Materials and methods used
All the chemicals and reagents used in the experimental part of the study were of analytical grade. Nutrient agar, nutrient broth, sabouraud dextrose agar, and sabouraud broth were purchased from HiMedia Laboratories (Mumbai, India). Aesculin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), anilines, hydrochloric acid were purchased from Sigma Aldrich (Germany), LobaChemie (Mumbai, India), and SRL (Mumbai, India). Reaction progress was checked by thin-layer chromatography (TLC) method. Standard streptomycin, ampicillin, ciprofloxacin, and fluconazole were obtained from Belco Pharma, Bahadurgarh (India). The standard microbial strains E. coli 45, S. aureus 3160, P. aeruginosa 1934, C. albicans 183 and A. niger 282 were obtained in lyophilized form from MTCC, Chandigarh (India). Melting point was recorded by the Sonar melting point apparatus. FTIR spectra were recorded on Perkin Elmer FTIR spectrophotometer, 1H NMR, and 13C NMR spectra were recorded on Bruker Avance II 400 NMR spectrometer. Mass spectra were recorded on Waters Micromass Q-ToF Micro instrument while the elemental analysis was done by Perkin Elmer 2400 elemental analyzer.
Molecular docking
The three-dimensional structures of aesculin derivatives were constructed by using Chemdraw ultra 8, and energy was minimized with the LigPrep tool of Schrodinger Maestro. The X-ray crystal structures of G-6-P synthase were downloaded from the Protein Data Bank (http://www.rcsb.org/pdb). PDB ID 1MOQ (resolution of 1.57 Å) was selected on the basis of the lowest resolution as well availability and water molecules (except those coordinated to metals and between the ligand–protein) were removed with the help of Schrodinger protein preparation wizard [14]. The energy-restrained of the protein structure target site optimization of targeted protein G-6-P synthase was done by using Optimized Potential for Liquid Simulations (OPLS-2005) as force field. The partial charges were computed according to the OPLS-2005 force field (32 stereoisomers, tautomers, and ionization) on biological pH. All the calculations were carried out by using Schrodinger, Inc. (New York, USA) software Maestro 11 with an induced fit docking (IFD) method. The ligands prepared after energy minimization was used for molecular docking studies. All the computational work was performed in Laboratory for preservation technology and Enzyme Inhibition Studies, Department of Pharmaceutical Sciences, M.D. University, Rohtak, INDIA, was used for all computational work [15, 16].
ADMET analysis
Quick prop from Schrodinger was utilized for in-silico prediction of ADME properties of proposed and synthesized aesculin derivatives. Various ADME parameters were calculated such as Log P, number of rotatable bonds, number of hydrogen acceptor, Log BB, and number of hydrogen bond donor atoms. Lipinski’s rule of five was also used for the prediction of a drug-like profile of newly synthesized derivatives.
Procedure for synthesis of aesculin derivatives
Aesculin derivatives were synthesized by some modifications in the procedure of Yang et al. 2006 as outlined in Fig. 2 [17]. The proposed derivatives were synthesized with substituted aniline (0.01 mol) taken in a round bottom flask. To this reaction mixture, concentrated hydrochloric acid was added dropwise with continuous stirring. Aesculin (0.01 mol) was dissolved in ethanol (50 mL) in equimolar concentration and was refluxed. Synthesis of derivatives was monitored by single spot TLC. On the completion of reaction the concentrated reaction mixture was precipitated. Recrystallization of the crude products was done by using alcohol. The final structure of the compounds was confirmed by FTIR, 1H NMR spectra, 13C NMR spectra, mass spectra and elemental analysis.
Spectral data
2-(3,4-dihydroxyphenyl)-3-(3-nitrophenylamino)chroman-5,7-diol (Compound 1)
Rf TLC mobile phase: Methanol: Chloroform (20:80) = 0.56; Yield = 35%; M.P. = 220–222 °C; M.Wt. = 429.14; IR (KBr pellets) cm−1: 1383 (–C–O–C), 1040 (–C–C–), 1684 (–C=N–), 2948 (–C–H–), 3387 (–OH–); 1H NMR (400 MHz, CDCL3): δ 9.93 (s, 1H), 7.66 (d, J = 9.2 Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.44 (s, 1H), 7.35 (d, J = 6.0 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 7.28 (t, J = 8.0 Hz, 1H), 6.87 (s, 1H), 5.04 (s, 1H), 4.95 (d, J = 8.1 Hz, 1H), 4.62 (d, J = 7.2 Hz, 1H), 4.59 (d, J = 9.7 Hz, 1H), 4.52 (d, J = 10.6 Hz, 1H), 4.13 (d, J = 8.0 Hz, 1H), 4.07 (d, J = 7.8 Hz, 1H), 3.73 (dd, J = 13.8, 7.5 Hz, 1H), 3.50 (q, J = 9.3, 8.8 Hz, 1H), 3.41 (s, 2H), 3.38 (q, J = 8.5 Hz, 1H), 3.25–3.23 (m, 1H); 13C NMR (400 MHz, CDCL3) δ 160.24, 149.30, 147.84, 145.58, 145.08, 137.34, 129.17, 127.71, 126.97, 114.97, 112.87, 111.52, 102.93, 101.32, 79.51, 74.14, 73.41, 54.69, 38.44, 25.96, 12.61, 10.1; MS ES + (ToF): m/z 429.14 [M++2]; CHNS: Calc (C23H23NO8): C, 61.53; H, 5.40; N, 3.26; O, 29.81; Found C, 61.54; H, 5.42; N, 3.27; O, 29.78.
2-(3,4-dihydroxyphenyl)-3-(naphthalen-1-ylamino)chroman-5,7-diol (Compound 2)
Rf TLC mobile phase: Methanol: Chloroform (20:80) = 0.60; Yield = 40%; M.P.; 168–170 °C; M.Wt. = 465.14;; IR (KBr pellets) cm−1: 1166 (–C–O–C), 1077 (–C–C–), 1457 (–C = C–), 1699 (–C=N–), 2936 (–C–H–), 3390 (–OH–); 1H NMR (400 MHz, CDCL3) δ 9.93 (s, 1H), 8.32 (d, J = 7.3 Hz, 1H), 7.95 (d, J = 8.9 Hz, 2H), 7.86 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.56 (d, J = 10.8 Hz, 2H), 7.54 (d, J = 8.1 Hz, 1H), 7.31 (s, 1H), 7.13 (d, J = 8.6 Hz, 1H), 6.75 (s, 1H), 5.04 (s, 1H), 4.95 (d, J = 8.1 Hz, 1H), 4.62 (d, J = 7.6 Hz, 1H), 4.13 (d, J = 8.1 Hz, 1H), 4.07 (d, J = 8.6 Hz, 1H), 3.73 (dd, J = 13.8, 7.5 Hz, 1H), 3.50 (q, J = 8.9 Hz, 1H), 3.41 (s, 2H), 3.38 (q, J = 8.5 Hz, 1H), 3.25–3.23 (m, 1H); 13C NMR (400 MHz, CDCL3) δ 163.20, 149.30, 146.42, 146.35, 145.76, 145.58, 133.27, 129.24, 128.14, 128.06, 126.64, 126.56, 123.70, 122.78, 122.06, 112.87, 111.70, 110.94, 103.36, 101.62, 77.41, 75.43, 73.23, 70.74, 62.21; MS ES + (ToF): m/z 467.16 [M++2]; CHNS: Calc (C25H23NO8): C, 64.51; H, 4.98; N, 3.01; O, 27.50; Found C, 64.53; H, 4.97; N, 3.02; O, 27.47.
In vitro evaluation of antioxidant potential of synthesized derivatives of selected leads using DPPH method
The ability of the synthesized aesculin derivatives to scavenge DPPH radicals was determined by DPPH free radical scavenging method. The aliquot of test compounds at different concentrations in methanol was mixed. The different concentration used for the evaluation antioxidant potential includes 12.5, 25, 50, 75 and 100 μg/mL. The 0.1 mM solution of DPPH was prepared in methyl alcohol, and 1 mL of this solution was further diluted to 3 mL both for the sample and standard. After 30 min of incubation in darkness and at ambient temperature, the resultant absorbance was recorded at 517 nm. The tests were performed in triplicate and the % inhibition of compounds was calculated by using the formula:
$$\% {\text{ Inhibition }} = \, \left( {{\text{A}}_{{\text{c}}} {-}{\text{ A}}_{{\text{s}}} } \right) \, \times { 1}00/{\text{A}}_{{\text{c}}}$$
Here, Ac was the absorbance of the control, and As was the absorbance of the sample [18].
In vitro evaluation of antimicrobial potential of synthesized derivatives of selected leads by using tube dilution method
The newly synthesized aesculin derivatives were further evaluated for their antimicrobial potential against various MTCC strains viz. E. coli 45, P. aeruginosa 1934, S. aureus 3160, P. mirabilis 3310, A. niger 282 and C. albicans by broth dilution method. The highest dilution of the test compound resulting in no growth of microorganism was recorded as their MIC value. Dilutions of test and standard compounds were prepared in double strength nutrient broth I.P. (bacteria) or Sabouraud dextrose broth I.P. (fungi) [19]. A 0.9% NaCl solution was used to adjust the turbidity of bacterial and fungal cultures. The CFU and density of microorganism was adjusted to 0.5 McFarland standards with the help of distilled water [20]. The samples were incubated at 37 °C for 24 h (bacteria), at 37 °C for 7 days (A. niger), and at 37 °C for 48 h (C. albicans), and the results were recorded in pMIC.
Evaluation of preservative efficacy of selected antimicrobial/antioxidant derivatives
White lotion USP and Aloe vera juice was used for evaluation of preservative efficacy of the selected aesculin derivatives. Selected derivatives of aesculin were used as preservatives in equivalent amount in cosmetic and the food product [21]. Aloe vera juice was prepared as per the method described by Ahlawat et al. with slight modifications. The aloe vera juice thus obtained was used for the testing of food preservative efficacy [22, 23]. White lotion USP was prepared as per the method of Narang et al. The compounds 1 and 2 in equimolar amount (0.0013 mol of methyl paraben) were used as novel preservatives by replacing standard preservatives sodium benzoate, methyl paraben and propyl paraben in both the preparations [24]. Standard strains of P. aeruginosa 1934, S. aureus 3160, E. coli 45, A. niger 282, and C. albicans 183 from MTCC were used as common microbial contaminants for evaluation of a preservative efficacy as per the protocol described in I.P., 2010 [25].
Test procedure
Aloe vera juice and White lotion USP were taken for preservative efficacy study and compound 1 was added as test preservative in equimolar quantity (0.0013 mol of methyl paraben) to that of standard preservative. A microbial cell count of 1 × 105–1 × 106 cfu/mL was used for microbial inoculation in a quantity of 0.5–1% to the volume of the product taken for study. Samples were incubated at room temperature for 28 days. On incubation the CFU/mL of the product was determined at 0 day, 7 days, 14 days, 21 days, and 28 days by using agar pour plate technique [26]. As per the USP standard protocol the log values of cfu/mL was calculated as not less than 2.0 log reduction from initial count at 14th day of incubation and no increase in microbial count from 14th day to 28th days for fungi [27].
Stability study of the selected preservatives as per ICH guidelines
From the results of preservative efficacy study, compound 1 was selected for further evaluation of its stability behavior as per the ICH guidelines. The compound 1 was added in the final containers containing the preparations of Aloe vera juice and White Lotion USP. Both the preparations having standard preservative and the test compound 1 were stored at 40° ± 2 °C at 75% RH ± 5% RH (as per ICH guidelines) and were analyzed for the change in pH and cfu/ml at the time interval of 0, 1, 2, 3, 4, 5 and 6 months.
Statistical analysis
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.