Effect of fertilization and liming on the content of secondary metabolites in Hydrocotyle umbellata L. var. bonariensis (Lam.) Mr. Spreng

The nature and the amount of secondary compounds produced by plant species depends on environmental factors, which can act directly on the synthesis of the secondary metabolites. Hydrocotyle umbellata L. var. bonariensis (Lam.) Mr. Spreng has been traditionally used for medicinal purposes, including for antinociceptive, antiinflammatory and anxiolytic-like effects, and phytochemical studies revealed its bioactive compounds. This work aimed to evaluate the effects of chemical and organic fertilization, and the soil base saturation correction in the H. umbellata crop in dystrophic yellow latosol soil in the production of the secondary metabolites (total phenolic, total flavonoid and hibalactone). The plant was cultivated in the soil of a rural property in the municipality of Anápolis (Goiás state). The experimental design was completely randomized in a controlled environment. The experiment with fertilization had five treatments (control; cattle manure; poultry manure; chemical fertilization; chemical and organic fertilization) and the experiment with liming included the correction of soil base saturation to 20%, 40%, 60% and 80%. The results in most of the two experiments were not statistically significant in the content of the metabolites studied. In the fertilization experiment, the control and manure treatments were statistically better in the content of total phenols in aerial mass analysis. Regarding the saturation correction experiment, the treatment without liming afforded higher levels of hibalactone content (considering the whole plant) and total phenolic content (considering the air mass). The treatment with correction of saturation to 40% afforded higher levels of total phenolic (considering the Research, Society and Development, v. 10, n. 13, e297101321337, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i13.21337 2 whole plant).


Introduction
The empirical knowledge about of medicinal plants has been transmitted from the ancient civilizations to the present day, and its uses has become a widespread practice in folk medicine (Soares et al., 2009). However, it is necessary scientific basis to evaluate the efficacy and possible risks of the plants, which involves botanical, chemical, pharmacological and toxicological studies, in addition to the development of appropriate pharmaceutical forms and techniques for quality control, in the case of the commercialization of plants as medicines (Paschoal, 1994).
The demand for phytomedicines have been caused a significant increase for this raw material. However, the market and commercialization of medicinal plants are complex and have peculiarities that make it necessary a detailed knowledge of the processes related to them in order to be successful in the sale of production (Scheffer & Correa Jr, 1998).
The cultivation of medicinal plants when poorly managed can favor plants with low content of bioactive compounds, making their commercialization unfeasible or in the opposite way, increasing the amount of substances considered toxic, making the product harmful and, therefore, of therapeutic use unfeasible (Freire, 2004).
There is little information on the nutritional aspects of native plants and their development in cultivated environments, and it is necessary to define behavior patterns for the optimization of production and future yields, especially for applications in medicinal areas (Martins et al., 1998).
It is known that both internal and external factors can influence the level of production of bioactive compounds from medicinal plants. In general, medicinal plants have a short cycle, rapid growth and are harvested in large quantities, thus requiring nutrient supplementation (Furlan, 1998). The fertilization and liming in traditional crops demonstrate a great productivity gain. The effect of soil saturation correction and chemical and organic fertilization is described in crops such as soybean, tomato, melon and orange.
The species Hydrocotyle umbellata L. var. bonariensis (Lam.) Mr. Spreng is an herbaceous perennial herb common in tropical regions and its habitat ranges from aquatic to semi-aquatic. This specie has high growth rate and reproductive capacity which enable it to quickly colonize large swaths of many habitats (Heneidy et al., 2019). It has great interest in phytotherapy, especially in Ayurvedic medicine, being recommended due to its antinociceptive, anti-inflammatory and anxiolytic activities (refs). Previous study showed the lignan hibalactone as a chemical marker linked to their activities (Oliveira et al., 2017). Nogueira (2000) verified that the attack of pests was not verified and the only disease identified so far is rust, caused by the fungus Uromices sp, and its attack was observed only in crops conducted inappropriately and without causing great economic damage.
Thus, to provide a greater knowledge of the H. umbellata cultivation, this study aimed to evaluate the effects of the chemical and organic fertilization, and the correction of saturation, in dystrophic yellow latosol of the species cultivated in municipality of Anápolis (Goiás state of Brazil), on the content of its secondary metabolites (total phenolic, total flavonoid and hibalactone).

Experimental planning:
The experiment started in August 2018, with several soil samplings being carried out and in October 2018 the soil of a rural property (-16.250727 Lat., -48.995298 Long.) with 1,100 m of altitude, in the municipality of Anápolis (Goiás state of Brazil). The soil presented the adequate characteristic for the study of saturation correction, being a dystrophic yellow latosol with base saturation of 6.83%, which was used for the experiments.
Soil saturation was correct in the first half of October. The period of action of limestone was almost three months.
From January 2019 it was conducted the fertilization, planting, crop conduction, harvest, evaluation of metabolites and statistical analysis of the data. The culture remained for 94 days in the pots for the experiment with chemical and organic fertilization and 98 days in the vessels for the saturation study.
After cultivation all plants were removed heavy and dried. Moving on to dry matter evaluation, metabolite evaluation, and statistical data analysis. The experiment was carried out in plastic pots of 45 cm x 15 cm x 15 cm, the plots, in which each vessel was a plot. All two experiments were with the treatments in number of five and each treatment had four replications, totaling 20 plots, per experiment.
In each pot, five seedlings were planted, with subsequent choice of three plants per pot, standardizing them. Irrigation was performed when necessary, the crop does not support excess water. The management was performed manually, with weed control through the arranquio and removal of some locusts that appeared in the environment.
All plant development was observed and at the end of cultivation all plants were removed and metabolite production was evaluated. The evaluation of secondary metabolites was performed from the aerial mass, separately from the underground mass. The evaluation process (the analyses of metabolites) was carried out at the Federal University of Goiás, in which the protocol and the study of metabolites are in patent mode by the Institution.
The experimental design was completely randomized, because it is the most suitable for the conditions of conducting this experiment (controlled environment) (Banzato & Kronka, 1989). The results of metabolite contents were submitted to variance analysis, and the doses were compared by the Tukey test, at the level of 5% probability.

Obtaining the vegetable drug:
The plant material from the cultivated plots was removed from the pots, previously described, washed with running water, dried in a greenhouse with forced air circulation at 40ºC and crushed. The plant drug obtained in the form of powder was packed under light and moisture and under refrigeration at -18°C.

Determination of total phenol and total flavonoid content
Total phenolic content (TPc) was determined, in triplicate, according to Mole & Waterman (1987). For that, 1 mL of extract was dissolved in 5 mL of ethanol 70% in a volumetric flask. Sample's spectrophotometer absorbance was measured at 510 nm. Standard curves were prepared with tannic acid. Total flavonoid content (TFc) was determined, in triplicate, following the method by Rolim et al. (2005). Sample's spectrophotometer absorbance was measured at 361 nm. Standard curves were prepared with rutin.

Determination of hibalactone content
The hibalactone quantifications were performed by High Performance Liquid Chromatography (HPLC) using the chromatographic conditions established by Oliveira et al. (2019). Hibalactone content (Hc) was determined by comparison with the standard (isolated hibalactone) obtained in our previous work (Oliveira et al., 2019). Stock solutions of the standard were prepared in the range of 10-150 μg mL -1 . The mean of the three calibration curves and the equation resulting from the linear regression were used to determine the Hc. The method was validated following the Brazilian National Health Surveillance Agency guidelines (data not shown) (Brasil, 2017).

Results and Discussion
The crop harvest of the experiment that evaluated the effect of chemical and organic fertilization on acariçoba crop was carried out at 94 days after planting, and the aerial mass was dried and evaluated, separately from the subterrant mass for the production of secondary metabolites (Hibalactone, Total Flavonoids and Total Phenols).
For the quantification of total phenolic compounds, a standard curve of tannic acid was constructed, as shown in For the quantification of total flavonoids, a rutin standard curve was constructed, as shown in Figure 2. From the equation obtained from the standard curve, it was possible to calculate the concentration (mg/mL) and total flavonoid content of the plant drug.
In the analysis of the selectivity of the HPLC method for quantification of hibalactone in the plant drug, the peak corresponding to the hibalactone pattern was observed with retention time of approximately 7 min, which was also verified in the extract. The absorbance spectrum in the ultraviolet region determined for the hibalactone pattern, through the DAD detector, reveals absorbance regions equivalent to the pattern with plant drug extract (Figure 3). The absorption spectra demonstrate that the method is capable of measuring the hibalactone compound in the presence of other constituents, being selective according to the definition of RDC no. 166/2017 (Brasil, 2017). The selectivity for the methanol diluent was also performed by scanning the DAD detector system and did not exhibit maximum absorption that could interfere in the detection of the hibalactone marker at 290 nm according to the co-validated method. Therefore, there was no interference in the chromatograms of the extract or hibalactone pattern.

Source: Authors
The calibration curve obtained is shown in Figure 4. The relative standard deviation between the peak areas was less than 5% at all points of the curve. According to RDC No. 166/2017(BRASIL, 2017, the minimum acceptable value for the correlation coefficient is 0.99. For this attribute the method was considered linear presenting a correlation coefficient equal to 1.0 (r = 1.0), which demonstrates that the results obtained are directly proportional to the concentration of the analyte in the sample. The following equation of the line was obtained: Research, Society and Development, v. 10, n. 13, e297101321337, 2021 (CC BY 4. The results that express the precision parameter at the repeatability level are described in tableau 1. For a relative standard deviation limit of no more than 5% (Brasil, 2017), the results showed a relative standard deviation equal to 1.13%, evidencing the reliability of the method developed and validated for quantification of hibalactone in the plant drug.  Table 1 shows the Hibalactone content (in the aerial mass, underground mass and the total of the plant); in Table 2 the total phenol content (in the aerial mass, underground mass and the total of the plant) and Table 3 the total flavonoid content (in the aerial mass, underground mass and the total of the plant) as a function of fertilization (T; EB, EA, AQ and AQO).  Where: witness (T); bovine manure (EB); poultry manure (EA); chemical fertilization (AQ) and chemical and organic fertilization (AQO). Source: Authors.
In Table 3 of mean total phenol content in the culture of acariçoba, we observed that the means of the air mass content are statistically different in the treatments studied and that the Control (without fertilization) and As (fertilization with Poultry Manure) present the best results, differing by the Tukey test to 5% in the means for the other treatments. Other authors such as Research, Society and Development, v. 10, n. 13, e297101321337, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i13.21337   Table 5 shows the Hibalactone content (in the aerial mass, underground mass and the total of the plant); in Table 6 the total phenol content (in the aerial mass, underground mass and the total of the plant) and Table 7 the total flavonoid content (in the aerial mass, underground mass and the total of the plant) as a function of the correction of saturation applied before planting. Where: no lime (SC) addition; Correction of base saturation to 20% (S20); Correction of base saturation to 40% (S40); Correction of base saturation to 60% (S60) and Correction of base saturation to 80% (S80). Source: Authors.
In Table 5 we have the Hibalactone content for the acariçoba crop as a function of the correction of soil saturation.
These means of these treatments did not differ statistically for air mass and ground mass, but the highest values for air mass were treatments SC (without cathe) and S40 (correction of base saturation to 40%) and as for the underground mass the SC treatment was the most responsive.
In Table 6, the behavior of the Total Phenols content varies according to the evaluated part of the acariçoba crop.
When analyzing the production of phenols in the aerial mass, there was a statistical difference and the best treatment was CS (without correction) being statistically superior to all treatments. This result is different than authors such as Mascarenhas et al. (1990); Mascarenhas et al. (1996); Faria et al. (2003); Silva et al. (2007); Ayres and Alfaia (2007) and Benedetti et al. (2009) because the behavior was the increase with the correction of base saturation and in this work the behavior is the decrease.
Being also different from the authors Oliveira Junior et al. (2006); Calgaro et al. (2007); Costa et al. (2007) and Souza et al. (2010) where the treatments were not significant in the production of Essential Oil with the use of soil pantic.
The results of Total Phenol sums when we evaluated the underground mass of Acariçoba had as results different values, the treatment S20 (saturation of 20%) was the highest value, but not statistically significant, corroborated by Oliveira Junior et al. (2006); Calgaro et al. (2007); Costa et al. (2007) and Souza et al. (2010) in which in their cather treatments were also not significant in the production of Essential Oil in the studied crops. Different from the works of Mascarenhas et al. (1990); Mascarenhas et al. (1996);Faria et al. (2003); Silva et al. (2007); Ayres and Alfaia (2007) and Benedetti et al. (2009) which generated an increase in production with soil dredging.
When we evaluated the production of total phenol levels of the whole of Acariçoba we have an interesting behavior, interesting because there was a statistical difference in the treatments having a peak, or rather, a higher and higher value for all when the saturation by soil bases was increased to 40%, corroborated by the authors Mascarenhas et al. (1990); Mascarenhas et al. (1996);Faria et al. (2003); Silva et al. (2007); Ayres and Alfaia (2007) and Benedetti et al. (2009) which the limeing favored and also had in some studies the peak in saturation of 50% as Silva et al. (2007). However, the authors Oliveira Junior et al.    Where: no lime (SC) addition; Correction of base saturation to 20% (S20); Correction of base saturation to 40% (S40); Correction of base saturation to 60% (S60) and Correction of base saturation to 80% (S80). Source: Authors.
Observing chart 7, the total flavonoid content for the acariçoba crop is observed. The treatments did not differ statistically in all results, but the production of Total Flavonoids for the Treatments SC (without correction) were the ones with

Conclusion
The two experiments conducted in Dystrophic Yellow Latosol soil with acariçoba culture showed favorable results to the conclusions. The levels of secondary metabolites evaluated showed different behaviors, and some were neither significant, thus we conclude that: For the experiment with Fertilization: 1. For hibalactone, chemical fertilization provided the highest values of the active ingredient, but not statistically differing from the other treatments; 2. For the total flavonoids all the results were not significant, but as the aerial mass the chemical fertilization provided the highest value, how much the control was the best value and how much the total production of the plant the fertilization with poultry manure was the best; 3. Analyzing the production of total phenols, poultry manure and without fertilization for the levels in the aerial mass of the plant were statistically better than the other treatments. As for the underground mass and the whole plant there is no statistical difference, but the chemical and organic fertilization had the highest value and the poultry manure was the one of the highest value for the whole plant.
For the base saturation correction experiment, we have: 1. For hibalactone, the levels in the aerial mass and underground mass the treatments did not differ statistically, but in the aerial the elevation to 40% saturation and the non-correction for the underground mass were the highest values.
As for hibalactone production in the plant, the whole non-correction was statistically higher than the other treatments; 2. For the total phenols of the aerial mass was not significant and higher value with the increase to 40% saturation.
As for the underground mass, the non-correction of saturation was statistically superior to the other treatments.
When we evaluated the total phenol contents of the plant, the increase to 40% of saturation was statistically the best treatment; 3. For the production of total flavonoids, the treatment without correction of saturation was better than the other treatments and none of the treatments differed statistically.
With the results, there is a need for more studies on Acariçoba, evaluating the effect of chemical and organic fertilization in eutrophic soil; planting density; of the harvest time in the production of secondary metabolites (total phenolics, total flavonoids and hybalactone).