Paper Title
Instrumental and Physical Characterization of Ellagitannin from Pomegranate Peel

Abstract
For optimization of the process variables (temperature and time), Response Surface Methodology (RSM) was applied (independent variables: temperature 19.6, 30, 55, 80 and 90.4oC and time 17.6, 30, 60, 90, 102.4 min) for maximum total ellagitannin (ETs) content (TEC) and antioxidant activity (AOA) (dependent variables) in each extraction method. RSM model predicted optimum temperature time combination of 90oC/19 min) at which TEC and AOA by physical and enzymatic method (43.90±1.64% as GAE, db & 64.71±1.05% RSA as DPPH and 45.01±0.85% as GAE, db & 63.74±1.28% RSA as DPPH, respectively) were the highest. Hence physical and enzymatic method were considered for further preparation of ellagitannin powder using 90oC/19 min. Extracts were purified through resin (XAD-16) packed column by using ethanol:water (80:20) as elution solvent. Solvent was evaporated in tray drier under vacuum (45±2oC & -18 to -22 mbar) to obtain the dark brown colour ellagitannin powder (ETP). ETP prepared from fresh pomegranate peel (FPP) and fermented pomegranate peel (FrPP) were characterized for functional group (acidic, phenolic, hydroxyl and hydrocarbon compounds) by FTIR, UV-Vis absorption of 10 ppm aqueous methanolic (1:1) solution exhibited λmax (213 nm), HPLC chromatogram confirms the characteristic peak of ellagic acid (EA), gallic acid (GA) and punicalagin (PU) in ETP from FPP while PU peak was not found at particular retention time in ETP from FrPP. LCMS scan spectra of ETP from FPP has confirmed the presence of EA (m/z 301), GA (m/z 169) and PU (m/z 541) along with pentoside (m/z 432), hexoside (m/z 770) and glucoside (m/z 472) of EA. ETP from FrPP also confirmed mass of EA and GA along with pentoside (m/z 403, 417) and other uncharacterized m/z compounds but PU was absent even at zoom view of spectrum which might be due to enzymatic breakdown of PU during fermentation. ETP form FPP and FrPP were having the color value (L,a,b: 49.42, 18.34, 9.89 and 45.07, 9.33 and 8.59), odour (characteristic), taste (stringent), pH of one percent aqueous solution (3.82 and 3.69), solubility at pH 2, 4, 6, 8, 10 (>99%), density (0.910 and 0.938 g/ml), wettability (144 and 124 second), hygroscopicity (3.86% and 4.18%), water activity (0.292 and 0.313), inorganic matter (0.03% and 0.04%) and antioxidant activity (94.47 and 91.16% RSA as DPPH) respectively. ETP from FPP is more heat stable (total mass change 44.33%) than the ETP from FrPP (mass change 54.75%) observed when heated up to 500oC by TGA. Glass transition temperature and heat capacity of ETP from FPP and FrPP were 152.5oC and 4.633 J/g and 66.2oC and 3.861 J/g respectively. Hence ETP from FPP is more suitable for high temperature processing. ETP from FPP was more effective than the ETP from FrPP against food pathogenic bacteria. Minimum inhibitory concentration (MIC) of ETP from FPP and FrPP were 2mg/ml and 4mg/ml (except Escherichia. coli) respectively against the Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and E. coli while MIC of ETP from FPP was 4 mg/ml against the Salmonella typhimurium. ETP from FrPP was not inhibited the growth of S. typhimurium and E. coli up to 10 mg/ml tested concentration. Cytotoxicity of ETP indicated IC50 of 92 mg/ml and 30 mg/ml of ETP from FPP and FrPP respectively. Hence ETP prepared from FPP is safer at low dose in comparison of ETP from FrPP. On the basis of above findings it can be stated that ETP from FPP have desirable physicochemical, thermal and antibacterial properties with low toxicity. Hence extraction of ETs from fresh pomegranate peel by physical method was recommended for preparation of ETP as a common agro-waste processing practice.