The genus Calea L . : A review of isolated compounds and biological activities

1 Laboratory of Medicinal Plants (LaPlaM/ PPGCA), Universidade do Extremo Sul Catarinense (UNESC), Avenida Universitária 1105, Bairro Universitário, 88806-000 Criciúma, SC, Brazil. 2 ISCR-UMR CNRS 6226, Faculté des Sciences Pharmaceutiques et Biologiques, Institut des Sciences Chimiques de Rennes, Université Rennes 1, 2 Av. du Pr. Léon Bernard, 35043 Rennes CEDEX, France. 3 Department of Pharmaceutical and Medicinal Chemistry, Royal College of Surgeons in Ireland, Dublin, Ireland.


INTRODUCTION
The Asteraceae family includes about 1,600 genera and 23,000 species around the world, which occur mostly in subtropical and tropical regions (Fernandes and Ritter, 2009).Calea L. is a large genus of this family (tribe Heliantheae Cass., subtribe Melampodiinae Less.) (Nascimento et al., 2002) containing approximately 125 species, distributed in tropical and subtropical regions of the Americas (Kadereit and Jeffrey, 2007;Roque and Carvalho, 2011).The genus ranges from Mexico through Central America into South America (Woodson Jr et al., 1975).Saslis-Lagoudakis et al. (2014), claim that an important point for change in history is the ability of humans to learn from others and transmit this knowledge to those who live around them, thus ensuring the transmission and conservation of information.In the context of medicinal plant use, this is no different, as traditional knowledge is usually transmitted orally and associated with families, communities or ethnic groups (Hamilton, 2004;Abbet et al., 2014).So it is important to report literature on the traditional uses of medicinal plants.
Although, many species within the genus Calea are used in folk medicine, only a few studies have reported their efficacy.Due to the common use of Calea species in folk medicine, coupled with the fact that the genus has some species with proven biological activity in previously published articles, this review was meant to consolidate known data within.The scope of this article is to review compounds isolated from the genus Calea and their biological activities.

METHODOLOGY
A selection of relevant data was made through a search using the keyword "Calea" in "Scopus", "Google Scholar", "Web of Science", "PubMed", "ScienceDirect" and Scifinder databases.About 167 articles were found with the word Calea for inclusion.In this review, search terms "Calea and biological activity" and "Calea and phytochemistry" were further used.In total, 96 publications describing the biological activity of extracts, fractionated extracts or isolated compounds from species of the genus Calea were used, excluding articles solely on botany and agronomy.The isolated compounds were categorized by species in Table 1 and biological activities were discussed by pathology.Reported biological activities include those of isolated compounds in addition to those of crude and fractionated extracts.Plant taxonomy was validated using the databases of Brazilian Flora and information about popular use and botany was obtained from published books and academic documents.

USE IN FOLK MEDICINE
Species of the genus Calea have been widely used in traditional medicine throughout their geographical range.
Calea glomerata Klatt has been described in Colombian folk medicine as an antihypertensive (Guerrero et al., 2002).In Mexican folk medicine, Calea integrifolia (DC.)Hemsl. is used as a hypoglycemic (Andrade-Cetto, 2015).In a Peruvian Amazonian ethnic group (Yanesha), Calea montana Klatt is used for skin infections and this was validated through testing for leishmanicidal activity; users apply onto infected wounds the mashed fresh leaves as a poultice (Valadeau et al., 2009).Calea serrata Less.popularly known as "erva-decobra", "chá-amargo" and "quebra-tudo", is an endemic species found in Southern Brazil (Ribeiro et al., 2008).It is used in traditional medicine to treat ulcers and liver problems (Camilotti et al., 2014) while also featuring in African-Brazilian religious rituals.Avancini and Wiest (2008) reported that C. serrata is popularly used in southern Brazil for skin diseases in humans and animals.Calea uniflora Less., commonly known as arnica-da-praia in the Southern region of Santa Catarina State, Brazil, has popular therapeutic indications including as an antiinflammatory, analgesic, in the treatment of hematomas, as an antiseptic (for mosquito bites), for rheumatism, treatment of urinary infections and flu (Ramos et al., 2016).
Calea hypoleuca Rob.& Greenm, is used by the Zapotec indigenous group of the Ocotlan district, within Mexico.In this context, the main use is postpartum, using the cooked leaves (decoction) in the form of baths.
One species of this genus with several indications is Calea zacatechichi Schltdl., known as "white bitter herb" by Mixe, and "bitter gum" by Popoluca peoples, inhabiting two areas in the Mexican states of Oaxaca and Veracruz, respectively, while both belonging to the Macro-Mayan linguistic stock.Within these populations, this plant is used for gastrointestinal purposes (stomach-ache, diarrhoea), dermatological/respiratory ailments (cough, asthma), and gynaecological indications by the Popoluca people; and gastrointestinal purposes (stomach-ache) and fevers by the Mixe people (Leonti et al., 2003).The rural people of Oaxaca, Puebla and Veracruz also use this plant to treat diabetes and biliary diseases (Zamora-Martinez, 1992).It is also used in Mexico as an "antidiabetic" (Ramos et al., 1992) and as an antiplasmodial in El Salvador (Köhler et al., 2002).Pereira and Pereira (2002) describes C. zacatechichi among Mayan medicinal flora for use in gastroenteritis, dermatitis, diarrhea and fever.

PHYTOCHEMISTRY
About 256 different compounds have been isolated and identified from Calea species with the aerial parts most thoroughly investigated.Of 257 compounds isolated from species of Calea, 116 are sesquiterpene lactones, 18 derived from p-hydroxyacetophenone, 14 phenolic compounds, 10 chromenes, 8 flavonoids, 7 benzofurans, and 5 chromanones.In this review, the inclusion of compounds has been based on chemical diversity.Such diversity is of primary interest for medicinal chemistry (Table 1).
Sesquiterpenoids are compounds containing 15 carbons, formed biosynthetically from three five-carbon isoprene units or may be synthesized industrially from monoterpenoid building blocks (Bauer et al., 1997).These secondary metabolites have significant roles in plants as deterrents against herbivores (Picman, 1986) and as anti-fungal and anti-bacterial allelopathic agents.Although the biological activities of these metabolites have not been completely elucidated as yet, anthelmintic, antibiotic, cytotoxic (Anke et al., 1989) and antiparasitic activities are known.
This review confirms that several Calea species traditionally utilized in folk medicine for the treatment of inflammation and various infections may serve as a biobank for the isolation of potential phytopharmaceuticals and act as leads in drug discovery.Collation of research reports on Calea and other plants is vital for a better understanding of how to develop rational therapeutics from ethnobotanical medicines.
A great number of natural compounds belonging to several families are present in the genus Calea.The most common are sesquiterpene lactones and chromenes.Nevertheless, chromanones and flavonoids are also represented in this genus.Unfortunately, few papers about the activity of chromanones are available.
Many natural compounds contain a chromene moiety, including tocopherols and flavonoids.Structural skeletons of chromane, 2H-chromene and 4H-chromene have in common the same benzofuran nucleus; of these, 2Hchromene compounds are predominantly found in the genus Calea.Such 2H-chromenes are known as antifungal compounds and some derivatives showed potential antidiabetic and antihypertensive activities as Na + -glucose co-transporter inhibitors and potassiumchannel activators, respectively (Thomas and Subin, 2013).They have also been revealed as efficient antiinflammatory compounds, acting as selective inhibitors of cyclooxygenase-2 (Wang et al., 2010) and of tumor necrosis factor α (TNF-α) production (Thomas and Subin, 2013).Mechanistically, no single mechanism of action is defined for these compounds as antimicrobial agents.Some chroman and chromene derivatives have exhibited DNA gyrase inhibition, and within the chromanones, a hydrogen bond donor/acceptor functionality at the 4position together with a lipophilic 2-alkyl moiety is believed to be important for antibacterial activity.Much interest has focused on the use of chromenes as antiparasitic agents, with chromen/chroman-4-ones suggested to exert their effects through pteridine reductase 1 (PTR1) inhibition within both Trypanosoma brucei and Leishmania species (Di Pisa et al., 2017).
Sesquiterpene lactones are a large group of over 5000 compounds and they are particularly abundant in plants of to the Asteraceae family, including the genus Calea.They are characterized by 15-carbon terpenoids consisting of three isoprene units and a lactone ring, with some having an α-methylene-γ-lactone motif with an exocyclic double bond conjugated with a carbonyl function (Zhang et al., 2005).Six skeletons of sesquiterpenoids termed germacranolides, eudesmanolides, guaianolides, pseudoguaianolides, xanthanolides and carabranolides can be found in plants.In the genus Calea, germacranolides, eudesmanolides and guaianolides in particular are found (Ferreira et al., 1980;Ober et al., 1984d;Ortega et al., 1989).The α-methylene-γ-lactone group is responsible for most of their biological effects via a Michael-type addition.Parthenolide is a representative sesquiterpene lactone of the anti-tumor agents largely described for this family.Nevertheless, several are in clinical or pre-clinical stages and one, arglabin, is actually used as anticancer drug in Kazakhstan.They exert their effects on the nuclear factor NF-κB but also on the redox equilibrium of malignant cells (Gach et al., 2015).Analogously with chromenes, they also exhibit antiinflammatory effects, notably those of the guaianolide family (Chadwick et al., 2013), with NF-κB involved through regulation of about 150 inflammatory genes.Besides already discussed antimicrobial effects, we can cite the antimalarial activity of artemisinin, whose activity is ascribed to the presence of an endoperoxide Calea hispida (DC.) Baker Brazil Air-dried plant material  Castro et al. (1989) 1β-Hydroxy-Bisabola-2,10-dien-4-one C124 1α-Hydroxy-Bisabola-2,10-dien-4-one C125 4-Hydroxy-3methoxybisabola-1,10-diene C126 1α-Hydroxy-8β-tigloyloxyguaia-3,9,11( 13  rather than an α-methylene-γ-lactone.Finally, they could have useful antihypertensive properties through increasing NO levels, resulting in vascular relaxation of the smooth muscle (Seca et al., 2015).Diverse biological activities have been attributed to the genus Calea.Notably however, several studies solely reported isolation of phytochemicals without parallel biological testing, revealing a gap in current knowledge.Known biological activities are discussed in the following paragraphs.
In contrast, another study showed that a mixture of two chromanones uniflorol-A (C215) and uniflorol-B (C216) (Table 1) from underground organs of C. uniflora had leishmanicidal activity.Dried and powdered underground parts of C. uniflora (200 g) were exhaustively extracted with dichloromethane at room temperature and 4.2 g of crude extract was obtained.Various chromatographies were performed, including TLC and HPLC, and NMR was used to deduce the structures.The inseparable mixture of uniflorol-A and uniflorol-B significantly inhibited Leishmania major promastigote growth in vitro by 54.8, 81.5 and 88.9% at concentrations of 25, 50 and 100 μg/ml, respectively (Nascimento et al., 2007a) (Figure 3).
Two flavonoids obtained from ethanolic extract of aerial parts of Calea clausseniana were evaluated for their in vitro trypanocidal activity against the trypomastigote forms of Trypanosoma cruzi, with neither compound showing trypanocidal activity (Nascimento and Oliveira, 2007b).

Acaricidal activity
One study was performed to evaluate the acaricidal properties of the essential oil obtained from C. serrata.This study demonstrated that both the essential oil and the isolated precocene II were toxic to the larvae of the tick Rhipicephalus microplus.The essential oil was obtained from fresh leaves of the plant by hydrodistillation using a Clevenger-type apparatus.The oil was analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS).For extraction and isolation of precocene II, air-dried and powdered plant material was extracted by repeated maceration with n-hexane.Precocene II (C182) (Table 1 and Figure 7) was isolated from this extract by column chromatography using silica gel 60 and n-hexane:  dichloromethane, in increasing polarities, obtaining some fractions rich in precocene II (Ribeiro et al., 2011).Precocene II (C182) had been previously reported in the literature (Steinbeck et al., 1997).

Anti-inflammatory effects
A study of C. prunifolia revealed two chemical compounds, one of which showed topical antiinflammatory activity.Both compounds were derived from p-hydroxyacetophenone.The compound that presented satisfactory anti-inflammatory activity was 1-(2-hydroxy-5-(1-methoxyethyl)phenyl)-3-methybut-2-en-1-one (C166) (Table 1 and Figure 9).Dried leaves of C. prunifolia were extracted by percolation with ethanol at room temperature and the extract was concentrated under vacuum at 35°C.The crude extract was chromatographed on a silica gel column with CH 2 Cl 2 obtaining five fractions.In the experiment, edema was induced in the ears of female mice by the topical application of 2.5 µg TPA to the ear surfaces.Subsequently, the right ear received the extracts at a concentration of 500 µg per ear, using indomethacin as reference substance in all cases.Although one of the compounds had anti-inflammatory activity, it was suggested that synthetic modification would be necessary to increase the anti-inflammatory activity of these compounds (Gómez and Gil, 2011).
The aqueous extract of C. zacatechichi showed potential antiiflammatory activity preventing the formation of edema after administration of carrageenan.Powdered leaves (1 g) were extracted with distilled water, heating for 10 min at 95°C.Following centrifugation at 100 g, the supernatant was adjusted to pH 7.4.For the biological assay, male Wistar rats (200 to 250 g) and Swiss albino mice (20 to 25 g) were used.The extracts were administered and compared with both indomethacin and a negative control.In this study, only crude extracts were evaluated for anti-inflammatory activity and no individual compounds were isolated.The study concluded that C. zacatechichi contains compounds with potential antiinflammatory activity and that this activity can be associated with the biosynthesis of prostaglandins and lipoxygenase products (Venegas-Flores et al., 2002).Other studies of C. zacatechichi have revealed the presence of sesquiterpene lactones (Herz and Kumar, 1980) and chromenes (Quijano et al., 1977) and these compounds are known to be associated with anti-

Cytotoxic effects
Cytotoxic activity was observed in a study of C. urticifolia.The major sesquiterpene lactones isolated from the acetone extract exhibited anti-melanotic activity in mouse B16 melanoma cells.The results suggested that the inhibitory effects of sesquiterpene lactones on melanin biosynthesis may be due to the suppression of tyrosinase expression.In particular, 2,3-epoxy-juanislamin (C233) (Table 1 and Figure 10) was notable for its potentinhibitory activity on melanogenesis through modulating the transcriptional machinery of tyrosinase mRNA (Ohguchi et al., 2009).
In another study, the anticancer activity of the dichloromethane crude extract obtained from C. pinnatifida was evaluated.Dried aerial parts ofC.pinnatifida were ground and an aliquot extracted by soxhlet with dichloromethane (DCE).The DCE showed high potency and selectivity for melanona and kidney cell lines.An in vivo study using Erlich ascites tumor and Erlich solid tumor further validated the cytotoxic effects of DCE.The substance(s) involved in the antitumor effect of C. pinnatifida DCE are unknown, although some compounds known from the extract, such as the sesquiterpene lactones, could explain these results (Marchetti et al., 2012).

Antimicrobial activity
Extracts of leaves and flowers of Calea platylepis have shown antimicrobial activity against bacteria and fungi.These activities were performed by the well diffusion method, evaluating compounds in the range of 50 to 1000 µg/ml.Bacitracin and ketoconazole were used as positive controls for bacterial and fungal strains, respectively.Certain compounds, namely (+)-4α,7β-Aromadendranediol (C155), euparin (C156), caleprunin B (C158) and euparone (C159) 1 and Figure 11), demonstrated a broad spectrum of action, inhibiting the growth of various strains of microorganisms (bacteria and fungi).
The essential oil of Calea fruticosa showed antimicrobial activity against Gram-negative bacteria (Proteus vulgaris), although activity proved to be selective as there was no activity against yeast strains.This study involved hydrodistillation of the leaves, GC/MS analysis of the essential oil and subsequent evaluation of the essential oil against a panel of microorganisms.C. fruticosa essential oil was characterized by 43 constituents, representing 62.9% of the total oil composition.The essential oil was dominated by the presence of oxygenated sesquiterpenes and sesquiterpene hydrocarbons, with caryophyllene (C69), α-cadinol (C76) and sellin-11-en-4-α-ol (C77) (Table 1) as the most abundant components (Carvalho et al., 2014).The antibacterial activity of the oil was ascribed to the synergistic effects of α-pinene and linalool (Sivasothy et al., 2011).
Antifungal activity of Calea clematidea was shown to be moderate against Trichophyton tonsurans, Trichophyton rubrum, Trichophyton menthagrophytes var.interdigitale, Epidermophyton floccosum, Microsporum gypseum, Microsporum canis and Microsporum nanum.The essential oils described were shown to inhibit fungal growth.Fresh leaves and flowers were subjected to steam distillation for 4 h using a Clevenger-type apparatus, followed by exhaustive extraction of the steam distillate with diethyl ether.The antifungal activities against pathogenic fungi, from patient isolates, were determined using the dilution technique.The minimum inhibitory concentration (MIC) was measured for the leaf essential oil and clemateol, and both showed moderate fungistatic and fungicidal action against dermatophytes (Flach et al., 2002).

Antidiarrheal and antinociceptive effects
Calea zacatechichi extracts exhibited antidiarrheal and antinociceptive effects in mouse models of irritable bowel syndrome.A methanolic macerate was further extracted with dichloromethane (DCM) to yield a solid extract.According to this paper, such extracts may be used as a source material to treat pain and diarrhea associated with irritable bowel syndrome (Salaga et al., 2015).
C. uniflora Less.was investigated for antinociceptive effects and cytotoxicity.Regarding antinociceptive activity, models produced significant results at doses corresponding to 100 and 300 mg/kg of the crude extract compared to the control.The rota rod model was favoured since the extract did not cause motor incoordination and sedation in the experiment.In the in vitro cytotoxic tests, both crude extracts and ethyl acetate and butanolic fractions produced IC 50 values greater than 58 µg/ml with the HaCaT lineage and 48 µg/ml with the B16-F1 lineage; thus, these values did not indicate cytotoxic effects.Phytochemical analyses verified the presence of flavonoids and sesquiterpenes within C. uniflora extracts (Torres et al., 2016).

Anti-obesity effects
A study of C. urticifolia indicated the possibility that germacranolides obtained from acetone extract of the leaves of this species have anti-obesity effects.The germacranolides inhibited preadipocyte differentiation in 3T3-L1 cells, suggesting this activity.Inhibition of the differentiation of 3T3-L1 cells to adipocytes is beneficial for the prevention of obesity complicated by atherosclerosis (Matsuura et al., 2005).

Conclusion
The genus Calea contains several potential pharmacophores for drug discovery programmes, notably chromenes as anti-inflammatory and anticancer agents but also for their diverse sesquiterpene lactone consituents.
However, prior to the application or recommendation of these species to prevent or treat disease states, additional pharmacological and toxicological studies are essential; notably, no literature reports exist on the safety and efficacy of the 37 species described in this paper.

Table 1 .
Species of the genus Calea used in traditional medicine and an overview of their isolated compounds.