*Review* **Mycosporine-Like Amino Acids (MAAs): Biology, Chemistry and Identification Features**

**Vanessa Geraldes 1,2 and Ernani Pinto 2,\***


**Abstract:** Mycosporines and mycosporine-like amino acids are ultra-violet-absorbing compounds produced by several organisms such as lichens, fungi, algae and cyanobacteria, especially upon exposure to solar ultraviolet radiation. These compounds have photoprotective and antioxidant functions. Mycosporine-like amino acids have been used as a natural bioactive ingredient in cosmetic products. Several reviews have already been developed on these photoprotective compounds, but they focus on specific features. Herein, an extremely complete database on mycosporines and mycosporine-like amino acids, covering the whole class of these natural sunscreen compounds known to date, is presented. Currently, this database has 74 compounds and provides information about the chemistry, absorption maxima, protonated mass, fragments and molecular structure of these UV-absorbing compounds as well as their presence in organisms. This platform completes the previous reviews and is available online for free and in the public domain. This database is a useful tool for natural product data mining, dereplication studies, research working in the field of UV-absorbing compounds mycosporines and being integrated in mass spectrometry library software.

**Keywords:** mycosporines; mycosporine-like amino acids (MAAs); mass spectrometry; database; photoprotective compounds; UV-absorbing compounds

#### **1. Ultraviolet Radiation and Natural UV-Absorbing Compounds**

The solar radiation reaching Earth is composed of infrared radiation (>800 nm), visible (photosynthetically active radiation, PAR, 400-750 nm) and ultraviolet radiation (UVR, 200–400 nm). UVR is divided into ultraviolet A (UVA, 320–400 nm), ultraviolet B (UVB, 280–320 nm) and ultraviolet C (UVC, 200–280 nm). Very small proportions of UVR contribute to the total irradiation on the Earth's surface: 0% of UVC (which is completely absorbed by the ozone layer), less than 1% of UVB and less than 7% of UVA. However, this part of the solar spectrum is highly energetic [1]. Photosynthetic organisms harness PAR to convert light into chemical energy. An obligate requirement for PAR results in prolonged exposure to UVR, which is detrimental for most sun-exposed organisms. Furthermore, due to the ozone depletion, the amount of UV reaching Earth tends to increase [2]. In order to circumvent the photodamage, several organisms have evolved biochemical and mechanical defenses [3]. Among these is the ability to synthesize UV-screening compounds such as phenylpropanoids and flavonoids (in higher plants), melanin (in animals), mycosporines, mycosporine-like amino acids (in cyanobacteria, fungi, algae and animals) and several other photoprotective compounds [4]. As well as providing protection against ambient UV radiation, these substances have other physiological roles. In previous studies, the characteristics of a wide diversity of UV-absorbing compounds are explored [4].

**Citation:** Geraldes, V.; Pinto, E. Mycosporine-Like Amino Acids (MAAs): Biology, Chemistry and Identification Features. *Pharmaceuticals* **2021**, *14*, 63. https:// doi.org/10.3390/ph14010063

Received: 27 December 2020 Accepted: 11 January 2021 Published: 14 January 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. Mycosporine-Like Amino Acids**

Mycosporines and mycosporine-like amino acids (MAAs) are a large family of natural UV-absorbing sunscreens [5–9], having evolved for protection against chronic UVR exposure in a wide variety of organisms such as cyanobacteria, microalgae, fungi, seaweeds, corals, lichens, as well as in freshwater and marine animals [5,8]. Their presence is evidence not only of their importance as natural UV-screening compounds but also of their early phylogenetic innovation [4]. MAAs were first reported in the 1960s [10–12]. Since then, many studies have been carried out and several MAAs have been identified as well as information on their structure, distribution, properties and functions.

#### *2.1. Physico-Chemical Characteristics of MAAs*

MAAs are low-molecular-weight (generally < 400 Da), colorless and water-soluble compounds. They are highly stable molecules under environmental conditions. They are composed of either an aminocyclohexenone or an aminocyclohexenimine ring, carrying nitrogen substituents. Aminocyclohexenone derivatives contain a cyclohexenone conjugated with an amino acid, such as mycosporine-glycine and mycosporine-taurine. Aminocyclohexenimine possesses a cyclohexenimine conjugated with a glycine or a methylamine attached to the third carbon atom and an amino acid or amino alcohol or enaminone chromophore to the first carbon atom (Figure 1) [13]. Glycosidic bonds or sulfate esters may occur within the imine group [14]. This group includes palythine, shinorine, porphyra-334, catenelline, hexose-bound porphyra-334, etc. MAA absorption maxima are between 268 and 362 nm, depending on their molecular structure, namely in the type of ring and substituents [15]. MAAs also have high molecular absorptivities (ε = 12.400–58.800 M−1·cm<sup>−</sup>1), and due to these characteristics, they are the strongest UVA-absorbing compounds in nature, and they are also effective against UVB radiations, which explains their potential role in photoprotection. MAAs are predominantly cytoplasmatic due to their high water solubility that enables MAAs to be easily dispersed in the cytoplasm [9]. Previous studies reviewed the physico-chemical properties of several MAAs [16].

**Figure 1.** Examples of mycosporine-like amino-acids (MAAs) structures: (**a**) mycosporine-glycine (oxo-mycosporine); (**b**) shinorine (imino-mycosporine).

#### *2.2. Occurrence and Distribution in the Environment*

Wittenberg et al. (1960) isolated, for the first time, compounds with high UV absorption from a siphonophore, *Physalia physalis* [10]. In 1965, mycosporines were discovered in fungal sporulating mycelia [12]. A few years later, MAAs have also been detected in corals and cyanobacteria from the Great Barrier Reef [11]. Since then, MAAs have been identified in a wide variety of organisms, such as heterotrophic bacteria [17], fungi [18], cyanobacteria [5,19], microalgae [20–22], macroalgae [23], lichens [6], invertebrates (e.g.,

dinoflagellates, sponges, corals, sea urchins and crustaceans) [2,24,25] and vertebrates (e.g., fishes) [26,27]. Several studies reported that animals can acquire MAAs from their food or through symbiosis and then subsequently accumulate them [28,29]. MAAs are not found in higher plants, in which UVR protection is provided by flavonoids, nor in higher vertebrates, in which the protective function is assumed by melanin [4]. MAAs are present especially in organisms that live in environments with high levels of UVR. Their composition varies according to the taxonomic group with the frequent coexistence of several MAAs with different absorption maxima allowing a more effective protective filter [8]. Several environmental factors, such as light, temperature, salinity and nutrients, influence the concentration of MAAs. Enhanced MAA contents, for example, were found in environments with a basic pH, a high ultraviolet radiation and high concentrations of phosphate and nitrate. Salinity, dissolved oxygen and variations of sea surface temperature also influence, in a secondary way, MAA content [30]. UV radiation is one of the most important factors that influences the accumulation of MAA and results in changes in the MAA profile of organisms. MAA synthesis is also affected by spectral variability and intensity [31,32]. Other factors, such as changes in salinity and nutrient availability, stimulate the production of MAAs. The content and composition of UV-absorbing MAAs are also affected by seasonal fluctuations [33,34]. These changes are mainly controlled by the solar radiation regime and nitrate regime. Guihéneuf et al. (2018) studies the temporal and spatial variability of mycosporine-like amino acids in seaweeds. An increase in total MAA contents in all species was induced by increasing daily light doses and irradiance levels, from winter to spring, but without clear significant correlations with light and/or temperature. Nutrient concentrations, in particular nitrate, appear to be a limiting factor for seaweed to accumulate MAAs when exposed to extreme light/irradiance stress [34].

#### *2.3. MAA Biosynthesis*

Cyanobacteria must have been the original MAA producer with the genes involved in MAA biosynthesis being transferred to other organisms, and MAAs may have been an early evolution to deal with cellular stress caused by UVR exposure [4]. Multiple lines of evidence support that MAAs are derived from conversion of the shikimate pathway [35], which is known for the synthesis of aromatic amino acids. The precursor of the six-membered carbon ring common to all MAAs is 3-dehydroquinate (3-DHQ). Three-DHQ transforms into gadusol and then 4-deoxygadusol (4-DG). However, contrasting evidence suggests that 4-DG is derived from conversion of the pentose phosphate pathway intermediate sedoheptulose-7-phosphate (SH-7P) [35–37]. Despite experimental data that support the pentose phosphate pathway, the use of the shikimate route inhibitors, such as glyphosate and tyrosine, has demonstrated the ability to abolish MAA biosynthesis in cyanobacteria [13] and corals [38]. Furthermore, the deletion of the gene encoding the enzyme cyclase-2-*epi*-5-*epi*-valiolone synthase (EVS) in cyanobacterium *Anabaena variabillis* ATCC 29413 still produced shinorine [39]. These results suggested that the shikimate pathway is the most predominant route for MAA synthesis in sufficient amounts to provide photoprotection, and the quantities of MAAs produced by the pentose phosphate pathway should have other biological functions [39]. However, there are clear links between the pentose phosphate and shikimate pathways. In both routes, 4-DG is the parent core structure of MAAs and the addition of glycine yielding mycosporine-glycine. This simple mono-substituted cyclohexenone-type MAA is a common intermediate in the production of di-substituted (aminocyclohexenimine-type) MAAs through the addition of a single amino acid residue (serine, threonine, etc.) yielding some common MAAs such as porphyra-334 and shinorine. This step encodes a nonribosomal peptide synthase (NRPS)-like protein or a D-alanyl-D-alanine ligase (D-ala-D-ala ligase) [13,36]. Figure 2 presents a proposed biosynthetic pathway of MAAs. Other MAAs are synthesized by modification in the attached side groups and nitrogen substituents (e.g., esterification, amidation, dehydration, decarboxylation, hydroxylation, sulfonation and glycosylation). The variations of amino acid side-chains are responsible for the difference in the absorption spectra of MAAs [15,40].

**Figure 2.** Proposed biosynthetic pathway of mycosporine-like amino acids. DAHP: 3-deoxy-D-arabino-heptulosonate phosphate, DHQS: 3-dehydroquinate synthase, 3-DHQ: 3-dehydroquinate, SH-7P: sedoheptulose-7-phosphate, EVS: cyclase-2-*epi*-5-*epi*-valiolone synthase, *O*MT: *O*-methyltransferase, 4DG: 4-deoxygadusol, ATP: adenosine triphosphate, NRPS: nonribosomal peptide synthase, D-ala-D-ala-ligase: D-alanyl-D-alanine ligase.

#### *2.4. Heterologous Expression*

The poor understanding of the biosynthesis pathways involved in the production of specific MAA is one of the reasons for the lack of widespread use of MAA in the industry in an economically viable way. Further understanding of these biosynthetic pathways can lead to easier large-scale production, for example, in a heterologous bacterial host [15,37]. Balskus et al. (2010) elucidated the biosynthesis of shinorine via heterologous expression in *Escherichia coli* [36]. The heterologous expression of MAAs in *Escherichia coli* also resulted in the production of 4-deoxygadusol, mycosporine-glycine, mycosporine-lysine and mycosporine-ornithine [36,37]. Shinorine and mycosporine-glycine-alanine have artificially been produced by heterologous expression in Actinomycetales [41]. Nowadays, most efforts are focused on the production of MAAs by genetically modified microorganisms as an alternative production of natural MAAs [9,37]. The heterologous expression of MAAs is significant from a biotechnological perspective, as MAAs are the active ingredient in next-generation sunscreens [37].

#### *2.5. Chemical Synthesis and Analogs*

The chemical synthesis of MAAs was motivated due to the low extraction yield from MAA-producing organisms and the need for large scale production. Several synthesized chemical structures were developed with interesting photoprotective and antioxidant properties [42–45]. The synthetic analogue of mycosporine-glycine, tetrahydropyridine, was considered hydrolytically and oxidatively stable for commercial application in sunscreens [46]. An efficient and environmentally friendly procedure by ultrasound and microwave for preparing MAA analogs was described by Andreguetti et al. (2013). These analogs showed high antioxidant effect [47]. Analogs with a high absorbance intensity in UVA and UVB regions, without in vitro cytotoxicity, were prepared by Nguyen et al. (2013) [48]. Recently, Losantos et al. (2017) developed a series of potential UV sunscreens with easy synthetic routes, providing a suitable source for their use in commercial products [42]. Bedoux et al. (2020) published a complete review of these chemical syntheses [49].

#### *2.6. MAA Extraction, Identification and Quantification*

#### 2.6.1. Extraction

Since MAAs are potential compounds to be used as sunscreens in cosmetic products, the extraction protocol must be optimized taking into account their physicochemical and absorption properties and must use environmentally friendly and inexpensive solvents [9,50]. Traditional extraction method is usually performed by a solid/liquid extraction from the raw material and is carried out on fresh or lyophilized samples with different temperature ranges [51,52]. Due to its high solubility in aqueous solution, extraction generally uses polar solvents, as aqueous ethanol or methanol. Chaves-Peña et al. (2020) compared the extraction in 20% aqueous methanol and in distilled water, and no significant differences were observed. According to their results, the drying and subsequent re-dissolution of the pellets declined total MAA concentrations [50]. Geraldes et al. (2020) developed a fast and efficient extraction protocol, without the need for pre-concentration procedures. This protocol uses only water and volatile additives as the extractor solvents, and the extracts were directly injected to a high-performance liquid chromatograph (HPLC). This extraction protocol is not only easy-to-handle but also uses solvents without certified toxic effects [32].

#### 2.6.2. Identification

HPLC was the most common method to separate and identify MAAs by using retention times and UV spectra. Although UV detection is sensitive because of high attenuation coefficients (ε) for MAAs, this method is poor in selectivity, since biosynthetic congeners can easily influence MAA identification [32]. Moreover, no commercial sources for standard compounds exist and few laboratories worldwide have the capacity to provide reference material against which structural elucidation of MAAs can be verified. In this sense, LC-MS is a good alternative to provide high sensitivity and selectivity for analysis of MAAs. A method for MAA identification was developed using an ultrahigh-performance liquid chromatography with diode array detection coupled to quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QTOFMS) with an electrospray ionization source (ESI) and showed to be fast, reliable and a powerful tool for identification and screening of MAAs in several organisms, such as cyanobacteria, dinoflagellates, macroalgae and microalgae [42]. Regarding MAA purification, HPLC is the most used method. Geraldes et al. (2020) published a protocol using a semi-preparative HPLC-DAD fitted to a Luna C18 (2) column and 0.2% (v/v) formic acid solution as buffer A [32].

#### 2.6.3. Quantification

MAAs were often quantified based on molar attenuation coefficients using HPLC techniques. However, nowadays, liquid chromatography coupled with mass spectrometry (LC-MS) is the most common technique, because this method provides high sensitivity and selectivity for analysis of MAAs [32,51]. Whitehead and Hedges (2002) published a quantitative method that allowed the quantification of MAAs based on molecular weights,

individual retention time and UV absorption maxima [53]. An LC-MS method using hydrophilic interaction chromatography (HILIC) was published by Hartmann et al. (2015) [54]. Although this method afforded good linear correlation coefficients, it did not account for some important validation parameters such as recovery and matrix effects. Geraldes et al. (2020) described a rapid quantitative method for LC-MS/MS analysis of MAAs that allowed the quantification of MAAs based on individual retention times, molecular weights and specific mass transitions using multiple reaction monitoring (MRM) experiments instead of a full scan [32]. This method has been thoroughly validated taking into account the ICH and EURACHEM guidelines for the following parameters: specificity, linearity, precision (repeatability and reproducibility within the laboratory), accuracy, extraction recovery, matrix effects and stability. In addition, the working range, as well as the limits of detection and quantification, were evaluated [32]. This technique improved the selectivity and sensitivity of the method, allowing mass distinction of isomeric compounds [16].

#### *2.7. MAA Structural Elucidation*

The structural elucidation of new derivatives of mycosporine-like amino acids is usually established by tandem mass spectrometry (MS/MS) and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy [9,55]. Previous studies focusing on the fragmentation patterns of MAAs showed a loss of mass 15 when analyzed by positive mode ESI–MS/MS. This highly characteristic loss is due to elimination of a methyl radical CH3 [56,57]. The detection of the production [M + H − 59]+ related to the elimination of the methyl radical and subsequently CO2 is also common in MAAs [56]. Thus, the screening of these eliminations could be a suitable tool to assign the presence of novel MAAs in different samples by neutral loss or product ion scans in mass spectrometry analyzers. For aminocyclohexenimines, the elimination of the remaining lateral chain can produce the product ion *m/z* 186. This ion can undergo a fragmentation process to produce the ion *m/z* 155. Then, this product ion loses either NH3 or H2O by neutral elimination to produce the ions *m/z* 138 and 137, respectively [57]. These product ions could provide additional information concerning the occurrence of new MAAs in a crude extract. However, the presence of some product ions may be reduced or not occur in certain MAA fragmentation processes [58]. Thus, the selection of the product ions should be carried out carefully in analytical methods for the analysis of extracts containing potential novel MAAs. Regarding NMR spectroscopic data, they show characteristic patterns allowing them to be comparable. For example, oxo-mycosporines and imino-mycosporines can be distinguished through the chemical shift of C-1 in 13C NMR experiments, which is more around 180 ppm for oxo-mycosporines and 160 ppm for imino-mycosporines. The 1H NMR data usually revealed the presence of the coupling of the pairs of duplets from the diasterotopic methylenes, with a geminal coupling constant around 17 Hz, corresponding to the four protons on C-4 and C-6 that show chemical shifts around 30–40 ppm between them. The hydroxymethyl group at C-5 and the methoxyl group at C-2 usually appear very close around 3.60 ppm as two singlets [9,13,59] (Figure 3).

**Figure 3.** Structure of an oxomycosporine with NMR results for (**a**) 13C (125 MHz, D2O) and (**b**) 1H (500 MHz, D2O). For iminomycosporine 13C NMR (125 MHz, D2O) δ<sup>C</sup> = 160 (C-1).

#### *2.8. MAA Photoprotective Role*

The MAA photoprotective role against UVR is due to their absorption spectra and molar attenuation coefficients. Their absorption gradient (268–362 nm) cover most of the UVR spectrum (~295–400 nm) that reaches the Earth's surface, while their high molar attenuation coefficients demonstrate how strongly MAAs absorb light at this wavelength range. The concentration of MAAs in organisms is directly related to their level of UVR exposure, which depends on latitude and altitude, seasonality and water depth [15,33,60,61]. They preferentially accumulate in tissues that receive the highest UVR exposure [62]. Photoprotection of MAAs has been demonstrated in a wide variety of species and prevents UVB-induced damage [63,64]. MAAs have been found in the cytoplasm of several cyanobacteria; however, in *Nostoc commune*, MAAs accumulate extracellularly, resulting in a more effective protection against ultraviolet radiation [65]. The presence of MAAs in animals supports their photoprotective role not only to producers but also to herbivores and even carnivores [8]. MAAs are also found in fossils that confirm their protective function against the UVR harmful effects in the early geological eras [66].

#### *2.9. MAA Additional Protective Roles*

Generally, MAA production is induced when organisms are exposed to UVR [67,68]. The photoprotective function is the most important role that MAAs play in nature. This is verified by the abundance of MAAs in organisms exposed to high UVR intensities [69]. However, MAAs can be produced constitutively in some species [5,13], and functionality could be linked to their individual structures [70]. Thus, MAAs may have additional protective roles in many other biological processes beyond their well-known UV sunscreen role. These compounds have convincingly demonstrated to possess physiologically relevant antioxidant properties [8,40]. Furthermore, MAAs are involved in osmotic regulation, desiccation and many other cellular functions [15,69]. An extensive review about these additional roles of MAAs has been carried out by Oren et al. (2007). However, the significance of these additional functions and the effects of different forms of stress on MAA synthesis are still poorly understood [69].

#### 2.9.1. Antioxidant and ROS Scavenging Function

UVR exposure generates oxidative stress and can produce reactive oxygen species (ROS) and DNA damage. This oxidative DNA damage can lead to mutations and inhibit DNA repair [71]. Antioxidants can reduce the harmful effects of ROS and can, thus, help to prevent oxidative stress [72]. Some MAAs may protect the cell not only by absorbing UVR and dissipating the energy as heat before it could reach the critical cellular targets

but also as scavengers of free radicals due to their antioxidant role [31,73]. This function has been demonstrated in several MAAs from a wide variety of organisms [74–79]. MAAs have also been shown to prevent lipid peroxidation and superoxide radicals, blocking the aftereffect of oxidative damage [80]. Several assays demonstrated that MAAs have antioxidant properties and efficiently prevent oxidative stress through filtering and direct and indirect quenching mechanisms [81,82]. The antioxidant abilities of MAAs (ORAC values, lipid peroxidation inhibition, DPPH and ABTS radical extinction, singlet oxygen quenching, superoxide anion radical scavenging, hydrogen peroxide extinction activities and physiological activities) have been provided in recent reviews [40,83]. However, the exact mechanism is yet to be elucidated.

#### 2.9.2. Osmotic Stress

The concentrations of intracellular solute are directly related to the concentration of salt in which the cell lives [69]. Thus, osmotic stress is one of the stressors MAAs seem to have an action against. In hypersaline environments, cyanobacteria usually contain high concentrations of MAAs suggesting that these compounds may have an osmotic function helping the cells to cope with the high salinity. In these environments, cell dehydration and reactive oxygen species (ROS) production can occur, leading to oxidative stress. Through the synthesis of MAAs, osmotic balance can be restored [31]. Oren (1997) reported that a halotolerant cyanobacterium, inhabiting a gypsum crust, has an extremely high concentration of MAA (≥98 mM), being responsible for about 3% of the cells' wet weight. It was observed that a reduction in the salt concentrations of its surroundings was accompanied with a rapid expulsion of MAAs [84]. Thus, MAAs may be involved in the adaptation of sea ice algae to osmotic variations. MAA production could be induced specifically either by exposure to UVB radiation or by osmotic stress, and a significant synergistic enhancement of MAA production was observed when both stress factors were combined. Although osmotic stress could induce MAA synthesis, MAAs play no significant role in attaining osmotic homeostasis [85]. Singh et al. (2008) proposed that salt treatment resulted in an increase in MAA content in the absence of UV radiation and had synergistic effects with UV stress [86]. Waditee-Sirisattha et al. (2013) recently demonstrated that the accumulation of MAAs, in a halotolerant cyanobacterium, was stimulated more under high salinity rather than under UVB radiation [87].

#### 2.9.3. Desiccation Stress

Desiccation is responsible for cell damage by affecting cytoplasmic components (such as DNA and proteins) and cell membrane fluidity. The production of polysaccharides and antioxidant compounds are some of the techniques to overcome the consequences of drying [64,88]. Few studies have been carried out to assess the effects on MAA concentration in microorganisms exposed to desiccation stress [63,64,89,90]. Desiccation stress leads to an increase in total MAA content. This group of compounds may be acting by modifying the structure of the extracellular matrix. Expulsion of the MAA is observed after rehydration. The combination of desiccation and irradiation stimulate MAA production. Colonies of fungi exposed to desiccation, UV radiation and nutrient scarcity contain high concentrations of mycosporine-glutaminol-glucoside. The survival and longevity potential of the vegetative hyphae of these fungi may be associated with the presence of these compounds [90]. Olsson-Francis et al. (2003) stated that cyanobacteria stressed experimentally by desiccation increased their MAA concentration. In this study, they suggest that the formation of an extracellular sheath may be related to desiccation [63]. Joshi et al. (2018) also proposed that desiccation together with UVB radiation led to an increase in the concentration of MAAs preventing and protecting cells from the harmful effects of these factors [64].

#### 2.9.4. Thermal Stress

There are a few reports of thermal stress protection by an increase in MAA induction in a range of organisms. Michalek-Wagner (2001) stated that MAA content was upregulated under heat stress, and their concentrations were further enhanced during simultaneous exposure to UV [91]. In contrast, some authors suggested that thermal stress had no effect on MAA production with or without UVR [85,86].

#### 2.9.5. Photosynthesis Accessory Pigments

An early study reported that MAAs may act as a photosynthetic accessory pigment due to its UVA absorption and subsequent production of small amounts of fluorescence at wavelengths close to the absorbance of chlorophyll-a. This result suggested that MAAs may increase photosynthetic efficiency. However, MAAs are only weakly fluorescent and are generally produced in environments of high irradiance, in which photosynthetic wavelengths are not the limiting factor for photosynthesis [92]. So far, this study has never been substantiated [69].

#### 2.9.6. Nitrogen Storage

MAAs are nitrogenous compounds that contain at least one nitrogen atom per molecule that can be released when required. Thus, MAAs may serve as an intracellular nitrogen storage [93]. A synergistic effect between ammonium ions and UVR was observed and resulted in an increase in MAA content [93]. If MAAs are accumulated as intracellular nitrogen storage compounds, nitrogen mobilization should occur whenever other suitable forms of nitrogen are absent. However, there is no evidence about the intracellular degradation of MAAs and the release of nitrogen atoms that support the proposal that MAAs may be nitrogen storage molecules [69].

#### 2.9.7. Reproductive Regulation

There is evidence that mycosporines and MAAs are involved in reproduction in fungi and marine invertebrates [69]. Mycosporines have been related to sporulating mycelia and were considered as biochemical markers for reproductive states of fungi or as reproduction markers [69,90]. Several studies reported that most MAAs reach their maximum concentration in ovaries and eggs at the time of ovarian reproductive maturity and spawning, which may be near the seasonal minima and maxima of solar irradiation [94,95]. Although a protective role is clearly demonstrated in marine invertebrate embryos, the exact function of MAAs in ovaries and eggs has not been determined [62].

#### 2.9.8. Ecological Interactions

Some MAAs play a role in ecological connectivity between organisms, as intraspecific alarm cues or cell-cell interaction tools, suggesting that MAAs can function as compounds of fundamental importance in marine ecosystems. The alarm cues are released in the ink secretion of sea hares and cause avoidance behaviors in neighboring conspecifics. The highest concentration of MAAs was concentrated in the defensive secretions and in the skin of these organisms [96]. In *M. aeruginosa* PCC 7806, shinorine was found to accumulate in an extracellular matrix, and this compound could be synthesized for its role in extracellular matrix formation and cell–cell interaction [97].

#### *2.10. Cosmetical Application of MAAs as Sunscreen*

As with other organisms, human exposure to ultraviolet radiation can cause damage, such as erythema or "sunburn" over the short term and premature skin aging and skin cancer over the long term [98]. Thus, protection against ultraviolet radiation is extremely important through the use of appropriate clothing and sunscreen, which are part of an overall prevention strategy [98,99]. Several photoprotective products are available on the market. They contain synthetic organic filters (e.g., oxybenzone, avobenzone, aminobenzoic acid), inorganic filters (e.g., titanium dioxide, zinc oxide) or a combination of both [100]. The frequent use of synthetic sunscreens can affect human health, causing allergic reactions, phototoxicity and endocrine disorders [101–103]. In addition, synthetic UVR filters are not environmentally friendly causing a negative impact in the marine life, including bioaccumulation in several species, hormonal changes and endocrine disruption in fish, hydrogen peroxide production and bleaching of corals [102,104–106]. This fact led Hawaii to ban some types of sunscreens, such as oxybenzone [107]. Subsequently, the Western Pacific Nation of Palau, the city of Key West, Florida and the US Virgin Islands have also passed similar bans [108]. Thus, there was a change in consumer trends with a strong demand in the cosmetics market for more natural products, since they are seen as safer and better and may be able to replace the existing ones. MAAs have been widely studied as natural alternatives to potentially toxic synthetic sunscreens and anti-aging products [8,15,83]. Several studies suggested that MAA have potential for the protection of human skin from a diverse range of adverse effects of solar UVR. Kageyama et al. (2019) provide an overview of MAAs, as potential anti-aging ingredients, which includes the molecular and cellular mechanisms through which MAAs might protect the skin. In addition to their UV-absorbing properties, these compounds have the potential to protect against skin aging, including antioxidative activity, anti-inflammatory activity, inhibition of protein-glycation and inhibition of collagenase activity [83]. MAAs are also highly stable over a wide range of temperature and pH [32,102]. These characteristics make them excellent cosmeceutical ingredients for skincare, cosmetics and pharmaceutical products [109,110]. However, only a few MAA products are currently available, and they still need to be exploited on a large scale. Mibelle AG Biochemistry developed a natural active compound, called Helioguard 365®, which contains MAA porphyra-334 and shinorine from the red seaweed *Porphyra umbilicalis*. Another MAA extract, named Helionori®, is also marketed by Gelyma [111]. Although these ingredients protect in UVA region, they provide minimal protection in the more damaging UVB range. Moreover, the MAA content in the formulation is usually very low when compared to the concentration of UVR filters in most sunscreen products. Thus, this ingredient ends up having a negligible influence on the SPF claims of the product [15]. Some MAA-based skin products have been marketed, such as Aethic Sôvée, a sunscreen with an environmentally friendly appeal. However, it is important to invest efforts to speed up the technological advancement of MAA application as an organic sunscreens [31].

#### *2.11. Other Biotechnological Applications of MAAs*

UV exposure alters the properties and durability of non-biological materials and affects their lifetime. MAAs have also nonmedical applications, as additives to protect plastics, paints and varnishes against UVR that exclusively consist of natural compounds [112]. These materials are biocompatible, photoresistant and thermoresistant and provide an efficient protection against UVA and UVB [112]. Figure 4 summarizes the main applications of MAAs including in cosmetics, biological functions and sources.

**Figure 4.** Mycosporine-like amino acids sources, biological functions and applications.

#### *2.12. Patents on MAAs*

Currently, there are already a large number of patents in international databases for several products and methods with MAAs. A list of patents on MAAs was obtained from patent databases such as the World Intellectual Property Organization, WIPO (http://www.wipo.int/patentscope/search/en/search.jsf), European Patent Office (http: //www.epo.org/searching/free/espacenet.html) and United States Patents and Trademark Office (http://www.uspto.gov/patents/process/search/index.jsp). The search yielded a total of 48 patents on MAAs products and methods that are summarized in Supplementary Materials (Table S1).

#### *2.13. MAA Database—MYCAS*

There are several software for searching for commercial natural products. However, they do not cover most mycosporine-like amino acids, and they are not in the public domain. Over time, some MAA databases have been developed [22,23,40]. Although, none of them contains complete and detailed information on all mycosporines and mycosporinelike amino acids identified in the bibliography. Sinha et al. (2007) constructed an excellent database that provides information on various mycosporines and MAAs reported in several organisms. However, this database is now out of date [22]. Wada et al. (2015) published a study focused on MAAs with radical scavenging activities. This work resumed the structures and the physical and chemical properties of these MAAs. Nevertheless, this database did not include all MAAs known to date [40]. Sun et al. (2020) developed a database that summarized the studies related to MAAs in marine macroalgae. However, this work does not contain information on MAAs present in organisms other than macroalgae [23]. Thus, the purpose of this work is to cover this gap, so we present a database on mycosporines and mycosporine-like amino acids, called MYCAS. Our study covers the

whole class of these natural sunscreen compounds known to date. This platform will be available online for free and will be in the public domain. It may also be incorporated into databases and search software of mass spectrometry for metabolomic and genomic studies. This platform provides information about their corresponding absorption maxima (λmax), molar attenuation (ε), molecular formula, exact mass, molecular structure, fragments and the organisms in which these compounds were found. Thus, MYCAS facilitate the search for mycosporine-like amino acids in different species and in dereplication studies. Our results are summarized in Supplementary Materials (Table S2).

#### *2.14. Main Tasks for Future Research and Perspectives*

The optimization of MAA production on an industrial scale is essential to enable the use of these photoprotective compounds in cosmetical field. Thus, it is extremely important that resources are invested to optimize the production of MAAs and to increase the concentration in organisms, including GMO. At the same time, alternatives should be researched, such as heterologous expression or organic synthesis of analogues. The isolation and purification methods of MAAs are other processes that require further research. These techniques should be as simple and sustainable as possible to allow their application on a large scale. The commercial applicability of these compounds, as well as their impact on the environment and human health, should also be investigated to confirm the feasibility of using these photoprotective compounds on an industrial scale and warranty their safety.

#### **3. Conclusions**

Mycosporines and mycosporine-like amino acids are a large family of natural UVabsorbing compounds. To date, more than 70 molecules have been identified in several organisms from different phyla (Arthropoda, Cnidaria, Chordata, Cyanobacteria, Echinodermata, Fungi, Lichen, Macroalgae—Chlorophyta, Phaeophyta and Rhodophyta, Microalgae—Bacillariophyta, Charophyta, Chlorophyta, Dinoflagellata, Miozoa, Ochrophyta and Mollusca). Although there are several reviews on these photoprotective compounds, none include all of the MAAs known to date. In addition, the information is dispersed, and there is no review that summarizes all the information, namely the structural information, spectrometric data and presence in organisms. Here, we present an extremely complete database on mycosporines and mycosporine-like amino acids, called MYCAS, that covers the whole class of these natural sunscreen compounds. Currently, MYCAS has 74 compounds, with structural information and spectrometric data and will be updated annually. This platform is available online (http://www.cena.usp.br/ernani-pinto-mycas), for free and in the public domain. MYCAS allows users to access to all MAAs already described in nature. Our database may also be incorporated into in-house libraries, as well as mass spectrometry software for metabolomic and genomic studies. Thus, MYCAS is a useful tool for scientists working with MAAs and researchers in the field of developing UV-protecting cosmetics from natural sources.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1424-8 247/14/1/63/s1, Table S1: Patents on mycosporines and mycosporine-like amino acids, Table S2: Mycosporines and mycosporine-like amino acids (MAAs) with their corresponding absorption maxima (λmax), molar absorptivities (ε), molecular formula, exact mass, molecular structure, fragments (underline font represents [M + H]+) and phylum.

**Author Contributions:** V.G. and E.P. have worked in the conception and design of this review. V.G. searched the data on mycosporine-like amino acids and summarized the database. V.G. analyzed the data and drafted the article. E.P. have made substantial contributions to conception of this manuscript and approve the final version for submission. Both authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP (FAPESP 2019/27707-3 and 2013/07914-8), University of São Paulo Foundation (FUSP) (Project#1979), the Coordination for the Improvement of Higher Education PersonnelCAPES (Project # 23038.001401/2018-92) and the National Council for Scientific and Technological Development—CNPq (311048/2016-1 and 439065/2018-6).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Plant Terpenoids as Hit Compounds against Trypanosomiasis**

**Raquel Durão 1,†, Cátia Ramalhete 1,2,† , Ana Margarida Madureira 1, Eduarda Mendes <sup>1</sup> and Noélia Duarte 1,\***


**Abstract:** Human African trypanosomiasis (sleeping sickness) and American trypanosomiasis (Chagas disease) are vector-borne neglected tropical diseases, caused by the protozoan parasites *Trypanosoma brucei* and *Trypanosoma cruzi*, respectively. These diseases were circumscribed to South American and African countries in the past. However, human migration, military interventions, and climate changes have had an important effect on their worldwide propagation, particularly Chagas disease. Currently, the treatment of trypanosomiasis is not ideal, becoming a challenge in poor populations with limited resources. Exploring natural products from higher plants remains a valuable approach to find new hits and enlarge the pipeline of new drugs against protozoal human infections. This review covers the recent studies (2016–2021) on plant terpenoids, and their semi-synthetic derivatives, which have shown promising in vitro and in vivo activities against Trypanosoma parasites.

**Keywords:** terpenoids; Trypanosoma; human African trypanosomiasis; sleeping disease; human American trypanosomiasis; Chagas disease

**Citation:** Durão, R.; Ramalhete, C.; Madureira, A.M.; Mendes, E.; Duarte, N. Plant Terpenoids as Hit Compounds against Trypanosomiasis. *Pharmaceuticals* **2022**, *15*, 340.

https://doi.org/10.3390/ph15030340 Academic Editor: Daniela De Vita

Received: 27 January 2022 Accepted: 4 March 2022 Published: 10 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Human African trypanosomiasis (sleeping sickness) and American trypanosomiasis (Chagas disease) are among the twenty Neglected Tropical Diseases (NTDs) defined as such by the World Health Organization (WHO). NTDs are a heterogeneous group of diseases including, among others, several parasitic, viral, and bacterial infections, responsible for high morbidity and mortality, and affecting more than one billion people globally [1–3]. Sometimes, the impact of NTDs on health can be underestimated because many infections are asymptomatic and associated with long incubation periods. Nevertheless, NTDs are recognized as a public health problem, particularly for people living in rural and conflict areas in developing countries [1]. These diseases are considered neglected due to the general lack of attention in developed countries and almost non-existent financial investment in the research and development of new drugs and vaccines [4]. In addition to all these problems, the COVID-19 pandemic has been affecting the programs of mass drug administration and other NTD control measures [5,6]. Currently, the pharmacological therapy of NTDs is not ideal, as it has some limitations that include severe side effects, unfavorable toxicity profiles, prolonged treatment duration, difficult administration procedures, and development of drug resistance [7–9]. The investment in these therapeutic areas by large pharmaceutical companies is not financially attractive due to the poor prospect of financial returns. Thus, the research of drugs against these diseases is not motivated by commercial reasons. Additionally, many pharmaceutical companies take an opportunistic approach to drug repositioning, using drugs that were previously developed and registered for other therapeutic indications and applying them in the treatment of NTDs. This strategy has obvious advantages, namely reduced development costs. However, the major disadvantage is the non-introduction of new specific drugs used in the treatment of these diseases [7].

Therefore, the discovery and development of new drugs is essential and urgent and should embrace the development of new therapeutic classes, the reduction in toxicity in the host, better administration processes, and the development of combined therapies [10]. One of the main strategies includes the phytochemical study of plants and other natural sources (marine organisms, animals, microorganisms, and fungi). Natural products have played an important role in the drug discovery and development processes [11]. However, in recent decades, most pharmaceutical companies have reduced their drug discovery and development programs from natural sources, largely due to the development of combinatorial chemistry programs [7]. Nevertheless, despite the large number of drugs derived from total synthesis, natural products and/or synthetic derivatives using their novel structures, contribute to the global number of new chemical entities that continue to be introduced on the market [12].

Over the last decade, several reviews have reported the bioactivity of natural products against protozoan neglected diseases [7,13–21]. However, natural products described in these reviews were obtained from different sources, including microbial [19], endophytes [20] and other fungi [21], marine [22], or animal origins [23,24]. Some reviews also present a mix of natural compounds origins [7,17]. Regarding natural products from plants, since 2016, there have been some reviews focusing exclusively on compounds from higher plants [16,18,25–30]. Nevertheless, the information is scattered amongst the diverse antiprotozoal diseases, compound families, and sources. To the best of our knowledge, a comprehensive review gathering the data concerning the most recent studies on terpenic compounds with antitrypanosomal activities is still missing. Therefore, in this work, a compilation of terpenes obtained from plants and evaluated for their activity against *T. brucei* and *T. cruzi*, covering the period from 2016 to 2021, will be presented and discussed. When available, data regarding in vivo activity and considerations about possible mechanisms of action will be also addressed.

The literature search was performed from June to December 2021 using Web of Science, ScienceDirect, PubMed, and some official websites (WHO, DNDi, CDC). An appropriate combination of keywords and truncation was selected and adapted for each database (for example, combinations of terpenoids or terpenes with Trypanosoma, Human African trypanosomiasis, and Chagas disease). Only peer-reviewed research articles or reviews in a six-year timespan (2016–2021) and in English language were considered. No restriction geographical origin of authors was applied. In particular cases, important reviews older than six years were also included. The literature was individually screened, applying as exclusion criteria, poor quality, inaccurate data, not considered relevant to the aim of the review, and articles reporting antitrypanosomal activity of extracts. Mendeley Reference Manager Software (2020) was used to manage the references and eliminate duplicates.

#### **2. Trypanosomiasis**

#### *2.1. Human African Trypanosomiasis (HAT)*

HAT is endemic in 36 African countries, with approximately 60 million people at risk, and approximately 10.8 million people living in areas of moderate to high risk of infection. In 1995, about 25,000 cases were detected and about 300,000 cases remained undetected. However, in 2001, the WHO launched an initiative to strengthen control and surveillance, and HAT declined in the following years. In 2019, less than 1000 cases were reported. It is noteworthy that this reduction is not due to a lack of control efforts as active and passive screening have been maintained at similar levels (about 2.5 million people screened per year) [31]. HAT is essentially present in poorer and rural areas, affecting populations dedicated to agriculture, fishing, livestock, and hunting, who are more exposed to the vector of transmission. Furthermore, their access to adequate health services is limited, thus lacking medical surveillance, also associated with difficulties in diagnosis and treatment [32]. This parasitic disease is transmitted mostly by the bite of the tsetse fly (*Glossina palpalis*), but other routes of transmission are possible, such as congenital transmission,

blood transfusion, and transplants, despite being poorly documented [33]. The life cycle of *Trypanosoma brucei* sp. is illustrated in Figure 1 and reviewed elsewhere [10,34].

**Figure 1.** The life cycle of *T. brucei*.

There are two subspecies of *Trypanosoma brucei* (*T. b*.), *T. b. gambiense*, and *T. b. rhodesiense*, with different geographic distributions. *T. b. gambiense* is found in 24 countries in West and Central Africa, accounting for more than 98% of reported cases. *T. b. rhodesiense* is present in 13 countries in East Africa, representing less than 3% of reported cases [32]. The two subspecies have different rates of progression and clinical characteristics. Infections by *T. b. gambiense* are characterized by a low parasitemia, with slow progression leading to the development of the chronic form of the disease, while *T. b. rhodesiense* progresses rapidly with high parasitemia, being characterized by the acute form. Both infections, if not diagnosed and treated, lead to death [34]. The clinical evolution of HAT has two phases. In the first phase, also known as the hemolymphatic phase, the parasite is found in the host's blood and lymphatic stream. This initial phase includes non-specific symptoms such as pyrexia, headache, muscle and joint pain, weight loss and even enlarged lymph nodes, usually in the neck area. The second or neurologic phase (meningoencephalitis) occurs when the parasite crosses the blood–brain barrier and reaches the central nervous system. The clinical manifestations are usually behavioral changes, such as anxiety and irritability, sensory, motor, and sleep cycle disorders [32,33].

#### 2.1.1. Antitrypanosomal Chemotherapy Targets and Current Drugs against HAT

Trypanosome-specific metabolic and cellular pathways represent excellent molecular targets. The ability to synthesize polyamines, putrescine, and spermidine, is of vital impor-

tance for the proliferation of bloodstream forms in trypanosomes. In this process, ornithine decarboxylase has a crucial function. This enzyme is considered the best-validated drug target in *T. brucei*, which is the target of eflornithine, a drug that is used clinically for the treatment of HAT [10,35]. In addition, the enzyme N-myristoyltransferase (NMT) has been well validated as a molecular target for HAT since its inhibition may lead to the death of the parasites. NMT catalyzes the covalent attachment of myristate, a 14-carbon saturated fatty acid, via amide bond to the N-terminal glycine residue of several proteins. NMT is also present in humans, but *T. brucei* is extremely sensitive to NMT inhibition, probably because endocytosis occurs at a very high rate in *T. brucei* [36]. Recently, significant progress in targeting the ubiquitin-proteasome system was reported [37]. The ubiquitin-proteasome system (UPS) is a crucial protein degradation system in eukaryotes and is essential for the survival of eukaryotes including trypanosomatids. There are promising inhibitors of this, but the overall success of clinical trials is low and therefore more drug candidates are needed. The bloodstream forms of *T. brucei* produce energy exclusively through glycolysis. Thus, inhibition of glycolytic enzymes, such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase, phosphofructokinase and pyruvate kinase, could be a potential therapeutic approach. However, there is little prospect of killing trypanosomes by suppressing glycolysis unless inhibition is irreversible or uncompetitive, owing to the enormous glycolytic flux through the system [10,35]. Regarding redox metabolism, a fundamental metabolic difference between host and parasite is the existence of trypanothione reductase in trypanosomes instead of glutathione reductase, which is essential for the parasite's survival. The inhibition of trypanothione reductase compromises the parasite's oxidative defenses, sometimes leading to its death. Unfortunately, until now, compounds suitable for clinical development have not been discovered [10].

Currently, there is no vaccination or chemoprophylaxis for HAT, and its combat is mainly conducted through prophylactic measures aimed at reducing the reservoir of the disease and controlling the vector. The latter is the main strategy in use, which aims to minimize human contact with the fly. The recommended measures in the most affected areas are the use of clothes with neutral colors, and the use of insecticide repellents [34]. Recently, rapid diagnostic tests have been also developed in order to detect the presence of the antigen, offering accurate and sensitive results [38]. The treatment of HAT depends essentially on the stage of the disease and the causative agent. Until recently, five drugs have been used to treat sleeping sickness, donated by manufacturers to WHO for free distribution [38]. For the first phase, suramin (Naganinum®, Naganol®) and pentamidine (Nebupent®, Pentam®) are the first line drugs. For the final stage of sleeping sickness, the treatment includes the use of melarsoprol (Arsobal®), eflornithine (Vaniqa®) and the nifurtimox (Lampit®) -eflornithine combination therapy (NECT) [39]. Recently, fexinidazole was approved by the European Medicine Agency (EMA) and the United States Food and Drug Administration (USFDA) as the first all-oral therapy for the treatment of phase-1 and phase-2 HAT [39]. Additionally, acoziborole a recently developed benzoxaborole, is currently in advanced clinical trials, for treatment of phase-1 and phase-2 caused by both *T.b. gambiense* and *T.b. rhodesiense*. Acoziborole is orally bioavailable, and importantly, curative with one dose [40].

#### *2.2. Human American Trypanosomiasis (Chagas Disease, CD)*

Human American trypanosomiasis, commonly known as Chagas disease (CD), is endemic in 21 countries in Latin America. However, human migrations have turned it into a global disease with a significant number of cases in non-endemic regions such as Canada or Europe, among others. CD is present in rural areas and affects populations living in poverty. The WHO estimates that about 6 to 7 million people worldwide are infected and there are approximately 70 million people at risk [41,42]. CD is caused by the protozoan parasite *Trypanosoma cruzi* (*T. cruzi*), and it is generally transmitted by vectors, such as the triatomine hematophagous insects of the *Reduviidae*family, usually known as "barbers". The other transmission routes are blood transfusions, transplantation, congenital transmission,

and oral transmission (breast milk), and also by ingestion of contaminated food. The life cycle of *Trypanosoma cruzi* is illustrated in Figure 2 and reviewed elsewhere [41–43].

**Figure 2.** The life cycle of *T. cruzi*.

CD has two successive clinical phases, an acute phase, and a chronic phase. The acute phase can be symptomatic or, most frequently asymptomatic. The initial acute phase occurs immediately after the infection, which can last for weeks or months. It is characterized by local manifestations such as the Romaña Sign, when the parasite penetrates the conjunctiva, or the skin, causing a skin lesion or a purplish swelling of the lids of one eye called Chagoma. After a period of 4 to 8 weeks, the parasitemia decreases and the clinical manifestations spontaneously disappear in 90% of the cases, when the disease enters the chronic phase [41,43]. During the chronic phase, the parasites are in the heart and gastrointestinal tract. Despite the long-lasting nature of the infection in which most individuals do not develop overt pathology, there are about 30% of people who can achieve the chronic phase, characterized by progressive heart and/or digestive disease. In most of these cases, it takes decades to become apparent. Cardiomyopathy is the most serious result of *T. cruzi* infection, and in many areas of South America, it is a major cause of heart disease. Digestive symptoms, including megaesophagus and megacolon, also have serious consequences and may require surgery [43–47]

#### 2.2.1. Antitrypanosomal Chemotherapy Targets and Current Drugs against CD

Infective trypomastigotes and intracellular replicative amastigotes are the clinically relevant life-cycle stages of *T. cruzi* that are potential targets for drug intervention [4,48]. *T. cruzi* requires specific sterols for cell viability and proliferation at all stages of the life cycle. The main sterol component of the parasite is ergosterol, while in the mammalian hosts it is cholesterol. Inhibitors of sterol biosynthesis have been shown antitrypanosomal in vitro activity [48]. Other trypanosomal targets are related to cysteine proteases that are involved in many crucial processes, including host cell invasion, cell division, and differentiation. *T.* *cruzi* contains a cysteine protease, cruzipain, which is responsible for proteolytic activity at all stages of the parasite's life. Although no inhibitors of this family of enzymes have progressed to clinical trials, the parasite cysteine proteases remain a promising area of research [47,48]. In addition, the trypanothione reductases and synthetases have also been considered key enzymes in the oxidative metabolism of the parasite. Although several potential inhibitors of the trypanothione reductase possess potent in vitro anti-*T. cruzi* activity, to date, none have achieved parasitological cure in animal models [47].

Only two old nitroheterocyclic drugs, benznidazole (Rochagan® or Rodanil®) and nifurtimox (Lampit®) have been available for the treatment of CD, as reviewed elsewhere [41,49]. They are effective for the acute phase of infection, but they have variable efficacy in the chronic phase of the disease, besides requiring prolonged treatment (60–90 days). In addition, significant problems of resistance have emerged with both drugs. In this context, there is an urgent need for more efficacious and safer drugs or drugs regimens, in particular for the treatment of the chronic stage of the infection. Presently, new benznidazole monotherapy regimens with reduced exposure to improve tolerability while maintaining efficacy, and combination regimens of benznidazole with fosravuconazole to improve efficacy are being developed [50].

#### **3. Terpenic Compounds with Antitrypanosomal Activity**

Herein, 150 terpenic compounds with antitrypanosomal activity, isolated from plants or obtained by derivatization, and reported in the literature from 2016 to 2021, are presented (Figures 3–15 and Tables 1 and 2). For clarity reasons, the terpenes are divided into four classes: monoterpenes and iridoids (C10, Figure 3), sesquiterpenes (C15, Figures 4–9), diterpenes (C20, Figures 10–12), and triterpenes (C30, Figures 13–15). The selected compounds were tested for their in vitro activity against *T. brucei* (*T. b. brucei, and T. b. rhodesiense*), and *T. cruzi*. Additionally, the in vivo results of some compounds were also described.

**Figure 3.** Structures of monoterpenes (**1**–**6**) and iridoids (**7**–**9**).

**Figure 4.** Structures of sesquiterpenes **10**–**27**.

**Figure 5.** Structures of sesquiterpenes **28**–**44**.

**Figure 6.** Structures of sesquiterpenes **45**–**50**.

**Figure 7.** Structures of sesquiterpenes **51**–**58**.

**Figure 8.** Structures of sesquiterpenes **59**–**71**.

2+

2

2

3

2&+ 2&+

2

The in vitro antitrypanosomal activities are expressed in micromolar concentrations (μM) and some were transformed into this unit to allow an accurate comparison. Furthermore, the in vitro cytotoxicity of these compounds on mammalian cells lines is also indicated, when evaluated simultaneously, allowing the assessment of the selectivity index (SI). SI is defined as the ratio between the half-maximal cytotoxic concentration against the mammalian cell line (CC50) and the half-maximal inhibitory concentration against the parasite (IC50) [51,52]. Although the SI values do not allow extrapolation to the in vivo condition, this parameter is valuable for the selection of compounds with selective activity against trypanosomes.

**Figure 10.** Structures of diterpenes **88**–**97**.

**Figure 11.** Structures of diterpenes **98**–**104**.

**Figure 12.** Structures of diterpenes **105**–**115**.

**Figure 13.** Structures of triterpenes **116**–**121**.

 5 + 5 2+

 5 2\$F 5 2+

**Figure 14.** Structures of triterpenes **122**–**135**.

**Figure 15.** Structures of triterpenes **136**–**150**.

Presently, there are general and specific criteria proposed by DNDi aiming at identifying hit and lead compounds for further development of drugs against trypanosomiasis, and other infectious diseases. Although these criteria are not strictly applied, they are very important to guide the development of hit and lead series, taking into account their potency, selectivity, toxicity, and chemical profile, among other requirements [51]. For a hit definition,

the criteria are divided into two main sets: the disease-specific criteria that focus on potency, efficacy, pathogenicity, and the compound-specific criteria that evaluate the chemical profile of the compounds, in silico pharmacokinetics and pharmacodynamics (DMPK), as well as the physical properties that are predictive of oral therapy [52]. Accordingly, a compound is considered active if it has an IC50 ≤ 10 μM in the in vitro assay against the bloodstream forms of *T. b. brucei* subspecies, and against the *T. cruzi* intracellular amastigote forms (TcVI (Tulahuen) or TcII/Y strain) [51,52]. The selectivity of the promising hit compound should be 10-fold higher for the parasite than for the mammalian cell line tested. On the other hand, a lead compound for a future drug against HAT or CD should display an IC50 value more than 10–20-fold higher than the IC50 value of the hit compound, and ideally, its selectivity should be ≥ 50 times higher for the parasite than for the mammalian cell line. Moreover, a significant reduction in parasitemia and/or increase in life-span should be observed in the acute mouse model of HAT at the end of the treatment with up to 4 doses at 50 mg/kg (*i.p* or *p.o)*. Concerning CD, the lead selection criteria include a hit that causes an 80% parasitemia reduction in organs or tissues, or no parasites detected at the end of treatment and an increase in lifespan with up to 10 doses at 50 mg/kg (*p.o*) in a mouse model [51,52].

#### *3.1. Monoterpenes and Iridoids*

Compound **1** is a limonene benzaldehyde-thiosemicarbazone derivative that showed in vitro antitrypanosomal activity and high selectivity against *T. cruzi* amastigotes (IC50 1.3 μM, CC50 795 μM, mammalian LLCMK2; SI = 611.2). It is believed that this compound act by inhibiting the proliferation of *T. cruzi* and inducing morphological changes that lead to the cell death of the parasite. In addition, a reduction in cell volume, depolarization of the mitochondrial membrane and an increase in production of reactive oxygen species (ROS) were also observed. Due to promising in vitro results, an in vivo study was performed on a murine model of acute Chagas disease, and a significant reduction in parasitemia in animals treated with **1** alone (100 mg/kg/day) or combined with benznidazole (5 mg/kg/day each) was found, when compared to the untreated animals. Moreover, it was observed that the survival rate of the animals treated with both compounds during the period of infection was the same that the group treated just with benznidazole, however, with only 5% of the dose used [53].

The essential oil of *Origanum onites* L. and its major components carvacrol (**2**) and thymol (**3**) were evaluated for their antitrypanosomal activity against *T. b. rhodesiense* trypomastigote forms (mammalian stage), and *T. cruzi* amastigotes. Good results were only observed against *T. b. rhodesiense*, and both compounds showed IC50 values of 1.0 μM and 0.73 μM, respectively, and a high selectivity for the parasite (SI = 327.5 and 454.4, respectively, L6 mammalian cell line). Additionally, in the in vivo *T. b. brucei* mouse model, only compound **3** extended the mean survival of animals, while none cured the infected animals when compared to the reference drug pentamidine [54].

The essential oils of some Apiaceae plants (*Echinophora spinosa* L., *Sison amomum* L., *Crithmum maritimum* L., *Helosciadium nodiflorum* (L.) W.D.J.Koch) were studied against *T. brucei* bloodstream forms (TC221 BSFs strain), showing IC50 values in the range of 2.7–10.7 μg/mL. From those, only the essential oil of *C. maritimum* had a good selectivity (SI = 13, mouse Balb3T3 fibroblasts cell line). Additionally, using the same parasite, the trypanocidal activity of the major compounds (**4**–**6**) of *C. maritimum* was also tested. Terpinolene (**4**) was the most potent showing an IC50 value of 0.26 μM with a SI = 180. Two other compounds displayed promising activities on the same model namely α-pinene (**5**, IC50 = 7.4 μM, SI > 100) and β-ocimene (**6**, IC50 = 8 μM, SI > 91) [55].

Three tetracyclic iridoids (**7**–**9**) were isolated from *Morinda lucida* Benth., a plant traditionally used to treat parasitic diseases in West Africa. Iridoids were evaluated for their in vitro activity against the bloodstream forms of *T*. *b. brucei*. Compound **7** was the most active (IC50 0.43 μM) and less toxic than **8** (IC50 1.27 μM), displaying CC50 values of 14.24 μM (SI = 33.1) and 4.74 μM (SI = 3.7), respectively [56]. The activity of compound **9** is

lower than **7** and **8** (IC50 3.75 μM), but it did not show cytotoxicity (CC50 ≤ 50 μM) [56]. The main structural differences between compounds **7**–**9** are the functional groups at C-4 on the side chain. Compound **9** has a carboxylic acid, while compound **7** and **8** have ethyl ester and methyl functional groups, respectively [56]. SI values of the three compounds showed that **7** and **9** are more specific against the parasite than compound **8**. Compound **7** was tested in vivo, and a complete clearance of parasitemia, with 100% cure for 20 days post infection, was observed, when 5 consecutive daily shots of 30 mg/kg of compound **7** were taken. It was concluded that compounds **7** and **9** suppressed the expression of paraflagellum rod protein subunit 2, and caused cell cycle alteration, which can preceded apoptosis induction in the bloodstream form of the parasite [56].

#### *3.2. Sesquiterpenes*

Two sesquiterpene lactones (**10** and **11**) were isolated from the methanolic extract of *Tithonia diversifolia* (Hemsl) A. Grey, and their activities were evaluated against the blood forms of T. brucei [57]. Compound **10** was the most active, exhibiting a very low IC50 value (0.012 μM), but displaying a high cytotoxicity on the mammalian fibroblasts cells (CC50 0.036 μM, SI = 3). Likewise, compound **11** showed antitrypanosomal activity (IC50 0.97 μM) and high cytotoxicity (CC50 1.27 μM, SI = 1.3) [57]. Some considerations can be made regarding the mechanism of action of these sesquiterpene lactones in the parasite cells. Due to the characteristic α,β-unsaturated lactone function present in these structures, which can act as a Michael acceptor, these compounds react with nucleophiles, such as thiol groups in proteins, leading to macromolecular dysfunction, oxidative stress and genetic mutations [57]. The presence of an extra carbonyl group conjugated with two double bonds in **10** can explain the higher antitrypanosomal activity of **10** when compared with **11**. In fact, the mechanism of action of these compounds against trypanosomes may be related with formation of thiol adducts with components found in the intracellular medium (namely trypanothione, glutathione and thiol groups in proteins). The parasite's cells become more vulnerable to oxidative stress with reduction in trypanothione [57].

Several sesquiterpene lactones were isolated from the dichloromethane extract of *Vernonia cinerascens* Sch.Bip., and evaluated for their in vitro activity against the blood forms of *T. b. rhodesiense* and for cytotoxicity on the L6 mammalian cell line [58]. Lactone **12** was the most active and selective exhibiting an IC50 value of 0.16 μM and SI = 35. Compounds **13** and **14** also showed activity against *T. b rhodesiense* (IC50 values of 0.5 μM and 1.1 μM, SI = 13 and 4.2, respectively), being lactone **13** the most selective [58]. Compounds **15** and **16** exhibited similar activities; however, compound **15** showed a higher selectivity (IC50 values of 4.8 μM and 5.0 μM, SI = 27 and 4.3, respectively). Moreover, lactones **12**–**16** displayed the lowest cytotoxicity in the cells tested [CC50 5.6 μM (**12**), CC50 6.9 μM (**13**), CC50 4.7 μM (**14**), CC50 128 μM (**15**), CC50 22 μM (**16**)] [58,59].

Two sesquiterpene lactones (**17** and **18**) isolated from the dichloromethane extract of *Tarchonanthus camphoratus* L. aerial parts, and twenty sesquiterpene lactones, including compounds **19**–**27** obtained from *Schkuhria pinnata* (Lam.) Kuntze ex Thell. were studied for their in vitro antitrypanosomal activity and cytotoxicity on mammalian L6 cell lines [60]. Lactones **17** and **18** were active against *T. b. rhodesiense* with IC50 values of 0.39 μM and 2.8 μM, respectively. Furthermore, **17** (SI = 18.6, CC50 7.2 μM) proved to be more selective although it was more cytotoxic than **18** (SI = 6.2, CC50 17.3 μM) [60]. Regarding compounds isolated from *S. pinnata*, most of them displayed antitrypanosomal activity with IC50 values ranging from 0.10 to 7.30 μM, Compounds **19** and **20** stood out for their high activities against the trypomastigotes, with IC50 values of 0.10 and 0.13 μM, respectively. However, they exhibited cytotoxicity on the cells tested (CC50 values of 2.10 and 3.90 μM, respectively) despite exhibiting some selectivity (SI = 20.5 and 29.7, respectively). Compounds **21** (IC50 0.35 μM, SI = 11.5) and **22** (IC50 0.52 μM, SI = 13) were particularly active, but cytotoxic against the mammalian cell lines assayed (CC50 values of 4.10 and 6.80 μM, respectively). Moreover, compounds **23** (IC50 0.60 μM, SI = 19.2), **24** (IC50 0.82 μM, SI = 13.4), **25** (IC50 0.91 μM, SI = 15.8) and **26** (IC50 0.92 μM, SI = 15.8) also showed very good activities. Finally, sesquiterpene **27**

display an IC50 value of 1.7 μM, and was the most selective and least cytotoxic in this group of compounds (SI = 31.1 and CC50 54.6 μM) [60].

Three sesquiterpene lactones (**28***–***30**) were isolated from the dichloromethane extract of *Achillea fragrantissima* (Forssk.) Sch.Bip. and tested against trypomastigote forms of *T. b. brucei* [61]. Lactone **28** was the most active (IC50 3.03 μM), while lactones **29** and **30** showed the same activity against the parasite with IC50 value of 10.97 μM. The authors did not assess the cytotoxicity of these compounds on mammalian cells [61].

Four sesquiterpene lactones were isolated by bioassay-guided fractionation from extracts of *Mikania variifolia* Hieron. and *Mikania micrantha* Kunth, and evaluated against the epimastigote, trypomastigote and amastigote forms of *T. cruzi*. Compounds **31** and **32** were found in both extracts (2.2% and 0.4% for *M. variifolia*, and 21.0% and 6.4% for *M. micrantha*, respectively, calculated based on the dry extract) [62]. Three of the isolated lactones (**31**, **32**, and **33**) showed trypanocidal activity, being active against the epimastigote form with IC50 values of 2.41 (SI = 31.9), 0.29 (SI = 992.5) and 8.55 (SI = 5.2) μM, respectively [62]. Compounds **31**, **32** and **33** also displayed activity against the trypomastigote form of the parasite with IC50 values of 7.24 (SI = 10.6), 5.43 (SI = 54.0) and 1.03 (SI = 49.0) μM, respectively. Finally, the activities of **31**, **32** and **33** against the amastigote forms were lower than those observed for the two previous forms of the parasite (IC50 15.5, 22.8 and 29.1 μM, and SI = 4.3, 12.5, and 1.5, respectively). From those compounds, **32** was the most selective for the human infective parasite, showing a SI of 54 when assayed on human monocyte leukemia THP1 cells. Due to its good selectivity, **32** was also tested in an in vivo model of *T. cruzi* infection, and was able to decrease the parasitemia and the weight loss associated with the acute phase of the parasite infection. Additionally, 70% of treated mice (1 mg/kg of body weight/day) survived, while all of the control mice died by day 22 after the infection. The authors also observed that this compound increased the production of TNF-*α* and IL-12 by macrophages [62].

The essential oils from different parts of *Smyrnium olusatrum* L. were evaluated against the bloodstream forms of *T. b. brucei*. All oils effectively inhibited the growth of the parasite [63]. From the main constituents of essential oils, sesquiterpene **34** exhibited a significant and selective inhibitory activity against the tested parasite (IC50 3.0 μM, SI = 30, mouse Balb/3T3 fibroblast)) [63].

From *Anthemis nobilis* L. dichlorometane extract, 19 sesquiterpene lactones, including 15 germacranolides, 2 seco-sesquiterpenes, 1 guaianolide sesquiterpene lactone, and 1 cadinane acid were obtained [64]. Among these compounds, thirteen were tested for their in vitro activity against the bloodstream forms of *T. b rhodesiense*, with compound **35** being the most potent and selective (IC50 0.08 μM, SI = 63.1). Compounds **36**–**38** exhibited also a significant anti-trypanosomal activity, but with lower selectivity (**36**, IC50 0.61 μM, SI = 8.3; **37**, IC50 0.36 μM, SI = 14.1; **38**, IC50 0.88 μM, SI = 8.3). Moreover, the compounds were assessed against *T. cruzi* intracellular amastigotes, and the best result was observed for compound **39** (IC50 2.8 μM), but a very low selectivity index was also observed (SI = 0.5). Compound **39** also exhibited a good activity against *T. b rhodesiense* (IC50 0.4 μM), but with a low selectivity due to its cytotoxicity on mammalian L6 cells (CC50 1.5 μM, SI = 3.8). Compound **40** was considered the one with higher selectivity for *T. cruzi* (IC50 4.2 μM, SI = 6.1) [64].

*Calea pinnatifida* (R. Br.) Less. is used in folk medicine as giardicidal, amoebicidal and to treat digestive disorders. Its phytochemical study led to the isolation of a furanoheliangolide sesquiterpene lactone (11,13-dihydroxy-calaxin, **41**) which showed a promising trypanocidal activity, displaying an IC50 value of 8.30 μM against *T. cruzi* amastigotes, and inhibiting the parasite growth in 94.3%. However, compound **41** presented a low selectivity for the parasite cells (CC50 < 15.60 μM on THP-1 cells) [65].

Three sesquiterpene lactones (**42***–***44**) were isolated from *Smallanthus sonchifolius* (Poepp.) H. Rob. and evaluated against *T. cruzi* epimastigotes, using benzonidazole as positive control [66]. Compounds **42** and **43** showed identical activities with IC50 values of 0.78 and 0.79 μM, respectively. Lactone **44** was also active exhibiting an IC50 value of 1.38 μM. All

compounds were more effective than benzonidazole (IC50 10.6 μM). The authors did not assess the cytotoxicity of these compounds on mammalian cells. Due to the high in vitro activity, compounds **42** and **43** were also tested on mice inoculated with *T. cruzi* trypomastigotes. A significant decrease in circulating parasites (50–71%) was observed, with no signs of toxicity in the dose administrated (1 mg/kg/day). Complementary studies showed marked ultrastructural alteration in trypanosome parasites when treated with these compounds [66].

Several sesquiterpenes isolated from the Cameroonian spice *Scleria striatinux* De Wild. were studied for their in vitro and in silico antiparasitic activity [67]. From those, sesquiterpene **45** exhibited the best activity against *T. cruzi* and *T. b. rhodesiense* bloodstreams forms, with IC50 values of 0.025 μM (SI = 0.74) and 0.002 μM (SI = 8.3), respectively, but the compounds were cytotoxic on HT-29 (human bladder carcinoma) cells. On the other hand, compound **46** showed better activity against *T. b. rhodesiense* than *T. cruzi*, with IC50 values of 0.025 μM (SI = 3.4) and 0.085 μM (SI = 1), respectively [67]. The in silico drug metabolism and pharmacokinetic parameters of these two sesquiterpene isomers were also studied, showing compound **46** a good solubility profile, moderate partition coefficient and acceptable in silico pharmacokinetic properties. Similar characteristics were observed for compound **45**, but with less optimal parameters, namely for the partition coefficient [67]. Nevertheless, despite the good pharmacokinetic features and the low IC50 values observed, SI values were very small, and compounds also showed a considerable cytotoxicity against HT-29 cell line, which reduces its possible application as a hit compound.

*Vernonia lasiopus* (O.Hoffm.) H.Rob. extracts were obtained with solvents of different polarities and evaluated in vitro for antiprotozoal activity [59]. The dichloromethane extract was shown to be particularly active against *T. b. rhodesiense*, and its phytochemical study led to the isolation and identification of six sesquiterpene lactones. These compounds were tested for their in vitro antitrypanosomal activity and cytotoxicity on L6 mammalian cells [59]. Compound **47**, the main component of the extract, was the most potent against *T. b. rhodesiense* trypomastigotes (IC50 0.185 μM); however, it displayed some cytotoxicity (CC50 2.68 μM and SI = 14.5). Moreover, compound **48** presented very similar values (IC50 0.26 μM, CC50 3.67 μM, SI = 14.4). Lactone **49** was the least selective (SI = 4.5) and displayed some cytotoxicity (CC50 2.26 μM), despite showing a considerable antitrypanosomal activity (IC50 0.51 μM). Compound **50** was the least cytotoxic compound in this group (CC50 34.6 μM, SI = 13.7) still showing a good activity against trypomastigotes (IC50 2.53 μM) [59].

On a recent work, the sesquiterpene lactones eupatoriopicrin (**51**), estafietin (**52**), eupahakonenin B (**53**) and minimolide (**54**) isolated from Argentinean Astearaceae species, which had previously showed activity against *T. cruzi* epimastigotes, were tested against other forms of the parasite [68]. On the bloodstream forms of *T. cruzi* the IC50 values obtained were 19.9 μM (**51**, SI = 12.9), 33.0 μM (**53**, SI = 10.4), and 21.0 μM (**54**, SI = 12.8). On the intracellular *T. cruzi* amastigotes the most active compound was **51** with an IC50 value of 6.3 μM (SI = 40.6). Moderate activities were observed for compound **54** (IC50 = 25.1 μM; SI = 10.7), and **53** (IC50 = 89.3 μM; SI = 3.8) against the same form of the parasite. The majority of compounds showed a significant selectivity for the parasite forms tested compared to Vero cells. The in vivo administration of eupatoriopicrin (**51**, 1 mg/kg/day) to mice infected with *T. cruzi* trypomastigotes, for five consecutive days, produced a significant reduction in the parasitemia levels in comparison with non-treated animals (area under parasitemia curves 4.48 vs. 30.47, respectively), being this reduction similar to that achieved with the reference drug, benznidazole. Authors also presented some information regarding the prevention of tissue damage during the chronic phase of the parasite infection, showing beneficial effects on skeletal and cardiac muscular tissues of infected mice treated with the sesquiterpenoid compound. Compound **52** was inactive [68].

The sesquiterpene lactone **55** (tagitinin C) isolated from leaves of *Tithonia diversifolia* (Hemsl.) A. Gray showed a high inhibition activity against the epimastigote forms of *T. cruzi*, with IC50 of 1.15 μM, being more active than benznidazole (35.81 μM). However, the cytotoxic concentration (6.54 μM) and the selectivity index (5.69) of compound **55** did

not show to be favorable. In an in vivo combination assay, it was observed a complete suppression of parasitemia and parasitological cure in all infected mice (100%) compared to those receiving benznidazole alone (70%). Moreover, despite its lower in vitro selectivity index, compound **55** was well tolerated during the in vivo assays. Interestingly, it was also found that tagitinin C was able to reduce myocarditis, especially when combined with benznidazole [69].

The antitrypanosomal potential of three sesquiterpene lactones (**56**–**58**) isolated from *Helianthus tuberosus* L. (Asteraceae) was evaluated against *T. b. rhodesiense* trypamastigote bloodstream form (**56**, IC50 0.077 μM; **57**, 0.26 μM; **58**, 0.92 μM) and *T. cruzi* trypomastigotes (**56**, IC50 1.6 μM; **57**, 3.1 μM; **58**, 5.7 μM); however, the selectivity index was not promising (CC50 between 0.52 and 3.9 μM, on L6 rat skeletal myoblasts) [70].

Seventeen sesquiterpene lactones were isolated from five plant species of Vernonieae tribe and assessed against *T. cruzi* epimastigotes [71]. The best trypanocidal effect was observed by elephantopus-type sesquiterpene lactones **59** (IC50 1.5 μM) and **60** (IC50 2.1 μM), obtained from *Vernonanthura nebularum* (Cabrera) H. Rob., and hirsutinolide **61** (IC50 2.0 μM), isolated from *Vernonanthura pinguis* (Griseb.) H.Rob. Furthermore, these compounds showed a high selectivity for the parasite (SI > 14) when compared to their cytotoxic effect against the mammalian Hela cells. Compounds **62**–**65**, also isolated from *V. nebularum*, showed a good antitrypanosomal activity on the same strain with IC50 values ranging from 3.7 to 9.7 μM, being compound **62** the most selective (IC50 = 3.7 μM and SI = 14.3). From *V. pinguis*, besides compound **61,** compounds **66** (IC50 10.7 μM; SI = 9.0) and **67** (IC50 8.1 μM; SI = 13.9) were also isolated and displayed a significant activity; however, it was lower than the observed to hirsutinolide (**61**). From the remaining species, compound **68** (IC50 6.8 μM; SI = 1.6) isolated from *Centratherum puctatum* ssp. Punctatum Cass. and compound **69** (IC50 4.7 μM; SI = 11.5) isolated from *Elephantopus mollis* Kunth also showed antitrypanosomal activity [71].

The sesquiterpene lactones eucannabinolide (**70**) and santhemoidin C (**71**), isolated from the dewaxed dichloromethane extract of *Urolepis hecatantha* (DC.) R.King & H.Rob., were active on *T. cruzi* epimastigotes with IC50 values of 10 μM and 18 μM, respectively. Both compounds showed low SI values (CC50 > 15 μM for **70** and CC50 = 15 μM for **71**) [72].

Goyazensolide (**72**) is a sesquiterpene lactone isolated from *Lychnophora passerina* (Mart ex DC) Gardn. that displayed promising results against the intracellular amastigote form of *T. cruzi* (IC50 = 0.181 μM/24 h, and IC50 = 0.020 μM/48 h), showing a higher selectivity index than the positive control benznidazol (SI = 52.82 and 915.0 for **72**, at 24 h and 48 h, respectively, and SI = 4.85 and 41.0 for benznidazol, at 24 h and 48 h, respectively). Further in vivo assays were performed and **72** showed an important therapeutic activity in mice infected with *T. cruzi*, which was demonstrated by the high percentage of negative parasitological tests employed by the authors in the successive post-treatment evaluations [73].

From the leaves of *Hedyosmum brasiliense* Mart. Ex Miq., five sesquiterpene lactones together with a sesterpene were isolated and tested against the amastigote and trypomastigote forms of *T. cruzi*. Among the assessed compounds, compound **73**, with a rare terpenoid structure, was the most active displaying an IC50 value of 21.6 μM and SI > 9 for the amastigote form, and an IC50 value of 28.1 μM and SI > 7 for the trypomastigote. The remaining compounds were inactive, excepting compound **74** that exhibited a very weak activity against both parasite forms tested, and a decrease in selectivity for the parasite when compared with selectivity of compound **73** [74].

The bio-guided fractionation of ethanolic extract of leaves of *Inula viscosa* (L.) Greuter (Asteraceae) led to the isolation of two sesquiterpenoids (**75** and **76**), which were tested against *T. cruzi* epimastigotes with IC50 values of 4.99 μM and 15.52 μM, respectively. Both compounds showed modest SI (3.67 and 3.38, respectively), when compared to murine macrophages cells [75]. A preliminary structure-activity study of these compounds demonstrated the importance of the lactone ring to the antiparasitic activity. Regarding the

mechanism of action, authors suggested that compounds induced programmed cell death in the tested parasite [75].

Costic acid (**77**), a eudesmane sesquiterpenoid isolated from the bio-guided fractionation of the n-hexane extract of *Nectandra barbellata* Coe-Teix. Twigs (Lauraceae) induced a trypanocidal effect with high selectivity for the intracellular amastigote form of *T. cruzi* (IC50 7.9 μM). A modest activity against *T. cruzi* trypomastigotes was also observed (IC50 37.8 μM). No cytotoxicity was observed on L929 human cells, revealing its selectivity for both forms of the parasite (CC50 > 200 μM, SI > 25 on amastigote forms and SI > 5 on trypomastigote forms). The authors suggested that costic acid (**77**) has a key action on the mitochondria activity of the parasite [76].

Some germacranolide sesquiterpene lactones were isolated from the aerial parts and flowers of *Tanacetum sonbolii* Mozaff. Compounds **78** and **79** were the most active showing an IC50 of 5.1 and 10.2 μM, respectively, against *T. b. rhodesiense* bloodstream forms, and SI values of 3.9 (**78**) and 4.0 (**79**) when compared with rat myoblast (L6) cells [77].

The bicyclic drimane-type sesquiterpene polygodial (**80**), firstly isolated from *Polygonum hydropiper* L. (Polygonaceae), and some natural and synthetic compounds of the same family were evaluated for growth inhibition against the amastigote, trypomastigote, and epimastigote forms of *T. cruzi*. The parent drug **80** exhibited a moderate inhibitory activity (GI50 = 34.4 μM amastigotes; GI50 = 68.2 μM trypomastigotes; GI50 = 51.0 μM epimastigotes). The best inhibition growth activities were observed for its synthetic derivatives, namely compound **81** (GI50 = 9.9 μM amastigotes; GI50 = 8.4 μM trypomastigotes; GI50 = 13.0 μM epimastigotes), **82** (GI50 = 6.7 μM amastigotes; GI50 = 6.4 μM trypomastigotes; GI50 = 12.3 μM epimastigotes), and **83** (GI50 = 8.3 μM amastigotes; GI50 = 6.9 μM trypomastigotes; GI50 = 7.2 μM epimastigotes). Selectivity index values were not determined. The synthetic *α*,β-unsaturated phosphonate (**83**) was favorably compared with the clinically approved drugs benznidazole and nifurtimox during a competition assay, being even effective against trypomastigotes, contrarily to benznidazole that showed no activity against this trypanosomal form. The effect of polygodial derivative **81** on the growth of the parasite in infected human retinal pigment epithelial (ARPE) cells was studied using confocal microscopy. A significant reduction in the intracellular parasites was observed, with no alterations of replication or viability of the cells [78]. Compound **80** was also previously isolated from the Chilean species *Drimys winteri*, and was tested on the same parasite forms with weak comparable results. On this work, the authors associated the trypanosomal activity of this compound with intracellular effects occurring in the parasite, namely, mitochondrial dysfunctions, ROS production and autophagic phenotype [79].

*Epi*-polygodial (**84**), isolated from the Brazilian plant *Drimys brasiliensis* Miers (Winteraceae), exhibited a high parasite selectivity towards *T. cruzi* trypomastigotes (IC50 = 5.01 μM, SI > 40 to NCTC cells). Authors correlated the antitrypanosomal activity of this compound with its effects on cellular membranes by the interaction of **84** with DPPE-monolayers (the Langmuir monolayers of dipalmitoylphosphoethanolamine) at the air–water interface, which affects the physical chemical properties of the mixed film [80].

The sesquiterpene (-)-T-cadinol (**85**) isolated from *Casearia sylvestris* Sw. displayed a moderate activity against amastigotes and trypomastigotes forms of *T. cruzi* with IC50 values of 15.8 and 18.2 μM, respectively, and no toxic effect on the mammalian cells was observed (CC50 200 μM, SI > 15). The mechanism of action was studied using different techniques, and it was observed that **85** affected the parasite mitochondria. However, additional studies are necessary in order to confirm this organelle as a candidate target [81].

A sesquiterpene glycoside ester (**86**) isolated from the flowers of *Calendula officinalis* L. have shown a moderate activity against *T. brucei* (IC50 16.9 μM). A closely similar compound (**87**), only differing in the type of sugar residue, did not display antitrypanosomal activity, suggesting the importance of sugar moiety conformation to the activity [82].

#### *3.3. Diterpenes*

Andrographolide (**88**) is a labdane-type diterpene, isolated from *Andrographis paniculate* (Burm. F.) Wall. Ex Nees, with reported anticancer, anti-inflammatory, antioxidant, cardioprotective and hepatoprotective properties [83]. To determine its effect on the viability of *T. brucei* procyclic trypomastigotes (the form of the parasite that differentiate in the insect gut), the parasites were incubated at different concentrations (0–200 μM) for 72 h. Compound **88** inhibited the growth of the parasite, exhibiting an IC50 = 8.3 μM and SI = 8.5. At this concentration, no cytotoxic effect was observed (CC50 70.5 μM) [83]. Giemsa staining of parasites treated with **88** allowed the observation of morphological changes, in particular, loss of integrity, damage to the cell membrane, general rounding, and loss of cells' flagella. Ultimately, the authors concluded that the trypanocidal activity of **88** is mediated by inducing the oxidative stress together with the depolarization of the mitochondrial membrane potential, generating an apoptosis-like programmed cell death [83].

The phytochemical study of aerial parts of *Baccharis retusa* DC., a medicinal plant used in Brazilian folk medicine to treat parasitic diseases, allowed the isolation and identification of the kaurane-type diterpene **89**. This compound was active against *T. cruzi* trypomastigotes (IC50 3.8 μM) with a high selectivity (SI = 50.0) due to its reduced cytotoxicity (CC50 189.7 μM) on NCTC cells [84].

*Ent*-kaurenoic acid (**90**) and *ent*-pimaradienoic acid (**91**) were used as starting material to obtain several derivatives. From those, the *ent*-kaurane derivatives **92** (IC50 < 12.5 μM) and **93** (IC50 26.1 μM) showed the highest antitrypanosomal activity when compared to compound **90** (IC50 225.8 μM). Regarding the ent-pimaradienoic acid (**91**, IC50 68.7 μM) set, compound **94** (IC50 3.8 μM) was the most active against trypomastigotes forms of *T. cruzi*. However, due to the lack of cytotoxicity data it is not possible to determine the selectivity index of these compounds [85].

Three quinone methide-type diterpenes (**95**–**97**) were isolated from the roots of *Salvia austriaca* Jacq. and tested for their in vitro activity against *T. b. rhodesiense* and *T. cruzi*. Cytotoxicity was determined on L6 cells [86]. The diterpene **95** was the most active and selective against *T. b. rhodesiense* trypomastigotes (IC50 0.05 μM and SI = 38). However, despite exhibiting an IC50 value of 7.11 μM against *T. cruzi* amastigotes, its selectivity was very low (SI = 0.27). Compounds **96** and **97** also showed activity against both parasites, being more active against *T. b. rhodesiense* with IC50 values of 0.62 μM (SI = 5.0) and 1.67 μM (SI = 2.4), respectively. Regarding their activity against *T. cruzi*, an IC50 value of 7.76 μM (SI = 0.4), and 7.63 μM (SI = 0.5) was observed for compound **96** and **97**, respectively [86], but the compounds were not selective to the parasite [86].

The phytochemical study of dichloromethane extract of *Aldama discolors* (Baker) E.E.Schill. & Panero leaves led to the isolation of four structurally and biosynthetically related diterpenes. These were evaluated for in vitro activity against *T. b. rhodesiense* trypomastigotes and *T. cruzi* amastigotes [87]. Among the isolated diterpenes, compound **98** showed a moderate in vitro activity with an IC50 value of 15.4 μM against *T. cruzi*, and an IC50 24.3 μM for *T. b. rhodesiense*. On the other hand, compound **99**, structurally similar to **98**, showed less activity against the amastigote forms of *T. cruzi* (IC50 19.4 μM), and no activity against the trypomastigote forms of *T. b. rhodesiense*. The selectivity of these compounds was very low, with SI values ranging from 2 to 4 [87].

The commercially available dehydroabietylamine (**100**), an abietane-type diterpenoid isolated in large amount from *Plectranthus* genus, was used as a starting material to produce a set of amides derivatives. Among these compounds, **100**–**103** were tested against *T. cruzi* amastigotes, showing compound **103** the highest antitrypanosomal activity and selectivity (IC50 0.6 μM; SI = 58). The remaining compounds, including **100**, in spite of displaying antitrypanosomal activity (IC50 values between 3.7 and 7.4 μM) were not very selective to the parasite, showing CC50 values ranging from 6.5 to 33.5 μM when tested on the human cell line (SI values between 1 and 6, L6 rat myoblasts) [88].

Leriifolione (**104**), isolated from the lipophilic extract of the roots of *Salvia leriifolia* Benth., showed high activity against *T. b. rhodesiense* (IC50 1.0 μM) and *T. cruzi* (IC50 4.6 μM), but an undesirable cytotoxicity on L6 cells (SI = 2.6 and 0.6, respectively) [89].

From the *n*-hexane extract of the roots of *Zhumeria majdae* Rech. F., eight abietane-type diterpenes were isolated. The antitrypanosomal activity was investigated for compounds **105**, **106**, and **107** against *T. b. rhodesiense* and *T. cruzi*. All the compounds showed a high activity against *T. b. rhodesiense*, (IC50 = 3.6 μM, 1.8 μM, and 0.1 μM to **105**, **106**, and **107**, respectively), being compound **106** the most selective for the parasite when compared with L6 cell line tested (**105**, SI = 1.7; **106**, SI = 21.9; **107**, SI = 15.4). None of the compounds were active against *T. cruzi* [90].

Seventeen diterpenes were isolated from the aerial parts of *Perovskia abrotanoides* Kar. These compounds were evaluated for antiprotozoal activity (*T. cruzi* amastigotes, and *T*. *b rhodesiense* trypomastigotes), and cytotoxicity was also assessed on rat skeletal myoblast L6 cell line. Most of the diterpenes were less active against *T. cruzi* than against *T. b rhodesiense* [91]. Compound **108** showed the best activity against *T. b. rhodesiense* (IC50 0.5 μM, SI = 10.5). However, it was inactive against *T. cruzi*, showing a lack of selectivity (IC50 58.7 μM; SI = 0.1, CC50 5.2 μM). With similar activity, but less cytotoxic (CC50 12.1 μM) than **108**, compound **109** displayed an IC50 value of 0.8 μM (SI = 14.9) against *T*. *b. rhodesiense* and an IC50 of 34.7 μM (SI = 0.3) against *T. cruzi*. Compounds **110**–**113** were particularly active against *T. b. rhodesiense*, displaying IC50 values ranging from 3.8 to 12 μM but low SI values (SI = 1.5–12.5) [91].

Bokkosin (**114**), a new cassane diterpene isolated from the Nigerian species *Calliandra portoricensis* Hassk. used in traditional medicine to treat tuberculosis, and helmintic diseases, showed a strong trypanocidal activity against the bloodstream forms of *T. b. brucei*, sensitive (IC50 = 1.1. μM) and resistant to pentamidine (IC50 = 0.5 μM). A highly favorable selectivity for the parasite strains was also observed, when compared with its cytotoxic effect on two mammalian cell lines (CC50 = 269 μM; SI = 246, on U937; CC50 = 230 μM; SI = 215, on RAW 246.7) [92].

From the *n*-hexane and ethylacetate extracts of the roots of *Acacia nilotica* L. several diterpenes were isolated. Among them, the cassane-type diterpenoid **115** was tested against the *T. b brucei* bloodstream form, exhibiting a high activity a (IC50 1.4 μM). Additionally, **115** was tested for its cytotoxic effect on human HEK cells (CC50 = 29.5 μM; SI = 21.1) displaying a significant selectivity for the parasite [93].

#### *3.4. Triterpenes*

Ursolic acid (**116**) was tested for antitrypanosomal activity against *T. brucei* trypomastigotes displaying an IC50 value of 3.35 μM. Similar IC50 values were also obtained by Catteau et al**.** (IC50 2.4 μM, CC50 > 11.1 μM). However, it is important to note that the compound showed a lack of selectivity for the trypanosoma parasite due to its cytotoxic effect against the mammalian WI38 cells used [94]. In order two find out a possible mode of action, in silico molecular modelling studies were also performed using the parasitic enzymes of the trypanosome, namely trypanothione reductase, methionyl-tRNA synthetase, and inosine-adenosine-guanosine nucleoside hydrolase [95]. Ursolic acid showed a good binding affinity for trypanothione reductase and methionyl-tRNA synthetase, which was higher than that obtain for the reference drug difluoromethylornithine. On the other hand, no inhibition was observed for inosine–adenosine–guanosine. These data may suggest that the inhibition of the two former enzymes may be responsible for the antitrypanosomal activity of compound **116** [95].

A new ursane-type triterpenoid glycoside (**117**) isolated from the dried roots of *Vangueria agrestis* (Schweinf. ex Hiern) Lantz exhibited a considerable growth suppressing effect against *T. brucei* trypomastigotes (IC50 11.1 μM and IC90 12.3 μM). However, no cytotoxicity studies on mammalian cell lines were performed [96].

Betulin acid (**118**), a lupane-type pentacyclic triterpene, and some semysynthetic amide derivatives were tested against *T. cruzi* trypomastigotes. Compound **118** showed a

moderate activity (IC50 19.5 μM and SI = 18.8), while an increasing activity was observed in derivatives **119** (IC50 1.8 μM, SI = 17.3), **120** (IC50 5.0 μM, SI = 10.7), and **121** (IC50 5.4 μM, SI = 5.3). The mechanism of action of compound **119** in trypomastigotes was studied and seemed to be associated with the death of the parasite by necrosis, characterized by the rupture of the membrane, flagellar retraction, and appearance of atypical cytoplasmic vacuoles and dilation of the Golgi cisterns. Furthermore, the amide derivatives of compound **118** act by reduction in the invasion process, as well as the development of the intracellular parasite in host cells [97]. Sousa et al**.** corroborated those mechanistic results, showing that betulinic acid was able to inhibit all the development forms of *T. cruzi* (namely epimastigotes, trypomastigotes and amastigotes) not only by using necrotic processes but also due to modifications on the parasite mitochondrial membrane potential and the increase in reactive oxygen species [98].

Six limonoids (**122**–**127**) obtained from the roots of *Pseudocedrela kotschyi* (Schweinf.) Harms were investigated for their trypanocidal activity using bloodstream forms of *T. brucei*, showing GI50 values ranging from 2.5 to 14.5 μM. The most active compound was **122** (GI50 2.5 μM) but showed some cytotoxicity on human HL-60 cells (GI50 31.5 μM). On the other hand, limonoid **125** exhibited a similar activity (GI50 3.18 μM) with no cytotoxic effect on the mammalian cell line (GI50 > 100 μM) [99].

From *Tabernaemontana longipes* Donn.Sm., baurenol acetate (**128**) was isolated and tested for its ability to inhibit the growth of *T. brucei* bloodstream forms, showing an IC50 value of 3.1 μM. Baurenol (**129**) displayed a higher activity against the same parasite (IC50 = 2.7 μM). Both compounds showed a low effect on cellular viability on Hep G2 cells (IC50 > 80 μM) [100].

Four new triterpenoids (**130**–**133**), with a rare scaffold, isolated from *Salvia hydrangea* DC. ex Benth. showed antitrypanosomal activity against *T. cruzi* amastigotes with IC50 values ranging from 3.5 to 19.8 μM, with no significant activity against *T. b. rhodesiense* trypomastigotes All the compounds showed a modest selectivity (SI ranging from 2.4 to 10.7; L6 cell line) [101].

Two lanostane-type triterpenoids, polycarpol (**134**) and dihydropolycarpol (**135**) were isolated from *Greenwayodendron suaveolens* (Engl. & Diels) Verdc., a plant traditional used in Congo to treat malaria. Both compounds were tested against *T. b. brucei* (IC50 = 8.1 μM; SI < 1.0, **134**; IC50 = 8.1 μM; SI = 2.4, **135**), and *T. cruzi* (IC50 = 1.4 μM; SI = 2.0, **134**; IC50 = 2.4 μM; SI = 8.1, **135**), showing good activities but very low selectivity [102].

From *Buxus sempervirens* L. leaf extract, several triterpenic-alkaloid derivatives were isolated, being the majority tested for their activity against *T. b. rhodesiense*. Cytotoxicity assays were performed on mammalian L6 cells. Compounds **136**–**142** displayed high activities with IC50 values < 3 μM. The highest activities and selectivities for the parasite strain tested were obtained for compounds **136** (IC50 = 1.5 μM; SI = 25), **138** (IC50 = 2.3 μM; SI = 42), **141** (IC50 = 2.4 μM; SI = 30), and **142** (IC50 = 1.3 μM; SI = 33). In spite of its promising activity against the parasite (IC50 = 1.1 μM), compound **146** showed a lower selectivity (SI = 12). The remaining compounds **143**–**150** displayed a significant activity with IC50 values ranging from 3.1 μM (compound **149**) to 9.0 μM (compound **144**), but a modest selectivity to the parasite (SI < 9) [103].



**Table 1.** Plant terpenoids with anti-*T. brucei* activity (2016–2021).







Some names are not indicated in the corresponding papers; Tryp: trypomastigotes bloodstream forms: n.d.: not defined/not

*Pharmaceuticals* **2022**, *15*, 340







Some names are not indicated in the corresponding papers; n.d.: not defined/not determined.

#### **4. Discussion**

Human African trypanosomiasis and American trypanosomiasis continue to be a major public health problem, affecting a significant proportion of the world's population, especially in tropical countries. Currently, the drugs used to treat these diseases are scarce and far from being ideal. Therefore, the discovery and development of new drugs and treatments should be a continuous process, and all possible approaches should be explored, mainly focusing on multidisciplinary collaborations. It is also important to stress that the new drugs should be affordable and easy to administrate, improving the adherence to the therapeutic protocol, and decreasing the need for patient hospitalization.

Several approaches have been considered for the development of new drugs against these diseases. Due to the high costs and slow pace of new drug discovery, one of the main strategies is the repositioning or repurposing of drugs that were developed and used to treat other diseases. Although several advantages can be addressed, including the lower risk of failing, reduced time frame for drug development, less investment and rapid return [104], drug repositioning have also major drawbacks. These include, for example, the existence of undesirable side effects, problems concerning a different target population, poor stability in conditions of high temperature and humidity, lack of oral bioavailability, and various regulatory issues and intellectual property barriers. Therefore, the development of a new drug that ideally would be suitable for combination therapy, increasing the clinical efficacy, and decreasing side effects and the development of resistance, is a goal of utmost importance [10]. Besides combinatorial chemistry, one of the main strategies for drug discovery is through the phytochemical study of plants or other sources of natural origin. The importance of natural products for the development of drug leads or actual drugs along the last three decades has been reported in various reviews [12].

The evaluation of compounds with anti-parasitic activity is usually performed by two main approaches: the target-based and the phenotypic approaches [10,104]. The targetbased methodologies (Sections 2.1.1 and 2.2.1) focus on the specific biochemical pathway of the parasites, and consist of the identification of possible molecular targets (e.g., enzymes) that are significantly involved in the disease and the screening of molecules that possibly interfere with these targets. However, regarding trypanosomiasis, restricted success has been achieved, possibly due to a lack of translation between the activity in the molecular target, and the result on the proliferation of the parasite. In fact, the number of robust and validated molecular targets against these diseases is very limited, and in addition, for several drugs currently used in clinic, the mode of action is not yet completely understood or it comprises several targets [10,104]. By far, the most widely used methodologies to identify anti-trypanosomal drugs are based on the phenotypic methods. They consist of the screening of the compounds directly against the different forms of the parasite, and most of the time, for the bioactive compounds, there is no knowledge regarding their underlying mode of action or their molecular target. Through this screening, the effects on the parasite and the host cell viability (toxicity) can be assessed simultaneously [10].

In this review, 150 terpenic compounds obtained by isolation or derivatization from different plant species, were grouped into four classes of terpenes, namely, monoterpenes and iridoids, sesquiterpenes, diterpenes and triterpenes. The scope of this review was limited to compounds that exhibited in vitro or in vivo activity against the diverse forms of *T. brucei* and/or *T. cruzi*, displaying IC50 values in low micromolar range, most of them below 10 μM.

Regarding the anti-*T. brucei* activity, it can be observed that most of the selected compounds were active on the trypomastigote bloodstream form of the parasite (in vitro assays), and several possible hits can be identified. A high number of compounds exhibited very low IC50 values (0.05 < IC50 < 3.0 μM), when tested against *T. b. rhodesiense* or *T. b. brucei*, also presenting low cytotoxicity on the mammalian cells (SI values higher than 10). Some of the most promising hits are depicted in Figure 16. It is interesting to note that the majority of the bioactive compounds are sesquiterpenes and more specifically, sesquiterpenic lactones. Indeed, the α,β-unsaturated lactone function is very common in biologically active

molecules, being responsible not only for their activity but also for the cytotoxicity of the compounds. The presence of this chemical function promotes the Michael-type addition to a suitable nucleophile (for example, the thiol group in proteins), and may irreversibly alkylate critical enzymes and transcription factors that control gene regulation, protein synthesis, cell metabolism, and ultimately, the cell division [105,106]. After the preliminary in vitro studies, two compounds (**3** and **7**) were further evaluated using in vivo studies on *T. brucei* mouse models achieving very good results.

Concerning *T. cruzi* assays, it is curious to notice that there are much fewer research papers reporting the bioactivity of compounds against this parasite, probably because the in vitro assays using intracellular amastigotes were not so straightforward. Nevertheless, it was possible to select some promising hits that were very active against *T. cruzi* amastigotes, according to the criteria established by DNDi (Figure 17). Some of the most active compounds (**1**, **51**, and **74**) were further evaluated in vivo using a *T. cruzi* mouse model. The best result was obtained for compound **51**, and a significant reduction in parasitemia levels was observed in treated mice (1 mg/Kg/day, for 5 consecutive days), similarly to that obtained with the control group treated with the reference drug benznidazole.

Although most of the reported compounds were remarkably active against some infectious forms of the parasites, some of them also displayed some cytotoxicity on the mammalian cells tested. Furthermore, there is a notorious lack of additional studies on structure-activity relationships (SAR) and on the possible mechanisms of action. In addition, some reasons could be addressed to justify the absence of results for in vivo assays, including that the majority of compounds are isolated in very low amounts, a fact that precludes this type of assay where a large amount of compound is always needed.

**Figure 16.** Promising hit compounds for further research into treating Human African Trypanosomiasis.

**Figure 17.** Promising hit compounds for further research into treating Human American Trypanosomiasis. All the selected compounds were tested against *T. cruzi* intracellular amastigote forms.

#### **5. Conclusions**

The data presented in this review gathered the recent scientific research and experimental evidence on the most promising terpenoids derived from plants, and active against *T. brucei* and *T. cruzi* parasites. These data represent the immense efforts of various research groups all over the world, and ultimately the collected information is highly pertinent and can be used, for example, to support the selection of other plants to study, using the chemotaxonomic approach. However, all of these studies are strictly academic and no further translation to drug development has been achieved. There is still a limited collaboration of academic institutions with the pharmaceutical industry, and importantly, obtaining opportunities for research funding is, nowadays, even more challenging. All these aspects have limited or made it difficult for academic researchers to advance promising hits for further development.

In the future, natural products research must be a multidisciplinary process combining phytochemistry with innovative technological resources, in a way that will be significantly different from the past. These technologies must include high-throughput screening and in silico methodologies, as well as new extraction and dereplication procedures, new analytical tools, metabolic engineering, omics-based analysis, informatics, and big data analysis, in order to overcome the constraints of the classic natural products research.

**Author Contributions:** Conceptualization, N.D.; writing—original draft preparation, R.D. and C.R.; writing—review and editing, N.D., C.R., E.M. and A.M.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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