biotecnia Biotecnia Biotecnia 1665-1456 Universidad de Sonora, División de Ciencias Biológicas y de la Salud 10.18633/biotecnia.v26.2233 00039 Articles In vitro and in silico antioxidant and antimicrobial activity of ethanolic extracts of Cnidoscolus chayamansa leaves Actividad antioxidante y antimicrobiana in vitro e in silico de extractos etanólicos de hojas de Cnidoscolus chayamansa 0009-0003-7542-35 Posada-Mayorga Karla Y. 1 0000-0001-8770-8180 Ruíz-Ruíz Jorge Carlos 2 0000-0001-6242-7964 Olivo-Vidal Zendy Evelyn 1 0000-0002-2837-5563 LobatoTapia Carlos Alberto 3 0000-0002-4637-2657 Pacheco-López Neith Aracely 4 0000-0001-6402-0850 Herrera-Pool Iván Emanuel 4 0000-0001-9914-1230 Irecta-Najera Cesar A. 1 0000-0002-8599-8150 Sánchez-Chino Xariss M. 5 * Departamento de Salud, El Colegio de la Frontera Sur-Villahermosa. Carretera a Reforma Km. 15.5 s/n. Ra. Guineo 2da. Sección, Villahermosa, C.P. 86280, Tabasco, México. Departamento de Salud El Colegio de la Frontera Sur El Colegio de la Frontera Sur Tabasco México karla.posada@posgrado.ecosur.mx Departamento de Salud, El Colegio de la Frontera Sur-Villahermosa. Carretera a Reforma Km. 15.5 s/n. Ra. Guineo 2da. Sección, Villahermosa, C.P. 86280, Tabasco, México. Departamento de Salud El Colegio de la Frontera Sur El Colegio de la Frontera Sur Tabasco México ozendy@ecosur.mx Departamento de Salud, El Colegio de la Frontera Sur-Villahermosa. Carretera a Reforma Km. 15.5 s/n. Ra. Guineo 2da. Sección, Villahermosa, C.P. 86280, Tabasco, México. Departamento de Salud El Colegio de la Frontera Sur El Colegio de la Frontera Sur Tabasco México cirecta@ecosur.mx Escuela de Nutrición, Universidad Anáhuac Mayab. Carr. Mérida Progreso Km. 15.5. 96 Cordemex, CP. 97310, Mérida, Yucatán, México. jorge.ruizr@anahuac.mx Universidad Anahuac Escuela de Nutrición Universidad Anáhuac Mayab Mérida Yucatán Mexico jorge.ruizr@anahuac.mx Universidad Politécnica Metropolitana de Puebla, Popocatépetl s/n, Reserva Territorial Atlixcáyotl, Tres Cerritos, 72480 Puebla, Pue., México. carlos.lobato@metropoli.edu.mx Universidad Politécnica Metropolitana de Puebla Universidad Politécnica Metropolitana de Puebla Puebla Mexico carlos.lobato@metropoli.edu.mx Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. Km 5.5 ca-rretera, Sierra Papacal - Chuburná, C.P. 97302, Chuburná, Yucatán. npacheco@ciatej.mx Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. Chuburná Yucatán Mexico npacheco@ciatej.mx Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. Km 5.5 ca-rretera, Sierra Papacal - Chuburná, C.P. 97302, Chuburná, Yucatán. npacheco@ciatej.mx Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. Chuburná Yucatán Mexico lvherrera_al@ciatej.edu.mx CONAHCYT-Departamento de Salud, El Colegio de la Frontera Sur-Villahermosa. Carretera a Reforma Km. 15.5 s/n. Ra. Guineo 2da. Sección, Villahermosa, C.P. 86280, Tabasco. xsanchez@ecosur.mx Departamento de Salud El Colegio de la Frontera Sur El Colegio de la Frontera Sur Tabasco Mexico xsanchez@ecosur.mx Author for correspondence: ariss M. Sánchez-Chino. e-mail: xsanchez@ecosur.mx

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

 2024 26 e2233 17 01 2024 21 03 2024 10 05 2024 This is an open-access article distributed under the terms of the Creative Commons Attribution License Abstract

Cnidoscolus chayamansa leaves - used in gastronomy and traditional medicine in Mexico - are rich in phenolic compounds, which may have antioxidant and antimicrobial activity. In this study we evaluated the in vitro antioxidant activity and in silico antibacterial activity, of ethanolic extracts of C. chayamansa leaves obtained by ultrasonication. Phenolic content was 14.37 mg GAE/mL. Guanosine nucleoside and coumaric acid, and kaempferol derivatives were identified through UPLC-PDA-ESI-MS. Evidence of antioxidant activity was demonstrated by the Cu2+ chelation activity (65.53 %) and the Fe3+ reducing antioxidant power (69.59 %). Although no antibacterial activity was found against E. coli and S. aureus, the in silico analysis revealed that the isolated phenolic compounds modify signaling pathways essential for the survival of the bacteria studied.

 Resumen

Las hojas de Cnidoscolus chayamansa - utilizadas en la gastronomía y la medicina tradicional en México - son ricas en compuestos fenólicos que pueden tener actividad antioxidante y antimicrobiana. En este estudio evaluamos la actividad antioxidante in vitro y la actividad antibacteriana in silico de extractos etanólicos de hojas de C. chayamansa obtenidos por ultrasonicación. El contenido de compuestos fenólicos fue de 14.37 mg GAE mL-1. Se lograron identificar compuestos como nucleósido de guanosina, ácido cumárico y los derivados de kaempferol mediante UPLC-PDA-ESI-MS. Los extractos tuvieron actividad antioxidante por medio de la quelación del Cu2+ (65.53 %) y el poder reductor del Fe3+ (69.59 %). Aunque no se encontró actividad antibacteriana contra E. coli y S. aureus, por medio de la inhibición de crecimiento en disco, el análisis in silico reveló que los compuestos fenólicos aislados modifican las vías de señalización esenciales para la supervivencia de las bacterias estudiadas.

 Keywords: Chaya leaves phenolic compounds molecular docking antioxidant activity antibacterial activity Palabras clave: Hojas de chaya compuestos fenólicos docking molecular actividad antioxidante actividad antibacteriana Introduction

Cnidoscolus chayamansa, commonly known as chaya is an endemic shrub of Tabasco and the Yucatan Peninsula in Mexico (Pérez-González et al., 2019) used in local gastronomy, in addition to traditional medicine (Rodrigues et al., 2021) presumably due to a high content of bioactive compounds (phenols, flavonoids, coumarins, and cyanogenic glycosides) in its leaves (Gutiérrez-Rebolledo et al., 2016; Bautista-Robles et al., 2020). The antioxidant activity of bioactive compounds plays a fundamental role through multiple pathways, preventing oxidative stress (OS)-related diseases (Pisoschi et al., 2021) such as diabetes mellitus and cardiovascular diseases (scavenging free radicals, increasing the activity of endogenous antioxidant enzymes, improvement of insulin resistance and enhancement of glucose uptake and metabolism) (Huang et al., 2020; Garcia and Blesso, 2021), as well as cancer, they induce apoptosis, by lowering the nucleoside diphosphate kinase-B activity (involved in nucleic acid replication), inhibiting cell-proliferation and cell cycle arrest by suppressing the NF-kB pathway in various cancers (Hazafa et al., 2020).

OS occurs when reactive oxygen species (ROS) and free radicals (FR) increase, which may cause cellular and tissue damage (Ouadi et al., 2017). Bioactive compounds such as polyphenols may act as antioxidants, anti-inflammatory, and antimicrobial agents through modulation of inhibitory receptors of inflammation and activators of anti-inflammatory enzymes (Kaabi, 2022).

A positive correlation exists between the content of phenolic compounds and the antibacterial capacity, including bacteriostatic and bactericidal properties. Phenolic compounds modify the bacterial cytoplasmic membrane permeability and inhibit signaling pathways involved in bacterial survival (Vazquez-Armenta et al., 2022). Research shows that ethanolic extracts of C. chayamansa leaves showed high antibacterial activity against S. aureus, B. Cereus, E. coli, K. pneumoniae and S. pyogenes (Elizabeth et al., 2023). Moreover, the extraction method can affect the concentration of phenolic compounds; for instance, modern extraction methods based on sonication - generated through the coupling of high-power and low-frequency ultrasound waves that travel through the liquid medium, causing cycles of low and high-pressure and creating acoustic cavitation bubbles that collapse releasing a large number of compounds present in the sample (Chemat et al., 2017; Fu et al., 2020). The current study aimed to evaluate the in vitro and in silico antioxidant and antibacterial potential of the Ultrasonic Assisted Ethanolic Extracts (UAEE) of C. chayamansa leaves.

 Materials and methods

Collection and preparation of samples. Leaves of C. chayamansa were collected on February 2022, from the edge of the Teapa River (17°33’49.3”N 92°57’09.7”W), Joyas del Pedregal, in the municipality of Teapa, Tabasco, Mexico, and studied in the herbarium from El Colegio de la Frontera Sur (code HET 2459, HET 2460, and HET 2461). Leaves were washed with drinking water, dried in a dehydrator (Model 32 100, Hamilton Beach) at a constant temperature of 41 ºC for 18 hours, and grounded until pulverized.

Ultrasonic Assisted Ethanolic Extracts (UAEE)

The ethanolic extraction comprised 10 g of dry leaves of C. chayamansa in 100 mL of aqueous ethanol (1:1) using a 40 kHz ultrasonic mechanical bath (1800, Branson, St. Louis, MO, USA) at 25 °C for 30 min. The extracts were filtered through Whatman #1 paper (150 mm diameter) and stored at 4 °C (Pérez-González et al., 2019).

 Quantification and profile of phenolic compounds from Ultrasonic Assisted Ethanolic Extracts (UAEE)

The Folin-Ciocalteu method (Ruiz et al., 2015) was used to estimate the concentration of phenolic compounds from C. chayamansa, with gallic acid as the standard (≥ 98.0, CAS: 5995-86-8, Fermont, Monterrey, Mexico). The analyses were carried out in triplicate, and results are expressed as mg of Gallic Acid Equivalents (mg GAE/mL).

The profile of phenolic compounds from Ultrasonic Assisted Ethanolic Extracts (UAEE) of C. chayamansa was determined through ultra-performance liquid chromatography coupled with a photodiode array detector and electrospray ionization mass spectrometry (UPLC-PDA-ESI-MS); using an ultra-performance liquid chromatograph (UPLC) (ACQUITY UPLC H-Class, Waters Corporation, Milford, MA, USA) equipped with a quaternary pump (UPQSM), and an automatic injector (UPPDALTC). Chromatographic separation was performed on a Waters’ ACQUITY UPLC BEH C18 column, 1.7 μm, 100 x 2.1 mm I.D (Milford, MA, USA) under similar conditions reported by Herrera-Pool et al. (2021). The Photodiode Array Detector (PDA) was set to scan within a wavelength (λ) range from 190 nm to 600 nm. The absorbance response was taken from channels A (290 nm) and B (350 nm). Mass spectra (Xevo TQ-S Micro, Waters, Chicago, IL, USA) were recorded in full scan negative ion mode at 50 - 2000 m/z. Compounds were identified by comparing the observed spectral fingerprint data with those reported in Pubchem and MassBank databases.

 <bold>Determination of antioxidant activities <italic>in vitro</italic> </bold> Chelating capacity of Cu<sup>2+</sup> and Fe<sup>2+</sup> <italic>in vitro</italic>

Chelating activity of Cu2+ was determined with the method reported by Saiga et al. (2003), mixing 250 µL of sodium acetate buffer (50 mM, pH 6.0) with 250 µL of 20 mM Cu2+ standard solution and 25 µL of 0.1 % violet pyrocatechol, reacted for 5 min at 25 ºC and then 250 µL of the blank (distilled water) or UAEE samples were added. The absorbances were measured at 632 nm in a spectrophotometer (VE5100UV, Velab, Pharr, TX, USA). All samples were performed in triplicate. The copper chelating activity was calculated as:

%   C C   C u 2 +   =   ( S a m p l e r   A b s   -   B l a n k   A b s ) / ( S a m p l e r   A b s ) × 100

Where % CC Cu2+ represents the percentage of copper chelated.

The Fe2+ chelating capacity was determined by the method used by Ruiz et al. (2015). Briefly, the absorbance of a blank and the UAEE samples were read, mixing 250 μL of sodium acetate buffer (100 mM, pH 4.9) with 250 μL of 20 mM Fe2+ standard solution, and 250 μL of water (in the case of the blank) or 250 µL of UAEE. Next, it was left to react for 5 min at room temperature and then 50 µL of 40 mM ferroxine solution were added. Absorbances were measured at 562 nm in a spectrophotometer (VE-5100UV, Velab, Pharr, TX, USA). All samples were processed in triplicate. The Fe2+ chelating activity is estimated as shown in Eq. 2:

%   C C   F e 2 +   =   ( S a m p l e r   A b s - B l a n k   A b s ) / ( B l a n k   A b s ) × 100

Where % CC Fe2+ represents the percentage chelating capacity.

 Fe<sup>3+</sup> reducing power

The Fe3+ reducing power was determined using the method described by Sudha et al. (2011). Briefly, absorbance measurements from a blank (distilled water) and UAEE were made. 250 µL of blank or sample were taken and 250 µL of phosphate buffer (0.2M, pH 6.6) and 250 µL of 1 % K3[Fe(CN)6] were added in each case, shaken for 5 sec in a vortex and incubated at 50 ºC for 20 min. Once the incubation was completed, 250 µL of 10 % C2HCl3O2 was added, 500 µL of this mixture was taken and deposited in a 2 mL Eppendorf tube, and then 400 µL of distilled water and 100 µL of 0.1% FeCl3 were added, mixed for 5 seconds in a vortex and incubated at 50 ºC for 10 minutes. Finally, the samples were centrifuged at 3000 rpm for 10 min in a centrifuge with a 10 cm rotor diameter (J-40, Solbat. Edo. Mex., Mexico), and the absorbances of the supernatant were read at 700 nm in a spectrophotometer (VE-5100UV, Velab, Pharr, TX, USA). All samples were analyzed in triplicate. The Fe3+ reducing power was estimated as shown in Eq 3:

%   P R   F e 2 +   =   ( S a m p l e r   A b s - B l a n k   A b s ) / ( S a m p l e r   A b s ) × 100

Where % PR Fe2+ represents the percentage reducing power of Fe3+.

 ABTS radical scavenging capacity

ABTS radical scavenging capacity was determined by the method reported by Ruiz et al. (2015) with some modifications. First, a 2.0 mM ABTS solution was prepared, then the ABTS+ radical cation was produced with a 70 mM K2S2O₈ solution, allowing the mixture to remain in the dark at 25 ºC for 16 hours before use. Subsequently, this solution was diluted with phosphate buffer (1.0 M, pH 7.4) until obtaining an absorbance of 0.800 ± 0.030 at 734 nm. Next, 10 µL of UAEE diluted 1:10 were taken and reacted with 990 µL of the ABTS+ radical diluted in phosphate buffer. Next, the absorbance at 734 nm was measured in a spectrophotometer (VE-5100UV, Velab, Pharr, TX, USA) after 1 and 6 min of reaction. The same procedure was performed with a blank sample using 50% ethanol. All samples were analyzed in triplicate. The ABTS+ radical scavenging percentage (% RS) of the samples were calculated as shown in Eq. 4:

%   R S   =   ( S a m p l e r   A b s - B l a n k   A b s ) / ( S a m p l e r   A b s ) × 100

Where % RS represents the percentage ABTS radical scavenging.

 DPPH radical scavenging capacity

The DPPH radical scavenging capacity was done according to the method proposed by Fukumoto and Mazza (2000), with some modifications. Briefly, a 0.1 mM DPPH solution in ethanol was prepared. UAEE samples diluted 1:10 and distilled water (as a blank) were analyzed. The procedure was the same for both. 100 µL of blank and 100 µL of extracts were taken individually and 1000 µL of the DPPH solution were added to each one, then shaked in a vortex for 10 seconds and allowed to react for 30 minutes in the dark. Next, their absorbances were read at 517 nm in a spectrophotometer (VE-5100UV, Velab, Pharr, TX, USA). All samples were analyzed in triplicate. The % uptake of RL DPPH was determined as shown in Eq. 5:

%   R S C   =   ( S a m p l e r   A b s - B l a n k   A b s ) / ( S a m p l e r   A b s ) × 100

Where % RSC represents the percentage DPPH radical scavenging capacity.

 Fe<sup>3+</sup> reducing power

The reducing power of Fe3+ was determined using the method described by Sudha et al. (2011). Briefly, absorbance measurements from a blank (distilled water) and UAEE of C. chayamansa were made. 250 µL of blank or sample were taken, mixed with 250 µL of phosphate buffer (0.2M, pH 6.6) and 250 µL of 1% K3[Fe(CN)6] in each case, shaken for 5 seconds in a vortex and incubated at 50 ºC for 20 minutes. Once the incubation was completed, 250 µL of 10 % C2HCl3O2 were added, then 500 µL of this mixture were taken and deposited in a 2 mL Eppendorf tube, 400 µL of distilled water and 100 µL of 0.1% FeCl3 were added, mixed for 5 seconds in a vortex and incubated at 50 ºC for 10 min. Finally, the samples were centrifuged at 3000 rpm for 10 min in a centrifuge with a 10 cm rotor diameter (J-40, Solbat. Edo. Mex., Mexico, and the absorbances of the supernatant measured read at 700 nm in a spectrophotometer (VE-5100UV, Velab, Pharr, TX, USA). All samples were analyzed in triplicate. The reducing power of Fe3+ was estimated as shown in Eq. 3:

%   P R   F e 2 +   =   ( S a m p l e r   A b s - B l a n k   A b s ) / ( S a m p l e r   A b s ) × 100

Where % PR Fe2+ represents the percentage reducing power of Fe3+.

 Antibacterial activity

The antibacterial activity of the UAEE of C. chayamansa was evaluated against Escherichia coli (G- ATCC 25922) and Staphylococcus aureus (G+ ATCC 25923). The agar disc diffusion method was performed on Muller-Hilton agar (MCD LAB, Cat 7131, Mex), prepared according to the manufacturer’s specifications and sterilized in an autoclave at 1.055 Kgf/ cm2 for 15 min. After that, 30 mL of agar were distributed in Petri dishes which were impregnated with 100 μL per box with the adjusted suspension of each indicator bacteria. Sixmm diameter sterile discs were impregnated with 30 μL of UAEE. As a positive control antibiogram discs were used with amoxicillin and clavulanic acid (AMC) at a concentration of 30 μg/mL. As negative controls, disks impregnated with 30 μL of sterilized water were used. All samples were analyzed in triplicate for each type of extract and were incubated at 37 °C for 24 h. Growth inhibition halos were measured with a Vernier Calliper (Calliper, Lenfech, 0 mm - 150 mm measuring range). The antibacterial activity was assessed according to Capitani et al. (2016) parameters.

 <bold> <italic>In silico</italic> antibacterial activity</bold>

For in silico antibacterial activity, the crystal structure of key receptors for E. coli (2WUB, 4XO8) and S. aureus (2W9S, 2ZCO) were retrieved from the Protein Data Bank (http://www.rcsb.org/). The structures were prepared using the Dock Prep Module of UCSF Chimera 1.14 (Pettersen et al., 2004) by removing water molecules, sidechains and ligands, adding hydrogens, and assigning partial charges. However, the Mg ion was kept due to its importance for the 4WUB protein function. Protein fragments were reconstructed by applying SWISS-MODEL (Waterhouse et al., 2018).

Ligands - Guanosine, Kaempferol-3-O-rutinoside, Kaempferol-3-(2G-glucosylrutinoside)-7-rhamninoside, Kaempferol-3-O-rhamninoside, Kaempferol-3-(2G-glucosylrutinoside) and Rutin - and control compounds (trimethoprim, farnesyl thiopyrofosfate, heptyl-α-D-mannopyrannoside, and phosphoaminophosphoric acid adenilate ester) were retrieved in Mole2 file format (.mol2) from PubChem (https://pubchem.ncbi.nlm.nih.gov/). Avogadro 1.2.0 (Hanwell et al., 2012) optimized f ligands’ molecular geometry and converted the input files to .pdb files, later prepared using the Chimera docking tool.

All structures were aligned on a grid box large enough to accommodate all the experimental ligands used for molecular docking analysis. The grid size and the grid box coordinates for each target were as follows: 2WUB, 25×25×25 Å (14.57, 19.81, -10.80); 4OX8, 30×30×30 Å (-43.84, 5.15, 3.86); 2W9S, 25×25×25 Å (2.67, -2.13, 44.93); and 2ZCO, 30×30×30 Å (53.86, 10.35, 51.81). Ten independent docking runs were executed for each structure with the Autodock Vina tool (Eberhardt et al., 2021). Additionally, ten replicates were performed for each combination of ligand and receptor, which were analyzed through LigPlot+ (Laskowski et al., 2011) and PyMOL (De Lano et al., 2002).

Docking results were validated by extracting the cocrystallised ligands of the 2W9S, 2ZCO, 4XO8, and 4WUB proteins and re-docking them into the same position. The ligands pose with the lowest energy obtained on re-docking, and the co-crystallised ligands were superimposed to calculate the RMSD values in PyMOL software. The RMSD values must be within a reliable range of 2 Å to validate the docking process (Jug et al., 2015). Table 1 summarizes the binding affinity between the C. chayamansa compounds, bacterial proteins, and ligand-amino acid interactions.

Binding affinities between <italic>C. chayamansa</italic> compounds and bacterial proteins and ligand-amino acid interactions.
Target Ligand Binding affinities (Kcal/mol) Amino acid interactions
  Guanosine -8.0 I14, G15, N18, Q19, K45, T46, I50, G94, Y98, T121
  Kaempferol-3-O-rutinoside -9.5 I5, A7, L20, W22, H23, D27, L28, T46, I50, F92, Y98
  Kaempferol-3-(2G-glucosylrutinoside)-7-rhamninoside -7.7 A7, L20, H23, I31, L32, T46, I50, L52, R57, F92, Y98
2W9S Kaempferol-3-O-rhamninoside -9.3 A7, Q19, L20, L28, I31, T46, I50, F92, Y98
  Kaempferol-3-(2G-glucosylrutinoside) -7.7 I5, A7, Q19, L20, T46, I50, L52, F92, Y98
  Rutin -9.2 A7, L20, W22, H23, D27, T46, S49, I50, F92, G94, Y98
  Trimethoprim -7.6 A7, I14, G46, N18, L20, D27, I31, T46, S49, F92, Y98, T121
  Guanosine -7.4 N17, H18, R45, D48, Q165, N168, D172, Y183, R265 H18, R45, D48, Y129, Q165, V133, I169, D172, Y183 D49, D52, V111, D114, Q165, N168, N179, R181, D172, D176, R265 H18, Y41, R45, D49, D114, Y129, D172, R181, Y183 R45, D48, D49, V111, D114, Q165, D172, D176, R181, Y183 D48, V111, D114, Y129, V133, Q165, N168, D176, R181, Y183 H18, F22, Y41, R45, A134, A157, L160, Q165, N168, R171, D172
  Kaempferol-3-O-rutinoside -10.1
  Kaempferol-3-(2G-glucosylrutinoside)-7-rhamninoside -10.4
2ZCO Kaempferol-3-O-rhamninoside -9.3
  Kaempferol-3-(2G-glucosylrutinoside) -8.9
  Rutin -9.8
  Farnesyl thiopyrophosphate -7.2
  Guanosine -6.6 F1, D46, D47, Y48, I52, D54, Q133, N135, D140, F142
  Kaempferol-3-O-rutinoside -6.9 D37, L76, S78, G79, V93, V94, Y95, L101, P102, P104, V105
  Kaempferol-3-(2G-glucosylrutinoside)-7-rhamninoside -5.9 A2, C3, L4, G8, A10, P12, F43, H45, D47, R98, D100
4XO8 Kaempferol-3-O-rhamninoside -6.9 F1, P12, H45, N46, D47, Y48, D54, R98, Q133, N135, D140, F142
  Kaempferol-3-(2G-glucosylrutinoside) -6.1 A2, C3, A10, I13, P12, F43, H45, D47, R98, T99, D100
  Rutin -6.8 D37, L76, S79, G79, V93, V94, Y95, L101, P102, P104
  Heptyl-α-D-mannopyranoside -6.5 F1, I13, N46, D47, Y48, I52, D54, Q133, N135, D140,
  Guanosine -8.3 N46, E50, D73, G77, I78, P79, I94, L103, Y109, V120, T165 N46, A90, V93, I94, G101, G102, L103, D105, N107, S108, Y109 E50, I78, H83, V93, G101, G102, L103, D105, N107, Y109, R136 N46, E50, R76, P79, H83, I94, G101, G102, D105, N107, S108, Y108 P79, H83, A90, G101, G102, L103, D105, N107, S108, Y109, R136 E50, D73, P79, H83, I94, G101, G102, L103, S108, Y109, G117 N46, D73, V97, A100, G102, L103, L115, H116, G117, V118, G119, V120, S121, Q335, L337
  Kaempferol-3-O-rutinoside -9.2
  Kaempferol-3-(2G-glucosylrutinoside)-7-rhamninoside -8.7
4WUB Kaempferol-3-O-rhamninoside -8.9
  Kaempferol-3-(2G-glucosylrutinoside) -8.5
  Rutin -10.1
  Phosphoaminophosphoric acid adenylate ester -11.1

Statistical analysis

Results were summarized by descriptive statistics using R Studio (V 4.2.1) and reported as mean ± standard error of the mean.

Results and discussion <bold>Phenolic compounds in ethanolic extracts of <italic>C. chayamansa</italic> </bold>

The concentration of phenolic compounds in the UAEE of C. chayamansa leaves was 143.7 mg of gallic acid equivalents (mg GAE)/g dry leaves. Guanosine nucleoside and different coumaric acid and kaempferol derivatives were identified (Table 2). Other compounds reported in extracts of C. chayamansa leaves are rutin, naringenin, chlorogenic acid, ferulic acid, protocatechuic acid, astragalin, caffeic acid, myristic acid, riboflavin, and β-carotene (Guzmán et al., 2020). Kaempferol has an antioxidant activity via free radical elimination (Hussain et al., 2021), coumaric acid (a hydroxycinnamic acid, i.e. a hydroxy metabolite cinnamic acid) has a high antibacterial, antioxidant, and anti-inflammatory potential related to the prevention of cardiovascular diseases (Liu et al., 2020).

Phenolic compounds detected in Ultrasonic Assisted Ethanolic Extracts (UAEE). The identified compounds that showed a higher signal intensity are shown in bold.
Peak # RT (PDA detector) λ max Molecular ion ([M - H]-) m/z Fragments m/z Tentative identification
1 6.93 253, 280sh 282 150 Guanosine
2 8.26 198, 266, 317 901 755, 593, 447, 355, 283 Kaempferol 3-(2G-glucosylrutinoside)-7-rhamnoside
3 8.39 221, 280sh, 311 355 209, 191, 147, 85 Coumaroyl aldaric acid (Isomer I)
4 8.45 197, 290sh, 312 355 209, 191, 147, 85 Coumaroyl aldaric acid (Isomer II)
5 8.64 221, 295sh, 311 355 209, 191, 147, 85 Coumaroyl aldaric acid (Isomer III)
6 8.69 210, 290sh, 316 355 209, 191, 147, 85 Coumaroyl aldaric acid (Isomer IV)
7 8.86 196, 300 355 209, 191, 147, 85 Coumaroyl aldaric acid (Isomer V)
8 8.95 197, 290sh, 312 355 209, 191, 147, 85 Coumaroyl aldaric acid (Isomer VI)
9 9.11 195, 262sh, 310 355 209, 191, 147, 85 Coumaroyl aldaric acid (Isomer VII)
10 9.25 197, 254, 268sh, 343 755 300, 284 Kaempferol 3-(2G-glucosylrutinoside)
11 9.57 210, 265, 346 739 284, 254, 227 Kaempferol 3-O-rhamninoside
12 9.72 203, 255, 268sh, 352 609 300, 271 Rutin
13 10.17 210, 265, 343 593 284, 254, 227 Kaempferol-3-O-rutinoside (Isomer I)
14 10.29 210, 265, 348 593 284, 254, 227 Kaempferol-3-O-rutinoside (Isomer II)
15 10.56 195, 266, 307 593 284, 254, 227 Kaempferol-3-O-rutinoside (Isomer III)
16 10.90 197, 265, 344 593 284, 254, 227 Kaempferol-3-O-rutinoside (Isomer IV)
17 11.22 210, 265, 321 447 284, 254, 227 Kaempferol-3-O-hexoside (Isomer I)
18 11.66 197, 265, 346 447 284, 254, 227 Kaempferol-3-O-hexoside (Isomer II)

RT: Retention time

In a study where the effect of the aqueous extract of chaya leaves (Cnidoscolus aconitifolius) in precarcinogenic lesions was evaluated, a minor concentration of total phenolic compounds (52.5 mg galic acid equivalents/g of dry leaf) was reported, which differs from our results. The presence of p-coumaric acid is reported, which together with rosmarinic acid, chlorogenic acid, resveratrol and luteoin are the major compounds in extracts obtained; other compounds identified were gallic acid, caffeic acid, vanilic acid, vanillin, resveratrol, apigenin y ferulic acid (Kuri-García et al., 2019).

Us-Medina et al. (2020) evaluated the in vitro antioxidant and anti-inflammatory activity of biologically active compounds from C. aconitifolius extracts, reporting a greater amount of phenolic compounds in aqueous extracts (706.1 mg galic acid equivalents/g of dry leaf) than those reported here; for ethanolic extracts, 351.3 mg galic acid equivalents/g of dry leaf were also reported in C. aconitifolius aqueous extracts. The concentration of phenolic compounds may vary according to the solvent used. Polar solvents are employed for plant extractions since they contain bonds between atoms that differ in electronegativity (e.g., O-H) and form hydrogen bonds; therefore, they are suitable for dissolving polar reactants such as ions (Li et al., 2018). Ethanol has a lower polarity than methanol, however, ethanol is Generally Recognized as Safe (GRAS) by the Food and Drug Administration (FDA).

In vitro antioxidant activity

The antioxidant potential of natural extracts is associated with the content of phenolic compounds. The main antioxidant potential of the UAEE of C. chayamansa leaves was obtained in the Cu2+ chelation activity assays (65. 53 ± 1.72) and Fe3+ reducing power (69.59 %). Regarding the ABTS and DPPH free radical trapping capacity, the antioxidant potential was less than 50 % (37.74 ±3.43 and 14.24± 0.22% respectively), and the Fe 2+ chelation activity was 15.71 ± 0.82%. However, the antioxidant activity by the DPPH method was higher than that reported by García-Rodríguez et al. (2013), which was 10.66% in ethanolic extract of C. chayamansa, and 254.04 µmol Fe2+/L for assay of ferric reducing power; this extract contained 35.7 mgEAG/g of leaf; the authors reported the presence of, coumarins, flavonoids, lignans and cyangenic glycosides. Among their findings, the authors reported that the ethanolic extracts of C. chayamansa also had anti-inflammatory activity in the in vivo model, although it was low, which was related to the concentration of phenolic compounds.

Antioxidant activity has also been reported in other species of the genus Cnidoscolus, although by other methods such as TEAC and ORAC, with values of 539 and 926 µmol Trolox equivalents/g of lyophilized extract respectively, in ethanolic extracts of C. aconitifolius leaf. These extracts had phenolic compounds (52.5 mg GAE/g) and flavonoids (41.6 mg catechin equivalent/g); the administration of these extracts in experimental animals protected against colon cancer in a model in which an oxidizing agent (azoxymethane) and an inflammatory agent (dextran sodium sulfate), through inhibiting cell proliferation and inflammation of colonic lesions by decreasing β-catenin and at long-term COX-2 reduction, although a high expression of NF-jB (Kuri-García et al., 2019).

 <bold> <italic>In vitro</italic> and <italic>in silico</italic> antibacterial activity</bold>

For in vitro antibacterial activity assay, the inhibition halos in positive controls showed high activity against Gram-negative and Gram-positive bacteria strains (Table 3). Conversely, the halos of inhibition size in UAEE of C. chayamansa leaves was less than 10 mm, hence, considered inactive (Capitani et al., 2016). This could have been mainly due to the solvent or extract concentration used, other authors reported that ethanol extract of C. chayamansa leaves contained flavonoids, saponins, cardenolides and polyphenols with antibacterial activity against S. aureus, with an inhibition zone of 13.2 mm, greater than that found in this study (9.96 mm). Also, for the test in E. coli the activity was greater (14.83 mm) than that reported by us (7.9 mm). On the other hand, they demonstrated that C. chayamansa extracts obtained with different solvents had activity agains Gram-positive pathogenic bacteria (B. Cereus and S. pyogenes) and Gram negative pathogenic bacteria (E. coli and K. pneumoniae), using ciprofloxacin as a control (Elizabeth et al., 2023).

Antibacterial activity of Ultrasonic Assisted Ethanolic Extracts (UAEE) from <italic>C. chayamansa</italic> leaves.
Escherichia coli Staphylococcus aureus
UAEE 7.90* 9.96*
AMC 20.05**** 42.93****
C- 7.28* 6.46*

UAEE: Ultrasonic Assisted Ethanolic Extract, AMC: amoxicillin with clavulanic acid C+; positive control)y C-: negative control *inactive, ** partially active, ***active **** very active (Capitani et al., 2016).

UAEE: Extracción Etanólica Asistida por Ultrasonido; AMC: Amoxicilina con ácido clavulánico; C+ Control positivo; C- Control negativo; *inactivo, ** parcialmente activo, ***activo y **** muy activo (Capitani et al., 2016).

For in silico assays, target proteins can be proposed for future tests, either in vitro or in vivo, in this sense, target proteins involved in the viability of S. aureus were selected for antibacterial potential evaluation of the extracted compounds against these microorganisms (Figure 1). Firstly, Dihydrofolate Reductase (DHFR, 2W9S) (https://www.rcsb. org/structure/2w9s) involved in the folic acid pathway in S. aureus, which promotes thymidylate biosynthesis essential for cell replication and proliferation (He et al., 2020; Bourne et al., 2010). Secondly, Dehydrosqualene Synthase (CrtM, 2ZCO) (https://www.rcsb.org/structure/2ZCO) responsible for synthesizing the golden carotenoid pigment staphyloxanthin of S. aureus, which provides its antioxidant properties, aiding bacteria survival within the host cell (Kahlon et al., 2010; Wu et al., 2019). Inhibitors targeting DHFR and CrtM potentially induce bacterial death and serve as effective targets for treating bacterial infections.

Two and three-dimensional representation of the hydrogen bonding and hydrophobic interaction between ligands within the binding cavity of receptors. a) Kaempferol-3-O-rutinoside-2W9S complex, b) Kaempferol-3-(2G-glucosylrutinoside)-7-rhamninoside-2ZCO complex, c) Kaempferol-3-O-rutinoside-4OX8 complex and d) Rutin-4WUB complex.

Molecular docking was conducted to determine the target protein-compound binding energy. All six characterized compounds were docked against 2W9S and 2ZCO proteins using Autodock Vina. Almost all the evaluated ligands showed higher affinities than the co-crystallized ligands found in the crystal structures of each target during re-docking. The most favorable ligand-target complexes were Kaempferol3-O-rutinoside-2W9S (-9.5 kcal/mol) and Kaempferol-3-(2Gglucosylrutinoside)-7-rhamninoside-2ZCO (-10.4 kcal/mol).

Two targets from E. coli were selected to evaluate the inhibitory potential of the compounds identified through the UAEE of C. chayamansa leaves. The first target is the FimH protein (4XO8), a bacterial adhesion lectin located at the tip of E. coli type 1 fimbriae or pili. These structures facilitate bacterial binding to surfaces that display mannose residues (Hartmann et al., 2011; Magala et al., 2020). The second target is the DNA gyrase B subunit (4WUB), which plays a crucial role in regulating the physiological function of the genome and providing the energy required for DNA supercoiling (Sissi et al., 2010). This enzyme is an ideal target for antibacterial drugs due to its potential for selective toxicity (Sissi et al., 2010; Fois et al., 2020).

Molecular docking evaluated the binding energy between the 4XO8 and 4WUB proteins, and the six compounds through Autodock Vina. The evaluated ligands showed similar affinities to the co-crystallized ligands, particularly with 4XO8. However, for 4WUB, the evaluated ligands exhibited slightly lower activity compared to the co-crystallized ligand, Phosphoaminophosphoric acid adenylate ester. The most favorable ligand-target complexes were Kaempferol-3-Orutinoside-4XO8 (-6.9 kcal/mol) and Rutin-4WUB (-10.1 kcal/ mol).

Due of the results obtained, it´s possible that the compounds from the C. chayamansa extract could exert a bacteriostatic effect on E. coli cultures during the in vitro antibacterial activity evaluation via inhibition of 4WUB, explaining the observed inhibition halos, such as Tang et al. (2022) and Biasi-Garbin et al. (2022) obtained with similar methodologies. On the other hand, 4XO8 inhibition was unclear in the in vitro evaluation; however, it suggests that the evaluated compounds can bind to these lectins, thereby obstructing bacterial adhesion to host tissues.

 Conclusions

The compounds present in UAEE from C. chayamansa leaves, are guanosine nucleoside and different coumaric acid and kaempferol derivatives. These compounds could be related to Cu2+ chelation activity and Fe3+ reducing antioxidant power. Although the antibacterial activity is not conclusive in the inhibition halos assays, the molecular docking results suggest that the identified compounds could intervene in metabolic processes necessary for the survival and replication of E. coli and S. aureus. Therefore, subsequent studies are necessary to evaluate the effect of different concentrations of C. chayamansa leaves extracts and their isolated compounds on bacterial strains.

 Acknowledgments

The authors acknowledge the School of Nutrition from Universidad Anahuac Mayab for providing the facilities, CONAHCyT for granting a research scholarship, and ECOSUR for financially supporting the project through the Master’s Thesis Support Program.

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