GC-MS Analysis, Antibacterial, and Anticancer Activities of Hibiscus sabdariffa L. Methanolic Extract: In Vitro and In Silico Studies

Abstract

The emergence of bacteria that are resistant to several antibiotics has represented a serious hazard to human health globally. Bioactive metabolites from medicinal plants have a wide spectrum of therapeutic possibilities against resistant bacteria. Therefore, this study was performed to investigate the antibacterial efficacy of various extracts of three medicinal plants as Salvia officinalis L., Ziziphus spina-christi L., and Hibiscus sabdariffa L. against pathogenic Gram-negative Enterobacter cloacae (ATCC13047), Pseudomonas aeruginosa (RCMB008001), Escherichia coli (RCMB004001), and Gram-positive Staphylococcus aureus (ATCC 25923), bacteria using the agar-well diffusion method. Results revealed that, out of the three examined plant extracts, the methanol extract of H. sabdariffa L. was the most effective against all tested bacteria. The highest growth inhibition (39.6 ± 0.20 mm) was recorded against E. coli. Additionally, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the methanol extract of H. sabdariffa were detected in the case of all tested bacteria. Moreover, an antibiotic susceptibility test revealed that all tested bacteria showed multidrug resistance (MDR). While 50% of tested bacteria were sensitive and 50% were intermediately sensitive to piperacillin/tazobactam (TZP) based on the inhibition zone but still less than the extract. Synergistic assay demonstrated the promising role of using a combination of H. sabdariffa L. and (TZP) against tested bacteria. A surface investigation using a scanning electron microscope of the E. coli treated with TZP, extract, or a combination of the two revealed extremely considerable bacterial cell death. In addition, H. sabdariffa L. has a promising anticancer role versus Caco-2 cells with IC₅₀ of 17.51 ± 0.07 µg/mL and minimal cytotoxicity upon testing versus Vero cells with CC₅₀ of 165.24 ± 0.89 µg/mL. Flow cytometric analysis confirmed that H. sabdariffa extract significantly increased the apoptotic rate of Caco-2-treated cells compared to the untreated group. Furthermore, GC-MS analysis confirmed the existence of various bioactive components in the methanol hibiscus extract. Utilizing molecular docking with the MOE-Dock tool, binding interactions between n-Hexadecanoic acid, hexadecanoic acid-methyl ester, and oleic acid, 3-hydroxypropyl ester were evaluated against the target crystal structures of E. coli (MenB) (PDB ID:3T88) and the structure of cyclophilin of a colon cancer cell line (PDB ID: 2HQ6). The observed results provide insight into how molecular modeling methods might inhibit the tested substances, which may have applications in the treatment of E. coli and colon cancer. Thus, H. sabdariffa methanol extract is a promising candidate to be further investigated for developing alternative natural therapies for infection treatment.

Keywords: GC-MC; MDR; antibacterial; anticancer; docking study; medicinal plants; scanning electron microscope.

Figure 1

Chord diagram showing antibacterial activity of different plant extracts against tested bacteria. WH: water extract of Hibiscus sabdariffa L.; MH: methanol extract of Hibiscus sabdariffa L.; EH: ethanol extract of Hibiscus sabdariffa L.; WZ: water extract of Ziziphus spina-christi L.; MZ: methanol extract of Ziziphus spina-christi L.; EZ: ethanol extract of Ziziphus spina-christi L.; WS: water extract of Salvia officinalis L.; MS: methanol extract of Salvia officinalis L.; ES: ethanol extract of Salvia officinalis L., and P: penicillin G (positive control).

Scheme 1

Diameter inhibition zone (mm) of different bacteria treated with various extracts of Hibiscus sabdariffa L.

Figure 2

Bar graph showing the resistance percentage for different bacterial strains toward tested antibiotics.

Figure 3

SEM of E. coli: (A) control; (B) treated by the most effective antibiotic (TZP); (C) treated by Hibiscus sabdariffa L. methanolic extract; (D) treated by a combination of Hibiscus sabdariffa L. methanolic extract and (TZP), magnification = 10,000×.

Figure 4

Anticancer activity of methanol extract H. sabdariffa L. versus Caco-2 cells where IC₅₀ = 17.51 ± 0.07 µg/mL: (A,B) microscopic examination of (A) untreated cells Cao-2 cells, (B) treated Cao-2 cells with H. sabdariffa L.; (a,b) flow cytometric analysis of (a) untreated Cao-2 cells, (b) treated Cao-2 cells with H. sabdariffa L., (C,D) untreated Vero cells, treated Vero cells with H. sabdariffa L. extract where CC50 = 165.24 ± 0.89µg/mL; (E) statistical analysis comparing untreated Caco-2 cells with treated cells. (Data are represented as means ± S.D where * p ≤ 0.05).

Figure 5

GC-MS chromatogram of bioactive compounds present in the methanol extract of H. sabdariffa L.

Figure 6

(A–C) The 2D-binding interaction profile for n-hexadecanoic acid, hexadecanoic acid, methyl ester, and oleic acid,3-hydroxypropyl ester, respectively, with E. coli (3T88) protein. (D–F) The 3D E. coli (3T88)-interaction mechanism for n-hexadecanoic acid, hexadecanoic acid, methyl ester, and oleic acid,3-hydroxypropyl ester, respectively.

Figure 6

(A–C) The 2D-binding interaction profile for n-hexadecanoic acid, hexadecanoic acid, methyl ester, and oleic acid,3-hydroxypropyl ester, respectively, with E. coli (3T88) protein. (D–F) The 3D E. coli (3T88)-interaction mechanism for n-hexadecanoic acid, hexadecanoic acid, methyl ester, and oleic acid,3-hydroxypropyl ester, respectively.

Figure 7

(A–C) The 2D-binding interaction profile for n-hexadecanoic acid, hexadecanoic acid, methyl ester, and oleic acid,3-hydroxypropyl ester, respectively, with colon cancer cell line (2HQ6) protein. (D–F) The 3D structure of colon cancer cell line (2HQ6)—n-hexadecanoic acid, hexadecanoic acid, methyl ester, and oleic acid,3-hydroxypropyl ester, respectively, shows the interaction mechanism.

Figure 7

(A–C) The 2D-binding interaction profile for n-hexadecanoic acid, hexadecanoic acid, methyl ester, and oleic acid,3-hydroxypropyl ester, respectively, with colon cancer cell line (2HQ6) protein. (D–F) The 3D structure of colon cancer cell line (2HQ6)—n-hexadecanoic acid, hexadecanoic acid, methyl ester, and oleic acid,3-hydroxypropyl ester, respectively, shows the interaction mechanism.