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

Glucosamine-6-Phosphate synthase enzyme has been targeted for development of better and safe preservative due to its role in microbial cell wall synthesis. In recent year’s demand of preservatives for the food, cosmetics and pharmaceuticals have increased. Although, the available synthetic preservatives have associated unwanted adverse effects, soa chain of naringin derivatives were schemed synthesized and judged for antioxidant, antimicrobial, preservative efficacy, stability study and topical evaluation. Molecular docking resulted with excellent dock score and binding energy for compound 7, compound 6 and compound 1 as compared to standard drugs. Resultant data of antimicrobial activity revealed compound 7as most potent antimicrobial compound for P. mirabilis, P. aeruginosa, S. aureus, E. coli, C. albicans, and A. niger, respectively, as compared to the standard drugs. The preservative efficacy test of compound 7 in White Lotion USP showed the log cfu/mL value within prescribed limit of USP standard. Compound 7 stabilize the White lotion USP from microbial growth for a period of six months under accelerated storage condition. Compound 7 was further evaluated for toxicity by using the Draize test in rabbits and showed no sign of eye and skin irritation. The outcome demonstrated that synthesized naringin compounds showed glorious antioxidant, antimicrobial, preservative efficacy, stable and safe as compared to standards.


Scientific Reports
| (2020) 10:20477 | https://doi.org/10.1038/s41598-020-77511-2 www.nature.com/scientificreports/ Ligand preparation. The three-dimensional structural of naringin derivatives were constructed by using the Chemdraw ultra 8 and were further preceded for energy minimization by using the LigPrep tool to gain the appropriate conformation through the addition or removal of hydrogen bonds. The partial charges were computed according to the OPLS-2005 force field at biological pH. ADME studies. In silico prediction for ADME properties of the synthesized compounds were calculated by quick prop from Schrodinger. Various ADME parameters such as Log P, number of rotatable bonds, Log BB, number of hydrogen acceptor and donor atoms were calculated. Lipinski's rule of five was used for the prediction of drug-likeness properties of synthesized naringin derivatives.
General procedure for the synthesis of naringin derivatives. The naringin derivatives were synthesized as per the procedure of Yang et al. and Saini et al. with slide modifications and are outlined in Scheme 1 47,48 . Substituted aniline (0.01 mol) was taken in a round bottom flask and concentrated hydrochloric acid drop wise was added. Equimolar concentration of naringin (0.01 mol) was dissolved in ethanol (50 mL) in equimolar concentration and was refluxed. Completion of the reaction was confirmed by single spot TLC. After the completion of reaction the concentrated reaction mixture was concentrated and the formed precipitated were filtered off desiccated. The crude products were recrystallized using alcohol yielded compound 1-8. The confirmation of the final compounds was made by physicochemical and spectral methods like FTIR, 1 H NMR and 13 C NMR spectra, Mass spectroscopy and elemental analysis.
Spectral data.   1            Antioxidant activity. DPPH radical scavenging assay. Antioxidant activity of the synthesized was evaluated by photocolorimetric assay by using DPPH (2,2-diphenyl-1-pycrilhydrazil hydrate) free radical scavenging method. Briefly, 0.1 mM solution of DPPH was prepared in methyl alcohol and 1 mL of this solution was added in to 1 mL of sample or standard. Discolorations were measured at 517 nm after incubation for 30 min at 30 °C in the dark. Lesser absorbance of the reaction mixture indicates the higher free radical scavenging potential. The test was performed in triplicate and the % inhibition values of all the synthesized compounds were calculated by using the formula:
Stability studies of the selected preservatives. From the results of preservative efficacy study, selected compound was further evaluated for its stability as per the protocol provided in ICH guidelines. The selected compound was added in the final containers containing White Lotion USP. The preparation having standard preservative and test compound was stored at 40 ± 2 °C at 75% RH ± 5% RH (as per ICH guidelines) and was analyzed for the change in pH and cfu/ml at the time interval of 0, 1, 2, 3, 4, 5 and 6 months.
Biological evaluation of selected preservatives. Finally, the selected most active compounds from preservative efficacy and stability study were evaluated for in vitro skin permeation, skin and eye irritation study. Naringin compound 7 was selected for their biological evaluation as per the following procedures: In vitro skin permeation study using Franz diffusion cell. The

Skin irritation test in rabbits.
Draize test has based on the principle of skin damage caused by direct toxic action of irritant substances. The Draize 24-h patch test in rabbits has been utilized as the most widely used animal test for testing of primary irritant substances 59 . Test substance (0.5 g) was smoothly applied over the previously shaved rabbit's skin on 6 cm 2 area and covered with gauze patch. If, no irritation has been observed in initial test, then confirmatory test will be conducted in another two rabbits, patch was removed after 4 h of contact and observations were made after 1, 24, 48 and 72 h after patch removal. The dermal irritation scores were recorded on the basis of type and severity of lesions, and graded as per standard Draize test scoring criteria 60 .
Eye irritation test rabbits. Test substance (100 mg/0.1 ml) was smoothly applied in rabbit's eyes. No irritation was observed in initial test, (after the 1 h of administration), then confirmatory test was conducted and observations were noted. The grades of ocular reaction (conjunctivae, cornea and iris) will be observed as per OECD guidelines and recorded at an interval of 1, 24, 48, and 72 h after the application of test substance. Different grades for eye irritation severity were recorded as per standard criteria.

Statistical analyses.
All the data was represented as mean ± standard deviation (SD) for three triplicates of each sample. One-way ANOVA test at a significance level of 0.05 (p < 0.05) using MS excel statistical tool was used to analyze the experimental data. Docking results of compound 7, showed the formation of four hydrogen bonds between residues Ala 602, Val 399, Cys 300 and Thr 302 with hydroxyl and oxygen atom of synthesized ligands. Hydrophobic interactions were seen among residues Ser 303 and Glu 488. Compound 6 has been attached to Gln 348, Thr 302 and Val 602 with hydroxyl as well oxygen atom of synthesized compounds by four hydrogen bonds and was also found to interact hydrophobically with Glu 488 and Leu 601 residues. The results of molecular docking for different ligands within G-6-P synthase pocket and their interaction with different amino acid residues have been shown in Table 1. Here, the inhibition of G-6-P synthase enzyme further evaluated by the outcomes of the inhibition likes antimicrobial activity. This further made the clearance behind the inhibition of G-6-P synthase enzyme by different proposed molecules. ADME study. Different ADMET parameters of proposed aesculin derivatives were determined in silico by QikProp application (Schrodinger LLC) and PreADMET software. The parameters were analyzed and were compared with the standard values. Here, QPPCaco descriptor determined the Caco-2 cell permeability and predicted value defined the barrier between gut and blood system. QPlogBB was the blood/brain partition coefficients determined to be used as a parameter contributing the entry of drugs to the central nervous system. QPPMDCK was used for the estimation of oral absorption. QPlogKp was the descriptor that determined the dermal penetration. The lower values logKp was considered to have low skin permeation and all the selected aesculin derivatives (marked yellow) were having the best suited values range and were to be selected as preservative for topical formulation. QPlogKhsa descriptor determined the binding of drugs to plasma proteins [61][62][63][64][65] . The different ADME parameters of proposed naringin derivatives have been represented in Table 2. All the results were expressed as mean ± standard deviation (n = 3) and results were found significant p < 0.05.

Ethics approval and consent to participate. Institutional Animal Ethical
Chemistry. Scheme  Antioxidant activity. DPPH radical scavenging activity. DPPH free radical scavenging assay confirmed that compounds 7, 6 and 1 possessed good antioxidant potential with IC 50 6.23 ± 0.03 µM, 7.03 ± 0.03 µM, 7.31 ± 0.06 µM, respectively as compared to standard l-ascorbic acid IC 50 , and A. niger, respectively). The results of antimicrobial activity revealed that the synthesized compounds have antimicrobial potential as compared to standard drugs as shown in Table 3. The probable mechanism of antimicrobial activity of naringin derivatives may be due to the better inhibition of G-6-Psynthase.
Preservative efficacy study. The highly active antimicrobial compounds 6 and 7 in the series were selected for the evaluation of preservative efficacy. The results of preservative efficacy testing were performed in triplicate and have been reported as mean values in Table 4. Compound 7 showed the values of log CFU/ mL reduction within the prescribed limit and the results were comparable to that of the standard preservatives sodium benzoate, propyl paraben and methyl paraben. Result of compound 6 showed a less than 2.0 log reductions from initial count on 14 days and number of CFU/ml values of preservative efficacy against some microbial strains increased on the 14th day to 28th day as compare to standard preservatives sodium benzoate, propyl paraben and methyl Paraben and found significant with p < 0.05.Preservative efficacy of compound 7 in White lotion USP and degree of microbial log reduction have been represented in Fig. 1. www.nature.com/scientificreports/ Stability study. Results of stability study revealed that the pH of White lotion USP samples were in range of 5.5-6.0.The results of the microbial study indicated that no microbial growth was observed in samples containing compound 7 over a period of six months period as per ICH guidelines. These results indicated that the product was stable as compared to standard preservative with added naringin compound 7 as preservative. The results of stability study were performed in triplicate and were reported as mean values. Results for microbial growth and pH changes also found to be significant at p < 0.05.

Biological evaluation of selected preservatives. In vitro skin permeation study using Franz diffusion
cell. In vitro skin permeation study of selected compound 7 was performed by using Franz diffusion cell having receptor and donor cell. Concentration of compound 7 at different time interval was calculated by standard plot. It was observed from the skin permeation data with different time intervals that maximum amount of drug was released within 120 min. It was concluded from the skin permeation data as shown in Table 5 that the maximum amount of drug released within first 120 min i.e. 45.05%. The total amount of selected compound 7 that was permeated through selected skin graft was found to be 4.27 mg (0.0013 mol of sample taken). www.nature.com/scientificreports/ Skin irritation test of naringin derivative in rabbits. The dermal irritation scores were evaluated by the type and severity of the lesions produced. All the rabbits were checked for the reversibility of any skin reaction up to 14 days, by evaluating skin irritation responses i.e. alopecia, hyperplasia, hyperkeratosis and scaling. Scoring of the dermal reaction was done with the standard OECD guideline-404. The test compound 7 did not showed any type of dermal reaction and was considered as non-irritant. No skin erythema and edema formation was observed during test. The irritation score for erythema and edema in all the rabbits were found have Score 0. Skin area of rabbit used for testing of compound 7 before and after application have been shown in Fig. 2. Table 2. Results of molecular docking and ADMET parameters for proposed naringin derivatives. QPPCaco: Caco-2 cell is a model for the gut-blood barrier; QPlogBB: Predicted brain/blood partition coefficient; QPPMDCK: MDCK cells are considered to be a good mimic for the blood brain barrier; QPlogKp: Predicted skin permeability; QPlogKhsa: Prediction of binding to human serum albumin; CNS: Predicted central nervous system activity.  did not showed any type of eye irritation reaction and was considered as non irritant for eyes. No ulceration, conjunctivae redness and chemosis were observed in eye irritation test. The irritation score for cornea chemosis, conjunctivae and iris in all the rabbits were found to have 0. Changes in produced in the eyes of the rabbits used for testing of compound 7 before and after application have been shown in Fig. 3.
Structure activity relationship (SAR) studies. Design approach of naringin derivative for G-6-P inhibition and antioxidant activity has been represented in Fig. 4. The structure activity relationship of the synthesized naringin derivatives with their antioxidant activity results have been summarized as: 1. The substitution of carbonyl group of naringin with aliphatic aliphatic amines decreased the biological activity i.e. compounds 3, 4, 5 shows lower activity as compared to compounds 1, 2 and 7. 2. The substitution of carbonyl group of naringin with aromatic amide ring attached directely to Naringin enhanced the biological activity.   www.nature.com/scientificreports/ 3. The presence of electronegative halides on ortho position of aromatic amide ringj enhanced the biological activity (compound 1 and 7). 4. The presence of nitro group on para position of aromatic amide ringj enhanced the biological activity (compound 6 and 7).

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.   www.nature.com/scientificreports/