Phytochemical Profiling of Allium subhirsutum L. Aqueous Extract with Antioxidant, Antimicrobial, Antibiofilm, and Anti-Quorum Sensing Properties: In Vitro and In Silico Studies

The present study was the first to evaluate the phytochemical composition, antioxidant, antimicrobial, antibiofilm, and anti-quorum sensing potential of Allium subhirsutum L. (hairy garlic) aqueous extract through in vitro and in silico studies. The phytochemical profile revealed the presence of saponins, terpenes, flavonols/flavonones, flavonoids, and fatty acids, particularly with flavonoids (231 ± 0.022 mg QE/g extract), tannins (159 ± 0.006 mg TAE/g extract), and phenols (4 ± 0.004 mg GAE/g extract). Gas chromatography–mass spectrometry (GC–MS) analysis identified 15 bioactive compounds, such as 5-hydroxymethylfurfural (37.04%), methyl methanethiolsulfonate (21.33%), furfural (7.64%), beta-D-glucopyranose, 1,6-anhydro- (6.17%), 1,6-anhydro-beta-D-glucofuranose (3.6%), trisulfide, di-2-propenyl (2.70%), and diallyl disulfide (1.93%). The extract was found to be non-toxic with 50% cytotoxic concentration higher than 30,000 µg/mL. The investigation of the antioxidant activity via DPPH (2, 2-diphenyl-1-picrylhydrazyl) and FRAP (IC50 = 1 μg/mL), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); IC50 = 0.698 ± 0.107 μg/mL), and β-carotene (IC50 = 0.811 ± 0.036 mg/mL) was assessed. Nevertheless, good antimicrobial potential against a diverse panel of microorganisms with bacteriostatic and fungistatic effect was observed. Quorum sensing inhibition effects were also assessed, and the data showed the ability of the extract to inhibit the production of violacein by the mutant C. violaceum strain in concentration-dependent manner. Similarly, the biofilm formation by all tested strains was inhibited at low concentrations. In silico pharmacokinetic and toxicological prediction indicated that, out of the sixteen identified compounds, fourteen showed promising drug ability and could be used as lead compounds for further development and drug design. Hence, these findings support the popular use of hairy garlic as a source of bioactive compounds with potential application for human health.


Introduction
In modern medicine, many bacteria and their associated infections are considered the major challenge in public health worldwide, causing annually 35,000 deaths in the United States and about 33,000 deaths in European Union [1,2]. Furthermore, the treatments with synthetic drugs are often associated with higher side effects and cannot be tolerated by some people at high doses. Contrary, the exploration of herbs and plant-based products remains important and one of the most applied pharmacological alternatives for the prevention and the treatment of several pathologies as well as a large number of illnesses due to their safety, affordability, and availability [3,4]. Their richness in bioactive molecules such as polyphenols and biochemical components of phytomedicines (alkaloids, flavonoids, phenolics, carotenoids, polysaccharides, lactones, and tannins, . . . ) gives them potent therapeutic benefits, allowing them to be a potential escort to the development of new drug candidates [5][6][7][8][9][10][11][12]. They have a powerful action in reducing the threat of numerous chronic diseases caused by free radicals, destroying the immune system, and translating into serious oxidative stress and oxygen (O 2 ) detoxification [12,13].
Besides that, the spread of infection disease, as well as the emergence of multidrugresistant bacteria and fungi, is increased recently due to the failure of chemotherapy and the indiscriminate or frequently uses of antibiotics, and their inhibition by alternative agents become an urgent need. Therefore, attention is now being shifted towards natural active components isolated from herbs along with phytomedicine which shares high antioxidant and antimicrobial effects with low cost and high efficiency. As a result, they have been used to avoid various curable infection diseases as the WHO has pointed out that "no action today means no cure tomorrow".
In this respect, Allium subhirsutum L. is a perennial plant that belongs to the garlic family. Recent literature conducted by our team reported on the ethno-pharmacological use of bulbs for therapeutic claims, including antioxidant, anti-inflammatory, and anticancer claims, as well as those relating to the inhibition of tumor angiogenesis in a murine model of skeletal metastases [14,15]. Based on an evaluation of our previous study, the phytochemical analysis of this plant has only been assessed by HR-LCM for methanolic extract; the results from this indicate that it contains various phytoconstituents, particularly polyphenols and flavonoids, along with several other bioactive compounds [15][16][17][18][19][20][21]. Whole plant is used in the popular medicine in Sardinia (Italy) due to its anti-hemorrhoidal, blood pressure regulation, and purifying action [22]. In addition, hairy garlic buds and leaves are cut into small pieces and eaten raw or used as flavoring agent in cooked dishes and salads [23]. It is also a good ingredient in the traditional Turkish flat bread called yufka [24]. Few studies have described the phytochemical composition of the essential oil and organic extracts from A. subhirsitum plant organs (bulbs, flowers, and leaves). Our team has demonstrated that methanolic extract from hairy garlic (bulbs) helps with antioxidant, anti-inflammatory, and anticancer claims, and can inhibit tumor angiogenesis in a murine model of skeletal metastases [16,20]. Nencini and colleagues reported the antioxidant activities of aged 15% aqueous ethanol extract from Italian A. subhirsutum leaves, flowers, and bulbs [21]. Emir and colleagues reported the cholinesterases and tyrosinase inhibition activities of Turkish A. subhirsutum (methanolic extract from bulbs and aerial parts) [18].
Hence, the present work was the first to quantitatively analyze the phytochemical constituents of the aqueous extract obtained from of A. subhirsutum bulbs. Additionally, the determination of its antioxidant, antimicrobial, anti-quorum sensing, antibiofilm, cyto-toxicity, and antiviral properties with the aqueous extract was also carried out. Moreover, to screen potential drugs/molecules from the above extract, pharmacokinetics and toxicological prediction were also performed.

Antimicrobial Activities
The hairy garlic aqueous extract was tested against a large collection of Gram-positive and Gram-negative bacteria, yeasts, molds, and coxsakievirus B3 (CVB3), and herpes simplex virus type 2 (HSV-2). Results of the antiviral activity showed that A. subhirsutum aqueous extract was not active against the two tested virus (CVB3) and (HSV-2).
Results of the antibacterial and antifungal activities of the hairy garlic aqueous extract using both disc diffusion and microdilution assays are summarized in Table 2. The mean diameter of the growth inhibition zone (mm ± SD) varied from 6.00 ± 0.00 to 15.66 ± 0.57. Using the scheme proposed by Parveen et al. (2010), our extract had low (1-6 mm) to high (11)(12)(13)(14)(15) mm) activity against all tested Gram-positive and Gramnegative bacteria. Most of tested microorganisms were more sensitive to the tested extract as compared to the standard antibiotic used (ampicillin). Using the microdilution assay, MIC values ranged from 6.25 to 12.5 mg/mL and concentrations around 50-100 mg/mL could kill the tested microorganisms (MBCs values). The MBC/MIC ratio ranged from 4 to 8, highlighting the bacteriostatic activity of the tested extract on all tested microorganisms. This extract was also active against yeast and mold strains at varying degrees. The Candida vaginalis strain was the most sensitive to hairy garlic extract with a mean diameter of about (15.66 ± 0.57 mm) in the growth inhibition zone. MIC values ranged from 1.62 to 6.25 mg/mL and MFCs values ranged from 25 to 50 mg/mL. The tested aqueous extract exhibited fungistatic activity against all Candida spp. strains tested (MFC/MIC ratio > 4).

Antioxidant Activities
The quantitative analysis of tannins, phenols, and flavonoids demonstrated that hairy garlic contains large quantities of flavonoids (231 ± 0.022 mg QE/g extract), followed by tannins (159 ± 0.006 mg TAE/g extract) and phenols (4 ± 0.004 mg GAE/g extract). The results of the free radical scavenging activities of A. subhirsutum L. aqueous extract, as compared to ascorbic acid and butylated hydroxyl-toluene (BHT), are reported in Table 3. The concentrations needed for scavenging 50% of radicals (IC 50% ) using hairy garlic aqueous extract were as follows: 1 mg/mL for DPPH and FRAP assays (respectively), (0.698 ± 0.107) mg/mL for the ABTS test, and (0.811 ± 0.036) mg/mL for the β-carotene experiment.

Anti-Quorum Sensing and Antibiofilm Activities
The ability of A. subhirsutum aqueous extract to inhibit the production of violacein, a pigmented molecule secreted by C. violaceum when bacterial concentration is reached (quorum sensing), was tested using the microtiter plate assay (mutant-C. violaceum CV026) and the Petri dishes test using Luria-Bertani agar medium (wild type C. violaceum ATCC 12472).
The results showed that the tested extract was able to inhibit the production of violacein by the mutant C. violaceum strain in a concentration-dependent manner ( Figure 1). In fact, the percentage of violacein production was inhibited by (24.05 ± 0.68)% at 2.5 mg/mL and about (37.43 ± 0.85)% at 5 mg/mL. No inhibition of violacein production was recorded at low concentrations of hairy garlic extract (1.25 and 0.625 mg/mL).

Anti-Quorum Sensing and Antibiofilm Activities
The ability of A. subhirsutum aqueous extract to inhibit the produc a pigmented molecule secreted by C. violaceum when bacterial concent (quorum sensing), was tested using the microtiter plate assay (mutant-C. and the Petri dishes test using Luria-Bertani agar medium (wild type C 12472). The results showed that the tested extract was able to inhibit t violacein by the mutant C. violaceum strain in a concentration-dependen 1). In fact, the percentage of violacein production was inhibited by (24. mg/mL and about (37.43 ± 0.85)% at 5 mg/mL. No inhibition of violacein recorded at low concentrations of hairy garlic extract (1.25 and 0.625 mg  Using LB Petri dish agar plates, the tested extract was able to inhibit the production of violacein by the wild-type starter strain (C. violaceum ATCC 12472). Furthermore, the diameter of growth inhibition zone ranged from (8 ± 1) mm at 1.25 mg/mL to (13 ± 0.5) mm at 5 mg/mL. All these data are summarized in Table 4. Anti-quorum sensing activity (mm) 13 ± 0.5 10 ± 1 8 ± 1 (−) In addition, two virulence properties controlled by the quorum-sensing system in the P. aeruginosa PAO1 strain (swarming and swimming) were inhibited at varying degrees when different concentrations of hairy garlic aqueous extract were used. In fact, the percentage of the inhibition of swarming activity ranged from (8.93 ± 0)% at 50 µg/mL to (23.66 ± 0.5)% at 100 µg/mL. Furthermore, the swimming activity was reduced by (13.67 ± 1)% at 100 µg/mL of the hairy garlic aqueous extract (Table 5). The antibiofilm activity of the tested extract was experimented on four bacteria and two Candida spp. strains at different concentrations, ranging from MIC/16 to MIC (from 0.312 to 10 mg/mL). Table 6 summarizes the percentage of biofilm inhibition when different concentrations of extract were used. Table 6. Anti-biofilm results (inhibition %) of A. subhirsutum L. aqueous extract tested against Gram-positive and Gram-negative bacteria and yeast strains. Interestingly, at an MIC value of (10 mg/mL), the percentage of biofilm inhibition depended on the tested strain and increased from (12. 18  respectively. The highest inhibition was recorded at an MIC value of (10 mg/mL of aqueous extract) with a percentage of inhibition at about (62.48 ± 5.50)% for C. albicans ATCC 10239 and (54.81 ± 4.08)% for C. tropicalis ATCC 13803. Based on Duncan's multiple-range test, there was no significant difference between the percentage of biofilm inhibition of the two Candida strains tested at 10 mg/mL.

Pharmacokinetic Properties and Toxicity Profile Prediction
During the time of the preclinical analysis trial in drug discovery and development, the assessment of absorption, distribution, metabolism excretion, and toxicity (ADMET) are very crucial for attractive molecules to possess the best chance to become an effective drug. Hence, based on ADME analysis, the identified phytocompounds from A. subhirsutum water extract were predicted for their pharmacokinetics, drug-likeness, and medicinal chemistry friendliness using the SwissADME web tool. As shown in (Table 7), all selected phytoconstituents did not violate the Lipinski's rule of five; therefore, they seem to be passed orally with suitable bioavailability scored at 0.55. They exhibited high gastrointestinal (GI) absorption with eight compounds being able to cross the blood-brain-barrier (BBB) permeant; this revealed that they have low to no central nervous system (CNS) side effects. Only ten compounds were found in the substrate for permeability glycoprotein (P-gp), meaning that they possess very little chance to efflux out of the cell. Moreover, compounds 1-7 did not inhibit all the tested cytochrome P450 isoenzymes which played a fundamental role in the biotransformation of drugs through O-type oxidation reactions.
The AMES toxicity and hepatotoxicity of the parameters of hERG I/II inhibitors were evaluated using the pkCSM online server which predicted whether the designed new molecules were toxic. Based on the predictive results (Table 7), except the compound 1, none of the others were expected to present any toxicity problems.
An estimation of drug-likeness properties based on bioavailability radar ( Figure 2) remains a powerful tool to understand identified compounds, as well as their lipophilicity, size, polarity, solubility, saturation, and flexibility behaviors. In addition, as shown in Figure 2, most of the identified compounds fit totally in the pink area, signifying their good predicted oral bioavailability.   To know more about both passive gastrointestinal absorption (HIA) and the BBB penetrating effect as a function of the position of the molecules in the WLOGP-versus-TPSA referential, the boiled egg model (Figure 3) has been established for the top ADME compounds. The results clearly indicate that compounds 2, 6, 10, 11, 14, and 15 were in the white zone, thus indicating the high probability of being passively absorbed by the gastrointestinal tract with only 11 appearing as a blue point, which was predicted as a substrate of the PGP+. On the other hand, compounds 1, 3, 4, 5, 7, 8, 12, and 13 were in the in the yolk region, which reflects their high probability to permeate through BBB to access CNS, and also appeared in the red point, suggesting that the substrate of the p-glycoprotein is actively effluxed by PGP+.
Most of the identified compounds in hairy garlic aqueous extract were previously described in the composition of different Allium species, as summarized in Table 9. Table 9. Review of the distribution of some identified bioactive compounds in some Allium plant species.

Bioactive Molecule
Allium Species/Variety References
No scientific report discussed the anti-quorum sensing and the antibiofilm activities of A. subhirsutum plant extracts. Interestingly, our extract was able to modulate the secretion of violacein by both C. violaceum mutant and wild type. The same extract inhibited the swarming and swimming motility mode of P. aeruginosa PAO1 in a concentrationdependent manner. Previous reports have focused on extracts from A. cepa and A. sativum plant species. Results showed that sulfur compounds (Ajoene, Iberin) from garlic and flavonoids (Quercetin) from red onion modulated the production of bioluminescence in Vibrio harveyi, violacein in C. violaceum, and pyocyanin/proteases/elastases and swarming motility in P. aeruginosa [38]. Allium cepa (95% methanolic) extract was also able to inhibit the production of the green pigment (Pyocyanin) by P. aeruginosa PAO1 and to reduce its motility by reducing swimming, twitching, and swarming abilities [39]. The same extract was able to reduce the production of violacein on agar medium with a mean diameter of about 10 mm in the growth inhibition zone and to prevent the formation of biofilm formed by P. aeruginosa (Isolate PA14) using the tube assay method [40].
A. sativum (methanolic and ethanolic) extracts were able to inhibit the biofilm formed by six pathogenic bacteria including S. aureus, Bacillus cereus, Streptococcus pneumoniae, P. aeruginosa, E. coli, and K. pneumoniae in a concentration-dependent manner [41]. Bhatwalkar and colleagues in 2019 [42] reported that the fresh garlic extract at 4% significantly inhibited the biofilm formed by Shiga-toxin-producing E. coli (STEC) strains. More recently, Caputo et al., [43] reported the antibiofilm activities of two landraces (Irsina and Contursi) of A. ampeloprasum var. holmense from south Italy (methanolic extract from bulbs and aerial parts) which were tested against Listeria monocytogenes ATCC 7644, entero-hemorrhagic E. coli DSM 8579, P. aeruginosa DSM 50071, Pectobacterium carovotorum DSM 102074, and S. aureus ATCC 25923.
The reported biological properties of the tested extract can be attributed to the presence of many phytocompounds with promising activities. In fact, it is well documented that the main identified compound in hairy garlic extract, 5-hydroxymethylfurfural, possessed antioxidant and antiproliferative activities [44], as well as anti-ischemic and antityrosine enzyme effects, improving blood rheology and affecting the role of glycyrrhizin metabolism [45]. It has been demonstrated that this compound (5-hydroxymethylfurfural) can protect human vein epidermal cell against H 2 O 2 and glucose and improve acute liver injury in mice [46].
Additionally, methyl methanethiolsulfonate which is produced in different amounts by Alliaceae members is an anti-oomycete agent with antimicrobial and antimutagenic characters [47]. In addition, this sulfur compound is known to inhibit colon tumor [48]. More recently, Vijayakumar and Ramanathan [49] demonstrated that 5-hydroxymethylfurfural showed interesting anti-quorum sensing and antibiofilm activities against C. violaceum, Streptococcus pyogenes, S. mutans, S. aureus, and S. epidermidis. In fact, at 100 µg/mL, this compound inhibited the production of violacein by 87% and reduced the biofilm formation by S. mutans and S. epidermidis strains by 86% and 79%, respectively. The streptococcal ad staphylococcal biofilm formation was also inhibited with high percentage at 125 µg/mL (up to 83% for S. pyogens, and 82% for S. aureus).
Furfural, a natural furan occurring compound, is known to possess antityrosinase and antimicrobial activities against Bacillus subtilis with an antimicrobial zone between 16 and 20 mm at 1.4 µM, MIC/MBC values about 0.027 µM [50]. This compound (furfural) was also active against Salmonella bacteria with an antimicrobial zone less than 15 mm at concentrations ranging from 0.35 to 1.4 µM, and MIC/MBC values of about 0.029 µM and 0.121 µM, respectively [50]. In addition, the identified sulfur compounds diallyl disulfide and diallyl trisulfide are known to be potent phytoconstituents for the prevention and treatment of several human diseases, such as endocrine system diseases, cardiovascular diseases, neurological diseases, infectious diseases, and cancerous diseases [51][52][53][54].

Plant Material Sampling and Extract Preparation
Hairy garlic bulbs ( Figure 4) were purchased from a local market in 2020 from Hail region (Saudi Arabia). A voucher specimen (AN03) was deposited at the herbarium in the Department of Biology (College of Science, University of Hail, Hail, Kingdom of Saudi Arabia). Briefly, 40 g of bulbs were macerated in 400 mL of distillated pure water at room temperature for 48 h and re-extracted three times using the same procedure. The yield expressed in percentage was calculated using the following equation (Equation (1)): (1) where W1 is the weight of extract after the evaporation of solvent and W2 is the dry weight of the sample.
Plants 2022, 10, x FOR PEER REVIEW 14 of 20 streptococcal ad staphylococcal biofilm formation was also inhibited with high percentage at 125 µg/mL (up to 83% for S. pyogens, and 82% for S. aureus). Furfural, a natural furan occurring compound, is known to possess antityrosinase and antimicrobial activities against Bacillus subtilis with an antimicrobial zone between 16 and 20 mm at 1.4 µM, MIC/MBC values about 0.027 µM [50]. This compound (furfural) was also active against Salmonella bacteria with an antimicrobial zone less than 15 mm at concentrations ranging from 0.35 to 1.4 µM, and MIC/MBC values of about 0.029 µM and 0.121 µM, respectively [50]. In addition, the identified sulfur compounds diallyl disulfide and diallyl trisulfide are known to be potent phytoconstituents for the prevention and treatment of several human diseases, such as endocrine system diseases, cardiovascular diseases, neurological diseases, infectious diseases, and cancerous diseases [51][52][53][54].

Plant Material Sampling and Extract Preparation
Hairy garlic bulbs ( Figure 4) were purchased from a local market in 2020 from Hail region (Saudi Arabia). A voucher specimen (AN03) was deposited at the herbarium in the Department of Biology (College of Science, University of Hail, Hail, Kingdom of Saudi Arabia). Briefly, 40 g of bulbs were macerated in 400 mL of distillated pure water at room temperature for 48 h and re-extracted three times using the same procedure. The yield expressed in percentage was calculated using the following equation (Equation (1)): Yield (%) = (W1 × 100)/W2 (1) where W1 is the weight of extract after the evaporation of solvent and W2 is the dry weight of the sample. The obtained extract was filtered, and water was lyophilized by using Millirock Technology apparatus (Kingston, NY, USA) to yield amorphous powder. The yield of extraction was about 27.366 ± 0.152%.

Phytochemical Profiling of Hairy Garlic Aqueous Extract
A Shimadzu Nexis GC-2030 Gas Chromatograph system equipped with a QP2020 NX Mass Spectrometer was used to identify the bioactive compounds in A. subhirsutum L. aqueous extract. Helium was used as a carrier gas in the constant flow mode at 1 mL/min. The initial temperature of the column was 70°C. It was maintained at this temperature for 2 min and was then gradually increased by 10°C up to 280°C. The oven temperature was The obtained extract was filtered, and water was lyophilized by using Millirock Technology apparatus (Kingston, NY, USA) to yield amorphous powder. The yield of extraction was about 27.366 ± 0.152%.

Phytochemical Profiling of Hairy Garlic Aqueous Extract
A Shimadzu Nexis GC-2030 Gas Chromatograph system equipped with a QP2020 NX Mass Spectrometer was used to identify the bioactive compounds in A. subhirsutum L. aqueous extract. Helium was used as a carrier gas in the constant flow mode at 1 mL/min. The initial temperature of the column was 70 • C. It was maintained at this temperature for 2 min and was then gradually increased by 10 • C up to 280 • C. The oven temperature was raised up to 280 • C at the increased rate of 5 • C/min and maintained for 9 min. The injection port temperature was 250 • C and the helium flow rate was 1 mL/min. The ionization voltage was 70 eV. Separation was achieved by a RTSvolatile column about 30 m long. A Quadrupole Mass Detector was employed to detect compounds when they were vented from the column. The temperature of the detector was 300 • C. Using MS data libraries, such as WILEY8.LIB and NIST08, the spectrum was analyzed, and compounds were identified.
The total phenols content was estimated using the Folin-Ciocalteu method [62]. The total contents of tannins and flavonoids were estimated using the techniques described by Broadhurst and Jones [63] and Benariba et al. [64], respectively.

Cytotoxicity Evaluation
The cytotoxicity of the obtained extract was evaluated based on the same protocol carried out by Snoussi et al. [65].
The disk diffusion assay was used for the determination of the diameter of the growth inhibition zone estimated on agar medium. The microdilution assay was used for the determination of the minimal inhibitory concentration (MICs) and the minimal bactericidal/fungicidal concentrations (MBC/MFC). To interpret the mean diameter of the growth inhibition zone obtained on agar media, the scheme proposed by Parveen et al. [67] was used. The results of the (MBC/MIC) and (MFC/MIC) ratios were interpreted using the scheme proposed by Gatsing et al. [68].
Coxsakievirus B-3 (CVB3) and herpes simplex virus type 2 (HSV-2) were used to test the antiviral potential of the aqueous extract using the procedure described by Alreshidi et al. [69].

Evaluation of Anti-Quorum Sensing Activity
The inhibition of violacein production was assayed using two starter strains: Chromobacterium violaceum ATCC 14272 and CV026, as previously described by Noumi et al. [73].
On the Lauria-Bertani agar plate, activity was interpreted as moderate when the inhibition zone < 10 mm and potent when zone > 10 mm) [74].
The effect of the A. subhirsutum aqueous extract to inhibit the motility of the Pseudomonas aeruginosa PAO1 strain was tested on 0.3% agar medium (swimming motility) and 0.5% agar medium (swarming motility), as described by Alreshidi et al. [69].
The percentage of biofilm inhibition (expressed in percentage) was calculated using the following equation (Equation (2)): Inhibition of biofilm formation (%) = [(OD Control − OD Sample) /ODControl] × 100 (2) where OD Control is the optical density of the control and OD Sample is the optical density of the sample.

Statistical Analysis
The average values of three replicates were calculated using the SPSS 25.0 statistical package for Windows. Differences in the means were calculated using Duncan's multiplerange tests for means with a 95% confidence interval (p ≤ 0.05).

Conclusions
Overall, we are the first to report the identification of several small peptides and bioactive molecules in the aqueous extract from A. subhirsutum L. using the GC-MS technique. The results obtained allowed the presence of flavonoids (231 ± 0.022 mg QE/g extract), followed by tannins (159 ± 0.006 mg TAE/g extract) and phenols (4 ± 0.004 mg GAE/g extract). Interestingly, several phytocompounds with high biological activities were identified, mainly including 5-hydroxymethylfurfural (37.04%), methyl methanethiolsulfonate (21.33%), furfural (7.64%), beta-D-glucopyranose, 1,6-anhydro-(6.17%), 1,6-anhydro-beta-D-glucofuranose (3.6%), trisulfide, di-2-propenyl (2.70%), and diallyl disulfide (1.93%). These compounds can act independently or synergistically, suggesting their potential application for the treatment of several chronic diseases. Moreover, our results showed significant antioxidant (IC 50 values; DPPH 1 mg/mL; ABTS 0.698 ± 0.107 mg/mL; β-carotene 0.811 ± 0.036 mg/mL; and FRAP 1 mg/mL) and antimicrobial properties, as well as antibiofilm properties (up to 62.48 ± 5.50 % against C. albicans ATCC 10239, and 56.21 ± 2.55% against S. aureus ATCC 25923). The tested extract was also able to inhibit the production of violacein by 37.43 ± 0.85% at 5 mg/mL. Swarming and swimming motility in P. aeruginosa PAO1 was inhibited by 23.66 ± 0.5% and 13.67 ± 1%, respectively. Our computational study on the major identified compounds revealed acceptable oral bioavailability of the extract, which can be useful in future bioassay studies. Finally, further study on the isolation of significant molecules in vivo studies is strongly recommended in order to evaluate the effectiveness of the isolates for the desired activity.