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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Industrial Crops and Products 38 (2012) 27–38 Contents lists available at SciVerse ScienceDirect Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop Review Marcela, a promising medicinal and aromatic plant from Latin America: A review Daiana Retta a , Eduardo Dellacassa b,∗ , José Villamil c , Susana A. Suárez d , Arnaldo L. Bandoni a a Cátedra de Farmacognosia, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 2◦ piso, Buenos Aires 1113, Argentina Cátedra Farmacognosia y Productos Naturales, Departamento de Química Orgánica, Facultad de Química, Universidad de la República, Gral. Flores 2124, Montevideo 11800, Uruguay c Instituto Nacional de Investigación Agropecuaria (INIA), Estación Experimental INIA Wilson Ferreira Aldunate, Las Brujas, Rincón del Colorado, Ruta 48 km 10, Canelones, Uruguay d Departamento de Ciencias Naturales, Facultad de Ciencias Exactas Fisicoquímicas y Naturales, Universidad de Río Cuarto, Ruta 36 km 601, Río Cuarto 5800, Córdoba, Argentina b a r t i c l e i n f o Article history: Received 28 August 2011 Received in revised form 26 December 2011 Accepted 10 January 2012 Keywords: Achyrocline satureioides Diversity Medicinal and aromatic plants Industrial applications a b s t r a c t Medicinal plants and their extracts are natural resources of compounds used for treatments in ethnomedicine and phytotherapy. They are also a source of natural products used in the development of new related compounds and drugs for conventional medicine. The increasing interest in use of herbal medicines requires a comprehensive assessment of research data in this field to help focus future efforts. Here we review the increasingly important role of Achyrocline satureioides (Lam.) DC (Asteraceae), marcela, which is used extensively in popular medicine. Like most medicinal plants, however, A. satureioides is generally not cultivated and most plants used commercially are harvested from ecologically and edaphically diverse natural habitats. We provide information on the current status of this promising medicinal and aromatic plant, and an overall view of its potential for production of material with more desirable physicochemical and phytochemical properties. © 2012 Elsevier B.V. All rights reserved. Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Botanical description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Powdered material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Distinction of species within Achyrocline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethnobotanical background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Analysis of main active components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacological activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aromatic properties of A. satureioides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Market potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cultivation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Cultivation conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Domestication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiplication by seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. 8.4. Multiplication by cuttings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5. Soil preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6. Spacing between plants and irrigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7. Cultural laboring and plant care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8. Fertilization and nutrient supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9. 8.10. Harvest, collection, drying and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11. Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ∗ Corresponding author. Tel.: +598 29244068; fax: +598 29241906. E-mail address: edellac@fq.edu.uy (E. Dellacassa). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2012.01.006 28 28 28 28 28 29 29 31 31 31 32 32 32 32 33 33 33 33 34 34 34 34 34 34 34 Author's personal copy 28 D. Retta et al. / Industrial Crops and Products 38 (2012) 27–38 1. Introduction Although the importance of plants may not be readily apparent in our increasingly urbanized society, the use of medicinal and aromatic plants in medical, cosmetic as well as well as other nonfood applications is expected to rise globally. This projected upward trend is explained in part by the ever increasing human population and the popularity of natural, environmentally friendly products. Latin America has historically been a notable reservoir of plant resources. These plants are used, among other applications, for the production of dyes, drugs, fibers, foods, forage, fuel, medicines, ornamentals, resins, spices and antidotes. Quina (Cinchona sp.), maca (Lepidium peruvianum), guaiacwood (Bulnesia sarmientoi), lemon verbena (Aloysia citriodora) and boldo (Peumus boldus) are examples of the widespread use of a number of native Latin American plants. A great number of such species having promising properties and countless potential industrial uses, however, remain largely unknown throughout the world. The development of timely and well-managed cultivation programs for production of some of these native plants is also a current challenge in agronomical research. There are some 40 species of Achyrocline spp., most of which are from tropical and subtropical America (25 were described from Brazil), and a few from tropical Africa and Madagascar (Deble, 2007). “Marcela,” known in Portuguese as “macela,” is Achyrocline satureioides (Lam.) De Candolle (family Asteraceae, tribe Inulae; synonymy: A. satureioides (Lam.) DC, Gnaphalium satureioides Lam., Achyrocline vargasiana DC, A. satureioides (Lam.) DC. var. citrina Lorentz (Cabrera, 1963, 1974; Gattuso et al., 2008). The word Achyrocline comes from the Greek achyros meaning undergrowth and kline meaning bed probably refers to the somewhat fimbriated receptacle. It is native to southeastern South America and grows in sandy or stony soils, on hilly or plain terrain. It is common in Brazil (from Minas Gerais to Rio Grande do Sul), Uruguay and central and northeastern Argentina (Lorenzo et al., 2000; De Souza et al., 2007). It is also found in Venezuela, Colombia as well as central and southern Bolivia, where it can grow at altitudes 3900 m above sea level (Girault, 1984), Paraguay and also Peru (Velasco Negueruela et al., 1995). In Argentina, it is found most frequently in humid areas and sandy soils, in the mountains of Córdoba, San Luis and Tandil and on coastal dunes of the Province of Buenos Aires (Giangulani, 1976). In Uruguay it is commonly found in stony soils and sandy coastal areas (Davies and Villamil, 2004). In addition to A. satureioides, the species Achyrocline flaccida and Gnaphalium gaudichaudianum also grow in these regions. These three plants are morphologically similar and are unfortunately often mistakenly collected together. Plants collected without carefully avoiding these other species can lead to sample adulteration. 2. Botanical description A. satureioides (Lam.) DC. is a suffrutex, perennial, aromatic plant that can grow to a height of 80 cm. Leaves are simple, alternate, sessile, entire, linear to linear-lanceolate, acute, smooth-edged, 5 cm long and up to 4 mm wide, pinnately nerved and downy. Flowers occur in numerous small cylindrical capitula forming dense, yellowish-gray or golden yellow terminal glomeruli. Marginal female flowers are infrequent, with toothed, filiform 4–5 corolla split at the apex; central flowers are only 1–2, perfect, with narrow tubular corolla, 5-toothed at the limb. The fruit type is achene and is attached to the plant by a pappus. The weight of a thousand seeds is 37.1 mg (Davies and Villamil, 2004). Five populations from the Brazilian state of Rio Grande do Sul were investigated and found to have a diploid number of chromosomes, 2n = 24 (Pereira et al., 2006). Mazzella et al. (2010) found four of the Brazilian Achyrocline species, including A. satureioides, to have a diploid number of 2n = 28, and also reported very few karyotype differences within the genus. 2.1. Powdered material The drug consists of powdered flowers, leaves, flower stalks and portions of stems. It is grayish-yellow and downy because of the abundance of flagelliform hairs. Characteristic features of diagnostic value for the identification of this species were initially defined by Gattuso and Gattuso (1998) and are discussed in more detail below. 2.2. Distinction of species within Achyrocline The identification of anatomical characteristics of A. satureioides specifically contrasting those of closely related species has been the subject of studies aimed at quantitating the occurrence of potential adulterants Cortadi et al. (2004). Giangulani (1976) reported that very similar, and sometimes controversial, morphological characteristics hinder the ability to distinguish between these species in a revision of the Argentinean species of the Achyrocline genus. Amat (1988) identified both common and species-specific leaf characteristics within the genus Achyrocline that should enable one to differentiate among these species. Adulteration and contamination of material during collection of plants from natural habitats continues to pose a difficult problem. In the Cuyo region of west-central Argentina, indiscriminate selection of A. satureioides and G. gaudichaudianum (Del Vitto et al., 1997), both from the tribe Inuleae in the family Asteraceae, was frequently a problem. Petenatti et al. (2004) conducted a study that established the macro- and micromorphological diacritical characteristics that could be used to distinguish these species. A complementary study by Gattuso et al. (2008) described floral characteristics that could be used to identify and distinguish A. satureioides, A. flaccida and G. gaudichaudianum, the three species that are most frequently mixed with each other during collection (Fig. 1). 3. Ethnobotanical background A. satureioides and its popular uses are mentioned in almost all ethnobotanical literature from countries where the plant can grow. González and Lombardo (1943) even refer to a certain worship of marcela being referred to as “a blessing from the Indians”. The origin of plant names is relevant from an ethnobotanical standpoint as these usually provide hints on the uses, traditions and virtues of a plant for a given culture. In the case of marcela, a lot of different applications over a long time in different places are already indicated. The Spanish common name “marcela” comes from the Portuguese word “macella,” which was used in early Brazil to refer to camomile. Use of this term was likely due to the similarity between the shape of the marcela flower bud and the Portuguese “maçã” apple, and thus “macella” would mean “small apple” (Bertoni, 1927; Houaiss, 2002). Similarly, other popular names used include: “marcela,” “vira-vira,” “marcela hembra,” “bira-bira,” “marcela del campo,” “juan blanco,” “marcelita,” “marcela blanca,” “alquitrán” (Argentina); “jate’i-caá” (Guarani language, Paraguay); “alkko wira wira,” “wira-wira” or “huira huira” (Quechua language, Bolivia); “macela,” “macelinha,” “macela amarela,” “macela da terra,” “macela do campo,” “chá de lagoa,” “carrapichinho de agulha,” “macela miuda” (Brazil); “yerba del chivo.” (Colombia); “marcela blanca,” “marcela hembra” (Uruguay); “viravilona” (Venezuela) (González et al., 1937; Hoehne, 1939; Saggese, 1959; Cárdenas, 1969; Arrillaga de Maffei, 1969; Garcia Barriga, 1975; Ratera and Ratera, 1980; Toursarkissian, 1980; Martínez Crovetto, 1981; Zardini, 1984; Oliveira Simoes et al., Author's personal copy D. Retta et al. / Industrial Crops and Products 38 (2012) 27–38 29 Fig. 1. Inflorescences of A. satureioides in their natural habitat. 1986; Correa and Bernal, 1990; Abdel-Malek et al., 1996; Alonso Paz et al., 2007; González Torres, 1997; Gupta, 1995). A. satureioides aerial parts and inflorescences are traditionally used as digestive, anti-inflammatory, antispasmodic, antidiabetic and anti-asthmatic agents (Ratera and Ratera, 1980; Toursarkissian, 1980; García et al., 1990; Heinzen et al., 2005), as a stimulant and emmenagogue (Paccard, 1905; Alonso Paz et al., 2007; González et al., 1993) and as a antipyretic agent (Parodi, 1979). This plant material also has tonic, stimulating and anthelmintic properties (Hieronymus, 1882), and is used for the treatment of digestive and intestinal disorders, colics, diarrhea, menstrual irregularities (Lorenzi and Matos, 2002). Some studies showed that the plant also has antibacterial activity (Zani et al., 1995). Plant material macerated in water is used as a slimming agent (Martínez Crovetto, 1981; Zardini, 1984; Dickel et al., 2007), and is also reported to have sedative and anxiolytic properties (Wannmacher et al., 1990). Other registered applications include the treatment of bronchial asthma (Manfred, 1958). Moreover, in Venezuela plant material is used as an antidiabetic infusion (Morton, 1975), to regulate blood pressure, for arthritis and as antipyretic agent (Hidalgo Báez et al., 1999). In Bolivia applications range from use as expectorant, sudorific and antipyretic agents; and boiled flower residues are used to relieve cough, even in children (Cárdenas, 1969; Bourdy et al., 1999). In Colombia, plant material has been used for the treatment of tumors (Gupta, 1995). 4. Phytochemistry A large number of compounds belonging to several phytochemical groups have been isolated from A. satureioides aerial parts and inflorescences. Polyphenols and flavonoids found in marcela include: caffeic acid, two esters of calleryanin (3,4dihydroxybenzyl alcohol-4-glucoside) with caffeic acid and protocatechuic acid, respectively, galangin, galangin-3-methyl ether, quercetin, quercetin-3-methyl ether (Dellacassa et al., 1993; Ferraro et al., 1981; Broussalis et al., 1989), gnaphalin and isognaphalin, quercetagetin, quercetin-3-methyl ether-7-diglucoside, tamarixetin, tamarixetin-7-glucoside, 3-caffeoylquinic acid, 4caffeoylquinic acid, 5-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid (Broussalis et al., 1988; Marques and Farah, 2009), luteolin, scoparol (Petrovick and Knorst, 1992), 5,8-dihydroxy-3,7-dimethoxyflavone (Haensel and Ohlendorf, 1971; Wagner et al., 1971), 3-methoxyquercetin (Simoes et al., 1988b), 3,5,7,8-tetramethoxy-flavone, 5,7,8-trimethoxyflavone, 7-hydroxy-3-5-8-trimethoxyflavone (Mesquita et al., 1986) and a new chalcone: achyrobichalcone (Holzschuh et al., 2010). Other compounds found in this plant include coumarins (Reinecke et al., 1995), polysaccharides (Puhlmann et al., 1992), achyrofuran (Carney et al., 2002), lactones (Schmeda Hirschmann, 1984; Kaloga et al., 1983) and polyacetylenides (Dembitsky et al., 2003). Mendes et al. (2006) identified five possible glycolipids in A. satureioides inflorescences. The essential oils, obtained by hydrodistillation, were studied using materials of different origins, which led to slight differences among results. In samples from Brazil, analyzed by GC/FID/MS. Lamaty et al. (1991) found ␣-pinene to be the most abundant compound (41–78%). Based on the composition of other compounds present, the authors classified the samples into 3 homogeneous groups, characterized, respectively, by: (a) a high content of (Z)- and (E)-␤-ocimene (3–12%) and ␤-caryophyllene; (b) the absence of ocimene isomers, low percentage of ␤-caryophyllene and presence of oxygenated monoterpenes, mainly pinocarveol and verbenone; and (c) a lower content of ␣-pinene, but high ␤-ocimene (27%) and 1,8-cineole (12%) content. Preliminary studies of this essential oil had already identified ␤-caryophyllene, 1,8-cineole, germacrene D and caryophyllene-1,10-epoxide (Ricciardi and Yunes, 1965; Akisue, 1971; Schmeda Hirschmann, 1984). In a subsequent study of plant material from central Argentina, 40% ␤-caryophyllene, 14% ␣-copaene and 9% ␦-cadinene was reported (Labuckas et al., 1999). Similar compositions were found in essences obtained from marcela aerial parts from southern Brazil and Uruguay, with ␤caryophyllene and ␣-pinene at 32 and 30%, respectively, in the samples from Brazil, and 11 and 46% in samples from Uruguay (Bauer et al., 1992; Lorenzo et al., 2000). ␤-Caryophyllene and ␣humulene were reported as major compounds in samples of leaves and young stems collected in Brazil obtained by hydrodistillation (Leal et al., 2006). 4.1. Analysis of main active components Numerous authors studied the content of active principles in species of Achyrocline spp. These were characterized by their high content of polyphenols and flavonoids. Many of these studies were designed based on what would be expected from traditional use. Sonaglio et al. (1986) developed a method for qualitative and quantitative analysis of A. satureioides inflorescence extract obtained by maceration with 60% ethanol. Separation was done by paper chromatography and quantitative determinations were based on analysis of quercetin by HPLC and UV spectrophotometry. Martino et al. (1989) also spectrophotometrically determined the content of caffeoylquinic acids in infusions and ethanolic extracts of aerial part of Achyrocline alata, A. satureioides and A. flaccida. Qualitative and quantitative analysis of caffeoylquinic derivatives from 8 commercial samples of marcela was done by López et al. (1996). In other study (Toursarkissian, 1980), chlorogenic acid, caffeic acid, ferulic acid, and isomers of isochlorogenic acid (3,4-dicaffeoyl quinic, 3,5-dicaffeoyl quinic and 4,5-dicaffeoyl quinic) were determined by HPLC in samples used as digestive Author's personal copy D. Retta et al. / Industrial Crops and Products 38 (2012) 27–38 30 Table 1 Biological activities tested. Activity Extract Observations Active constituents References Hepatoprotective and choleretic (digestive) Central depressant action 5% (w/v) infusion aerial parts Aqueous extract of leaves and stems Aqueous (macerate and decoction) and ethanolic extracts of inflorescences, leaves and stems Ethanolic extract of inflorescences Hydroalcoholic extract In vivo Flavonoids, caffeic and protocatechuic acids Kadarian et al. (2002) Whole-plant extract 80% ethanol macerate of inflorescences Essential oil In vivo In vivo Ethanolic extracts of aerial parts Ethanolic Hexanic and ethanolic extracts of inflorescences and leaves Aqueous In vivo Ibrahim and Zaki (1998); Park et al. (2005); Jaenson et al. (2006); Trongtokit et al. (2005); Yang et al. (2004); Gillij et al. (2008) Macêdo et al. (1997) In vivo In vivo Mendes et al. (1999) Rojas de Arias et al. (1995) In vivo Brandelli et al. (2009); González and Marioli (2010) Morquio et al. (2005) Desmarchelier et al. (1998); Gugliucci and Menini (2002a); Polydoro et al. (2004); Asolini et al. (2006); Grassi-Zampieron et al. (2009); Chiari et al. (2010) Leal et al. (2006) Antiinflammatory, analgesic, antispasmodic, constipating, miorelaxant and sedative Antispasmodic and miorelaxant Antiulcer gastric Antihyperglycemic Use in dysentery and diarrhea Repellency against mosquitoes Larvicidal Molluscidal Insecticidal and trypanocidal Antiparasitic In vivo In vivo Flavonoids Simoes et al. (1986, 1988a,b); De Souza et al. (2007) In vitro Flavonoids In vivo Terpenoids and flavonoids Achyrofuran Langeloh and Schenkel (1982); Langeloh (1988) Santin et al. (2010) In vivo Photoprotection Antioxidant and free radical-scavenging capacity, antiatherosclerotic Ethanolic extract Infusion, ethanolic and methanolic extracts In vivo In vitro Antioxidant Essential oil and ethanolic extracts Infusion of aerial parts Aqueous In vitro In vitro In vitro Antimicrobial Decoction In vitro Antimicrobial Alcoholic and essentials oil Aqueous extracts Alcoholic extract In vitro Cytoprotective Immunostimulant and antiinflammatory Antimicrobial Antiviral Antifungal Cholinomimetic and cholinolytic, miorelaxant Antiglycation Antiallergic Aqueous Simoes et al. (1986) Flavonoids Flavonoids Polysaccharides In vitro In vitro In vitro In vitro Infusion Leaves and flowers decoction Aqueous Ethanolic macerate of aerial parts In vitro In vitro Vasorelaxant Aqueous Protection of neuronal cells Antitumoral Infusion Methanolic extract of aerial parts, flowers Modification of the intracellular availability of calcium and the participation of NO In vitro In vitro Miorelaxant Miorelaxant Flavonoids In vitro In vitro (cholagogue-choleretic) and antispasmodic agents, for the treatment of hepatic disorders, and as bitters in aperitifs. De Souza et al. (2002) determined the flavonoid concentration by HPLC, expressed as luteolin. Schneider Cezarotto (2009) found composition variation in extracts collected throughout the year Quercetin and quercetin-3-methyl ether Carney et al. (2002) Rocha et al. (1994) Arredondo et al., 2004 Wagner and Ott (1991); Wagner et al. (1985); Puhlmann et al. (1992); Santos et al. (1999); Maldonado et al. (2001); Calvo et al. (2006), Cosentino et al. (2008); Alaniz et al. (2010) Anesini and Perez (1993); Calvo et al. (2006); Schneider Cezarotto (2009); Joray et al. (2011) da Silva Nina et al. (2007); Teixeira Duarte et al. (2005, 2007) Mota (2008) Zanon et al. (1999); Bettega et al. (2004) Vogt et al. (2010) Simoes et al. (1986); Filot da Silva and Langeloh (1994) Gugliucci and Menini (2002b) Maldonado et al. (2007) Simoes et al. (1986) Hnatyszyn et al. (2004) Vecchio et al. (2002); Gorzalczany et al. (2005) Flavonoids Blasina et al. (2009) Ruffa et al. (2002); Arisawa (1994) also corresponding to differences in antioxidant and antimicrobial activities. Quantitative analysis of flavonoids has been an area of focus since these compounds have been reported to have activities that account for several popular uses of the plant (Retta et al., 2011). These compounds include quercetin as anti-inflammatory and as an Author's personal copy D. Retta et al. / Industrial Crops and Products 38 (2012) 27–38 antihepatotoxic, antispasmodic and antiulcer agent (Simoes, 1988; Husain et al., 1987; Morand et al., 1998; Harborne and Willians, 2000; Díaz and Heinzen, 2006), luteolin as an antiplatelet and vasodilating agent (Santos et al., 1999; di Carlo et al., 1999) and 3-O-methylquercetin as having antiviral properties (Formica et al., 1995). Total polyphenols were determined by UV spectrophotometry in an infusion of an A. satureioides sample from Uruguay (Arredondo et al., 2004), using the Folin–Ciocalteu method (Singleton and Rossi, 1965) with caffeic acid as standard. The flavonoid content was quantified by HPLC, after hydrolysis of the glycosides. The chromatographic profile showed the presence of quercetin, luteolin, and 3-O-methylquercetin as major components, both in glycosylated forms and as aglycones. Debenedetti et al. (1993) and Lopez et al. (2006) evaluated qualitative and quantitative content of caffeoylquinic acid derivatives in A. satureioides, Achyrocline tomentosa, A. flaccida and A. alata aerial parts using HPLC. These studies showed extracts of A. flaccida to have the lowest percent content of caffeoylquinic acid derivatives. Different authors studied the effect of different extraction methods, and found that depending on the method, variations were found in dry extract yield, the flavonoid profile and the amount of free quercetin (Díaz and Heinzen, 2006; Takeuchi et al., 2010). In a recent study by Marques and Farah (2009) the content of chlorogenic acids and related compounds was determined in A. satureioides from Brazil. Caffeic, ferulic, and p-coumaric acids are trans-cinnamic acids occurring naturally in their free form or as a family of mono- or diesters with quinic acid, are collectively termed chlorogenic acids. The content of chlorogenic acids in methanolic extracts determined by HPLC was (mg/100 g): 3-caffeoylquinic acid (6.6), 4-caffeoylquinic acid (9.7), 5-caffeoylquinic acid (33.6), 3,4-dicaffeoylquinic acid (57.1), 3,5-dicaffeoylquinic acid (30.9), 4,5-dicaffeoylquinic acid (24.2), caffeic acid (3.4). Chlorogenic acids compositions in the 0.5% infusions (mg/200 ml) were: 3-caffeoylquinic acid (0.05–0.12), 4-caffeoylquinic acid (0.03–0.14), 5-caffeoylquinic acid (0.16–0.69), 3,4-dicaffeoylquinic acid (0.14–0.94), 3,5-dicaffeoylquinic acid (0.57–1.12), 4,5dicaffeoylquinic acid (0.27–0.53), caffeic acid (0.07–0.09). Del Vitto et al. (2009) analyzed the content of oligoelements in leaves and infusions of A. satureioides and A. tomentosa. Both species have similar composition, although A. satureioides contains a higher amount of essential minerals. The yields of essential oils obtained by hydrodistillation were, in general, low. In eight samples from Brazil, the average yield of essential oils was found to be 0.4% (v/w) (Lamaty et al., 1991). In related experiments Labuckas et al. (1999) reported yields lower than 0.2% and Lorenzo et al. (2000) reported yields between 0.3 and 0.45% (w/w). The study of seasonal variations of the oil composition from aerial parts was also investigated (Cezarotto et al., 2011). The results showed that yield and chemical composition of the essential oil could change according to the plant collection period. 5. Pharmacological activities Pharmacological activities of A. satureioides have been the subject of numerous studies in the past few years (Table 1). Many of these studies were focused on the high content of known active principles, such as flavonoids and polyphenols. Other studies were focused on its most relevant ethnobotanical uses and aimed at providing scientific evidence to support such uses, and some studies reported results of pharmacological screenings. The large volume of related literature and the positive results reported by most authors reflects the undoubted potential of this plant (Fraga et al., 1987; Carini et al., 1992; Sanz et al., 1994; Laughton et al., 1989; Petrovick et al., 2001; Morquio et al., 2005; Dajas and Heinzen, 31 2004; Dajas, 2005; Dajas and Heinzen, 2001; Heinzen and Dajas, 2003; Mototsugu et al., 1998; Gomes, 2006; Jia et al., 2003; Chiari et al., 2010; Del Solar, 2008). Despite the widespread use of marcela in the MERCOSUR (Common Southern Market) region, European standards remarkably prohibit its use in food products, in compliance with Belgian Law (Moniteur Belge, 2006; European Food Safety Authority, 2007, 2009). The exclusion of the use of marcela from European markets was not consistent with previous determination of toxicological properties published for marcela (Simoes et al., 1988a; Carney et al., 2002; González et al., 1993; Fachinetto et al., 2007; Rojas de Arias et al., 1995; González et al., 1993; Bettega et al., 2004; Ferrari et al., 1993; Rivera et al., 2004; Polydoro et al., 2004; Arredondo et al., 2004; Vargas et al., 1990, 1991). There was still some concern on the part of European agencies in this regard since a low toxicity was noted in vivo and a marginal toxicity in vitro, in some test using Artemia salina or the Ames test. These results, however, were interpreted as promising pharmacological activities rather than as a health risk. In spite of European agency concerns, the use of marcela is apparently very safe by its widespread use since ancient times, which has never been associated with toxicity or compromising healthy in any way. For these same reasons marcela can be used as a flavoring agent under MERCOSUR (MERCOSUR, 1993) and as an official drug in the Brazilian Pharmacopoeia (Farmacopea Brasileira, 2001). 6. Aromatic properties of A. satureioides The overall olfactive note of marcela is herbaceous, earthy and spicy, reminiscent of lovage, celery and fenugreek. Olfactive evaluation of marcela essential oils revealed an aromatic profile distinct from the olfactive characteristics of the plant itself. This suggested that the volatile compounds extracted from the plant may not be exactly the same as those that account for the olfactive characteristics of marcela. Within the framework of the COTEPA Project involving the European Union and Uruguay, sensory evaluations of marcela essential oils were conducted by several European perfume companies. The aromas of marcela were compared with that of Helicrysum italicum (Davies and Villamil, 2004) and marcela was valued as a potential perfume ingredient. Tests conducted on a marcela resinoid obtained by ethanol extraction demonstrated that the typical aromatic fraction of A. satureioides is best maintained as an extract of this type (Bandoni, 1992). Despite various attempts to characterize the compounds that define the olfactive profile of marcela, no such compounds have as yet been identified (Fernandes et al., 1996). Aqueous and hydroalcoholic marcela extracts are highly complex, aromatic, and bitter in taste. Studies involving experienced testers enabled identification of differences according to extract origin, e.g., aerial portion extracts or flower-derived extracts (Retta et al., 2010). Marcela extracts are used in several products marketed in the MERCOSUR region for their characteristic complex, bitter, aromatic flavor (Ares et al., 2010). The same authors (Ares et al., 2009) also studied how to reduce the bitterness, astringency and characteristic flavor of extracts of marcela, Milk was the most effective of these inhibitors tested with A. satureioides extracts. Marcela is increasingly used in the fragrance, flavoring and soft drink industries, and additional applications might include production of alcoholic drinks, sauces as well as tobacco flavoring. 7. Market potential The inclusion of A. satureioides in the official Brazilian Pharmacopoeia in 2001 (Farmacopea Brasileira, 2001) was the result of the dedicated efforts of numerous research teams who Author's personal copy D. Retta et al. / Industrial Crops and Products 38 (2012) 27–38 32 Fig. 2. Inflorescences of A. satureioides collected in Argentina (A) and adaxial side (B) of the leaf with glands containing essential oil. established a basic framework for better understanding the possible pharmacological, pharmacotechnical and agronomical applications of marcela. Inclusion in the Pharmacopoeia also constituted recognition of the widespread traditional use of this plant species throughout Brazil’s southern region. This is reflected in several marcelaand marcela extract-based products available on the Brazilian market ranging from herbal infusion drinks to cosmetics. In Argentina marcela-based products are used in conventional medicine, in phytotherapeutic formulations and in the manufacture of food products that require its bitter aromatic flavor (Bastianello Campagnol et al., 2011). It is estimated that the demand for this plant in Argentina is ca. 20 tn/year. In Uruguay, it is marketed as an everyday tea in local shops and restaurants and is also used in the manufacture of marcela-containing cosmetics by virtue of its antioxidant and anti-inflammatory properties and UV-blocking action. Likewise, in Paraguay, most herbal infusion products include Yaetí-kaá or marcela as a traditional ingredient. It has been included in the European standards as an ingredient for use in cosmetics under the name “Achyrocline satureioides flower oil” (European Commission, 2010, CAS 92346-81-1, EINECS 296165-3). 8. Cultivation 8.1. Cultivation conditions Whereas marcela is collected on an extractive basis in most of the countries in Latin America, several efforts for domestication of the plant have been initiated. The efforts were made with the objective of improving the quality and homogeneity of the marketed product. Bourdy et al. (1999) cite the cultivation of marcela managed by some local indigenous (Tacana) villages in northwestern Bolivia, reflecting the importance of marcela in some regional cultures. Experience in the management of marcela crops and the major agroclimatic factors that impact production are discussed in following section. is determined primarily by its content of specific secondary metabolites the abundance of which is a result of ecological relationships between the plant and its biotic and abiotic environments or stress conditions (Piñol et al., 2000; Wagner et al., 2006). Effective domestication of commercially valuable plants thus provides new challenges and perhaps greater complexity than for many food crops. Marcela is better adapted to moderate climates, and grows during the coldest months of the year. Its leaves are characterized by a spongy parenchyma on the abaxial side, which has lower photosynthetic efficiency than the palisade parenchyma on the adaxial side (Fig. 2). This would be consistent with the adaptation to a mesophytic environment, free of water-stress. However, the presence of glandular trichomes (associated with the production of essential oils) and nonglandular trichomes (associated with a adaptation to low temperatures, evapotranspiration and herbivory) suggests physical and metabolic adaptation to a range of environments involving different types of stress. Consistent with these observations, studies on the distribution of marcela showed that the plant is best adapted to moderate climates and is constrained by low temperature, as it is not found beyond 41◦ S (Suárez et al., 2010). In the case of an allogamous species like marcela, there is great genetic variability, which is expressed in commercially relevant characters such as biomass productivity, as well as leaf morphology and architecture, flowering season, the content of active substances and the percentage of propagation by cuttings (Magalhaes, 1997, 2000). Over the past 15 years, these and other characters have been explored as part of a domestication program for this species in southern Brazil. These efforts have resulted in the generation of hybrid lines such as higher AS-2 and AS-3 (Montanari Jr, 1997; Magalhaes, 2000). Other experimental plantations are developing breeding programs in the central and mesopotamian regions of Argentina, Paraguay and Uruguay. In Argentina, two populations from widely contrasting environments in the hills of Comechingones, Córdoba (i.e., different latitude and altitude) were grown under the same conditions. Factorial experiments (population and density) enabled characterization of the two populations as distinct ecotypes (Cardoso et al., 2009, 2010). 8.2. Domestication 8.3. Multiplication by seeds The method of collection for marcela is extremely exploitative and destructive, posing severe threat to its biodiversity. Therefore, a sustainable collection strategy needs to be adopted that will conserve the valuable medicinal plants. Alternatively the cultivation of marecla is a promising option as there is availability of experience generated (Davies and Villamil, 2004). Management of marcela as a crop is intended not only for the purpose of domestication, but also maintaining and protecting of wild populations and natural biodiversity of the species. The commercial value A. satureioides The literature includes vegetative propagation studies as well as general data relevant to different alternatives (Marques and Barros, 1995, 1996, 2001; Magalhaes, 2000; Serdiuk et al., 2000) as in vitro propagation cultivation (Barros and Castro, 1995; Diefenthaeler et al., 1996) of marcela. Marques and Barros (1999) also found that the germinative power of seeds increased from 68% to 71% after 6 months’ storage at room temperature (19–23 ◦ C) in a dry place. In contrast, a high ambient humidity has a negative influence (Fig. 3). Author's personal copy D. Retta et al. / Industrial Crops and Products 38 (2012) 27–38 33 Compounds found in A. Satureioides OH OH OH O OH HO O OH HO OH O OH O HO OH 2 OH O OH O 3 OH 1 OH OH OH OH HO HO O O 6 O OH OH O O 5 4 7 8 1, chlorogenic acid; 2, caffeic acid; 3, quercetin; 4, quercetin-3-methyl ether; 5, luteolin; 6, α- pinene; 7, -caryophyllene; 8, achyrofuran Fig. 3. Compounds found in A. satureioides. 8.4. Multiplication by cuttings The use of apical branch cuttings is recommended for vegetative propagation (Davies, 1993, 1997). To avoid early flowering of the resulting plantlets, material from the mother plant should be obtained during vegetative stages of development, avoiding material cut from the mother plant during flowering or just prior to flowering (Davies, 1993, 1997). Several preliminary studies were made on the in vitro vegetative micropropagation of marcela (Ikuta and de Barros, 1992; Gattuso et al., 2007; Severin et al., 2008). Fungal and bacterial contamination problems were found, mainly caused by Pseudomonas spp. The positive effect of some phytoregulators that promoted the growth (in length) of axillary buds was also studied. marcela crops, leading to considerable damage unless kept under control. 8.6. Spacing between plants and irrigation Plants should be spaced by a distance of 30–45 cm, and at a density of 40,800 plants/ha and using a 35 cm × 70 cm planting frame for single-row planting. Spacing can be increased 30% if two-row bed planting is used. Araújo et al. (2009) studied a crop consociated with Plantago major, without any interference between species. Irrigation is essential during months when plants are kept in a nursery. Once transplanted, irrigation is necessary until plants become established. Symptoms of hydric stress are manifested as withering of apical branches; yet, A. satureioides is robust and withstands a water stress. 8.5. Soil preparation 8.7. Cultural laboring and plant care Although marcela can thrive in widely different soil types, ranging from sandy to clay soils, cultivation soil should have excellent drainage. Problems commonly found in orchard crops, such as disease resulting from fungi like Botritis and Sclerotinia, also occur in Experience with crops in Brazil indicated that marcela can easily be managed, with the exception of the major care needed during transplanting (Magalhaes, 2000). A. satureioides extract production, in addition to production being correlated with Author's personal copy 34 D. Retta et al. / Industrial Crops and Products 38 (2012) 27–38 favoring crop growth, has been reported to show allelopathy, i.e., it inhibits the germination of weed that might otherwise compete for resources (Ungaretti et al., 1997; Aquila et al., 1999). 8.8. Fertilization and nutrient supply Although marcela does not have special requirements regarding fertilizers, organic fertilization with 3.0 kg/m2 of stockyard manure or organic compost, or 1.5 kg/m2 of poultry manure, is sufficient and recommended (Correa et al., 1994). Other experiments (Davies, 1997) showed, however, that productivity can be increased considerably by nitrogen fertilization in pots. Flower dry weight could be increased by ca. 45% with the addition of the equivalent of 30 N units/ha and by 122% with the addition of 60 N units/ha. These results were interesting and experiments should be repeated in cultivation plots having the same density of plants normally used for production. Leite et al. (2009) found that both phosphorous and poultry manure fertilizer had a positive effect on flavonoid content produced in marcela. Bottega et al. (2009) also found that the quality essential oil in marcela varied with the addition of manure or phosphorous fertilizers. 8.9. Diseases During the vegetative growth period, plants can be affected by Botritis and Sclerotinia; while in the reproductive phase, a blackening of inflorescences may be caused by fungi such as: Alternaria spp., Cladosporium spp. and Epiccocum spp. Secondary (non pathogenic) fungi were identified and were associated with high-humidity conditions during flowering. If harvest is performed in these conditions, these fungi appear as seed contaminants and primarily affect the quality of harvest (germination, vigor, etc.). 8.10. Harvest, collection, drying and storage The crop normally allows for two harvests: the first one is 1 year alter field transplanting, between March and April (Uruguay) and the second one is the following year at the same time of year. Radaelli et al. (2009) reported variation in the quality of marcela essential oil depended on the time of harvest. Collection can be done manually, using a scythe to cut the inflorescences or pruning scissors to cut leaves. Also a “comb” may be used to separate the inflorescences (Magalhaes, 1997, 2000). Flower and leaf drying may be done in air-circulation dryers at a temperature not exceeding 40 ◦ C. Dried marcela material should be stored in a cool dry place, away from sunlight. 8.11. Yield Flower dry weight yield of two lines developed in Brazil (AS2 and AS-3), planted using a 1.0 m × 1.0 m distance, was 336 and 570 kg/ha, respectively (Magalhaes, 2000). In Uruguay, yield values of 1218 and 920 kg flower dry weight/ha was obtained for these lines using 30 cm and 45 cm spacing between plants, respectively (Davies, 1997). In another study, using a plantation density of 40,800 plants/ha, 10,040 kg green matter/ha was obtained, corresponding to 2774 kg/ha of dry matter. In the same study, flower dry weight was also increased by 45 and 122%, respectively, in plots treated with 30 and 60 kg urea/ha, respectively. Soares et al. (2007) demonstrated the economic feasibility of marcela cultivation in Brazil, associated primarily with small-scale production. 9. Concluding remarks Marcela is a good example of a plant well-established by longterm use by native populations. As a result it has been included in many conventional medicinal products, cosmetics and foodstuffs, and was formerly accepted by its inclusion in the Brazilian Pharmacopoeia as well as within MERCOSUR for specific usage as a food product. The extensive bibliography on their biological activities and the existing patents on its various applications are also consequences of its widespread use. There is, however, a general lack of awareness of the potential of marcela in global markets in spite of it attributes (essence and flavor) that have significantly influenced dramatic expansion in commercialization recent years. Marcela has not yet succeeded in generating a demand beyond the South America countries of origin, possibly for two main reasons: (a) insufficient or ineffective marketing in global communities that could benefit from use of marcela-derived products and (b) lack of a sustainable supply chain complying with established international standards for both quality and quantity. Regarding this last objective, we should emphasize the need to encourage multidisciplinary studies in order to improve the agronomic management of marcela guiding the crop to the production of a material with the more desirable physicochemical and phytochemical properties in terms of marketable industrial use. Acknowledgments Part of this article was supported by projects Universidad de Buenos Aires (BO 14) and PICT 2008-1969. References Abdel-Malek, S., Bastien, J., Mahler, W., Jia, Q., Reinecke, M., Robinson Jr., W., Shu, Y., Zalles-Asin, J., 1996. Drug leads from the Kallawaya herbalists of Bolivia. 1. 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