Phytochemical characterization , and antioxidant and antibacterial activities of the hydroethanolic extract of Anadenanthera peregrina stem bark

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The Brazilian Cerrado biome consists of a great variety of endemic species with several bioactive compounds, and Anadenanthera peregrina (L.) Speg is a promising species. In this study, we aimed to perform phytochemical characterization and evaluate the antioxidant and antibacterial activities against Staphylococcus aureus and Escherichia coli of the hydroethanolic extract of A. peregrina stem bark. The barks were collected in the Botanical Garden of Goiânia, Brazil. The hydroethanolic extract was obtained by percolation and subjected to physicochemical screening, total phenolic content estimation, high-performance liquid chromatography (HPLC) fingerprinting, and antioxidant (IC50 values were calculated for the 2,2-diphenyl-1-picrylhydrazyl assay DPPH) and antibacterial activity determination. The pH of the extract was 5.21 and density was 0.956 g/cm3. The phytochemical screening indicated the presence of cardiac glycosides, organic acids, reducing sugars, hemolytic saponins, phenols, coumarins, condensed tannins, flavonoids, catechins, depsides, and depsidones derived from benzoquinones. The extract showed intense hemolytic activity. The total phenolic content was 6.40 g GAE 100 g-1. The HPLC fingerprinting analysis revealed the presence of gallic acid, catechin, and epicatechin. We confirmed the antioxidant activity of the extract. Furthermore, the extract did not inhibit the growth of E. coli colonies at any volume tested, but there were halos around S. aureus colonies at all three volumes tested. These results contribute to a better understanding of the chemical composition of A. peregrina stem bark and further support the medicinal applications of this species.

The Cerrado, considered a hotspot of global biodiversity, is the second largest biome in South America, covering approximately 22% of the national territory. It is recognized as the richest savanna in the world, with 11.627 native plant species. In addition to environmental aspects, the Cerrado has social importance to the local population that uses its natural resources, including 220 medicinal species (Brasil, 2019). Some species in the Cerrado, such as Stryphnodendron adstringens (barbatimão) (Almeida et al., 2010;Ribeiro et al., 2014;Queiroz et al., 2021), Macairea radula (capuchina), and Pterodon emarginatus (sucupira) (Vila Verde et al., 2018), have been studied.
The genus Anadenanthera has two species, Anadenanthera colubrina, which has a wide geographical coverage, and Anadenanthera peregrina, typical to the Brazilian Cerrado (Morim, 2015;Carvalho, 2003). Anadenanthera peregrina is distributed in the drainage areas, gallery forests, and rocky fields in the Cerrado. Popularly known as angico, A. peregrina, a rustic species of canopy-forming trees, resists drought and fire due to its thick bark that protects the plant (Souza et al., 2014). According to Mota et al. (2017), the bark of A. peregrina is a potential source of polar extracts, enabling the extraction of tannins that represent approximately 17% of the bark (173.3 mg CE g -1 bark) and 59% of the hydroalcoholic extract (in catechin equivalents). The bark and seeds of Anadenanthera are used to treat wounds (Pessoa et al., 2012(Pessoa et al., , 2015 and respiratory volume testado, mas houve halos em torno das colônias de S. aureus nos três volumes testados. Estes resultados contribuem para uma melhor compreensão da composição química da casca de A. peregrina e apoia ainda mais as aplicações medicinais desta espécie.

Preliminary phytochemical screening
Phytochemical screening was performed according to the procedure described by , for each phytochemical test, 3 mL of the extract was used. The reaction intensity was visually determined using the cross test: (+++) highly positive, (++) moderately positive, (+) less positive, and (-) negative (Marín et al., 2018). Hemolytic activity was determined at 1 and 10 min of reaction of the extract with 5% red cell suspension. Optical micrographs were obtained to observe the hemolysis reaction.

Determination of phenolic compounds
The total phenolic compounds were determined as described by , using the colorimetric method with Folin-Ciocalteu reagent. The results are expressed as gallic acid equivalent (GAE) 100 g -1 dry weight.
For the analysis, 1 mL of the extract was diluted in 5 mL of methanol in a volumetric flask. To identify the peaks separated by HPLC, stock solutions (0.1 mg mL -1 in methanol) of the following standards, procured from Sigma Aldrich, were used: caffeic acid, caffeine, catechin, chlorogenic acid, ellagic acid, epicatechin, gallic acid, hesperidin, kaempferol, p-coumaric acid, quercetin, and rutin. The chromatograms were recorded at the wavelengths of 254, 327, and 366 nm, according to the different absorptions of each compound evaluated. The compounds were identified by comparing the HPLC chromatograms of the extract and those of the pure standards based on the retention time (R t ) and UV spectra in the wavelength range of 190-400 nm. Before injection, the solutions were filtered through a 0.45 μm PVDF membrane.

Antioxidant activity determination
The antioxidant activity was determined as described by . The results are expressed as the extract concentration at which 50% of 2,2-diphenyl-1picrylhydrazyl (DPPH) free radicals were inhibited (IC 50 ).

In vitro antibacterial activity of A. peregrina extract
Under a laminar flow hood, Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 samples were thawed to room temperature, and then transferred to trypticase soy broth (TSB) liquid culture medium for sample dilution and incubated at 37 °C for 4 h. The activated strains were inoculated on Cled Agar and incubated at 37 °C for 24 h for the isolation of colonies. Using a sterile loop, the colonies were transferred to selective media, MacConkey for E. coli and mannitol salt agar for S. aureus; after 24 h, the isolated colonies were verified.
Using sterile loops, the isolated colonies were collected from each selective medium, and then a bacterial suspension in saline solution (0.85% NaCl) was prepared for each strain until the turbidity reached 0.5 on the McFarland scale. For this procedure, a McFarland 0.5 calibrated tube was used as the reference. A swab soaked in bacterial suspension solution was inoculated (for each sample) on Mueller-Hinton agar, covering the entire plate. Immediately, wells of diameter 10 mm were created in the agar plate using autoclave and ultraviolet light-sterilized glass tubes. Each well was identified with letters A, B, and C and filled with 50, 100, and 200 μL A. peregrina extract, respectively. Meropenem discs were used as the positive control and 200 μL of saline solution as the negative control. The plates were incubated in a bacteriological oven for 24 h. There were five replicates for each microorganism on different days (Silveira et al., 2009).

Preliminary phytochemical screening
The extract was clear, homogeneous, and dark brown. Table 1 presents the results of the preliminary phytochemical screening. The hydroethanolic extract of A. peregrina stem bark was positive for glycosides, based on the moderate-intensity reaction with Kedde and Keller-Kiliani reagents, and a highly positive reaction with Raymond-Marthoud reagent. However, the result of Baljet reagent test was negative. The reaction with Baljet and Kedde reagents was positive due to the presence of compounds with cardenolide unsaturated pentagonal lactone ring. The reaction with Keller-Kiliani reagent was positive due to the presence of deoxygenating compounds (deoxysugar) with a free end. The reaction was positive with Raymond-Marthoud reagent due to the presence of an aglycone (genin), a non-glycidyl group that forms a part of glycosides.
The extract showed negative results in the tests for alkaloids, including the Libermann-Bouchardat, Wagner, and Mayer tests. The test for organic acids was positive with medium intensity according to the cross test. The test with Fehling's reagent for reducing sugars was also positive. The test for coumarins was positive. Foamed saponins were not observed in the extract.
A strong hemolysis was observed in a short time, that is, 1-10 min after incubation of the hydroethanolic extract with red blood cell suspension ( Figure 1). Furthermore, in the micrographs, erythrocyte hemolysis was apparent.
The extract showed a positive reaction for condensed tannin compounds and intense reaction in the tests for catechins and flavonoids. Benzoquinone and depside and depsidone derivatives were detected in the extract, based on the positive results with an intense reaction in the respective tests. The tests for purine compounds, steroids, triterpenoids, and sesquiterpene lactones were negative.

HPLC fingerprinting analysis
The data obtained from the HPLC fingerprinting analysis revealed that the following compounds were present in the extract: gallic acid (the R t for extract and standard was 6.735 and 6.648 min, respectively), catechin (the R t for extract and standard was 16.375 and 16.479 min, respectively), and epicatechin (the R t for extract and standard was 21.335 and 21.387 min, respectively); the UV spectra of the extract and standard were identical. The chromatogram is shown at the wavelength of 254 nm, at which all compounds identified can be visualized ( Figure 2). Table 2 presents the results of the physicochemical analysis and antioxidant activity assays, reduction of DPPH free radical and total phenol content expressed in g of gallic acid 100 g -1 extract. The pH of the hydroalcoholic extract of the stem bark of A. peregrina was 5.21. The relative density was 0.956 g/cm 3 . The antioxidant activity expressed as IC 50 was 44.13 mg mL -1 for extract and 0.25 mg mL -1 for butylated hydroxy toluene (BHT). Although the IC 50 value of BHT was lower than that of the extract, the results showed that the plant material possess antioxidant activity. The total phenolic compound content was 6.40 g GAE 100 g -1 extract.

Antibacterial activity
The antibacterial activity of the extract is presented in Table 3. After 24 h of incubation, the extract did not inhibit E. coli colonies at any extract volume tested. There were halo regions around S. aureus colonies at all three volumes tested. Using a millimeter ruler, the diameter of inhibitory zones was measured, excluding the diameter of the wells.

Discussion
The hydroethanolic extract of A. peregrina stem bark showed a positive result in the Raymond-Marthoud, Kedde, and Keller-Kiliani tests for glycosidic compounds, although the result was negative in the Baljet test. Aglycones or genin compounds are characterized by the cyclopentanoperhydrophenanthrene structural core. There are two groups of cardiac glycosides: (1) cardenolides, 23-carbon chain compounds in which the unsaturated lactone ring is attached to the pentacyclic C-17 and (2) bufadienolides, compounds with 24 hexacyclic carbons (Kloss et al., 2016). These cardiotonic compounds directly

Pb(C 2 H 3 O 2 ) 2 ++
Purines -  Phytochemical properties, and antioxidant and antibacterial activities of Anadenanthera peregrine act on the myocardium to alleviate heart failure and intoxication (Vickery and Vickery, 1981), a beneficial characteristic for medicinal plants.

Sesquiterpenolactones
In the present study, alkaloids, non-reducing sugars, foamed saponins, polysaccharides, purines, steroids, triterpenoids, and sesquiterpenelactones were not detected in A. peregrina stem bark extract. There are numerous factors that influence the production of certain class of compounds by plants, including the seasonality, circadian rhythm, and full development of the plant. Therefore, frequent sample collection in different seasons is necessary (Gobbo-Neto and Lopes, 2007). It is noteworthy that the alkaloid 5-Hydroxy-N,N-dimethyltryptamine (bufotenine) has been identified in the seeds of A. peregrina (Blackledge and Phelan, 2006). Foamed saponins were not present in the extract, and it is possible that during the sample collection period, this class of compounds was produced in minimal detectable quantities or was not produced (Ndamba et al., 1994).
Here, we observed erythrocyte hemolysis at different time points, suggesting that the extract is toxic to the hematopoietic system. The interaction of the extract  with the erythrocyte membrane constituent sterols leads to the formation of pores in the membrane causing hemolysis, resulting in hemoglobin dispersion to the external environment (Sousa et al., 2018); furthermore, the platelets aggregated in small clusters. Further studies should be performed to evaluate the cytotoxic action of the stem bark extract of A. peregrina with respect to its action on platelets.
Starch and mucilage are the well-known polysaccharides with phytotherapeutic action against pneumological inflammation ). In the present study, polysaccharides were not observed, although A. peregrina extract has been reported to exhibit this function .
The absence of purine compounds, steroids, triterpenoids, and sesquiterpene lactones in the stem bark extract of A. peregrine reinforces seasonal variations in the production of secondary metabolites (Gobbo-Neto and Lopes, 2007). Other important factors associated with the production of secondary metabolites are age, development, and different organs of plants, influencing the content and relative proportion of components in Phytochemical properties, and antioxidant and antibacterial activities of Anadenanthera peregrine the extract (Hendricks et al., 1997). Purine compounds with at least one structural nitrogen (N) atom have potential use in the production of new drugs, owing to their antiangiogenic and cytotoxic activities; thus, these compounds are important to the pharmaceutical and agricultural industries (Palkar et al., 2015). Furthermore, purines are associated with the wound-healing property of A. peregrina extract, favoring angiogenesis. The potential agronomic benefits of the extract are associated with steroidal and triterpene compounds, which are involved in pollen tube growth, internode elongation, and plant growth regulator production (Carvalho et al., 2015).
In the presented study, organic acids were detected in the extract; they, especially malate, citrate, and oxalate, play a role in the tolerance mechanisms of plants to aluminum silicates (Hartwig et al., 2007). Citrate is the most common organic acid in plants; it is a tricarboxylated anion that can form chelates with Al 3 with stable bonds (Jian Zheng et al., 1998;Piñeros et al., 2002).
In the present study, the extract was positive for reducing sugars and coumarins. Glucose and fructose are the reducing sugars that are present in diverse plant organs. Glucose acts synergistically on the central nervous system supplying energy and on the gastrointestinal system. Fructose acts as a source of energy to the musculoskeletal system (Barreiros et al., 2005;Araújo and Martel 2009). Among the coumarins, phytoalexins exhibit phytopathogenic activity as a fungicide. Phytoalexins act on cytoplasmic granulation systems, disorganize cellular content, cause plasma membrane disruption, and inhibit fungal enzymes, and thereby reduce and inhibit mycelial growth (Schwan-Estrada et al., 2000); these findings validate their antimicrobial use .
In the present study, we detected tannins in the extract. Tannins are categorized as flavanones, procyanidins, and condensed and hydrolysable tannins. Condensed tannins include true non-hydrolysable tannin compounds, and they are more resistant to fragmentation and are associated with flavonoid pigments, with the flavan-3-ol polymeric structure. Tannins are responsible for the reddish coloration of the stem bark (SBFGONOSIA, 2009). Tannins are known for their medicinal properties, including antioxidant and bactericidal activities (Ogawa and Yazaki, 2018;Cruz et al., 2020).
During the rainy season, grasses produce higher quantities of tannins, which are considered an antinutritional factor, reducing the consumption of grasses by ruminants, causing nutritional deficit (Nepomuceno et al., 2013). The ingestion of large amounts of tannin compounds can interfere with the digestibility, absorption, and bioavailability of nutrients (Lamy et al., 2011). However, in granular sorghum under short and medium cycle intercropping, condensed tannins improved grain yield, ranging from 1.285 to 8.710 kg ha -1 , dietary protein content, growth rate, fertility, and animal welfare (De Souza et al., 2019;Cuitiño and Vera, 2016).
In the present study, catechins were detected in the extract. Catechins include a diverse group of allelopathic compounds involved in plant-plant interaction, and they are widely used as an insecticide and a natural herbicide. These findings highlight the potential use of the extract as a natural defensive agent (Rabaioli and Silva, 2016).
Here, benzoquinone and depside and depsidone derivatives were also detected in the extract. The p-benzoquinone sorgoleone acts as a natural herbicide, via allelopathic effects on sorghum (Carvalho et al., 2015). This compound acts as a potent inhibitor of mitochondrial respiration and photosynthesis, via its action in the electron transport chain of photosystem II, competing for the same site of action of synthetic herbicides (atrazine and diuron) (Gonzalez et al., 1997). Depsides are polyketides and are produced via the biosynthetic reaction of orselinic acid synthase, where chain cyclization occurs in the formation of this acid. The derivatives of this compound have potential anticancer and anti-inflammatory activities (Kamiya et al., 2018).
In the present study, the test for phenols was positive and the total amount was evaluated in GAE. Phenolic compounds are important for the therapeutic properties of plants. The relationship between polyphenols and human health has been explored with an emphasis on cardiovascular diseases and metabolic syndrome, highlighting the relevance of these bioactive compounds (Durazzo et al., 2019). Phenolic compounds can alleviate the deleterious effects of free radicals acquired or internally produced in an organism. Several plant phenols effectively protect cells under oxidative stress. Studies have explored the potential of phenolic compounds and its derivatives in the treatment of inflammatory diseases, via autophagy mechanisms (Zenkov et al., 2016) and antimicrobial activity (Pinheiro et al., 2018).
In the present study, gallic acid, catechin, and epicatechin were detected in the extract by HPLC fingerprinting; they are common precursors of tannins. Thus, it is possible that the extract contained hydrolyzable tannins (gallotannins) and condensed tannins (catechin tannins and proanthocyanidins). These findings corroborated with those of the preliminary phytochemical screening, contributing to a better understanding of the chemical composition of this species. According to Monteiro et al. (2005), Azêvedo et al. (2017), and Dai et al. (2020), the proportion of hydrolyzable and condensed tannins varies considerably, irrespective of whether both groups are present in the same plant; their content is influenced by natural factors (such as rain, soil, soil nutrition, and solar radiation), as well as by factors between species of the same group, genus, or family.
The qualitative analysis by HPLC of A. colubrina aerial parts indicated the presence of quercetin and low levels of gallic acid, catechin, and p-coumaric acid (Araújo et al., 2019). Gallic acid, catechin, and epicatechin have been shown to possess a wide variety of pharmacological activities, including antimicrobial, anti-inflammatory, and antioxidant activities (Abdulah et al., 2017;Kahkeshani et al., 2019;Pedro et al., 2019;), elucidating the therapeutic uses of A. peregrina.
Although other compounds evaluated were not found in the extract in the present study, it does not necessarily indicate that they are not produced by this species, as several factors may be involved in the biosynthesis of secondary metabolites, including environmental and genetic factors (Gobbo-Neto and Lopes, 2007;Pavarini et al., 2012). Further studies may highlight the potential of A. peregrina stem bark as an alternative source of these compounds, which may also be used as chemical markers of the species for quality control.
In the present study, the DPPH assay revealed the antioxidant activity of the hydroethanolic extract of A. peregrina stem bark. Although the IC 50 value of BHT was lower than that of the extract, the results showed that the plant possesses antioxidant activity. The chemical compounds produced by plants can be altered by several abiotic factors, which interfere in the expression of allelopathic compounds (Pilatti et al., 2019). It is suggested that the antioxidant activity may be related to the presence of phenolic compounds, especially tannins that have antioxidant activity, and they were identified in the present study. Moreover, the dark color of the extract might be due to the presence of high levels of chlorophyll A and B pigments, and these pigments possibly masked the colorimetric reaction to reduce the purple coloration of the radical. Mota et al. (2017) evaluated the hydroethanolic 50% (v/v) extract of A. peregrina bark. They reported a high content of total phenolic compounds (583 mg of GAE g -1 extract) and antioxidant activity of moderate intensity with an average IC 50 value of 13 μg mL -1 compared with 2 mg mL -1 for Trolox. Furthermore, the Trolox equivalent antioxidant capacity was 237.6 mg Trolox g -1 . The phenol content in the bark extracts is highly variable between species, and the phenol content reported by Santos et al. (2012) in the bark extracts of Eucalyptus grandis (386 mg of GAE g -1 ), E. urograndis (347 mg of GAE g -1 ), and E. maidenii (204 mg of GAE g -1 ) was lower than that reported on the bark of A. peregrina by Mota et al. (2017). Moreover, the methanolic extract obtained from eucalyptus barks is reported to exhibit antioxidant activity, validated by the presence of phenolic compounds and flavonoids (Mishra et al., 2010;Srivastava and Vankar, 2012). Just like as eucalyptus barks, A. peregrina barks are a source of polar extracts due to the presence of tannins and other phenolic compounds and can be used in the pharmaceutical sectors due to their antioxidant potential (Sartori et al., 2013).
In conclusion, the phytochemical analysis of the hydroethanolic stem bark extract of A. peregrina showed the presence of a wide variety of chemical compounds with importance in the pharmaceutical, food, and agricultural fields. The HPLC fingerprinting analysis revealed the presence of gallic acid, catechin, and epicatechin in the extract. The extract showed hemolytic action, necessitating further toxicological assessment. Furthermore, the extract showed antibacterial and antioxidant activities. The results contribute to a better understanding of the chemical composition of A. peregrina stem bark extract and further strengthen its application in traditional medicine practices. ALMOHAWES, Z.N. and ALRUHAIMI, H.S., 2020. Effect of Lavandula dentata extract on ovalbumin-induced asthma in male guinea pigs. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 80, no. 1, pp. 87-96. http://dx.doi.org/10.1590Biologia, vol. 80, no. 1, pp. 87-96. http://dx.doi.org/10. /1519 Biota Amazônia,vol. 5,no. 3,