Antibacterial activity of organic extracts from

Solidago graminifolia leaves

Original Article

Actividad antibacteriana de extractos orgánicos de hojas de Solidago graminifolia

Guadalupe Tobías1 , José Ramírez De León2 , Juan Francisco Castañón Rodríguez2 , Francisco A. Paredes- Sanchez1 , Alma D. Paz-González3 , Gildardo Rivera3 and Verónica Herrera-Mayorga1*

1 Unidad Académica Multidisciplinaria Mante, Universidad Autónoma de Tamaulipas, 89840, El Mante, Tamaulipas, México.

2 Unidad Académica de Trabajo Social y Ciencias para el Desarrollo Humano, Universidad Autónoma de Tamaulipas, Centro Universitario, 87120, Cd. Victoria, Tamaulipas, México.

3 Laboratorio de Biotecnología Farmacéutica, Centro de Biotecnología Genómica, Instituto Politécnico Nacional, 88710, Reynosa, Tamaulipas, México.

ABSTRACT

Solidago graminifolia (syn. Euthamia graminifolia (L.) Nutt) is a native species plant from North America, with abundant flavonoids, diterpenes, and polyacetylenes metabolites, that have shown cholinesterase enzyme inhibitory activity and antimicrobial activity. The aim of this study was to de- termine the antibacterial activity of Solidago graminifolia leaf extracts obtained with ethanol, dichloromethane, and hexane solvents. The S. graminifolia extracts were tested against Escherichia coli, Salmonella enterica, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The chemical composition of each extract was analyzed by UPLC-MS/MS. The extracs yields in ethanolic, dichlorometh- ane and hexanoic solvents were 20.39 %, 18.34 %, and 5.3

%, respectively. The secondary metabolites identified were flavonoids, hyperoxide, quercetin, kaempferol, and some phenolic acids, such as chlorogenic acid and solidagoic acid derivatives. The ethanolic extract inhibited the five strains in all concentrations (15 mg/mL, 10 mg/mL, 5 mg/mL, and 2.5 mg/mL). The ethanol extract has a MIC of 2.0 mg/mL against S. aureus and 1.5 mg/mL for the Gram-negative bacteria E. coli, S. enterica, P. aeruginosa, and K. pneumoniae; the dichloromethane extract has MIC values of 2.5 mg/mL for Gram-negative strains and 2.0 mg/mL for S. aureus. This study showed that the ethanolic extract had the best antibacterial activity, and its biological activity can be attributed to its richness in polyphenolic compounds.

Keywords: plant, secondary metabolites, gram-negative.

RESUMEN

Solidago graminifolia (sin. Euthamia graminifolia (L.) Nutt) es una especie nativa de Norteamérica; es una especie abundan- te en flavonoides, diterpenos y policacetilenos, con actividad antimicrobiana e inhibitoria sobre la enzima acetilcolineste- rasa. El objetivo fue determinar la actividad antibacteriana de tres extractos (etanol, diclorometano, hexano) de la planta

S. graminifolia sobre Escherichia coli, Salmonella enterica, Staphylococcus aureus, Pseudomonas aeruginosa y Klebsiella pneumoniae. La composición química de cada extracto fue analizada mediante UPLC-MS/MS. El rendimiento en los ex- tractos de etanol, diclorometano y hexano fue 20.39 %, 18.34

*Author for correspondence: Verónica Herrera-Mayorga e-mail: evherrera@docentes.uat.edu.mx

Received: March 8, 2024

Accepted: September 7, 2024

Published: October 8, 2024

% y 5.3 %, respectivamente. Los metabolitos secundarios identificados fueron flavonoides, hiperósidos, quercetina, ka- empferol y ácidos fenólicos, derivados del ácido clorogénico y solidagoico. El extracto etanólico inhibió las cinco cepas en todas las concentraciones (15 mg/mL, 10 mg/mL, 5 mg/mL, 2.5 mg/mL). El extracto etanólico tuvo una CMI de 2.0 mg/ mL contra S. aureus y 1.5 mg/mL contra las bacterias Gram negativas E. coli, S. enterica, P. aeruginosa y K. pneumoniae; el extracto de diclorometano tuvo valores de CMI de 2.5 mg/ mL para las cepas Gram negativas y 2.0 mg/mL para S. aureus. Este estudio mostró que el extracto etanólico tiene la mejor actividad antibacteriana y su actividad biológica puede ser atribuida a la presencia de compuestos polifenólicos.

Palabras clave: plantas, metabolitos secundarios, gran- negativa.

INTRODUCTION

Plants are an important source of secondary metabolites with a wide chemical diversity and different biological properties (Starks et al., 2010). However, the type and concentration of secondary metabolites vary according to the plant species, environmental conditions, stress factors, and other elements that condition their production (Isah, 2019).

1

In traditional medicine, the different parts of plants are used against diverse pathologies since their extracts have various biological activities, such as antiproliferative (Nkuimi et al., 2020), anti-inflammatory (Yoo et al., 2020), antiproto- zoal (De Mieri et al., 2017), antibacterial (Wasihun et al., 2023), among others. Particularly, plants of the Artemisia genus have shown antibacterial activity against Staphylococcus aureus with a Minimum Inhibitory Concentration (MIC) of 3.0 mg/ mL (Zhang et al., 2022). Aqueous extracts of Alkanna tinctoria leaves, and Punica granatum peel extracts have antibacterial activity against multidrug-resistant pathogens such as Acine- tobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus with a MIC values between 12.5 mg/ml and 25 mg/mL (Khan et al., 2017). Interestingly, Soli- dago graminifolia has an important antifungal effect against yeasts such as Candida albicans and Candida parapsilosis and an antibacterial effect against Staphylococcus aureus, with a MIC of 0.048–3.12 mg/mL (Toiu et al., 2019).

Volume XXVI

DOI: 10.18633/biotecnia.v26.2277

Journal of biological and health sciences http://biotecnia.unison.mx

Universidad de Sonora

ISSN: 1665-1456

The genus Solidago (Asteraceae) includes about 130 plant species worldwide. In particular, Solidago graminifolia (syn. Euthamia graminifolia (L.) Nutt) is a native species from North America, but there are no reports from the central region of Mexico. It is a perennial herbaceous plant with yellow flowers that has been described as a species abun- dant in flavonoids, such as quercetin, rutin, and astragalin metabolites, and terpenes, labdanum, diterpenes, and polyacetylenes obtained from extracts of the aerial part and roots of the plant (Szymura and Szymura, 2016; Móricz et al., 2020). Another similar species, Solidago virgaurea L., also has antioxidant, anti-inflammatory, analgesic, antifungal, and antiparasitic potential (Fursenco et al., 2020). In addition, its extracts have been associated with antimicrobial activity against the strains Bacillus subtilis F1276, Bacillus subtilis sub- sp. spizizenii, and Aliivibrio fischeri. The Solidago graminifolia extracts have been previously described with a promising antimicrobial effect on Staphylocccus aureus and Candida albicans species, these evaluations have been carried out in countries such as Romania and Poland (Kołodziej et al., 2011; Toiu et al., 2019). However, in our country it has not been carried out; therefore, it becomes necessary to know the biological capacity of the extracts in bacteria that impairs hu- man health. Hence, the aim of this research was to evaluate the antibacterial activity of three organic extracts of Solidago graminifolia against strains of Escherichia coli, Pseudomonas, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylo- coccus aureus, and Salmonella enterica, and to determine the secondary metabolites involved.

‌MATERIAL AND METHODS

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 Auton- omous University of Tamaulipas. The collected specimens were sent to the Institute of Ecology A.C. for genus and species identification, consulting specialized botanical liter- ature 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 dry- ing 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 spec- trometry (UPLC-MS) analysis

One milligram of crude extract sample was weighed, dis- solved 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 Wa- ters 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 au- tosampler 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 compo- sition 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 % aga- rose 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 deter- mined 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 Eppen- dorf 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 so- lidify. 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 antibac- terial 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 in- oculum 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 corre- sponding 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 mix- ture 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).

RESULTS AND DISCUSSION

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 sol- vents, 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 dichloro- methane and as a non-polar solvent petroleum ether while we used hexane. Another variant was the time and tempera- ture 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 mol- ecules 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 metabo- lites 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 in- hibition 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 in- terruption of fatty acid biosynthesis, and of the formation of bacterial biofilms in strains of Mycobacterium, Pseudomonas

Table 1. Secondary metabolites in organic extracts identified by molecular weight of the plant Solidago grami- nifolia by UPLC-MS.

Tabla 1. Metabolitos secundarios identificados mediante sus pesos moleculares en los extractos orgánicos de la planta Solidago graminifolia realizado mediante UPLC-MS.

NR: Not reported.

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 mech- anisms 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 soli- dagoic 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 pres- ence of these constituents has been associated with antimi- crobial 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 ge- nus, 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, lab- dane diterpenes, and glycosidic derivatives. As reported by Toiu et al. (2019), there is a large difference between the con- tent 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 com- pounds 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 entero- pathogenic strains (Table 2). The organic extracts were eval- uated 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 con- centration of 2.5 mg/mL, which allowed the growth of four bacteria. Finally, it was not possible to determine the antibac- terial 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, depres- sive disorder, anxiety disorder, and others (Silva et al., 2021).

Table 2. Bacterial strains isolated and molecularly identified.

Tabla 2. Cepas bacterianas aisladas e identificadas molecularmente.

Minimum Inhibitory Concentration (MIC)

The MIC of the extracts against E. coli, K. pneumoniae, S. en- terica, 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 de- scribed by Toiu et al. (2019). They obtained MIC values between

0.048 and 3.12 mg/mL for ethanol extracts of the S. gramini- folia 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).

*NI: Not identified.

Table 3. Antibacterial activity of organic extracts of S. graminifolia.

Tabla 3. Actividad antibacteriana de los extractos orgánicos de S. graminifolia.

ND: not determined; (-): showed null growth; (+): showed growth; EtOH: ethanol; DCM: dichloromethane; Hex: hexane; [ ]: concentration.

Table 4. Minimum Inhibitory Concentration (mg/mL) of the organic extracts from S. graminifolia.

Tabla 4. Concentración Mínima Inhibitoria (mg/mL) de los extractos orgáni-

cos de S. graminifolia.

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 me- tabolites such as quercetin may exert a possible antibacterial activity with an ability to eliminate biofilm formation in Bacil- lus 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 inter- est in recent years due to their notable antibacterial, antifun- gal, 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

ACKNOWLEDGMENTS

The authors want to thank the support of the “Unidad Aca- démica Multidisciplinaria Mante-Universidad Autónoma de Tamaulipas” and M.C. Óscar Hinojosa Espinosa, Dr. Arturo Mora Olivo for the taxonomic identification of S. graminifolia.

CONFLICTS OF INTEREST

The authors have no conflict of interest to declare.

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