Anadenanthera peregrina subsp. extract, (L.) Speg., Speg.

Marinho, T. A., Oliveira, M. G., Menezes-Filho, A. C. P., Castro, C. F. S., Oliveira, I. M. M., Borges, L. L., Melo-Reis, P. R. & Silva-Jr, N. J., 2022, Phytochemical characterization, and antioxidant and antibacterial activities of the hydroethanolic extract of Anadenanthera peregrina stem bark, Brazilian Journal of Biology (e 234476) 82, pp. 1-12 : 3-4

2.7. 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).

3. Results

3.1. 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.

3.2. 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 ).

3.3. Physicochemical properties and antioxidant activity

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.

3.4. 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.

Figures

Figure 1. Erythrocyte hemolysis in a 5% red blood cell suspension by the hydroethanolic extract of A. peregrina stem bark. (A) 5% suspension of red blood cells; (B) hemolysis after 1 min of reaction;(C) advanced hemolysis after 5 min; and (D) completely hemolyzed red blood cells within 10 min of reaction. Bars: At (A) 1.000×; (B) 500×; (C) 650×; and (D) 1.800×.

Figure 2. HPLC-PDA chromatographic profiles (λ = 254 nm) of: (A) sample extract; (B) gallic acid standard; (C) catechin standard; and (D) epicatechin standard, followed by UV spectra (190-400 nm).