In Vitro and In Silico Screening of Anti-Vibrio spp., Antibiofilm, Antioxidant and Anti-Quorum Sensing Activities of Cuminum cyminum L. Volatile Oil

Cuminum cyminum L. essential oil (cumin EO) was studied for its chemical composition, antioxidant and vibriocidal activities. Inhibition of biofilm formation and secretion of some virulence properties controlled by the quorum sensing system in Chromobacterium violaceum and Pseudomonas aeruginosa strains were also reported. The obtained results showed that cuminaldehyde (44.2%) was the dominant compound followed by β-pinene (15.1%), γ-terpinene (14.4%), and p-cymene (14.2%). Using the disc diffusion assay, cumin EO (10 mg/disc) was particularly active against all fifteen Vibrio species, and the highest diameter of growth inhibition zone was recorded against Vibrio fluvialis (41.33 ± 1.15 mm), Vibrio parahaemolyticus (39.67 ± 0.58 mm), and Vibrio natrigens (36.67 ± 0.58 mm). At low concentration (MICs value from 0.023–0.046 mg/mL), cumin EO inhibited the growth of all Vibrio strains, and concentrations as low as 1.5 mg/mL were necessary to kill them (MBCs values from 1.5–12 mg/mL). Using four antioxidant assays, cumin EO exhibited a good result as compared to standard molecules (DPPH = 8 ± 0.54 mg/mL; reducing power = 3.5 ± 0.38 mg/mL; β-carotene = 3.8 ± 0.34 mg/mL; chelating power = 8.4 ± 0.14 mg/mL). More interestingly, at 2x MIC value, cumin EO inhibited the formation of biofilm by Vibrio alginolyticus (9.96 ± 1%), V. parahaemolyticus (15.45 ± 0.7%), Vibrio cholerae (14.9 ± 0.4%), and Vibrio vulnificus (18.14 ± 0.3%). In addition, cumin EO and cuminaldehyde inhibited the production of violacein on Lauria Bertani medium (19 mm and 35 mm, respectively). Meanwhile, 50% of violacein inhibition concentration (VIC50%) was about 2.746 mg/mL for cumin EO and 1.676 mg/mL for cuminaldehyde. Moreover, elastase and protease production and flagellar motility in P. aeruginosa were inhibited at low concentrations of cumin EO and cuminaldehyde. The adopted in-silico approach revealed good ADMET properties as well as a high binding score of the main compounds with target proteins (1JIJ, 2UV0, 1HD2, and 3QP1). Overall, the obtained results highlighted the effectiveness of cumin EO to prevent spoilage with Vibrio species and to interfere with the quorum sensing system in Gram-negative bacteria by inhibiting the flagellar motility, formation of biofilm, and the secretion of some virulence enzymes.


Introduction
Infectious diseases, reinforced by the emergence of antibiotic resistant pathogens are known as a high leading cause of death in the world lead causing higher mortality and morbidity and increased healthcare costs [1,2]. Antimicrobial resistance (AMR) represents the acquired ability of pathogens to withstand antimicrobial treatment is an increasing global concern results from the abuse and misuse of antibiotics have been recognized as  Table 1 summarized the phytochemical composition of cumin EO obtained by hydrodistillation technique of seeds. Twenty chemical compounds were identified representing 99.1% of the total identified phytoconstituents. This volatile oil was dominated by oxygenated monoterpenes (51.3%) and monoterpene hydrocarbons (46.7%). The main compounds identified in cumin EO were cuminaldehyde (42.4%), β-pinene (15.1%), γ-terpinene (14.4%), p-cymene (14.2%), and α-terpin-7-al (5.2%).  Table 2 summarizes the results of the antioxidant activities of cumin EO as compared to well-known standard molecules evaluated by using DPPH, reducing power, β-carotene, and chelating power assays. The obtained results reveal promising antioxidant activities at low concentrations as compared to ascorbic acid (AA), butylated hydroxytoluene (BHT), and butylate hydroxyanisole (BHA). In fact, IC 50 for the DPPH test was about 8 ± 0.54 mg/mL, 3.8 ± 0.34 mg/mL for the β-carotene test, and 8.4 ± 0.14 mg/mL for the chelating power test. Table 2. Antioxidant activities of cumin EO. The letters (a-c) indicate a significant difference between the different antioxidant methods according to the Duncan test (p < 0.05).

Antimicrobial Activity
The ability of the obtained cumin EO was tested against fifteen Vibrio species. Results revealed a bacteriostatic action of the tested oil (MBC/MIC ratio > 4). The growth of almost all Vibrio species on liquid media was inhibited at low concentrations ranging from 0.023 to 0.046 mg/mL. In addition, the same bacteria were completely killed by low concentration of cumin EO varying from 1.5 to 12 mg/mL. The mean diameter of growth inhibition zone obtained by the disc diffusion agar test at 10mg/disc confirms the high activity of cumin EO against almost all Vibrio species with mean diameter of inhibition zone (mZI) of approximately 34 Table 3.

Biofilm Inhibition
Cumin EO was tested for its ability to inhibit the biofilm formation on polystyrene 96 well-plate by four Vibrio species including V. cholerae, V. vulnificus, V. parahaemolyticus, and V. alginolyticus by using XTT technique. Results showed that the examined oil was able to inhibit the biofilm formation of the tested Vibrio species in a concentration-dependent manner. In fact, at 2xMIC, the inhibition was about 9.96 ± 1.00% against V. alginolyticus ATCC 33787) and 18.14 ± 0.30% against V. cholerae ATCC 9459. Interestingly, at 50 mg/mL, the highest percentage of biofilm formation inhibition was recorded for all strains reaching a percentage between 66.29 ± 3% (V. cholerae ATCC 9459) and 76.29 ± 4%. All these data are summarized in Figure 1.

Qualitative and Quantitative Violacein Inhibition Estimation
The ability of cumin EO and its major compound (cuminaldehyde) to inhibit the production of violacein by C. violaceum CV026 was tested at 2 mg/mL ( Figure 2). The inhibition zone of the EO was about 32 mm and about 35 mm for its main compound (cuminaldehyde). Meanwhile, the anti-QS sensing zone of cuminaldehyde was interestingly higher than the EO (35 mm and 19 mm, respectively). More interestingly, quantitative estimation on the effect of various concentration of cumin volatile oil on the growth of C. violaceum, showed a MIC value about 5 mg/mL and the VIC50% was about 2.746 mg/mL. Meanwhile, for the main compound (Cuminaldehyde), MIC and VIC50% values were about 1.25 mg/mL about 1.676 mg/mL, respectively.

Qualitative and Quantitative Violacein Inhibition Estimation
The ability of cumin EO and its major compound (cuminaldehyde) to inhibit the production of violacein by C. violaceum CV026 was tested at 2 mg/mL ( Figure 2). The inhibition zone of the EO was about 32 mm and about 35 mm for its main compound (cuminaldehyde). Meanwhile, the anti-QS sensing zone of cuminaldehyde was interestingly higher than the EO (35 mm and 19 mm, respectively).

Qualitative and Quantitative Violacein Inhibition Estimation
The ability of cumin EO and its major compound (cuminaldehyde) to inhibit the production of violacein by C. violaceum CV026 was tested at 2 mg/mL ( Figure 2). The inhibition zone of the EO was about 32 mm and about 35 mm for its main compound (cuminaldehyde). Meanwhile, the anti-QS sensing zone of cuminaldehyde was interestingly higher than the EO (35 mm and 19 mm, respectively).  More interestingly, quantitative estimation on the effect of various concentration of cumin volatile oil on the growth of C. violaceum, showed a MIC value about 5 mg/mL and the VIC 50% was about 2.746 mg/mL. Meanwhile, for the main compound (Cuminaldehyde), MIC and VIC 50% values were about 1.25 mg/mL about 1.676 mg/mL, respectively.

Anti-Swarming Activity
The starter strain (P. aeruginosa PAO1) was used to test the effect of cumin EO and cumin aldehyde at different concentrations on its motility on semi-solid agar plates. The  Table 4. At 10 mm/mL, the motility of this bacterium was more inhibited by cuminaldehyde (by 70.99 ± 0.57%) as compared to the EO (64.20 ± 0.57%). At higher concentration (500 mg/mL), the percentage of motility inhibition was about 89.77 ± 0.00% for cuminaldehyde and 90.12 ± 0.57% for the cumin EO.

Elastase and Protease Inhibition
Pseudomonas aeruginosa is able to produce several virulence factors responsible for its pathogenecity like alkaline proteases, elastases, and collagenase. Our results showed that the obtained cumin EO and its main compounds are able to modulate the production of elastase and protease with different degree and in a concentration dependent manner ( Figure 3). In fact, cumin EO and cuminaldehyde decreased the production of protease by 68.32% and 71.09% respectively at 0.05 mg/mL. Similarly, at high concentration (2.5 mg/mL), cumin EO inhibited the production of protease by 82.14%, and by 83.43% for cuminaldehyde. More interestingly, cumin EO inhibited the production of elastase by 46.08% for cuminaldehyde and by 43.34% for the volatile oil. At 2.5 mg/mL, elastase production in P. aeruginosa PAO1 was inhibited by 63.14% and 62.12% respectively for cumin EO and its main compound (cuminaldehyde). The starter strain (P. aeruginosa PAO1) was used to test the effect of cumin EO and cumin aldehyde at different concentrations on its motility on semi-solid agar plates. The results obtained are summarized in Table 4. At 10 mm/mL, the motility of this bacterium was more inhibited by cuminaldehyde (by 70.99 ± 0.57%) as compared to the EO (64.20 ± 0.57%). At higher concentration (500 mg/mL), the percentage of motility inhibition was about 89.77 ± 0.00% for cuminaldehyde and 90.12 ± 0.57% for the cumin EO. Table 4. Swarming inhibition on Lauria Bertani (0.5% agar-agar) by cumin EO and cuminaldehyde. The letters (a-f) indicate a significant difference between the diameter of colony tested at different concentrations according to the Duncan test (p < 0.05).

Elastase and Protease Inhibition
Pseudomonas aeruginosa is able to produce several virulence factors responsible for its pathogenecity like alkaline proteases, elastases, and collagenase. Our results showed that the obtained cumin EO and its main compounds are able to modulate the production of elastase and protease with different degree and in a concentration dependent manner ( Figure 3). In fact, cumin EO and cuminaldehyde decreased the production of protease by 68.32% and 71.09% respectively at 0.05 mg/mL. Similarly, at high concentration (2.5 mg/mL), cumin EO inhibited the production of protease by 82.14%, and by 83.43% for cuminaldehyde. More interestingly, cumin EO inhibited the production of elastase by 46.08% for cuminaldehyde and by 43.34% for the volatile oil. At 2.5 mg/mL, elastase production in P. aeruginosa PAO1 was inhibited by 63.14% and 62.12% respectively for cumin EO and its main compound (cuminaldehyde).

ADMET Analysis
The in silico ADMET prediction of the selected major compounds (Table 5) revealed a good permeability on intestinal Caco-2 cells and is easy to be absorbed, with values in the range of 1.373-1.517, and high intestinal human absorption (above 94%), with only 16 and 17 exhibited low skin permeability. All phytocompounds were expected to not act on P-glycoprotein, are likely to cross the blood-brain barrier (BBB) with 9, 16 and 17 are able to slightly access to the central nervous system (CNS). Another important parameter used in distribution named distribution volume which characterize the distribution of drugs in various tissues in vivo. Predictive data showed that compound 4 was well distributed, 7 and 9 were moderately, but 16 and 17 were relatively lower distributed. Cytochrome P450s is an important enzyme system for drug metabolism in liver, with the most important where subtypes are CYP2D6 and CYP3A4. Results indicate that none of the selected compounds will be metabolized by the cytochrome P450s enzymes. Regarding toxicity parameters, our phytocompounds may not inhibit the hERG channel and have no AMES nor hepatotoxicity profile.

Molecular Docking Analysis
In order to assess the potential of cumin EO to inhibit the growth of pathogenic microorganisms and to reduce hydrogen peroxide and alkyl hydroperoxides, molecular docking study was performed to gain insight into the most preferred binding mode of compound into the enzyme binding active site. Ligands have been selected based on their abundance in the EO (%) and their lowest binding score.

Discussion
Cumin seeds are largely used as a flavoring and food preservative agent due to their richness in bio-compounds with a large array of biological activities [5].
In this study, the volatile oil extracted from cumin seeds by hydrodistillation is a rich source of cuminaldehyde (42.4%). In fact, it is well documented that the chemical composition of cumin seeds depends on several endogenous (cultivar, genetic traits) and exogenous factors (geographical region, harvesting time, and extraction procedures). Different percentages of cuminaldehyde were reported from cumin seeds around the word as summarized in Table 7. Table 7. Review of the chemical composition of C. cyminum EO from seeds.
[48]   [52] Our results revealed that the obtained EO was active against fifteen Vibrio species with different degrees. The diameter of growth inhibition zone ranged from 11 ± 00 mm (V. diazotrophicus ATCC 33466) to 41.33 ± 1.15 mm (V. fluvialis ATCC 33809). Cumin EO oil exhibited bacteriostatic activity against all Vibrio species with MICs and MBCs values ranging from 0.023-0.046 mg/mL and 1.5-12 mg/mL, respectively. Our results are in agreement with previous study who demonstrated that cumin EO is active against a wide spectrum of microorganisms [32,33,47]. More recently, it has been reported that cumin EO from Iran (cuminaldehyde 38.26%) was active against multidrug resistant Staphylococcus aureus (S. aureus) strains with MICs and MBCs values ranging from 5 to 10 and 10 to 20 µL/mL, respectively [38].
In addition, our results showed that cumin EO (Chemotype cuminaldehyde) was able to inhibit the biofilm formation of V. alginolyticus ATCC 33787, V. parahaemolyticus ATCC 17802, V. vulnificus ATCC 27962, and V. cholerae ATCC 9454 at MICs value ranging from 9.96 to 18.14%. The biofilm formation by these strains was highly inhibited at MBCs values, and at 50 mg/mL. Similar results have reported the effectiveness of EO from P. crispum, O. basilicum, M. spicata, C. carvi to inhibit the biofilm formation by the same strains [53,[67][68][69]. It has been also demonstrated that clove, garlic, and thyme volatile oils are able to inhibit the formation of biofilm by V. parahaemolyticus at 8xMIC (0.56% for clove, 0.16% for thyme, and 0.72% for garlic) after 30 min of application of the volatile oils. Cumin EO was described to inhibit the biofilm formation by clinical Klebsiella pneumoniae on semiglass lamellas [81] and the attachment of E. coli MTCC 40, Salmonella spp. MTCC 1163 and S. aureus MTCC 7443 strains on microtiter plate by 52.11% [82].
In this work, we evaluated the effect of cumin EO and cuminaldehyde to inhibit the production of violacein by C. violaceum using both qualitative and quantitative methods. Previous reports have shown that C. cyminum exhibited potent inhibition of violacein production in C. violaceum at low concentration (0.5 mg/mL of methanolic extract), as well as swarming and swimming motility in P. aeruginosa PAO1 at 60 µg/mL [83]. In addition, methanolic extract of cumin seeds was able to inhibit the violacein production by C. violaceum, exopolysaccharide production, flagellar motility, and biofilm formation [84].

Plant Material and Extraction Procedure
Cumin seeds (Cuminum cyminum L.) were purchased from a local market in August 2021. The taxonomic position was evaluated by Dr. Zouhair Noumi, University of Sfax, Tunisia (Voucher No: AN-0005). The volatile oil was extracted by using hydrodistillation technique [53].

Sceening of the Anti-Vibrio spp. Activity
Fifteen Vibrio species (17 bacteria) commonly isolated from aquatic environment ant their associated organisms were used in this study. Semi-quantitative disc diffusion technique on Mueller Hinton-1%NaCl Petri dishes was used to estimate the growth inhibition zone around sterile Whatmann disc impregnated with 10 mg of cumin EO [53,67,68]. For the experiment, Vibrio strains were grown on Mueller-Hinton supplemented with 1% NaCl. Fresh Petri dishes were inoculated using bacterial suspension (optical density was adjusted to 0.5 McFarland) by cotton swab technique. Sterile filter paper disks (6 mm in diameter, Biolife, Milan, Italy) were impregnated with 10 mg of cumin EO and then placed on the inoculated Petri dishes. After sitting overnight at 37 • C, the diameter of growth inhibition zone around the disks was estimated using a 1-cm flat ruler.
The determination of the lowest concentration able to inhibit the growth and/or to kill the tested Vibrio species was estimated by using microdilution technique as reviously described by Snoussi et al. [58]. In fact, a twofold serial dilution of cumin EO in DMSO-5% was prepared in 96-well plates, starting from 25 µL/mL (23.125 mg/mL), in Mueller-Hinton Broth-1% NaCl. Five microliters of microbial inoculum were added to each well containing 100 µL of the serially diluted volatile oil. After incubation at 37 • C, the minimum inhibitory concentration (MIC) was defined as the lowest concentration able to inhibit the growth of a specific microorganism. To determine the minimum bactericidal concentration (MBC), 3 µL from all the wells with no visible growth were point-inoculated in Mueller-Hinton (1% NaCl) agar. After 24 h of incubation, the concentration at which the Vibrio spp. strain presents no growth is recorded as the MBC value.

Evaluation of the Antioxidant Activities
The antioxidant activity experiments were carried out by using four different assays: DPPH, β-Carotene bleaching, and reducing/chelating power assays by using the protocols previously described Ghannay et al. [53].

Inhibition of Violacein
Chromobacterium violaceum (CV026) strain was selected to study the effect of cumin EO against the production of violacein by using disc diffusion assay on LB-agar Petri dishes (2 mm/disc). Twofold serial dilutions of cumin EO were prepared in 96-well plates starting from 5 mg/mL in LB broth and inoculated with C. violaceum ATCC 12472 [88].

Biofilm Inhibition
The ability of the tested cumin EO to inhibit the biofilm formation by four Vibrio species (V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae) on a 96 well plate was tested at different concentrations ranging from 2xMIC to 50 mg/mL by using the same protocol described by Ghannay et al. [53].

Effect on Flagellar Motility
Pseudomonas aeruginosa PAO1 was used to study the effect of cumin EO at different concentrations on its motility on semi-solid Lauria Bertani (LB-0.3% agar-agar) by using the same protocol described by Snoussi et al. [88].

Elastase and Protease Inhibition in P. aeruginosa PAO1
The effect of cumin on the production of elastase by P. aeruginosa PAO1 was tested in Elastin Congo Red buffer supplemented with 0.05, 0.5, 0.625, 1.25, and 2.5 mg/mL of the volatile oil. For the protease inhibition, 3 mg of azocasein (Sigma, Tokyo, Japan) was used as enzyme.

Computational Approach
The receptor proteins (PDB ID: 1HD2, 1JIJ, 2UV0, and 2QP1) were selected from the RSCB protein data bank (http://www.rcsb.org/ accessed on 15 December 2021). Water molecules and co-crystal ligands were removed from each of the protein. AutoGrid was used to create a grid map using a grid box. The grid size and grid dimensions were set for each protein according to the binding pocket are as follow:

ADMET Predicted Properties
The ADMET predictor remains one of the powerful tools for the enhancement of drug design [89][90][91][92][93]. In order to discover effective compounds with better ADMET and drug-likeliness properties, the ADMET profiles of the top major identified compounds were predicted using ADMET SAR online server (http://lmmd.ecust.edu.cn:8000/ accessed on 15 December 2021).

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

Conclusions
In summary, our results indicated that cuminaldehyde, β-pinene, γ-terpinene, and p-cymene were the main phytoconstituents identified in cumin EO by GC/MS technique. This chemovar was particularly active against planktonic and biofilm forming V. alginolyticus, V. cholerae, V. vulnificus, and V. parahaemolyticus species. The same EO and its main compound (cuminaldehyde) were able to modulate the expression of violacein production in C. violaceum in a concentration dependent manner. At low concentrations, cumin EO and cuminaldehyde were able to inhibit the flagellar motility of P. aeruginosa PAO1 strain and attenuate the production of elastase and protease. Further analyses are necessary to elucidate the mechanism of action of cumin EO and its role in the prevention of seafood product contamination by spoilage bacteria belonging to Vibrio genus.