The Genus Tripleurospermum Sch. Bip. (Asteraceae): A Comprehensive Review of Its Ethnobotanical Utilizations, Pharmacology, Phytochemistry, and Toxicity

This review provides a comprehensive overview of the botany, traditional uses, phytochemistry, pharmacology, and toxicity of the genus Tripleurospermum. Tripleurospermum, a prominent genus within the family Asteraceae, is recognized for its therapeutic potential in treating various ailments, including skin, digestive, and respiratory diseases; cancer; muscular pain; and stress and as a sedative. Through extensive phytochemical studies regarding the Tripleurospermum species, numerous chemical compounds have been identified and classified into distinct classes, predominantly encompassing terpenes, hydrocarbons, steroids, hydrocarbons, oxygenated compounds, flavonoids, tannins, alcohols, acids, melatonin, and fragrant compounds. The findings from this review highlight the presence of bioactive compounds within the Tripleurospermum species that possess significant medicinal properties.


Introduction
In recent years, there has been a growing trend in the utilization of natural medicines to treat illnesses due to their reduced adverse effects, cost-effectiveness, and wide availability [1]. Although several articles have already explored the medicinal potential of active compounds, namely active molecules, derived from the genus Tripleurospermum in various therapeutic areas [2][3][4][5][6], it is imperative to conduct further investigations into the therapeutic and toxicological properties of this plant genus. The aim of this review was to comprehensively analyze the botanical characteristics, traditional uses, phytochemistry, pharmacology, and toxicity profiles of different Tripleurospermum species. Moreover, we intended to facilitate and guide future research endeavors by documenting the ethnopharmacological applications of Tripleurospermum. Noteworthy scientific contributions have been made on diverse facets of the genus Tripleurospermum, such as its pharmacology [2], toxicity [2], biochemical properties, the pharmaceutical potential of proteins and peptides derived from this genus [7][8][9][10][11][12], as well as its chemical constituents [2,13,14]. Lastly, this review provides a concise summary and outlines future research directions in the field of the Tripleurospermum genus. In the subsequent sections, an overview of the taxonomy, stomatal characteristics, achene, as well as the anatomical features of leaves and stems observed in various species of Tripleurospermum is presented. The genus Tripleurospermum is a member of the tribe Anthemideae, in the family Asteraceae, comprising nearly 40 species. It is closely related to the genus Matricaria L. which consists of approximately seven well-defined taxa [15]. In the geographical region of Turkey, Tripleurospermum is reported to encompass 40 species and 32 taxa [3,[16][17][18][19][20][21], of which 12 exhibit distinct leaf and achene/cypsela anatomical characteristics [22]. Recent taxonomic research by Ref. [23] identified Tripleurospermum eskilensis as a new species, thus expanding the number of Tripleurospermum taxa in Turkey Life 2023, 13, 1323 2 of 17 to 33. Notably, Turkey has been recognized as the primary source of the cultivated variety of Tripleurospermum [24,25]. In Russia, the genus Tripleurospermum comprises 17 species [26], while Iran is home to 6 species of Tripleurospermum [13,27].
The taxonomic classification of the genus Tripleurospermum remains a subject of contention, particularly due to its species' distinct characteristics. Initially, the genus Tripleurospermum was classified under the genus Matricaria L. but was later reclassified as a separate genus based on two distinguishing factors: the unique shape of its achene and the prevalence of tetrasporic embryo sacs [4]. However, it should be noted that Refs. [5,6] have erroneously reported Matricaria L. as part of the Tripleurospermum genus. Subsequent investigations confirmed the association of Tripleurospermum with Anthemis L. rather than with Matricaria L. [28]. In addition, limited chromosomal information is available for both Tripleurospermum and Anthemis L. The ploidy levels observed in Tripleurospermum range from 2n = 2x = 18 to 2n = 3x = 27 to 2n = 4x = 36. There are four ploidy levels (2x, 3x, 4x, and 5x) in all Tripleurospermum species [29]. In contrast, Matricaria L. was found to have a single ploidy level (2x) [8]. Polyploids have been proposed as a potential mechanism contributing to the diversification and divergence of the genus Tripleurospermum [3,25]. Furthermore, the outermost layer of this genus' leaves has been identified as a significant factor in taxonomic differentiation and to play a role in the development of stomatal complexes, including those found in achenes [30,31]. It is worth noting that anatomical features such as stomatal length, vascular bundle size, and palisade sclerenchyma thickness are important for distinguishing between Tripleurospermum species and for establishing a correlation between ploidy level and anatomical structures across different Tripleurospermum species [22,32].
The stomata of Tripleurospermum species are distributed on both the upper and lower surfaces of their leaves, with equal stomatal density reported [22]. Stomatal characteristics such as stomatal frequency, guard cell length, and stomata plastids' diversity have been used as morphological indicators for assessing ploidy levels in different plant species [33][34][35][36]. Studies have demonstrated that polyploid plants exhibit longer stomata than diploid plants [37,38]. Furthermore, a positive correlation was observed between ploidy level, stomatal size, and altitude in Tripleurospermum species [3]. Taxonomically, stomatal and vascular bundle sizes are associated with the degree of polyploidy, which holds significance in distinguishing different Tripleurospermum species, particularly within the Turkish endemic species [30].
The leaf anatomy of Tripleurospermum species bears similarities to that of Matricaria L. [22]. The leaf anatomy of Turkish Tripleurospermum taxa typically consists of lower and upper epidermis, parenchymatous mesophyll, and a vascular bundle [46]. The epidermal cells are isodiametric, resulting in nearly straight walls in both the lower and upper epidermis [30]. The leaf surfaces are covered with nonglandular uniseriate and multicellular trichomes, although T. corymbosum has been reported to be glabrous [22,30]. The mesophyll blades primarily comprise dorsiventral and palisade parenchyma cells, similar to the mesophyll structure observed in the Asteraceae family [22,47,48]. In addition, all taxa of Tripleurospermum exhibit either one major vascular bundle or two medium-sized vascular bundles with a secondary vascular bundle [22]. It is worth noting that the large vascular bundle, which constitutes 50% of the mesophyll's area, holds taxonomic significance in the delimitation of the taxa [22,30,47].

Habitat, Distribution, and Ecology
The Asteraceae family represents the largest and most prominent family of plants [52]. The related extensive research has focused on the remarkable diversity of the species and genera, global distribution, and effective plant species [42].
Tripleurospermum Sch. Bip., a genus within the family, has been cultivated in various temperate regions of Europe and Asia alongside other species found in north Africa and North America [12,15,20,22,25] (Figure 1). Species belonging to this genus are characterized as herbaceous, either annual or perennial in nature [14].

Habitat, Distribution, and Ecology
The Asteraceae family represents the largest and most prominent family o [52]. The related extensive research has focused on the remarkable diversity of th and genera, global distribution, and effective plant species [42] .
Tripleurospermum Sch. Bip., a genus within the family, has been cultivated in temperate regions of Europe and Asia alongside other species found in north Af North America [12,15,20,22,25] (Figure 1). Species belonging to this genus are ch ized as herbaceous, either annual or perennial in nature [14] .

Phytochemical Compounds of the Genus Tripleurospermum
The essential oil composition of Tripleurospermum decipiens flowers has bee ously investigated, and the main reported compounds are matricaria esters [44 2). Matricaria esters have also been identified as compounds of T. inodorum [54 disiforme [55] (Figure 2).

Phytochemical Compounds of the Genus Tripleurospermum
The essential oil composition of Tripleurospermum decipiens flowers has been previously investigated, and the main reported compounds are matricaria esters [44] (Figure 2). Matricaria esters have also been identified as compounds of T. inodorum [54] and T. disiforme [55] ( Figure 2).
In addition, the hot water extracts of T. parthenium and T. disciforme revealed the presence of melatonin in flowers [67]. Another study reported the detection of an acetylene derivative of dioxaspiran in the chloroform extract of T. disciforme [68].
The amounts of β-farnesene, β-sesquiphellandrene, p-methoxy-β-cyclopropylstyrene, heptadecane, and benzene acetaldehyde were reported to be highest during the flowering stage and decreased after flowering and seed maturation.
It is worth noting that a study analyzing the chemical composition of T. disciforme [55] found a relatively small amount of β-esquiphellandrene (0.22%), which deviated from previous data. Another recent study [74] employed techniques such as NMR, electron impact mass spectroscopy, column chromatography, and thin layer-chromatography (see Figure 2) to identify three triterpenoids-taraxasterol, lupeol, and betulinic acid-in the dichloromethane extract of T. disciforme.
Research on Tanacetum parthenium, another member of the Asteraceae family, has shown significant antibacterial activity, potentially attributed to sesquiterpene lactones, such as parthenolide and flavonoids. T. parthenium has been used as a neurotonic and antipyretic agent [89], as a relaxant and for muscle ache alleviation, stress management [90] and as a hair dye [91,92]. Additionally, T. disciforme extract has demonstrated antimicrobial effects against S. aureus and S. epidermidis [93,94]. The antioxidant activity of different parts of T. disciforme was assessed by evaluating their ability to inhibit linoleic acid peroxidation. Notably, the chloroform extract exhibited a substantial antioxidant effect, slightly lower than that of the standard α-tocopherol [88,95]. Moreover, a study [96]. showed that extracts of T. parviflorum and T. monticola were effective in treating cough and stomachache and as antipyretic agents. Tripleurospermum parviflorum also demonstrated efficacy in the treatment of pharyngeal illnesses and vaginitis [97]. Traditional medicinal practices involve the use of the entire plant of T. limosum for the treatment of gastritis [2,96,97]. Additionally, T. sevanense has been utilized for hair-care purposes [98].
Tripleurospermum callosum flowers have been recommended to treat urinary tract disorders, kidney stones, shortness of breath, common cold, asthma, and bronchitis and as a panacea [70]. Furthermore, T. auriculatum was found to be effective in the treatment of neuromuscular blockade and mild hypoglycemia [72]. T. inodorum is known for its efficacy in alleviating gastrointestinal pain and its anti-inflammatory properties [99]. Moreover, T. inodorum was historically utilized in prehistoric painting techniques, and it is important to mention that apigenin, a compound found in this species, contributes to its yellow coloration [100].

Antioxidant Activity
The antioxidant activity of the essential oils from the newly discovered species T. insularum, belonging to the genus Tripleurospermum [19], was investigated using 2diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing activity (FRAP) assays [59]. The essential oil's ability to scavenge stable free radicals and reduce metal intermediates may be attributed to the substantial presence of sesquiterpenes, which have known antioxidant properties [59].
The antioxidant activity of T. insularum is comparable to that of other Tripleurospermum species, such as T. disciforme and T. oreades [91], which are recognized for their abundance of caffeoyl derivatives, which are regarded as significant compounds for the antioxidant potential of the Asteraceae family [101]. The aqueous extract of T. oreades also showed antioxidant activity [91].
It is important to note that the low temperatures at higher altitudes represent a crucial environmental factor contributing to the enhanced biosynthesis of several antioxidants, despite the genetic differences between different species [103,104]. Among the essential oil compounds of T. inodorum, matricaria ester exhibited modest antioxidant activity [105]. Recent findings [2] demonstrated the antioxidant activity of T. limosum, with notable effectiveness observed in aqueous, methanol, and ethanol extracts.

Antimicrobial Activity
Previous studies have investigated the antibacterial properties of Asteraceae plant species and have found a significant inhibitory effect [89,107]. The inhibitory effects of different essential oils have also been examined (Table 2). Most authors have reported a stronger inhibitory effect of essential oils against Gram-positive bacteria than against Gramnegative bacteria [73,92,108,109]. Interestingly, it was found that the levels of flavonoids in Gram-negative bacteria are higher than those in Gram-positive bacteria.
A noteworthy study explored and elucidated the antiyeast activity of T. disciforme, which is attributed to the presence of farnesol [92], a crucial compound in the essential oil of the Tripleurospermum genus. Additionally, flavonoids were identified as antimicrobial agents, exhibiting direct antibacterial activity, synergy with antibiotics, and disease elimination in various studies [110]. The methanol extract of the genus T. disciforme showed antimicrobial activity against S. aureues and S. epidermidis [88]. In addition, the essential oil of T. disciforme showed antimicrobial activity against Staphylococcus subtilis and Bacillus cereus [63]. The phytochemical composition analysis revealed that compounds such as modephene, cis-β-farnesene, β-sesquiphellandrene, anisole, and p-1-cyclohexen-1-yl are responsible for the antibacterial activity in the essential oil of T. disciforme. Additionally, these essential oil compounds are present in high concentrations during the flowering period.
Furthermore, the antibacterial activity of essential oils is not solely dependent on their main compounds; minor compounds can also exhibit significant efficacy. Sesquiterpenes, for instance, have demonstrated inhibitory effects. Additionally, a study [111] highlighted the antibacterial activity of flavonoids, specifically kaempferol and quercetin, against Propionibacterium acnes.
Apigenin was identified as a compound responsible for suppressing S. typhi, Proteus mirabilis, and P. aeruginosa [112]. Another study investigated the eradication of S. aureus, MRSA, and methicillin-sensitive S. aureus using apigenin and luteolin [113]. Researchers provided evidence supporting the therapeutic and disinfectant properties of T. disciform in improving acne and resolving skin issues, particularly in teenagers [88]. Moreover, a recent report showed the strong antibacterial activity of T. parthenium essential oil against Aspergillus brasiliensis [73]. Previous findings demonstrated the strong antibacterial effects of all extracts from T. Parviflorum, including ethanol, methanol, and ethyl acetate extracts against Staphylococcus aureus ATCC 29213. However, none of the tested extracts showed any effect on Candida albicans [114]. Furthermore, another investigation reported the high inhibitory activity of T. disciforme essential oil against Klebsiella pneumoniae, Shigella dysenteriae, Escherichia coli, and Candida albicans [73].

Anti-Inflammatory Activity
T. disciforme was shown to have significant anti-inflammatory and analgesic effects by inhibiting the release of prostaglandins and other mediators [115]. Notably, fatty acids have been recognized for their protective properties and diverse biological activities, including anti-inflammatory effects [87,99,108,116].
Previous studies reported the remarkable inhibitory effect of ethyl acetate extracts from T. tenuifolium and T. parviflorum on overall in vivo anti-inflammatory activity [117]. Palmitic acid is the major and important component of T. tenuifolium. The substantial presence of linoleic acid and palmitic acid accounts for the significant anti-inflammatory activity observed (Table 3). The anti-inflammatory and analgesic activities of T. disciforme were assessed using carrageenan-induced edema, formalin, and the tail-flick test. Extracts of T. disciforme demonstrated remarkable anti-inflammatory and analgesic properties in the tested models [118]. An extract of T. disciforme was shown to be nontoxic at analgesic doses. Furthermore, the administration of large amounts of T. disciforme further confirmed its potent anti-inflammatory effects [118].
Studies on the hydroalcoholic extracts of T. disciforme have revealed its remarkable ability to protect against ulcer formation in rats with pyloric ligation, indicating the involvement of additional mechanism(s) beyond its acid-reducing activity. Additionally, high doses of T. disciforme exhibited effectiveness upon administration via injection [87]. The intensity of ulceration is typically assessed by parameters such as ulcer score or ulcer incidence [117,119]. Flavonoids and essential oils containing important secondary metabolites in the flowers of T. disciform were identified as contributors to the anti-inflammatory activity of the genus T. disciform [120]. Furthermore, a study [121] identified the major flavonoid compounds of T. disciform, including apigenin, apigenin-7-glucoside, apigenin-7-glucuronide, luteolin, luteolin-7-glucoside, luteolin-7-glucuronide, quercetin, quercetin-7-glucoside, and chrysoeriol, which demonstrated effective anti-inflammatory activity [122]. Table 3. Anti-inflammatory activities of the genus Tripleurospermum.

Part of Plant Identified Compounds Extract Biological Activity
Aerial part Linoleic and palmitic acids n-Hexane Aqueous, ethyl acetate, and methanol Anti-inflammatory, by carrageenan-and serotonin-induced paw edema acetic-acid-induced increase in capillary permeability models [114] Flower Flavonoids such as apigenin, quercetin, patuletin, luteolin, and their glucosides Water Analgesic and anti-inflammatory, evaluated by formalin test, by inhibiting the cyclooxygenase(COX)-mediated conversion of arachidonic acid to prostanoids [118] Flower Flavonoids and essential oil compounds [122] Hydroalcoholic and chloroform Antiulcerogenic potential [68,103]; anti-inflammatory, by carrageenan induced edema, formalin, and tail-flick test [114] Flower Flavonoids such as flavones and flavonols, tannins, and essential oil Hydroalcoholic Protection against gastric ulcers in pylorus-ligated rats [87]
Moreover, the dichloromethane extract of T. disciforme demonstrated antitumor activity against human gastric carcinoma (AGS) and mouse skin fibro sarcoma (WEHI-164) cell lines [74]. However, T. disciforme did not exhibit cytotoxic effects on Pc12 cells [124]. Notably, the essential oil of the genus T. inodorum contains a compound called matricaria ester, known for its potent cytotoxicity against Artemia salina. [105].
Recent findings revealed that the methanol extract of T. limosum exhibited low cytotoxicity at the lowest concentration tested and no cytotoxicity at the other three concentrations [2].
Traditionally, these plants have been commonly employed for the treatment of gastrointestinal disorders, inflammation, throat ailments, vaginitis, dysentery, fever, skin diseases, urinary tract disorders, kidney stones, shortness of breath, common cold, asthma, bronchitis, and muscular pain, and as a sedative, a gastro tonic, an antihemorrhagic, a carminative, a relaxant, stress-relieving agents, hair colorants, and as panaceas. This review provided an overview of the medicinal uses of Tripleurospermum species across different countries, thus highlighting their remarkable potential for various biological activities. Notwithstanding, further future research is required to identify the individual bioactive compounds present in Tripleurospermum species and elucidate their mechanisms of action.
Author Contributions: Conceptualization, A.P.D. and P.S.; methodology, P.S.; data curation, P.S.; writing-original draft preparation, P.S.; writing-review and editing, P.S. and A.P.D.; visualization; supervision, A.P.D. All authors have read and agreed to the published version of the manuscript.
Funding: This work was partially supported by CICS-UBI, which was financed by the Portuguese Funding Agency for Science Research and Technology (FCT) and by FEDER under the scope of PORTUGAL 2020 and CENTRO 2020, within the projects UIDB/00709/2020 (core and programmatic fundings).