Sample procurement and extracts preparation

About 100 A. sativum adult plants were obtained from a local farmer in Ksar Mezouar (36°46'60" N et 9°19'60" E), Beja Governorate, in North Tunisia in early May. In Tunisia, no permits are required to conduct scientific research on phyto resources within the country. However, permission is mandatory if samples need to be sent abroad. The plant materials were identified based on Tunisian flora literature and morphological characteristics of leaves and bulbs described in descriptors for Allium spp. [9,28,29]. The garlic bulb was washed, peeled, and crushed using a mortar. Bioactive compounds were then extracted via maceration using solvents of varying polarities (water, methanol, ethyl acetate, and hexane), following the methodology outlined by Shetty [30]. Approximately 100 g of crushed garlic was extracted using 400 mL of each solvent using an automatic shaker for 24 hours at room temperature. This approach was designed to selectively isolate compounds based on their differential solubilities in solvents.

The mixture was then filtered using a Buchner funnel, followed by centrifugation for 10 min at 3000 rpm. The upper phase was then evaporated to dryness using a rotary evaporator (Heidolph Instruments, Schwabach, Germany). The resulting dry residues were reconstituted in methanol and adjusted to a concentration of 40 mg/mL. The aqueous extract macerate was filtered through filter paper, dried in SpeedVac vacuum concentrators SPD1030 (Thermo Scientific, Lissieu, France), and stored at +4°C until use. All samples were processed in triplicate.

Chemicals and standard solutions

Extraction solvents: methanol, ethyl acetate, and hexane were of analytical grade, and LC-MS-grade methanol, formic, and acetic acids were obtained from VWR International (Rosny-sous-Bois, France). Analytical standard solutions of phenolic acids, flavonoids, and vitamins (purity ≥ 97%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Purified water was obtained using a Multi Q system from Millipore (Darmstadt, Germany).

Fungal strains and human cancer cell origins and culture conditions

The fungal strains tested for antifungal activities were Aspergillus flavus strain SRRC 1000A and Aspergillus niger strain CBS 513, hereafter referred to as A. flavus and A. niger, respectively. These strains were previously isolated from food samples, identified using conventional methods and ITS1-5.8S-ITS2 region sequencing of the nuclear ribosomal DNA, and stored in the laboratory of Toxicology, Centre Mahmoud Yaakoub of Urgent Medical Assistance, Tunis, Tunisia. Antifungal activities were investigated using a Dichloran Rose Bengal Chloramphenicol (DRBC) medium (Scharlau, Barcelona, Spain).

To study the extracts’ anticancer activities, two cell lines were used: U266 human multiple myeloma cells, generously provided by Professor Brigitte Sola [31], and MDA-MB-231, a human breast cancer cell line purchased from ATCC (ATCC HTB-26) [32]. Both cell lines were maintained under standard culture conditions (at 37 °C and 5% CO₂) using RPMI 1640 and DMEM media, respectively. Cell culture media were supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin 0.1 μg/μL/streptomycin 0.1 μg/μL solution, obtained from PAN-Biotech (Aidenbach, Germany).

LC-MS analysis

The quantitative analysis of phenolic acids, flavonoids, water-and fat-soluble vitamins across diverse fractions of A. sativum was conducted employing an 8020 LC-MS system including an LC-20AD XR binary pump system, SIL-20 AC XR autosampler, CTO-20 AC column oven and quadrupole mass spectrometer equipped with an electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) modules and piloted by LabSolutions software (Shimadzu, Kyoto, Japan). Previously validated methods were applied [33]. Chromatograms and mass spectra for single-component standard solutions were employed for analyte identification, based on their relative retention times and specific molecular ions. Calibration curves were established and validated prior to sample analysis using freshly prepared calibration solutions and were employed for quantitative determination. The biomolecule contents in the analyzed extracts are reported as mean values ± standard deviation in mg/g of garlic fresh weight (fw).

Phenolic compound LC-MS identification and quantification.

The quantification of 15 phenolic acids and 15 flavonoids was carried out on A. sativum extracts. A reverse-phase C18 column (100 x 2.1 mm x 3 µm), maintained at a consistent temperature of 40°C, was used for chromatographic separation at a 0.4 mL/min flow rate. The mobile phase consisted of an aqueous solution of 20 mmol ammonium formate and 0.1% formic acid (A) and a solution of 0.1% formic acid in methanol (B). The separation was performed using an elution gradient optimized as follows: 10% −100% B for 45 min., 100% B for 45−50 min., and return to the initial condition (10% B) over 5 min. The mass spectrometer operated under the following conditions: Nitrogen served as the nebulizing gas at a flow rate of 1.2 L/min, with a drying line temperature maintained at 250°C. The capillary voltage was set at −3.5 V, while the detector voltage was set at 1.1 V. Mass spectra were collected in negative ion mode across a mass-to-charge ratio (m/z) range of 50–1200. The sought molecular ions used for SIM are described in Table 1.

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Table 1. Retention time and sought molecular Ions (M/Z) used for SIM of explored polyphenolic compounds.

https://doi.org/10.1371/journal.pone.0325227.t001

Water-soluble vitamins LC-MS identification and quantification.

Chromatographic separation of nine water-soluble vitamins was executed utilizing a mobile phase composed of an aqueous mixture of 20 mmol ammonium formate and 0.1% aqueous formic acid (A) and 0.1% formic acid in methanol (B). The linear gradient profile (A: B) began with 2% B and was maintained over 2 min. Subsequently, it linearly decreased to 55% B for 13 min, followed by a 2-minute hold at 55% B. Finally, it further decreased to 100% B during the next 3 min. The flow rate was set to 0.4 mL/min, with a total runtime of 20 min. The column temperature was maintained at 40°C, and the injection volume was 5 µL.

The mass detector was operated in negative ion conditions in the SIM mode, scanning the range of 50–1200 m/z. The specific molecular ions (m/z) applied for SIM are summarized in Table 2. The ESI source parameters were a capillary voltage of −3.5 V, and a detector voltage of 1.2 V, desolvatation line and heat block temperatures of 250°C and 500°C, respectively, a nebulizing gas flow of 1.5 L/min, and a drying gas flow of 15 L/min.

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Table 2. Explored water- and fat-soluble vitamins.

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Determination of antioxidant activity

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was employed to evaluate the antioxidant potential, as described by Kirby and Schmidt [34]. Briefly, 1 mL of extracts at variable concentrations (2.5, 5, 10, 20, and 40 mg/mL) was added to 1 mL of a DPPH solution (0.2 mM in ethanol) as the free radical source and incubated for 30 min at room temperature. The reduction of DPPH by the samples was measured at 517 nm using a PG Instruments T60 spectrophotometer (Lutterworth, UK). The scavenging activity was calculated as a percentage of inhibition (PI) by comparing it to the negative control and using the formula: , where A₀ represents the absorbance of the control and A₁ represents the absorbance of the test sample. L-ascorbic acid was used as the positive control, the IC₅₀ was evaluated for each extract and the assay was performed in triplicate.

Determination of antifungal activities

With minor adjustments, the disc diffusion method, as reported by Jorgensen and Turnidge [35], was employed to evaluate the antifungal activity of A. sativum extracts. Briefly, a paper disk was loaded with 15 µL of various extract concentrations (2.5, 5, 10, 20, and 40 mg/mL) in 2% DMSO before being placed on agar plates seeded with the tested fungal strain. Paper discs soaked with 2% DMSO alone were included as controls in each plate. Plates were then incubated for 48–72 h at 25°C. Negative control plates contained disks without plant extract, and other plates with disks saturated with 15 μl of carbendazim solution (10 mg/mL) served as a positive control was also prepared and incubated.

Antifungal activity was assessed by measuring the inhibition-zone diameter around the disks in cm, with each experiment conducted in triplicate. Antifungal efficacy was presented as the mean inhibition zone (cm) ± standard deviation. The minimum inhibitory concentration (MIC) of the studied extracts was determined through broth macro-dilution in 1 mL standard test tubes, following the method described by Arendrup and colleagues with slight modifications [36,37].

Determination of cytotoxicity activities

Cell viability was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay obtained from Sigma-Aldrich, following a validated protocol [38]. In brief, U266 and MDA-MB-231 cells were exposed to varying concentrations (15.56, 31.125, 62.25, 125, 250, and 500 μg/mL) of distinct solvent extracts for either 24 or 48 h. At the end of the experiment, 20 μL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for 4 h at 37 °C. The resultant purple/blue MTT formazan precipitate was dissolved in 100 μL of 10% DMSO. Absorbance was measured at 490 nm using an ELISA plate reader (Model EXL800; BioTek, USA). The growth inhibition percentage was calculated using the formula: . Experiments were performed in triplicate, and all values are presented as mean ± SD. The obtained results were also used to determine the IC50 values.

Statistical analysis

Each experiment was conducted with three independent analyses to ensure the robustness and reliability of the results. Statistical analysis was conducted using SPSS version 20 and GraphPad Prism 5 software (for MTT assay). From this analysis, mean values and standard deviations (SD) were calculated. Statistical significance between the results was assessed using the student’s t-test with a probability threshold of p < 0.05, and correlation studies between the biomolecule levels and the antioxidant, antifungal, and antiproliferative activities were tested based on Pearson’s and Spearman’s correlation coefficients.

Yield indices from various extractions

Following the application of various individual solvent-based extraction methods, the total yield rate of each extract was determined. The calculation results revealed that the aqueous extracts exhibited the highest yield rate (5.80 ± 3.11%). Similarly, the yield rate of the methanolic extract was notably high, reaching 5.31 ± 2.24%. Conversely, ethyl acetate and hexane extracts displayed the lowest extraction rates, 0.42 ± 0.31% and 0.14 ± 0.08%, respectively. Aqueous and methanolic extracts demonstrated substantially higher yield rates compared to ethyl acetate and hexane extracts. This suggests that the choice of solvent significantly impacts the efficiency of the extraction process, potentially influencing the overall composition and bioactivity of the obtained extracts. The selection of hexane, ethyl acetate, methanol, and water as extraction solvents is guided by their polarity and affinity for specific bioactive compounds in A. sativum. Each solvent targets compounds with varying solubilities. Water and methanol efficiently extract hydrophilic and polar compounds like polyphenols and vitamins, yielding higher amounts, while ethyl acetate and hexane target moderately polar and lipophilic compounds (e.g., flavonoids, terpenoids, and fat-soluble vitamins) with lower yields. The Solvent choice is critical for optimizing extraction efficiency and composition [39–41].

Phenolic acid content in samples

Table 3 presents the LC-MS analysis of sixteen phenolic acids in garlic samples extracted using four different solvents. The varied phenolic acid content across extracts can be attributed to compound responses to distinct extraction conditions. Quantitatively, the aqueous extract exhibited the highest content (96.25 mg/kg fw), followed by methanolic and ethyl acetate extracts (48.10 and 10.36 mg/kg fw), while hexane extract had the lowest content (0.82 mg/kg fw). Significant differences (p < 0.05) were observed between all extract types. The aqueous extract is the most promising for applications requiring high polyphenol content, such as those leveraging antioxidant properties. Previous studies conducted by Chakki and collaborators on Tunisian A. sativum collected in autumn from a different region revealed interesting findings regarding phenolic content. Results showed that the 70% methanolic extract contained a total phenolic content of 10.6 mg GA/g, while the 80% methanolic extract, within 72 h of incubation, yielded 5 mg GA/g [22]. It is well known that the composition of garlic bulbs is strongly influenced by soil characteristics, broad climatic conditions, and weather variations. Additionally, differences in experimental protocols and conditions may further explain the observed discrepancies [42].

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Table 3. Phenolic acids contents (mg/kg fw) in the various A. sativum extracts.

https://doi.org/10.1371/journal.pone.0325227.t003

Qualitative analysis demonstrated that the methanol extraction recovered seven out of the sixteen phenolic acids. Water and ethyl acetate extraction were also effective, retrieving four compounds each. Notably, trans-ferulic acid was the most abundant phenolic acid, found in aqueous and ethyl acetate extracts (92.18 and 7.52 mg/kg fw, respectively), followed by syringic acid, exclusively detected in the methanol extract (17.27 mg/kg fw). Interestingly, syringic acid and p-hydroxybenzoic acid derivatives were most abundant in the methanolic extract, followed in descending order by ethanol, acetone, and hexane during the extraction process from two Egyptian garlic varieties [43]. Quinic acid (16.52 mg/kg fw in methanol extract) and its derivatives were identified, with varying concentrations in methanol, aqueous, and ethyl acetate extracts. However, according to the literature, quinic acid has rarely been reported in studies on garlic bulbs, where another metabolite, chlorogenic acid, is more commonly detected. For instance, Nagella et al. identified chlorogenic acid in three out of five aqueous extracts of garlic cultivated in different locations in Korea [44]. Interestingly, Chahbani and colleagues reported that quinic acid was the predominant phenolic acid in all dried samples (772.02–1026.56 µg/g extract) from Tunisian garlic leaves [45]. Moreover, p-coumaric acid was also identified in methanol, water, and ethyl acetate extracts, with concentrations of 5.54, 2.9, and 1.9 mg/kg fw, respectively. According to Szychowski and collaborators, the analysis of nine aqueous extracts from raw garlic grown in diverse regions-including Poland, Spain, China, Portugal, Burma, Thailand, and Uzbekistan- revealed the presence of p-coumaric acid in all cultivars, with concentrations ranging from 1 to 3.03 μg/g of raw garlic [46]. Contrary to our findings, where gallic acid was not detected, their study reported the presence of this compound in all nine garlic varieties. Methanol proved particularly effective for extracting caffeic acid (2.33 mg/kg fw compared to 0.63 mg/kg fw in ethyl acetate). For instance, Nagella et al. identified this compound in five aqueous extracts of Korean garlic [44]. Additionally, caffeic acid has been described as one of the major phenolic acids in ten Spanish garlic varieties from different regions, with an average concentration of 2.9 mg/kg [47].

Flavonoid content in extracts

The LC-MS analysis of flavonoids in the four types of A. sativum extracts was conducted and the outcomes are displayed in Table 4. Methanol proved to be the most efficient solvent, exhibiting the highest recovered flavonoid concentration (424.87 mg/kg fw).

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Table 4. Flavonoids contents (mg/kg fw) in the various A. sativum extracts.

https://doi.org/10.1371/journal.pone.0325227.t004

The quantity of flavonoids detected in this extract was nearly nine times higher than the phenolic content, resulting in a total polyphenol content of 473 mg/kg fw. This finding establishes methanol as the most effective solvent for polyphenol extraction, followed by the aqueous extract, which yielded approximately 175 mg/kg fw. These results are consistent with literature [44,48], such as the findings reported by Chakki and colleagues, who identified 59.6 QE mg/100 g of flavonoids in the 70% methanolic extract of Tunisian garlic (compared to 10.6 GAE mg/100 g of phenolic content) versus 13.2 QE mg/100 g in the ethanolic extract (compared to 43.6 mg GAE/100g of phenolic content). Similarly, Elhafez’s work highlighted methanol’s superiority in extracting the highest total polyphenol content, estimated at 402.64 and 383.90 µg/g for two Egyptian garlic varieties, outperforming both acetone and hexane, with the latter showing the weakest extraction efficiency. [43]. In contrast, Sergiova et al. reported significantly higher total phenolic content values, ranging from 797.11 to 1183.98 mg GAE/kg dm (dry material), and total flavonoid content ranging from 15.96 to 28.18 mg CE/kg dm [49]. Interestingly, a recent Tunisian study analyzing garlic leaves from southern Tunisia via LC–ESI–MS revealed flavonoids as the dominant category among identified compounds, representing 63% in fresh samples and up to 89.40% in samples dried at 40 °C [45]. In our study, Cirsiliol was identified as the predominant flavonoid in all four extracts, with concentrations of 417.26, 77.7, 10.63, and 0.78 mg/kg fw, for methanolic, aqueous, ethyl acetate and hexane extracts, respectively. Notably, this compound was also identified in Tunisian garlic leaves in recent work by Chahbani et al., further supporting our findings [45].

Qualitatively, the methanolic extract recovered the highest number of flavonoid compounds (six), followed by the ethyl acetate extract (five). These two extracts notably facilitated the identification of cirsilineol and apigenin-7-O-glucoside. Although luteolin was detected in methanolic, aqueous, and ethyl acetate extracts, its concentration remained relatively low (1.69 mg/kg fw vs. 0.89 and 0.04 mg/kg fw, respectively). Additionally, quercetin was exclusively detected in trace amounts in the ethyl acetate extract, whereas rutin and apigenin were detected in the methanolic extract. The flavonoids detected in this study were also identified in the leaves of Tunisian garlic, albeit in varying amounts, as reported by Chahbani et al. [45]. Such differences may be attributed to geographical variations, climate, and harvest season. Moreover, some flavonoids observed in other studies were not detected in our extracts. For instance, Elhafez et al. reported catechin and epicatechin as prominent flavonoids in methanolic extracts of Egyptian garlic [43]. These discrepancies could stem from differences in genetic makeup, environmental conditions (notably climatic variations and drought observed in recent years), solvent choice, and extraction methods [42,45]. Additionally, factors such as plant maturity at harvest and post-harvest handling likely influenced the flavonoid profile, contributing to the observed variations [42,50].

Water-soluble vitamins content

The results of water-soluble vitamin quantification in extracts from different fractions of A. sativum revealed significant variations as presented in Table 5. In terms of total vitamins, extracts showed overall similar concentrations, slightly higher in methanolic and aqueous extracts (36.42 mg/ Kg), emphasizing the diversity of vitamin profiles in garlic extracts. Among the scrutinized vitamins, ascorbic acid and pyridoxine were the most relevant in all extracts. Vitamin C concentrations were high in methanolic and aqueous extracts, while hexane and ethyl acetate extracts showed lower concentrations. Similarly, pyridoxine concentrations were significantly higher in ethyl acetate, and aqueous extracts compared to hexane and methanolic extracts. In contrast, thiamine was not detected in any extracts, and nicotinamide was measurable at the lowest levels. These results align with previous work, which reported higher concentrations of vitamin C, ranging from 9.7 to 15.6 mg/100 g fw, followed by B vitamins, with vitamin B6 being the most abundant specifically in Italian A. sativum cultivar (2.04 mg/100 g fw) [51].

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Table 5. Water-soluble vitamins contents (mg/kg fw) in the various A. sativum extracts.

https://doi.org/10.1371/journal.pone.0325227.t005

The results of the vitamin concentration analysis in different extracts of A. sativum present intriguing insights into the nutritional composition and potential health benefits of garlic. The variations in vitamin content across different extraction methods highlight the importance of selecting the appropriate extraction solvent to maximize the recovery of specific vitamins. For instance, methanolic extraction appears particularly efficient in extracting thiamine and pantothenic acid, while riboflavin concentrations are notably higher in hexane and ethyl acetate extracts. The presence of vitamins such as pyridoxine and ascorbic acid across various extracts underscores garlic’s potential as a source of these essential nutrients [52,53].

These findings have implications for dietary supplementation and functional food development, as different extraction methods can yield garlic extracts with diverse vitamin profiles, tailored to specific nutritional needs and health objectives. Furthermore, understanding the variations in vitamin content among different extracts can guide the development of optimized extraction protocols to enhance the nutritional value of garlic-based products. Further research exploring the bioavailability and biological activities of these vitamins from garlic extracts would provide valuable insights into their potential health-promoting effects and therapeutic applications.

Fat-soluble vitamins content

Table 6 displays the fat-soluble vitamin contents of A. sativum hexane and ethyl acetate extracts. Of the seven vitamins examined, only vitamins D3 and K3 were absent from both extracts. Compared to the hexane extract (2.85 ± 0.68 mg/kg fw), the ethyl acetate extract had the highest overall vitamin content (20.21 ± 4.01 mg/kg fw). Riboflavin (vitamin D2) was the most concentrated vitamin in both extracts, with an average concentration of 19.05 mg/kg fw for the ethyl acetate extract and 2.14 mg/kg fw for the hexane extract. Similar results have been reported by Okoro and associates, who found 0.05 mg riboflavin, 0.24 mg thiamine, 10 mg ascorbic acid, and 0.4 mg niacin in 100 g of garlic bulbs [10]. Another study quantified thiamine, riboflavin, and niacin in four A. Sativum cultivars in Italy from the Viterbo and Alvito areas in Italy, with respective mean concentrations of 0.26, 0.02, and 0.84 mg/100 g fw [51].

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Table 6. Fat-soluble vitamins contents (mg/kg fw) in the various A. sativum extracts.

https://doi.org/10.1371/journal.pone.0325227.t006

Antioxidant potential of A. sativum extracts

The antioxidant effect of A. sativum extracts was evaluated using DPPH radical scavenging assay. Table 7 illustrates the percent inhibition (PI) of DPPH radical scavenging activity at different extracts concentrations.

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Table 7. Percentage inhibition of DPPH scavenging activity and IC₅₀ values of the A. sativum extracts.

https://doi.org/10.1371/journal.pone.0325227.t007

Overall, these findings underscore the potent antioxidant activity of A. sativum extracts, with the aqueous extract exhibiting the highest antioxidant potential, followed by the methanolic, ethyl acetate, and hexane extracts, respectively. The median inhibitory concentrations (IC50) of extracts obtained using different solvents show significant variations in terms of efficacy. The hexane extract exhibits the lowest inhibitory activity, with an IC50 of 75.26 mg/mL, indicating a lower ability to inhibit the tested target. In contrast, the ethyl acetate extract demonstrates better efficacy, with an IC50 of 51.08 mg/mL. The methanolic extract and the aqueous extract prove to be even more effective, with IC50 values of 37.70 mg/mL and 23.75 mg/mL, respectively, the latter being the most active among all evaluated extracts.

These results align with previous studies highlighting the antioxidant potential of garlic extracts. For instance, Wani and Basir attributed superior antioxidant activity to methanolic extracts of Indian garlic, reporting 51% PI, compared to ethanolic (45%) and aqueous extracts (24%) [54]. Similarly, Awan et al. found that methanolic extracts of Pakistani garlic exhibited a PI of 61.59 ± 1.58%, outperforming ethyl acetate and aqueous extracts [55]. This variation in antioxidant potential could be attributed to differences in plant variety, geographical origin, and extraction protocols, all of which influence the phytochemical composition [42,56,57].

Among the bioactive constituents of garlic, certain polyphenols and flavonoids, such as trans-ferulic acid, have garnered attention for their remarkable antioxidant properties [58]. Notably, the aqueous extract stands out for its elevated concentration of trans-ferulic acid (92.18 mg/kg fw), indicative of robust antioxidant activity. The methanolic extract, particularly abundant in flavonoids, also demonstrates significant antioxidative potential. The substantial presence of cirsiliol (417.26 mg/kg fw) and apigenin-7-O-glucoside (1.11 mg/kg fw), recognized for their antioxidative properties, in the methanolic extract, underscores its potent antioxidant capacity. These results align with prior research highlighting the antioxidative potentials of A. sativum extracts, attributing them to the presence of phenolic and other bioactive compounds [59]. The correlation between the detected biomolecule concentrations and the antioxidant effects of different extracts was assessed using Pearson’s and Spearman’s correlation coefficients. Phenolic compounds exhibited a strong and significant positive correlation with antioxidant activity (r = 0.99, with a p-value of 0.014). Spearman Correlation Coefficient (ρ) for this compound = 1.0 that confirmed a perfect monotonic relationship). Similarly, water-soluble vitamins showed a strong positive correlation (r = 0.85, ρ = 1.0, ρ = 1.0), though the relationship was not statistically significant (p = 0.15).

The high concentrations of cirsiliol and trans-ferulic acid in the polar extract suggest that this extract exhibits strong antioxidant activity. It is also plausible that organosulfur compounds, known for their antioxidative effects, contribute to this activity. Unfortunately, these compounds were not quantified in the present study, which represents a limitation. Further investigation into these bioactive molecules is warranted to better elucidate their roles in the observed antioxidant activities [60].

Antifungal effects of different A. sativum extracts

The antifungal activity of A. sativum extracts was evaluated by measuring the diameter of the inhibition zones after 48 h of incubation with fungal strains. Table 8 displays the results observed at various concentrations of the extracts. Among the tested extracts, the aqueous extract demonstrated the most pronounced antifungal activity, with inhibition zones of 2 cm for both A. flavus and A. niger, surpassing carbendazim (0.55 cm and 0.40 cm, respectively) at a concentration of 10 mg/mL. The methanolic extract also showed notable antifungal activity (1.50 cm for A. flavus and 1.17 cm for A. niger) at the same concentration, followed by the ethyl acetate extract, which showed 0.70 cm and 0.47 cm inhibition zones, respectively, for the same strains, while the hexane extract exhibited relatively smaller inhibition zones.

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Table 8. A. sativum extracts antifungal activities (expressed as inhibition zone (cm)).

https://doi.org/10.1371/journal.pone.0325227.t008

This trend aligns with previous studies highlighting the antifungal efficacy of A. sativum [10,26,61–63]. Research on Pakistani A. sativum extracts similarly reported strong antifungal activity across various solvents, including aqueous, acetone, ethanol, and methanol, with inhibition zones ranging from 7.4–10.3 mm for A. flavus and 9–12.5 mm for A. niger [64]. Additionally, Ashraf et al. demonstrated the significant antifungal potential of garlic essential oil, achieving maximum inhibition zones of 18.3 ± 0.29 mm for A. flavus and 16.1 ± 0.43 mm for A. niger after three days of treatment with 100 μL of essential oil [65].

The minimum inhibitory concentration (MIC), of each garlic extract was tested, and results were presented in Table 9. The aqueous extract exhibited the most potent inhibition of fungal growth, with MIC values of 1.5 mg/mL for A. flavus, and 3 mg/mL for A. niger. The methanolic extract also exhibited significant efficacy, with MIC values of 5 mg/mL and 10 mg/mL for the respective strains. In contrast, the ethyl acetate and the hexane extracts demonstrated weaker inhibitory fungal activities with MIC values of 10 mg/mL and 20 mg/mL, respectively.

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Table 9. Minimum inhibitory concentration (mg/mL) of tested A. sativum extracts.

https://doi.org/10.1371/journal.pone.0325227.t009

Our findings reveal a possible close relationship between the polyphenol content and the observed antifungal activity. Specifically, the aqueous extract emerged as the richest in polyphenols and displayed the strongest antifungal activity. The correlation analysis showed that phenolic acids had a strong and significant positive correlation with antifungal activities against A. flavus (Pearson’s r = 0.973, p = 0.027) and A. niger (Pearson’s r = 0.999, p = 0.001). Water-soluble vitamins also exhibited strong correlations, particularly with Spearman’s ρ = 1.0 for both fungi. In contrast, flavonoids displayed weak and non-significant correlations.

These findings highlight the significant antifungal potential of the aqueous and methanolic extracts, which are richer in polyphenols. However, the antifungal efficacy was not strictly proportional to the polyphenol content, suggesting the involvement of other bioactive compounds. This may be attributed to organosulfur compounds, widely recognized for their potent antifungal properties [10] but not quantified in this study. For instance, previous research demonstrated a correlation between higher allicin content and enhanced antifungal activity in water extracts compared to ethanol extracts [42]. This limitation underscores the need for future investigations to identify and characterize these compounds in the extracts.

Cytotoxic effects of garlic extracts

The cytotoxic effect of A. sativum extracts was assessed using the MTT assay following 24 and 48 h of treatment of U266 (refractory multiple myeloma) and MDA-MB-231 (triple-negative breast cancer) cells with varying concentrations (15.56 μg/mL - 500 μg/mL). The results allowed the determination of IC₅₀ values, representing the concentration required to achieve 50% inhibition of cell viability. Among the extracts tested, methanolic extract demonstrated moderate cytotoxicity, with maximum inhibition not exceeding 55% at the highest concentration (500 μg/mL) for both cell lines (Fig 1). The IC50 value for the methanolic extract after 48 h of treatment on MDA-MB-231 cells was estimated at approximately 418.8 ± 1.14 μg/mL. Interestingly, significant differences in cytotoxic effects were observed between extraction methods. The hexane extract exhibited greater potency, with IC50 values of 69.14 ± 1.2 μg/mL and 45.29 ± 1.08 μg/mL after 24 and 48 h, respectively. Ethyl acetate extract demonstrated the highest efficacy against the metastatic breast cancer cell line, with IC50 values of 26.38 ± 1.24 μg/mL and 17.47 ± 1.09 μg/mL after 24 and 48 hours of treatment, respectively (Fig 1).

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Fig 1. Cytotoxic effect of A. sativum extracts on MDA-MB-231 cells.

The MTT assay was conducted to assess cytotoxicity in vitro against MDA-MB-231 cells. Dose-response curves were generated to evaluate the inhibition of proliferation after 24 h and 48 h of treatment with escalating concentrations (15.56, 31.125, 62.25, 125, 250, and 500 μg/mL) of different solvent extracts: Hex (hexane), EtOH (ethyl acetate), and MeOH (methanol). Three independent experiments were performed in triplicate. All values are expressed as mean ± SD (n = 3) and are presented as a percentage relative to the vehicle control. The IC50 for the 24 h exposure to the methanol extract was not determined due to an incomplete curve. All concentration points were statistically significant compared to the negative control (unless P < 0.05), except for the first point (15.56 μg/mL) of the methanol extract after 48 h of treatment. Statistical comparisons were as follows: a, P < 0.05 ethyl acetate vs. hexane; b, P < 0.05 ethyl acetate vs. methanol; and c, P < 0.05 methanol vs. hexane.

https://doi.org/10.1371/journal.pone.0325227.g001

Previous studies have shown a correlation between the cytotoxic activity of garlic extracts and their composition [66]. However, few studies have explored the impact of different extraction solvents on cytotoxicity [67]. Recent literature has associated garlic consumption with a reduced risk of breast cancer, while garlic-derived compounds have demonstrated inhibitory effects on breast cancer cells [68,69]. Notably, Isbilen and Volkan demonstrated that ethanolic A. sativum extract exhibited an inhibitory effect against MDA-MB-231 cells, with an IC50 value of 2874.2 μg/mL after 48 h of treatment [70]. Comparatively, the IC50 value observed in this study for methanolic extract (418.8 μg/mL) suggests greater efficacy of our extract. The most striking finding of this work, however, lies in the exceptional potency of the ethyl acetate extract, which achieved significantly lower IC50 values compared to other extracts. This highlights the potential superiority of ethyl acetate as a solvent for isolating bioactive compounds, such as lipophilic or sulfur-containing compounds, known for their anticancer properties, although these specific compounds were not quantified in our study. For instance, the works of Malki et al. and Na et al., highlight the promising inhibitory effect of diallyl trisulfide (DATS) on the MCF-7 human breast cancer cell line through an apoptotic death mechanism [71,72]. Similarly, Sato’s recent work confirmed the potential of diallyl disulfide (DADS) and DATS as promising agents for preventing and treating breast cancer by decreasing STS protein expression and suppressing active estrogen levels in MCF-7 cells [73]. Interestingly, Marni et al. demonstrated that DADS and DATS exhibited high cytotoxicity against two paclitaxel-resistant triple-negative breast cancer cell lines, MDA-MB-231 PR and MDA-MB-468 PR, by blocking the cell cycle [69]. These findings underscore the potential of garlic-derived compounds in breast cancer treatment and highlight the need to further explore ethyl acetate as a solvent for isolating active compounds with enhanced cytotoxic activity.

For U266 cells, the IC50 values for the hexane extract were 184.9 ± 1.19 μg/mL and 116.4 ± 1.27 μg/mL after 24 and 48 h, respectively. Remarkably, when extraction was performed in ethyl acetate, the cytotoxic effect on refractory myeloma cells was higher (Fig 2a) with IC50 values at 24 and 48 h of 78.4 ± 1.23 μg/mL and 75.7 ± 1.31 μg/mL, respectively. Observations from phase-contrast inverted microscopy corroborated the MTT assay results (Fig 2b), showing no visible reduction in the number of untreated U266 cells (control) compared to those treated with methanolic extract. However, a significant decrease in cell viability was observed in cells treated with 250 µg/mL of A. sativum extracted in hexane and ethyl acetate, indicating a potent cytotoxic effect.

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Fig 2. Effect of A. sativum extracts on U266 multiple myeloma cells.

(a) The cytotoxic effects of different solvent extracts Hex (hexane), EtOAc (ethyl acetate), and MeOH (methanol)—were evaluated after 24 h and 48 h of treatment with escalating concentrations (15.56–500 μg/mL). Values are expressed as mean ± SD (n = 3) from three independent assays and presented as percentages relative to the vehicle control. All concentration points were statistically significant compared to the negative control (P < 0.05), except for the first point (15.56 μg/mL) of the hexane and methanol extracts, and the second point of the methanol extract after 48 h of treatment. Statistical comparisons are indicated as follows: a, P < 0.05 ethyl acetate vs. hexane; b, P < 0.05 ethyl acetate vs. methanol; and c, P < 0.05 methanol vs. hexane. (b) Representative phase-contrast inverted microscopy images of U266 cells treated or untreated (vehicle control) with 250 μg/mL of the different extracts (magnification, × 100).

https://doi.org/10.1371/journal.pone.0325227.g002

According to the literature, no studies have been found on multiple myeloma using garlic extracts, with only two studies investigating DADS and DATS. The first study suggested that co-treatment with DATS and dexamethasone inhibited the proliferation of two multiple myeloma cell lines. Last year, Wei Hu et al. demonstrated for the first time that DADS induces apoptosis in multiple myeloma cells through a mitochondria-dependent pathway and enhances the activity of two anti-myeloma drugs via synergistic effects [74]. These findings, along with ours, suggest that garlic-derived compounds or extracts could represent promising candidates for complementary approaches in multiple myeloma treatment. However, further studies are necessary to clarify the mechanisms of action and identify the specific bioactive components responsible for these effects.

The bioactivities of the extracts, including antioxidant, antifungal, and anticancer effects, are driven by the synergistic actions of molecules like polyphenols, phenolic acids, flavonoids, and vitamins. These compounds interact with cellular pathways to exert their effects. Polyphenols neutralize reactive oxygen species (ROS) and free radicals, boosting antioxidant enzymes like SOD and CAT. Vitamins C and E enhance antioxidant defenses, protecting cellular structures from oxidative damage. They also disrupt fungal cell membranes, inhibit ergosterol biosynthesis, and boost immune responses. In anticancer mechanisms, polyphenols and flavonoids regulate apoptosis, cell cycle arrest, and angiogenesis, while phenolic acids suppress tumor growth. Vitamins C and E repair oxidative DNA damage and promote cancer cell apoptosis [69,74].