Tagetes spp. Essential Oils and Other Extracts: Chemical Characterization and Biological Activity

Tagetes (marigold) is native to America, and its cultivation currently extends to other countries in Africa, Asia, and Europe. Many species of this genus, such as T. minuta, T. erecta, T. patula, and T. tenuifolia, are cultivated as ornamental plants and studied for their medicinal properties on the basis of their use in folk medicine. Different parts of the Tagetes species are used as remedies to treat various health problems, including dental, stomach, intestinal, emotional, and nervous disorders, as well as muscular pain, across the world. Furthermore, these plants are studied in the field of agriculture for their fungicidal, bactericidal, and insecticidal activities. The phytochemical composition of the extracts of different Tagetes species parts are reported in this work. These compounds exhibit antioxidant, antiinflammatory, and enzyme inhibitory properties. Cultivation and the factors affecting the chemical composition of Tagetes species are also covered. In the current work, available literature on Tagetes species in traditional medicine, their application as a food preservative, and their antimicrobial activities are reviewed.


Introduction
The human species used plants for treating diseases throughout history. The therapeutic properties and efficacy of each plant were mainly based on popular observations. This approach significantly contributed to their prescription, even if their chemical constituents were not always completely known [1][2][3][4][5]. Commercial drugs substituted medicinal plants; however, worldwide, people still use natural products for primary healthcare [6][7][8][9][10].
Tagetes (marigold) is an important genus belonging to the Asteraceae family and consists at least of 56 species [30]. It is a plant which is native to America, but it is naturalized in other countries in Africa, Asia, and Europe [31,32]. Tagetes spp. can be cultivated as ornamental plants or can be found as wild species [33]. There are many species of this genus, such as T. minuta, T. erecta, T. patula, and T. tenuifolia, that are studied because of their application in the field of agriculture, where they exhibit fungicidal, bactericidal, and insecticidal activities, as well as anticancer properties [34][35][36], resulting in their exploitation as beverages and condiments in folk medicine [37,38].
Marigold extracts are characterized by the presence of diverse compounds with different properties, namely phenylpropanoids, carotenoids, flavonoids, thiophenes, and others [39]. It is known that the compounds produced by plants could vary based on many factors, including the part of the plant from which it is extracted, harvesting seasons, plant development stage, and geographical sources [39,40].
T. patula presented antioxidant and cytotoxic activity in previous studies; however, the extract did not display good results for antimicrobial action against fungi (Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Microsporum anisopliae, and Beauveria bassiana) [41]. Conversely, T. minuta essential oil (EO) showed inhibition against Aspergillus niger and Candida albicans in addition to Gram-positive bacteria [42]. T. erecta showed that its compounds have antiinflammatory potential [20] and have anticancer activity [43], as well as T. minuta, which also presented cytotoxic [42] and antiinflammatory activities.
Collectively, the studies showed the potential of Tagetes spp. as alternatives to a wide variety of drugs. The many alternative uses for Tagetes spp. resulted in the depletion of natural sources because its extracts and specific metabolites have such a high demand. Thus, the crop needs systematic cultivation in tropical, subtropical, and temperate agroclimatic zones worldwide [44,45]. Marotti et al. [39] evidenced that most Tagetes spp. could be cultivated in environmental conditions different from those typical of the genus.

Tagetes Genus Plant Cultivation
Tagetes spp. are used in different areas for various purposes such as cosmetic preparation, medicine, and ornamentals. They are found in different colors and fragrances [46]. Tagetes species grow in the temperate forests and mountainous regions of most countries in the world. They originated in South America and spread throughout the world as weeds. It was reported that South Africa, Australia, Nigeria, India, Uruguay, Brazil, France, Chile, Bolivia, France, Kenya, and Argentina and are the main producing countries of Tagetes oil in the world. In India, Tagetes is found in the western Himalayas between altitudes of 1000 and 2500 m [47]. Himachal Pradesh and the hills of Uttar Pradesh are the main growing regions where Tagetes species occur in their natural habitat. The wild growth of T. minuta in these regions forms the most important source of "Tagetes oil" in India [45].
Mild climates are most suitable for the luxuriant growth of Tagetes species. Flowering in these species greatly improves during mild climates in temperatures ranging from 14.5-28.6 • C. However, higher temperatures (26.2-36.4 • C) adversely affect flower production. The species of Tagetes successfully adapted to different soil conditions. However, the most desirable conditions for enhanced production are a deep, fertile, friable soil having good water-holding capacity, being well drained with a pH close to 7.0-7.5. Sandy loam is ideal for the cultivation of Tagetes spp. The crop of Tagetes is suitable for cultivation in the plains, as well as on the hills, as a monocrop or intercrop in orchards/forest aromatic trees or as widely spaced crops such as rose-scented geranium. It is amenable for integration with traditional agricultural or aromatic crops in suitable field rotations. Cool temperatures induce germination, whereas high temperatures encourage profuse vegetative growth and flowering. Direct seeding (2.0-2.5 kg seeds/ha) or transplanting of 100-200-mm-long 30-60-day-old seedlings (0.50-0.75 kg seeds/ha for raising the nursery) with (300-600) × (150-300) mm spacing is practiced. A closer spacing of 300 × (150-300) mm for direct broadcasted or line-sown seeded crops and a wider spacing of (450-600) × 300 mm for transplanted crops are recommended. In less fertile soils, a closer spacing of 300 × 300 mm is suggested. Seeds germinate in 7-10 days [48]. Young seedlings are susceptible to weed competition. Two to three manual weeding operations are necessary until the crop establishes. Fully grown plants can smother the weeds. Nipping apical buds 50-60 days after seed sowing or 30-45 days after transplanting promotes the growth of branches and produces a crop canopy with a higher proportion of leaves and flowers. The crop can withstand short periods of moisture shortage. Phosphorus and potassium are usually applied basally, while nitrogen is applied in three equal splits upon planting, active vegetative, and flower bud initiation stages. The crop is occasionally affected by wilt (caused by Sclerotiana sclerotium, for which seed treatment with Thiram is suggested), S. rolfsii (seed treatment with Thiram is recommended), little leaf (caused by phytoplasma, for which spraying with Streptomycin is useful), marigold mosaic (caused by virus, infected plants are burnt) diseases, and collar rot (caused by S. sclerotium). Three to five irrigations at depths of 25-30 mm or at 0.5 IW:CPE ratio of irrigation scheduling [45] are sufficient for raising a good crop. The crop responds to applications of 20-30 tons of Farm Yard Manures (FYM),  kg N, 60 kg P 2 Os, 60 kg K 2 O/ha. Wild marigold is harvested manually with sickles during full flowering [49,50] (4-7 months duration) or seed setting [47,50] stages 200-300 mm above ground level. Crop duration is short (main 10 days, ratoon 30-60 days) in south India irrespective of whether the crop is planted in rainy (July/September) or winter (December) seasons [45,49]. In the rainy seasons, weeds become the major problem for the yield of marigold. If the weeds are not removed in time, great loss occurs in terms of growth and productivity.

Phytochemical Composition of EOs Obtained from Tagetes Genus Plants
The Tagetes genus is notorious for species rich in aromatic compounds and resinous exudate [51]. For 15 Tagetes species, it was possible to find information on the chemical composition of the EO contained in the aerial parts, capitula, or leaves.
Generally speaking, the oils are rich in monoterpene hydrocarbons (ocimenes, limonene, terpinene, myrcene, etc.) and in acyclic monoterpene ketones (tagetone, dihydrotagetone, and tagetenone) which are the primary odorants, in addition to lower amounts of sesquiterpene hydrocarbons and oxygenated compounds. Within these groups, the chemical diversity is quite high. The only striking differences in EO composition come from T. lucida and T. filifolia, whose EOs are dominated by phenylpropanoids such as methyleugenol, methylchavicol, and anethole. The EO from the leaves of T. tenuifolia contained 2.2% methylchavicol. Figure 1 presents the main chemical structures of the compounds found in the EOs, numbered for ease of reference in the other tables.

Components of T. patula
According to some taxonomical authorities [52,53], T. patula is to be considered a synonym of T. erecta. However, given the relative large amount of literature on its EO chemistry, where it is always considered as a separate entity, it is treated here as a close relative but a separate taxon from T. erecta.

EOs from Aerial Parts (Comprising Capitula, Leaves, and Stems)
According to Tisserand and Young [54], the major components of the EOs from the aerial parts of the plants found in India, Egypt, and South Africa are summarized in Table 1.

Components of T. patula
According to some taxonomical authorities [52,53], T. patula is to be considered a synonym of T. erecta. However, given the relative large amount of literature on its EO chemistry, where it is always considered as a separate entity, it is treated here as a close relative but a separate taxon from T. erecta.
Another paper [62] reported the main components of leaf EOs from Brazil as piperitone (45.7%), limonene (9.7%), and piperitenone (5.9%). There were ambiguous data relative to the presence of phototoxic thiophenes in these plants. According to Marques and colleagues [62], a thiophene derivative, α-terthienyl, was present in the roots of T. erecta. This compound is able to generate singlet oxygen in organic solvents and superoxide radical anions in an aqueous medium. Gupta [55] mentioned the presence of β-terthienyl and α-terthienyl in T. patula. The data on the EOs from various parts of T. erecta are summarized in Table 3. Table 3. Comparison of the EO components from various parts of T. erecta.

Molecules
Aerial Parts Capitula Leaves

Molecules Aerial Parts
Capitula Leaves X γ-muurolene X γ-terpinene X X means that the chemical compound was detected in the EOs obtained from that specific part of the plant.

EOs from Aerial Parts
According to Tisserand and Young [54], the major components of EOs from the aerial parts of plants in India, South Africa, and Egypt are collected in Table 4.
According to Burfield [68], the major components of EOs from Madagascar are subject to considerable variation, since both ocimenes and dihydrotagetone can vary from 0.1% to 99%.
Two previous reviews explored the chemistry of T. minuta EOs [55,56] and identified (Z)-β-ocimene, dihydrotagetone, (E)-β-ocimene, (Z)-and (E)-tagetones, and (Z)-and (E)-tagetenones as the major components of the aerial parts. The (E)-β-ocimene content varied quite extensively across the studies. In one study, it was observed that drying the plant material prior to distillation changed the chemistry of the EO. Both EOs contained (E)-tagetenone, (Z)-β-ocimene, and (Z)-tagetone; however, the fresh plant contained larger amounts of (Z)-tagetenone, limonene, and allocimene, while the dried plant was characterized by a high content of dihydrotagetone.
According to Singh and colleagues [56], eight types of EOs from capitula or whole flowering plants can be recognized. These types were clustered into two larger supergroups, one made of EOs dominated by (Z)-β-ocimene, and a second made of EOs dominated by dihydrotagetone (Table 5).
The chemical diversity could be independent of geographical origin, but could depend on plant phenology. A paper on an Indian EO revealed that EOs from plants harvested in the winter (December-January) were dominated by ocimenes (just like the first group above), and those from plants harvested in autumn (October-November) were dominated by dihydrotagetone (just like the second group above), while plants harvested in the summer (June) were dominated by tagetone, and a tagetenone-rich oil was obtained in the winter if the seeds were sown in September [45].

EOs from Capitula
Two previous reviews [55,56] proposed the main components of EOs from capitula of T. minuta as the compounds listed in Table 9. Table 9. List of EO components of T. minuta from capitula.
The main components are reported in bold with their corresponding percentages; tr: traces.
Notable points in the composition of the EOs analyzed by Gila and colleagues [81] were the very high amount of (E)-β-ocimene, and the huge quantities of α-phellandrene, as well as the absence of (Z)-tagetone and (E)-tagetone.
Some papers evaluated the variation in EO content according to phenological stage. According to Worku and Bertoldi [83], who observed the evolution of EO composition from the pre-flowering stage to the immature seed stage, the content of (Z)-β-ocimene increased through the process from 7.2% to 37.5%, and the content of (Z)-tagetenone declined from almost 40% to 13.1%, while little difference was observed for (Z)-and (E)-tagetone. Chalchat and coworkers [84], measuring the same variations, found that the content of (Z)-β-ocimene increased dramatically from the pre-flowering (2%) to early flowering (20.4%) stage, and then decreased slightly. The content of (Z)-tagetenone increased from 0.1% to 8.3% at the early flowering stage, and then decreased again. Dihydrotagetone increased from 16.5% to 33.4%, (E)-tagetone decreased from 16.9% to 3.7%, (Z)-tagetone remained stable at levels of 18.2-23.4%, and (E)-tagetenone was stable around 0.2% apart from a peak at the early flowering stage of 2.2%.
Looking at the same variations, Lawrence [85] reported different results. Dihydrotagetone was the major compound and it decreased in content from 51% to 14%; (Z)-β-ocimene was the second most common compound, and its content increased from the pre-flowering to the post-flowering stage from 16.9% to 45.9%. The content of (Z)-tagetone saw a dramatic decrease from the pre-flowering (18.5-22.4%) to the early flowering (1.3%) stage, before a steep increase to 16.9-20.4%, while the content of (E)-tagetone varied between 1.4 and 2.1%. On the other hand, the (Z)-tagetenone content was variable with higher values in the early and full flowering stages (8.6%) and a lower content at the seed stage (0.3-0.4%), while (E)-tagetenone was stable at 1.5-3.6%.
Moreover, two papers analyzed the variations in EO content relative to various agronomical parameters [87,88], and they are presented in Table 13.

Traditional Medicine Uses of Tagetes Genus: Ethnopharmacological Relevance
Traditionally, different parts of some Tagetes species are used as remedies to treat various health problems across the world. In Bangladesh, the leaves of T. patula are applied on boils and carbuncles and used against kidney troubles, muscular pains, and piles. Their juice is prescribed for earache and opthalmia [107]. In Pakistan, both leaves and flowers are collected and used as an antipyretic [108]. Other uses were recorded for T. filifolia in Mexico, where the Pima tribe prescribes a cup of tea prepared with its branches for stomachache [109], and in Argentina, where it is recommended for infected wounds [110]. Wounds and sores are also healed with leaf and flower decoctions or infusions of T. minuta [111], while a topical application of its sap is used in Kenya [112]. A particular use of T. minuta for wound healing in dental disorders was reported by Rahman et al. [113]. Ata and coworkers [114] attributed a general use in skin diseases. In Argentina, Bolivia, Brazil, Paraguay, and Peru, T. minuta infusions and decoctions are considered as digestives, appetizers, cholagogues, carminatives, gastric sedatives, antidiarrheics, and vermifuges. They are administered against food poisoning as antiparasitics and to cure dyspepsia, gastritis, intestinal colic, and flatulence, while the chewed fresh leaves are recommended for removing bad breath. The leaf decoction is prepared as an expectorant or an antiabortive, and is also used in order to reduce milk secretion. The infusion regulates menstrual flow and is used for vaginal washes in cases of infected flows. The whole plant is a febrifuge and diuretic [111]. T. minuta leaf and flower infusions are now incorporated in home medicines of the descendants of Polish migrants in Argentina as a prophylaxis after labor [115]. Ijaz and coworkers [116] documented new Pakistani uses of T. minuta leaves against cough and stomach disorders. Furthermore, their use against children's cough (three decoction teaspoons thrice per day for a week) and headache is rooted in Southern Uganda [117]. Both in new and old world countries, the leaves or the entire plant are indicated for liver diseases through internal (tea or juice) or external (poultice) administration [118]. Although scarcely used, leaves and stems without sap are externally applied in Turkey for earache [119], while flower tea is drunk for musculoskeletal ailments in Morocco [120]. T. minuta and T. lucida are recommended for treating emotional and nervous disorders as part of a mixture with other anxiolytic plants. In Bolivia, the infusion of the T. minuta is used as a tonic for nerves [111], while, in Brazil, it is used as a sedative to drink before sleeping [121]. T. lucida, known to the Aztecs as a remedy for fever, diuresis, and epilepsy, was also used to treat tumors and age-related brain disorders such as dementia and fear [122,123]. It is sold instead of Hypericum perforatum in different regions of Mexico, where the aerial parts are still consumed orally in infusions and hydroalcoholic extracts to soothe anxiety, depression, irritability, and sadness [124,125]. Mexican traditional medicine prescribes T. lucida for "nervios" and "susto", two culture-bound syndromes described as illnesses characterized by a "state of bodily and mental unrest" able to decrease the ability to achieve daily goals and as a condition of being frightened and "chronic somatic suffering stemming from emotional trauma", respectively [124,125]. There are several minor uses for the treatment of gastrointestinal, respiratory, and urogenital systems, and against rheumatism, ulcers, and inflammation. Moreover, T. lucida is recommended as a stimulant of the immune system and decoctions of its aerial parts fight infections caused by some helminthes and protozoa (e.g., ascaridiasis and giardiasis) [123][124][125][126]. T. lucida, together with T. erecta and T. tenuifolia, is an important plant for treating folk illnesses considered cold or "friadad" such as "frío en el estómago" (cold in the stomach), "calor en el estómago" (heat in the stomach), and "empacho" (indigestion), as well as constipation, baby and child diarrhea, and eye irritation [127]. The remedies prepared with aerial parts of these plants are ointments employed in topical administration, in seat baths and in specific sweat lodges, in addition to flower infusions and tinctures consumed orally [127,128]. The use of T. erecta was documented in phytomedicine from Guatemala to cure the respiratory system against pneumonia, asthma, and tuberculosis, to cope with colic, for use as an antibiotic, analgesic, and antileukemic, and for wound healing, and immune system stimulating, as well as against headache, tetanus, and various parasites [129]. The efficacy of T. erecta on parasitic infections, as well as in the puerperium, is increased by combining all its aerial parts with those from other aromatic species in a Mexican syrup. Only the petals, however, are collected for the preparation of remedies useful in the affections of the central nervous system [128]. The infusion of T. erecta flowers covers a wide range of other actions against flu, fever, body pain, rash, sore throat, heart attack, and arthritis [130]. Among plants collected for medical purposes in India, T. erecta flowers are claimed to treat several skin diseases (sores, wounds, burns, ulcers, eczema, boils, and carbuncles), as well as earache, piles, and muscular pains [131]. Its extract-two teaspoons twice daily for 8-10 days-combined with common salt and minerals treats kidney problems, specifically removing blocked urine [131,132]. The leaves are used to relieve pain and remove inflammation [133]. T. erecta is used in Spanish and French herbal medicine as an external detersive, resolutive, and vesicant [134]. The inhabitants of Madagascar recognize that T. erecta has antimalarial properties, while the people of Rodrigues Island cure fever due to infection by drinking one cup per day of an infusion of three flowers [135,136]. Mauritians suggest a glass of T. lucida flower decoction in the case of abdominal pain related to diseases of the circulatory system, and in the case of neonatal jaundice for breastfeeding mothers [137]. Indian folk veterinary medicine applies drops of T. erecta flower extract thrice a day to cows and buffalos for otitis [138], and applies leaves to limit bleeding and to cure broken horns, external injury, and eye diseases [139]. In southern Ethiopia, leaves and stems of T. minuta are chopped, mixed with water, and given orally to cattle and sheep affected by anthrax, blackleg, and amoebiasis [140].

Food Preservative Applications of Tagetes Genus Plants
Metabolites synthesized by plants belonging to the genus Tagetes show significant effects as antioxidants, enzyme inhibitors, precursors of toxic substances, and pigments. In addition, these bioactive compounds are involved in photosensitization and energy transfer, actions of plant growth hormones and regulators, control of respiration and photosynthesis, and defense against parasites, bacteria, fungi, and some insects. On the other hand, some of the secondary metabolites, especially those contained in the flowers, are responsible for the attraction of pollinators. It is believed that the activity of the secondary metabolites in the Tagetes species, like in the case in other plants, is related to their composition, concentration, and environmental conditions affecting their content. Thus, plant extracts or EOs obtained from different parts may show different biological abilities, and therefore, can be used in a variety of industries, including cosmetic, pharmaceutical, or food production [141,142]. Although the extracts from these plants are not so popularly used as preservatives, Tagetes spp. are characterized with high potential in the field of agriculture. According to the approach "from the field to the fork", the use of Tagetes spp. as bioactive agents in plant protection against micro-and macro-organisms is directed toward the first stages of the production, mainly in the plant breeding or post-harvest stages.

Tagetes spp. as Potential Plants in Agriculture
One of the first papers on marigold and its potential agricultural uses was published in 1958 by Uhlenbroeck and Bijloo, who studied the nematicidal activity of extracts obtained from the roots [143]. The results of their research indicated that prepared formulations were active against Heterodera rostochiensis, Ditylenchus dipsaci, and Anguina tritici. In later years, Swarup and Sharma [144] showed that T. erecta root extracts showed cidal or inhibitory activities against other plant-pathogenic nematodes, Meloidogyne javanica and M. arenaria, while Munoz et al. [145] indicated the potential use of T. halisciencis roots against M. incognita. The results of the study were important due to the fact that these nematodes are major agricultural pests of a wide range of crops cultivated in tropical and subtropical regions, as well as in greenhouses in temperate climates. T. patula showed activities against Caenorhabditis elegans and Pratylenchus penetrans, which are nematodes known as worms of gardens, compost piles, and rooting fruits [146]. Tagetes spp. produce a number of potentially bioactive compounds; however, thiophene derivatives and particularly α-terthienyl derivatives seem to be responsible for these nematocidal properties [147].
In addition, extracts and EOs obtained from the Tagetes genus were reported as insecticidal and larvicidal natural agents. Generally, T. erecta showed insecticidal activity against Tribolium spp., which are considered to be common pests of cereal silos, mills, and warehouses. The presence of these insects in stored food directly affects its quantity and quality [148,149]. Extracts obtained from T. erecta showed good insecticidal properties against aphids (Aphidoidea) and fall armyworm (Spodoptera frugiperda), which are responsible for the destruction of wild varieties of crops [150,151]. According to dos Santos et al. [152,153], the species T. erecta and T. patula showed larvicidal and insecticidal properties with the possibility of the use of Tagetes extracts as an alternative to synthetic insecticides used against rice weevil (Sitophilus zeamais).
From another point of view, Zoubiri and Baaliouamer [154] documented the high effectiveness of EOs of T. minuta against mosquitoes of Anopheles gambiae, which are responsible for malaria transmission. The results were comparable to commercial, synthetic, insecticidal agents. What is more, T. minuta oil possesses phytotoxicity toward common weeds, including green amaranth (Amaranthus viridis), vegetable amaranth (A. tricolor), hairy beggars tick (Bidens pilosa), little seed canary grass (Phalaris minor), nettle leaf goosefoot (Chenopodium murale), and barnyard grass (Echinochloa crus-galli) [155]. Going further, Tagetes spp. are potential chromium hyperaccumulators. According to Coelho et al. [156], at Cr(III) concentrations up to 0.12 mmol/L, the plants accumulated levels above that proposed for hyperaccumulators while still maintaining considerable growth and even flourishing.

Tagetes spp. as Antimicrobial Agents
Despite the lack of a clear and direct reference of extracts and EOs from Tagetes spp. as food preservatives, the activity of compounds from Tagetes spp. was noted against various microorganisms (Table 14).
In research conducted by Tereschuk et al. [157], extracts obtained from leaves of T. minuta showed antimicrobial activity against both Gram-positive and Gram-negative bacteria. The major component of the extract, quercetagetin-7-arabinosyl-galactoside, showed significant antimicrobial activity against tested pathogen microorganisms. In studies conducted by Tereschuk et al. [158], almost all of the tested strains were susceptible to T. terniflora extracts at concentrations of 200 mg/mL, except for bacteria Zymomonas mobilis and Lactobacillus plantarum, and yeast Saccharomyces cerevisiae. Moreover, these results were generally comparable with chloramphenicol at 100 mg/mL. Hernández et al. [159] indicated that T. lucida extracts, containing 5,7,4 -trimethoxyflavone, showed wide antibacterial activity. T. lucida extracts also showed high activity against Gram-negative bacteria and phytopathogenic fungi in the study conducted by Cespedes et al. [160]. Antifungal activities of the EOs against species belonging to Candida, Penicillium, and Aspergillus were also described [161,162]. What is more, Gakuubi et al. [79] documented the antibacterial activity of T. minuta EOs against phytopathogenic bacteria Pseudomonas savastanoi pv. phaseolicola, Xanthomonas axonopodis pv. phaseoli, and X. axonopodis pv. Manihotis, which are responsible for different plant diseases. The results obtained confirmed the biopesticidal nature of EOs of Tagetes spp. and their potential uses as cheap, safe, and effective alternatives to chemical bactericides used for the protection of agricultural products. Recent research on the antimicrobial potential of T. minuta was done by dos Santos [141], correlating the extract of this plant with traditionally used antibacterial, insecticidal, biocide, disinfectant, anthelminthic, antifungal, and antiseptic agents. T. terniflora E. coli, S. aureus, S. epidermidis, P. aeruginosa, M. luteus, Z. mobilis, L. plantarum, and S. cerevisiae [158] The results indicated that Tagetes spp. may potentially play an important role in food preparation and food preservation, as well as for use as an excellent food spice. Even from a traditional point of view, the nature of Tagetes spp. and their composition affect the quantity and quality of the extracts [172]. Despite promising results obtained in vitro, more detailed studies of the mechanisms of action of Tagetes spp. extracts and EOs would be beneficial for reaching their potential in biotechnology. It was documented that EO components, especially terpenoids such as dihydrotagetones, tagetones, and ocimenones, are sufficient to account for the observed antimicrobial activity [171]. Further progress in determining the active components of the EOs may be achieved by fractionating the EOs of Tagetes spp. and determining the antimicrobial activity of each component individually. Caution is required in this approach, as both synergistic and antagonistic antimicrobial interactions between constituents of EOs can occur, giving the whole extract a different activity from the sum of the individual components.

Tagetes spp. as Functional Food Additives
The concept of the development of a functional food may involve the integration of healthpromoting substances into a multifunctional ingredient, which could be used to design a wide range of novel food products [173][174][175]. Tagetes spp. present compounds such as flavonoids and carotenoids that are not part of the EOs but have several functions as biologically active compounds. For example, Tagetes spp. flavonoids were recognized as antiallergic, antiinflammatory, antiviral, antiproliferative, and anticarcinogenic substances. Infusions, tinctures, and juice from aerial parts of Tagetes spp. are used as traditional food additives worldwide. In turn, T. terniflora is a native plant known as "suico-suico" or "quichia", widely used in Argentina as a condiment in soups [158,176]. T. minuta can be used in hot or cold refreshing beverages.
Current epidemiological studies indicate that high flavonoid consumption is associated with reduced risk of chronic diseases, such as cardiovascular diseases. The global publicity of traditional medicine during the past two decades, together with advances in ethnopharmacological knowledge, increased the interest in flavonoids and their interaction with mammalian cells and tissues. Leaf infusions from members of Tagetes are used in folk medicine in the treatment of stomach and intestinal diseases. An important factor is that EOs and extracts from Tagetes spp. show no antimicrobial activity against human microflora, regarded as central to human immunity, such as Lactobacillus species. An absence of antimicrobial activity against nonpathogenic human bacteria could be beneficial for intestinal disease treatments, in which the intestinal flora must be preserved [177].
Tagetes EOs are potential agents for protecting food crops on the farm and during storage, thereby increasing food security, particularly in undernourished communities of the world. These EOs also provide an opportunity for developing environmentally friendly and nontoxic acaricide agents to enhance increased production of milk, beef, and hides/skin in the livestock industry [172].
Tagetes species were originally used as a source of EOs, with applications as flavoring in the food industry, extracted from leaves, stalks, and flowers. What is more, marigold pigments have potential as a natural food colorant. The flowers and their extracts are rich in orange/yellow carotenoids [141]. The status of marigold as a source of natural carotenoids was reviewed by Verghese [178,179]. The stability of these pigments was studied for oil-in-water emulsions or arabic gums. It was concluded that the composition of the emulsifying agents and the pH level have important roles in determining the effectiveness of the emulsions against color loss and coalescence kinetics. Anaerobic and lactic acid treatments of fresh flowers are promising in terms of pigment stability. What is more, compared with other natural sources of yellow and orange color, like turmeric, chill, and saffron, marigold is a cheaper and easily available source. Factors influencing the qualitative and quantitative profiles of carotenoids in flowers of marigolds are their storage conditions. It is widely known that carotenoids are beneficial for human health. However, the biological functions of many individual carotenoids like zeaxanthin, cryptoxanthin, antheraxanthlin, and neoxanthin, which are present in a large amount in marigold flowers, are not known. Although marigold as a colorant offers a strong intense orange to yellow color, no toxicity data are available in the literature on marigold extract, whether partially purified or raw, which makes it unusable in food.
It is worth noting that marigold flowers can be a cheaper source as a starting material for the isolation of lutein, a valuable natural pigment that can also serve as a nutraceutical. The deficit of this compound in the human body is probably responsible for the age-related impairment of vision. Dried flowers of T. erecta contain 0.1-0.2% carotenoids in dry matter, out of which 80% are lutein diesters [180]. In T. erecta and T. minuta, lutein was isolated, identified, and approved by the Food and Agriculture Organization (FAO) and the European Union [181][182][183]. This bioactive compound can find application as a food colorant and flavor in various foodstuffs. Additionally, the orange pigment extracted from the petals of marigold is in great demand for poultry feed.
Another valuable compound in marigold is quercetagetin. The in vitro antioxidant activity of this flavonoid and its potential in the control of diabetes mellitus and obesity were investigated by Wang et al. [184] and compared to quercetin and rutin. It was documented that quercetagetin has potential antidiabetic and antilipemic activities, showing inhibitory effects of α-glucosidase, α-amylase, and pancreatic lipase. These results may be essential scientific support for the application of quercetagetin as a nutraceutical for the treatment of diabetes and obesity. What is more, according to the newest research, both flavonoids and carotenoids, primarily patuletin, constituents of T. patula extract can protect Jurkat cells (human T lymphoblastoid cell line) from hydrogen peroxide responsible for oxidative stress. These findings were presented by Chkhikvishvili et al. [185] and they are in line with the antioxidant and antiinflammatory properties of marigold preparations used in folk medicine.

Antimicrobial Ethnomedicine of the Tagetes Genus
Despite being a genus with a considerable number of species (more than 50, according to The Plant List [52]), only four species were found for the treatment of symptoms associated with bacterial and fungal diseases, namely T. erecta, T. filifolia, T. lucida, and T. minuta (Table 15). All plants are commonly indicated for diseases of the digestive system, and only T. filifolia is not indicated for the treatment of diseases of the skin and subcutaneous tissue. T. erecta and T. lucida are used against diseases of the respiratory system [186]. Oral/local application for wounds and dental problems; oral as leaf juice; local application: paste of leaves used in the treatment of ulcers and wounds; topical: leaves boiled in water to wash affected area and to relieve itchiness and rash [130,[187][188][189][190][191][192] Mexico, India, Belize, Bangladesh Topical [113,140,196] Ethiopia, Pakistan, Argentina The use of these species crosses continents such as America, Asia, and Africa, and, according to registries, the whole plant or only parts of the plant can be used, with no reports found for the use of the root. The form of use can be both internal and/or external, and the plant is used from a raw to a cooked (decoction) state. In the scientific milieu, a constant need to validate popular knowledge through assays that seek to ascertain whether the medicinal plant has any influence on the growth of microbial populations is seen, whether reducing or decimating them. In the following sections, the results of research in which plants from the Tagetes genus were investigated for their effect on pathogenic bacteria and fungi are discussed.

Antibacterial Activity of Plants from the Tagetes Genus
Igwaran et al. [197] evaluated the properties of the EO from T. minuta flowers and confirmed its antibacterial activity through microdilution considering the minimum inhibitory concentration (MIC). The EO showed good antibacterial activity against all tested strains. The MIC of 0.06 mg/mL was shown for Vibrio spp., E. coli, Enterobacter cloacae, and Listeria ivanovii, while the MICs against S. aureus, Mycobacterium smegmatis, and Streptococcus uberis were higher (0.125 mg/mL). As a positive control, the antibiotic ciprofloxacin was used and showed activity against M. smegmatis (0.06 mg/mL), E. coli (0.125 mg/mL), and Vibrio spp. (0.25 mg/mL).
Lambrecht et al. [198] tested the EO and hydroalcoholic extract from the aerial parts of the plant through a broth microdilution assay to determine the MIC. Although all plant samples showed antimicrobial activity, the hydroalcoholic extracts showed the most satisfactory results with all bacterial species. The T. minuta EO showed antimicrobial activity against all Gram-positive bacteria with EO percentage activities of 1.3% for S. aureus, 0.6% for Staphylococcus coagulase (+), 2.5% for S. coagulase (−), and 0.8% for S. uberis. On the other hand, for Gram-negative bacteria, the percentage activity values were 4.0% for P. aeruginosa and 5.0% for E. coli.
Two species, T. patula and T. erecta, were evaluated by Ayub et al. [199], who observed the antimicrobial activity of the petal hexane and methanolic extracts by disc diffusion (100 µL of the extract) and microdilution (0.03 to 72.0 mg/mL). The tests demonstrated antibacterial activity against the E. coli, Pastrulla multocida, B. subtilis, and S. aureus strains, with T. patula presenting inhibition zones with values varying from 12.4 mm to 20.2 mm for the methanolic extract and 8.2 mm to 11.4 mm for the hexane extract. Meanwhile, the T. erecta species showed inhibition zones varying from 10.0 mm to 17.5 mm (methanolic extract) and 8.3 mm to 10.0 mm (hexane extract). The MICs for T. patula were from 0.19 mg/mL to 4.05 mg/mL (methanolic extract) and from 4.05 mg/mL to 24.0 mg (hexane extract), while T. erecta displayed MICs from 0.63 mg/mL to 7.6 mg/mL (methanolic extract) and from 6.8 mg/mL to 24.4 mg/mL (hexane extract).
Shahzadi and Shah [200] tested the antimicrobial activity of crude, aqueous, ethyl acetate and butanol T. minuta extracts, in addition to leaf and stem extracts extracted with ethyl acetate and butanol. The antimicrobial activity was determined from cavity diffusion (20 µL) against Gram-positive and Gram-negative strains. Inhibition halos of the crude extract were from 5 mm to 6.5 mm against Pseudomonas picketti, S. aureus, and B. subtilis strains. The aqueous extract had inhibition halos only against B. subtilis (5.7 mm). However, the ethyl acetate extract from the flowers and seeds presented activity against S. aureus (3.0 mm) and P. picketti (5.0 mm), while the butanolic extract was active against P. picketti and B. subtilis (5.2 mm and 7.6 mm, respectively). The inhibitory results from the leaf and stem extracts showed activity against S. aureus (3.1 mm), P. picketti (6.3 mm), and B. subtilis (6.1 mm) for ethyl acetate extracts, and 1.0 mm and 5.0 mm against M. luteus and B. subtilis from the butanolic extract. For all assays, ampicillin was used as the control.
The species T. elliptica and T. minuta were evaluated by Ramirez et al. [201] against bacteria causing periodontal diseases. The MIC of the T. elliptica ethanolic extract obtained against Lactobacillus acidophilus was 125 mg/mL. This extract had the highest inhibition halo of 13.83 mm against L. acidophilus and 14.23 mm against Porphyromonas gingivalis, both at the concentration of 500 mg/mL. The T. minuta ethanolic extract showed a mean inhibition halo of 16.0 mm (500 mg/mL) only against P. gingivalis. Using the T. minuta EO, Shirazi et al. [42] evaluated its antibacterial activity against Gram-negative and Gram-positive bacteria, with MICs found against S. typhi, E. coli, S. aureus, and B. subtilis of 150 µg/mL, 165 µg/mL, 67 µg/mL, and 75 µg/mL, respectively.

Antifungal Activity of Plants from the Tagetes Genus
The benefit of antimicrobial activity is one of the most proven targets by several authors through studies on the antifungal capacity of plants from this genus [202].
Ayub et al. [199] carried out an experiment to verify the antifungal potential of the T. erecta and T. patula species. The petals were collected, dried, and ground for the preparation of hexane and methanolic extracts. Spores of the species Ganoderma lucidum and Alternaria alternata were used. The disc diffusion method was used, with the drug flumequine (30 µg/disc) used as a control. MIC was determined by microdilution with flumequine (1.0 mg/mL) as the control. The results of the test showed that the values for the inhibition zones varied from6.4-8.5 mm and in the control from 20.6-21.8 mm. The MIC was established between 7.5 and 13.3 mg/mL, while it was 0.21-0.30 mg/mL in the control. The T. patula species showed higher antifungal activity compared to T. erecta, with inhibition zones ranging from 6.5 to 7.3 mm and MIC values from 0.19 to 24 mg/mL.
Romagnoli et al. [203], extracted the EO from dried T. patula flowers and investigated its antifungal effect on Penicillium digitatum and Botrytis cinerea strains. The EO showed remarkable activity in both fungi, reaching 100% inhibition, even at the lowest concentrations. The MIC of the EO against P. digitatum presented a low value (1.25 µL/mL), with no colonies being observed at this concentration. B. cinerea showed dose-dependent growth inhibition with an MIC of 10 µL/mL. Still with the same species, T. patula, Sesan et al. [204] using the hydroalcoholic extract at concentrations of 10% and 5%, analyzed its action on the isolated Trichoderma viride (TV 82) strain, a fungal biocontrol agent that can cause infections in humans. The extract was made from fresh biomass such as stems, leaves, flowers, shoots, and bulbs. The T. patula extract inhibited the development of the fungus, even when applied at low concentrations (10% and 5%), with inhibition varying between 50 and 54%.
Thembo et al. [162] used the aerial parts from T. minuta against isolates from four fungi species of agricultural and clinical importance: Fusarium verticillioides, F. proliferatum, Aspergillus flavus, and A. parasiticus. The extraction solvents used were hexane, dichloromethane, methanol, and water. The concentration of the extracts was 10 mg/mL. The drug amphotericin B and the agricultural fungicide Cantus were used as positive controls. The MIC was determined by microdilution and some isolates from F. verticillioides and F. proliferatum strains were sensitive to hexane (0.02-2.5 mg/mL), dichloromethane (0.02-0.32 mg/mL), and methanol (0.02-2.5 mg/mL) extracts with fungistatic action. The aqueous extract had no activity on the fungal strains.
The Tagetes pusilla EO was investigated against the C. albicans strain using the cavity diffusion method. Alzamora et al. [205] used micoral (100 mg), sporostantin (330 mg), mycostatin (10,000 UI/mL), and oxonasol (200 mg) as controls. The analysis resulted in an inhibition halo greater than 20 mm in diameter, suggesting that the C. albicans strain was extremely sensitive to the EO. Ali et al. [90], using the same methodology, tested the EO from T. minuta leaves against C. albicans. The EO showed good anticandidal activity with a 26-mm inhibition zone, compared to nystatin.
Dutta et al. [206] tested the EO from the T. patula leaf on a C. albicans strain by disc diffusion. Miconazole (1000 µg/mL) and clotrimazole (1000 µg/mL) were used as controls. The MIC was determined by microdilution with the EO dissolved in 5% dimethyl sulfoxide (DMSO) and then diluted in Sabouraud dextrose broth. The result recorded an inhibition zone of 7.7 mm, a result similar to the inhibition determined for miconazole (10 mm) and clotrimazole (9.3 mm). The MIC for the EO was 3180 µg/mL. A. niger and C. albicans strains were used by Shirazi et al. [42] to perform experiments with the T. minuta EO. MIC was determined, and ketoconazole (10 µg/mL) was used as a control. The MICs for A. niger and C. albicans were 135 and 115 µg/mL, respectively.
Politi et al. [41] used 70% (5 mg/mL) ethanolic extracts from the aerial parts (stems, leaves, and flowers) of T. patula to evaluate the antifungal potential of clinically important fungi by microdilution using amphotericin B (16 mg/mL) as a positive control. Extracts from the aerial parts with flowers, without flowers, and with flower extracts were evaluated. The best results verified against T. rubrum were recorded using the extract from aerial parts with flowers (254 µg/mL), whilst against T. mentagrophytes, the ethanolic extract from aerial parts with and without flowers were more effective, with MIC values of 573 µg/mL and 625 µg/mL, respectively. Additionally, for M. canis, the flower extract (195 µg/mL) stood out, also displaying the lowest inhibitory concentration of all the extracts tested. Metarhizium anisoplia and B. bassiana were not sensitive to the extracts, which is a good result, considering that these entomopathogenic microorganisms are important for biological control, despite the latter being responsible for hyalohyphomycoses in humans.
Tests with the methanolic extract from the T. patula plant against the fungi B. cinerea, Fusarium moniliforme, and Pythium ultimum were performed by Mares and coworkers [207]. The extract was tested in solid medium at concentrations of 5, 10, and 50 µg/mL. Treatments were performed with sunlight (Biolux lamps), ultraviolet (UV)-A, and in the dark with scanning electron microscope readings. For B. cinerea under solar irradiation, the inhibition was dose-dependent reaching 39.3% growth in colonies treated with the maximum dose (50 µg/mL). At the same dose, irradiation with UV-A improved the action of the extract with 57.4% inhibition, and, in the dark, a value of 24.8% was observed. For F. moniliforme under sunlight, the inhibition observed at 50 mg/mL had the value of 50.9%, while it was 47.3% for UV-A and 33.8% in the dark, while the results for P. ultimum were 72.6% (solar), 62.7% (UV-A), and 51.4% (dark).

Ethnobiology of Tagetes Antimicrobial Activity
The scientific literature discussed here revealed the antimicrobial potential of species from the Tagetes genus. Four species presented antifungal effects and five presented antibacterial effects. In this sense, species of the Tagetes genus had an effect on 17 different fungal strains and 15 different bacterial strains which cause infections in humans. From ethnobiological reports, the T. filifolia and T. lucida species, indicated for the treatment of diseases related to fungi and bacteria, are yet to be evaluated by the scientific community regarding their antifungal and antibacterial potentials. None of the species which had antimicrobial bioactivity were evaluated in combination with commercial antifungals or antibacterials, where a synergistic effect is often seen as a positive interaction from this association. This investigation was also not mentioned in the methodology and, consequently, results from this ethnobiological research are lacking, which is of the utmost importance, since many people use teas and medications concomitantly.

Conclusions and Future Perspectives
The Tagetes genus is rich in aromatic compounds and resinous exudate, and the EOs of these plants are rich in ocimenes, limonene, terpinene, myrcene, tagetones, dihydrotagetone, and tagetenones, which are the primary odorants, and lower amounts of sesquiterpene hydrocarbons and oxygenated compounds. However, the chemical EO composition is high dependent on several endogenous and exogenous factors, including genetic traits, plant organs (leaves, stems, capitula, or roots), growing, drying, and storage conditions, and stress factors such as adverse climatology and diseases affecting the plant. Moreover, one crucial aspect of the EO composition is the type of extraction, the solvents utilized, and the standardization of the extracts. Thus, plant extracts from different parts of these plants may show different biological abilities, and therefore, can be used in a variety of industries, including cosmetic, pharmaceutical, or food production, due to the presence of biologically active compounds such as 5,7,4 -trimethoxyflavone (apigenin trimethyl ether), patuletin (O-methylatedflavanol), quercetagetin and its 7-arabinosyl-galactoside, and other flavonoids, carotenoids (lutein), and thiophene derivatives (α-terthienyl).
A clear-cut and feasible correlation of the biological effects reported for the various EOs and other extracts with their chemical composition is not evident, but some associations can be made. For example, the antimicrobial activity of limonene for medical purposes is well known [208], and its therapeutic effects involving anti-inflammatory, antioxidant, antinoceptive, anticancer, antidiabetic, and antihyperalgesic effects, among others, were extensively studied [209]. In an in vitro study [210], antibacterial and antifungal activities of α-pinene and β-pinene enantiomers were evaluated, showing that positive enantiomers had antimicrobial activity against C. albicans, C. neoformans, Rhizopus oryzae, and methicillin-resistant S. aureus (MRSA), while also showing that pinene is useful in formulating strategies to limit C. albicans biofilm formation. Marchese et al. [211] proposed a mechanistic viewpoint of antimicrobial activity, against both planktonic and sessile cells belonging to food-decaying microorganisms and human pathogens, of eugenol and EOs containing eugenol. Eugenol interferes with membrane functions or suppresses virulence factors, including toxins, enzymes, and the formation of bacterial and fungal biofilms. Interestingly, the authors suggest a synergist effect of eugenol and other compounds present in EOs such as thymol, carvacrol, and menthol (compounds also present in Tagetes EOs). Some studies were conducted with major and common compounds present in the extracts; however, much research is missing with the minor and uncommon compounds, with a lack of investigation into the mechanisms of action. However, in this review, we evidenced the antimicrobial activity of Tagetes spp. extracts.
Antioxidant components are commonly used as food preservative applications. In the agriculture field, these plants have high potential uses because their bioactive compounds are involved in defense against parasites (bacteria, fungi, and some insects) and the attraction of pollinators. The Tagetes genus is commonly cultivated as an ornamental plant around the world. On the other hand, this plant is used in folk medicine, especially in countries where this plant is native. Today, this plant is used in folk medicine in several countries against signs and symptoms related to bacterial and fungal infection. However, further clinical studies are needed to corroborate these effects in humans.
Among the bioactivities reported for these plants, their properties are interesting to be exploited in the industry. In addition, the use of Tagetes products in foods seems to be promising as natural antioxidant agents and antimicrobial preservatives, particularly as novel components of active packaging systems. Finally, extraction methods should be improved to obtain compounds or optimized EO compositions, using food-grade solvents and green extraction methods, e.g., supercritical fluid extraction using CO 2 or pressurized water extraction. C. gathered, organized, and wrote the "Antimicrobial Ethnomedicine of the Tagetes Genus", "Antibacterial Activity of Plants from the Tagetes Genus", "Antifungal Activity of Plants from the Tagetes Genus", and "Ethnobiology of Tagetes Antimicrobial Activity" sections of the manuscript. S.V. gathered, organized, and wrote the section of "Traditional Medicine Use of Genus Tagetes". D.K. and H.A. gathered, organized, and wrote the sections of "Food Preservative Applications of Tagetes Genus Plants", "Tagetes spp. as Potential Plants in Agriculture", "Tagetes spp. as Antimicrobial Agents", and "Tagetes spp. as Functional Food Additives". N.C.C.S. gathered, organized, and wrote the introduction. Z.Y. gathered, organized, and wrote the section "Tagetes Genus Plant Cultivation". All authors read and approved the final manuscript, providing important intellectual content.
Funding: This research received no external funding.