RESEARCH WORK
Qualitative characterization of the content of secondary metabolites in the edible fraction of Tithonia diversifolia (Hemsl.) A. Gray
¹Universidad de Ciego de Ávila "Máximo Gómez Báez",Carretera Morón Km 91/2. Ciego de Ávila, Cuba
E-mail: pfa_yohanka@agronomia.unica.cu
²Estación Experimental de Pastos y Forrajes "Indio Hatuey", Matanzas, Cuba
³Centro Nacional de Sanidad Agropecuaria, Mayabeque, Cuba
ABSTRACT
In order to characterize the phytochemical composition in the edible fraction of Tithonia diversifolia a study was conducted at the EEPF "Indio Hatuey", during the rainy (RS) and dry seasons (DS), with two stages of the physiological cycle (30 and 60 days). The evaluated plant parts were leaves, fresh stems and leaves plus fresh stems. For detecting secondary metabolites phytochemical sieving was used, from an initial methanolic extract. The groups of compounds identified through the method were primary and secondary amino groups, free phenols, tannins, tripertenes and/or steroids, which were detected in low quantities in both periods. Alkaloids were the ones with higher amounts in all the plant fractions during the RS, except in stems and leaves plus stems after 60 days; in the DS they appeared in moderate quantities in all fractions for both ages, except in stems after 60 days. No cardiotonic glycosides, flavonoids, leucoanthocyanidins or saponins were detected, for any plant part. T. diversifolia was concluded to show secondary metabolites in the edible fraction, after 30 as well as 60 days, where alkaloids remarkably stand out during both seasons. In general, the presence of the other metabolites was small and no differences were detected in their appearance, with regards to season and stage of the physiological cycle. To conduct further quantitative studies of these compounds is recommended, to determine the inclusion of this plant in diets for animal feeding.
Key words: metabolites, Tithonia diversifolia.
INTRODUCTION
Tithonia diversifolia (Hemsl.) A. Gray is a non-leguminous plant belonging to the Asteraceae family, which stands out for its excellent capacity to produce edible biomass of high feeding quality. Since the 90's its forage potential began to be evaluated and its use was recommended in cut and carry systems for sheep, goats and cattle, as well as for monogastric animals (Alonso et al.,2010; La O et al., 2010).
According to García et al. (2008), this plant contains 1 200 classes of secondary compounds. However, because of their high diversity not all of them have been studied, although some groups are more known, such as polyphenols, cyanogenic glycosides, saponins, steroids and phytohemoagglutinins.
In this regard, Valdés and Balbin (2000) define secondary metabolism as the biosynthesis, transformation and degradation of endogenous compounds of plant species, which, under certain circumstances, may cause diverse and even contrasting effects on animal physiology, related to forage acceptability and digestion inhibition, by affecting the catalytic activity of some enzymes, which may limit feed absorption (Ahn et al., 1997).
Nevertheless, the existing knowledge about secondary metabolites, present in the edible parts, as well as the effect of the factors which can influence their variations, is not enough. For such reason, the objective of the work was to evaluate the influence of the stage of the physiological cycle (30 and 60 days) on the presence of secondary metabolites, in the edible fraction of T. diversifolia.
The study was conducted at the EEPF "Indio Hatuey", which is located at 22º 48'87'' latitude north and 81º 32'2'' longitude west, at 19 masl, Perico municipality, Matanzas province. The soil has plain topography and is classified as lixiviated Ferralitic Red (Hernández et al., 2003).
The samplings were conducted at three moments of the rainy RS- (May-October) and dry seasons DS- (November-April), respectively, corresponding to 2009 and 2010.
The plantation of T. diversifolia had a total area of 1 248,0 m2, composed by 960 plants, which were planted with a frame of 1,0 m between plants and 1,30 m between rows. Neither irrigation nor fertilization was applied.
The design was completely randomized, and the treatments corresponded to the stages of the physiological cycle of 30 and 60 days in each season. Three manual cuttings were made, with pruning shears, at a fixed height of 50 cm above the soil level. Before the first cutting in each season, a homogenization cut was performed.
The evaluated plant material was the edible fraction of T. diversifolia (leaves, fresh stems and leaves plus fresh stems). From 10 plants, randomly selected in the evaluated areas after eliminating the edge effect, 10 samples were manually taken from each fraction, of 300 g each; they were taken to the laboratory of chemical analysis, where they were dried and packed, and transferred to the National Center of Agricultural Health for their phytochemical analysis.
The secondary metabolites were qualitatively characterized using the phytochemical sieving suggested by Rondina and Coussio (1969), modified by Alfonso et al. (2000) and based on the fractioning of an initial methanolic extract of the plant parts, which was obtained by maceration during 24 hours and by reflux for two hours. On the obtained fractions, coloration and/or precipitation reactions with different specific or selective chromophore agents were done, for the following functional groups: primary and secondary amines, phenols, tannins, tripertenes and/or steroids, cardiotonic glycosides, alkaloids, flavonoids, leucoanthocyanidins and saponins (table 1). In the qualitative analysis the system of crosses was used and the presence or absence of secondary metabolites in the samples was specified, according to the criterion expressed by García (2003). Pattern compound solutions were previously used for reagent control (table 1).
Tables 2 and 3 show the phytochemical characterization of T. diversifolia in the RS and DS, respectively. The groups found were: primary and secondary amines, free phenols, tannins, tripertenes and/or steroids and alkaloids; while cardiotonic glycosides, flavonoids, leucoanthocyanidins and saponins were not detected in the essays conducted, in both seasons.
The primary and secondary amines were present in low quantities in all plant parts, in both seasons. However, this result differs from the report by Galindo (2009), who did not found these compounds in the same forage species.
Phenols had a low presence in almost all fractions in both seasons, because they were not detected in the stems after 30 days, or in leaves and stems after 60 days, in the DS. The presence of phenols revealed the typical black coloration shown by this test, which is characteristic of extracts that contain a large diversity of hydroxylated structures. These results differ from the ones reported by García and Medina (2005), who did not found phenols; however, they stated that, from the nutritional point of view, the presence of polyphenolic fractions in such forage plants as Leucaena leucocephala and A. cyanophylla is positive, because they have hydroxyl groups (OH) in their structure. In this sense, Ben Salem et al. (2000) found phenols in these plants, with bactericide and fungicide activity, at different concentrations.
Tannins were present in low quantities in the RS, and were not detected in the leaves in both ages or in the fraction leaf and stem after 30 days. In the DS they were found in low quantities in all fractions and ages, except in stems after 30 days, where they were not detected, which could have been related to the use of the jelly assay to induce precipitation, or to the molecular weight of tannins. Only those precipitation assays where a remarkable molecular weight is present, allowing protein-binding and its insolubilization are considered to be positive (García and Medina, 2006).
From the nutritional point of view, the moderate presence of tannins in the diet of ruminants was favorable, increasing the possibility of forming bypass protein not degraded in the rumen-, which facilitates post-ruminal digestibility and contributes to an adequate digestive functioning and higher animal response. This mechanism may be explained by the ability of tannins to form complexes with feed or endogenous proteins, through multiple bindings, which creates a complex tridimensional structure of low digestibility at ruminal level. For such reason, forage plants that contain tannins and, also, adequate protein content constitute excellent supplementary sources for ruminant feeding in the tropics (García et al., 2006).
On the contrary, when tannin levels in forage plants are very high, they can cause nutrient losses and poor utilization of rations in ruminants, as well as acute toxicity in monogastric animals (Abdulrazak et al., 2000). The active principles of tannins are also characterized by being astringent, due to their protein precipitation capacity, which gives them antidiarrheal, vasoconstrictor, antimicrobial and fungicide properties (Mahecha et al., 2007).
The tripertene and/or steroid group was detected in both periods, except in leaves and stems after 30 days, with moderate presence in the latter after 30 days. From the applied essay, the intense greenish blue color indicating the presence of several sterols in plant tissue was revealed, which has been reported in the edible part of T. diversifolia and forage trees, by Murgueitio et al. (2009), from moderate to considerable amounts.
Some integral studies have determined that sesquiterpene lactones constitute, within tripertenes and/or steroids, one of the structures of higher phytochemical interest, due to their pharmacological potential, because they are used as diuretic, expectorant, cicatrizant and anti-inflammatory (Villalba and Provenza, 2005). These compounds give this forage an acutely bitter taste (García et al., 2008), which could influence their acceptability. Nevertheless, no toxicity-related problems or adverse physiological effects have been observed, in species fed experimental diets based on this shrub (Lauser et al., 2006).
Alkaloids showed the highest quantities in the RS for all the plant parts, except in stems and leaves plus stems after 60 days, where they appeared in low amounts. In the DS this metabolite had a moderate performance in all the edible fractions; it showed slightly in stems after 60 days, which could be ascribed to the fact that the growing plant parts, especially the youngest, show higher concentration of secondary compounds (or their reactivity) as compared to old tissue (Bagnarello et al., 2009). These groups of metabolites are the ones with the widest natural distribution corroborated in forage trees and, particularly, in forage legumes (Baldizán et al., 2006).
The presence of alkaloids in a plant provides a distinctive character to the crop, which has little variation before such ecological factors as climate, seasons and water availability. In addition, it has been stated that these compounds may be used as nitrogen reserve material for aminoacid synthesis, and as protection against external agents such as fungi and herbivores (La O et al., 2010).
There are reports of the presence of other secondary metabolites in the edible fraction of this species, which were not determined in this study, such as: coumarins (possibly colinin), cytotoxic compounds tagitinin and hispidulin and flavonoids, which are ascribed cytotoxic activity in carcinogenic leukemic cells (Owoyele et al., 2004; Kuroda et al., 2007).
García et al. (2008) determined the substantial presence of terpenoids, lactones and pyrans in the edible part of T. diversifolia, which is a topic that could be further developed in future studies, although the cytotoxic effect of only a small group of metabolites present in this species has been reported.
T. diversifolia was concluded to show secondary metabolites in the edible fraction, after 30 as well as 60 days; the alkaloids considerably and remarkably stood out during the RS and DS. In general, the presence of the other metabolites was low and no differences were detected in their appearance, regarding season and stage of the physiological cycle. To conduct further quantitative studies of secondary metabolites is recommended, to determine the inclusion of this plant in diets for animal feeding
