Investigating Grape Seed Extract as a Natural Antibacterial Agent for Water Disinfection in Saudi Arabia: A Pilot Chemical, Phytochemical, Heavy-Metal, Mineral, and CB-Dock Study Employing Water and Urine Samples

Abstract

Microorganisms remain in water from various sources after desalination and other treatments, posing health risks. We explored alternative natural disinfection agents, focusing on grape seed extract (GSE). We collected local grape seeds in Saudi Arabia and analyzed their chemical components. Using gas chromatography–mass spectrometry and inductively coupled plasma mass spectrometry, we identified essential phytochemicals in the GSE, including polyphenols, flavonoids, and alkaloids. Notably, the GSE was free from bacteria and heavy-metal contamination and rich in beneficial nutrient metals. We conducted qualitative analyses on local water and urine samples to detect bacterial infections, heavy metals, and minerals. To assess GSE’s antibacterial potential, we performed molecular docking analysis. Our results reveal a strong binding energy between GSE and bacterial protein receptors, parallel to that of standard antibiotics. Additionally, the results of the laboratory pilot investigations align with those of computational analyses, confirming GSE’s efficacy. Agar well diffusion tests demonstrated significantly greater zones of inhibition for the crude oil extract compared with both diluted GSE and the positive control against the bacteria detected in the water and urine samples. Furthermore, we identified contamination by four bacterial strains and heavy metals in water samples and female urine samples, highlighting the need for effective water disinfectants. GSE shows promise as a safe and potent natural water disinfectant.

1. Introduction

Many microorganisms, such as Pseudomonas aeruginosa, can be found in seawater and in petroleum oil in the coastal areas of Saudi Arabia; they have an impact on the biodegradation of oil and infect the water supply. Furthermore, many bacterial populations, such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus faecalis, Proteus vulgaris, and Proteus mirabilis, have been detected in tap water, wells, and local desalinated water [1,2,3]. Many countries, including Saudi Arabia, need to find ways to meet the growing need for clean water, especially in the current context of global warming-related issues that affect water consumption. Water is essential to human life, and its quality is vital to human health. Therefore, research on water safety is very important. As presented in previous publications, many studies have revealed intestinal contamination with microorganisms that are present in many water samples from Saudi Arabia and are related to many waterborne diseases [4,5,6].

Saudi Arabia relies on many technologies for the desalination of seawater and the disinfection of well water to supply fresh water for domestic and agricultural purposes that is safe to use. However, many microorganisms remain in the water from various sources after local desalination and other treatments [7,8]. Various technological devices are used for detecting and removing water microorganisms in Saudi Arabia, such as the QIAstat-Dx platform and Luminex 200 [5]. For Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus epidermidis, there are many disinfection agents, including chloramines, ozone, chlorine, and chlorine dioxide. Chlorine is used as a pre-treatment disinfection chemical to control microorganism growth and biofouling in desalination plants in Saudi Arabia. However, its use has a side effect: chlorine reacts with organic material, yielding trihalomethanes [THMs], haloacetic acids [HAAs], and haloacetonitriles [HANs], which cause many health issues and various consequences [9,10].

Nanoparticles, due to their unique physicochemical properties, have emerged as promising therapeutic agents in the fight against antibiotic-resistant bacteria, and those made of gold, silver, zinc, copper, and iron support the delivery of novel antimicrobial agents. These antimicrobial nanoproducts physically disrupt cell membranes, preventing the emergence of drug-resistant microorganisms, and interfere with DNA replication and protein synthesis. Additionally, these nanosized particles can serve as diagnostic agents, targeted drug delivery vehicles, and noninvasive imaging tools [11,12,13,14]. For example, silver nanoparticles are known for their ability to be combined with broad-spectrum antibacterial agents. Oxide nanoparticles have strong effects against Gram-positive and Gram-negative bacteria. Copper nanoparticles can encapsulate antibiotics for targeted delivery and are used in wound healing and infection prevention; they also have many applications in addition to antimicrobial action, such as coatings for medical devices to reduce infection risk. Despite the many benefits of the application of nanoparticles, it is essential to consider both their efficacy and potential side effects; for example, Ag nanoparticles (AgNPs) have adverse effects including nephrotoxicity, renal insufficiency, and cardiotoxicity [12,13,14].

Currently, finding alternative natural disinfection agents is vital to providing fresh, clean water for safe use and consumption in Saudi Arabia. Grape fruit seed extract is used to disinfect wounds, sterilize fruits and vegetables, sanitize dishes, and inhibit bacterial and viral growth [15,16]. Furthermore, grape seeds, considered agricultural and wine by-products, are used as fuel, cattle feed, and a source of oil for human consumption and skin treatment [15,16]. Furthermore, the authors of a previous publication reported on green nanoparticles produced from grape seeds using the extract of Ives cultivar (Vitis labrusca) pomace that can be used as a natural antimicrobial water agent and water cleaner [17]. In this context, we collected local grape seeds in Saudi Arabia, identified their chemical components, and analyzed their phytochemical characteristics with gas chromatography–mass spectrometry and inductively coupled plasma mass spectrometry. We further performed qualitative analyses on local water samples and urine samples for the detection of bacteria, heavy metals, and minerals. With this work, we aimed to determine the possibility of studying grape seed oil extract as an antibacterial receptor, and we propose its potential use as a natural disinfection agent based on the potential binding energy results obtained via the molecular docking analysis. Figure 1 shows the experimental design.

2. Materials and Methods

2.1. Plant Sample Collection

We obtained samples of local grapes (Vitis vinifera) from Saudi Arabia for this investigation. Grape seeds were crushed and extracted with a solvent (1: 6 w/v), where the solvent components were hexane and ethanol (1:1). After solvent extraction for 20 min at room temperature (25 ± 3 °C), the extracts were concentrated with a 40–45 °C rotary evaporator [16,23]; we obtained a total GSE (grape (Vitis vinifera) extract) yield of 15% [16,23]. Figure 1 demonstrates the experimental design.

2.2. Water Sample Collection

We obtained three samples of desalinated water and three samples of tap water from different places in Saudi Arabia and stored them separately in sterile syringes at 25 °C for further studies.

2.3. Urine Sample Collection and Preparation

As part of our previous work [22], we collected 100 urine samples from 100 females (21–45 years of age) with a body mass index (BMI) of 32.45 ± 3.5. We excluded participants affected by a known disease, pregnant females, and smokers from this study. The relevant institution approved and accepted our protocol, which satisfied the Declaration of Helsinki as revised in 2013 (protocol code HAPO-02-K-012-2022-06-1110; 12 June 2022). We obtained consent from all of the female volunteers.

2.4. Gas Chromatography–Mass Spectrometry Analysis

We employed the same method we recently described for the GC–MS analysis in this study to determine the phytochemical content of the GSE with a gas chromatography system (G3440B; Agilent Technologies, Santa Clara, CA, USA). We re-dissolved the GSE in ethanol/hexane (1:5) and then purified the mixture by using a nylon membrane filter with a pore size of 0.45 µm. Then, we added 5 µL of the purified GSE into the GC-MS system and analyzed the samples by using helium as a carrier gas with a 1 mL/min flow rate. Then, we used the WILEY and National Institute of Standards and Technology (NIST) mass spectral libraries to determine the phytochemicals in the GSE, as reported in detail in our previous publication; see Figure 1 [16,23].

2.5. Phytochemical Determination

We analyzed the phytochemical contents of the GSE, including flavonoids, total phenolics, tannins, and alkaloids, with a UV–Vis spectrophotometer (1000 series; CECIL Instruments Limited, Milton Technical Centre Cambridge, UK) according to the method described in our previous reports [24,25].

2.6. Metal Analyses

We determined the heavy metals and minerals present in the GSE, urine, and water samples according to our previous publication by using inductively coupled plasma–mass spectrometry (ICP–MS; 7500 cx; Agilent Technologies, Santa Clara, CA, USA) [22]. We pre-treated the GSE and urine samples with nitric acid (1:1 v/v) in a microwave digestion system (Ethos 1; Milestone, Fremont, CA, USA); then, we diluted the GSE, urine, and water samples (1:4 v/v) with nitric acid to analyze the contents of lead (Pb), cadmium (Cd), aluminum (Al), potassium (K,) sodium (Na), calcium (Ca), iron (Fe), zinc (Zn), and chromium (Cr) [16,22,23,24,25,26].

2.7. Bacterial Population Analysis

We incubated 1 mL each of water, urine, and GSE samples in 5 mL MacConkey broth-containing tubes (Sigma–Aldrich, St. Louis, MO, USA) at 37 °C for 48 h to observe bacterial growth. We identified both Gram-positive and -negative bacteria in each sample following the 48 h incubation by using standard analytical profile index (API) identification kits (Biomerieux, Marcy L’etoil, France) according to the manufacturer’s instructions [14,16] and the aid of the ID System manual; see Figure 1.

The API provides a standardized, miniaturized version of existing identification techniques; until now, these techniques were complicated to perform and their results difficult to read. In API 20E, the plastic strip holds twenty mini-test chambers containing dehydrated media having chemically defined compositions for each test. Figure 1 shows the experimental design. Each test corresponds to a biochemical reaction for the detection of enzymatic activity related to the fermentation of carbohydrates or the catabolism of proteins or amino acids performed by the bacterial organisms incubated in the suspension. During incubation, these metabolic reactions produce color changes that are either spontaneous or revealed following the addition of reagents. All positive and negative test results are put together to obtain a profile number, which is then compared with the profile numbers in a commercial manual codebook to determine the identification of the bacterial species, as demonstrated in Figure 1 [18,19,20,21,22].

2.8. Docking Analysis

The preparation of the modeled bacterial protein receptor based on the Protein Data Bank (PDB) is reported in Supplementary Table S1. Additionally, the preparation of grape seed oil extract chemical components and ligands for docking is illustrated in Supplementary Table S2, which includes the properties of the 15 ligands studied and the chemical structure of standard antibiotics derived from PubChem compound identifier (CID) [16].

We obtained bacterial protein receptors (Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus epidermidis) from the PDB portal site (https://www.rcsb.org/), accessed on 1 January 2024 (Supplementary Tables S1 and S2) and used the protein–ligand docking method on CB-Dock (https://www.rcsb.org/), a web server for cavity detection-guided protein–ligand blind docking. The protein–ligand docking method on this website (http://cao.labshare.cn/cb-dock/) is based on AutoDock Vina (accessed on 15 May 2024) (Figure 1). The program is designed to accurately detect potential binding cavities on a protein and sort them according to their binding energy rather than just performing docking on the protein surface as a whole. The procedure includes performing the following nine times: sorting the detected blind protein cavities according to their binding energy and cavity size according to AutoDock Vina and then performing molecular docking; this exhaustive procedure gave us more chances of determining the best binding cavity size with the highest binding energy, which is indicated by the ordering of the Vina scores. Then, we compared the chemical composition of the GSE with a standard antibiotic (doxycycline) against the detected bacterial receptors (Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus epidermidis) [16,22,27,28,29,30,31].

2.9. Antibacterial Activity

The antimicrobial activity was evaluated by using a modified agar well diffusion technique with the disk diffusion assay, which showed the production of zones of inhibition. In this study, we tested the bacteria in 10 mL of fresh medium. Next, we applied 100 μL of the bacterial culture (102 cells/mL of bacteria) onto nutrient agar plates to assess susceptibility by using the well diffusion method. Nutrient agar promotes bacterial growth, and agarose provides structure to solid media for well diffusion assays. Circular holes with a diameter of six millimeters were created in the agarose gel, and crude grape seed oil extract was added to the wells at doses of 10, 20, 30, 50, and 100 mg/mL. The Petri dishes were then incubated for 24–48 h at 37 °C for bacterial strains and for 48 h at 28 °C for other microbes, such as fungi. Inhibition zones were measured in millimeters to determine the antimicrobial efficiency. Additionally, we included positive controls based on conventional medications, i.e., 30 mcg of the standard antibiotic doxycycline, an example of an antibacterial drug [32].

2.10. Statistical Analysis

We analyzed the data with one-way analysis of variance (ANOVA) by using IBM SPSS software package version 20.0. (Armonk, NY, USA: IBM Corp). The values were expressed as the means ± standard deviation (SD) [22].

3. Results

3.1. GC-MS and Analyses of GSE

Table 1. Results of gas chromatography–mass spectrometry analysis of GSE (grape seed oil extract).

No.	Name	Base Peak	RT (min)	Chromatogram
1.	9-Octadecenamide, (Z)-	59	12.351–12.426
2.	Stigmastan-3,5-diene	396.4	18.213–18.272
3.	9,12-Octadecadienoic acid (Z,Z)-, methyl ester	81.1	12.308–12.351
4.	Oleamide	59	15.121–15.218
5.	Hexadecanamide	59	12.928–12.961
6.	Phosphoric acid, trimethyl ester	110	3.151–3.296
7.	Dodecyl acrylate	55	9.564–9.591
8.	Pentanoic acid, 5-hydroxy-, 2,4-di-t-butylphenyl esters	191.2	8.227–8.297
9.	Methyl stearate	74	12.485–12.511
10.	1-Pentadecyne	55	12.720–12.789
11.	11,13-Dimethyl-12-tetradecen-1-ol acetate	207	14.645–14.688
12.	4-Methyl-2,4-bis(4′-trimethylsilyloxyphenyl)pentene-1	207	21.919–22.198
13.	1,3-Benzenediol, o-(4-methylbenzoyl)-o′-(2-methoxybenzoyl)-	135.1	5.644–5.697
14.	Cyclotrisiloxane, hexamethyl-	207	17.411–17.469
15.	Isooctyl 3-mercaptopropionate	57	11.891–11.923

RT: retention time (minutes); PA: peak area (%). The GC–MS investigation was performed on hexane/ethanol (1:6) extracts with Agilent Technologies equipment (G3440B; Santa Clara, CA, USA). The phytochemical contents of the GSE were standardized with computer simulations in the commercial libraries of Wiley and the NIST (National Institute of Standards and Technology).

shows the chemical composition of GSE (grape (Vitis vinifera) seed oil extract) identified by using GC-MS [16].

3.2. Phytochemical Components and Metal Contents of GSE

Supplementary Table S3 shows the quantified phytochemical components and metal contents of GSE (grape seed oil extract). As indicated in the table, phytochemicals such as tannins (in mg/100 g TAE) were not detected, while the content of polyphenols was 125.1 mg/100 g GAE, that of flavonoids 57.0 mg/100 g CE, and that of alkaloids 22.5 mg/g AE. Furthermore, the content of Na was 21.0 µg/g, that of Zn 9.1 µg/g, that of K 172.0 µg/g, that of Ca 27.2 µg/g, and that of Fe 29.1 µg/g, while Cr, Cd, Pb, and Al were not detected.

3.3. Metal Analysis in Water Samples

Figure 2A shows the concentrations in µg/L of lead (Pb), cadmium (Cd), aluminum (Al), potassium (K,) sodium (Na), calcium (Ca), iron (Fe), zinc (Zn), and chromium (Cr) in the water samples. These concentrations were analyzed in each of the water samples separately in the present study (sample 1 = Tap Water 1; sample 2 = Tap Water 2; sample 3 = Tap Water 3; sample 4 = Desalinated Water 1; sample 5 = Desalinated Water 2; sample 6 = Desalinated Water 3). The outcomes analyzed according to the Pareto curve rule (80/20) [22] showed that the greatest concentration, among the metals and minerals considered, in the studied samples was that of Zn, followed by that of K, Fe, Na, Ca, and Cr, in this order (Figure 2B).

3.4. Analysis of Bacterial Species in Water and GSE Samples

The results of the analysis of the bacterial species in the water samples indicate the presence of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus epidermidis as the main bacteria identified; their presence in the urine samples further supported the bacterial contamination of the water samples. Furthermore, the present outcomes also indicate the absence of bacteria in the GSE. The results indicate that Klebsiella pneumoniae is a Gram-negative bacterium (5215773), Staphylococcus epidermidis a Gram-positive bacterium (6706113), Pseudomonas aeruginosa a Gram-negative bacterium (2200004), and Staphylococcus aureus a Gram-positive bacterium (6736153); no other organisms or fungi were detected. Figure 3 shows the percentage of Klebsiella pneumoniae, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Staphylococcus aureus in each water sample and in the GSE, which were separately analyzed in the present study (sample 1 = Tap Water 1; sample 2 = Tap Water 2; sample 3 = Tap Water 3; sample 4 = Desalinated Water 1; sample 5 = Desalinated Water 2; sample 6 = Desalinated Water (Brand) 3; sample 7 = GSE). The report outcomes indicate that, according to the Pareto curve cumulative frequency rule (80/20), the frequency percentage of contamination by the studied bacteria in water samples 1 to 6 and the GSE in descending order was as follows—sample 4, sample 6, sample 5, sample 2, sample 1, and sample 3—with the GSE showing 0%.

3.5. Screening of Urine Biological Samples for Heavy Metals, Other Elements, and Bacteria

Supplementary Table S4 shows the data, represented as means ± SD, for the concentrations of heavy metals and elements in µg/L as follows: Na, 0.007 ± 0.001 µg/L; Cr, not detected; Zn, 0.0032 ± 0.002 µg/L; K, 0.0045 ± 0.021 µg/L; Cd, not detected; Pb, 0.0023 ± 0.031 µg/L; Al, 0.0012 ± 0.021 µg/L; Ca, 0.0029 ± 0.011 µg/L; Fe, not detected. Moreover, Supplementary Table S5 and Figure 4 illustrate the presence of bacteria (expressed as a percentage) detected in the urine samples; group 1 was positive for Klebsiella pneumoniae, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Staphylococcus aureus, while group 2 was negative for the same species. Specifically, in the urine analysis study of 100 volunteers (n = 100), the results were as follows: for Klebsiella pneumoniae, we obtained 97% positive vs. 3% negative results; for Staphylococcus epidermidis, we obtained 52% positive vs. 48% negative results; for Pseudomonas aeruginosa, we obtained 23% positive vs. 77% negative results; and finally, for Staphylococcus aureus, we obtained 10% positive vs. 90% negative results.

3.6. CB-Dock Analysis

Table 2. Comparison between the chemical components of GSE (grape (Vitis vinifera) seed oil extract) and standard antibiotic (doxycycline) in potential CB-Dock interaction scenarios with selected protein receptors in the bacterial species detected.

	Ligand	Target Protein Receptor (PDB)
1.	CID 525918 Stigmastan-3,5-diene	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
2.	CID 5284421 9,12-Octadecadienoic acid (Z,Z)-, methyl ester	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
3.	CID 5283387 Oleamide	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
4.	CID 69421 Hexadecanamide	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
5.	CID 10541 Phosphoric acid, trimethyl ester	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
6.	CID 75084 Dodecyl acrylate	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
7.	CID 605777 Pentanoic acid, 5-hydroxy-, 2,4-di-t-butylphenyl esters	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
8.	CID 8201 Methyl stearate	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
9.	CID 69825 1-Pentadecyne	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
10.	CID 549821 11,13-Dimethyl-12-tetradecen-1-ol acetate	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
11.	CID 91715040 1,3-Benzenediol, o-(4-methylbenzoyl)-o′-(2-methoxybenzoyl)-	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
12.	CID 104386 Isooctyl 3-mercaptopropionate	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP
	Ligand	Target Protein Receptor (PDB)
13.	CID 54671203 Standard antibiotic (doxycycline)	Klebsiella pneumoniae
		5HFT	4HWM
		Pseudomonas aeruginosa	3OS prokaryotic ribosoma (binding unit of antibiotic)
		4F1R	3OS3
		Staphylococcus aureus
		1DUA	1DUE
		8EXP

shows potential CB-Dock interaction scenarios according to the chemical composition of GSE (grape seed oil extract) identified with GC-MS. The extract components were compared with a standard antibiotic (doxycycline) in the interaction with selected proteins and receptors in the detected bacterial species identified in the water and urine samples. The docking interaction was simulated with CB-Dock and AutoDock Vina based on the binding energy potential of the ligands. The procedure included three stages: (1) curvature determination of the protein surface, (2) cavity detection based on clustering, and (3) docking with AutoDock Vina. The results were sorted by the Vina score according to the cavity size as well as accuracy in predicting the binding sites of the PDB target whole proteins, not only surface binding. Figure 5 shows the highest binding energy between the studied bacterial receptor proteins and the ligands. The higher the binding energy, represented with negative symbols, the higher the binding affinity (kcal/mole).

The binding energy values between Klebsiella pneumoniae target protein receptors PDB:5HFT and PDB:4HWM (Table 2, Figure 5) and the studied ligands, i.e., CID 525918 (stigmastan-3,5-diene), CID 5284421 (9,12-octadecadienoic acid (Z,Z)-, methyl ester), CID 5283387 (oleamide), CID 69421 (hexadecanamide), CID 10541 (phosphoric acid, trimethyl ester), CID 75084 (dodecyl acrylate), CID 605777 (pentanoic acid, 5-hydroxy-, 2,4-di-t-butylphenyl esters), CID 8201 (methyl stearate), CID 69825 (1-pentadecyne), CID 549821 (11,13-dimethyl-12-tetradecen-1-ol acetate), CID 610040 (4-methyl-2,4-bis (4′-trimethylsilyloxyphenyl) pentene-1), CID 91715040 (1,3-benzenediol, o-(4-methylbenzoyl)-o′-(2-methoxybenzoyl)), CID 10914 (cyclotrisiloxane, hexamethyl-), CID 104386 (isooctyl 3-mercaptopropionate), and CID 54671203 (standard antibiotic (doxycycline)), were as follows: for PDB:5HFT, 7.2, −5.7, −5.8, −5.3, −3.5, −5.5, −5.9, −5.5, −5.4, −6.4, 0, −7.9, 0, −5.2, and −8.4, respectively; for PDB:4HWM, −8.1,−5,−5.1,−5.1,−3.6,−5.1,−6.2,−4.7,−4.3,−5.2, 0, −7.3, 0, −4.4, and −7.2, respectively.

Additionally, the binding energy values between the Pseudomonas aeruginosa target protein receptor PDB:4F1R (Table 2, Figure 5) and ligands CID 525918, CID 5284421, CID 5283387, CID 69421, CID 10541, CID 75084, CID 605777, CID 8201, CID 69825, CID 549821, CID 610040, CID 91715040, CID 10914, CID 104386, and CID 54671203 were as follows: −7.2, −4.8, −4.6, −4.3, −4, −4.2, −6.6, −4.3, −3.7, −4.8, 0, −8.1, 0, −4.4, and −8.4, respectively.

Furthermore, the prokaryotic ribosome (binding unit of antibiotic) PDB:3OS3 (Table 2, Figure 5) showed the following binding energy values with ligands CID 525918, CID 5284421, CID 5283387, CID 69421, CID 10541, CID 75084, CID 605777, CID 8201, CID 69825, CID 549821, CID 610040, CID 91715040, CID 10914, CID 104386, and CID 54671203: −8.3, −6.5, −5.7, −6.3, −3.7, −5.7, −6.9, −5.6, −5.1, −6.8, 0, −9.8, 0, −5.1, and −9.3, respectively.

Finally, the Staphylococcus aureus target protein receptors PDB:1DUA, PDB:1DUE, and PDB:8EXP (Table 2, Figure 5) showed the following binding energy values with ligands CID 525918, CID 5284421, CID 5283387, CID 69421, CID 10541, CID 75084, CID 605777, CID 8201, CID 69825, CID 549821, CID 610040, CID 91715040, CID 10914, CID 104386, and CID 54671203: for PDB:1DUA, −7.5, −5, −4.7, −4.8, −3.9, −4.4, −5.7, −4.4, −4.1, −4.5, 0, −7.5, 0, −4.2, and −7.8, respectively; for PDB:1DUE −7.3, −4.7, −4.7, −4.7, −3.8, −4.1, −5.8, −4.4, −3.9, −4.6, 0, −7.2, 0, −4.2, and −6.8, respectively; and for PDB:8EXP, −8.2, −6.1, −5.9, −5.5, −4.2, −6.2, −6.8, −6.1, −6.2, −6.2, 0, −9, 0, −5.3, and −8.1, respectively.

3.7. Antibacterial Effects at Different Concentrations of GSE

We analyzed the production of zones of inhibition (in mm). The results indicate that the highest antimicrobial effect was achieved with GSE at the crude concentration of 100% against Klebsiella pneumoniae (Gram-negative), where the inhibition zone was 9.5 mm, followed by Staphylococcus aureus (Gram-positive; inhibition zone of 9.2 mm), Pseudomonas aeruginosa (Gram-negative; inhibition zone of 8.7 mm), and Staphylococcus epidermidis (Gram-positive; inhibition zone of 8.6 mm), with the lowest effect being obtained at a GSE concentration equal to 10% (Figure 6).

4. Discussion

Plant waste, including seeds, represents a significant amount of biomass. Specifically, industrial waste such as grape seeds stands out due to its antioxidant properties. Research on recycling such waste is gaining traction, especially in the context of green treatment approaches [16,17,23,25,33,34].

In our previous publication [16], we highlighted the importance of unlocking the potential of fruit seed waste, given the substantial waste generated during fruit processing, as a green approach to developing potential anti-coronavirus ligands. The discarded stones from fruits such as grapes, prunes, apple, Ajwa dates, pomegranate, and avocado contribute significantly to this waste stream, posing environmental challenges. However, these seemingly overlooked remnants harbor untapped dietary and therapeutic potential, which could alleviate the burden of environmental pollution, including river water toxicity. We also reported that oil extract has many health benefits, such as anti-inflammatory, anti-diabetic, anti-obesity, and anti-apoptosis properties, as well as protective skincare properties. Additionally, we suggest that the studied seed oil could serve as a promising candidate for future research on its use as a nanoparticle against viruses and its use in vaccines and psychiatric drugs. By harnessing the power of these underutilized waste materials, we contribute to both environmental sustainability and scientific advancement. Previous research has explored the potential of grape seed extract as a stabilizing–reducing agent, transforming grape waste into green chemistry. This approach relies on less harmful chemicals from renewable sources. Notably, grape seed extract has demonstrated synergistic antimicrobial activity against both Gram-positive and Gram-negative bacteria in water. Our results agree with previous findings [16,17,23,25,33,34].

In the context of maintaining a clean water supply worldwide, in our study, we determined the presence of phytochemicals in grape seeds, and by using chromatography–mass spectrometry and inductively coupled plasma mass spectrometry, we identified these compounds. Furthermore, we conducted qualitative analyses on local water and urine samples to detect bacterial infections, heavy metals, and minerals. Our findings suggest a potential scenario for investigating grape seed oil extract as an antibacterial agent that targets bacterial receptors. Additionally, we analyzed binding energy with CB-Dock molecular docking analysis, and the results highlight the potential disinfection properties of natural agents derived from grape seed oil.

Our results indicate that grape seed extract is free from heavy-metal contamination (such as Cr, Cd, Pb, and Al) and rich in essential phytochemicals such as polyphenols, flavonoids, and alkaloids. Additionally, GSE contains healthy nutrient metals (Na, K, Zn, Ca, and Fe; Table 1 and Table 2), making it a safe choice for use in applied scientific fields, particularly as a water disinfectant. The strong binding energy observed between grape seed oil extract and microbes, confirmed based on CB-Dock analysis (Table 2 and Figure 5), underscores its potential efficacy. In the present study, we identified contamination by four bacterial strains and heavy metals in the studied water samples and in the urine samples from female individuals, a result that confirms the presence of harmful microorganisms and substances in water and the human body (Supplementary Tables S3 and S4, Figure 3 and Figure 4). In the agar well diffusion tests, the crude oil extract exhibited significantly greater zones of inhibition than the diluted GSE, parallel with the positive control. Furthermore, our chemical identification based on GC-MS revealed GSE’s effectiveness against specific bacteria: Klebsiella pneumoniae (Gram-negative; ID: 5215773), Staphylococcus epidermidis (Gram-positive; ID: 6706113), Pseudomonas aeruginosa (Gram-negative; ID: 2200004), and Staphylococcus aureus (Gram-positive; ID: 6736153). These findings emphasize GSE’s antimicrobial benefits and its relevance for water quality and human health (Table 2, Figure 5 and Figure 6).

CB-Dock, a computational tool for blind docking, is used for predicted binding sites within proteins. The workflow includes the following steps: (1) cavity detection, where CB-Dock identifies potential binding sites on the protein surface by selecting larger cavities for further analysis; (2) the sorting of the detected cavities, with the top candidates being chosen based on size, serving as potential binding sites for the ligand; (3) center and size calculations, where the docking center coordinates are determined and the docking box size is adjusted for subsequent molecular docking; (4) the AutoDock Vina method, where the ligand docks into the selected cavities, generating bound poses; (5) docking score reranking, where the bound poses are ranked based on docking scores, with the highest-scoring conformation representing the optimal binding pose; and (6) optimal binding site identification, where the site associated with the top-ranking conformation is determined to be the best binding site for the query ligand. CB-Dock’s Perl script automates the entire process after submitting the input files. As the number of elucidated protein structures continues to grow, the efficient utilization of structural information becomes crucial for biological and pharmaceutical purposes. Predicting binding sites and affinity between proteins and ligands is essential for computer-aided drug discovery. While existing docking tools often rely on preset binding sites provided by users, CB-Dock takes a different approach, as described above. Furthermore, small-molecule binding involves binding inside protein pockets or cavities as a result of high affinity, which can only be simulated with sufficiently large interaction interfaces. CB-Dock searches for concave surfaces to detect these cavities. Briefly, the tool generates a set of points representing the solvent-accessible surface and calculates the curvature factor for each point by using the method described earlier. These points on the concave surface (curvature factor > 8) are clustered by using a density-peak-based algorithm. As a result, several clusters of points representing cavities on the protein surface are obtained. In this study, we present our work on identifying ligands from the GSE database based on GC-MS and comparing them with an antibiotic to study their potential interactions as antibacterial agents in water and urine samples [30,31].

Our results indicate strong binding energy between all the studied ligands and bacterial protein receptors, compared with the binding site of the standard antibiotic. However, an exception was observed for 1,3-benzenediol, o-(4-methylbenzoyl)-o’-(2-methoxybenzoyl)-, and cyclotrisiloxane, hexamethyl-. Figure 5 and Table 2 indicate that grape seed extract (GSE) can be considered an excellent antibacterial agent for further chemical analysis investigation. Furthermore, our laboratory pilot investigation results align with CB-Dock’s analysis results in terms of the global binding energy of the highest binding affinity (kcal/mole) of the studied receptors, with the greatest enlarged angle for ligand binding, as shown in the protein visual representations. Our findings demonstrate that the highest antimicrobial effect occurred when GSE was used at a crude concentration of 100%. Specifically, against Klebsiella pneumoniae (Gram-negative), the inhibition zone was 9.5 mm; for Staphylococcus aureus (Gram-positive), it was 9.2 mm. Pseudomonas aeruginosa (Gram-negative) followed, with an inhibition zone of 8.7 mm, and Staphylococcus epidermidis (Gram-positive) exhibited an inhibition zone of 8.6 mm. Conversely, the lowest effect was observed at a GSE concentration of 10% (see Figure 6), which may have been due to the phytochemical components of the GSE, such as polyphenols, flavonoids, and alkaloids, and their individual chemical composition as identified with GC-MS (Table 1 and Table 2).

Heavy metals, often considered pseudo-elements, are hazardous due to their negative impact on the body’s metabolic processes. Toxic effects depend on exposure duration, dose, and route. These metals can lead to lipid peroxidation, genetic mutations, and interactions with proteins such as albumin and cell receptors. Heavy metals are implicated in human toxicity, infertility, chronic diseases, immune disorders, DNA alterations, and other health issues. Regular screening and monitoring for environmental toxicity, especially heavy metals, remain crucial [22,35]. Reference ranges vary based on factors such as population, race, age, lifestyle, sex, geographical region, and analytical methods [22,35].

In our previous investigation [22], we confirmed the presence of heavy metals in the serum, hair, and nails of female university students and in selected food, drink, water, and cosmetic products. Notably, we observed a relationship between heavy metals and the biological profile of complete blood count (CBC) and the cholinesterase enzyme, as confirmed with AutoDock Interaction [22,35]. Furthermore, we highlighted the interactions between heavy metals and the human body. These metals, through electron loss and the formation of metal cations, bind to various proteins, genes, and DNA. The affinity sites on macromolecules are affected, impacting tissues and organs. Direct exposure to heavy metals from sources such as food, beverages, cosmetics, soil, air, and water can disrupt internal biological systems, leading to acute and chronic health issues. Neurological, brain, liver, kidney, bone, skin, and fetal diseases may result due to the interactions of metals with different protein receptors. In our previous studies based on AutoDock analysis, we specifically investigated the effects of the strong binding energy of heavy metals on cholinesterase, P53, dopamine, metallothionein, estrogen, keratin, protein kinase enzymes, beta-amyloid, ATPase, albumin, MAO, adrenaline, cortisol, TNF-α, IL-1β, COX-2, and LOX-1, shedding light on potential health consequences [22,34].

The present results indicate that GSE may be considered a good choice for water disinfection from free bacteria and heavy metals. Therefore, it could be used to prevent side effects of bacteria such as inflammation. Bacterial diseases, such as pneumonia, skin infections, kidney issues, and bone inflammation, are often associated with Gram-negative bacteria. Klebsiella pneumoniae and Pseudomonas aeruginosa, particularly in hospital-acquired cases, can cause pneumonia, especially when the body’s immune system is not powerful. Moreover, Staphylococcus aureus, a Gram-positive pathogen, leads to skin infections, pneumonia, endocarditis, and osteomyelitis. Some Staphylococcus aureus strains produce toxins that result in health issues such as gastroenteritis and toxic shock syndrome. Additionally, Pseudomonas aeruginosa, another Gram-negative bacterium, is commonly associated with pneumonia, and Staphylococcus epidermidis, a Gram-positive bacterium found on human skin, is generally harmless but can cause infections, especially in immunocompromised individuals. It is crucial to be aware that antibiotics may have adverse effects, including kidney issues, skin reactions, and bone inflammation. Therefore, it is essential to find natural, side-effect-free ways, such as using grape seed extract, in agreement with the present results, to combat bacteria in water and the human body [36,37,38,39].

Many reports [16,40,41,42,43,44,45], including our previous publication, indicate that grape seed extract (GSE) contains various bioactive compounds, including 9-octadecenamide; 9,12-octadecadienoic acid; oleamide; and hexadecanamide. These compounds exhibit a sedative effect by interacting with neurotransmitters during sleep deprivation, making them potential treatments for mood, depression, and sleep disorders [16,40,41,42,43,44,45]. Additionally, they bind to cannabinoid receptor type 1 (CB1) as full agonists. GSE also contains stigmastane, which serves as a biomarker for initial eukaryotes, and stigmastanol derivatives, which inhibit cholesterol absorption. Acrylates found in GSE have multifunctional properties, including use in the polymer industry and as antimicrobial agents. The extract also contains non-genotoxic, non-carcinogenic phosphoric acids used in water treatment and other applications. Valeric acid, also present in GSE, is produced by gut microbiota and has implications for gut health. Methyl stearate, another component, is associated with reduced LDL cholesterol. GSE’s secondary metabolites, such as sulfurous acid and 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), exhibit antibacterial, antifungal, and anti-diabetic properties. Overall, GSE offers a diverse range of bioactive compounds with potential health benefits [16,40,41,42,43,44,45].

Grape seed extract (GSE) contains antioxidants, including oligomeric proanthocyanidins (OPCs), which indirectly support detoxification. While GSE is not specifically used as a heavy-metal removal agent, its antioxidant properties may help manage heavy-metal exposure. Research [46,47] suggests that GSE can ameliorate the effects of heavy metals such as cadmium and lead by reducing the number apoptotic cells and regulating protein levels. However, for targeted heavy-metal removal, specialized methods or products may be more effective. Researchers found that GSE effectively inhibits corrosion in mild steel exposed to acidic conditions. As the concentration of GSE increases, the inhibition efficiency improves, suggesting the chemisorption of inhibitive species. A surface analysis revealed reduced quantities of corrosion products and enhanced surface film integrity, likely attributed to GSE’s antioxidant activity [46,47].

Grapes, widely consumed worldwide, offer not only vitamins and fiber but also valuable bioactive compounds found in their skin and seeds. Among these, proanthocyanidins stand out as potent polyphenols present in grape seeds. GSE, derived from these seeds, has garnered attention for its potential health benefits, and grape seed extract has been used as a wound disinfectant, fruit and vegetable sanitizer, and cattle feed. In this study, we collected local grape seeds from Saudi Arabia and analyzed their chemical components. By using gas chromatography–mass spectrometry and inductively coupled plasma mass spectrometry, we identified essential phytochemicals in GSE, including polyphenols, flavonoids, and alkaloids. Notably, GSE was free from bacterial and heavy-metal contamination (e.g., Cr, Cd, Pb, and Al) and rich in beneficial nutrient metals (Na, K, Zn, Ca, and Fe). Our results are in agreement with previous findings [48,49], according to which GSE has demonstrated beneficial effects against various diseases. It reduces inflammation, prevents cardiovascular disease, and helps to manage blood pressure and glycemic control. It also exhibits antioxidant properties, as demonstrated by its use in cancer patients’ diets during chemotherapy to decrease the side effects of the latter, such as gastroenteritis and constipation, thanks to its gastroprotective effects. Additionally, it showed antimicrobial activity [48,49]. Furthermore, GSE contains proanthocyanidins, flavonoids, and other bioactive compounds and, as confirmed by the results of this study, is free from heavy-metal contamination (e.g., chromium, cadmium, lead, and aluminum) and provides essential minerals (sodium, potassium, zinc, calcium, and iron). Beyond its use as a nutraceutical or cosmeceutical, GSE could complement existing drugs. It is a safe and potent natural extract that could be used to develop pharmaceutical formulations, holding promise for better health outcomes [48,49].

The findings of this study highlight the potential of grape seed extract (GSE) as a natural antibacterial agent for water disinfection in Saudi Arabia. However, several avenues for further research and practical implementation have to be explored. First, comprehensive laboratory toxicity assessments are essential in in vivo animal studies to ensure GSE’s safety for human consumption and environmental impact. Long-term studies should evaluate any potential adverse effects and address concerns related to its widespread use. Additionally, field trials and real-world applications are necessary to validate GSE’s efficacy in large-scale water treatment facilities, for which a pilot investigation was conducted in the present study. Collaborations with relevant financial institutions, including government agencies, public health organizations, and industry partners, can facilitate the adoption of GSE as a green, sustainable, and safe water disinfection solution. While GSE itself is not a new substance, and its use draws upon traditional knowledge and practices, its application in water disinfection represents an innovative approach aligning with the global trend toward natural and eco-friendly solutions. By integrating indigenous wisdom (i.e., GSE’s historical use) with modern scientific methods, we can create innovative approaches to address pressing environmental and health challenges. Water scarcity and contamination remain critical global challenges. In Saudi Arabia, where water desalination is vital, finding safe and effective disinfection methods is crucial. Researchers, policymakers, and practitioners should collaborate to bridge the gap between traditional practices and evidence-based scientific methods. In doing so, we can unlock the full potential of natural resources such as grape seeds while safeguarding public health and promoting sustainable water management practices [8,15,16,19,35].

In conclusion, GSE boasts antibacterial properties and is rich in phytochemicals such as polyphenols and flavonoids. GSE’s efficacy against bacteria, its safety profile, and its rich phytochemical composition make it a promising candidate for water treatment. In our study, we explored GSE’s potential as a water disinfectant, revealing strong binding energy with bacterial receptors, and agar well diffusion tests confirmed its efficacy against the detected bacteria. GSE thus represents a promising, eco-friendly alternative for clean water access [8,15,16,19,35].

5. Conclusions

In this study, we investigated grape seed extract (GSE) as a potential natural antibacterial agent for water disinfection in Saudi Arabia. Our findings indicate that GSE exhibits a strong binding energy with bacterial protein receptors compared with standard antibiotics. Additionally, GSE is free from heavy-metal contamination and is rich in essential phytochemicals. Agar well diffusion tests confirmed its efficacy against bacteria. Given its safety profile and promising antibacterial properties, GSE holds potential as an effective water disinfectant. Further research and practical applications are warranted to harness GSE’s benefits for clean water supply in Saudi Arabia.