RESEARCH WORK

Qualitative profile of secondary metabolites in the edible fraction of woody species selected by cattle in a semi-deciduous forest

Á. Ojeda¹, N. Obispo², J. L. Gil² and Irana Matute²

¹Instituto de Producción Animal, Facultad de Agronomía, Universidad Central de Venezuela. Maracay 2101, Venezuela.
²Instituto Nacional de Investigaciones Agrícolas. Centro Nacional de Investigaciones Agropecuarias. Maracay 2101, Aragua. Venezuela.
E-mail: ajojeda99@yahoo.com

ABSTRACT

In order to characterize qualitatively the profile of secondary metabolites of the edible fraction of woody plants selected by cattle in a silvopastoral system of a tropophilous semi-deciduous forest of Venezuela, 14 Brahman x Holstein bulls of 390,4 ± 18,0 kg of LW were used. They were managed under continuous grazing on a surface of 81 ha, from which 50 ha corresponded to a semi-deciduous forest and the rest, to natural herbaceous vegetation (Cynodon nlemfuensis, Sporolobus indicus, Axonopus sp., Mimosa pudica and Hyptis suaveolens). The composition of the woody biomass selected by the animals was studied through the evaluation of the epidermal fragments present in the feces collected from the rectum, every 15 days. From these results, samples were taken from five plants per woody species, in the dry and rainy seasons. Fifteen groups of metabolites were determined: alkaloids, á-amines, soluble carbohydrates, cardenolides, cyanogens, sterols, total phenols, flavonoids, lectins, mucilages, quinones, saponins, extractable (total and condensed) tannins and terpenoids. Most of the biomass corresponded to species from the Fabaceae family, which showed all the evaluated metabolites. In both seasons levels of sterols, terpenoids, total phenols and extractable tannins which varied from slight to abundant, were detected. Independently from the season, 66,7 % of the species showed between 10 and 11 secondary metabolites in their edible biomass. It is recommended to conduct studies for the quantification of the secondary metabolites present in woody plants of forage value, for this silvopastoral system.

Key words: Fabaceae, silvopastoral systems.

It is estimated that in America there are 519 597 km² of savannas, from which 51 % is located in the south of the continent. From this surface, Venezuela has 19 396 km², associated in 80 % to formations of tropical dry forest, where farmers promote the entrance of cattle to the forest areas as the dry season advances, in an empirically developed model, in order to guarantee the increase of the quantity and quality of the forage offer (Baldizán and Chacón, 2007; Portillo-Quintero and Sánchez-Azofeifa, 2010).

To design sustainable management strategies of silvopastoral systems in tropical forests, it is necessary to conduct further studies related to the characterization of plant resources, with particular emphasis on their chemical and nutritional evaluation. Although at local level studies have been performed about some forage woody plants, as well as evaluations of silvopastoral systems in formations of dry deciduous forests (Benezra et al., 2003; Baldizán et al., 2006; García et al., 2006), there is no available information about the profiles of the secondary metabolites present in the biomass of woody plants which are selected by cattle when grazing in dry semi-deciduous forests.

The term «secondary metabolites» is used to identify ecologically efficacious substances, of wide chemical diversity and which can exert opposite effects on the optimal nutrition of the animals, due to the affectation of the digestive and metabolic processes (Kumar, 1992). Secondary metabolites exceed 80 000 compounds, among which the following stand out: 29 000 terpenoids, 12 000 alkaloids, 8 000 polyphenols, 600 non protein amino acids, 100 amines and 100 cyanogens. These substances have functional importance for plant tissues, because they act as allelopathic agents and against fungal invasions; offer protection against ultraviolet radiation and dehydration, with nitrogen reserve; and establish mutualism and toxicity relations with predators, among other benefits (Mazid et al., 2011).

The ingestion of plant biomass containing these metabolites can generate adverse effects on the voluntary intake, nutrient digestion and absorption, the intermediary metabolism and functioning of organs or systems (Sorensen et al., 2005); although it has been stated that moderate concentrations are beneficial for ruminant nutrition, because an improvement of the pattern of synthesis of volatile fatty acids and an increase of the flow of nutrients to the posterior tract occur, as well as a reduction of meteorism, gastrointestinal parasites and enteric methane emissions (Rochfort et al. 2008).

The objective of this study was to characterize qualitatively the profile of secondary metabolites of the edible fraction of woody plants selected by cattle under silvopastoral system conditions, in a tropophilous semi-deciduous forest of Venezuela.

MATERIALS AND METHODS

The study was conducted at the Research Station San Nicolás (8º 49' 58'' North latitude and 69º 48' 00'' West longitude), belonging to the Central University of Venezuela. A surface of 81 ha was selected, with soils classified as Fluventic Haplustepts and Vertic Endoaquepts, of mixed fine, non acid and isohyperthermal clayey texture (Abarca, 2005).

During the experimental period the climate unit of the station recorded 1 253,8 mm of rainfall; 5,9 ± 1,01 hours of insolation; 27,7 ± 0,62 ºC of temperature; 1 832,5 mm of potential evapotranspiration and 72,2 ± 14,3 % of relative humidity. The rainfall-temperature relation allowed to define a rainy season (May-November) and a dry season (December-April).

Fourteen whole crossbred Brahman x Holstein bulls, of 390,4 ± 18,0 kg of LW were used, under continuous grazing. From the considered surface, 50 ha corresponded to a tropophilous semi-deciduous forest, of low anthropic intervention level; while the rest was occupied by herbaceous vegetation (Cynodon nlemfuensis, Sporobolus indicus, Axonopus sp., Mimosa pudica and Hyptis suaveolens, among others), with a yield of 969,3 ± 28,6 kg DM/ha, and a leaf:stem ratio of 0,53 ± 0,15. This information was obtained from grids of 0,25 m², placed every 10 m on zigzag transects, approximately 120 m long, which were located depending on the plant variation and the topography of the sampling area.

The animals received water ad libitum and a commercial mixture of salts and minerals, composed by: 20 % Ca; 10 % P; 9 % Na; 0,6 % S; 0,25 % Cu and 80 ppm of I; among others.

To allow the selectivity of the animals under the conditions of the usual stocking rate for the natural savannas of the zone, and depending on the grass biomass, additional bulls were used, in order to guarantee a grazing pressure of 6 kg DM/100 kg LW, and a weight variation of 450 ± 35 g/animal/day. Fifteen days before the beginning of the evaluation, they were dewormed with levamisole hydrochloride (1 mL/50 kg of weight) and 1 mL/100 kg LW of a multivitamin complex (500 000 IU/mL vit. A, 75 000 IU/mL vit. D₃ and 50 IU/mL vit. E) was applied.

The selectivity of the woody biomass was recorded during the dry season, because only in this time lapse farmers allow grazing in the forests, to promote then the recovery of the ecosystem during the rainy season. The structural composition of the woody biomass was estimated through the evaluation of feces samples, individually collected directly from the rectum, between 8:00 and 10:00 h, every 15 days.

The samples were placed in plastic bags and stored at -18 ºC, until their dehydration in a forced air stove at 65 ºC, during 48 h. Afterwards they were ground in sieve of Ø 1 mm. From this material 2 g were taken, which were subject to processes of rehydration with 50 % v/v ethylic alcohol, sieving (180 µ) and removal of the plant pigments with sodium hypochlorite; the lay out was made on semi-permanent glass slides, at a rate of three 24 x 24 mm slides for each sample (Holeckek, 1982).

The epidermal fragments of the woody biomass were observed through a binocular microscope, at 400 X, and were compared with digital photographic patterns obtained from the leaf epidermal tissue of the woody plants present in the forest, which were botanically identified in the Herbarium Víctor Manuel Badillo (MY), of the Central University of Venezuela.

The design was completely randomized and samples were taken from five plants for every ligneous species selected by the animals. The samples were grouped per plant, according to the two seasons of the year (dry and rainy), and were monthly collected using pruning shears and at a rate of 200 g/plant. For the purpose of sample taking, the plant structures with diameter lower than 6 mm and maximum height of 2 m were considered susceptible to browsing by adult cattle.

The qualitative profile of secondary metabolites was determined according to the methodology proposed by Rondina and Coussio (1969), with the modifications suggested by Galindo et al. (1989) and García (2003). For that purpose, 25 g of each sample were taken and macerated with 25 mL of 98 % ethanol during 4 h, in a Soxhlet extractor. The obtained extract was filtered in paper of 4 µm diameter up to a volume of 50 mL. From this solution, and by the successive processing of filtrates and solid residues, a total of six fractions were obtained. Then the qualitative determinations of 15 groups of metabolites were conducted: alkaloids, á-amines, soluble reducing carbohydrates, cardenolides, cyanogens, sterols, total phenols, flavonoids, lectins, mucilages, quinones, saponins, extractable (total and condensed) tannins and terpenoids. The presence or absence of these compounds was systematized from: the abundant (+++), moderate (++) or slight (+) presence, and the absence (-). In the case of saponins, the foam height was used as criterion and the following levels were considered: abundant (> 14 mm), moderate (10-14 mm) or low (< 10 mm), and absent (0 mm).

RESULTS AND DISCUSSION

Tables 1 and 2 show the qualitative profiles of secondary metabolites corresponding to the edible fraction of the woody plants selected by the bulls under grazing conditions, in the two seasons of the year.

The browsed plants belonged to the following botanical families: Boraginaceae (Rochefortia spinosa), Fabaceae (Enterolobium cyclocarpus, Inga laurina, Machaerium humboldtianum, Pithecellobium lanceolatum, Samanea saman and Senna sp), Polygonaceae (Coccoloba caracasana) and Sterculiaceae (Guazuma ulmifolia).

In a floristic inventory carried out in the semi-deciduous forest, Ojeda et al. (2012) reported that in a density of 463 plants/ha there were four species (G. ulmifolia, P. lanceolatum, S. saman and Guadua angustifolia), which represented 80,2 % of the population of the woody plants; from them, the first three accumulated the highest records for the Importance Value Index (IVI). IVI is a variable related to voluntary intake, because, in the absence of physical or chemical limitations, the spatial distribution has influence on the opportunity that have the grazing animals of having contact with a particular species (Provenza and Balph, 1988), which allows to explain that 66,7 % of the selected species in this evaluation were Fabaceae.

The preference of ruminants for this family under silvopastoral system conditions has been documented by other authors (Benezra et al., 2003; Mayer et al., 2003; Rutter, 2006). Even though the highest components of the ration tend to maintain its prevalence under different management circumstances, it should be stated that the variations in the botanical composition of the diet can be influenced by the climate conditions that regulate time and spatial variability of the forage offer; as well as by some intrinsic factors of the plant and animals, among which the nutritional value, architecture, animal age and physiological status, grazing intensity, feeding experience, social learning (individual and collected) and neophobic response, stand out (Marquardt et al., 2009).

On the other hand, the 15 evaluated secondary metabolites were found in the edible fraction of the woody plants. Likewise, independently from the season, the plants showed sterols, terpenoids, total phenols and extractable (total and condensed) tannins.

In the dry season, the sterols and terpenoids had slight to moderate concentrations, with a higher presence in E. cyclocarpus and P. lanceolatum; while in the rainy season they prevailed in I. laurina, P. lanceolatum and S. saman.

Likewise, mixtures of steroids (blue shades) and poly-glycosylated triterpenoids (green and yellow colorings) were identified, which indicated the presence of â-citosterol and stigmasterol, metabolites which are related to the processes of activation of plant growth and biosynthesis of steroidal (C₂₇) and triterpenic (C₃₀) saponins of potential toxic effect, (Marcano and Hasegawa, 2002). As a group, sterols are abundant in Morus alba (García, 2003); while in Venezuela they are reported in variable concentrations in Acacia flexuosa, Acacia glomerosa, Acacia macracantha, Albizia lebbeck, Albizia caribaea, Calliandra crugieri, Cassia grandis, Cassia fistula, Pithecellobium dulce, S. saman, Gliricidia sepium, Leucaena macrophylla, Lysiloma latisiliquum and Enterolobium contortisilicum (Baldizán et al., 2006).

Regarding terpenoids, Marcano and Hasegawa (2002) state that these metabolites show biocide, sedative and anthelmintic activity (monoterpenes); allelopathic, allergenic and toxic effects on rumen microorganisms (sesquiterpenes); hypersensitivity, dyspnea. hypotension, skin and mucosae irritations, neuropathy and alopecia (diterpenes); and could alter the blood pressure too (triterpenes).

Saponins, although not found in P. lanceolatum, G. ulmifolia and C. caracasana, had slight to moderate levels in the other plants, independently from the season. Of steroidal or triterpenic nature, they are linked to alterations of the cell membranes, for which they are associated to the destruction of ruminal protozoa and facilitate the flow of microbial protein to the posterior tract. In high quantities, saponins are related to meteorism as they are potent foaming agents; as well as because they can also reduce blood and liver cholesterol and growth rate, inhibit the activity of the smooth muscle and some enzymes, and regulate the nutrient absorption (Kerem et al., 2002).

Total phenols had from slight to abundant presence, which was higher during the rainy season, particularly in I. laurina and S. saman. These aromatic compounds do not always constitute antinutritional factors, as they are part of all the vascular plants of importance in animal production, in free form (seeds, fruits or dead tissues) or polymerized, as in the case of tannins and lignin. In this sense, the presence of total phenols has been reported in genera in common with the forest under study, such as: Acacia, Albizia, Cadaba, Caesalpinia, Calliandra, Cassia, Copernicia, Enterolobium, Gliricidia, Inga, Lablab, Leucaena, Lysiloma, Macroptilium, Moringa, Morus, Pithecellobium and Trichantera, among others (Romero et al., 2000; García, 2003; Baldizán et al., 2006; García and Medina, 2006).

In the case of tannins, they showed a distribution pattern similar to that of total phenols, with an increase during the rainy season of the condensed tannin fraction in I. laurina, P. lanceolatum, S. saman and G. ulmifolia. In some vascular plants their increase is evident, as a mechanism of defense from predators during the periods of active growth. Although tannins are frequently identified as antinutritional factors, due to their potential negative effect on voluntary intake, nutrient digestion and animal response, they are considered to have low nutritional impact when their presence is of 2-4 % of dry ingested biomass. Beneficial properties are even ascribed to them for the grazing ruminant, because they reduce the ruminal degradation of the ingested protein and increase its flow to the posterior tract. In addition, along with saponins, they act against gastrointestinal parasites and reduce the enteric methane emissions (Ramírez-Restrepo and Barry, 2005; Goel and Makkar, 2012).

On the other hand, alkaloids are highly associated to the genotype and are little dependent on such ecological factors as climate, seasons or period of the year and water availability. Thus, in this study they were absent in P. lanceolatum and G. ulmifolia, and with slight presence in the other evaluated plants. These metabolites are frequently considered as part of the defense mechanism of plants, because of their neurotoxic character; in addition, they act as nitrogen reserves and in the fixation of certain metals, due to their chelating capacity (Marcano and Hasegawa, 2002). The levels found in the selected woody plants suggest that the ruminal detoxification capacity is able to avoid the adverse effects to the macroanimal, and that they can be used as sources of nitrogen and minerals for microbial growth.

Independently from the season, the soluble reducing carbohydrates were absent in 55,6 % of the selected woody plants, with moderate presence in I. laurina. In silvopastoral systems schemes, such as the ones in this study, it is not expected that these intermediaries of energy metabolism cause problems to the animal, for which they should constitute a contribution of easily fermentable energy to the ruminal medium.

The other metabolites showed a slight presence or were absent independently from the evaluated season, which indicates the little effect they exerted on the ruminants under silvopastoral system conditions in this forest formation, as a consequence of the variety in selected biomass. In this sense, the moderate levels of the á-amine and mucilage groups, during the dry season, in E. cyclocarpus and G. ulmifolia, respectively, stand out; as well as in R. spinosa and E. cyclocarpus (á-amines) and G. ulmifolia (flavonoids and mucilages), during the rainy season.

Some á-amines are considered secondary metabolites, due to their capacity to generate anomalous proteins; while mucilages, restricted in this study to their exclusive presence in G. ulmifolia, are identified as soluble fiber of viscous nature, with analogous characteristics to those of gums. In literature it is not reported (Marcano and Hasegawa, 2002) that these compounds constitute antinutritional factors, with the exception of those that show caustic and irritating properties, which were not identified in the plants of this study.

Flavonoids (flavones, flavonols, anthocyanidins, leucoanthocyanidins, catechins, charcones, isoflavones and quinones; among others) were found in only three species (S. saman, Senna sp. and G. ulmifolia), and constitute a group that depends on the genetic component. In addition, they are highly polar and are associated to the free radical sequestering (antioxidants), anti-senescent, diuretic and antimicrobial function (García, 2003).

According to the different combinations of secondary metabolites identified in the edible fraction of the woody plants selected by the bulls, the lowest combination corresponded to P. lanceolatum, with 6 different metabolites; and the highest, to I. laurina and Senna sp., with 11 metabolites in each species (fig. 1). In relative terms, and independently from the season, 66,7 % of the species showed between 10 and 11 secondary metabolites in their edible biomass.

When comparing Fabaceae to the other botanical families, they were higher regarding the number of secondary metabolites, which were concurrently in the same species.

In general, nitrogen-fixing species synthesize a higher diversity of secondary structures, as a defensive strategy in an environment that is particularly aggressive, because defoliators select them for their higher biological value, which is frequently associated to the offer of nitrogen fractions. This relation, derived from the co-evolutional processes between plants and animals, constitutes a fundamental aspect to understand the chemo-taxonomic differences among the woody species and their potential impact on the nutritional plane of the ruminant under silvopastoral system conditions (Launchbaugh et al., 2001).

Finally, it should be stated that some authors prove the existence of interactions among secondary metabolites, traditionally called antinutrient-antinutrient interactions. Thus, in the case of the tannins-saponins interactions, an additive effect is indicated on the decrease of the true digestibility and the in vitro gas production rate, when both metabolites are present in the ration (Makkar et al., 1995). Likewise, it is indicated that the presence of lectins in plants with tannins allows to eliminate the adverse effects of these phenolic compounds on amylase (Fish and Thompson, 1991), or that tannins reduce the deleterious effect of cyanogenic glycosides, by interfering in the release of cyanhydric acid (Goldstein and Spencer, 1985).

CONCLUSIONS

The evaluation of the qualitative profile of secondary metabolites in the edible fraction of woody species selected by cattle under silvopastoral system conditions, in a tropophilous semi-deciduous forest of Venezuela, allowed to identify a wide variety of compounds present in the selected biomass, some of potential toxic effect for ruminants. This should be considered in future evaluations, to define their forage value within the framework of sustainable strategies of silvopastoral systems, under tropical conditions.

ACKNOWLEDGEMENTS

The authors thank the Council of Scientific and Humanistic Development of the Central University of Venezuela, for the financial support to conduct this research (Project CDCH Nº PG-01-36-4995-2002).