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. 2023 May 18;31(7):1237–1243. doi: 10.1016/j.jsps.2023.05.015

Production of antibacterial compounds using Bacillus spp. isolated from thermal springs in Saudi Arabia

Omaish Alqahtani a,b,⁎, Paul Stapleton b, Simon Gibbons c
PMCID: PMC10239688  PMID: 37284417

Abstract

Seventeen water samples were collected from four different thermal springs in Saudi Arabia. Microbiological assays were used to assess the antibacterial activities of bacterial colonies against antibiotic-resistant and susceptible-bacterial strains, and 16S rRNA gene sequencing was used to identify the genus and species of these antibiotic-producing bacteria. Chromatography and spectroscopy were used to separate the active compounds and help figuring out what their structures were. Four compounds were isolated using bacteria: N-acetyltryptamine (1), isovaleric acid (2), ethyl-4-ethoxybenzoate (3) and phenylacetic acid (4). Compounds 1, 2 and 4 were produced from Bacillus pumilus and 3 was from Bacillus licheniformis (AH-E1). The outcomes of the minimum inhibitory concentrations (MICs) showed that all pure compounds produced in this work had antibacterial activities against Gram-positive pathogens (between 128 mg/L and 512 mg/L compared to the control) and compound 2 had activity against E. coli.

Keywords: Bacterial resistance, Antimicrobial agents, Thermal springs, 16S rRNA gene sequencing, Bacillus spp.

1. Introduction

A considerable global health crisis that threatens communities has been attributed to the emergence of resistant bacterial infections (Khameneh et al., 2016). The dissemination of resistance within bacterial pathogens results in a gradual decline in the effectiveness of antibiotics over time (Rossolini et al., 2014). As a result, the search for novel antibiotics from different natural sources has increased (Alrumman et al., 2018). Some microorganisms are able to produce secondary metabolites for protection against competition, and bacteria are superlative at producing clinically valuable antibiotics.

One of the most critical and niche habitats for bacteria is geothermal springs, which have been used for spas as well as for treating dermatological infections (Hussein and Loni, 2011). In the Kingdom of Saudi Arabia, several thermal springs have been used for hundreds of years for both domestic and irrigation purposes. Examples include the Ghomygah and Al-Aridhah springs, and microorganisms that live in these habitats are either thermophilic or hyperthermophilic.

Because of their harsh environment, it is thought that thermophilic bacteria are a biodiverse reservoir to produce bioactive secondary metabolites (Alrumman et al., 2018). Thermophiles also produce numerous valuable products such as antibiotics, thermostable enzymes, and anticancer compounds, making this source of considerable global interest (Salem et al., 2016, Shakhatreh et al., 2017).

Thermophilic Bacillus have the ability to produce a large number of extra cellular thermostable enzymes that have considerable industrial importance including lipases, amylases, cellulases and proteases (Lele and Deshmukh, 2016). Furthermore, several Bacillus species are able to create various secondary metabolites which are diverse in function and structure (Athukorala et al., 2009). For example, peptide antibiotics such as gramicidins, bacitracins, and tyrocidines are produced by members of Bacillus (Esikova et al., 2002).

B. paralicheniformis is a facultative anaerobic, motile Gram-positive bacterium, which is closely related to B. sonorensis and B. licheniformis based on phylogenetic analysis (Du et al., 2019). Fengycin is a peptide antibiotic drug that has been discovered from B. paralicheniformis (Du et al., 2019) and the lantibiotic formicin is also a peptide produced by B. paralicheniformis (Collins et al., 2016).

Both B. licheniformis and B. paralicheniformis have been utilized for years in the biotechnology sector in order to synthesize antibiotics, enzymes as well as consumer and biochemical products (Du et al., 2019). Moreover, they both widely used commercially for plant protection, as live organisms in feed applications and in aquaculture (Agersø et al., 2019).

Four hot springs in Saudi Arabia, some of which had never been studied before, were selected in this work to isolate bacterial strains capable of producing antibacterial metabolites and evaluate their biological activity as antimicrobial agents.

2. Materials and methods

2.1. Sample collection and bacterial isolation

Water samples were collected in June 2016 from four different hot springs in Saudi Arabia. Each sample was collected in a 20 mL sterile tube. Thirteen samples were collected from the Aseer region, KSA, and four from the Jazan region, KSA. All were transported to the School of Pharmacy at UCL for further work and stored at 4 °C until analysis.

2.1.1. Growth and isolation of bacteria

1 mL of each water sample was filtered using a 0.22 µm syringe filter (microfiltration) into a new reaction tube and then 150 µL of each was transferred onto a blood agar plate (Oxoid, UK) and spread using a disposable cotton swab. Subsequently, plates were incubated at 37 °C for 72 h (Genlab). After incubation, each colony with a different color or morphology was collected with a disposable sterile loop and streaked onto a new blood agar plate, and stored at 4 °C until analysis.

2.1.2. Bacterial culture

Each isolated bacterium was transferred onto a blood agar plate and streaked in different directions in order to ensure its purity and to make a bacterial culture, which was used as a stock culture.

2.2. Bacterial identification

2.2.1. Extraction of genomic DNA from isolated bacteria

Four bacterial strains were cultured on blood agar (each strain on one plate) and incubated at 37 °C for 18 h preparation for DNA extraction. The DNeasy® UltraClean® Microbial Kit (Qiagen, Germany) was used for DNA extraction according to the manufacturer’s protocol. The isolated DNA was analyzed by gel electrophoresis and nanodrop (Thermo) to ensure of purity and measure its concentration, before being stored at 4 °C for further analysis.

2.2.2. Biochemical tests and molecular identification

Isolates were identified at the genus level using motility test, Gram stain, endospore test, catalase test, oxidase test, thermo-tolerance test and anerobic growth test (Holt et al., 1994). The 16S rRNA sequence was used for identification at the species level. Four isolated DNA samples from different bacterial strains were subjected to PCR to amplify them for 16S rRNA gene sequencing and the primers are detailed in Table 1. PCR amplification was performed according to the conditions recommended by the manufacturer for the Phusion High-Fidelity DNA Polymerase (New England Biolabs – Massachusetts, USA). Each bacterial colony was suspended in 1 mL of distilled water prior to analysis. The reaction mixture for each sample (50 µL) contained: 1 µL of the isolated DNA, 10 µL of a 5X Phusion buffer, 1 µL of dNTPs (10 mM), 0.5 µL of each primer (FD1 + RP1) (10 µM), 0.5 µL of Phusion polymerase and 36.5 µL of microbiological H2O. Reaction conditions for amplification were 94 °C for 5 min, 35 cycles [94 °C for 30 secs, 62 °C for 30 secs, 72 °C for 30 secs], and 72 °C for 5 min (Bio Rad). The PCR products (a little bit above 1517 bp) were purified using a QIAquick PCR Purification Kit (Qiagen, Germany) according to the manufacturer’s protocol. All tubes were then analyzed by gel electrophoresis and nanodrop and stored at 4 °C for further analysis. Phylogenetic analysis was performed using MEGA X software, a multiple alignment of 12 partial sequence of 16S rRNA closely matches isolates were retrieved from GenBank and were aligned using Clustal W software. E. coli was used as outgroup.

Table 1.

Primers used in this work.

Primer Sequence Primer site Reference
Forward PCR Primer (FD1) 5′-AGAGTTTGATCCTGGCTCAG-3′
(Epicenter Technologies – Wisconsin, USA)
8–27		 Devereux and Willis, 1995
Reversed PCR Primer (RP1) 5′-ACGGTTACCTTGTTACGACTT-3′
(Epicenter Technologies – Wisconsin, USA)		 1512–1492
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2.2.3. 16S rRNA gene sequencing

30 ng/µL from each pure PCR product was prepared in EB solution for sequencing. All samples were sent to the UCL Sequencing Facility for 16S rRNA gene sequencing using the following primers: FD1 and RP1. The chromatograms of obtained nucleotide sequences of PCR products were manually assessed for quality using SnapGene Viewer 3.0.1. (GSL Biotech LLC, USA). Quality was assessed using Bioedit 7.2.5. (Ibis Biosciences, USA). The sequence data was saved as a FASTA file and then converted to a translated sequence and compared for homology using BLASTx (https://blast.ncbi.nlm.nih.gov, National Center for Biotechnology Information, USA).

2.3. Extraction and isolation of active compounds

2.3.1. General experimental procedures

IR spectra were measured on a Perkin Elmer 100 FT-IR spectrometer. 1D 1H, 13C and DEPT-135, and 2D HMQC, HMBC, COSY and NOESY NMR spectroscopic data were acquired on a Bruker Avance 500 MHz NMR spectrometer. Deuterated solvents and NMR tubes were purchased from Cambridge Isotope Laboratories and Sigma-Aldrich, respectively. Bruker Topspin, version 3.2 software was used to process NMR data. Low-resolution mass spectra were acquired on a LCQ Duo Ion-Trap mass spectrometer (Thermo Fisher Scientific). Column chromatography was performed on silica gel 60 (0.04–0.063 mm; Merck) and TLC on Silica gel 60 F254 (Merck) plates. Vanillin-sulfuric acid reagent was used to visualize the TLC plates. All chemicals used were HPLC grade. Chemicals were supplied by either Sigma-Aldrich or Fisher Scientific.

2.3.2. Solid-state fermentation

One single colony was inoculated onto one blood agar plate in order to be cultured. The plate was then incubated at 37 °C for 24 h. After incubation, colonies were transferred into a reagent bottle containing 30 mL of sterile PBS as a stock solution. Then, each 1 mL was transferred into a vial containing 20 mL of PBS. 1 mL was then inoculated onto one blood agar plate and incubated for one week at 37 °C. 600 plates were used as a large-scale fermentation for each bacterium and 20 blood plates were used as control.

2.3.3. Extraction and isolation of bioactive compounds

After seven days of incubation, the agar was cut into small cubes (approximately 1 cm in length of each side) and scraped away and transferred into a conical flask (500 mL). (3 × 300 mL) of chloroform was first utilized to kill bacteria as well as to extract compounds from the agar. Next, (3 × 300 mL) of methanol and (3 × 300 mL) of water were used to extract the active compounds, respectively. All extractions were performed by agitation on a rotatory shaker (IKA) at 160 rpm/min, at 37 °C for 24 h. Subsequently, all extracts were sonicated (Grant) at 40 °C for 30 min and filtered into a round-bottom flask. Chloroform and methanol extracts were evaporated using a rotatory evaporator (Buchi). However, the water extract was evaporated via freeze-drying (Savant). All extracts were weighed and kept at 4 °C until analysis.

2.3.4. Separation of active compounds by TLC and bioautography

Active fractions from extracts were applied on a normal or reverse phase TLC plate (5 × 10 cm) in order to separate the active compound. Three solvent systems were used for this method: the first (n-hexane: ethyl acetate: methanol) (80:20:1) was used for the fractions of AH-E1. For the fractions of TH-C4, TB-A3 and KH-A1, (chloroform: methanol) (90:10) was used. 50 mg from each fraction was dissolved in 1 mL of a suitable solvent (methanol or chloroform) and then 5 drops from each were spotted onto the TLC plate at least 1 cm from its bottom edge. Next, the TLC plate was developed in a twin trough glass chamber saturated with the mobile phase solvents for 30 min up to a distance of 80 mm. After solvent development, the plate was visualized under UV TLC visualizer at UV254 nm and UV366 nm to ensure that the separation was of sufficient quality. The TLC plate was then directly placed into a sterile cabinet for drying for at least one hour, and then transferred into a sterile petri dish. A bacterial inoculum of S. aureus with a density of 5 × 105 CFU/mL was prepared and added to 20 mL of molten nutrient agar. After gentle shaking (to avoid air bubbles), the molten agar was poured over the TLC plate and incubated for 18 h at 37 °C. After incubation, the plate was treated with a 1 mg/mL solution of MTT in methanol and incubated for 30 min at 37 °C to reveal zones of inhibition. Living bacteria were seen as a purple color, whereas a yellow zone indicated an active compound able to inhibit the bacterial growth.

2.4. Antibacterial assay

2.4.1. Test organisms

Five Gram-positive and three Gram-negative bacteria were used in this project as test organisms: Staphylococcus aureus (ATCC-25923), SA 1199B, eMRSA-15, XU212, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

2.4.2. Broth dilution assay

A bacterial inoculum of each test organism with a density of 5 × 105 CFU/mL was prepared. 51.2 mg from each extract was dissolved in 1 mL of DMSO and then 10 µL of this solution was dissolved in 500 µL of broth for the MIC assay. Pure DMSO was used as the control. A 96-well plate (Thermo Scientific) was used and 200 µL of each sample was transferred to the first column of this plate. 100 µL of MH broth was pipetted into the wells from column 2 to column 9 in order to be used for serial dilution. 100 µL from each well in the first column was then transferred to the second column and mixed well. This step was repeated until column 9. After that, the excess amount was transferred to column 12 as a sterile control for each sample. 100 µL of a test organism was added to column 11 to be used as a growth control. Norfloxacin (Fluka BioChemika) was used as a positive control in row 8 with all tested strains except for tetracycline, which was used with SA 1199B strain. 2% of DMSO was added to column 10 to ensure it had no positive effect on inhibition of bacterial growth. MHB was added to row 7 as a blank. 100 µL of the tested strain was added to each well and then the plate was incubated at 37 °C for 18 h. After the incubation, 20 µL of a 1 mg/mL solution of MTT in methanol was added to each well and incubated for 30 min at 37 °C. A blue color indicated growth and a yellow color indicated a lack of growth. MIC values were recorded as the lowest concentration at which no color change was observed. The MIC experiment was done in duplicate per plate and repeated in at least three independent experiments.

3. Results

3.1. Isolation and identification of bacterial strains

In this study, strains KH-A1, TB-A3, AH-E1 and TH-C4 (Table 2) were isolated from four different springs in Saudi Arabia. The strains were named according to the spring source that they were isolated from. For example, the first two letters are from the spring’s name, the third letter from the sample’s name and the numbers indicate the colony number from each sample. Bacterial identification showed that all isolates were classified as closely of Bacillus genus (Table S7). The findings of the 16S rRNA gene sequencing illustrated those strains KH-A1 (97.79%), TB-A3 (97.09%) and TH-C4 (97.48%) had the highest homology with Bacillus pumilus. Strain AH-E1 had the highest homology with Bacillus licheniformis (97.02%) (Table S6). According to phylogenetic analysis of 16S rRNA gene (Fig. 1), it was possible to identify each strain at the species level.

Table 2.

Bacterial isolates from each spring.

Spring Location Antibiotic-Producing Bacteria
Tharban spring (TH) 183 Km northwest of Abha city, (41°67E-19°03N) TH-C4
Al-Khobh spring (KH) 81 Km southeast of Jazan city, (43°07E- 16°45N) KH-A1
Al-Haridhah spring (AH) 132 Km west of Abha city, (42°05E- 18°01N) AH-E1
Thebah spring (TB) 158 Km southeast of Abha city, (43°15E- 17°62N) TB-A3
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Fig. 1.

Fig. 1

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Comparative analysis of partial 16S rRNA gene sequences of the isolates obtained from thermal springs in Saudi Arabia. The phylogenetic tree was inferred using the neighbor-joining method with a bootstrap analysis of 1000 repetitions.

3.2. Isolation and identification of active compounds

Four known compounds (1–4) (Fig. 2) were isolated from the different bacterial strains. 1 was isolated from the methanol extract of KH-A1 and identified as N-acetyltryptamine based on its NMR, MS and IR spectral data (Table S1), which correlated well with the published 13C data of Zhang et al., (2013). The molecular formula of 1 was determined as C12H14N2O and the molecular weight of the compound was calculated as 202, which corresponded to a pseudo molecular ion peak at m/z 225.13 [M+Na]+ in its ESI-MS.

Fig. 2.

Fig. 2

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Structures of 1–4.

Natural product 2 was isolated from the methanol extract of strain TB-A3 and its molecular formula was determined as C5H10O2 on the basis of a pseudo molecular ion at at m/z 101.0 [M−H]− in its ESI-MS. It was readily identified as isovaleric acid by comparison of its NMR spectral data (Table S2) with that published (Bal et al., 2008).

Compound 3 was isolated from the methanol extract of the AH-E1 strain and identified as ethyl-4-ethoxybenzoate based on the findings of its NMR (Siddique, 2019), MS and IR spectral data (Table S3). The molecular formula of 3 was determined as C11H14O3 and the molecular weight of the compound was calculated as 194, which was supported by an ion in the ESI-MS at m/z 195.10 [M+H]+.

Secondary metabolite 4 was isolated from a methanol extract of strain TH-C4 and identified as phenylacetic acid based on its NMR (Holmes and Lightner, 1995), MS and IR spectral data (Table S4). Its molecular formula was confirmed as C8H8O2 with a molecular weight of 136, which corresponded to an ion in the ESI-MS at m/z 135.04, attrributable to [M−H]−.

3.3. Antibacterial activity of isolated compounds

As illustrated in Table 3, compounds 1, 3 and 4 had antibacterial activity (narrow-spectrum activity) against only the Gram-positive bacteria. In contrast, compound 2 illustrated moderate broad-spectrum activity toward both Gram-positive and Gram-negative strains.

Table 3.

MIC values of (1–4) and standard antibiotics in µg/mL.

S. aureus SA-1199B eMRSA-15 XU212 B. subtilis E. coli P. aeruginosa K. pneumoniae
1 128 256 256 512 >512 >512 >512 >512
2 256 256 512 512 512 512 >512 >512
3 256 512 512 >512 >512 >512 >512 >512
4 128 256 256 512 512 >512 >512 >512
Nora 1 – 1 16 1 1 1 1
Tetb – 16 – – – – – –
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a

Norfloxacin.

b

Tetracycline.

4. Discussion

Bacillus spp. are one of the most prominent genera of thermophilic bacteria that have been studied (Lele and Deshmukh, 2016). This genus currently comprises 377 species and can be found in aquatic environments, soil, food as well as in the gut microbiota of mammals and arthropods (Caulier et al., 2019). Bacillus spp. are known as rod-shaped, Gram-positive bacteria (Sumi et al., 2015). Additionally, Bacillus can be found singly or in pairs, and in chains or as long filaments (Logan and Vos, 2015) and the thermophilic bacilli may survive better at temperatures between 45 and 70 °C (Adiguzel et al., 2009).

B. licheniformis has been discovered from Tharban (Alrumman et al., 2018) and Al-Khobh springs (Yasir et al., 2019), but this is the first time it was isolated from the Al-Haridhah spring. Furthermore, B. pumilus has been previously found in the Al-Khobh spring (Yasir et al., 2019). However, its discovery from the Tharban and Thebah springs is reported here for the first time.

Several studies have recorded the isolation of N-acetyltryptamine from various microbes, for example, from different bacteria such as Corallococcus corraloides (Böhlendorf et al., 1996), Intrasporangium strain N8 (Okudoh and Wallis, 2012), Streptomyces spp. (AC-2) (Lin et al., 2008), Streptomyces spp. strain TN58 (Mehdi et al., 2009), Streptomyces djakartensis (Zhang et al., 2013) and Streptomyces spp. MBT76 (Wu et al., 2016). Furthermore, it has also been found in some fungi, including Penicillium vitale (Vinokurova et al., 2000), Penicillium solitum (Antipova et al., 2018) and Fusarium poae (Nagia et al., 2012). It has also been isolated from a Micromonospora spp. strain associated with the marine sponge Tethya aurantium (Tuan et al., 2017) as well as from Microbispora aerata from Antarctica (Bratchkova and Ivanova, 2011). In Bacillus spp., B. cereus readily produces N-acetyltryptamine as a metabolite of tryptamine (Hutzinger, 1969), but it is reported here for the first time from B. pumilus.

Numerous studies have described the isolation of isovaleric acid from microbes. For example, it has been isolated from Peptostreptococcus anaerobius (Guerrant et al., 1982), Pseudoalteromonas haloplanktis strain (Hayashida-Soiza et al., 2008) and Megasphaera hexanoica spp. nov (Jeon et al., 2017). Furthermore, it has also been isolated from Bacillus spp. including B. amyloliquefaciens (Chen et al., 2017), B. subtilis (Hong et al., 2017, Mumtaz et al., 2019), B. velezensis (Calvo et al., 2020), B. cereus and Paenibacillus polymyxa (Mumtaz et al., 2019).

In this study, isovaleric acid was isolated from B. pumilus from a Saudi spring, which supports a study stating that B. pumilus was able to produce isovaleric acid (de la Cochetière-Collinet and Larsson, 1984). In general, isovaleric acid and other simple acids such as isocaproic and 2-methylbutyric acids are thought to be induced by L-leucine catabolism in anaerobic bacteria (Díaz-Pérez et al., 2016).

Ethyl-4-ethoxybenzoate has been isolated from both plants and microbes. From microbes, it has been isolated from the hexane extract of B. licheniformis (Sharma et al., 2010) as well as from Paenibacillus spp. (Tahir et al., 2019).

Ethyl-4-ethoxy benzoate was isolated from B. licheniformis from the Al-Haridhah spring and this is consistent with the study of Sharma et al., (2010), who isolated the same compound from this species.

There are numerous studies on the isolation of phenylacetic acid from microbes. For instance, it has been isolated from Streptomyces zhaozhouensis (Lacret et al., 2015), Streptomyces humidus (Hwang et al., 2001), Proteus mirabilis (Erdmann, 1987), Azospirillum brasilense, Enterobacter cloacae, Thauera aromatica and the nitrogen-fixing bacteria Frankia (Cook, 2019), Bacteroides ruminicola, Bacteroides melaninogenicus subsp. macacae, Clostridium botulinum type G and in other species of Clostridium (Mayrand and Bourgeau, 1982). Moreover, phenylacetic acid has been produced by the fungi such as Rhizoctonia solani, Sporobolomyces roseus and Schizophyllum commune (Cook, 2019). It has also been found in the brown alga, Undaria pinnatifida (Cook, 2019). Furthermore, it has been reported that phenylacetic acid was found in some Bacillus species. For example, B. fortis (Akram et al., 2016), B. megaterium, B. cereus strain HY-3 and B. subtilis strain HY-16 (Hwang et al., 2001) as well as B. licheniformis (Kim et al., 2004).

This is the first report of phenylacetic acid being produced by B. pumilus. However, it has been isolated from some Bacillus spp. such as B. licheniformis (Kim et al., 2004) and B. cereus (Hwang et al., 2001). Moreover, it was previously identified in the same spring (Tharban), but from a different organism, Streptomyces spp. (Al-Dhabi et al., 2019). Phenylacetic acid (also known as benzene acetic acid or α-toluic acid) has drawn the attention of the pharmaceutical industry due to its various biological properties such as antioxidant, anti-inflammatory and antifungal properties as well as its ability to inhibit quorum sensing in P. aeruginosa (Musthafa et al., 2012). It has a prevalent distribution in nature, and formed metabolically from L-phenylalanine (Burkhead et al., 1998).

In comparison with the literature, N-acetyltryptamine has been reported to have antibacterial activity against B. subtilis, B. cereus, S. aureus, E. coli, MRSA and Pseudomonas syringae pv. actinidiae (Zhang et al., 2013). Furthermore, it has been published as an antifungal and antibacterial agent with activity against Fusarium spp., Verticillium dahlia and the Gram-positive bacterium Micrococcus luteus (Mehdi et al., 2009).

Moreover, it has been reported that isovaleric acid has antibacterial properties (Hayashida-Soiza et al., 2008, Huang et al., 2011). Ethyl-4-ethoxybenzoate has been described as an anti-biofilm agent (Campbell et al., 2019). In addition, phenylacetic acid has been shown to demonstrate antibacterial and antifungal activities against E. coli, MRSA, A. fumigatus and C. albicans, (Lacret et al., 2015). Moreover, it demonstrated antimicrobial activity against E. coli and Ralstonia solanacearum (Zhu et al., 2011), S. aureus, E. coli, C. albicans, Pseudomonas aeruginosa and B. subtilis (Kim et al., 2004). It has also been found as an antibacterial agent (Erdmann, 1987) and antifungal compound (Hwang et al., 2001).

5. Conclusion

In conclusion, seventeen water samples were collected from four different hot springs in Saudi Arabia. Several biological and microbiological assays were used to assess the antibacterial activity of samples against resistant- and susceptible-bacterial strains, and to identify the genus of the bacteria. Four antibacterial-producing bacterial strains were isolated from these springs and identified as Bacillus licheniformis (one strain) and Bacillus pumilus (three strains). Additionally, four compounds were produced using these bacteria: N-acetyltryptamine, isovaleric acid, ethyl-4-ethoxybenzoate and phenylacetic acid. These compounds have been isolated from these springs for the first time except phenylacetic acid. The findings of this study revealed that Bacillus spp., isolated from hot springs in Saudi Arabia, could be used to produce a variety of antibacterial compounds.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Acknowledgment

The authors are thankful to Najran University, School of Pharmacy at UCL and the Saudi Culture Bureau for facilitating doing this project.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding

The authors are thankful to the Deanship of Scientific Research, Najran University, Najran, Saudi Arabia, for funding this research under the General Research Funding program grant code (NU/DRP/MRC/12/17).

Footnotes

Peer review under responsibility of King Saud University.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jsps.2023.05.015.

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (4.6MB, docx)

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