*2.5. Helenalin*

Helenalin (**5**) (C15H18O4) (Figure 6), a 5/7-fused bicyclic sesquiterpene lactone that belongs to the pseudoguaianolide subclass. It is very abundant in *Arnica montana* L. but is also identified in other species from *Arnica* and *Helenium* genera and from *Centipeda minima* (L.) A.Braun and Asch. [132–136].

**Figure 6.** Structure of helenalin (**5**).

Alcohol extracts containing helenalin (**5**) and its derivatives have been used as a staple of traditional herbal anti-inflammatory medicine for many decades [134]. For instance *Arnica montana* L. solutions are used to treat rheumatism, arthritis, inflammation, hematoma, soreness, sprains, swelling and muscle spasms from athletic activity etc., seasonally triggered arthritis, arteriosclerosis, angina pectoris, postoperative conditions, and joint pain. The plant is used externally in creams, alcoholic tincture, and ointment form but also taken highly diluted in homeopathic remedies [134]. Many such preparations, even hair oil and shampoo, are commercially available from a range of suppliers in healthfood shops and pharmacies almost worldwide [137]. Likewise, clinical trials have aimed to assess topical *Arnica* applications, regarding possible reduction of laser-induced minor hematomas and osteoarthritis-type symptoms. Orally administered homeopathic formulations are also widely employed in the clinical setting to treat and manage conditions such as carpal tunnel, slow knee-surgery recovery, tonsil and wisdom tooth extractions, facelifts, neuralgia, hysterectomy, venous surgery, hallux valgus, heart-valve surgery, hemarthrosis, prolonged intravenous perfusion, joint sprains and strains, muscle pain, etc. [133].

Helenalin (**5**) inhibits NF-κB transcription of inflammatory cytokines, which have an essential role in both inflammation and cancer [138]. It also efficiently inhibits cancer cell proliferation through a variety of action modes, e.g., telomerase inhibition [139], DNA and protein synthesis attenuation, apoptosis induction and promoting reactive oxygen species generation [140]. It is noteworthy that inhibition of NF-κB activation associated with another sesquiterpene, helenin, occurs in T-cells, B-cells and epithelial cells in response to four different stimuli, nullifying kB-driven gene expression. Since this activity does not affect transcription factors Oct-1, TBP, Sp1 or STAT 5, this NF-κB activation is probably inhibited selectively [141,142]. Lyss et al. [141] described how helenalin modifies the NF-κB/IκB complex, preventing IκB release. They proposed a molecular action mode for the anti-inflammatory influence, which is different from other nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and indomethacin. Furthermore, it targets Cys38 on p65, ablating DNA-binding [143], inhibiting neutrophil chemotaxis and migration as well as 5-lipoxygenase and leukotriene C4 synthase activities [144].

Experiments performed by Schröder et al. [145] demonstrate that compound **5** inhibits platelet aggregation induced by collagen, 5-hydroxytryptamine secretion and thromboxane formation, depending its on concentration (between 3–300 μM). They concluded that helenalin inhibits platelet function via interaction with platelet sulfhydryl groups in a way most likely associated with reduced phospholipase A2 activity.

Helenalin (**5**) inhibited complete Freund's adjuvant-induced arthritis and carrageenan-induced paw edema in the rat [146]. Topical application of Arnica 3D gel (10%), combined with a 10 mA microcurrent for 2 min also significantly improved wound healing in a linear incision wound model in the rat back [147]. The evidence was consistent with a higher percentage of mature collagen fibers and a significantly larger total number of cells in the wound, as assessed by structural and morphometric analysis. According to this, different proportions of *Arnica montana* extracts alone or combined with other plants have been patented for their therapeutic potential [137]. Widrig et al. [148] performed a randomized, double-blind study in 204 patients with osteoarthritis (OA) to compare the e ffects of ibuprofen (5%) and *Arnica montana* gel (50 g tincture/100 g) containing helenalin (**5**), 11 <sup>α</sup>,13-dihydrohelenalin and its ester. The results show that short-term use, up to three weeks, of Arnica gel improves pain and hand function in OA, indistinguishably from ibuprofen gel. Substantial antiosteoarthritic activity by blocking transcription factors NF-κB and NF-AT is attributable to helenalin and derivatives.

Boulanger et al. [149] demonstrated that helenalin (**5**), intraperitoneally delivered at 20 mg/kg in lactating-Balb/C mice 9 and 3 h prior to infection, reduced intracellular growth of *Staphylococcus aureus* in mouse mammary glands This suggests the compound interferes with host molecular mechanisms and not directly with *Staphylococcus aureus* growth. The authors conclude helenalin might be worth investigating as a potential treatment for *Staphylococcus aureus*-induced mastitis in bovine species. There is however some concern regarding this treatment, mainly regarding helenin persistence in the animal's milk, and whether or not the therapeutic doses pose short- and long-term toxicity risks. It is therefore imperative to further study and characterize its safety and pharmacological properties.

Valan et al. [150] showed that helenalin (**5**) has a significant biphasic positive inotropic e ffect on the myocardium of guinea pigs at doses of 10−5–10−<sup>3</sup> mol. Nevertheless, concentrations above 10−<sup>3</sup> mol. cause an irreversible negative inotropic action leading to a blocking of muscle contraction.

The skin is susceptible to environmental damage by multiple agents, particularly solar ultraviolet (UV), which induces skin hyperpigmentation disorders. Expression of heat shock proteins (HSPs, particularly HSP70) is receiving consideration in the field of cosmetics, to reduce skin damage and signs of aging. Usui et al. [151] isolated AM-2 (helenalin 2-methylbutyrate) from *Arnica montana* as a good inductor of HSP70, with low cytotoxicity. Treatment of cultured mouse melanoma cells with AM-2 or *Arnica montana* extract up-regulated the expression of HSP70 in a dose-dependent manner. It also activated the transcription factor for hsp genes, i.e., heat shock factor-1. They concluded that both *Arnica montana* extract and AM-2 are likely to show beneficial e ffects if incorporated in hypopigmenting cosmetics.

Acute liver injury is a life-threatening syndrome frequently associated with hepatocyte damage and characterized by oxidative and inflammatory responses. Li et al. [136] recently observed that intragastric administration of helenalin (**5**) for 10 days significantly ameliorated hepatic injury induced previously in mice with LPS/D-GalN. These results were evidenced by the attenuation of histopathological changes and the decrease in serum aminotransferase and total bilirubin activities. Therefore, helenalin (**5**) shows a hepatoprotective e ffect against damage induced by LPS/D-GalN. This in turn may be associated with reduced hepatocyte apoptosis, by protection of mitochondrial function and oxidative stress inhibition by Nrf2 pathway activation, as well as attenuating inflammation by inhibiting NF-κB activation. The co-authors of that study [152] submitted Patent No. CN 110283151 in 2019 as a method for isolation of helenalin from *Centipeda minima* and its application for treating hepatic fibrosis and inflammation. Furthermore, the same authors [153] patented it for inhibiting hepatic stellate cell activation, showing the advantages of reducing collagen deposition and synthesis of inflammation-related proteins, promoting death of stellate cells, and its application in liver fibrosis treatment.
