Rock nettle B. insignis belongs to the Loasaceae family (order Cornales), popularly known as “electric shock plant” or amor seco in the southern hemisphere, widely distributed in its native region including Brazil, Uruguay, and Argentina. The genera Blumenbachia (11 spp.) (Acuña Castillo et al. 2019) is a morphologically quite homogeneous group for vegetative characters, with complex floral morphology and function (Henning et al. 2015). Adult and complete specimens of B. insignis were identified and collected from General Madariaga. Province of Buenos Aires (37° 06′ 67″ S and 56° 98′ 33″ W) and harvested in October 2018 (Additional file 1: Table S1). The taxonomic identification of the plant was confirmed by Ing. Agr. Cardinali at the Centre of Plant Taxonomy, National University of Mar del Plata (Argentina). All the parts of the plant material, such as flower, fruit, leaf, stem, and root, were analyzed separately. Sampled plant material with its geographic origin and the herbarium voucher are given in the Appendix.
Preparation of the extracts
Dried flowers, fruits, leaves, stems, and roots (3 g each) were extracted with 50 mL ethanol 50% (v/v) for 24 h at room temperature and then filtered through a paper filter to remove insoluble residuals. The solvent was evaporated under vacuum using the rotary evaporator Büchi Rotary evaporator RII (Germany) and the extracts were stored at 4 °C for further analysis.
The preparation of the extract for gas chromatography–mass spectroscopic (GC–MS) analysis was carried out by assisted extraction in an ultrasonic bath at 25 °C, using sequentially n-hexane, ethyl acetate, and methanol as solvent. Briefly, 10 g of powdered material was soaked in 25 mL of hexane, sonicated for 30 min, and filtered. Subsequently, the residue was treated in the same way with ethyl acetate and with methanol. The three extracts were dried using a rotary evaporator and redissolved using ethyl acetate that showed redissolution capacity for all residues, and the etile acetate fractions were combined for analysis by GC–MS.
Total phenolic content
The total polyphenols content of the samples was determined according to the Folin–Ciocalteu colorimetric method (Fangio et al. 2019). The resulting absorbance was measured at 765 nm. Gallic acid solutions (0–100 μg/mL) were used to perform the calibration curve. Total polyphenols contents were expressed as mg of gallic acid equivalents (GAE) per gram of extracts. The values are presented as the mean of analyzes performed in triplicate ± standard deviation.
Total flavonoid content
Flavonoid content was determined by the method of Woisky and Salatino 1998 with some modifications. The absorbance was measured at 420 nm. Quercetin solutions between 2.5 and 25 μg/mL were used to construct the calibration curve. The content of total flavonoids was calculated as mg equivalents of quercetin (QE)/g of extract. The values are presented as the mean of the analysis carried out in triplicate ± standard deviation.
Total saponin content
A qualitative analysis was performed. Quantification of total saponin content was carried out using oleanolic acid as a reference. The standard curve was made with 0.00, 0.25, 0.5, 0.75, 1.00, and 1.25 mL of oleanolic acid. The absorbance was read at 530 nm. The results were reported as mg saponins/g extract (Le et al. 2018). The values are presented as the mean of the analysis carried out in triplicate ± standard deviation.
The antioxidant activity of plant extracts was determined according to Fangio et al. 2019 with slight modifications using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. For each extract and solution of the synthetic antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), an aliquot was added to an ethanolic solution of 60 μM DPPH. Absorbance measurements were read at 517 nm at different times. A sample containing the same amount of ethanol and DPPH solution was taken as the blank control. The results were expressed as µmol Trolox equivalent antioxidant capacity (TEAC) per gram.
The minimum inhibitory concentration (MIC) of the plant extracts was evaluated by the microdilution test according to the Clinical and Laboratory Standards Institute (CLSI, 2015) (Weinstein and Patel 2018). Extracts were filtered through membranes of 0.22 µm (Titan syringe filters; Sri Scientific Resources Inc., USA) and added to the culture medium to achieve different final concentrations. Bacterial suspensions of the strains Escherichia coli ATCC 25,922 and Staphylococcus aureus ATCC 25,923 (105–106 CFU/mL) were added to the wells with Mueller–Hinton Broth, while a suspension of Paenibacillus larvae (ERIC I genotype) was added to Mueller–Hinton, yeast extract, glucose and sodium pyruvate broth (Fangio et al. 2019). All microtiter plates (with positive and negative controls) were incubated at 35 ± 0.5 °C for 24–48 h according to each strain. The MIC was determined as the lowest concentration inhibiting the visible growth of each strain (Kowalska-Krochmal and Dudek-Wicher 2021). The minimum bactericidal concentration (MBC) was determined by subculturing the broth dilutions from the microdilution test. The broth dilutions that inhibited the growth of the bacterial organism were streaked onto Mueller–Hinton agar or Mueller–Hinton, yeast extract, glucose, and sodium pyruvate agar and incubated for 24 to 48 h, according to each strain. The MBC was determined as the lowest dilution of antimicrobial that prevents the growth of the organism on the agar plate (Ferreira et al. 2018).
Samples were analyzed using a Shimadzu GCMS-QP2100ULTRA-AOC20i with a column of 0.25 mm, 30 m, and 0.1 µm phase thickness Zebron ZB-5MS. Samples were injected at pulsed splitless mode, and the injection volume was 2 mL. The interface and the ionization source were kept at 300 °C and 230 °C, respectively. Helium chromatographic grade (99.9999%) was used as the carrier gas with a constant linear velocity of 52.1 cm/sec. The oven temperature program started at 50 °C, where it was held for 2 min and then increased to 300 °C at 15 °C/min where it was held for 4 min. Electron impact ionization (EI) was used at 70 eV in a full scan. The identification of compounds was achieved by analyzing the retention indexes (relative to C8–C24 n-alkanes) and the mass spectra were also matched to those of standards available in the National Institute of Standards and Technology (NIST) 05 and Wiley 08 mass spectrum libraries. Retention time (RT) is reported in Table 3 in order to simplify comparisons. The relative percentage contribution as a function of concentration (peak area %) of each compound was calculated from the chromatograms by a computerized integration assuming all the response factors were 1. Analyses were carried out in triplicate.
Statistical analysis was carried out using analysis of variance (ANOVA) with post hoc Tukey’s honestly significant difference (HSD) test. All experiments were performed in triplicate. Results were presented as a value ± standard deviation. Significant levels were defined at p < 0.05. All the analyses were carried out by using SPSS 15.0 (SPSS Inc., Chicago, Ill., USA) for Windows.