Pasture Science and other Crops

Effect of growth age on the polyphenol content of materials from Tithonia diversifolia (Hemsl.)

0000-0002-9516-7182Idania Scull^{1  *}  , 0000-0001-9880-0310Lourdes Savón¹  , 0000-0002-1690-1140T. E. Ruiz¹  , 0000-0002-2641-1815Magaly Herrera¹ 

¹Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba.

ABSTRACT

The effect of growth age (30, 60 and 90 days) on the polyphenol content of Tithonia diversifolia (Hemsl.) materials collected in eastern Cuba was evaluated. A completely random design with 10 repetitions was applied. Total polyphenols and total condensed tannins were determined in seven tithonia materials (2, 3, 12, 14, 17, 23 and 24) in the rainy and dry seasons. An analysis of variance was performed, according to a completely random design in a 3 x 7 factorial arrangement. The factors were the growth age (30, 60 and 90 days) and the T. diversifolia materials (2, 3, 12, 14, 17, 23 and 24). There was an increase in phenolic compounds with growing age and the highest values were reached at 90 days. Material 3 showed the highest content of total polyphenols (4.62 % DM) in the dry season, and 12 reached the highest concentration of total condensed tannins (2.33 % DM) in the rainy season. The studied materials showed differences in the content and type of phenolic compounds in both seasons of the year. This experiment will help to select the materials with the greatest biological potential for animal feeding. Future researches are suggested to identify the biological activity of these substances and their relation with the beneficial effects in animals.

Key words: secondary metabolites; tannins; buttercup; phytochemical composition

Tithonia diversifolia is a fast-growing shrub, native to Central America, with appropriate agronomic characteristics for its exploitation and use in the edaphoclimatic conditions of Cuba (^{Ruiz et al. 2014}). Its forage, of high nutritional value, is the subject of physiological, biochemical, productive and health researches to promote its use as animal food (^{Rodríguez et al. 2018} and ^{Rodríguez et al. 2019}).

Numerous secondary compounds, which are considered essential to prevent disease and other zootechnical damage, are reported in its composition (^{Rivera et al. 2018} and ^{Ladeska et al. 2019}). However, these can limit the use of the plant when are in high amounts. ^{Chagas-Paula et al. (2012)}, ^{Gonzalez et al. (2019)} and ^{Ladeska et al. (2019)} indicated the phenolic compounds among the majority groups of this species. However, these can limit the use of the plant when they are in high amounts. ^{Chagas-Paula et al. (2012)}, ^{Gonzalez et al. (2019)} and ^{Ladeska et al. (2019)} showed the phenolic compounds among the majority groups of this species.

Phenolic compounds, also called polyphenols, are a large group of secondary metabolites found in most plant-based foods.

Current literature refers that diets rich in polyphenols can improve health and reduces the incidence of diseases in animals and humans (^{Prescha et al. 2018} and ^{Scull et al. 2020}). These compounds are attributed antioxidant, antimicrobial, anti-inflammatory and antiviral properties (^{Rejeki et al. 2017} and ^{Omokhua et al. 2018}).

The objective of this study was to determine the effect of growth age on the polyphenol content of Tithonia diversifolia materials, collected in the eastern part of Cuba.

Materials and Methods

Location, soil and climate. The study was carried out from 2017-2018, at the Miguel Sistachs Naya Experimental Center, from Instituto de Ciencia Animal de Cuba (ICA), located at 22°,81ʼ North latitude and 82°,01ʼ West longitude, in San José de Las Lajas, Mayabeque province (^{Academia de Ciencias de Cuba 1989}). The samples of tithonia materials studied are in the Institute germplasm bank. The soil in the area is red ferralitic, rapidly drying, clayey and deep on limestone (^{Hernández et al. 2019}), moderately acidic with pH=6.1. The content of Ca, Mg and K was 7.86, 0.70 and 1.38 cmol/kg. Likewise, the P₂O₄ was 81.64 kg/ha and the OM, 1.38 %. According to the data from ICA Meteorological Station, rainfalls in the experimental period averaged 84.3 mm, with a minimum monthly value of 0 mm in December and a maximum value of 231 mm (July). Figures 1 and 2 show the monthly distribution of rainfalls and days with rain in the experimental stage, as well as the historical mean.

Figure 1 Performance of rainfalls in the experimental period 

Rainfalls -Historical mean - Experimental periodJanuary- February- March- April- May- June- July- August- September- October- November- DecemberDays with rainfalls

Figure 2 Performance of the days with rainfalls in the experimental period 

Treatments and collection design of vegetative material. The treatments consisted of the evaluation of seven materials (2, 3, 12, 14, 17, 23 and 24) of Tithonia diversifolia (Hemsl.) Gray (tithonia), belonging to Asteraceae family, collected in Granma area, in eastern Cuba, through a completely random design, with ten repetitions. These materials were selected from the evaluation of 24 of them, according to their promising characteristics, agronomic response, chemical composition and ability to activate fermentative processes (^{Ruiz et al. 2018}).

Experimental procedure. The sowing was carried out in the rainy season, at a distance between rows of 3.0 m and 50 cm between cuttings. Cuttings were used for planting, taken from the middle part of the stem, with an age of 80 days, a diameter of 2 cm and a length of 50 cm, in furrows 15 cm deep. The area was clean of weeds using a hoe and in dry conditions.

To start the experiment, the plantation was cut at a height of 15 cm, 120 d after sowing. A total of 10 plants (leaves and petioles) were random sampled for each of the ages (30, 60 and 90 d). The materials were dried in a forced air oven at 60 °C for 48 hours. Then they were milled to a particle size of 1 mm. The forage meal of each material was stored in amber- glass bottles, which were hermetically sealed until the time of performing the chemical analyses.

Determination of total polyphenols. The extraction of the phenolic compounds was carried out in an ultrasonic bath (Randelin Sonorlex brand, series 2000) for 15 min., with an acetone solution at 70 % v/v. The content of total polyphenols (TP) was determined by using the Folin-Ciocalteu reagent, according to ^{Makkar (2003)} method. Analyzes were performed in triplicate. To determine the concentration of TP, a reference solution of tannic acid (Sigma Aldrich) of concentration (0.5 g/L) was used. Concentrations of 10 to 30 g/L of the reference solution were used in the preparation of the calibration curve.

Quantification of total condensed tannins. Total condensed tannins (TCT) were quantified from the extract of phenolic compounds, according to ^{Makkar (2003)} methodology. For the determination, the reagent butanol/HCl 95:5 v/v was used in the presence of heat. The absorbance of the solution was read in a spectrophotometer at 550 nm, using the unheated mixture as reference.

Statistical analysis. For the analysis of the results, the statistical system Infostat (^{Di Rienzo et al. 2012}) was used. An analysis of variance was performed, according to a completely random design in a 3 x 7 factorial arrangement. Growth age (30, 60, 90 d) and T. diversifolia materials (2, 3, 12, 14, 17, 23, 24) were considered as factors. Duncan's test was applied to compare the differences between means (^{Duncan 1955}). Where the interaction was non-significant, the main effects were reported.

Results and Discussion

The content of total polyphenols in the dry season (table 1) showed interaction between the factors growth age (30, 60, 90 d) and T. diversifolia materials (2, 3, 12, 14, 17, 23, 24). However, in the total condensed tannins there were not interactions. The main effects are shown in figures 3 and 4.

Table 1 Total polyphenols content in T. diversifolia forage meal in the dry season 

Indicators, % DM	Growth age Materials	30	60	90	SE (±) Signif.
Total polyphenols	2	0.95^r	2.15^h	2.43^f	0.01 P < 0.0001
	3	1.14^p	1.99^i	4.62^a
	12	1.67^l	1.85^j	2.21^g
	14	1.02^q	1.82^k	2.58^e
	17	1.60^m	1.65^l	4.04^c
	23	1.21^o	1.84^jk	3.57^d
	24	1.26^n	1.66^l	4.35^b

Different letters indicate significant differences for P < 0.05, according to ^{Duncan (1955)}

In the dry season, the TP content differed between the materials. It increased (P < 0.0001) with the growing age and the highest values were reached at 90 d. Material 3 showed the highest concentrations.

The concentration of TCT did not differ between the materials studied in this period. However, the evaluation of the growth age factor showed variations between days. The highest values (P = 0.0008) were recorded at 90 d.

Condensed tannins (%DM)-Materials- SE

Figure 3 Total condensed tannin content in T. diversifolia materials in the dry season 

Condensed tannins (%DM)-Growth age - SE

Figure 4 Total condensed tannin content of T. diversifolia materials with different growth ages in the dry season 

In the rainy season, there were interactions between the tithonia materials and the days of growth for TP and TCT (table 2)

Table 2 Content of total polyphenols and total condensed tannins in the forage meal of T. diversifolia materials in the rainy season 

Indicators, % DM	Growth age Materials	30	60	90	SE (±) Signif.
Total polyphenols	2	0.35l^m	0.45^k	0.49^j	0.01 P < 0.0001
	3	0.45^k	0.64^i	0.74^fg
	12	0.74^fg	0.78^de	0.92^a
	14	0.34^m	0.47^jk	0.75^efg
	17	0.21^n	0.39^l	0.77^def
	23	0.77^ef	0.87^b	0.83^c
	24	0.68^h	0.73^g	0.81^cd
Condensed tannins	2	0.18^n	0.38^m	0.42^l	001 P < 0.0001
	3	0.52^k	0.74^i	1.22^e
	12	0.33^m	0.88^h	2.33^a
	14	0.40^l	0.63^j	0.98^g
	17	0.34^m	1.02^f	1.34^d
	23	0.62^j	1.45^c	1.67^b
	24	0.65^j	1.23^e	1.44^c

Different letters indicate significant differences for P < 0.05, according to ^{Duncan (1955)}

In the rainy season, the concentrations of TP and TCT differed between materials and increased (P < 0.0001) with the growth age. The highest values were reached at 90 d. In material 12 the highest levels of these metabolites were found (0.92 and 2.33 % DM).

The content of TP and TCT in this experiment showed an increase with the growth age, regardless of the season of the year. ^{Boukhris et al. (2015)} studied the effect of the phenological state on the chemical composition of essential oils from plants of the Pelargonium graveolens species. These authors pointed out that the expression and activity of the genes and enzymes involved in secondary metabolism vary with the different stages of plant development.

^{Verdecia et al. (2018)}, when studying the chemical composition of T. diversifolia, reported that this performance can be related to the increase in the synthesis of phenolic compounds during the maturation stage of the plant. These authors found an increase in the concentration of TP and TCT up to 180 d (0.64 and 1.45 % DM) in the rainy season. The TP content was lower than that obtained in this study. While the TCT levels were lower than those found in the rainy season, and higher than those recorded in the dry season. ^{Omokhua et al. (2018)} evaluated the phytomedicinal potential of T. diversifolia cultivated in Africa, and found TP concentrations of 1.49 % DM. These results were higher than those obtained in this study in the rainy season and lower than those of the dry season. These differences could be controlled by other factors, which also affect the development of the plant in the ecosystem: environmental variations, genotype, growth rate, nutritional conditions of the soil, and predation (^{Sampaio and Da Costa 2018}).

In this study, the variability in the concentration and class of phenolic compounds that the materials had between the experimental periods can be attributed to the environmental conditions in which the plants were developed and their genetic characteristics. ^{Sampaio et al. (2016)}, when researching the influence of different environmental factors on the metabolite profile of T. diversifolia, argued that plants under stress conditions, induced by climatic factors, can cause changes in the production of different types of metabolites. In addition, the mentioned authors point out that stress due to solar radiation, specifically UV-B (280-320 nm), affects the production of phenolic compounds: tannins, anthocyanins, flavonoids and derivatives of cinnamic acid. These compounds help dissipate solar energy and protect against the deleterious effects of UV radiation.

^{Herrera et al. (2020)}, when studying the secondary metabolites of T. diversifolia related to climate, found high correlations of phenolic compounds with temperatures (maximum, minimum and average). The best correlations were obtained in total condensed tannins and free condensed tannins, with temperatures, rainfalls and its distribution.

During the development of the experiment, the volume of rain fell considerably, as well as the number of rainy days with respect to the historical average (figures 1 and 2), which undeniably had an effect on the development of the plants.

Water stress could cause a reduction in the photosynthetic rate and, consequently, an increase in the production of reactive oxygen species (ROS). To overcome stress limitations, plants adopted alternative mechanisms. These involved the increase in the production of phenolic substances (natural antioxidants) to eliminate ROS, maintain redox balance and be able to survive in adverse conditions (^{Khare et al. 2020}). According to ^{Morales et al. (2017)}, anthocyanins, substances with high antioxidant activity, accumulate in plant tissues and provide resistance to drought conditions. Phenolic compounds are metabolites that are characterized by having one or more aromatic rings in their molecular structure, joined to at least one hydroxyl group (^{Huajun et al. 2016}). This configuration is what gives them their high antioxidant capacity, so they can eliminate free radicals by donating hydrogen atoms and protect the plant from the damage they cause (^{González et al. 2019}). However, relatively high concentrations of these metabolites can hinder growth, in response to different types of stress (^{Khare et al. 2020}). The results of this experiment corroborate this hypothesis. Material 3 showed the highest concentration of TP in the dry season. ^{Ruiz et al. (2018)}, when evaluating the growth of these T. diversifolia materials, showed that material 3 highlighted in the slow recovery group, at 30 d. Also, it did not change in its growth over time (30, 60 and 90 d), and was always part of the group with the lowest growth.

The differences found in the responses of each plant material under the same environmental conditions suggest that genetic characteristics also influenced on the composition of these compounds. Chemical variation in a species can influence on its climatic adaptation and protection against different stress factors. This supports their ability to survive and resist (^{Del Val-Días et al. 2017}).

The equality in the content of TCT, which was observed in the materials in the dry season, could indicate that they reacted in a similar way to environmental stress in this climatic season. The materials emitted similar responses to physiological changes associated with adverse environmental conditions, which led to the synthesis of similar concentrations of CT (^{Valares 2011}). The CT levels found in this study did not exceed the limits in which intake and digestibility can be affected (^{García et al. 2008}). Considering the beneficial effects associated with these compounds, the incorporation of forages in ruminant diets could reduce methane emissions, as well as improve weight gain, some meat quality parameters and milk production of productive animals (^{Jenko et al. 2018} and ^{Ku-Vera et al. 2020}).

Conclusions

The T. diversifolia materials showed differences in the content and type of phenolic compounds in both seasons of the year, with their concentrations increasing as plant growth progressed. These studies will contribute to selecting the materials with the greatest biological potential for animal feeding. Future researches are recommended, which can elucidate the biological activity of these substances and their relation with the beneficial effects in animals.

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Received: December 15, 2021; Accepted: April 29, 2022

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Conflict of interests: The authors declare that there is no conflict of interest between them.

Authors contribution: Idania Scull: Conceptualization, Investigation, Methodology, Writing-original draft. Lourdes Savón: Conceptualization, Methodology. T. E. Ruiz: Conceptualization, Methodology. Magaly Herrera: Formal analysis.