Journal of biological and health sciences http://biotecnia.unison.mx
Universidad de Sonora
ISSN: 1665-1456
1 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.
2 Escuela de Nutrición, Universidad Anáhuac Mayab. Carr. Mérida Progreso Km. 15.5. 96 Cordemex, CP. 97310, Mérida, Yucatán, México.
3 Universidad Politécnica Metropolitana de Puebla, Popocatépetl s/n, Reserva Territorial Atlixcáyotl, Tres Cerritos, 72480 Puebla, Pue., México.
4 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.
5 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.
Actividad antioxidante y antimicrobiana in vitro e in silico de extractos etanólicos de hojas de Cnidoscolus chayamansa
Cnidoscolus chayamansa leaves – used in gastronomy and traditional medicine in Mexico – are rich in phenolic com- pounds, which may have antioxidant and antimicrobial acti- vity. 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. au- reus, the in silico analysis revealed that the isolated phenolic compounds modify signaling pathways essential for the survival of the bacteria studied.
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 creci- miento en disco, el análisis in silico reveló que los compuestos fenólicos aislados modifican las vías de señalización esencia- les para la supervivencia de las bacterias estudiadas.
king molecular; actividad antioxidante; actividad antibacte- riana.
Cnidoscolus chayamansa, commonly known as chaya is an endemic shrub of Tabasco and the Yucatan Peninsula in Mexi- co (Pérez-González et al., 2019) used in local gastronomy, in addition to traditional medicine (Rodrigues et al., 2021) pre- sumably due to a high content of bioactive compounds (phe- nols, 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 (sca- venging 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), inhibi- ting 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 re-
Volume XXVI
DOI: 10.18633/biotecnia.v26.2233
*Author for correspondence: ariss M. Sánchez-Chino
Received: January 17, 2024
Accepted: March 21, 2024
Published: May 13, 2024
ceptors of inflammation and activators of anti-inflammatory enzymes (Kaabi, 2022).
A positive correlation exists between the content of phenolic compounds and the antibacterial capacity, inclu- ding bacteriostatic and bactericidal properties. Phenolic compounds modify the bacterial cytoplasmic membrane permeability and inhibit signaling pathways involved in bac- terial 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 phe- nolic 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 Ethano- lic Extracts (UAEE) of C. chayamansa leaves.
Collection and preparation of samples. Leaves of C. cha- yamansa 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 was- hed 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.
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).
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 de- termined through ultra-performance liquid chromatography coupled with a photodiode array detector and electrospray
ultra-performance liquid chromatograph (UPLC) (ACQUITY UPLC H-Class, Waters Corporation, Milford, MA, USA) equip- ped with a quaternary pump (UPQSM), and an automatic injector (UPPDALTC). Chromatographic separation was per- formed 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 data- bases.
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 (dis- tilled water) or UAEE samples were added. The absorbances were measured at 632 nm in a spectrophotometer (VE- 5100UV, Velab, Pharr, TX, USA). All samples were performed in triplicate. The copper chelating activity was calculated as:
% CC Cu2+ = (Sampler Abs - Blank Abs)/(Sampler Abs)×100 (Eq 1)
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:
% CC Fe2+ = (Sampler Abs-Blank Abs)/(Blank Abs)×100 (Eq 2)
Where % CC Fe2+ represents the percentage chelating capacity.
The Fe3+ reducing power was determined using the method described by Sudha et al. (2011). Briefly, absorbance mea- surements 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 % K [Fe(CN) ]
ionization mass spectrometry (UPLC-PDA-ESI-MS); using an 3 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 com- pleted, 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 diame- ter (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 analy- zed in triplicate. The Fe3+ reducing power was estimated as shown in Eq 3:
% PR Fe2+ = (Sampler Abs-Blank Abs)/(Sampler Abs)×100 (Eq. 3)
Where % PR Fe2+ represents the percentage reducing power of Fe3+.
ABTS radical scavenging capacity was determined by the method reported by Ruiz et al. (2015) with some modifica- tions. 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 dilu- ted 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:
% RS = (Sampler Abs-Blank Abs)/(Sampler Abs)×100 (Eq. 4)
Where % RS represents the percentage ABTS radical scavenging.
The DPPH radical scavenging capacity was done ac- cording to the method proposed by Fukumoto and Mazza (2000), with some modifications. Briefly, a 0.1 mM DPPH solu- tion 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 analy- zed in triplicate. The % uptake of RL DPPH was determined as shown in Eq. 5:
% RSC = (Sampler Abs-Blank Abs)/(Sampler Abs)×100 (Eq. 5)
Where % RSC represents the percentage DPPH radical scavenging capacity.
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 se- conds 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:
% PR Fe2+ = (Sampler Abs-Blank Abs)/(Sampler Abs)×100 (Eq. 3)
Where % PR Fe2+ represents the percentage reducing power of Fe3+.
The antibacterial activity of the UAEE of C. chayamansa was evaluated against Escherichia coli (G- ATCC 25922) and Sta- phylococcus 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. Six- mm 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.
In silico antibacterial activity
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, Ka- empferol-3-O-rhamninoside, Kaempferol-3-(2G-glucosylruti- noside) 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 con- verted 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 mo- lecular docking analysis. The grid size and the grid box coor- dinates 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 per- formed 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 co- crystallised 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 calcu- late 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.
Results were summarized by descriptive statistics using R Studio (V 4.2.1) and reported as mean ± standard error of the mean.
The concentration of phenolic compounds in the UAEE of C. chayamansa leaves was 143.7 mg of gallic acid equivalents
Table 1. Binding affinities between C. chayamansa compounds and bacterial proteins and ligand-amino acid interactions.
Tabla 1. Afinidades de unión entre compuestos de C. chayamansa y proteínas bacterianas e interacciones ligando-aminoácido.
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, Q16 | |
Kaempferol-3-O-rutinoside | -10.1 H18, R45, D48, Y129, Q1 | |
Kaempferol-3-(2G-glucosylrutinoside)-7-rhamninoside | -10.4 D49, D52, V111, D114, Q165 | |
2ZCO | Kaempferol-3-O-rhamninoside | -9.3 |
Kaempferol-3-(2G-glucosylrutinoside) | -8.9 H18, Y41, R45, D49, D11 | |
Rutin | -9.8 R45, D48, D49, V111, D114, |
5, N168, D172, Y183, R265
65, V133, I169, D172, Y183
, N168, N179, R181, D172, D176, R265
4, Y129, D172, R181, Y183 Q165, D172, D176, R181, Y183
Farnesyl thiopyrophosphate | -7.2 | H18, F22, Y41, R45, A134, A157, L160, Q165, N168, R171, D172 | |
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 | ||
Kaempferol-3-O-rutinoside | -9.2 N46, A90, V93, I94, G101, G102, L103, D105, N107, S108, Y109 | ||
Kaempferol-3-(2G-glucosylrutinoside)-7-rhamninoside | -8.7 E50, I78, H83, V93, G101, G102, L103, D105, N107, Y109, R136 | ||
4WUB | Kaempferol-3-O-rhamninoside | -8.9 N46, E50, R76, P79, H83, I94, G101, G102, D105, N107, S108, Y108 | |
Kaempferol-3-(2G-glucosylrutinoside) | -8.5 P79, H83, A90, G101, G102, L103, D105, N107, S108, Y109, R136 | ||
Rutin | -10.1 E50, D73, P79, H83, I94, G101, G102, L103, S108, Y109, G117 | ||
Phosphoaminophosphoric acid adenylate ester | -11.1 N46, D73, V97, A100, G102, L103, L115, H116, G117, V118, G119, V120, S121, Q335, L337 | ||
D48, V111, D114, Y129, V133, Q165, N168, D176, R181, Y183
(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. chaya- mansa leaves are rutin, naringenin, chlorogenic acid, ferulic acid, protocatechuic acid, astragalin, caffeic acid, myristic acid, riboflavin, and β-carotene (Guzmán et al., 2020). Kaem- pferol 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 antibac- terial, antioxidant, and anti-inflammatory potential related to the prevention of cardiovascular diseases (Liu et al., 2020).
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 pheno- lic 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 rosma- rinic 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 accor- ding 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 reac- tants 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).
The antioxidant potential of natural extracts is associated with the content of phenolic compounds. The main anti- oxidant potential of the UAEE of C. chayamansa leaves was obtained in the Cu2+ chelation activity assays (65. 53 ± 1.72)
Table 2. Phenolic compounds detected in Ultrasonic Assisted Ethanolic Extracts (UAEE). The identified compounds that showed a higher signal intensity are shown in bold.
Tabla 2. Compuestos fenólicos detectados en Extractos Etanólicos Asistidos por Ultrasonido (UAEE). Los compuestos identificados que mostraron una mayor intensidad de señal se muestran en negrita.
RT (PDA | Molecular ion Fragments ([M – H]-) m/z Tentative identification | ||||
m/z | |||||
1 | 6.93 | 253, 280sh | 282 | 150 | Guanosine |
2 8.26 198, 266, 317 901 755, 593, 447, 355, Kaempferol 3-(2G-glucosylrutinoside)-7-rhamnoside | |||||
Peak #
detector) λ max
283 | |||||
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 10 | 9.11 9.25 | 195, 262sh, 310 197, 254, | 355 755 | 209, 191, 147, 85 300, 284 | Coumaroyl aldaric acid (Isomer VII) Kaempferol 3-(2G-glucosylrutinoside) |
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) |
268sh, 343 | |||||
11 | 9.57 | 210, 265, 346 | 739 | 284, 254, 227 | Kaempferol 3-O-rhamninoside |
RT: Retention time
and Fe3+ reducing power (69.59 %). Regarding the ABTS and DPPH free radical trapping capacity, the antioxidant poten- tial was less than 50 % (37.74 ±3.43 and 14.24± 0.22% res- pectively), 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 re- ported 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 Tro- lox equivalents/g of lyophilized extract respectively, in etha- nolic extracts of C. aconitifolius leaf. These extracts had phe- nolic 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 inhi- biting 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).
In vitro and in silico antibacterial activity
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 ex- tract concentration used, other authors reported that etha- nol 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
Table 3. Antibacterial activity of Ultrasonic Assisted Ethanolic Extracts (UAEE) from C. chayamansa leaves.
Tabla 3. Actividad antibacteriana de extractos etanólicos obtenidos por ul-
trasonicación (UAEE) de hojas de C. chayamansa.
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 clavu- lanic 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).
reported by us (7.9 mm). On the other hand, they demonstra- ted that C. chayamansa extracts obtained with different sol- vents 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).
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 com- pounds 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 trea- ting bacterial infections.
Molecular docking was conducted to determine the tar- get 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 Kaempferol- 3-O-rutinoside-2W9S (-9.5 kcal/mol) and Kaempferol-3-(2G- glucosylrutinoside)-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 bet- ween the 4XO8 and 4WUB proteins, and the six compounds through Autodock Vina. The evaluated ligands showed simi- lar 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-O- rutinoside-4XO8 (-6.9 kcal/mol) and Rutin-4WUB (-10.1 kcal/ mol).
Figure 1. 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-rhamni- noside-2ZCO complex, c) Kaempferol-3-O-rutinoside-4OX8 complex and d) Rutin-4WUB complex.
Figura 1. Representación bidimensional y tridimensional de los enlaces de hidrógeno y la interacción hidrofóbica entre ligandos dentro de la cavidad de unión de los receptores. a) Complejo Kaempferol-3-O-rutinósido-2W9S, b) Complejo Kaempfe-rol-3-(2G- glucosilrutinósido)-7-ramninósido-2ZCO, c) Complejo Kaempfe-rol-3-O-rutinósido-4OX8 y d) Rutin-4WUB complejo.
Due of the results obtained, it´s possible that the com- pounds from the C. chayamansa extract could exert a bacte- riostatic effect on E. coli cultures during the in vitro antibac- terial 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 methodolo- gies. 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.
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 replica- tion 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.
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.
The authors declare no conflict of interest.
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