Antibacterial, Antibiofilm, Antiswarming, and Antioxidant Activities of Flavonoids Isolated from <i>Allium colchicifolium</i> Leaves

Majnooni, Mohammad Bagher; Ghanadian, Syed Mustafa; Mojarrab, Mahdi; Bahrami, Gholamreza; Mansouri, Kamran; Mirzaei, Arezoo; Farzaei, Mohammad Hossain

doi:https://doi.org/10.1155/2023/5521661

Journal of Food Biochemistry

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Abstract Introduction Materials and Methods Results and Discussion Conclusion Data Availability Disclosure Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 5521661 | https://doi.org/10.1155/2023/5521661

Antibacterial, Antibiofilm, Antiswarming, and Antioxidant Activities of Flavonoids Isolated from Allium colchicifolium Leaves

Mohammad Bagher Majnooni,^1,2Syed Mustafa Ghanadian ,³Mahdi Mojarrab,²Gholamreza Bahrami,^2,4Kamran Mansouri,⁴Arezoo Mirzaei,⁵and Mohammad Hossain Farzaei²

Academic Editor: Charalampos Proestos

Received22 Feb 2023

Revised05 May 2023

Accepted01 Jul 2023

Published18 Aug 2023

Abstract

Allium (A.) species are one of the most widespread plants in the world, which have played a special role in terms of nutrition, treatment, and economy since long ago. This study is the first report on the phytochemical and biological activities of A. colchicifolium. Five flavonoids, including isorhamnetin (compound 1), quercetin (compound 2), morin (compound 3), isorhamnetin-3-O-glucoside (compound 4), and quercetin 3-O-glucoside (compound 5), were isolated and purified for the first time from A. colchicifolium leaves. All isolated flavonoids showed antibacterial and antibiofilm activities. Compound 3 revealed prominent antibacterial activities against Staphylococcus (S.) aureus and Proteus (P.) mirabilis. Also, compounds 3 and 5 showed the highest antibiofilm activities on S. aureus and P. mirabilis, respectively. The docking study results showed that compounds 3, 4, and 5 had the most robust interactions with two critical proteins in biofilm formation, including staphylococcal accessory regulator A (SarA) and mannose-resistant Proteus-like fimbriae H (MrpH). Besides, compounds 2 and 3 revealed significant antioxidant activities in 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and ferric (Fe³⁺)-reducing antioxidant power (FRAP) tests compared to the other compounds and positive controls. Also, the ADMET (absorption, distribution, metabolism, excretion, and toxicity) prediction assay showed that compounds 1, 2, and 3 have suitable physicochemical and pharmacokinetic properties. The results of this study confirm that the flavonoids isolated from the leaves of A. colchicifolium can be promising candidates for use in the pharmaceutical and food industries.

1. Introduction

The genus Allium (A.) of the family Liliaceae, with 800 to 900 species, is one of the largest monocotyledonous plants. The Allium species have fantastic nutritional, therapeutic, and economic value. Onion (A. cepa), garlic (A. sativum), shallot (A. ascalonicum/A. hirtifolium), leek (A. ampeloprasum), Welsh onion (A. fistulosum), bear’s garlic (A. ursinum), and chives (A. schoenoprasum) are among the most important edible species of this genus, which since ancient times (2800–3200 B.C) until now as medicine and part of the people’s diet [1–3]. Steroidal saponins, organosulfur compounds, and fructooligosaccharides are the major compounds that are isolated from flowers, leaves, and bulbs of Allium species [4, 5]. Besides, the plants of the Allium genus are known as one of the rich natural sources of polyphenolic compounds, especially flavonoids such as quercetin, isorhamnetin, kaempferol, apigenin, myricetin, luteolin, and their glycosylated derivatives [6–9]. Kaempferol-3-O-(6″-feruloyl)-sophoroside and quercetin-3-O-(6-trans-feruloyl)-β-D-glucopyranosyl-(1 ⟶ 2)-β-D-glucopyranoside-7-O-β-D-glucopyranoside from A. tuberosum shoots [10, 11], kuwanon K, xanthomicrol, and rhamnazin from A. cepa bulbs [12], and kaempferol-3-O-neohesperidoside-7-O-glucuronide from A. microdictyon [13] are among the flavonoids that have recently been isolated from the Allium species. Also, several biological and pharmacological activities, including anticancer, antihypercholesterolemia, cardioprotective, neuroprotective, anti-inflammation, and antidiabetic, have been reported for Allium flavonoids [14–16]. Also, the Allium flavonoids showed prominent antioxidant and antimicrobial activities in several investigations [3, 17]. Besides, Allium spp and its flavonoids showed antibacterial activities via inhibiting the bacterial defence mechanisms, including quorum sensing, biofilm formation, and swarming [18, 19]. Quecan and coworkers reported that quercetin and its glucoside derivatives, including quercetin 4-O-glucoside and quercetin 3,4-O-diglucoside, found in Allium cepa revealed the antiquorum sensing, antibiofilm, and antiswarming effects on Pseudomonas aeruginosa and Serratia marcescens. This study showed that quercetin 4-O-glucoside and quercetin 3,4-O-diglucoside had a high affinity with CviR and LasR as two main quorum sensing proteins [20]. Taxifolin and apigenin [21], kaempferol and naringenin [22], myricetin [23], and luteolin [24] are other Allium flavonoids that showed antiquorum-sensing activities. On the other hand, Allium flavonoids revealed high antioxidant potential. Kim and coworkers isolated four antioxidant flavonoids, including kaempferol-3-O-β-D-glucopyranoside, quercetin 3-O-β-D-glucopyranoside, isorhamnetin 3-O-β-D-glucopyranoside, and quercetin 3-O-β-D-xylopyranoside from Allium sativum shoots and leaves [25]. Also, three flavonoids, including chrysoeriol, chrysoeriol-7-O(2″-O-E-feruloyl)-β-D-glucoside, and isorhamnetin-3-β-D-glucoside isolated from Allium vineale showed antioxidant properties [26]. Another study showed that kaempferol glycoside derivatives, luteolin, and apigenin identified in Allium roseum flowers and leaves have prominent antibacterial and antioxidant activities [27]. On the other hand, A. colchicifolium is one of the native species of Allium in western Iran, especially in Kermanshah province, which is commonly known as Koul in Kurdish. A. colchicifolium (synonym = A. haussknechtii and A. straussii) leaves are used raw as a garnish and salad and cooked as local food. It is also used as an antirheumatoid, antilipidemic, and anti-infective in folk medicine [28–30]. To the best of our knowledge, there are no reports of phytochemical studies and biological/pharmacological effects of A. colchicifolium. Therefore, due to the critical roles of Allium flavonoids mentioned above, the flavonoids of A. colchicifolium leaves were isolated and identified using chromatographic and spectroscopic techniques in the present study using one- and two-dimensional nuclear magnetic resonance (NMR) and mass spectrometry (MS). Also, these flavonoids’ antibacterial and antioxidant activities were evaluated, and an in-silico study investigated the antibiofilm mechanisms of these compounds.

2. Materials and Methods

2.1. General Experimental Procedures

The NMR spectra were conducted on a Bruker Avance AV 400 ¹H-NMR (400 MHz, DMSO-d6) and ¹³C-NMR (100 MHz, DMSO-d6). The ¹³C-NMR multiplicities of resonances were specified by the DEPT spectrum. Heteronuclear multiple-bond correlation spectroscopy ¹H-¹³C connections were identified with the HMBC spectrum. Mass analysis was done by the Agilent 1200 series liquid chromatography (LC) system (Agilent Technologies, Germany) coupled with an Agilent 6410 triple quadrupole MS and electrospray ionization (ESI) with a capillary voltage of 4,000 V (Agilent Technologies, Palo Alto, CA, USA). Thin layer chromatography (TLC) was conducted on the Merck TLC silica gel (SiO₂, Germany) with chloroform/methanol (CHCl₃: MeOH, 9 : 1) and discerned by spraying 1% natural product reagent (2-aminoethyl diphenylborinate, Merck, Germany) and 1% ceric sulfate (Merck, Germany) solution in 10% sulfuric acid (Mojalali, Iran), followed by heating using hair dryer for about 2-3 minutes. Column chromatography (CC) was performed on polyamide SC6 (Roth, Germany) and Sephadex-LH (Pharmacia fine chemicals, Uppsala, Sweden). CHCl₃ and MeOH (Merck, Germany) were used as elution solvents in developing columns.

2.2. Plant Material

The whole plant of A. colchicifolium was bought on April 2021 from local vegetable markets in Kermanshah province, Iran. The plant was identified by Prof. Seyed Mohammad Masoumi (Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran) and confirmed by comparison to the voucher specimen (No. 160-036-001-001) already deposited in the Department of Pharmacognosy, Faculty of Pharmacy, Kermanshah University of Medical Sciences. Also, its morphological characteristics, including the arrangement of inflorescences, the colour of flowers, and the shape of its leaves and bulbs, were consistent with the information provided by Fritsch and Abbasi [28, 31].

2.3. Extraction and Isolation

The air-dried leave powders (7 kg) of the A. colchicifolium were extracted at room temperature with hexane, CHCl₃, CHCl₃/MeOH (9 : 1), and MeOH (each 20 L for 3 days), consecutively. To extract the flavonoid content, the CHCl₃/MeOH extract (100 g) as a semipolar part was suspended in bicarbonate solution (1%, 1 L) and extracted by diethyl ether (1 L, two times) in a separation funnel to remove the lipophilic content. The pH of the aqueous solution was adjusted to 3.0 by adding HCl (0.2 N) and re-extracted using ethyl acetate (1 L, three times) [32, 33]. The ethyl acetate extract rich in phenolics was concentrated and submitted on polyamide SC6 column chromatography using a stepwise gradient solvent system of CHCl₃: MeOH (98 : 2, Fr.1; 96 : 4, Fr.2; 94 : 6, Fr.3; 92 : 8, Fr.4; 90 : 10, Fr.5; 88 : 12, Fr.6; 86 : 14, Fr.7; 84 : 16, Fr.8; 82 : 18, Fr.9; 80 : 20, Fr.10; each 500 mL). According to the TLC profile (SiO₂, CHCl3: MeOH, 9 : 1) visualized by natural product reagent (2-aminoethyl Diphenylborinate 1%), Fr.6 and Fr.7 with yellow spots were selected as fractions rich in flavonoids. Fr.6 was submitted on a Sephadex LH-20 column (3 × 80 cm; Methanol) in 10 mL collection size tubes from Fr.6.1 to Fr.6.4. Fr.6.2 was obtained in a pure state as compound 4 (58 mg). Fr.6 − 1, Fr.6 − 3, and Fr.6 − 4 were recrystallized in cold methanol to take compounds 1 (21 mg), 2 (25 mg), and 3 (15 mg). Fr.7 was submitted on the same column and yielded compound 5 (32 mg).

2.4. Antibacterial, Antibiofilm, and Antiswarming Activities

2.4.1. Microbial Strains and Culture Maintenance

Staphylococcus aureus (ATCC 25923) and Proteus mirabilis (ATCC 7002) as a member of gram-positive and gram-negative bacteria were chosen and provided by the Microbiology Department of Medical Sciences University of Isfahan, Iran. Strains were cultured on blood, eosin-methylene blue (EMB), and MacConkey agar and enriched in trypticase soy broth (TSB) and trypticase soy agar (TSA) agar (Himedia, India) plates at 37°C.

2.4.2. Antibacterial Activities

The antibacterial activity of the crude extract and isolated flavonoids was assayed by the minimum inhibitory concentration (MIC) values using the broth microdilution method with 96-well microtiter plates according to Mirzaei et al. [34]. Two-fold serial dilutions of the samples (500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.9, and 1.9 μg/ml) were prepared in 1 ml TSB. The inoculum of the strains was prepared in TSB-containing glucose (1% w/v) (Himedia, India), and the turbidity was adjusted to 0.5 McFarland and 1/1000 diluted to obtain final turbidity, approximately 0.5 × 10⁵colony-forming units (CFU)/ml. 75 μl of the solution of the compounds and 75 μl of the bacterial inoculum were inserted into the wells of a microtiter plate and incubated at 37°C for 24 h. The results of MIC were visually assayed, and the minimum concentration of bacteria growth was reported as MIC [34]. To find the exact dilution for MIC₅₀, an ELISA microplate reader evaluated the bacterial growth at 595 nm. The results were reported as MIC₅₀, which is equivalent to the minimum concentration that inhibits 50% of the bacterial growth [35].

2.4.3. Antibiofilm Activities

The antibiofilm activities of the crude extract and isolated flavonoids were investigated by the modified crystal violet assay. Twofold serial dilutions of the compounds in the LB broth were prepared from 500 μg/ml to 1.96 μg/ml in sterile 96-well microtiter plates. A 10 μl of overnight incubation strains suspension (0.5 McFarland) was added to each well containing 100 μl LB broth and different concentrations of crude extracts and the flavonoid compounds. Then, they were incubated at 37°C for 48 h. The positive control included LB media with bacteria, and the negative control included LB media without bacteria and compounds. After incubation, the wells were rinsed with 200 μl of phosphate-buffered saline to remove the loosely attached bacteria. After that, the wells were fixed with 200 μl of 96% ethanol for 15 minutes. The wells were dried for 30 min at 37°C, and biofilms formed by adherent cells in the wells were stained with 1% (w/v) crystal violet for 10 min. Then excess crystal violet was gently washed five times with deionized water. Optical densities (OD₅₉₅) of the stained adherent bacteria were determined after adding 200 μl acetone-ethanol (33%–80%) with the proportion of 1 : 1 using an ELISA microplate reader, and absorbance was recorded. The antibiofilm activity ratio of the compounds was calculated with the following formula and reported as MIBC₅₀ equivalent to the minimum concentration inhibiting 50% of bacterial biofilm formation [35]. The positive control was the untreated bacteria. The test was done in triplicate.

2.4.4. Antiswarming Activities

For antiswarming effects, various concentrations of the crude extract and isolated flavonoids ((500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.9, and 1.9 μg/ml) were mixed with 10 ml of the LB agar medium containing overnight cultures of P. mirabilis. After incubating the plates at 37°C for 3 days, the inhibition of swarming activities was recorded by measuring the area of the colonies in mm [34, 36].

2.5. Antioxidant Activities

2.5.1. DPPH Assay

The antioxidant activity of the samples was measured by the ability of bleaching of the purple-coloured methanol solution of DPPH [37]. The different concentrations of the crude extract and test compounds were prepared by serial dilution in methanol. Then, 1 ml of DPPH (0.5 mM in methanol) was added with 2 ml of 0.1 M sodium acetate buffer (pH 5.5). The mixtures were vigorously shaken and kept at room temperature in a dark place for 30 min. The absorbance was measured at 517 nm against the blank (containing all reagents except the test compound). The ascorbic acid was used as a positive control. The percentage of scavenging (% scavenging) of DPPH radical was calculated using the following equation:

In this formula, A0 is the absorbance of the blank and as is the absorbance of the test compounds. The IC50 value represents the concentration of the test compounds causing 50% scavenging.

2.5.2. Reducing Power

The FRAP assay was performed based on the total antioxidant capacity assay kit (Naxifer™, Navandsalamat, Iran). In brief, 250 μL working solution (acetate buffer (pH 3.6), 2,4,6-tripyridyl-s-triazine (TPTZ, 40 mM) solution, and FeCl₃·6H₂O solution (20 mM) (5 : 1:1, v/v/v)) was added to 5 μL of compounds (1–5) and the crude extract and kept at 37°C for 5 minutes. Then, the absorbance was measured at 593 nm using a microplate reader. The standard calibration curve of the FeSO₄ was obtained at different concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1 μM), and the total antioxidant capacity of the compounds was calculated as FeSO₄μM equivalent. All determinations were done in triplicate and compared with butylated hydroxyanisole as a positive control [38].

2.6. Molecular Docking

The X-ray crystal structures of the two target proteins staphylococcal accessory regulator A (SarA) (PDB ID: 2FNP) and mannose-resistant Proteus-like fimbriae H (MrpH) (PDB ID: 6Y4E) were downloaded from the Protein Data Bank (PDB; https://www.RCSB.org) [39]. The cocrystalized ligand, cofactor, and water molecules were removed. Polar hydrogens were added by AutoDock tools version 1.5.6, and energy minimization was performed using the Molegro Virtual Docker [40]. To prepare the structures of the ligands generated by ChemScketch, adding hydrogens and energy minimization were done using Chimera software [41]. To identify the binding site of SarA, the Computed Atlas of Surface Topography of proteins (CASTp) server (https://sts.bioe.uic.edu/castp/) was utilized. The residues of the predicted binding site include Phe110, Leu113, Ser114, Thr117, Tyr118, Lys121, Glu223, and Leu224 in chain A and Thr141, Tyr142, Glu145, Asn146, His159, and Leu160 in chain B. The binding site of MrpH was determined based on its cocrystallographic ligand (Arg118, Thr116, Ile140, His74, Asn82, His72, His117, and Gly81) [42]. Docking was conducted using AutoDock Vina in PyRx 0.8. Grid boxes were set with dimensions (Angestrom) of X = 28.2522, Y = 24.6015, and Z = 25.7223 and coordinates of X = −5.8249, Y = −8.0013, and Z = −1.8428 and with dimensions of X = 23.2163, Y = 22.5185, andZ = 20.4395 and coordinates of X = 4.7575, Y = 8.4108, and Z = −6.4855 for SarA and MrpH, respectively. After docking calculations, LigPlot+ was used to analyze protein-ligand interactions [43].

2.7. In Silico ADMET Studies

ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties are dominant factors that should be evaluated in the early stages of drug discovery [44]. In this study, the physiochemical properties, including the molecular weight, the number of H-bond receptors and H-bond donors, TPSA (topological polar surface area), and lipophilicity (MLOGP) and pharmacokinetic parameters including gastrointestinal (GI) absorption, blood-brain barrier (BBB) permeability, P-glycoprotein (P-gp) substrate, and CYP450 2C9 (CYP2C9) inhibitor of isolated flavonoids (compound 1–5) were predicted by SwissADME web tool (https://www.swissadme.ch). Also, drug likeness was investigated according to Lipinski’s rule of 5 (Ro5). Drug likeness assesses the chances for a molecule to become an oral drug with respect to bioavailability [45, 46]. Besides, toxicity, carcinogenicity, and cardiotoxicity endpoint such as human ether-a-go-go-related gene (HERG) inhibition of compounds (1–5) were predicted using the admetSAR database (https://lmmd.ecust.edu.cn/admetsar1/) [47].

3. Results and Discussion

3.1. Identification of the Isolated Pure Compounds

Edible plants, including Allium spp, have long been the focus of researchers to discover new medicinal molecules due to their high safety. They could be used in the food industry as safe and low-risk preservatives, one of the richest natural sources of polyphenols, especially flavonoids [48–50]. Therefore, in this study, the extraction, purification, and phytochemical investigation of flavonoids in A. colchicifolium, widely used for nutritional and therapeutic purposes, have been addressed. In the present study, the CHCl₃/MeOH extract (9 : 1) with a yield of 12% was selected for phytochemical analysis, which resulted in the isolation and identification of five flavonoids (Figure 1). Compound 1 was obtained as a pale-yellow solid with a positive reaction to flavonoid natural product reagent (2-aminoethyl diphenylborinate 1%). The ¹H-NMR spectra showed five protons in the aromatic region, two meta-coupled doublets at δ_H 6.31 (1H, d, J = 2.02 Hz) and 6.60 (1H, d, J = 2.02 Hz) corresponding to H-6 and H-8, two ortho-coupled proton signals at δ_H 7.06 (1H, d, J = 8.41 Hz) and 7.80 (1H, dd, J = 8.41, 2.06 Hz) related to H- 5′ and H-6′, and a doublet at δ_H 7.87 (1H, d, J = 2.06 Hz) related to H-2′ proton, as well as a methoxy resonance at δ_H 3.95 (3H, s), with HMBC correlation with C-3′ (δ_C 135.77). It also showed signals of four hydroxy groups at δ_H 9.57, 9.89, 10.95, and 12.58 that corresponded to 3-OH, 4ʹ-OH, 7-OH, and 5-OH, respectively. LC-MS/MS analysis showed molecular ions (m/z) at 314 (M-H)⁻, 284 (M-H-OCH3), 271 (M-H-OH-CO), and 257 (M-H- OH -CO- CH3), and finally compound 1 was identified as 3′-O-methylquercetin in agreement with isorhamnetin aglycone isolated by Fattorusso and coworkers from A. ascalonicum [51]. Compound 2 showed NMR resonances similar to 1 but with no methoxy group in NMR spectra, which is in agreement with quercetin data in the literature [52]. Compound 3 showed a fragmentation pattern similar to flavonoids, including molecular ions (m/z) at 301 (M-H)⁻, 283 (M-H- OH), 229 (M-H-C2HO2-OH), and 151 (M-H- C8H6O3). The ¹H-NMR spectrum displayed two meta doublets at δ_H 6.17 (1H, d, J = 2.10) and 6.29 (1H, d, J = 2.10) related to H-8 and H-6 at ring A, with an ABX spin system of noncatechol types at δ_H 6.34 (1H, dd, J = 8.40, 2.30, H-5ʹ), 6.39 (1H, d, J = 2.30, H-3ʹ), and 7.22 (1H, d, J = 8.40, H-6ʹ) corresponding to H-5′, H-3′, and H-6′ of B ring, respectively. The ¹³C-NMR spectra showed 15 signals at δ_C 176.17 (C-4), 163.63 (C-7), 160.88 (C-5), 160.39 (C-2ʹ), 156.76 (C-9, 4ʹ), 148.98 (C-2), 136.20 (C-3), 131.68 (C-6ʹ), 109.15 (C-1ʹ), 106.74 (C-5ʹ), 103.50 (C-10), 102.87 (C-3ʹ), 97.99 (C-6), and 93.32 (C-8) similar to those reported for 2′,3,4′,5,7-pentahydroxyflavone (morin) [53], which was previously reported in other Allium species including A. cepa peel [54], A. ampeloprasum leaves [55], and A. nigrum and A. subhirsutum bulbs and aerial parts [56]. Compound 4 showed positive reaction to 2-aminoethyl diphenylborinate 1% (flavonoid natural product reagent), with UV spectrum absorption maxima at 251 and 356 nm, characteristic of flavone derivatives. The NMR spectrum of the aglycone moieties showed two meta-coupled doublets at δ_H 6.210 (1H, d, J = 2.14 Hz) and 6.43 (1H, d, J = 2.14 Hz) corresponding to H-6 and H-8, in addition to two ortho-coupled proton signals at δ_H 6.91 (1H, d, J = 8.37 Hz) and 7.49 (1H, dd, J = 8.37, 2.05 Hz) related to H-5′ and H-6′ as well as δ_H 7.95 (1H, d, J = 2.05 Hz) related to H-2′. ¹³C-NMR resonances at 100.78 (C-1″), 77.48 (C-5″), 76.42 (C-3″), 74.36 (C-2″), 69.81 (C-4″), and 60.59 (C-6″) ppm were correspondent to the glucopyranosyl group. Also, δ_c at 98.81, 93.77, and 177.40 ppm was correspondent to (C-6), (C-8), and (C-4), respectively. The glucosyl linkage at C-3 was confirmed by HMBC (Figure 2) correlations between anomeric sugar proton H-1″ as follows: δ_H 5.56 (1H, d, J = 7.6) and carbon C-3 (δ_C 132.89), and the configurations were deduced to be β-form based on coupling constants of 7.10 Hz. OCH3 resonance at δ_H 3.83 (3H, s) showed HMBC correlation with C-3′ (δ_C 149.37), and finally 4 was identified as 3′-O-methylquercetin-3-O-β-D-glucopyranoside [57]. It was further confirmed by the negative ESI mass spectrum (Figure 3) at m/z 477 (M-H)⁻, 447 (M-OCH3)⁻, and 315 (M-Glucosyl)⁻. The isorhamnetin 3-O- glucoside and other isorhamnetin glycoside derivatives were isolated before from Allium spp, including Allium macrostemon, Allium microdictyon, and Allium cepa [51, 58, 59].

LC-MS/MS analysis of compound 5 revealed a molecular ion at (M-H)⁻ 463 m/z. The fragmentation of 463 m/z resulted in the production of (M-H- glucose) 301 m/z, 283 m/z, 270 m/z, and 151 m/z, which is similar to the fragmentation of quercetin glucoside [60]. The ¹H-NMR spectra exhibited δ_H: 3.08–3.80 (overlapped, H-2″- H-6″) and the anomeric hydrogen of glucoside moiety at δ_H 5.46 (1H, d, J = 7.10, H-1ʹʹ) and 5 protons in the aromatic region in accordance with the quercetin aglycone [61]. The ¹³C-NMR showed similarities to 4 except for lack of methoxy group. Therefore, it was identified as quercetin 3-O-β-D-glucopyranoside in agreement with the literature [62], which is reported in other Allium species including A. Macrostemon and A. myrianthum [58, 63]. Compound 1: yellow powder, ¹H-NMR in DMSO-d6 (400 MHz) ppm δ_H: 3.95 (3H, s 3ʹ-OCH3), 6.31 (1H, d, J = 2.02, H-6), 6.60 (1H, d, J = 2.02, H-8), 7.06 (1H, d, J = 8.41, H-5ʹ), 7.80 (1H, dd, J = 8.41, 2.06, H-6ʹ), 7.87 (1H, d, J = 2.06, H-2ʹ), 9.57 (1H, bs, 3-OH), 9.89 (1H, bs, 4ʹ-OH), 10.95 (1H, bs, 7-OH), 12.58 (1H, bs, 5-OH); ¹³C-NMR (100 MHz, DMSO-d6) δ_C: 175.83 (C-4),165.87 (C-7), 160.63 (C-5), 156.11 (C-9), 148.78 (C-3ʹ), 147.31 (C-4ʹ), 135.77 (C-3), 121.91 (C-6ʹ), 121.66 (C-1ʹ), 115.47 (C-5ʹ), 111.58 (C-2ʹ), 102.97 (C-10), 98.16 (C-6), 93.56 (C-8), and 55.69 (3ʹ-OCH₃). Negative ESI mass (m/z): (M-H)⁻ 314 m/z, 284 m/z, 271 m/z, 257 m/z, and 151 m/z. Compound 2: pale yellow powder, ¹H-NMR in DMSO-d6 (400 MHz) ppm δ_H: 6.20 (1H, d, J = 1.75 Hz, H-6), 6.43 (1H, d, J = 1.75 Hz, H-8), 6.92 (1H, d, J = 9.92 Hz, H-5ʹ), 7.58 (1H, dd, J = 9.92, 1.7 Hz, H-6ʹ), 7.70 (1H, d, J = 1.7 Hz, H-2ʹ), 12.49 (1H, bs, 5-OH); ¹³C-NMR (100 MHz, DMSO-d6) δ_C: 175.52 (C-4),164.43 (C-7), 160.63 (C-5), 156.13 (C-9), 147.75 (C-4ʹ), 146.63 (C-2), 145.08 (C-3ʹ), 135.62 (C-3), 121.87 (C-6ʹ), 119.90 (C-5ʹ), 115.60 (C-2ʹ), 114.98 (C-1ʹ), 102.73 (C-10), 98.29 (C-6), and 93.38 (C-8). Negative ESI mass (m/z): (M-H)⁻ 301 m/z, 285 m/z, 271 m/z, 255 m/z, 243 m/z, 155, and 151 m/z. Compound 3: yellow powder, ¹H-NMR in DMSO-d6 (400 MHz) ppm δ_H: 6.17 (1H, d, J = 2.10, H-6), 6.29 (1H, d, J = 2.10, H-8), 6.34 (1H, dd, J = 8.40, 2.30, H-5ʹ), 6.39 (1H, d, J = 2.30, H-3ʹ), 7.22 (1H, d, J = 8.40, H-6ʹ), 12.62 (1H, s, 5-OH), 10.73 (1H, s, 7-OH); ¹³C-NMR (100 MHz, DMSO-d6) δ_C: ¹³C-NMR (101 MHz, DMSO-d6) δ 176.17 (C-4), 163.63 (C-7), 160.88 (C-5), 160.39 (C-2ʹ), 156.76 (C-9, 4ʹ), 148.98 (C-2), 136.20 (C-3), 131.68 (C-6ʹ), 109.15 (C-1ʹ), 106.74 (C-5ʹ), 103.50 (C-10), 102.87 (C-3ʹ), 97.99 (C-6), and 93.32 (C-8). Negative ESI mass (m/z): (M-H)⁻ 301 m/z, 283 m/z, 229 m/z, and 151 m/z. Compound 4: yellow powder, ¹H-NMR in DMSO-d6 (400 MHz) ppm δ_H: 3.10–3.80 (overlapped, H-2″- H-6″), 3.83 (3H, s, 3ʹ-OCH3), 5.57 (1H, d, J = 7.10, H-1ʹʹ), 6.20 (1H, d, J = 2.14, H-6), 6.43 (1H, d, J = 2.14, H-8), 6.91 (1H, d, J = 8.37, H-5ʹ), 7.49 (1H, dd, J = 8.37, 2.05, H-6ʹ), 7.95 (1H, d, J = 2.05, H-2ʹ), 12.62 (1H, s, 5-OH); ¹³C-NMR (100 MHz, DMSO-d6) δ_C: 177.40 (C-4),164.45 (C-7), 161.24 (C-5), 156.44 (C-2), 156.28 (C-9), 149.42 (C-3ʹ), 146.90 (C-4ʹ), 132.95 (C-3), 122.04 (C-6ʹ), 121.10 (C-1ʹ), 115.23 (C-5ʹ), 113.48 (C-2ʹ), 103.97 (C-10), 100.78 (C-1ʹʹ), 98.81 (C-6), 93.77 (C-8), 77.48 (C-5″), 76.42 (C-3″), 74.36 (C-2″), 69.81 (C-4″), 60.59 (C-6″), and 55.68(3ʹ –OCH₃). Negative ESI mass (m/z): (M-H)⁻ 477, 314, 299, 284, 271, 257, 243, and 151. Compound 5: yellow powder, ¹ H-NMR in DMSO-d6 (400 MHz) ppm δ_H: 3.08–3.80 (overlapped, H-2″- H-6″), 5.46 (1H, d, J = 7.10, H-1″), 6.19 (1H, d, J = 2.10, H-6), 6.39 (1H, d, J = 2.10, H-8), 6.83 (1H, d, J = 8.86, H-5ʹ), 7.57(1H, dd, J = 8.86, 2.40, H-6ʹ), 7.58 (1H, d, J = 2.40, H-2ʹ), 12.64 (1H, s, 5-OH). ¹³C-NMR (100 MHz, DMSO-d6) δ_C: 177.39 (C-4),164.22 (C-7), 161.18 (C-5), 156.31 (C-2), 156.15 (C-9), 148.43 (C-4ʹ), 144.78 (C-3ʹ), 121.58 (C-6ʹ), 121.11 (C-1ʹ), 116.12 (C-5ʹ), 115.17 (C-2ʹ), 133.24 (C-3), 103.88 (C-10), 100.78 (C-1″), 98.67 (C-6), 93.52 (C-8), 77.49 (C-5″), 76.42 (C-3″), 74.04 (C-2″), 69.85 (C-4ʹʹ), and 60.88 (C-6″). Negative ESI mass (m/z): (M-H)⁻ 463, 301, 283, 271, and 151.

3.2. Antibacterial, Antibiofilm, and Antiswarming Activities

The result of antibacterial and antibiofilm activities of the crude extract and isolated flavonoids is shown in Table 1. Our results revealed that the crude extract and compounds 3 and 4 showed the highest antibacterial activities against S. aureus with MIC₅₀ of 25 ± 2.23 μg/ml, 60 ± 3.70 μg/ml, and 73.50 ± 3.20 μg/ml and against P. mirabilis with MIC₅₀ of 37 ± 3.11 μg/ml, 97.50 ± 6.80 μg/ml, and 133 ± 6.02 μg/ml, respectively. Among the isolated flavonoids, compound 3 showed the most potential against S. aureus biofilm formation (MIBC₅₀ = 79 ± 5.02 μg/ml) and compound 5 exhibited the most against P. mirabilis biofilm formation with (MIBC₅₀ = 102 ± 11.01 μg/ml), which was less than the crude extract (Table 1). Besides, our observation showed that compounds (1–5) have no considerable effects on the antiswarming activities of P. mirabilis although the total extract partially inhibited the swarming of P. mirabilis (Figure 4). Flavonoids show antibacterial activities by disrupting bacterial membrane, obstructing bacterial nucleic acid replication, reducing bacterial efflux pump activity, and blocking adenosine triphosphate synthase of bac teria [64, 65]. The presence of hydroxyl groups at positions 5 and 7 and the simultaneous presence of hydroxyl groups in the 2′ and 4′ positions, such as compound 3, enhance the antibacterial activities of flavonoids. Besides, the presence of glycoside groups attached at position 3, such as compound 4, increases the effectiveness of flavonoids against bacteria compared to their aglycone form (compound 1) [66, 67]. Also, compound 5 had the most significant effect on preventing biofilm formation by P. mirabilis, which is confirmed by the results of other studies [68].

On the other hand, the formation of biofilm as the production of an extracellular polymer layer by bacteria to adhere to each other and surfaces is a bacterial defence mechanism that increases their resistance to antibacterial agents and causes chronic infections [69]. Flavonoids reveal antibiofilm effects by penetrating biofilm layers and inhibiting bacterial growth and surface adhesion. The presence of a hydrophilic part in the chemical structure of flavonoids, including glycoside and hydroxy groups, such as compound 5, improves penetration in the biofilm structure and increases antibiofilm activities [64]. However, it seems that compound 3 showed more substantial antibiofilm effects on S. aureus by inhibiting growth and killing the bacteria [70]. On the other hand, the higher antibacterial and antibiofilm activities of the crude extract can be related to the presence of other compounds with more potential effects and the synergistic effects of the crude extract compounds [71].

3.3. Antioxidant Activities

All compounds showed prominent antioxidant activities (Table 2 and Figure 5). The DPPH scavenging IC50 of the crude extract and compounds 3 and 2 was 0.60 ± 0.05 μg/ml, 0.90 ± 0.03, and 1.02 ± 0.03 μg/ml, respectively, more than the ascorbic acid as a positive control (IC50 = 1.21 ± 0.09 μg/ml). The DPPH scavenging IC50 (μg/ml) of compounds 4 and 5 was 1.61 ± 0.1 μg/ml and 1.26 ± 0.04 μg/ml, respectively, lower than that of other compounds, crude extract, and ascorbic acid. The higher free radical scavenging potential of compounds 2 and 3 compared to compounds 4 and 5 can be due to the presence of more free hydroxyl groups in their structures [72]. Besides, the crude extract and compound 2 revealed more FRAP values than other compounds and BHA as a positive control in all assayed doses (Figure 5). The FRAP method can show the power of antioxidant compounds in increasing the total antioxidant capacity of serum. The FRAP method principle is based on reducing Fe³⁺ to Fe²⁺ in the presence of antioxidants. Therefore, the reducing power directly relates to the electron-donating properties of antioxidant compounds. The presence of the hydroxyl group in the 5, 2′, and 3′ positions of compound 2 strengthens the resonance power and enhances electron-donating properties, thereby creating a more stable flavonoid radical of compound 2 against active free radicals [72, 73]. Also, our antioxidant results confirmed with the Nantitanon and Okonogi study. Their study showed that the ability to scavenge free radicals and the reducing power of quercetin is greater than morin, which is related to the more electron-donating properties of quercetin [74].

The synergistic effect of the compounds in the crude extract, as well as the possible presence of strong electron-donating compounds in it, has enhanced the potential power of the antioxidant effect of the crude extract compared to the isolated compounds [26].

3.4. Molecular Docking Results

3.4.1. Molecular Docking of SarA

SarA has a critical role in biofilm formation by Staphylococcus aureus [75]. As shown in Table 3, compound 1 with a binding affinity of −7.90 kcal/mol formed 4 hydrogen bonds (Table 3). O2 and O3 in the ether and the hydroxyl groups formed hydrogen bonds with His159 and Tyr142 in chain B, respectively (bond length: 3.01 and 2.70 Å, respectively). O5 and O6 in the hydroxyl groups formed hydrogen bonds with Ser114 and Glu223 in chain A, respectively (bond length: 2.85 and 2.76 Å, respectively). Moreover, Lys121, Leu224, Thr117, and Phe110 in chain A and Glu145 and Asn146 in chain B are involved in these interactions hydrophobically. Compound 2 formed three hydrogen bonds with a binding affinity of −7.8 kcal/mol bound to the SarA. O5 and O6 in hydroxyl groups formed hydrogen bonds with Ser114 and Glu223 in chain A (bond length: 2.92 and 2.75 Å, respectively). Also, a hydrogen bond was found between O1 in the ether group and His159 in chain B (bond length: 3.09 Å). This compound interacted with Lys121, Leu224, Thr117, and Phe110 in chain A and with Tyr142, Glu145, and Asn146 in chain B. Compound 3, with a binding affinity of −8.10 kcal/mol, formed 4 hydrogen bonds (Table 3, Figure 6(a)). O6 in the hydroxyl group formed two hydrogen bonds with Lys121 and Glu223 (chain A) with bond lengths of 3.12 and 2.81 Å, respectively. Two hydrogen bonds were found between O5 in the hydroxyl group and Ser114 (chain A) (bond length: 2.81 Å) and O1 in the ether group and His159 (chain B) (bond length: 3.13 Å). Thr117, Phe110, and Leu224 in chain A and Glu145, Asn146, and Tyr142 in chain B are involved hydrophobically in the binding. Compound 4 with a binding affinity of −8.1 kcal/mol bound to SarA (Table 3). This compound, through O2 in the ether group and O8 in the hydroxyl group, formed two hydrogen bonds with His 159 (chain B) (bond length: 2.98 and 3.12 Å respectively; Figure 6(b)). Also, O1 in the carbonyl group and O4 in the ether group formed hydrogen bonds with Lys121 (chain A) (bond length: 2.80 and 2.91, respectively). Two other hydrogen bonds were found between O11 and O5 in the hydroxyl groups and Ser114 and Glu223 (chain A) with bond lengths of 3.23 and 3.10 Å, respectively. In addition, this compound hydrophobically interacted with Leu113, Phe110, Leu224, and Thr117 (chain A). Compound 5 has a binding affinity of −7.70 kcal/mol bound to the SarA binding site (Table 3). O1, O4, and O8 in the carbonyl, ether, and hydroxyl groups formed three hydrogen bonds with Lys121 (chain A) (bond length: 2.80, 2.89, and 3.24 Å, respectively). O5 and O11 (in the hydroxyl groups) formed hydrogen bonds with Glu223 and Ser114 (in chain A) with bond lengths of 3.05 and 3.25 Å, respectively. In addition, a hydrogen bond was found between O2 (in the ether group) and His159 (in chain B) (bond length: 3.00 Å). Also, this compound formed several hydrophobic interactions (Leu113, Phe110, Leu224, and Thr117; chain A). The results obtained from the antibiofilm effects of compounds isolated from A. colchicifolium on S. aureus indicate that compounds 3 and 4 (Table 1) are more effective than other compounds, which are consistent docking study results of these compounds. Therefore, the strong interaction of compounds 3 and 4 with SarA is probably one of the most important mechanisms for preventing the biofilm formation of these compounds. The study conducted by Chemmugil et al. also showed the interaction of morin (compound 3) with SarA [70].

(a)

(b)

3.4.2. Molecular Docking of MrpH

MrpH, a new member of mannose-resistant/Proteus-like (MR/P) fimbriae (MR/P), has a vital role in the biofilm formation by P. mirabilis [76]. Compound 1, with a binding affinity of −5.50 kcal/mol, bound to SarA and hydrophobically interacted with Asn82, Arg118, and Thr116 in the binding site (Table 3). Compound 2 with a binding affinity of −5.70 kcal/mol bound to the binding site of MrpH (Table 3). O5 in the hydroxyl group formed two hydrogen bonds with Thr116 (bond length: 3.16 and 2.87 Å). Also, Asn82, Arg118, and His72 are hydrophobically involved in the binding. Compound 3 with a binding affinity of −5.50 kcal/mol bound to the binding site of MrpH (Table 3). O5 and O7 in the hydroxyl groups formed hydrogen bonds with Asn82 and Thr116 (bond length: 3.16 and 2.96 Å, respectively). This compound hydrophobically interacted with Arg118. Compound 4 with a binding affinity of −5.80 kcal/mol bound to MrpH (Table 3). This compound formed 6 hydrogen bonds, but only one of them was in the binding site (Asn82 with O8 in the hydroxyl group; bond length: 3.14 Å). It interacted with Thr116 and Arg118 hydrophobically. Compound 5, with higher affinity (−6.20 kcal/mol) compared to others, bound to the binding site of MrpH (Table 3) and formed 4 hydrogen bonds with Asn82 by O6, O8, and O10 in the hydroxyl groups and O4 in the ether group (bond length: 3.24, 3.31, 2.84, and 3.10 Å, respectively; Figure 7). Also, a hydrogen bond was found between His74 and O12 (in the hydroxyl group; bond length: 3.08 Å). Moreover, it hydrophobically interacted with Arg118 and Ile140. The results obtained from the studies of the antibiofilm activities of compounds isolated from A. colchicifolium on P. mirabilis indicated that compound 5 was more effective than other compounds, which is consistent with the results of the strong interaction of this compound with MrpH. So far, no study has been reported on the interaction of the present study compounds with MrpH.

3.5. ADMET Prediction

The results of the compound’s ADMET properties are shown in Tables 4 and 5. Compounds 1, 2, and 4, based on Lipinski’s rule of 5 (Ro5) [46], are drug likeness, i.e., these compounds have molecular weight (MW) < 500, number of H-bond acceptors (HBA) ≤ 10, number of H-bond donors (HBD) ≤ 5, and MLOGP (lipophilicity) < 4.15. Compounds 4 and 5 with two violations (NorO > 10, NHorOH > 5) are not drug likeness. Also, compounds 1, 2, and 3 have topological polar surface area (TPSA) < 140 Å2, which indicates good absorption in the intestine [77]. However, none of these compounds can pass from BBB. Among them, only compound 4 is the P-gp substrate. P‐gp mediates the export of a wide variety of chemically diverse compounds out of cells, consequently resulting in limited bioavailability. Also, all of them are noncarcinogens and a weak inhibitor of hERG. The inhibition of hERG channel (cardiac potassium channel) is associated with the prolongation of QT interval and arrhythmia [78]. Taken together, compounds 1, 2, and 3 as flavonoid aglycones have better physicochemical and pharmacokinetic properties.

4. Conclusion

In addition to the nutritional value, Allium plants often have a special place in traditional and complementary medicine in the world. The present study is the first report on the phytochemical investigation and antibacterial, antibiofilm, and antioxidant effects of the A. colchicifolium as a native plant in the western regions of Iran from the Allium genus. In this study, three flavonoid aglycones, including isorhamnetin (compound 1), quercetin (compound 2), and morin (compound 3), and two glucosylated flavonoids, including isorhamnetin-3-O-glucoside (compound 4) and quercetin 3-O-glucoside (compound 5), were isolated and purified from the CHCl₃/MeOH (9 : 1) extract of A. colchicifolium leaves for the first time. On the other hand, compounds 3 and 5 and the total CHCl₃/MeOH (9 : 1) extract had more antibacterial and antibiofilm effects on two pathogenic bacteria, S. aureus and P. mirabilis. Also, in this study, the interaction of the isolated compounds with two effective proteins in bacterial biofilm formation of SarA and MrpH was investigated. Compounds 3 and 4 showed a strong interaction (−8.1 Kcal/mol) with SarA, and compound 5 showed the highest interaction (−6.2 Kcal/mol) with MrpH. The isolated flavonoids showed low antiswarming activities. Compounds 2 and 3 showed high antioxidant activities in DPPH and FRAP assay. Besides, the ADMET prediction assay showed high GI absorption and safety for compounds 1, 2, and 3. According to the obtained results, the flavonoids in the A. colchicifolium leaves can be promising candidates for their use in the pharmaceutical and food industries as antibacterial and antioxidant compounds with natural sources. However, more comprehensive research studies in this regard are suggested for the future.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon reasonable request.

Disclosure

This work was part of the thesis of Mohammad Bagher Majnooni (Pharmacognosy Ph.D. student), Kermanshah University of Medical Sciences, funded with grant number (no. 4000661).

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors greatly appreciate the Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Iran, for their valuable technical assistance.

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