Chemical assessment, antioxidant and antimicrobial of leafs extracts of Virola sebifera, an Amazonian medicinal plant

The high biodiversity of Amazon forest implies in a great number of plants with ethnopharmacological utilization. V. sebifera is one of the most important species of Virola genus, used in treatment of rheumatism, arthritis, dyspepsia, malaria, muscle pain and erysipelas. This study aimed to investigate its chemical composition, antioxidant and antimicrobial properties. For this, leafs extracts i) 70% ethanol in ultrasound bath (CEU); ii) 70% ethanol in Soxhlet (CES); and iii) sequential extraction in Soxhlet apparatus, starting with hexane (HE), followed by methanol (ME), and 70% ethanol extract (EE). Phenolic concentration, total flavonoid and antioxidant activity were assessed. The highest phenolic and total flavonoid contents were found in CEU and EE showed the best antioxidant activity. The most relevant substances identified by GC-MS analysis were the Kusunokinin, Hinokinin and catechol, among others first time related in V. sebifera. The antimicrobial activity was tested against Staphylococcus aureus, S. epidermidis, Salmonella typhimurium, Escherichia coli, and Candida albicans. The CEU, CES, EM, and EE obtained positive results against S. aureus and S. epidermidis. CES and EM also inhibited S. typhimurium and E. coli. Based on these results, V. sebifera can be recognized as a promising source of antioxidant and antimicrobial compounds.


Introduction
Brazil has several biomes and one of the greatest biological diversities in the world. In this context, the region called the Brazilian Legal Amazon stands out, covering an area of 4.196.943 km² (Somavilla et al., 2020). It constitutes a surface that covers nine Brazilian states: Acre, Amazonas, Amapá, Maranhão, Mato Grosso, Pará, Rondônia, Roraima and Tocantins (Saraiva et al., 2020), which corresponds to approximately 61% of the Brazilian territory, it is revealed by the largest continuous reserve of tropical rainforest with one of the greatest biodiversity in the world, which covers part of the country's three largest biomes, Amazon, Cerrado and Pantanal (Saraiva et al., 2020;Ministry of Environment, 2018).
Among the nine component states of this region, the State of Tocantins stands out for being located in an ecotonal region between the Amazon Forest, Cerrado and Pantanal (Coutinho et al., 2019). The biological diversity of the Amazon is one of the most important natural heritages of humanity, with approximately 40 thousand species of plants (Santiago et al., 2019). Likewise, the Cerrado is considered a biodiversity hotspot, qualifying it as the richest savanna in the world (Castuera- Saraiva et al., 2020).
Due to the great plant biodiversity and morphological characteristics of the species in this biome, it appears that its chemical constituents are diversified and with enormous biological potentials (Castuera- . However, these resources have still been little studied even though the flora is threatened by deforestation and the advance of the agricultural frontier that leads to the loss of medicinal biodiversity (Roquette et al., 2019;Castro et al., 2019). Such loss, together with the traditional knowledge associated with it, denotes an urgent need to guarantee the registration of this knowledge and biodiversity (Roquette et al., 2019). This legitimizes the need for research with Amazonian medicinal plants, as these studies bring popular knowledge closer to scientific knowledge, they help to preserve the forest and contribute to the sustainable use of biological resources (Moraes et al., 2019).
The therapeutic potential of medicinal plants, perpetuated over the generations, is justified by the presence of bioactive components that have many proven biological activities (Moraes et al., 2019). Phytochemical research aims to verify the presence of groups of secondary metabolites, study the structural characterization, evaluate the properties and investigate the biosynthesis of natural compounds (Bicalho et al., 2012;Garcia et al., 2019;Martínez-Francés et al., 2017).
One of the therapeutic effects of secondary metabolites of high importance is the antioxidant activity, that is, protection against damage caused by free radicals, preventing or postponing the onset of various diseases (Garcia et al., 2019), a biological role attributed mainly to compounds phenolics, especially flavonoids (Sarwar et al., 2015;Rezende et al., 2005).
Another biological activity of interest is antimicrobial. The search for plant-based antimicrobials has advanced due to concerns about microbial resistance confirmed in recent years (Ghuman et al., 2016). Medicinal plants are used for their bioactive properties, most without any toxicological study . Among these plants stands out the Virola sebifera (Aubl.), popularly known as ucuuba, urucuba and bicuíba (Rodrigues, 1980), V. sebifera is a species of the Myristicaceae family that occurs in the savannahs of Central and South America (Santamaría-Aguila et al., 2019).
However, it is worth emphasizing the need for the search for new molecules with biological action. The present work investigates the main classes of secondary metabolites, the antioxidant, antimicrobial and cytotoxic activities of V. sebifera in order to validate the empirical therapeutic indications for this plant and highlight its potential for the exploration of its molecules by the productive sector.

Plant material
The leaves of the species Virola sebifera (Aubl.) were collected on the banks of sub-basin of Ribeirão São João in Porto Nacional, state of Tocantins, Brazil, between coordinates S 10º25'12" and O 48º16'47" in June 2018. The access was registered at SISGEN under number A53BF6B. One voucher specimen was produced and deposited at the Herbário Tocantins (HTO) linked to the Federal University of Tocantins, Campus of Porto Nacional, under registration number HTO 1202.

Preparation of extracts
The leaves of V. sebifera were dried and stabilized in an oven at 50°C, then milled in a Willye knife mill (model star FT 50, Fortenox brand). Five extracts were prepared by two methods, extraction by ultrasound-assisted and soxhlet apparatus.
The Soxhlet extraction was performed based on the method described by Soares et al. (2017) with modifications, using 5g of leaf powder in the extractor and 200 mL of solvent in the boiling flask heated to the boiling temperature of the solvent, for a period of six hours. An extraction was performed with only 70% ethanol giving rise to the Crude Soxhlet Extract (CES). By this same method, sequential extraction was performed, starting with hexane, for maximum removal of nonpolar substances, followed by methanol and 70% ethanol, giving rise respectively to the hexane extract (HE), methanol extract (ME), and ethanol extract (EE). Each time before performing the extraction with the next solvent, the powder was allowed to dry at room temperature for 24 hours. In ultrasound-assisted extraction, the powder from the leaves was mixed with 80 ml of 70% ethanol in a beaker, which was immersed in an ultrasonic cleaning bath (USC1600, ultrasonic cleaner, frequency 40 kHz, 135 W) programmed for 1 hour cycles. The process was repeated 10 times and the supernatant was collected and renewed in each cycle, combined and filtered at the end, originating the Crude Ultrasound Extract (CEU). The solvents of the all extracts were removed in a rotary evaporator at -600 mmHg at 45 o C, after that the extracts were lyophilized and stored in sterile flasks until analysis.

Phytochemical Prospecting
The characterization of the main classes of metabolites was carried out through color and precipitation reactions, with tests for tannins, flavonoids, anthraquinones and saponins following the methodology of Mouco et al. (2003) and phytosterols by the method described by Lin et al. (2009).

Determination of the total content of phenolic compounds
Total phenols were quantified using the Folin-Ciocalteu method, as described by Amorim et al. (2008), with modifications, using tannic acid as a reference standard. Briefly, 0.2 mL of methanol solutions of V. sebifera extracts CEU, CES, EH, EM, and EE (1 mg/mL, w/v) or the tannic acid standard (3-100 μg/mL, w/v) were mixed, 1 mL of calcium carbonate (7.5%, w / v), 0.5 mL of Folin-Ciocalteu reagent (10%, v/v) and 8.3 mL of distilled water, gently stirred and kept for 30 min in the dark. Absorbance was measured using a spectrophotometer (760 nm) and distilled water was used as a blank. The total phenolic content was determined by interpolating the absorbance of the samples against a standard curve constructed with different concentrations of tannic acid in methanol (y = 0.005x + 0.007, adjusted r 2 = 0.980). The result was expressed in mg of tannic acid equivalents (TAE) per gram of extract of the studied plant (mg TAE/g). The analysis was performed in triplicate.

Determination of the total flavonoid content
The content of total flavonoids was quantified using the method described by Soares et al. (2014) with modifications, using the rutin as a reference standard. Methanol solutions (0.5 mL) of V. sebifera leaf extracts (1 mg/mL, w/v) or rutin standard (8-400 mg/mL, w/v), aqueous solution were mixed with acetic acid (0.5 mL at 60%, v/v), aluminum chloride (1 mL at 5%, w/v), methanol pyridine solution (2 mL at 20%, v/v) and 6 mL distilled water. The blank was prepared by replacing aluminum chloride with methanol. The mixtures were stirred and kept in the dark for 30 min, their absorbance measured at 420 nm in a spectrophotometer. The content of total flavonoids was determined by interpolating the absorbance of the samples against a calibration curve (y = 0.002x + 0.012, adjusted r 2 = 0.999), constructed with different concentrations of rutin and expressed in milligrams of rutin equivalents (RE) per gram of dry extract (mg RE/g). The reactions were carried out in triplicate.

Evaluation of antioxidant activity
The evaluation of antioxidant capacity was determined by the 1,1-diphenyl-2-picryl-hidrazila (DPPH) method established by Brand-Williams et al. (1995), and modified by Peixoto Sobrinho et al. (2011), with the rutin pattern as a positive control. 0.5 mL of different concentrations of extracts or standards (20 -200 μg/mL, w/v) was mixed with a methanol solution of DPPH (3 mL at 40 μg/mL, w/v). The blank was prepared by replacing DPPH with methanol in the reaction medium. The reaction complex and the white were stirred and kept in the dark for 30 min, the absorbances measured at 517 nm in a spectrophotometer calibrated with methanol. As a negative control, the DPPH solution at 40 μg/mL was used. The activity of removing free radicals or antioxidant activity was expressed as the percentage of inhibition determined by the equation: % AA = ABScn -(ABSsample -ABSwhite) / ABScn × 100 % AA is the percentage of antioxidant activity; ABScn, the absorbance of the negative control; ABS sample, the absorbance of the sample; ABS white, white absorbance. The IC50 (μg/mL) was obtained using the calibration curves obtained by plotting the different concentrations in relation to % AA. The assays were carried out in triplicate.

Characterization by gas chromatography mass spectrometry (GC-MS)
The extracts were analyzed by GC-MS using a Shimadzu® chromatograph model QP2010 Ultra equipped with a ZB-5HT column (30 m x 0.25 mm x 0.25 µm). It was carried out under the following conditions: heating at 60°C for 1 min, until reaching 280 °C in 35 min. Injection temperature: 280°C; Interface temperature: 280°C; carrier gas (Helium): 1 mL/min; the electron energy was 70 eV and the temperature of the ion source was 280 °C; scan mode. 1 µL of each extract was injected, in which the constituents were identified by comparison with the mass spectra of the NIST 14 library.

Antimicrobial activity
For the bioassays reference strains ATCC (American Type Culture Collection) obtained from the collection of the National Institute for Quality Control in Health, Fundação Oswaldo Cruz (INCQS/FIOCRUZ, Rio de Janeiro, RJ) were used.
The strains selected for the study were Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 25922), and the fungus Candida albicans (ATCC 10231). The microorganisms Staphylococcus epidermidis (NEWP 0128), Salmonella typhimurium (NEWP 0028) were purchased from the Newprov Laboratory. The strains were stored under freezing at -80 ºC in Brain Heart Infusion (BHI) broth. For reactivation, they were grown in nutrient agar and incubated for 24 hours at 35ºC.

Well diffusion test
Antimicrobial activity was performed using the well diffusion method, in triplicate and with 3 replicates (CLSI, 2012). The extracts were diluted in a mixture with 10% dimethyl sulfoxide (DMSO) in the concentrations (200, 100 and 50 mg / mL). The inoculum solutions were prepared using isolated strains, diluting in 0.85% saline solution until reaching the turbidity corresponding to 0.5 on the MacFarland scale, obtaining about 1,5 × 10 8 (UFC/mL) of bacteria and 2,0 × 10 6 UFC mL -1 of yeast. For the positive control, 2 mg/mL chloramphenicol was used for the bacteria and 1,5 mg/mL albocresil for the fungus, for the negative control the 10% DMSO solution (Oliveira et al., 2017 with adaptations). After 24h of incubation at 37 ºC, the results were analyzed by measuring the diameter of the inhibition halos observed around the discs with a digital caliper model Starret 799.

Determination of Minimum Inhibitory Concentration (MIC)
The tests were performed in a 96-well sterile microplate, in triplicate. In each microplate, the extract for a microorganism was tested (NCCLS, 2003), a step made after the screening performed by the well diffusion test. 100 μL of the extracts were added to each well at final concentrations of 100; 50; 25; 12,5; 6,25; 3,12; 1,56; 0,78 mg/mL. For the positive control, 2 mg/mL of chloramphenicol, 1,5 mg/mL of albocresil and inoculum were used, as a negative control, CMH, 10% DMSO solvent and inoculum were used. For growth control, 5 μL of 10 7 UFC/mL bacteria suspension was used. The plates were incubated at 37 ºC for 24 hours .
After the incubation period, 0.03% (m/v) resazurin microplate wells were added to each well, which were reincubated for approximately 1h. After this period, the reading was performed, showing that the presence of blue color represented the absence of growth and pink color, presence of bacterial growth (Palomino et al., 2002;Li et al., 2017). MIC was considered the lowest concentration of the extract capable of inhibiting bacterial growth.

Statistical analysis
The experiments carried out in triplicate had the results expressed as mean ± standard deviation. The data obtained were submitted to statistical analysis using the program SISVAR version 5.6 (Ferreira, 2008) and GraphPad Prism 8. The Analysis of variance (ANOVA) was used to compare the average values obtained in the analysis. The values p <0.05 were considered statistically significant by the Tukey test.

Phytochemical prospecting
Phytochemical prospecting revealed only flavonoids and alkaloids in HE (Table 1). In the other extracts obtained in this experiment, these classes of substances were found to be associated with tannins, flavonoids, flavonols and anthraquinones (Table 1). Such secondary metabolites can act synergistically as good antimicrobials and antioxidants (Kosalec et al., 2013;Rammohan et al., 2019).

Content of Phenolic and Flavonoids
As shown in Table 2, the values of total phenolic compounds and total flavonoids in the leaf extracts of V. sebifera varied significantly (p<0.05).
The highest concentration of phenolic and flavonoid compounds was found in the CEU extract followed by the CES and EE extract, respectively. At the same time that the data obtained allow us to observe the effect of polarity on the extraction of antioxidants from this plant, with less affinity for nonpolar hexane solvent, it was also found that for an efficient extraction of non-destructive bioactive molecules from the leaves of V. sebifera it is necessary to consider the temperature of the extraction system, with cold extraction being ultrasound-assisted which best preserves the polyphenols. The effect of polarity on the extraction of phytochemicals has also been observed by Nawaz et al. (2020) who studied the effect of solvent polarity on extraction yield and antioxidant properties of phytochemicals in bean seeds. Cold extraction, on the other hand, is very well mentioned by Dzah et al. (2020), who, when evaluating various extraction parameters in different studies, found that extraction temperatures above 50°C can degrade polyphenols and that ultrasound-assisted extraction can favor the extraction yield and better preserve the biological activity of the extracts when compared to traditional maceration and Soxhlet extraction.

Evaluation of Antioxidant activity
Flavonoids are widely distributed in the plant kingdom, representing one of the most important and diverse phenolic groups among products of natural origin (Simões et al., 2017) and constitute a potential alternative as therapeutic agents in the face of inflammatory processes (Coutinho et al., 2009), they help to protect against UV rays and attack by microorganisms, and the phenolic compounds help to protect against attack by fungi (Díaz, 2015). In addition, several studies show that the presence of phenolic compounds can contribute to antioxidant activity (Toiu et al., 2018).
All extracts from the leaves of V. sebifera showed sequestering activity of the DPPH radical with the exception of EH extract. The DPPH radical scavenging activity, expressed as IC50, is presented in Table 3. The EH showed antioxidant activity values below 50%, making it impossible to determine the IC50. The weak antioxidant activity of EH can be justified by the low content of total phenols (Table 2). This evaluation demonstrates that the extract (EE) extracted sequentially was more pure in terms of the presence of antioxidants, because even though it was lower than the CEU extract in the concentration of phenols and flavonoids, it presented the best IC50, with a value close to that of the reference substance rutin.  Figure 1 presents the data obtained in the analysis of the total antioxidant activity in three concentrations of the extracts and the reference substance rutin. The data presented are consistent with the concentration of total phenolics and total flavonoids and demonstrate that the plant is an excellent source of natural antioxidants, since in concentrations of 60 and 120 µg/mL they had a total antioxidant activity equal to the rutin standard. It was found that ultrasound extraction was efficient in obtaining antioxidants, while sequential extraction in Soxhlet can favor the removal of non-antioxidant interferents in the EE extract, which showed a higher percentage of elimination of DPPH radicals in relation to other extracts at a concentration of 30 µg/mL. These data are in agreement with those obtained by Soares et al. (2017), with Siparuna guianensis, in the sequential extraction was able to eliminate interference and favor the extraction of phenolic compounds. Source: Authors.
The antioxidant activity V. sebifera was also determined by García et al. (2019), who found 81.08% antioxidant activity with an IC50 of 1.5 mg/ml in the methanol extract. In another study, Rezende et al. (2005) determined that the antioxidant activity of ariltetralone lignans isolated from V. sebifera seeds, as well as catechol derivatives obtained by demethylene lignans, indicated better antioxidant activities when compared to the standard α-tocopherol.
These results point to a promising profile of antioxidant compounds, which may, in the future, be used to obtain new products, since phenolic substances in plants have effects in the reduction of cancer and an inverse relationship between cardiovascular diseases and the ingestion of food sources of phenolic compounds, probably due to their antioxidant properties (Bernardes, 2010).
2-(hydroxymethyl)-2-nitro-1,3-propanediol or tris(hydroxymethyl)nitromethane (8), (Figure 2), was found in extracts EM (12.43%) and EE (7.98%), both obtained by the sequential extraction method. Sequential extraction favored the extraction of this compound, since it was not found in the crude extracts. Tris(hydroxymethyl)nitromethane is a biocide used in microbial control (Yin et al., 2018) in addition to being used in organic synthesis as a precursor to a series of reactive nitrogen compounds (Choteau et al., 2012;Morin & Sello, 2010).
The compounds 3, 5-10 were described by the first time in V. sebifera species. The results obtained in the analysis of the extracts showed a significant difference in the amount and type of compound identified in each extract, showing the importance of choosing the method and the solvent to be used in the extraction of bioactive compounds. García et al. (2019), found in the methanolic fractions of V. sebifera folic acid, 3,5-diterbutio-4-hydroxyanisole (phenolic compound), karyophylene (bicyclic sesquiterpene) and spatulenol (alcoholic sesquiterpene), compounds recognized as antioxidants. Tonelli et al. (2014), in his study with the same plant found quercetin-3-O-α-ramnopyranoside as the main constituent. Pereira et al. (2018) reported that in the essential oils obtained from the leaves, bark/pulp and seed of V. sebifera, collected at the Research Campus of the Museu Paraense Emilio Goeldi, in Belém, Brazil, 28 chemical constituents were identified, being the majority (E, E) -α-farnesene, bicyclogermacrene, (E) -β-ocimene, 6-methyl-5-hepten-2-one, α-pinene, Ekaryophylene.

Antimicrobian activity
The antimicrobial activity of V. sebifera leaf extracts was carried out against four bacterial strains, two Gram-positive, Staphylococcus aureus and Staphylococcus epidermidis, and two Gram-negative strains, Salmonella typhimurium and Escherichia coli, and one yeast, Candida albicans, using chloramphenicol as a positive control for bacteria and albocresil, for the fungus (Table 5). (-) there was no inhibition in the tested concentrations. Positive control 2.0 mg / mL of chloramphenicol (to bacteria strains) and 1.5 mg / mL of albocresil (to fungi strain). Source: Authors.
The inhibition tests in the shaft showed some inhibition of the microorganisms tested, however this inhibition was not significant with 0.05% significance by the Tukey test compared to the positive control. In spite of this, we consider the well diffusion test only as a screening, since this methodology has great limitations, when the diffusion pattern of the extract in the previously established culture medium does not exist, as it is routinely performed for antibiograms and antifungigrams (CLSI, 2012). Considering this, we can see that the CEU extract exhibited a halo against S. aureus and S. epidermidis, the CES and EM extracts inhibited S. aureus, Sal. typhimurium and S. epidermidis and the EE extract inhibited both S. aureus and S. epidermidis (Table 5). Only the EH extract was not able to inhibit any of the tested microorganisms. It was also observed that S. aureus and S. epidermidis were the microorganisms that were most inhibited by the tested extracts. Both S. aureus and S.
epidermidis are the only Gram-positive bacteria tested. The evaluated yeast, C. albicans was not inhibited by any tested extract as well as the Gram-negative bacteria E. coli. and Sal. typhimurium, which is also a Gram negative, was only inhibited at concentrations equal to or greater than 100 mg/ml of the extracts.
From these results, the minimum inhibitory concentration (MIC) was determined for all extracts that produced an inhibition halo against some microorganism (Table 6 and Supplementary Figure S6-S9). The antimicrobial activity of plant extracts is related to some requirements such as, for example, the species and strain of the microorganism evaluated, the extractive method and the type of solvent, as the polarity of the solvent has a great influence on the extraction of plant metabolites (Girondi et al., 2017). This corroborates with the observed result, that the hexane extract, being nonpolar, did not extract substances with antimicrobial action, or at least in sufficient quantity.
According to Akhtar et al. (2018), the absence of antimicrobial activity may occur due to the smaller amount of compounds or their actions were antagonized by the presence of other compounds.
Among the microorganisms evaluated, S. aureus was inhibited by the extracts obtained by the CES, EM and EE hot method and by the CEU cold method in all tested concentrations, showing greater sensitivity to the extracts with MIC 1.56 mg/mL, showing the antimicrobial potential of V. sebifera against this pathogen. S. epidermidis, similarly to S. aureus, was also inhibited by all extracts evaluated in the MIC test, and the most efficient extract in inhibiting the growth of this bacterium was the EE extract. Sal. Typhimurium was inhibited by the CES and EM extracts and the ME extract was able to inhibit growth at the lowest concentration evaluated (1.56 mg/mL). It is observed that flavonoids, tannins were detected in all these extracts and it is deduced that these metabolites are responsible for the antibacterial activity obtained.
According to Vieira et al. (2018), natural products have better action on Gram-positive bacteria. According to Araújo et al. (2015), among the phenolic compounds, flavonoids and tannins are capable of inhibiting microorganisms and virulence factors.
Only the extracts obtained by the CES and EM hot method showed sensitivity to Sal. typhimurium with MIC of 6.25 mg/mL and 12.5 mg/mL, respectively. This result indicates that degreasing favored the extraction of polar compounds. The CES did not differ statistically from the EM at a concentration of 200 mg/mL. According to Exner et al. (2107), Gram negative microorganisms, due to their complexity, are less susceptible to antimicrobials.
The flavonoid class has antibiotic activity due to the ability to complex with proteins and the membrane of bacterial cells (Cowan, 1999;Ullah et al., 2019), there are reports of the cytotoxic activity of flavonoids against different tumor strains (Costa-Lotufo et al., 2003). Tannins are other compounds with antimicrobial activity (Monteiro et al., 2005;Ullah et al., 2019) and alkaloids, according to Kokoska et al. (2019), inhibit the growth of some pathogenic microorganisms. Velasco et al. (2005) evaluated the antibacterial activity of extracts of the species V. sebifera (Aubl.) against S. aureus and 28 strains of MRSA (Methicillin-resistant Staphylococcus aureus) from infected patients. The results showed that the aqueous extract showed greater antibacterial activity, with a sensitivity of 89.3% of the MRSA strains evaluated, followed by ethanolic (67.2%) and acetone (39.3%), inferring that the active substances present in V. sebifera are of a nature polar.
In their research, Barreto et al. (2011) reported that extracts prepared with ethyl acetate and chloroform from the sap of V. sebifera showed excellent antimicrobial activity against the species of Enteroccus faecalis and Pseudomonas aeruginosa.
The oil extracted from the leaves of V. sebifera, according to Costa et al. (2013), showed a significant inhibition against the fungus Corynespora cassicola.
In studies with extracts of V. sebifera, Baquero et al. (2015) isolated the compound methylpluviatilol that was active with a percentage of proliferation inhibition greater than 90% of Mycobacterium tuberculosis at 128 µg/mL.
According to Manandhar et al. (2019), it is difficult to compare antimicrobial activity of plant extracts, as many variables can influence the result, such as climatic aspects that influence the chemical composition of the plant, part of the plant studied, the age of the plant or its parts sampled, how to prepare the extract. However, the discovery of new antimicrobial substances is of considerable interest due to the occurrence of bacteria resistant to antibiotics currently available for the treatment of various infections (Braquehais, 2016).

Conclusion
The chemical characterization of the leaves of the V. sebifera has identified the presence of phenolic compounds in the phytochemical screening. The results show the species as a good source of natural antioxidants. The EE ethanol fraction showed statistically superior antioxidant activity in relation to the other tested extracts, which demonstrates that sequenced extraction may be a promising methodology for the extraction of antioxidant compounds and other pharmacological products.
There are few studies that demonstrate the biological potential of V. sebifera, a typical species of the cerrado, mainly in the Legal Amazonian region of Tocantins State. The CEU, CES, EM and EE extracts of this medicinal plant are very promissory, and must be more studied with the aim in to obtain an antimicrobial substance, especially against Gram positive bacteria like S. aureus and S. epidermidis. This study can advise the correct utilization of this medicinal plant by the population, showing the correct indication and preparation mode. Thus, the continuity of studies is essential to complement and prove the antimicrobial and antioxidant potential of this plant.