Naringin derivatives as glucosamine-6-phosphate synthase inhibitors based preservatives and their biological evaluation

The study addresses the need for safer, effective preservatives for aqueous-based foods, cosmetics, and pharmaceuticals, given concerns about adverse effects associated with widely used synthetic preservatives (e.g., parabens, benzoates, benzalkonium chloride). It explores naringin, a citrus-derived flavonoid with reported antioxidant and antimicrobial properties, as a scaffold for developing novel preservatives. The research targets glucosamine-6-phosphate (G-6-P) synthase, a key enzyme in microbial cell wall biosynthesis, leveraging in silico docking against the enzyme (PDB ID: 1moq) to guide synthesis of naringin derivatives. The purpose is to synthesize and evaluate these derivatives for antioxidant activity, antimicrobial potency, preservative efficacy in a model topical formulation (White Lotion USP), stability under ICH conditions, skin permeation, and topical/ocular safety.

The paper reviews the widespread use of synthetic preservatives and their reported associations with adverse health effects (e.g., irritation, allergies, neurological effects). It highlights interest in natural alternatives, with prior preservative-related activity reported for phenolics such as ferulic, gallic, caffeic, and p-coumaric acids. Naringin and related derivatives (naringenin, prunin, alkylprunin esters) have shown antibacterial activity against pathogens including Listeria monocytogenes, E. coli, and S. aureus. G-6-P synthase has been validated as an antibacterial/antifungal target important for peptidoglycan biosynthesis, and availability of its crystal structure enables docking-driven inhibitor design. This background motivates derivatization of naringin to enhance antimicrobial and preservative properties while ensuring safety.

- In silico docking: Conducted using Schrodinger Maestro 10 (Glide) with OPLS-2005 force field. Protein: G-6-P synthase (PDB ID: 1moq, 1.57 Å). Protein preparation with Prepwiz; waters not coordinating metals or bridging ligand-protein were removed. Receptor grids generated; ligands (designed naringin derivatives) built in ChemDraw and prepared with LigPrep (biological pH, OPLS-2005 partial charges). Binding energy calculated as ΔE = Ecomplex – Eligand – Eprotein. - ADME prediction: QikProp (Schrödinger LLC) and PreADMET assessed descriptors including QPPCaco, QPlogBB, QPPMDCK, QPlogKp, QPlogKhsa, CNS activity, Lipinski’s Rule of Five, and in silico toxicity flags (Ames test, mouse/rat carcinogenicity). - Synthesis: Naringin derivatives (compounds 1–8) synthesized via Schiff-base formation adapting methods of Yang et al. and Saini et al. Substituted anilines or aliphatic amines (0.01 mol) were acidified with conc. HCl; equimolar naringin in ethanol (50 mL) was refluxed. Reaction progress monitored by TLC; products isolated by filtration and recrystallized from alcohol. Structures confirmed by FTIR, 1H/13C NMR, MS, and elemental analysis (CHNS). Representative spectral data reported for each compound. - Antioxidant assay: DPPH radical scavenging measured colorimetrically at 517 nm after 30 min incubation at 30 °C in the dark. 0.1 mM DPPH in methanol; samples/standards mixed 1:1 (v/v). Percent inhibition = (Ac−As)×100/Ac. Triplicate measurements; IC50 values determined. L-ascorbic acid as standard. - Antimicrobial MIC (pMIC): Tube dilution method against S. aureus MTCC 3160, P. aeruginosa MTCC 1934, E. coli MTCC 45, P. mirabilis MTCC 3310, C. albicans MTCC 183, A. niger MTCC 282. Inocula standardized to 0.5 McFarland in 0.9% NaCl; serial dilutions (1.56–50 µg/mL) in nutrient or sabouraud dextrose broth. Incubation: bacteria 37 °C/24 h; C. albicans 37 °C/48 h; A. niger 25 °C/7 days. CFU determined by pour-plate method; pMIC reported (µM/mL). - Preservative efficacy (challenge test): White Lotion USP prepared per Khatkar et al. Standard preservatives (sodium benzoate, methyl and propyl paraben) were replaced with equimolar amounts of synthesized compounds (0.0013 mol equivalent to methyl paraben) to test efficacy. Inoculation with 0.5–1% v/v microbial suspensions (10^5–10^6 CFU/mL) of challenge organisms (E. coli, P. aeruginosa, S. aureus, C. albicans, A. niger). CFU/mL measured on days 0, 7, 14, 21, 28. USP criteria: ≥2.0 log reduction in bacteria by day 14 with no increase to day 28; no increase for fungi at days 14 and 28. - Stability study: Formulations with selected compound were stored under ICH accelerated conditions (40 ± 2 °C; 75% ± 5% RH) for 6 months. Periodic assessment (0–6 months) of pH and CFU/mL. - In vitro skin permeation: Franz diffusion cell with Strat-M synthetic membrane; receptor (10 mL) filled with phosphate buffer pH 7.4 at 37 °C with stirring. Donor loaded with 1 mL of 0.1% solution of compound 7. Sampling at 5, 10, 15, 30, 60, 120 min with UV absorbance at 291 nm; cumulative release and percent permeated calculated. - Skin irritation (Draize test, OECD 404): 0.5 g test substance applied to shaved rabbit skin under occlusion for 4 h. Erythema/edema scored at 1, 24, 48, 72 h; follow-up to 14 days for reversibility. - Eye irritation (OECD 405): 100 mg/0.1 mL instilled into rabbit eyes. Ocular reactions (conjunctivae, cornea, iris) scored at 1, 24, 48, 72 h; follow-up up to 21 days. - Statistics: Data reported as mean ± SD (n=3). One-way ANOVA, p<0.05 considered significant.

- Molecular docking: Compounds 6 and 7 showed the best Glide docking scores and binding energies against G-6-P synthase: compound 7 score −8.45, binding energy −69.22 kJ/mol; compound 6 score −7.98, −65.35 kJ/mol. These outperformed standards ciprofloxacin (−5.18; −37.16 kJ/mol), ampicillin (−5.06; −25.41 kJ/mol), and fluconazole (−5.12; −23.15 kJ/mol). Key interactions for compound 7 included H-bonds with Ala602, Val399, Cys300, Thr302 and hydrophobic contacts with Ser303, Glu488; compound 6 formed H-bonds with Gln348, Thr302, Val602 and hydrophobic interactions with Glu488, Leu601. - ADME (in silico): Predicted descriptors for selected derivatives fell within acceptable ranges for permeability and protein binding; low predicted skin permeability (e.g., compound 7 QPlogKp −7.20). PreADMET flags indicated potential Ames mutagenicity for some derivatives (e.g., compound 7 AT-M), whereas others showed non-mutagenic profiles; some standards showed differing carcinogenicity predictions (table data provided in the paper). - Antioxidant activity (DPPH): Compounds 7, 6, and 1 exhibited superior radical scavenging with IC50 6.23±0.03 µM, 7.03±0.03 µM, and 7.31±0.06 µM, respectively, outperforming L-ascorbic acid (8.11±0.06 µM). Other IC50 values: compound 2 (14.52±0.40), 3 (10.62±0.01), 4 (18.77±0.06), 5 (11.32±0.16), 8 (20.9±0.26) µM; naringin 6.36±0.36 µM. - Antimicrobial activity (pMIC, µM/mL): Compound 7 was the most potent across test strains: P. mirabilis 2.07, P. aeruginosa 2.37, S. aureus 2.07, E. coli 2.37, C. albicans 1.77, A. niger 1.77. Comparators: ciprofloxacin (bacteria) 1.12–1.42; ampicillin 0.84–1.74; fluconazole (fungi) 1.08–1.38. Several other derivatives showed activity but were less potent than compound 7. - Preservative efficacy (White Lotion USP): Compounds 6 and 7 were evaluated; compound 7 achieved log CFU/mL reductions within USP acceptance criteria across all challenge organisms and was comparable to sodium benzoate, propyl paraben, and ethyl/methyl paraben. Compound 6 failed to meet criteria in some cases (less than 2.0 log reduction at day 14 and increases between day 14 and 28). Representative CFU data (log CFU/mL) for compound 7: E. coli 2.03±0.03 (day 14), 2.71±0.02 (day 28); P. aeruginosa 2.63±0.02 (day 14), 2.96±0.03 (day 28); S. aureus 2.11±0.01 (day 14), 2.39±0.04 (day 28); C. albicans 2.44±0.08 (day 14), 2.58±0.08 (day 28); A. niger 2.33±0.04 (day 14), 2.99±0.01 (day 28). - Stability (ICH accelerated): Formulations containing compound 7 maintained pH 5.5–6.0 and showed no microbial growth over 6 months at 40±2 °C/75%±5% RH. - Skin permeation (Franz cell, Strat-M): Compound 7 exhibited cumulative release of 45.07% by 120 min; total permeated amount reported as 4.27 mg from the tested dose. - Safety (rabbits): Compound 7 was non-irritant on skin (Draize score 0 for erythema/edema up to 72 h; reversible up to 14 days) and non-irritant in eyes (scores 0 for cornea, conjunctivae, iris up to 72 h; monitored to 21 days).

The study demonstrates that rationally designed naringin derivatives can inhibit G-6-P synthase in silico and translate to broad antimicrobial activity in vitro. Compound 7, featuring ortho-chloro and para-nitro substitutions on the aromatic imine moiety, showed the strongest predicted binding to the enzyme and the best experimental antimicrobial and antioxidant profiles. Its performance as a preservative in a White Lotion USP model met USP antimicrobial effectiveness criteria and sustained product stability under accelerated conditions, supporting its utility as a preservative. Skin permeation was limited (low QPlogKp; partial release across Strat-M), aligning with desired low systemic exposure for topical preservatives. Absence of dermal and ocular irritation in rabbits suggests a favorable local safety profile. Collectively, these findings support the hypothesis that G-6-P synthase–targeting naringin derivatives, especially compound 7, can serve as effective and potentially safer alternatives to conventional synthetic preservatives in topical and possibly other aqueous formulations.

Compound 7 emerged as the most active naringin-derived G-6-P synthase inhibitor, combining potent antioxidant and antimicrobial activities with effective preservative performance in a USP White Lotion, maintaining acceptable pH and preventing microbial growth over six months under ICH accelerated conditions. It showed no dermal or ocular irritation in rabbits and exhibited limited skin permeation. The work supports naringin derivatives, particularly compound 7, as promising preservative candidates for food, cosmetic, and pharmaceutical products, consistent with docking-guided design and SAR indicating enhanced activity with aromatic imine substitution and electron-withdrawing groups. Future work could expand preservative testing to additional formulation types, broader microbial panels, comprehensive toxicology (including systemic and genotoxicity studies), and mechanistic validation of G-6-P synthase inhibition in cellular/biochemical assays.