Bruguiera gymnorhiza (L.) Lam. at the Forefront of Pharma to Confront Zika Virus and Microbial Infections-An In Vitro and In Silico Perspective

Abstract

The recent emergence of Zika virus (ZIKV) in Brazil and the increasing resistance developed by pathogenic bacteria to nearly all existing antibiotics should be taken as a wakeup call for the international authority as this represents a risk for global public health. The lack of antiviral drugs and effective antibiotics on the market triggers the need to search for safe therapeutics from medicinal plants to fight viral and microbial infections. In the present study, we investigated whether a mangrove plant, Bruguiera gymnorhiza (L.) Lam. (B. gymnorhiza) collected in Mauritius, possesses antimicrobial and antibiotic potentiating abilities and exerts anti-ZIKV activity at non-cytotoxic doses. Microorganisms Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 70603, methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA), Salmonella enteritidis ATCC 13076, Sarcina lutea ATCC 9341, Proteus mirabilis ATCC 25933, Bacillus cereus ATCC 11778 and Candida albicans ATCC 26555 were used to evaluate the antimicrobial properties. Ciprofloxacin, chloramphenicol and streptomycin antibiotics were used for assessing antibiotic potentiating activity. ZIKV^MC-MR766NIID (ZIKV^GFP) was used for assessing anti-ZIKV activity. In silico docking (Autodock 4) and ADME (SwissADME) analyses were performed on collected data. Antimicrobial results revealed that Bruguiera twig ethyl acetate (BTE) was the most potent extract inhibiting the growth of all nine microbes tested, with minimum inhibitory concentrations ranging from 0.19-0.39 mg/mL. BTE showed partial synergy effects against MRSA and Pseudomonas aeruginosa when applied in combination with streptomycin and ciprofloxacin, respectively. By using a recombinant ZIKV-expressing reporter GFP protein, we identified both Bruguiera root aqueous and Bruguiera fruit aqueous extracts as potent inhibitors of ZIKV infection in human epithelial A549 cells. The mechanisms by which such extracts prevented ZIKV infection are linked to the inability of the virus to bind to the host cell surface. In silico docking showed that ZIKV E protein, which is involved in cell receptor binding, could be a target for cryptochlorogenic acid, a chemical compound identified in B. gymnorhiza. From ADME results, cryptochlorogenic acid is predicted to be not orally bioavailable because it is too polar. Scientific data collected in this present work can open a new avenue for the development of potential inhibitors from B. gymnorhiza to fight ZIKV and microbial infections in the future.

Keywords: ADME; antimicrobial; antiviral; envelope protein; mangrove plants; pharmacokinetics.

Figure 1

Cytotoxicity of B. gymnorhiza extract on A549 cell lines. A549 cells were incubated with different concentrations of B. gymnorhiza extract for 48 h. Cell viability was determined using the metabolic activity by MTT assays. Results are given as means ± SEM of four independent experiments and expressed as relative values in contrast to untreated cells.

Figure 2

BFA and BRA exert antiviral activity against ZIKV. A549 cells were infected with ZIKV^GFP at MOI of 2 in the presence of B. gymnorhiza extracts at 200 µg/mL. After 24 h of infection, flow cytometric analysis of GFP fluorescence was conducted. The results are given as means ± SD of three independent experiments and expressed as relative values in contrast to untreated infected cells. **** p < 0.0001 indicates significant differences in the extracts.

Figure 3

BFA and BRA extracts exert a dose–response antiviral activity against ZIKV. (A) A549 cells were incubated with B. gymnorhiza extracts (400 to 12.5 µg/mL) for 72 h. Cell viability was assessed by MTT assay. Results are given as means ± SD of four independent experiments and expressed as relative values compared to vehicle. (B) A549 cells were infected with ZIKV^GFP at MOI of 2 in presence of B. gymnorhiza extracts (400 to 12.5 µg/mL). After 24 h of infection, flow cytometric analysis of GFP fluorescence was conducted. The results are given as means ± SD of four independent experiments and expressed as relative value in contrast to untreated infected cells.

Figure 4

B. gymnorhiza extracts inhibit ZIKV entry in human cells. (A) Schematic diagram representing the time-of-drug-addition assay. The green segments in the arrows show the presence of extract during infection. (B) Flow cytometric analysis of GFP expression in A549 cells infected with ZIKV^GFP at MOI of 2 PFU/cell under the experimental conditions as illustrated in (A). Results are given as means ± SD of three independent experiments and expressed as relative values in contrast to the control. **** p < 0.0001 indicates significant differences in the extracts; n.s. = not significant.

Figure 5

Non-bonding interactions of highest binding affinity against ZIKV envelope protein using docking calculations.

Figure 6

Three-dimensional structure of cryptochlorogenic acid at the active site of ZIKV envelope protein using docking calculation.

Figure 7

The bioavailability radar plot of cryptochlorogenic acid. The pink area represents the optimal range for each property (lipophilicity: XLOGP3 between −0.7 and +5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å2, solubility: log S not higher than 6, saturation: fraction of carbons in the sp3 hybridization not less than 0.25, and flexibility: no more than 9 rotatable bonds). Cryptochlorogenic acid is predicted not to be orally bioavailable because it is too polar.