Mueller-Hinton agar and broth (Bioxon^®) were from Becton Dickinson (BD) brand. 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, quercetin, 2,2-difenil-1-picrilhidrazil, chloramphenicol, 2,4,6-Tris(2-pyridyl)-s-triazine, potassium persulfate, trolox, iron (III) chloride, sodium acetate, sodium hydroxide anhydrous, gelatin, alpha-naphthol, acetic anhydride, Folin & Ciocalteu’s phenol reagent, gallic acid and sodium carbonate used in the experiments were from Sigma-Aldrich brand. Dragendorff reagent Supelco brand and 2,3,5-Triphenyl-tetrazolium chloride solution Merck Millipore brand. Sulfuric, acetic, hydrochloric and phosphoric acids, as well as solvents used (hexane, acetone, methanol, acetonitrile, and water), were analytical grade and HPLC grade from J.T. Baker.

Turnera diffusa was collected in August 2022, in the municipality of Santa María Ixcatlán, in the state of Oaxaca, Mexico. It is located at 17˚48'90.7" North latitude and 17˚00'61.8" West longitude. The species was identified in the IZTA herbarium of Facultad de Estudios Superiores Iztacala, of the Universidad Nacional Autonoma de Mexico. A voucher specimen (3260 IZTA) was deposited at the herbarium.

The aerial part of T. diffusa was dried at room temperature. The extracts were obtained with hexane, acetone and methanol, the extraction was carried out sequentially. 316.7 g of dry and powdered plant material were placed in a flask, 1 L of hexane was added and left to stand for 48 hours, the extract was filtered and concentrated in a rotary evaporator (Heidolph Laborota 4010). Later the same was done with acetone and finally with methanol. The yields obtained from the extracts were: hexane (1.95 g, 0.62%), acetone (7.09 g, 2.24%) and methanol (14.91 g, 4.71%). The extracts were stored at 4˚C and in the dark, for later use in tests.

2.4.1. Microorganisms

The bacterial strains used in bioassays were: a) Gram negative bacteria: Escherichia coli ATCC 25922, E. coli ATCC 53218, Pseudomonas aeruginosaATCC 27853, Salmonella typhi ATCC 19430 (donated by the Laboratory of Microbiology Laboratory of Superior Studies Cuautitlan), Enterobacter aerogenes, E. coli 1249 MR, E. coli 182 MR, E. coli 28 MR (donated by the FES Iztacala Clinical Analysis Laboratory), Klebsiella pneumoniae (isolated from a clinical case and donated by the Angeles Metropolitan Hospital, Mexico). b) Gram positive bacteria: Staphylococcus aureus ATCC 12398, S. aureus ATCC 29213, S. aureus (isolated from a clinical case), S. aureus 75 MR, S. aureus 83 MR, S. epidermidis. These strains were maintained at 4˚C on Mueller-Hinton agar (Bioxon^®).

2.4.2. Agar Diffusion Test

The antibacterial activity of extracts was evaluated by Kirby-Bauer method [22]. Inoculums were prepared in 10 mL of Muller-Hinton broth (Bioxon^®) and adjusted with 0.5 McFarland standard (10⁸ CFU/mL). Inocula were spread on surface of Muller-Hinton agar plates, and triplicate Whatman paper discs of 5 mm diameter, impregnated with 2 mg of extract, were placed on plates. Discs containing 25 μg of chloramphenicol were used as a positive control, and discs containing 10 μL of solvents used (hexane, acetone, and methanol) as negative control. Plates were incubated at 37˚C for 24 h, and inhibition zones were measured and reported in mm.

2.4.3. Agar Dilution Test

The minimum inhibitory concentration (MIC) on susceptible strains was determined by broth dilution method [23]. 100 μL of different concentrations of extracts (0.25 to 3.0 mg/mL), chloramphenicol (1.0 to 20.0 μg/mL), and a control group with 100 μL of Muller-Hinton broth were plated in triplicate in 96-well plates. 100 μL of inocula adjusted to 10⁵ CFU/mL were added to each well. Plates were incubated for 24 h at 37˚C and developed with 100 μL/well of tetrazolium chloride (0.1%). MIC was the lowest concentration of extract that visibly inhibited the growth of tested microorganisms.

2.4.4. Bacterial Kinetics Test

The effect of extracts on kinetics of bacterial growth was evaluated by method of Candelaria-Dueñas et al., 2021 [24] on strains that showed sensitivity. Different concentrations of extracts were placed in triplicate in tubes with 10 mL of Muller-Hinton broth (Bioxon^®) (1.0 to 4.0 mg/mL) and control (without extract). 100 mL of inoculum (10⁵ CFU/mL) was added to each tube and placed in incubation at 37˚C. 50 mL samples were taken from each tube at different time intervals (0, 2, 4, 6, 8, 12 and 24 hours), plated on Muller-Hinton agar plates and incubated for 24 hours. CFU/mL were counted to determine the extract concentration and the time required to eliminate or attenuate the growth of microbial population.

2.5.1. DPPH Free Radical-Scavenging Activity

The DPPH free radical-scavenging capacity of extract was determined using reagent 1,1-diphenyl-2-picryl hydrazyl and following method of Baliyan et al., 2022 [25], with some modifications. Assays were carried out in 96-well plates. 50 µL of extracts at different concentrations (20 to 200 μg/mL for acetone and methanol extracts and 100 to 1000 μg/mL for hexanic extract) and 150 µL of a methanolic DPPH solution (250 µM) were added in triplicate. Plates were incubated in the dark and with constant shaking for 30 min, at 37˚C. Absorbance was measured at 515 nm in an ELISA reader (SLT Spectra ELISA reader). Different concentrations of quercetin (1.5 to 15 µg/mL) were used as a reference standard. Percentage of reduction of the radical was calculated from absorbance data using following formula:

where: Ac = Absorbance of control (DPPH without extract); As = Absorbance of sample (extract or quercetin).

Linear regression model was obtained from the data on reduction percentages of extract and quercetin concentrations, which determined mean inhibitory concentration (IC₅₀), which is concentration that reduces DPPH radical by 50%.

2.5.2. ABTS Free Radical-Scavenging Activity

The antioxidant capacity of extracts on cationic radical ABTS was carried out using the method of Re et al., 1999 [26], with some modifications. The radical was generated by an oxidation reaction of ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt 7 mM) with potassium persulfate (2.45 mM). 250 μL of extracts at different concentrations (20 to 200 μg/mL for acetone and methanol extracts and 100 to 1000 μg/mL for hexanic extract) and 2250 μL of ABTS radical (Absorbance = 0.7 ± 0.02 units, at 734 nm) were added in triplicate to test tubes. After 7 minutes of reaction in dark and at room temperature, the absorbance at 734 nm was read (VELAB^TM Sspectrophotometer VE-5100UV). Trolox was used as a reference standard at different concentrations (5 to 50 μg/mL). The percentage of reduction of the ABTS radical was calculated with the formula:

where: Ac = Absorbance of control (ABTS without extract); As = Absorbance of sample (extract or Trolox).

The data were graphed, linear regression model was obtained, and median inhibitory concentration (IC₅₀) was calculated, which represents 50% reduction of ABTS radical, which is expressed as milligrams equivalent of Trolox per gram of extract (mgET/g).

2.5.3. Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP method was performed using the method of Benzie and Strain, 1999 [27], which measures the ability of antioxidant compounds to donate an electron to Fe³⁺ to form Fe²⁺. Colorless 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) complex is reduced to colored ferrous complex. The FRAP reagent was prepared by mixing acetate buffer pH 3.6 (300 mM), TPTZ (10 mM) in 40 mM HCl and FeCl₃ (20 mM), in a 10:1:1 (v/v/v) ratio. The reagent was kept throughout process in a bath at 37˚C. A standard curve of Trolox was prepared at different concentrations (5 to 50 µg/mL). Samples of 250, 500 and 1000 µg/mL of hexane, acetone and methanol extracts respectively were prepared. In test tubes, 250 µL of Trolox and extract concentrations and 2250 µL of FRAP reagent were added in triplicate. Samples were incubated for 10 minutes at room temperature and in dark, the absorbance was read at 593 nm (VELAB^TM Spectrophotometer VE-5100UV). Trolox absorbance data were graphed, linear regression model was obtained, number of equivalent milligrams of Trolox per gram of extract (mgTE/g) and percentage of ferric reduction were calculated.

The identification of the main groups of secondary metabolites in extracts was carried out by means of reactions that indicate presence of coumarins, phenols, alkaloids, flavonoids, tannins, glycosides, steroids and terpenes. The reagents used were: NaOH-HCl, ferric chloride, Dragendorff, NaOH, gelatin, α-naphthol-HCl and Liebermann-Burchard, respectively [28].

The total phenol in extracts was determined by Folin-Ciocalteu method [29]. A standard curve was prepared with gallic acid (0.02 - 0.12 mg/mL). Standard solutions of each extract were prepared (0.2 mg/mL). 300 µL of the gallic acid and extract concentrations, 1800 µL of distilled water and 150 µL of the Folin & Ciocalteu’s phenol reagent were transferred to test tubes in triplicate. After five minutes of incubation, 450 µL of a Na₂CO₃ solution (200 g/L) were added. The samples were incubated for one hour at room temperature, and absorbance was read at 760 nm (VELABTM Sspectrophotometer VE-5100UV). The gallic acid data were graphed (concentration vs absorbance) and the linear regression model was obtained. The extract absorbance data were used to calculate the milligrams of gallic acid equivalents per gram of extract (mgGAE/g).

The polyphenol profile of the T. diffusa extracts with highest antioxidant activity was determined by high-performance liquid chromatography (HPLC-reverse phase) using a Hewlett-Packard model 1100 with a quaternary pump and diode array detector-DAD. Sample analysis was performed using an Allsphere ODS-1 C18 column (250 × 46 mm, 5 μm). The mobile phase was isocratic [methanol:acetonitrile:water (30:5:65) and 1% phosphoric acid]. Signal analysis of the chromatograms was obtained in the ultraviolet spectrum (254 nm) using Chemstation A.09.03 software.

The results of tests were analyzed using a one-way ANOVA to determine differences in antibacterial and antioxidant effects between extracts and controls. Linear regression analysis was also performed to determine IC₅₀ values of extracts on DPPH and ABTS radicals, as well as mg equivalent concentrations of gallic acid and Trolox, in tests for total phenols and FRAP, respectively. In all cases, results were considered significantly different with a P < 0.05. Statistical tests were performed with Microsoft^® Excel for Mac software, version 16.91.

The acetone extract of T.diffusa showed highest antibacterial activity, inhibiting growth of seven strains (4 Gram-negative and 3 Gram-positive) (Table 1). The largest inhibition zones were observed with chloramphenicol, which was much greater than those of extracts, indicating significant differences between effects of extracts and positive control (p < 0.01). However, the results can be considered good, since extracts are complex mixtures in which bioactive compounds are found at variable concentrations, which can be very low or can have antagonistic interactions [30], making it necessary to use high extract concentrations to observe their inhibitory effect on microorganisms.

Regarding acetone extract, largest inhibition zones were observed for K. pneumoniae ATCC 13883, P. aeruginosa ATCC 27853, and S. epidermidis cc (10 ± 1.00 and 13.66 ± 1.15 mm). Relevantly, acetone extract inhibited P. aeruginosa, a strain that was resistant to positive control (chloramphenicol). The MIC values of extracts for susceptible strains were 2.0 and 3.0 mg/mL (Table 1). These concentrations are high compared to positive control, which has MIC and MBC values of 1.0 to 12.0 µg/mL. The results of present work coincide with those obtained by other authors, who mention antibacterial effect of T. diffusa extracts on S. aureus,

Table 1. Antibacterial activity of T. diffusa extracts.

Halo of inhibition (mm). The extracts were tested with 2 mg of extract/disk. na: no activity was observed. Average values of three repetitions ± SD. *Statistically significant compared to positive control (p < 0.01). MIC: Minimum Inhibitory Concentration in mg/mL.

E. faecalis, E. coli, K. pneumoniae and C. albicans [20], on 12 bacterial strains related to gastrointestinal diseases (Hernández et al. 2003), as well as inhibitory effect of essential oil on Mycobacterium tuberculosis [15].

In bacterial kinetic tests of acetone extract of T. diffusa, the Gram-negative strain most susceptible was E. coli cc, as a bactericidal effect was observed at concentrations of 3.0 and 2.0 mg/mL, i.e., death of 99.9% of microorganisms at 2 and 6 hours of exposure to extract (Figure 1). The same effect was observed in K. pneumoniae ATCC 13883 and P. aeruginosa ATCC 27853 at 24 hours of exposure to extract at concentration of 3.0 mg/mL. Only a bacteriostatic effect was observed in S. marcescens ATCC 14756, as the highest concentrations of extract (3.0 and 4.0 mg/mL) showed a decrease in microbial growth but not its elimination.

Figure 1.Effect of acetone extract of T. diffusa on bacterial death curves in Gram negative strains. The control was tested without extract. Data are represented as the mean ± S.E. (n = 3). In all extract concentrations evaluated, there were statistically significant differences compared to the control.

Of the Gram-positive bacteria, S. aureus cc was the most susceptible strain to acetone extract, with a bactericidal effect observed 2 hours after exposure to extract at a concentration of 3.0 mg/mL (Figure 2). The same effect was observed in E. epidermidis after 12 hours of exposure. E. faecalis was more resistant, with a bacteriostatic effect observed 24 hours after exposure to 3.0 mg/mL. The difference in time periods in which acetone extract eliminate different microbial populations can be attributed to factors such as microbial morphology and resistance mechanisms of each microorganism, which can vary, even in strains of same species [31]. E. coli, from Gram-negative group, and S. aureus cc, from Gram-positive group, were strains most susceptible to acetone extract, this effect is significant because these species are cause of a large number of fatal infections worldwide, and they are also among species that have developed a high percentage of resistance to antibiotics such as methicillin, third-generation cephalosporins, ampicillin, clotrimoxazole, and fluoroquinolones [32]. The mechanisms by which this effect occurs remain to be resolved; however, according to chemical composition of acetone extract, which is mainly composed of phenolic compounds such as phenylpropanoids, flavonoids and tannins (Table 3 and Table 4), it is likely that compounds such as those mentioned are responsible for effect. The antibacterial activity of phenolic compounds is due to a combination of mechanisms, such as enzyme inhibition, disruption of cell membrane, induction of oxidative stress, and interference with metabolic processes of microorganisms [33].

Figure 2.Effect of acetone extract of T. diffusa on bacterial death curves in Gram positive strains. The control was tested without extract. Data are represented as the mean ± S.E. (n = 3). In all extract concentrations evaluated, there were statistically significant differences compared to the control.

In antioxidant activity tests, all three extracts of T. diffusa showed a reducing effect on DPPH and ABTS radicals. The methanolic extract showed a greater effect at lowest concentrations (IC 50 = 29.99 ± 1.84 and 106.02 ± 0.81 µg/mL, respectively) (Table 2), this effect is directly related to the content of phenolic compounds, which was higher in the methanolic extract (60.52 mg GAE/g, Figure 3) compared to the acetone and hexane extracts. This relationship has been mentioned in several studies [34]-[36].

Although the methanolic extract showed a reducing effect on DPPH and ABTS radicals, this was greater for DPPH, presenting a lower IC₅₀ value (29.99 ± 1.84 vs 106.02 ± 0.81 µg/mL, respectively), which suggests that there is a difference in the way the compounds contained in extracts react with radicals. In DPPH assay, the radical has a greater affinity for hydrophilic antioxidant components, while ABTS^•+ radical can interact with both hydrophilic and lipophilic antioxidant compounds [37].

In the ferric reduction assay (FRAP), the methanolic extract also was the most effective, with 569.25 ± 6.78 mgET/g of extract, corresponding to 56.93% iron reduction (Fe³⁺ to Fe²⁺) (Table 2), suggesting that the phenolic components in extract have the capacity to chelate iron. This has biological importance because, although iron is an important constituent in the function of hemoproteins, such as hemoglobin and myoglobin, cytochromes in electron transport chain, and other proteins, an excess of iron in the body can be toxic to cells and cause oxidative stress [38].

Table 2.Antioxidant activity of T. diffusa extracts.

Data expressed as mean ± SD. n = 3. DPPH and ABTS significantly different compared to reference standard (quercetin) (*P < 0.001) by one-way ANOVA. Different letters denote significant differences (P < 0.05). mgET/g: equivalent milligrams of Trolox per gram of extract.

The results of iron-reducing antioxidant power of extracts suggest that polyphenols they contain, mainly in methanolic extract, have the property of binding with iron. This reaction is related to presence of catechol and galloyl groups [39] found in polyphenols such as flavonoids and tannins, which are groups of secondary metabolites identified in methanolic extract of T. diffusa in this work (Table 3).

In secondary metabolite groups identification tests, flavonoids and steroids were found in all extracts (Table 3). Acetone and methanol extracts presented similar groups (phenols, flavonoids, tannins and steroids). These secondary metabolites in T. diffusa have been reported in other works, where it is mentioned that it contains flavonoids, terpenes [40], tannins and steroids [41].

Table 3.Groups of secondary metabolites present in T. diffusa extracts.

Cum: coumarins, Phe: phenols, Alk: alkaloids, Flv: flavonoids, Tan: tannins, Gly: glycosides, Str: steroids, Ter: terpenes. √ positive test. – negative test.

The methanolic extract showed the highest concentration of phenols (Figure 3), with 60.52 mgEAG/g, corresponding to 6.05%, while hexane extract had lowest (0.525%). This result is explained by the fact that polar solvents, such as methanol, dissolve equally polar and ionic solutes [42], including phenolic compounds, whose polarity is due to various OH groups in their structure [43].

In this study, methanolic extract, due to its higher phenol concentration, exhibited highest antioxidant activity, demonstrating the direct relationship between phenol content and antioxidant activity, as previously mentioned in several studies [44]. This occurs because phenols can reduce and stabilize free radicals, which are harmful to cells. Their antioxidant mechanism of action consists of the transfer of hydrogen atoms, electrons, and chelation of transition metals [45].

Figure 3.Total phenols of T. diffusa. Data expressed as mean ± SD. n = 3. Different letters denote significant differences (P < 0.05). mgEGA/g: equivalent milligrams of gallic acid per gram of extract.

The phenol groups identified in this work were phenylpropanoids and flavonoids, as two large phenolic subgroups. In HPLC analyzes carried out on extracts, it was obtained that phenylpropanoids were most abundant and diverse, 9 were detected in acetone extract and 6 in methanolic extract (Table 4). While flavonoids, although detected, were found in lower quantities (1 in acetone extract and 3 in methanolic extract). These analyzes were carried out by comparing the ultraviolet absorption patterns of mentioned phenol groups. In both extracts, the abundance of phenylpropanoids was more than 90% (1505.4 mUA = 99.56% phenylpropanoids for acetone extract; 953.2 mUA = 94.18% for methanolic extract).

Table 4.Reverse-phase HPLC of acetone and methanol extracts of T. diffusa (DAD, λ = 254 nm).

RT: retention time, php: phenylpropanoid, flv: flavonoid, λmax: maximum absorbance, mAU: milliabsorbance units.

Phenylpropanoids are formed by an aromatic ring and a propane group [46] and may also have hydroxyl groups. They are part of group of simplest phenolic compounds and are precursors in biosynthesis of flavonoids, stilbenes, and coumarins, which together are among the main groups of secondary plant metabolites with pharmacological effects [47]. Phenylpropanoids and their derivatives have shown a potent effect on bacteria of genera Escherichia, Pseudomonas, Staphylococcus, among others. An antioxidant effect has also been reported, due to their ability to stabilize free radicals, inhibit lipid peroxidation, and chelate metal ions [48], effects that have been corroborated in this work. However, a more specific analysis is still needed to elucidate the precise chemical structure of each of phenylpropanoid and flavonoid constituents in bioactive extracts, corresponding to acetate extract, which was more effective against bacteria, and the methanolic extract, which showed greater antioxidant activity.