Effect of environmental and phenological factors on the antimicrobial activity of Cochlospermum regium ( Schrank ) Pilg . roots

Cochlospermum regium (Bixaceae) is a medicinal plant species native to the Brazilian savannah, known as the Cerrado. Local communities use its roots to treat infections of the female reproductive system. This study evaluated how seasonality, phenological stage, geographical location, plant age, and plant cultivation substrate affect the antimicrobial activity of Cochlospermum regium roots against Candida albicans. Although the plant displayed antimicrobial action in all the assayed conditions, the roots collected during fall and winter inhibited C. albicans the most effectively. The geographical location in which the plant material was found did not influence its biological response. Plant age and cultivation substrate did impact the biological response; those grown in sand showed fungicidal activity, while those grown in Cerrado soil fertilized with cow dung showed fungistatic activity during certain periods. The inner bark and core of the root were the main structures underlying the antimicrobial activity of Cochlospermum regium.


Introduction
Cochlospermum regium (Schrank) Pilg., Bixaceae, is a medicinal plant native to the Brazilian savannah, or the Cerrado.According to ethnopharmacological data, its roots are applied to treat infections of the female reproductive system (Nunes, Silva, & Rezende, 2003;Souza & Felfili, 2006;Moreira & Guarim-Neto, 2009).Cochlospermum regium has been widely investigated for two main reasons: its medicinal potential and its existence in the Cerrado, a hotspot biome that occupies 21% of Brazilian territory and contains 4,400 endemic plant species (Zachos & Habel, 2011).
In this context, this work aimed to investigate how seasonality, phenological stage, geographical location, plant cultivation substrate, and plant age influence the antimicrobial activity of Cochlospermum regium roots against the pathogenic microorganism Candida albicans.In addition, we aimed to investigate the specific root structure (bark, inner bark and core) responsible for the fungicidal effect.
In the research laboratory, the roots were washed with detergent, running water, and they were brushed.Next, they were separated into bark, inner bark, and core.They were then dried in a circulating air oven at 45ºC for 48h, milled in a knife mill, and sieved (48 mesh).Whole roots were also used in the assays.The activities of C. regium root bark, inner root bark, root core, and whole root were compared in all of the experiments, except for the experiment conducted in a greenhouse, which evaluated the effect of age and substrate in the antimicrobial activity of roots.
The STAMP (Screening Test for Antibiotics in Medicinal Plants) (Inácio et al., 2013) method and the microorganism Candida albicans ATCC 10231 were employed to evaluate the antimicrobial activity.This method uses powdered plant materials instead of extracts.The raw plant material was dried in an oven at 45ºC under circulating air, ground in a knife mill and sieved to standardize the particle size (48 mesh).The mass of dry powdered plant material deposited in each well (28 mg) was calculated from its density and the volume of the well.
A totally random statistical design based on four specimens was used to assess the influence of geographical location, and six specimens were used to assess the other parameters.Statistical analyses were conducted using a Tukey's test with 0.5% probability.The program SISVAR aided the statistical calculations (Ferreira, 2011).

Effect of phenological stage on the antimicrobial activity of C. regium roots
To evaluate how the phenological stage affects the antimicrobial activity of C. regium roots, the roots of specimens growing in the municipalities of Altinópolis (São Paulo State), Rifaina (São Paulo State) and Sacramento (Minas Gerais State) were collected in the morning during the vegetative/pre-flowering and flowering stage.Six specimens were assessed for each phenological stage and population.

Effect of seasonality on the antimicrobial activity of C. regium roots
To examine how seasonality impacts the antimicrobial activity of C. regium roots, specimens were always collected at the end of summer (March), spring (December), fall (June), and winter (September) in the municipality of Altinópolis (São Paulo State), also in the morning.Six specimens were collected at the end of each season.

Effect of geographical location on the antimicrobial activity of C. regium roots
To assess how geographical location influences the antimicrobial action of C. regium roots, the roots of specimens growing in the municipalities of Altinópolis (São Paulo State), Rifaina (São Paulo State), Sacramento (Minas Gerais State), Campo Alegre de Goiás (Goiás State) and Terenos (Mato Grosso do Sul State) were collected during the vegetative stage.Four specimens were assayed for each population.
Meteorological data (temperature, relative humidity and rainfall) on the Cochlospermum regium collection months and location were obtained from automated stations of the Instituto Nacional de Metereologia (Brazilian Meteorology Institute, INMET, http://www.inmet.gov.br).

Effect of plant age and cultivation substrate on the antimicrobial activity of C. regium roots
To assess how plant age and cultivation substrate affect the antimicrobial activity of C. regium roots, plants were cultivated in a greenhouse in Ribeirão Preto city (São Paulo State -Latitude: 21°10'40", Longitude: 47°48'36", Altitude: 546m); seeds were collected in the same municipalities as the roots.The seeds were grouped into a single lot, which was used for cultivation.Two cultivation substrates were employed: 1) Cerrado soil (oxisol soil) mixed with cow dung (1:1) and 2) sand, which represents the extreme concentration of soil nutrients.The plants were watered manually twice a day.Analyses were performed on plants aged two, four, six, nine, and twelve months.To perform the experiments, thirty whole plant roots collected at different ages were grouped into a single lot of ten roots each, and the antimicrobial tests were carried out in triplicate.

Effect of soil constituents on the antimicrobial activity of C. regium roots
A simple correlation helped to evaluate how soil constituents influenced the antimicrobial action of C. regium roots.Analysis of the main components (Principal Components Analysis -PCA) was also accomplished with the aid of the program Genes VS 2009.7.0 (Cruz, 2006).The antimicrobial activity data used in this analysis were obtained during the assay that evaluated the effect of geographical location.The analysis of the main components included 17 variables: biological activity, organic matter (OM), pH, phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S-SO 4 ), total bases (TB), potential acidity (H + Al), cation exchange capacity (CEC), bases saturation (V), boron (B), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn).

Effect of phenological stage on the antimicrobial activity of C. regium roots
The phenological stage did not elicit a standard antimicrobial response (Table 1).For the plants collected in Altinópolis, whole roots and inner root barks of specimens in the vegetative/pre-flowering stage displayed higher antimicrobial activity compared with specimens in the flowering stage.Concerning the plants collected in Rifaina, specimens in different phenological stages (vegetative and flowering) did not differ significantly in terms of antimicrobial action.As for the roots collected in Sacramento, their response contrasted with the response of the specimens collected in Altinópolis: all of the root structures, as well as the whole root collected during the flowering stage, exhibited higher antimicrobial activity compared with those collected in the vegetative stage.The means followed by the same small letter in the column and the same capital letter in the row do not differ statistically on the basis of Tukey's test at 0.5% probability.

Effect of seasonality on the antimicrobial activity of C. regium roots
The different seasons interfered with the antimicrobial response of the whole root and of the root bark, inner bark, and core (Table 2).In all samples, with the exception of the root bark, fall and winter favored antimicrobial activity; winter elicited the most action in the root bark compared with the other seasons.With respect to the root bark, there was an accumulation of biologically active metabolites over seasons: in the spring, the root bark was not active, but this activity started increasing in the summer and fall; it peaked in the winter (14.32 mm -Table 2).The means followed by the same small letter in the column and the same capital letter in the row do not differ statistically on the basis of Tukey's test at 0.5% probability.
Two seasons occur in the Cerrado: the dry season (fall and winter) and the rainy season (spring and summer); temperatures do not vary significantly.At the start of fall, C. regium leaves begin the process of senescence; in winter, almost 100% of the plants are deciduous.The plant experiences a higher level of stress during fall and winter, which may be observed when the humidity is low and leaves are absent.Consequently, the antimicrobial compounds migrate to the root bark to protect the root.The higher antimicrobial action verified in other root parts during fall and winter demonstrates that secondary metabolites accumulate in these parts during the drier seasons.In general, the substances produced in the secondary metabolism are more concentrated when plants are under drought stress (Selmar & Kleinwaechter, 2013).One explanation for this finding relates to the stoma closure in response to this stress.When this event happens, no CO 2 absorption occurs, and much of the NADPH + H + that the Calvin cycle ceases to consume participates in the synthesis of highly reduced compounds such as isoprenoids, simple and complex phenols, and alkaloids (Selmar & Kleinwaechter, 2013).
During the dry period, leaves are absent in C. regium, so CO 2 absorption all but ceases to occur.As a result, active compounds accumulate in the roots.Moreover, although the active compound has not been identified yet, C. regium and other species of this genus are known to be rich in flavonoids, which are complex phenols (Sólon, Brandão, & Siqueira, 2009;Sólon et al., 2012) that can protect the plant during the dry season (Ramakrishna & Ravishankar, 2011).
This work has verified that both the whole root and the root parts exhibit similar antimicrobial activity regardless of the season.When considering the fall and winter, the seasons that accounted for most of the observed antimicrobial activity, we find that, statistically, the activity in the whole root was similar to that of other structures (Table 2).Thus, there is no need for the local population to separate the root parts before using the plant.
Effect of geographical location on the antimicrobial activity of C. regium roots C. regium populations located in different geographical positions displayed similar antimicrobial activity (Table 3).The fungicidal activity was statistically similar between the different locations and different structures (except for root bark of plants collected in Rifaina and Altinópolis).Therefore, the active compounds present in C. regium are constitutive, and the environmental factors assessed in this work do not influence the antimicrobial action.Again, this assay revealed that it is best to use the whole root as a phytotherapic medicine.
It is noteworthy that the root core, inner root bark, and root bark represent 57.11% (±7.23), 26.82% (±4.64), and 16.07%(±6.87) of the root dry mass, respectively.Judging from the data obtained in the present work, the use of an isolated root part would lead to a great loss of active material.
Meteorological data from the time of plant collection that was obtained from Instituto Nacional de Metereologia (Brazilian Meteorology Institute, INMET) verified that temperature and relative humidity varied.The monthly averages of temperature and relative humidity varied the most in Terenos (State of Mato Grosso do Sul) and Campo Alegre do Goiás (State of Goiás), respectively (Table 4).Nonetheless, all of the roots collected in these areas acted as antimicrobials, which reinforced the notion that the active compounds are constitutive.The means followed by the same small letter in the column and the same capital letter in the row do not differ statistically on the basis of Tukey's test at 0.5% probability.

Effect of plant cultivation substrate and age on the pharmacological activity of C. regium roots
Roots that grew in sand and in Cerrado soil fertilized with cow dung displayed the expected medicinal effect because the soil is naturally sandy.Roots aged two and four months that grew in Cerrado soil fertilized with dung did not present any antimicrobial action; roots aged six, nine, and twelve months that grew in Cerrado soil fertilized with dung exhibited fungistatic activity (Figures 2 and 3).
In sand, the fungistatic activity was evident in the four-month-old roots.Thereafter, a fungicide effect continued until the plant was 12 months old.Hence, the active substances do exist in roots cultivated in soil fertilized with dung, but they occur at lower concentrations compared with roots grown in sand, which prevents detection of the fungicidal effect that occurred in the former case.Fungistatic activity has gained prominence in the treatment of infections because it elicits lower toxicity (Graybill, Burgess, & Hardin, 1997) and stimulates the host's immune system to act on the infection.Nevertheless, even though many studies have included results on the fungistatic activity of extracts and isolated compounds, their behavior in microorganisms and humans is not known.

Figure 3 .
Figure 3. Average value of fungistatic activity (mm) of Cochlospermum regium roots cultivated in (A) Cerrado soil fertilized with cow dung (1:1) for nine months and (B) in sand for four months.(C) Fungicidal activity of Cochlospermum regium roots cultivated in sand for 12 months.

Figure
Figure 4. Corre analysis in Cochlos As for the components c variation.Th variables of bi the first and Hence, these observed varia m.Agronomy

Table 1 .
Effect of phenological stage on the antimicrobial activity of Cochlospermum regium whole roots as well as root bark, inner bark, and core collected in the municipalities of Altinópolis (State of São Paulo), Rifaina (State of São Paulo), and Sacramento (State of Minas Gerais) against Candida albicans ATCC 10231.

Table 2 .
Effect of seasonality on the antimicrobial activity of Cochlospermum regium whole roots as well as root bark, inner bark, and core collected in the municipality of Altinópolis (State of São Paulo) against Candida albicans ATCC 10231.

Table 3 .
Effect of geographical location on the antimicrobial activity of Cochlospermum regium whole roots as well as root bark, inner bark, and core collected in the municipalities of Rifaina (State of São Paulo), Altinópolis (State of São Paulo), Sacramento (State of Minas Gerais), Campo Alegre do Goiás (State of Goiás) and Terenos (State of Mato Grosso do Sul) against Candida albicans ATCC 10231.