Amygdalin based G-6-P synthase inhibitors as novel preservatives for food and pharmaceutical products

G-6-P synthase enzyme has been involved in the synthesis of the microbial cell wall, and its inhibition may lead to the antimicrobial effect. In the present study, we designed a library of amygdalin derivatives, and two most active derivatives selected on the basis of various parameters viz. dock score, binding energy, and ADMET data using molecular docking software (Schrodinger’s Maestro). The selected derivatives were synthesized and evaluated for their antioxidant and antimicrobial potential against several Gram (+ ve), Gram (−ve), as well as fungal strains. The results indicated that synthesized compounds exhibited good antioxidant, antimicrobial, and better preservative efficacy in food preparation as compared to the standard compounds. No significant differences were observed in different parameters as confirmed by Kruskal–Wallis test (p < 0.05). Docking results have been found in good correlation with experimental wet-lab data. Moreover, the mechanistic insight into the docking poses has also been explored by binding interactions of amygdalin derivative inside the dynamic site of G-6-P synthase.

General procedure for the synthesis of amygdalin derivatives. The amygdalin derivatives were synthesized by using the procedure outlined in Scheme 1 by enzyme catalysis. Here, 40 ml of acetone containing 0.1 mol/L amygdalin and 0.3 mol/L of sinapic and syringic acid in 1 g of Novozyme 435 were taken in incubator shaker at 200 rpm at 45 °C (48 h). The filtration of the mixture terminated the reaction. Both the compounds in the series were synthesized according to the standard procedures as outlined in Scheme 1 (Fig. 1). The 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 1-2. The confirmation of the final compounds was made by physicochemical and spectral methods like FTIR, 1H NMR, 13C NMR spectra, CHN and mass analysis. (6-((6-(cyano(phenyl) 25 .

Spectral data. (E)-
Preservative effectiveness study. The selected most active antioxidant/antimicrobial compounds were further evaluated for their preservative potential in the cosmetic product, White lotion USP and food products; such as fresh aloe vera juice the cosmetic product as per the procedure mentioned in USP 2004 26 .
Preparation of aloe vera juice. Aloe vera leaves were cleaned with distilled water and cleaned with 0.5% chlorine water. The leaf base and tip were chopped 1.5 inches and 3 inches, respectively. Margins of leaves were removed with the help of a stainless steel knife. The pulp was washed 2-3 times with distilled water to remove the exudates and homogenized with the help of a blender, and then filtered through an autoclave muslin cloth. The aloe vera juice thus obtained was used for the testing of food preservative efficacy 27 .
Preparation of white lotion USP. Ingredients: Zinc sulfate 40 gm, sulfurated potash 40 gm and purified water q.s. to 1,000 mL. Firstly, zinc sulfate and sulfurated potash were dissolved in 450 mL of water separately and filtered. Then, sulfurated potash solution was added to zinc sulfate with stirring. At last, the required amount of water was added and mixed thoroughly and sterilized. For preservative efficacy testing, the White lotion USP was prepared using the equimolar amount of compounds 1-2 as novel preservatives by replacing sodium benzoate, methyl paraben and propyl paraben from the formula 28  Preservative efficacy procedure. Aloe vera juice. Preservative efficacy of the selected compound 1 and sodium benzoate (standard) was estimated in freshly prepared aloe vera juice as per the method with minor modifications as described by Ahlawat et al. 30 . A concentration of 600 mg/kg or 600 ppm of sodium benzoate in aloe vera juice was taken as per food safety and standard guidelines. Equimolar quantity (0.0004 mol) of selected compound was taken as a preservative system in test samples. Challenged microbial cell suspension was inoculated the juice preparation with inoculum size never exceed more than 1%. After inoculation with microbes juice was incubated at room temperature for consecutive 28 days, and samples were collected on the 14th and 28th day of the experiment. The viable count of microorganisms was performed on nutrient agar (bacteria), and sabouraud dextrose agar (fungi) plates 31 . Each experiment was done in triplicate and log cfu/ml of juice was determined with comparison to standard.
White lotions USP. White lotions USP was added in final containers and were used in the challenge test. The preparation was inoculated with a 0.5-1% volume of microbial inoculum having a concentration of 1 × 10 5 -1 × 10 6 CFU/mL 32 . Inoculated samples were mixed thoroughly to ensure homogeneous microorganism distribution and incubated. The CFU/mL of the product was determined at an interval of 0 days, 7 days, 14 days, 21 days, and 28 days in agar plates. Log CFU/mL of white lotion USP was calculated as not as not less than 2.0 log reductions from initial count on 14th day of incubation and no increase in CFU from 14th day count to 28th day in case of bacteria and no increase from the initial calculated count on 14th day and 28th day in case of fungi 33 .
Stability study. Compound 1 was selected for stability study in their final container containing the formulation of Aole vera gel and White Lotion USP. Formulation having different preservatives i.e., standard and compound 1 were stored at 40° ± 2 °C at 75% RH ± 5% RH (as per ICH guidelines) and were analyzed for the pH and cfu/ml of the product at 0, 1, 2, 3, 4, 5, and 6 months.
Statistical analyses. Data

Results and discussion
Molecular docking study. The selection of amygdalin for further evaluation as preservative has been made on the basis of our prior evaluation in docking and absorption, distribution, metabolism and excretion (ADMET) study data 34 . Then the proposed library of amygdalin derivatives was again evaluated for their molecular docking behavior with the help of Schrodinger's Maestro docking software 35 . Molecules were docked with the help of the G-6-P synthase crystallographic complex having pdb id 1moq. The Induced Fit docking (IFD) method of docking was utilized for the same. The predicted binding pattern revealed that synthesized ligand binds within the catalytic cavity of G-6-P synthase firmly via hydrogen bond formation, pi-pi stacking, and hydrophobic interactions. Two compounds 1 and 2, were selected via docking score, binding energy, and ADMET study parameters.  Table 1.
ADMET study. The compounds selected from docking have been further evaluated for their ADME parameters so that the selection of final preservative becomes easy. The evaluation of different ADMET parameters of selected amygdalin derivatives has been represented in Table 1. All the synthesized compounds fulfilled the standard Rule of Five 36 . All the synthesized compounds qualified the conditions for various descriptors like lipo- Antioxidant activity. DPPH radical scavenging activity. The plant-based antioxidants can be used in food and pharmaceuticals to enhance their shelf life against oxidation 40 . In the present study, DPPH radical scavenging assay method was utilized for the evaluation of the antioxidant profile of the synthesized compounds 41 . In this screening, compound 1 was observed as the most potent antioxidant compound (IC 50 values 5.54 ± 0.03 µM) as compared to reference standard L-ascorbic acid (IC 50 values 8.11 ± 0.0.69 µM). However, compound 2 showed moderate antioxidant activity (IC 50 value 6.51 ± 0.04 µM). The antioxidant activity of the amygdalin was found 7.72 ± 0.03 µM 42 . Here, the better antioxidant property of amygdalin derivative shall be useful in the preservation of food, cosmetics, and pharmaceuticals 43 . All the results were expressed as mean ± standard deviation (n = 5) and results were found significant with Krukal-Wallis test (p < 0.05).
Antimicrobial activity. MIC 1) revealed that the synthesized compounds have better antimicrobial potential as compared to standard ciprofloxacin, ampecillin, and fluconazole. The probable mechanism of antimicrobial activity of amygdalin derivatives may be due to the better inhibition of G-6-P synthase. www.nature.com/scientificreports/ Preservative efficacy. The results of preservative efficacy study of the aloe vera juice and White lotion USP were performed and were reported as mean ± standard deviation. Results of microbial growth at 14th day and 28th day were found to be significant with p < 0.05 as confirmed by Kruskal-Wallis test.
Aloe vera juice. The results of preservative effectiveness have been summarized in Table 2. The log CFU/ml for compound 1 revealed that the values were within the prescribed limit as per USP criteria. The selected compound 1 reduced the growth of microbes on the 14th day from the initial count and found to be effective on the 28th day and results were also comparable to sodium benzoate. The preservative efficacy of the amygdalin compound 1 has been represented for number of days vs. degree of microbial log reduction and has been shown graphically in Fig. 3.
White lotion USP. The highly active antimicrobial compound 1 was selected for evaluation of its preservative efficacy. Result showed a less than 2.0 log reductions from initial count on 14th day and number of CFU/ml in some samples increased on the 14th day to 28th day as compared to that of the standard preservatives sodium benzoate, propyl paraben and ethyl paraben. The log CFU/ml ( Table 2) for compound 1 revealed that the values were within the prescribed limit as per USP criteria 28 . The graphical representation of preservative efficacy  www.nature.com/scientificreports/ of amygdalin compound 1 in white lotion USP has been presented between the numbers of days vs. degree of microbial log reduction and graphically same has been represented in Fig. 4.
Stability study. The results of stability testing were performed in triplicate, and were reported as mean values in Table 3. Results revealed that the pH of Aloe vera juice and White lotion USP samples were in range of 7.6-8.0, which indicated the stability of compound 1 ((E)-(6-((6-(cyano(phenyl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2yl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-yl)methyl3-(4-hydroxy-3,5 dimethoxyphenyl) acrylate) as preservative over, the six months period as compared to that of the standard preservatives sodium benzoate, propyl paraben, and methyl paraben. The results of the microbial study indicated that no microbial growth was observed in samples containing compound 1, over 6 months period. These results indicated that the products were found stable over 6 months with added preservatives. Results for microbial growth and pH changes also found to be significant at p < 0.05.

Conclusion
It has also been reported in our previous study that amygdalin can act as an active inhibitor of G-6-P synthase enzyme based upon the results of molecular docking and ADMET data 43    www.nature.com/scientificreports/ of mechanism for the inhibition with visual binding interactions. The derivatives of amygdalin ((E)-(6-((6-(cyano(phenyl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2yl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-yl)methyl3-(4-hydroxy-3,5 dimethoxyphenyl) acrylate) showed antioxidant, antimicrobial, better preservative efficacy and prevent the change in pH as well microbial count of formulation for food as well as pharmaceutical products, which were in agreement with the results of molecular docking and highlight the mechanism of their preservative activity. Therefore, the synthesized amygdalin derivatives can be used as novel food and pharmaceutical preservatives to prevent them from microbial degradation.

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