Efficient Enrichment of Total Flavonoids and Antibacterial Activity of the Ethyl Acetate Fraction of Croton blanchetianus Baill. (Euphorbiaceae) Leaves

Abstract

Background/Objectives: This study investigated the flavonoid enrichment and antimicrobial activity of the ethyl acetate fraction (EAF) obtained from Croton blanchetianus (Euphorbiaceae) leaves against Staphylococcus aureus, including the methicillin-resistant strains (MRSA) that were isolated, as well as its possible mechanism of action. Methods: Croton blanchetianus leaves were extracted with ethanol:water (50%), then the extract was spray-dried and partitioned (8×) with ethyl acetate. Phytochemical analysis was performed using thin layer chromatography (TLC), while antibacterial activity was conducted using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods. Results: Chemical profiling (TLC) confirmed multiple flavonoid bands and the presence of hyperoside; the total flavonoid content in the EAF reached 25.3% (≈2.28× the spray-dried extract and 6.65× the aqueous fraction). The MIC and MBC assays against S. aureus ATCC 29213 and six clinical isolates showed an MIC of 4–32 μg/mL and an MBC of 16–64 μg/mL for EAF. The combination of EAF with chloramphenicol showed a complete synergistic effect for S. aureus ATCC 29213 and S. aureus UFPEDA 705, a partial effect for S. aureus UFPEDA-659 and S. aureus UFPEDA-671, antagonistic effect for S. aureus UFPEDA 731 and S. aureus UFPEDA 802, and no effect for S. aureus UFPEDA-691. Growth curves indicated time- and concentration-dependent inhibition. Membrane integrity assays revealed K⁺ efflux and release of DNA/RNA and proteins, suggesting bacterial membrane destabilization as a likely mechanism. Conclusions: The flavonoid-rich fraction of C. blanchetianus exhibits potent anti-S. aureus activity, including MRSA. Furthermore, it was observed that EAF has a synergistic effect with chloramphenicol and acts through membrane damage, making it a candidate for a phytoderived adjuvant in antimicrobial therapies.

1. Introduction

Antibiotics have been playing an important role in the treatment of infectious diseases; however, the occurrence of resistant bacteria has been increasing over the years [1,2]. The challenge of antibiotics is met by the diversity of resistance mechanisms, which makes therapy a serious public health problem [3,4].

According to this scenario and the need for new antimicrobial agents, medicinal plants and their derivatives appear as a source of several bioactive molecules with therapeutic potential [5,6]. According to records in the literature, flavonoids stand out for being part of a broad and diverse class of phytochemicals with antimicrobial properties [7]. Several species of the genus Croton have in their constitution a diversity of polyphenols, especially flavonoids. Among these species, Croton blanchetianus Baill., popularly known as “marmeleiro”, an endemic species in the Brazilian Caatinga biome, is used in traditional medicine to treat gastrointestinal disorders such as stomach problems, dysentery, diarrhea, stomach pain, and indigestion [8,9,10].

In a previous study, it was reported that the main components of the ethyl acetate fraction from C. blanchetianus were quercetin-3-O-(2-rhamnosyl) rutinoside, hyperoside, quercetin rutinoside pentoside, and quercetin hexoside deoxyhexoside [11]. Cruz et al. [12] isolated the flavonoid kaempferol 7-O-β-D-(6″-O-cumaroyl)-glucopyranoside from Croton piauhiensis leaves that showed a more synergistic effect against S. aureus. Other Croton species, such as C. argyrophylloides, C. heliotropiifolius, C. hondensis, C. killipianus, and C. smithianus, also showed positive effects against the pathogenic bacteria S. aureus [13,14,15].

Considering the therapeutic potential of the genus Croton and the context of antibiotic resistance, in these studies, we investigated the antimicrobial potential and its possible mechanism in strains of S. aureus from a flavonoid-rich fraction of C. blanchetianus leaves from the Brazilian Caatinga biome.

2. Results and Discussion

2.1. Chemical Analysis

The thin layer chromatography (TLC) analysis was developed by evaluating several modifications in the parameters, such as the mobile phase composition and number of partitioning, until achieving the condition of maximum extraction efficiency. The spray-dried extract (SDE) was partitioned eight times with ethyl acetate, which generated a fraction (EAF) and a residual aqueous fraction (AqF) from C. blanchetianus. With reference to the chromatographic analysis by TLC of samples and standard, the TLC-fingerprints, the chromatogram of the SDE, presented a series of known and unknown bands. In total, the SDE and AqF samples showed nine and five bands, respectively. EAF samples showed 12 bands with greater color intensity (Figure 1).

The analysis of the chromatograms revealed the presence of hyperoside and suggested the presence of cinnamic derivates (blue color bands). Additionally, there are several bands of typical flavonoid, with colors characteristic of the metabolite class after derivatization (yellow, orange, and greenish bands). The fractionation of the SDE provides fractions with typical and enriched profiles in comparison with the starting material (SDE). Thus, the aqueous fraction (AqF) showed the intensification of the cinnamic derivatives, while the EAF presented a significant improvement in the flavonoid’s concentration. In addition, the presence of hyperoside in the SDE and EAF was verified and confirmed, as they each present a band of orange color and an Rf value (0.5) similar to the hyperoside standard.

In order to evaluate and confirm the flavonoid enrichment process, the determination of total flavonoids by UV-Vis spectrophotometry was performed in the SDE and the fractions (EAF and AqF). Thus, the procedure resulted in the enrichment of the EAF when the substances of interest were observed. Thus, the content of total flavonoids in the SDE, EAF, and AqF was 11.1 (0.94%), 25.3 (1.25%), and 3.8% (4.17%), respectively, calculated as rutin. These results demonstrate that EAF has about 2.28× more flavonoid content in relation to SDE and 6.65× more in relation to the aqueous fraction. These data reinforce the efficiency of the enrichment process and indicate that EAF contained the highest content of total flavonoids, implying that EAF was a flavonoid-enriched fraction.

In previous studies, it was possible to identify the presence of flavonoids and other phenolic compounds in EAF using HPLC and LC-MS techniques [11,16]. Quercetin-3-O-(2-rhamnosyl) rutinoside was the main constituent identified in EAF using the LC-MS technique [11]. Other major compounds, such as quercetin 3-O-β-D-galactopyranoside (hyperoside), quercetin rutinoside pentoside, and quercetin hexoside deoxyhexoside, were also identified in this sample. These compounds present in EAF were associated with antinociceptive effects in mice, acting on the opioid and cholinergic systems [11]. According to Oliveira et al. [16], the flavonoid hyperoside was identified in EAF by the HPLC-DAD technique, corroborating our TLC results (Figure 1). Using the HPLC technique, Dantas et al. [17] identified rutin as the major compound in the spray-dried extract of C. blanchetianus, with a content of 0.86 mg/g (1.82%). This extract showed gastroprotective and antifungal potential in in vivo and in vitro models, respectively. Taken together, these data reinforce that C. blanchetianus is a plant rich in flavonoids and stands out as a promising source for the treatment of various diseases.

2.2. Antibacterial Activity

To our knowledge, this is the first report of a flavonoid-rich fraction from C. blanchetianus leaves with antimicrobial activity. Therefore, the antimicrobial potentials and the synergistic effect with chloramphenicol from EAF were evaluated against antibiotic-resistant clinical isolates of S. aureus. A reference strain of S. aureus (ATCC 29213), five clinical isolates of S. aureus MRSA (UFPEDA-659, UFPEDA-671, UFPEDA-705, UFPEDA-731, and UFPEDA-802), and one isolate of S. aureus MSSA (UFPEDA-691) were used according to the resistance profile described in Table S1—Supplementary Material. Staphylococcus aureus is the most prevalent bacterial species in human infections, presenting high rates of morbidity and mortality, mainly due to its virulence factors and its ability to acquire antibiotic resistance [18]. The results of determining the susceptibility of clinical isolates of S. aureus to EAF are shown in

Table 1. Minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of the ethyl acetate fraction of Croton blanchetianus (EAF) and chloramphenicol against different strains of Staphylococcus aureus.

Strains	EAF		Chloramphenicol
	MIC (µg/mL)	MBC (µg/mL)	MIC (µg/mL)	MBC (µg/mL)
S. aureus ATCC 29213	4	16	8	32
S. aureus UFPEDA 659	32	64	4	32
S. aureus UFPEDA 671	16	64	32	64
S. aureus UFPEDA 691	16	64	64	128
S. aureus UFPEDA 705	16	32	8	32
S. aureus UFPEDA 731	32	64	128	256
S. aureus UFPEDA 802	32	64	256	512

EAF: ethyl acetate fraction of Croton blanchetianus; MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration. The experiments were performed in triplicate.

. The MIC against S. aureus ranged from 4 to 256 μg/mL and 4 to 32 μg/mL for chloramphenicol and EAF, respectively. In the case of MBC, EAF presented values of 16 to 64 μg/mL, while chloramphenicol obtained values of 32 to 512 μg/mL. EAF showed high activity against S. aureus, including MRSA. This activity can be attributed to the high concentration of the glycosylated flavonoid present. Studies have shown that extracts rich in glycosylated flavonoids act by altering the plasma membrane, causing structural changes and leading to bacterial death [19,20]. Furthermore, the antibacterial activities of many flavonoids use mechanisms different from those of conventional drugs, so flavonoid-rich extracts may be more effective than antibiotics [21].

The antimicrobial effect of EAF was evaluated by the growth curve assay and is presented in Figure 2A. The negative control group showed a rapid increase in the number of bacteria, unlike what was observed in the groups treated with different concentrations of EAF, where growth was suppressed. Comparing the different treatments, it is possible to observe that at the 1× MIC concentration, growth was lower than the control throughout the time, while the 2× MIC concentration proved to be more effective than 1× MIC, especially in the first 8 h. The dynamic interaction of EAF with S. aureus demonstrated that the antimicrobial effect of EAF is time- and concentration-dependent, providing relevant information about the antibacterial action of EAF on the exponential growth process of S. aureus. There are already reports of the involvement of Croton species in antimicrobial activity, with an effect dependent on the exposure time or the concentration used [22,23]. Flavonoids have been shown to interact with the bacterial plasma membrane [24]. Therefore, the flavonoid-rich fraction of C. blanchetianus was evaluated for its ability to cause membrane damage. For this purpose, S. aureus was subjected to assays analyzing the extravasation of potassium ions, genetic material, and proteins.

Potassium ions, due to their low molecular weight, are the first to be released in the event of membrane damage [25]. When S. aureus was exposed to different concentrations of EAF, potassium ions were released into the medium. Figure 2B shows that during the first 30 min, there was no difference between the control and 1× MIC groups (p > 0.05). However, a marked increase in K⁺ ion release was observed in the 2× MIC group, significantly different (p < 0.05) compared to the control and 1× MIC groups. Throughout the period, the release was continuous, so that there was a difference between the three groups (control, 1× MIC, and 2× MIC) up to 120 min.

Another method used to assess the leakage of cytoplasmic material resulting from membrane damage in S. aureus is through the quantification of nucleic acids released into the medium, considered a sensitive indicator of membrane damage [26]. Figure 2C shows that there was no significant difference between the absorbances of the cultures of the 1× CIM and 2× CIM groups in the first 4 h of exposure (0.18 ± 0.03–0.36 ± 0.02 and 0.23 ± 0.01–0.39 ± 0.02 nm, respectively). The control group presented low OD260 at all analysis times (0.02–0.13 nm), being significantly different in relation to the 1× CIM and 2× CIM groups. Thus, the groups treated with EAF promoted the release of cellular constituents with a significant increase in concentration in relation to time, and an OD260 reached a maximum point at 24 h, with results of 0.51 ± 0.02 and 0.68 ± 0.03 nm for 1× CIM and 2× CIM, respectively.

Protein leakage analysis was used to assess the extent of damage to bacterial membrane integrity [27]. As shown in Figure 2D, EAF can cause protein leakage through the S. aureus membrane at the tested concentrations. Initially (time 1 h), bacterial protein leakage in the control was 0.33 μg/mL, while in the EAF-treated groups it was 0.84 and 1.00 μg/mL at 1× MIC and 2× MIC, respectively, showing no statistical difference between the EAF-treated groups (p > 0.05) and different from the control (p < 0.05). Protein leakage from S. aureus cells treated with EAF increased as treatment time increased, with a significant concentration-dependent difference between the three groups (p < 0.05) observed between 2 and 12 h of treatment.

The plasma membrane is extremely important for the vital processes of microorganisms, and even minor damage to this structure impairs its functions, leading to bacterial death [28]. Therefore, in the present study, different methods were used to evaluate the interaction of EAF with the bacterial membrane. The results indicate that EAF disrupted the S. aureus membrane, resulting in increased potassium ion efflux and the release of nucleic acids and proteins.

2.3. Synergistic Effect

The results of the synergistic effects of EAF with chloramphenicol are presented in

Table 2. Synergistic interaction between ethyl acetate fraction of Croton blanchetianus (EAF) and chloramphenicol against Staphylococcus aureus.

Strains	FIC		ΣFIC	Effect
	EAF	CLO
S. aureus ATCC 29213	0.125	0.06	0.18	Total synergism
S. aureus UFPEDA 659	0.5	0.06	0.56	Partial synergism
S. aureus UFPEDA 671	0.5	0.25	0.75	Partial synergism
S. aureus UFPEDA 691	0.5	0.5	1	No synergism
S. aureus UFPEDA 705	0.25	0.12	0.37	Total synergism
S. aureus UFPEDA 731	1	1	2	Antagonist
S. aureus UFPEDA 802	1	2	3	Antagonist

EAF: ethyl acetate fraction of Croton blanchetianus; CLO: chloramphenicol; FIC: fraction inhibitory concentration index. The experiments were performed in triplicate.

, demonstrating that the combination resulted in a 2- to 64-fold reduction in the concentration of chloramphenicol and/or EAF. The interaction showed a complete synergistic effect for S. aureus ATCC 29213 and UFPEDA 705, a partial effect for S. aureus UFPEDA-659 and S. aureus UFPEDA-671, an antagonistic effect for S. aureus UFPEDA 731 and S. aureus UFPEDA 802, and no effect for S. aureus UFPEDA-691. An alternative to combat bacterial resistance to antibiotics is combination therapy, where there is interaction between antibiotics and plant extracts [12]. The present study demonstrated that when EAF was combined with chloramphenicol, there was a decrease in MIC concentrations, demonstrating that the combination could be used to inhibit the growth of S. aureus, including MRSA. Plants rich in flavonoids have the ability to modify bacterial resistance, making them more susceptible to antibiotic action [29]. This modification, influenced by flavonoids, may involve the inhibition of antibiotic-degrading enzymes [30], efflux pumps [31], or alteration of membrane permeability [32].

Croton species are known to demonstrate antimicrobial activity and also modulate antibiotic activity. Obey et al. [33] obtained activity against several pathogenic microorganisms using extracts of Croton macrostachyus. Cruz et al. [12] isolated a flavonoid from Croton piauhiensis leaves that was effective in antimicrobial treatment against S. aureus and E. coli strains. The essential oil from Croton limae leaves showed an MIC of 512 μg/mL and synergism with amikacin against S. aureus strains [34]. Studies involving extracts, fractions, and compounds isolated from plants have demonstrated antimicrobial potential; however, few studies provide information on the duration of action of these compounds on bacteria, as well as their mechanism of action [35,36].

3. Materials and Methods

3.1. Plant Material

Leaves of C. blanchetianus were collected in April 2019, at the municipality of Santa Terezinha, Pernambuco (07°22′40″ S, 37°28′48″ W). The access was recorded (AEC4F6E) in the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen—Brazil), and a voucher specimen (number 93062) was deposited at the herbarium “Dárdano de Andrade Lima” in the Instituto Agronômico de Pernambuco (IPA). After that, the leaves were stabilized at 40 °C for 7 days using a circulating air oven (Luca-82/480, Lucadema^®, São José do Rio Preto, SP, Brazil) and ground in a Willye-type mill (TE-680, Tecnal^®, São Paulo, SP, Brasil).

3.2. Preparation of the Flavonoid-Rich Fraction of Croton blanchetianus

The ground leaves of C. blanchetianus were extracted by turbo extraction using a hydroethanolic solution (50%, v/v) at a ratio of 10% (w/v). The extract was dried using a lab-scale spray dryer (MSD 1.0, Labmaq^®, Ribeirão Preto, SP, Brazil) under the following preliminary conditions: inlet temperature = 140 °C, feed flow = 0.6 L/h, and air flow = 1.65 m³/min; air flow = 40 L/min to obtain the spray-dried extract (SDE). The dry residue content was determined by an infrared balance (series ID—V.18, Marte^®, São Paulo, SP, Brazil) at a temperature of 130 °C and used for the performance of the operation. The dry residue obtained was 2.84% and the operating yield reached was 13.55% for the spray-dried extract (drying volume 600 mL).

To obtain the flavonoid-rich fraction, the SDE was dissolved in water (1 g/10 mL) and partitioned eight times with 10 mL of ethyl acetate. The final aqueous residue and the ethyl acetate fraction were concentrated in an evaporator (50 °C), frozen, and lyophilized (L101, Liotop^®, São Carlos, SP, Brazil) to obtain the EAF and the AqF [37,38].

3.3. Phytochemical Analysis

To evaluate the efficiency of the process of enrichment, extract and fractions were submitted to TLC tests and total flavonoid content.

3.3.1. Analysis by Thin Layer Chromatography (TLC)

The SDE and the fractions (AqF and EAF) were prepared at a concentration of 1 mg/mL in methanol P.A. The standard used was hyperoside at a concentration of 0.5 mg/mL in methanol P.A.

The fingerprint was obtained using F₂₅₄ silica gel plates (10–12 μm particles) for TLC (Macherey-Nagel^®, Düren, Germany). The application was carried out with semiautomatic equipment (Linomat V, Camag^®, Muttenz, Switzerland) controlled by the WinCATS^® software, version 1.4.8.2031. Aliquots of 30 μL of the samples and 15 μL of the hyperoside standard were applied in bands with a width of 10 mm and a space between the bands of 5 mm. The chromatogram was developed in a double vertical glass chamber (20 cm × 10, Camag^®) after saturation for 30 min, with the mobile phase consisting of ethyl acetate–formic acid–water (90:5:5, v/v/v). At the end, the plate was derivatized with natural reagent A and evaluated under UV at 366 nm. The observation of the plate under UV light and image acquisition was performed using a MultiDoc-It^® 125 TLC imaging system (model 125), with a Canon^® camera (Rebel T3, EOS 1100D) and UVP^® software, version 1.0.6.

3.3.2. Total Flavonoid Content (TFC)

To evaluate the total flavonoid content, 0.25 g of SDE, AqF, and EAF were weighed in a 25 mL volumetric flask. Then, a 4 mL aliquot of each solution was transferred to a 25 mL volumetric flask, 2 mL of aluminum chloride (AlCl₃; 5%, w/v, in ethanol) was added, and its volume was checked with 50% ethanol (v/v). After 20 min, the absorbance was measured at 410 nm in a spectrophotometer (Evolution 60S, Thermo Fisher Scientific^®, Waltham, MA, USA) using the sample as a blank solution without adding AlCl₃. The total flavonoid content was calculated in % of rutin, according to the equation

$$\mathrm{T}\mathrm{F}\mathrm{C}={\displaystyle \frac{\mathrm{A}\times \mathrm{D}\mathrm{F}}{\mathrm{w}\times {\mathrm{E}}_{1\mathrm{c}\mathrm{m}}^{1\mathrm{\%}}}}$$

where TFC = total flavonoid content, expressed in grams of the standard per 100 g of the residue; A = sample absorbance; DF = dilution factor; w = mass of extract or fraction (g); and ${\mathrm{E}}_{1\mathrm{c}\mathrm{m}}^{1\mathrm{\%}}$ = specific absorption for the rutin-AlCl₃ complex.

3.4. Evaluation of Antibacterial Activity

3.4.1. Determination of Minimal Inhibitory (MIC) and Bactericidal (MBC) Concentrations

The antibacterial activity of EAF was determined by the methods of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in a 96-well plate described by İşçan et al. [39]. For this, Staphylococcus aureus strains obtained from the collection of microorganisms from the Federal University of Pernambuco were S. aureus (ATCC 29213) and six antibiotic-resistant isolates (UFPEDA-659, UFPEDA-671, UFPEDA-691, UFPEDA-705, UFPEDA-731, and UFPEDA-802) (Table S1—Supplementary Material). Bacterial cells were adjusted to a final concentration of 5 × 10⁵ CFU/mL using the McFarland scale. Thus, the Mueller–Hinton growth medium was used and added to each well, followed by a solution of bacterial cells and, finally, increasing concentrations (1–1024 µg/mL) of EAF or chloramphenicol (reference antibiotic). The growth control wells (100%) contained only the treatment-free bacterial inoculum. After plating, an incubation for 24 h at 37 °C followed. OD₆₀₀ was measured at zero time and at 24 h using a microplate reader. MIC was defined as the lowest concentration capable of promoting a reduction in optical density, in comparison with the 100% growth control. To determine the MBC, an aliquot (10 μL) of the suspension from each well was transferred to the Mueller–Hinton agar plates and incubated for 24 h at 37 °C. The MBC was determined as the lowest concentration of EAF capable of preventing bacterial growth. Each trial was performed in triplicate, and three independent experiments were performed.

3.4.2. Growth Curve

The bactericidal effects of EAF were assessed using the growth curve test method described by Zhou et al. [40]. Strains of S. aureus (5 × 10⁵ CFU/mL) were incubated with 1× MIC, 2× MIC of EAF, or DMSO (1%) at 37 °C with shaking at 180 rpm at different times (0, 1, 2, 4, 8, 12, or 24 h). After each time interval, the supernatant was analyzed on a UV-Vis spectrophotometer (OD₆₀₀), and the growth curve was determined. Three independent tests were performed in triplicate.

3.4.3. Integrity of Cell Membrane

Efflux of Potassium Ions Through the Membrane

Previously, suspensions of S. aureus were incubated in brain heart infusion broth (BHI) at 37 °C for 12 h. Then, the cells were washed three times (4500 rpm, 15 min) and resuspended in deionized water to obtain a concentration of 1 × 10⁵ CFU. The bacterial cells were immediately treated with EAF at 1× MIC, 2× MIC, or DMSO (1%) at 37 °C at different times (30, 60, 90, and 120 min) and centrifuged at 13,400 rpm for 15 min [41]. The determination of the amount of leakage of K⁺ was measured in a selective ion analyzer (9180 Electrolyte Analyzer (Roche)) in triplicate, and the results were expressed in mEq/L.

Leakage of DNA and RNA Through the Membrane

Initially, the bacteria were incubated in nutrient broth (NB) at 37 °C for 12 h. The cells were adjusted to 0.5 on the McFarland scale and treated with EAF (1× MIC and 2× MIC) or DMSO (1%, negative control) and incubated at 37 °C for 1, 2, 4, 8, 12, or 24 h, followed by centrifugation (13,400 rpm for 15 min). Then, the supernatant was filtered through a 0.22 μm Millipore membrane, and the DNA and RNA released was measured at OD₂₆₀ nm [42].

Leakage of Proteins Through the Membrane

The method of releasing proteins in bacteria was as described by Meng et al. [33]. Briefly, S. aureus (0.5 McFarland scale) was treated in different concentrations of EAF (1× MIC, 2× MIC) or 1% DMSO (negative control) and incubated at 37 °C for 1, 2, 4, 8, 12, and 24 h. Then, S. aureus was centrifuged (10,000× g for 5 min at 4 °C). The supernatant was collected, and the concentration of released proteins OD₅₉₅ was measured, following the methodology of Bradford [43].

3.4.4. Synergism Assay

In a 96-well plate, 20 μL of cell suspension (10⁵ CFU/mL), 80 μL of Mueller–Hinton broth, 50 μL of chloramphenicol, and 50 μL of EAF in different concentrations (4× MIC initial solution) were incubated at 37 °C for 24 h. The evaluation of the interaction between the different treatments was measured by the fraction inhibitory concentration index (ΣFIC) described by Nafis et al. [42], with the following formula:

$$\mathsf{\Sigma }\mathrm{F}\mathrm{I}\mathrm{C}={\displaystyle \frac{\mathrm{M}\mathrm{I}\mathrm{C} \mathrm{o}\mathrm{f} \mathrm{E}\mathrm{A}\mathrm{F} \mathrm{i}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{m}\mathrm{b}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}{\mathrm{M}\mathrm{I}\mathrm{C} \mathrm{o}\mathrm{f} \mathrm{E}\mathrm{A}\mathrm{F} \mathrm{a}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{e}}}+{\displaystyle \frac{\mathrm{M}\mathrm{I}\mathrm{C} \mathrm{o}\mathrm{f} \mathrm{C}\mathrm{l}\mathrm{o} \mathrm{i}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{m}\mathrm{b}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}{\mathrm{M}\mathrm{I}\mathrm{C} \mathrm{o}\mathrm{f} \mathrm{C}\mathrm{l}\mathrm{o} \mathrm{a}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{e}}}$$

where MIC: minimum inhibitory concentration; EAF: ethyl acetate fraction; Clo: chloramphenicol.

The combinations were classified as total synergism (FIC ≤ 0.5), partial synergism (0.5 < FIC ≤ 0.75), no effect (0.75 < FIC ≤ 2), or antagonist (FIC > 2) [44].

3.5. Statistical Analysis

Experimental results are presented as mean ± standard deviation (SD) and analyzed using GraphPad Prism^® software version 8.0. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey’s test. All in vitro tests were performed at least three times in triplicate. Differences were considered statistically significant at a probability of less than 5% (p < 0.05).

4. Conclusions

Our results revealed that a flavonoid-rich fraction from Croton blanchetianus exhibited antimicrobial activity against resistant S. aureus, promoting reduced bacterial growth. Furthermore, EAF exhibited a synergistic effect with chloramphenicol and possibly acts by destabilizing the bacterial membrane, promoting the release of intracellular contents and bacterial death. This contributes to scientific knowledge about C. blanchetianus in the search for new plant-based products with antimicrobial potential.