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Introduction
The plants contain numerous organic compounds called "primary metabolites", among which are the first, sugars or carbohydrates, which are produced because of photosynthesis. On the other hand, different chemical compounds called, in general, "secondary metabolites" are synthesized in variable concentrations, since they do not directly intervene in their metabolism, or at least, their function in the plant is not well known and it is assumed that they are used as a defense mechanism. They belong to different groups such as: essential oils, alkaloids, coumarins, steroids, phenols, flavonoids, glycosides, gums, iridoids, lignans, mucilages, pectins, quinones, saponins, tannins and terpenes, among others (Paumier et al. 2018 and Pinard et al. 2019).
These substances are synthesized as a response of plants to the attack by fungi, bacteria, pests, allelopathic effect, pollution, adverse effects of climatic factors and herbivorous animals. These chemical defenses are found in variable concentrations in nature and depend on the metabolite type, the characteristics of the plant and the conditions to which they are subjected. The defense based on these compounds causes toxicity to its predators and influences on the forage palatability, causing rejection when intake it (Reyes-Silva et al. 2020 and Verdecia et al. 2020a).
In the last ten years, the use of trees, shrubs and legumes or other species has become very important, as a supplement to the diet of ruminant animals, to improve the quality and quantity of the diet. These plants contain secondary metabolites, which can modify the degradation rate and passage of nutrients through the gastrointestinal tract as a result of a direct effect on ruminal ecology. By considering these effects, it is necessary to study their levels in plants that can be used for animal food (Huang et al. 2020). In accordance with the above, the objective of this research was to determine the content of primary and secondary metabolites of six species of trees, shrubs and herbaceous legumes, as well as to establish the groups with similar values of their content.
Materials and Methods
Research area, climate and soil. Six experiments were carried out, one for each species, simultaneously in the same agricultural area (soil and climate) from the Departamento Docente Productivo de la Universidad de Granma. This facility is located in the southeast of the country, in Granma province, Cuba.
The experiments were developed during two years, dividing the study into two seasons: the rainy (May-October) and the dry (November-April) for each of the years. During the rainy season the precipitations were 893.67 mm; the average, minimum and maximum temperature registered values of 26.73, 22.31 and 33.92 ºC, respectively, and the average, minimum and maximum relative humidity was 80.78, 51.02 and 96.22 %, respectively. In the dry season the precipitations reached values of 364 mm; the temperature was in the order of 24.05, 18.29 and 31.58 º C for the average, minimum, maximum, respectively, and the minimum, average, and maximum relative humidity reached averages of 76.21, 44.16 and 97.03 %, respectively. These values are within the range of historical values for the region.
The soil of the experimental area was Brown with carbonate (Hernández et al. 2015), with a pH of 6.2. The content of P2O5, K2O and total N was 2.4, 33.42 and 3 mg/100g of soil, respectively and 3.6 % of organic matter.
Treatments and experimental design. A randomized block design was used in each experiment, with six agronomic replications for each of the treatments. These consisted of regrowth ages of 60, 120 and 180 days for Leucaena leucocephala, Gliricidia sepium, Erythrina variegata and Tithonia diversifolia; and 30, 45, 60, 75 and 90 days for Teramnus labialis and Neonotonia wightii.
Procedures. At the beginning of each seasonal period, a uniformity cut was made at 1m in height in the plots (0.5 ha) of Leucaena leucocephala, Gliricidia sepium and Erythrina variegata and at 15 cm above the soil for Tithonia diversifolia. At each of the regrowth ages (60, 120 and 180 days), 10 plants/plot were randomly harvested and leaves, petioles and stems with diameter less than 2 cm were manually separated for laboratory analysis.
For the case of Teramnus labialis and Neonotonia wightii, in each season, at the beginning of the evaluation, the uniformity cut was made at 5 cm from the soil. Plots of 25 m2 were delimited, corresponding to the regrowth ages (30, 45, 60, 75 and 90 days), which were manually cut, after eliminating 50 cm of border effect and 200 g were taken for the laboratory analysis. The area was not irrigated or fertilized during the experimental stage.
Determination of primary and secondary metabolites. The samples were dried at room temperature in a dark and ventilated room for 12 days, then they were milled to a 1 mm particle size and stored in amber bottles at room temperature until their analysis in the laboratory.
The N was determined according to AOAC (2016) and for glucose (Glu), fructose (Frut), sucrose (Suc), raffinose (Raf), verbascose (Verb) and stachyose (Stac), 1 g of the dry and milled sample was taken, 9 mL of 70 % methanol were added and it was heated for 20 minutes at 100 °C, then it was cooled to room temperature and filtered by Watman 42, later 0.2 mL of Carrez I [K 4Fe (CN)] solution at 15 % in distilled water and Carrez II [Zn (CH3COO)] at 30% in distilled water were added and it was made up to 10 mL with the 70 % methanol solution . It was centrifuged at 4000 rpm for 15 minutes. A total of 5 mL of the supernatant were taken and 5 mL of dichloromethane were added, stirring vigorously in Vortex. The two phases were subsequently allowed to define and the upper phase was taken with a pipette. Two more extractions were made with dichloromethane equal to previous described. The sample was filtered with a 0.45 µm pore filter. For detection, a 2410 HPLC (WATERS) was used, a refractive index detector using the software (Empower Pro 2002), the chromatographic method used was the isocratic (constant flow) of 1 mL/min with water mobile phase + 0.01 N sulfuric acid, 50 °C column temperature, the column used was ion exclusion BioRad Amino HPX-87M 300 mm x 7.8 mm. Standards of renowned analytical quality were used.
The analysis of total phenols (TF) and total tannins (TT) was performed by the Folin-Ciocalteu method, before and after the treatment of the extracts with polyvinylpolypyrrolidone (PVPP) according to Makkar (2003), while the total condensed tannins (TCT), free condensed tannins (FCT) and total bound condensed tannins (TBCT) were determined by the nbutanol /HCl/Fe3 + method (Porter et al. 1986). The flavonoids (Flv) according to Boham and Kocipai-Abyazan (1994). Saponins (Sap) by the method described by Obdoni and Ochuko (2001). Triterpenes (Trit) according to Jie-Ping and Chao-Hong (2006). Steroids (ST) by Galindo et al. (1989) and the alkaloids (Alk) by the method described by Muzquiz et al. (1994). The analyzes were carried out in the departamento de Producción Animal, de la Facultad de Medicina Veterinaria de la Universidad de León, Spain. All the analyses were performed in duplicate in the six replications of each age for all species and seasonal period.
Statistical analysis. The results of the two years were grouped by seasonal period (rainy season and dry season) and a cluster analysis was performed in each of them (Visauta 1998) to establish groups with similarity in their content of primary and secondary metabolites. The sequence used by Vargas et al. (2013) was fallowed, which includes two phases: in the first, the Ward's hierarchical grouping method was used, in order to determine a preliminary number of groups (clusters) to form. Progressive grouping levels were explored and the optimal level was defined as the best distribution of the cases under study, according to the formed groups. In the second phase of the analysis, the definitive grouping of the cases was obtained; the non-hierarchical K-means method was used, specifying as a starting point the number of clusters identified as optimal in the previous step and the experience of the researcher.
Results and Discussion
In the rainy season, seven groups (figure 1) were formed N. wightii (30, 45, 60, 75 and 90 days), T. labialis (30 and 45 days), E. variegata (60 and 120 days) and G. sepium (60 days) were located in the first group characterized by low levels of Verb and ST. The G. sepium (120 and 180 days) in the second group showed the lowest FCT value and the highest Sap value. The E. variegata (180 days) with the highest amounts of Verb, Alk and Trit, and the lowest amounts of Stac formed the third group. T. diversifolia (60, 120 and 180 days) formed the fourth group with the lowest values of TCT, TBCT, Glu and Trit, and the highest of Stac. L. leucocephala (120 and 180 days) in the fifth group showed the highest concentrations of TT, TF, TCT, TBCT, Stac, Glu, Frut, Suc and Flv. In the sixth, T. labialis (60, 75 and 90 days) was located with the lowest values of TF, Raf, Frut, Suc, Flv, Alk and Sap. In the seventh L. leucocephala (60 days) had the lowest results in TT and the highest in Raf (table 1).
Figure 1 Dendrogram of the content of primary and secondary metabolites of trees, shrubs and herbaceous legumes during the rainy season
Table 1 Concentration of primary and secondary metabolites according to the groups obtained by cluster analysis in the rainy season.
| PrimaryAnd secondary metabolites, g/kg | Groups | ||||||
|---|---|---|---|---|---|---|---|
|
1- |
2- |
3- |
4- |
5- |
6- |
7- |
|
| 117.4±5.01 | 134.7±1.00 | 107.8 | 133.2±6.81 | 171.1±2.55 | 90.88±1.77 | 197.9 | |
| 4.09 ±1.94 | 13.57 ±1.02 | 11.17 | 3.05 ±2.41 | 21.49 ±0.75 | 3.08 ±1.02 | 2.33 | |
| 11.44 ±4.35 | 33.25 ±2.10 | 21.05 | 8.75 ±3.23 | 43.59 ±0.06 | 6.64 ±0.65 | 17.7 | |
| 61.56 ±4.93 | 49.77 ±1.13 | 115.72 | 14.19 ±0.46 | 129.07 ±1.61 | 109.42 ±4.16 | 126.9 | |
| 60.64 ±4.80 | 49.15 ±1.10 | 113.2 | 10.58 ±0.95 | 119.5 ±0.35 | 108.25 ±4.08 | 118.00 | |
| 0.95 ±0.34 | 0.62 ±0.03 | 2.50 | 3.62 ±0.62 | 9.59 ±1.26 | 1.17 ±0.18 | 8.88 | |
| 0.0007 ±0.0001 | 0.0014 ±0.001 | 0.007 | 0.001 ±0.0001 | 0.004 ±0.01 | 0.0008 ±0.001 | 0.002 | |
| 0.0008 ±0.002 | 0.0011 ±0.002 | 0.0003 | 0.0004 ±0.0002 | 0.0045 ±0.01 | 0.0005 ±0.0003 | 0.0018 | |
| 0.0011 ±0.0003 | 0.0013 ±0.000 | 0.0009 | 0.0014 ±0.0003 | 0.0019 ±0.001 | 0.0003 ±0.0001 | 0.0022 | |
| 0.004 ±0.0001 | 0.0057 ±0.01 | 0.0009 | 0.0008 ±0.0001 | 0.024 ±0.01 | 0.0012 ±0.0002 | 0.013 | |
| 0.0005 ±0.001 | 0.0005 ±0.000 | 0.0007 | 0.0008 ±0.01 | 0.003 ±0.001 | 0.00012 ±0.002 | 0.0019 | |
| 0.004 ±0.0004 | 0.0062 ±0.01 | 0.0015 | 0.0016 ±0.004 | 0.0027 ±0.01 | 0.0013 ±0.001 | 0.014 | |
| 2.58 ±1.09 | 11.67 ±3.96 | 7.35 | 24.77 ±13.60 | 68.44 ±12.88 | 2.21 ±0.59 | 30.30 | |
| 1.09 ±1.11 | 0.34 ±0.07 | 5.09 | 0.92 ±0.14 | 2.97 ±0.11 | 0.30 ±0.001 | 2.73 | |
| 1.13 ±1.39 | 11.63 ±2.96 | 1.30 | 1.61 ±0.55 | 9.73 ±1.38 | 0.43 ±0.10 | 5.48 | |
| 8.01 ±0.66 | 8.41 ±0.38 | 11.13 | 7.43 ±1.08 | 9.18 ±1.53 | 7.75 ±0.41 | 8.83 | |
| 0.52 ±0.17 | 0.80 ±0.13 | 1.39 | 10.71 ±3.16 | 7.07 ±1.75 | 0.57 ±0.12 | 5.26 | |
The low Verb contents in group one (figure 1 and table 1) coincide with the results reported by Velásquez-Holguín et al. (2019) in Erythrina and also found the Verb in lower concentration than Raf and Stac.
While Verdecia et al. (2020a) showed that in E. variegata the secondary metabolite concentrations increase with age and lower values at 60 days of regrowth. Ávila-Hernández et al. (2017) and Reyes-Silva et al. (2020) reported values lower than 0.62 g/ kg of ST in S. humboldtiana, S. alba and P. alba, which are agree with those reported in this study, so it is unlikely that the proportion of ST (0.52 g/kg) found in the species from the first group is of importance for its use in animal diets, although the literature shows that the toxic effects of this metabolite are limited (Paumier et al. 2020 and Verdecia et al. 2020b). However, in T. diversifolia studies should be carried out on the biological response and animal behavior, since its value is higher than the rest of the evaluated species. Nevertheless, Holopainen et al. (2018) when evaluating the effects of climate change on secondary compounds (polyphenols, alkaloids and terpenes) of trees, found that CO2 levels of 4-6.7 favor polyphenol values in foliage and decrease terpenoids, while with the normal values (1.5-3) of CO2 are obtained high contents of terpenes with decrease of the polyphenols.
On the other hand, Holopainen et al. (2018) and Estrada-Jiménez et al. (2019) pointed out that the presence of secondary compounds is related to the age of the material, the defense mechanisms of the plant, the content of primary nutrients and the influence of the soil and climate. In the complexity of the ecosystem where the plants develop, essentially the climate and the soil exert a great influence with regard to the active principles and secondary compounds. The humidity, the salts content, soil nutrients, light, temperature and rainfalls, mainly, have a marked effect on their presence in plants, since their deficiencies and excesses cause stress situations that cause the synthesis of these substances; for example, wind speed, a factor little studied experimentally, is decisive in many cases since it is known that its action increases the evaporation of essential oils and in the case of the tropane alkaloids, the increase in transpiration in the plants increases the content of liquid that rises from the roots.
In the second group, G. sepium (120 and 180 days, in the rainy season) showed the lowest concentrations of FCT and the highest of Sap. Cabrera-Núñez et al. (2019) showed values of 2.71 g/kg of FCT, Sandoval-Pelcastre et al. (2020) reported 13.8 g/kg of Sap, so that G. sepium could be considered, according to Rodríguez et al. (2014) and Ramos-Trejo et al. (2016), as the tropical legume with the lowest content of condensed tannins, compared to Eucalyptus grandis, Arabidopsis thaliana, Acacia mangium and Callyandra sp. (Pinard et al. 2019 and Huang et al. 2020).
The Sap contents, although they were the highest in the rainy season (table 1), are lower than those reported by Sirohi et al. (2009) in Acacia concinna (34 g/kg). For their part, García et al. (2006) in several species from the genera Albizia, Cassia and Pithecellobium, found between 12.8 and 38.5 g/kg of Sap with the highest values for the Pithecellobium genus. Therefore, the Sap content (11.63 g/kg) found in this study can be classified as light.
E. variegata at 180 days was part of the third group with the highest values in Verb, Alk and Trit, and the lowest in Stac. Similar performance to those obtained by Verdecia et al. (2020a) who reported Verb, Alk, Trit and Stac values of 0.007, 5.09, 11.13 and 0.0029 g/ kg, respectively in the foliage of Erythrina variegata, and stated that the contents of Verb, Stac and Trit in the different parts of plants are variable depending on the characteristics of each species and the phenological phase of the plant, since these compounds show mobility between the different organs.
On the other hand, Noudou et al. (2018) and Herlina et al. (2019) identified alkaloids, hypaphorin, erisodin, sterols, stigmasterol and isoflavonoids in Erythrina poeppigiana seed, while Patti et al. (2019) in E. suberosa reported that the seeds contain a large amount of organic acids, steroids and alkaloids, and they also isolated erythraline, erisodin, erisothrin and hypaphorin, for which they concluded that, due to the pharmacological properties of these compounds in different extracts, they may have anticancer activity. Therefore, it would be appropriate to carry out future researches on this topic in Erythrina variegata seeds.
With the best results, comprehensively during the rainy season, T. diversifolia appeared in the fourth group, with the lowest concentrations of TCT, TBCT, Glu, Trit and higher concentrations of ST. Santacoloma-Varón and Granados (2012) found in this plant between 0.43 and 2.6 g/kg of TF and from 3.1 to 4.9 g/kg of CT while, Medina et al. (2009) and Verdecia et al. (2018) reported from 6.4 to 8.7 g/kg of TF, although the Trit and ST were higher with 10.2 to 13.9 and from 25.4 to 29.2 g/kg, respectively. Aspects, which denote that the content of metabolites is controlled by genetic aspects of each species, mainly, and secondly by the environment. Although it is important to highlight that, the contents in this species are lower than those reported in L. leucocephala, G. sepium and N. wightii.
The fifth group composed of L. leucocephala, from the point of view of the concentration of secondary metabolites, was the one with the worst comprehensive results with the highest amounts of TT, TF, TCT, TBCT, FCT, Stac and Flv, although it showed the highest values of Glu, Frut and Suc. These results coincide with those reported by Verdecia et al. (2020b) in L. leucocephala, who when evaluating the effect of secondary metabolites on digestibility found relations higher than R2 = 0.80 for TT, TCT and Sap. However, although the concentration of TT (21.49 g/kg) found in this group could be a positive element when evaluating, entirely, the antinutritional characteristics of the polyphenolic fraction of this species, since they are in the range where, possibly, the ruminal ecosystem is not affected and the probability of bypass protein formation increases and it would facilitate postruminal nitrogen digestibility (Albores-Moreno et al. 2019).
The soluble carbohydrate content is linked to the morpho-structural development of plants. The reserves of these compounds, in smaller quantities, in the growth points (buds) favor the foliar concentrations of saccharides after the regrowth emission. However, although these aspects are generally described from the physiological point of view, the performance of energy metabolites as a function of morpho-structural variations depends on the species, nutritional status and edaphoclimatic conditions in which it is grown (Cao et al. 2011).
T. labialis at 60, 75 and 90 days in the sixth group (table 1) showed the lowest concentrations of TF, Raf, Frut, Suc, Flv, Alk and Sap. Similar results were obtained by Scull and Savón (2003) when reporting 3.32-6.9 g/kg of TF in Vigna unguiculata and Pinto et al. (2002) found TF values of 3-6 g/kg in Ipomoea triloba, Stizolobium deeringianum and Stizolobium pruri, so it could be considered that the varieties of these legumes have low total content of polyphenols and condensed tannins, and in general , of secondary metabolites if they are compared with the other species of legumes reported in this research. However, the previous requires future researches that include a higher number of legumes.
Scull et al. (2012) found in Lablab purpureus that Suc and Frut concentrations (0.041 and 0.083 mg, respectively) were low when compared with those reported in T. labialis in the sixth group during the rainy period, while Paumier et al. (2018) showed similar results to those of this research in G. sepium. The content of monosaccharides in legumes is usually low, because these sugars are used in the biosynthesis of disaccharides and oligosaccharides during plant development for the formation of tissues, flowers and seeds (Verdecia et al. 2020b).
In the seventh group, L. leucocephala at 60 days had low TT content and high Raf content. Rodríguez et al. (2014) and Morales-Velasco et al. (2015) also found low levels of TT (3.01 g/kg) in Albizia lebbekoides and Acacia cornigera. This performance at early ages of the trees describes that the species with the highest proportion of fiber have a high concentration of hydroxylated metabolites, aspects that coincide with that stated by numerous authors about the negative effect of the cell wall compounds and tannins on the nutrition animal (Barragán-Hernández et al. 2019).
In the dry season, eight groups were obtained according to the similarity of the primary and secondary metabolites (figure 2). T. labialis (30, 45, 60, 75 and 90 days) and N. wightii (90 days) formed the first group and were characterized by low values of Raf, Frut and ST. In the second E. variegata (180 days) showed the highest values of TT, Alk and Trit. N. wightii (30, 45, 60 and 75 days) in the third group showed the lowest amounts of Verb, Flv, Alk, Sap and Trit. In the fourth was E. variegata (60 and 120 days) with the lowest results for Stac and the highest for Frut. G. sepium (60, 120 and 180 days) with the lowest concentration of FCT occupied the fifth group. The T. diversifolia (60, 120 and 180 days) in the sixth group had the lowest values of TT, TF, TCT, TBCT, Glu and Suc, and the highest of ST. In the seventh group, L. leucocephala (60 and 120 days) revealed the highest amounts of Verb, Stac, Raf, Glu and Suc. L. leucocephala at 180 days occupied the eighth group and showed the highest concentrations of TF, TCT, TBCT, FCT, Flv and Sap (table 2).
Figure 2 Dendrogram of the content of primary and secondary metabolites of trees, shrubs and legumes during the dry season
Rescaled distances of the clusterT. labialis (30, 45, 60, 75 and 90 days) and N. wightii (90 days) in the first group (table 2) were characterized by low contents of Raf, Frut and ST. These results could be related to what was stated by Salas et al. (2015) and Méndez et al. (2018) who in Phaseolus vulgaris and Moringa oleifera found that carbohydrate values were closely correlated with the succession of phenological events, from early growth to flower formation and flowering. From the latter, the general decrease in carbohydrates began, while oligosaccharides showed a highly variable performance during the study, where Stac and Verb were the majority.
Table 2 Concentration of primary and secondary metabolites according to the groups obtained by cluster analysis in the dry season.
| Primary and secondary metabolites ,g/kg | Groups | |||||||
|---|---|---|---|---|---|---|---|---|
|
1- |
2- |
3- |
4- |
5- |
6- |
7- |
8- |
|
| 82.19±3.59 | 121.1 | 98.30±0.47 | 154.4±4.30 | 148.1±5.80 | 151.3±6.09 | 198.97±3.51 | 161.9 | |
| 4.40 ± 1.23 | 35.29 | 2.24 ± 0.45 | 27.30 ± 3.58 | 13.28 ± 2.85 | 2.22 ± 0.88 | 26.89 ± 5.18 | 34.10 | |
| 8.85 ± 0.9 | 48.09 | 7.24 ± 0.48 | 43.79 ± 5.18 | 31.88 ± 3.94 | 5.87 ± 0.56 | 46.15 ± 2.91 | 50.53 | |
| 103.19 ± 8.56 | 97.52 | 68.08 ± 7.49 | 65.07 ± 0.2 | 38.88 ± 4.49 | 11.56 ± 1.40 | 133.42 ± 8.59 | 142.04 | |
| 101.79 ± 8.7 | 94.73 | 67.24 ± 7.30 | 63.28 ± 0.01 | 38.19 ± 4.50 | 9.42 ± 0.68 | 123.77 ± 9.61 | 130.48 | |
| 1.39 ± 0.43 | 2.79 | 0.85 ± 0.21 | 1.79 ± 0.21 | 0.69 ± 0.1 | 2.13 ± 0.76 | 9.65 ± 1.01 | 11.56 | |
| 0.0006 ± 0.0002 | 0.0007 | 0.0004 ± 0.0001 | 0.0006 ± 0.0001 | 0.0008 ± 0.0002 | 0.0005 ± 0.0002 | 0.0025 ± 0.0002 | 0.0012 | |
| 0.0006 ± 0.0002 | 0.0002 | 0.0007 ± 0.0001 | 0.0002 ± 0.000 | 0.0007 ± 0.0001 | 0.0002 ± 0.000 | 0.0032 ± 0.0006 | 0.0002 | |
| 0.0004 ± 0.0005 | 0.0009 | 0.001 ± 0.0003 | 0.001 ± 0.0001 | 0.0007 ± 0.0003 | 0.0014 ± 0.0002 | 0.0018 ± 0.0005 | 0.0012 | |
| 0.0017 ± 0.0007 | 0.0009 | 0.0031 ± 0.0005 | 0.0021 ± 0.0013 | 0.0039 ± 0.0007 | 0.0006 ± 0.0001 | 0.02 ± 0.0049 | 0.010 | |
| 0.0001 ± 0.0001 | 0.0009 | 0.0003 ± 0.0001 | 0.0011 ± 0.0001 | 0.0004 ± 0.0003 | 0.0005 ± 0.0002 | 0.0005 ± 0.0003 | 0.0004 | |
| 0.0018 ± 0.0007 | 0.0017 | 0.0034 ± 0.0005 | 0.0032 ± 0.0012 | 0.0043 ± 0.0009 | 0.0011 ± 0.0002 | 0.02 ± 0.0042 | 0.010 | |
| 2.30 ± 1.11 | 10.05 | 1.99 ± 0.67 | 5.38 ± 2.66 | 11.25 ± 4.83 | 29.75 ± 14.61 | 54.13 ± 10.37 | 86.95 | |
| 0.28 ± 0.07 | 5.22 | 0.25 ± 0.06 | 4.65 ± 0.42 | 0.33 ± 0.06 | 0.99 ± 0.2 | 2.87 ± 0.15 | 3.25 | |
| 0.42 ± 0.15 | 1.38 | 0.35 ± 0.11 | 0.79 ± 0.14 | 10.30 ± 3.13 | 1.97 ± 0.37 | 9.82 ±3.68 | 15.17 | |
| 7.79 ± 0.57 | 10.11 | 7.68 ± 0.30 | 9.29 ± 0.07 | 8.05 ± 0.11 | 7.69 ± 1.34 | 7.76 ± 0.96 | 8.43 | |
| 0.50 ± 0.12 | 1.43 | 0.54 ± 0.08 | 1.01 ± 0.31 | 0.77 ± 0.23 | 11.59 ± 3.39 | 5.00 ± 1.18 | 8.65 | |
In the second group, in the rainy season, only E. variegata was present at 180 days with the highest values of TT, Alk and Trit. The TT content obtained in this research are comparable with those reported by Herlina and Supratman (2016) and Herlina et al. (2018) in Erythrina poeppigiana who found 4.6-35.9 g/kg of TT, and this last value was higher than those of the rest of the studied trees, although it did not exceed the concentration from which ruminal fermentation begins to be affected (Makkar 2003). It is important to highlight that these authors also identified isoflavonoids, flavonoids, and cuersentin.
The Alk in Erythrina are distributed throughout the plant, largely in the seeds, stem bark and flowers, although there are also reports of their presence, in lower proportions, in roots and leaves (Herlina et al. (2018). The results of this research are within the range reported by Verdecia et al. (2019, 2020a) in E. variegata and J. curcas from 3.4 to 10.4 g/kg.
N. wightii at 30, 45, 60 and 75 days, in the dry season, in the third group showed the lowest values of Verb, Flv, Alk, Sap and Trit. The Verb values are low as they are influenced by the plant age and different authors (Salas et al. 2015 and Espejo and Morales 2019) showed that the individual content of each α-galactoside depends not only on the type of legume and on the genotypes , but also on the growing conditions.
The concentrations of Flv found (1.5 g/kg) coincide with that reported by Vázquez et al. (2017), but they are lower than those obtained by Mori et al. (2015) in the stems (7.5-17.2 g/kg) and leaves (15-17.6 g/kg) of Leucaena leucocephala and Mucuna gigantea. The higher concentration in the leaves is due to its function in vegetables, because of its importance for the development and good functioning of plants by protecting them against external agents, such as UV radiation, microorganisms, herbivorous animals and the environment. They can also act as chemical markers, showing to insects the appropriate plant for their feeding.
The fluctuations in non-structural carbohydrates of E. variegata at the ages of 60 and 120 days in the fourth group during the dry season could be due to genetic factors, weather conditions, the land where they grow and cultivation techniques used. The plant maturity is important when evaluating the carbohydrates content, taking into account that the frut is synthesized during the first stages of growth and the highest amounts of α-galactosides, as is the case of the Estq, are found during flowering and fruiting (Sánchez-Mendoza et al. 2016, Kannan et al. 2018 and Shibata et al. 2018).
With the best results in the fifth group, G. sepium at the ages of 60, 120 and 180 days in the dry season, showed the lowest FCT values and this agrees with that reported by La O et al. (2018) and Canul-Solis et al. (2020), who recommend this species for use as excellent quality forage.
Santacoloma-Varón and Granados (2012) found a low amount of tannins in this species, which could be related to the taking of samples, which were carried out during the dry season, where environmental conditions can affect the photosynthetic rate and in consequently the synthesis of secondary metabolites. In addition, it must taken into account that these substances are synthesized in the plant and fulfill functions such as defense against fungi, bacteria and virus, protection against ultraviolet radiation, and serve as a mechanism to avoid dehydration of tissues, an aspect that could affect their concentration (Silva et al. 2017).
T. diversifolia (60, 120 and 180 days) showed, like G. sepium, the best comprehensive results. The lower concentrations of TT, TF, TCT, TBCT, Glu and Suc, and the higher of ST are mainly due to that during the dry season the leaves intercept a lower proportion of light and consequently lower production of primary and secondary metabolites , due to the slower speed of biochemical reactions in the plant (González-Sierra et al. 2019 and Rodríguez et al. 2019), while Santacoloma-Varón and Granados (2012) when studying the content of TF, TT and CT in this species in several thermal floors there were not significant differences. On the other hand, Lezcano et al. (2012) reported low contents of TF, TT, Trit and ST. This little variability found could be due to that the volume and speed of biochemical reactions can increase with temperature and most reactions in plants have a characteristic thermal optimum, which decreases both at higher and lower temperatures. This is due, in the first instance, that the enzymatic activity and the integrity of cell membranes are affected by extreme temperatures.
Estrada-Jiménez et al. (2019) reported 12 g/kg of ST in T. diversifolia, results similar to those obtained in this study, a response that may be given by the similarity in climatic and soil conditions, aspects that directly influence on the production of secondary compounds. Corroborating this statement, Herrera (2020) reported a close relation between this metabolite and maximum temperature and rainfalls.
L. leucocephala at 60 and 120 days, during the dry season, was in the seventh group and showed the highest values of oligosaccharides (Verb, Stac and Raf) and of Glu and Suc. Although, it should be noted that Glu and Suc concentrations in vegetables are higher than those of oligosaccharides. This performance was pointed out by Almeida (2003) and Vilhena et al. (2003) who reported higher concentrations of these ones during the vegetative growth stage, which could be determined by the speed of photosynthetic activity and the phenological state of plants where carbohydrates are widely used in plant growth and development.
Aimard et al. (2003) when evaluating the effect of water stress on meadow grasses, they found that Raf, Stac and Verb increased, while Glu and Suc remained stable and Frut decreased more than 50%. It is probably that this response also occurs in legumes, although it would be necessary to design specific researches to validate this hypothesis.
In the eighth group, only L. leucocephala was at 180 days, in the dry season, and it was characterized by showing the highest concentrations of TF TCT, TBCT, FCT and Flv. These results are similar to those reported by Verdecia et al. (2020b) who found values of 43, 130, 119, 10 and 77 g/kg for TF, TCT, TBCT, FCT and Flv in this species in Valle del Cauto, respectively. In Jatropha curcasVerdecia et al. (2019) reported CT values in the range of 65-98 g/kg, of these 90% are TBCT and about 70 % are protein-bound, mainly because as higher TCT concentration, higher will be the amount of these coupled to proteins by the affinity they have for this nutrient.
Dago-Dueñas et al. (2020) obtained values from 22.2 to 33.6 and from 105 to 238 g/kg of TF and TBCT, respectively and related it to the high proportions of tannins bound to the reduction of the cell wall digestibility. Aspects highlighted by Herrera et al. (2017) when reporting correlations higher than R2 = -0.71 between polyphenols and DMD, IVDMD and NDFD.
Conclusions
The age had a marked effect on the content of primary and secondary metabolites, as the former decreased and the latter increased as maturity advanced. This performance was evidenced by verifying, through cluster analysis, which the groups where the highest contents of secondary compounds were found were made up of the most advanced ages of the Neonotonia wightii, Teramnus labialis, Erythrina variegata and Leucaena leucocephala species, in both seasonal periods. Aspects of vital importance for future studies where the associative effects of species with lower content of these compounds (Tithonia diversifolia and Gliricidia sepium) were analyzed both in the ecosystem and in the animal response.
