,100] and in vivo, appearing to be linked to NF-κB pathway inhibition [101]. For example, costunolide (**3**) is able to suppress inflammatory angiogenesis [102], alleviating gastric ulcers [103], and acute lung and liver damage [104–107].

Recently, studies have shown that costunolide (**3**) also exhibited anti-osteoarthritic e ffects [108]. In fact, treatment in rats with osteoarthritis causes attenuation of cartilage degeneration compared to the control osteoarthritic group. The observed e ffect has been attributed to the inhibitory action of this compound on the Wnt/β-catenin and NF-κB signaling pathways, and on the expression of matrix metalloproteinases [108].

Costunolide (**3**) seems to be also a powerful antiasthmatic [109]. In this work, the researchers treated asthma-induced mice with 10 mg/kg (b.w.) before an immune challenge. Results showed a 61.8% inhibition of asthma-associated eosinophil increase, as well as significantly reducing lung inflammation scores and mucin production [109].

Recently, it has been shown that costunolide (**3**) is an e ffective inducer of hair growth in mice [110]. For this assay, the researchers implanted mouse dermal cells, treated with 3 mM costunolide (**3**) for two days. Results showed a 2.5-fold increase in induced hair follicles in the implanted treated cells, and topically applied costunolide (**3**) significantly and visibly improved hair growth. The authors claim this might be due to activation by compound **3** of key follicle-cell cycle pathways, including the Wnt/b-catenin, Shh/Gli, and TGF-β/Smad pathways. It is worth mentioning the TGF-β/Smad pathway was also mentioned in previous studies related to anti-inflammatory action of costunolide (**3**) [106].

Costunolide (**3**) derivatives have also proven to be highly interesting, with remarkable in vivo effects. In recent work by Cala et al. in an agro-research context [111], many di fferent functional groups were added to the sesquiterpene backbone, yielding, among others, two amino and two methyl ether derivatives with strong herbicidal activity. The etiolated wheat coleoptile assay indicated treatment with these costunolide (**3**) derivatives had dose-dependent growth inhibition responses equivalent to a widely used synthetic herbicide used as positive control. These results show there is potential for some of these derivatives as bio-herbicides, but further studies are necessary. There was also another interesting result of this work which the authors did not address, but is worth pointing out: two tested thiol derivatives boosted coleoptile growth instead of inhibiting it, with one compound increasing coleoptile length by ~30% with a 30 μM treatment. The general implication of these results is that costunolide (**3**) modification can be powerful and versatile, capable of yielding diametrically opposed bioactive compounds depending on the functional group added.

In conclusion, costunolide (**3**) is a highly multitalented and potent bioactive compound. Similar to other sesquiterpene lactones such as the previously discussed alantolactone (**1**), much of its potential for future drug development lies in its anticancer and anti-inflammatory e ffects. It is interesting that there appears to be more work done with costunolide (**3**) derivatives than alantolactone (**1**) derivatives, which in general suggests greater interest of the scientific community in costunolide (**3**) as opposed to alantolactone (**1**). With so many relevant publications in 2019 alone, we expect to see costunolide (**3**) research ramping up to a pre-clinical stage very soon.
