Plant material collection

Leaves of Solidago graminifolia were collected from the municipality of Villa de Cos, Zacatecas, and Santo Domingo, San Luis Potosí, with the latitude coordinates 23.2592960-102.2226380. The plant material was placed in airtight bags and transferred to the Chemistry-Biochemistry laboratory of the Mante Multidisciplinary Academic Unit of the Autonomous University of Tamaulipas. The collected specimens were sent to the Institute of Ecology A.C. for genus and species identification, consulting specialized botanical literature and specialists of the Asteraceae family. The ITS region was amplified for molecular identification using the primers ITS-20F 5’-TCGCGTTGACTACGTCCCTGCC-3’ and ITS-262R 5’ -ATTCCCAAACAACCCGACTCG-3’ with the PCR reaction and sequencing conditions described by ^{Herrera-Mayorga et al. (2022)}.

Obtention of organic extracts

The collected leaves were placed on aluminum trays for drying in an oven at 60 ˚C for 2 days. Subsequently, the leaves were manually pulverized until a small particle size was obtained. Solvents were used in a polarity gradient (ethanol, dichloromethane, and hexane) to obtain the extracts; 100 grams of pulverized leaves were placed in a 1 L flask with 500 mL of the corresponding solvent under constant stirring for seven days protected from light. Afterward, the solvents were filtered under a vacuum to eliminate plant material remains. The crude extract was obtained by placing the sample in a rotary evaporator at a temperature no higher than 40 °C.

Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis

One milligram of crude extract sample was weighed, dissolved in 1 mL of HPLC grade solvent, and filtered through a 0.45 μm syringe filter for analysis. The UPLC-MS/MS was carried out with an ACQUITY UPLC system coupled to a Waters QDA® mass detector (Milford, MA, USA) and an ACQUITY UPLC CORTECS® C18 1.6 µm column 3.0 x 100 mm in positive Ion mode. The column temperature was 40 °C, and the autosampler temperature was 15 °C. Elution was achieved with 0.1 % formic acid in water (Phase A), acetonitrile (Phase B), and 5 mM ammonium acetate (Phase C). The flow rate was 0.3 mL/min, and the injection volume was 5 μL. The composition of the solvents over time was initial A: 5 %; B: 85 %; C: 10 %, at 3.0 min increase, A: 15 %; B: 75 %; C: 10 %, changing at 10.0 min to A: 5 %; B: 85 %; C:10 %. The running time was 15.0 min.

Bacteria isolation and identification

The bacteria in this study were isolated from agricultural bean and corn fields soil samples, at the municipality of Fresnillo de González Echeverria (23°12’ N, 103° 30’W). The bacteria were identified molecularly by amplifying the 16S ribosomal gene and bidirectional sequencing (^{Herrera-Mayorga et al., 2023}). Genomic DNA was extracted using the commercial Promega Wizard® Genomic kit (Promega A1120, USA) according to the protocol described by the manufacturer. The endpoint polymerase chain reaction was carried out using the primers Bac1-FW 5’-AGAGAGTTTGATCVTGGCTCAG-3’ and 16S-1400 RV 5’-GCGGGTGTGTGTACAAGGCCCG-3’ (Criollo et al., 2012), with a final reaction volume of 25 µL. The reaction was carried out with an amplification program that consisted of an initial denaturation cycle at 94 °C for 3 min, followed by 30 cycles at 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 3 min in a Labnet MULTIGENE TM MINI thermocycler. The amplicons were visualized on 1.5 % agarose gel using PROMEGA’s 100 bp molecular weight marker (Madison, Wis., USA). The quality of the PCR products was analyzed with a photodocumenter using the Alphalmager HP system.

The PCR product was purified using the ExoSAP-IT® protocol (Affymetrix, Santa Clara, CA). Subsequently, the bidirectional sequencing reaction was carried out using the conditions indicated in the Big Dye Terminator v.3.1 Cycle Sequencing Kit of the ABI 3130 system (Applied Biosystems, Foster City). The electropherogram obtained was visualized, edited, and assembled with the Chromas Lite 2.1 program (Technelysium) and SeqMan from the commercial DNASTAR suite of Lasergene 8 (Madison, WI). Finally, the assembled nucleotide sequences were compared with the NCBI nr/nt database using the BLASTn program for identification of the selected bacteria ClustalW (homology > 99%) (^{Koolivand et al., 2019}).

Evaluation of antibacterial activity

The antibacterial activity of the crude extracts was determined with the agar diffusion technique at four different concentrations: 15 mg/mL, 10 mg/mL, 5 mg/mL, and 2.5 mg/mL using chloramphenicol (50 μg/mL) as a positive control (^{Mojica et al., 2015}). The preparation consisted of weighing the corresponding amount of each extract in a 1.5 mL Eppendorf tube, and then dissolved in 2 % DMSO. Once dissolved, it was placed in a vortex to stir until a homogeneous mixture was obtained, which, with the help of a micropipette, was added to the center of the sterile petri dish. The liquid agar was immediately poured, and the box was covered, mixing with rotary movements. Finally, each box was allowed to solidify. Each evaluation was done in triplicate and under sterile conditions (^{Ramírez and Marín, 2009}).

A bacterial suspension was prepared for the inoculum in 0.85 % saline solution from a 24 h culture at 35 °C in nutrient agar. The inoculum solution was adjusted to tube number 5 McFarland using a spectrophotometer at a wavelength of 530 nm, obtaining a suspension at a concentration of 1 x 10⁶ CFU/mL. From this suspension, 1 µL of each bacterial suspension was taken with a micropipette and sterile tips, and placed in the corresponding quadrant, trying not to pierce the agar and placing the drop as central as possible. After inoculating each box (except the negative or sterility control), it was incubated at 35 °C for 24 h. It is important to mention that each evaluation was done in triplicate and under sterile conditions. From the last two concentrations, dilutions were worked out for the measurement of MIC for each of the strains.

Minimum Inhibitory Concentration (MIC) determination

The MIC was determined only for the extracts with antibacterial activity (ethanol and dichloromethane). The evaluation was carried out with 5 concentrations (5.0 mg/mL, 2.5 mg/mL, 1.0 mg/mL, 0.5 mg/mL, and 0.1 mg/mL) to which the inoculum was added at 1x10⁶ CFU/mL. Duplicates were worked with the same concentrations but without adding the inoculum to compare the turbidity of the medium. The corresponding sample was weighed in the tube with the highest concentration and subsequently dissolved in 2 % DMSO to prepare the samples with extract. The desired concentrations were adjusted in nutritious broth once a homogeneous mixture of the extract and solvent was obtained. The MIC was defined as the lowest concentration of the extract capable of total inhibition compared to the 100 % growth control (^{Da Silva et al., 2019}).

Plant identification

The plant was identified as Solidago graminifolia (syn. Euthamia graminifolia (L.) Nutt) by botanical experts, and genotypically had a sequence homology of 96 % (744/779) with the Solidago graminifolia MT610936.1 sequence. A specimen of the plant was identified taxonomically and deposited in the herbarium of Francisco González Medrano with the code UAT-22866. This species is native from North America, however, to our knowledge, this is the first report of its presence in Mexico in the states of San Luis Potosi and Zacatecas. Currently, the identification of plants by molecular biology is an easy and unexpensive tool that could be used as complementary to the traditional identification.

Organic extracts yield

The yield of organic extracts obtained by maceration from S. graminifolia leaves had a value of 20.39 % with ethanol, followed by dichloromethane with 18.34 %. The hexanoic extract had the lowest yield with 5.3 %. In general, the yields obtained could be considered low because, in previous research, ^{Toiu et al. (2019)} mention that the extracts from the aerial part of S. graminifolia with polar solvents, such as methanol and ethanol, produce a higher yield with values ranging from 28.01 % and 31.17 %, respectively. Additionally, they mention that polar solvents produce a high yield of total polyphenol content (192.69 mg/g extract) and flavonoids (151.41 mg/g extract), compared to chloroform (40.5 mg/g) and petroleum ether (121.2 mg/g) as non-polar solvents. This can be attributed to factors such as the solubility in the solvents, since in our study absolute ethanol was used and the authors worked with a 70 % ethanol ratio 1:20; as a medium polarity solvent, chloroform was used instead of dichloromethane and as a non-polar solvent petroleum ether while we used hexane. Another variant was the time and temperature of extraction which was worked by the authors at 60 °C with a time of 50 min, while our conditions were stirring at room temperature for seven days.

UPLC phytochemical analysis

The UPLC analysis of the organic extracts from S. graminifolia leaves allowed the detection of some previously reported secondary metabolites (Table 1). The most representative metabolites were flavonoids, among which were quercetin and kaempferol. In general, flavonoids are a group of molecules with greater abundance in plants, and this group of metabolites are considered to be of low toxicity and a high pharmacological capacity (^{Tafroji et al., 2022}). Both metabolites have been described for their oxidative properties and for being involved in the growth inhibition of bacteria and other microorganisms, which have made them an alternative for the development of new drugs whose mechanism of action has been described in Gram positive and negative bacteria, such as Micrococcus luteus and Escherichia coli, where the greatest damage has been observed in the cell membrane causing rupture, activation of apoptosis and inhibition of the synthesis of nucleic acids and proteins. It has also been reported that combined, these two metabolites enhance the antibacterial activity by participating in the interruption of fatty acid biosynthesis, and of the formation of bacterial biofilms in strains of Mycobacterium, Pseudomonas aeruginosa and Vibrio cholerae (^{Nguyen and Bhattacharya, 2022}; ^{Periferakis et al., 2022}). Other constituents detected were phenolic acids, such as chlorogenic and solidagoic acid derivatives, this group of metabolites are produced by many plants to defend themselves against bacteria. Their mechanisms involve the alteration of physiological pathways for biofilm formation, membrane destruction, and alterations of cellular transport (^{Chen et al., 2022}; ^{Bozsó et al., 2024}).

According to their polarity, some secondary metabolites identified in the ethanolic extract were quercetin and solidagoic acid derivatives (E, G, and H). These metabolites are highly polar, which justifies their extraction and have been identified in S. virgaurea and S. gigantea extracts, the presence of these constituents has been associated with antimicrobial activity (^{Starks et al., 2010}; ^{Jaisinghani, 2017}). In the hydroalcoholic or polar extractions of the roots and leaves of the genus Solidago, large quantities of clerodane diterpenes such as solidagoic acids have been described; however, this group of metabolites, despite being very specific to this genus, has not been described in the phytochemical profile of Solidago graminifolia (^{Toiu et al., 2019}). This may be due to the fact that this group of constituents are produced by the plant as a defense mechanism against high temperatures, which is an environmental factor in the region where it was sampled. The most abundant secondary metabolites in the dichloromethane and hexane extracts were coumarins, labdane diterpenes, and glycosidic derivatives. As reported by Toiu et al. (2019), there is a large difference between the content of extracted metabolites and the order of polarity, where substances such as ethanol achieve greater efficiency and diversity of metabolites due to their diffusion capacity and solubility, about four times greater than non-polar solvents such as petroleum ether or chloroform, however, solvents such as hexane reduce the matrix between polar compounds and make the extraction of non-polar or semi-polar compounds more efficient.

Other secondary metabolites that were identified in this work have been obtained from the aerial parts and roots of plants from the genus Solidago by different authors, such as acetylenes (esters of feverfew and dehydromatricaria), clerodane diterpenes (kingidiol and solidagoic acid A), labdane diterpenes (solidagenone and presolidagenones), benzyl benzoate and terpene derivatives, which have shown antibacterial activity and pharmacological interest (^{Toiu et al., 2019}; ^{Baglyas et al., 2022}; ^{Bozsó et al., 2024}).

Bacteria identification and Antibacterial activity

The isolates obtained from agricultural soils were enteropathogenic strains (Table 2). The organic extracts were evaluated against the strains identified at four concentrations to determine their potential antibacterial activity (Table 3). The ethanolic extract had antibacterial activity against the five strains at every evaluated concentration. A similar result was found with the dichloromethane extract, except at the concentration of 2.5 mg/mL, which allowed the growth of four bacteria. Finally, it was not possible to determine the antibacterial activity in the hexanoic extract due to poor solubility in the aqueous solution. This finding may be associated with the type of non-polar components, such as kaempferol, one of the extract’s most abundant secondary metabolites. This metabolite is described as a very hydrophobic molecule, practically insoluble in water, and whose main associated biological activity is its antioxidant nature in neurological diseases such as Parkinson’s, Alzheimer’s, epilepsy, depressive disorder, anxiety disorder, and others (^{Silva et al., 2021}).

Minimum Inhibitory Concentration (MIC)

The MIC of the extracts against E. coli, K. pneumoniae, S. enterica, S. aureus and P. aeruginosa, are shown in Table 4. The ethanolic extract of S. graminifolia had lowest MIC value (2.0 mg/mL for S. aureus and 1.5 mg/mL for the Gram-negative bacteria E. coli, S. enterica, P. aeruginosa and K. pneumoniae). The MIC of the dichloromethane extract was slightly higher than the ethanolic extract in the case of four bacteria (2.5 mg/mL) and had the same MIC value for S. aureus.

Our biological activity results are similar to those described by ^{Toiu et al. (2019)}. They obtained MIC values between 0.048 and 3.12 mg/mL for ethanol extracts of the S. graminifolia plant and values of 0.096 and 3.12 mg/mL for methanol extracts, against Gram-positive and negative bacteria such as Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium and Escherichia coli. Our results and those of these authors suggest moderate activity against the bacterial strains evaluated since it is estimated that MIC levels around or less than 0.5 mg/mL suggest good antibacterial activity (^{Salvat et al., 2004}).

The extract with the best antibacterial activity was the ethanolic extract of S. graminifolia possibly due to its richness in polyphenolic compounds (^{Alves et al., 2013}), where metabolites such as quercetin may exert a possible antibacterial activity with an ability to eliminate biofilm formation in Bacillus subtilis FB17, and Enterococcus faecalis MTCC2729 strains. In addition, the quercetin molecule causes suppressing adhesion expression in the strains S. aureus ATCC 6538 and ATCC 25923 (^{Yang et al., 2020}).

Other representative secondary metabolites (ethanol and dichloromethane) in the extracts were phenolic acids such as solidagoic acid derivatives. Clerodane diterpenes identified by UPLC-MS as solidagoic acid have sparked interest in recent years due to their notable antibacterial, antifungal, antitumor, antifeedant for insects, and other biological activities (^{Li et al., 2016}). Four bioactive diterpenes have been described in S. gigantea plant extracts: solidagoic acid E, solidagoic acid F, solidagoic acid H, and solidagoic acid I; the latter two have acted with moderate antibacterial activity against Gram-positive Bacillus subtilis subsp strains spizizenii and Rhodococcus fascians with IC₅₀ values of 32.3-64.4 µg/mL (^{Baglyas et al., 2022}).