Advanced glycation end products formation inhibition through standardized crude extract of punica granatum L. stem bark

Abstract

Formation of advanced glycation end products (AGEs) under hyperglycemic condition in diabetes mellitus results in micro/macro-angiopathy disorders. Juice, leaves, or peel of pomegranate have shown antioxidant or antiglycation effects. Pomegranate stem barks which are hugely wasted during the pruning season could be a good source of phyto-based anti-AGEs. This study evaluated standardized pomegranate stem barks extract in term of antioxidant activity, antiglycation potential and also its effect on lipid formation and glucose consumption in 3T3-L1 cells.Various extraction conditions were performed including types of solvents, time and type of extraction methods. Phytochemical analysis of extracts was carried by highperformance liquid chromatography-pulsed amperometric detector (HPLC-PAD), gas chromatography-mass spectroscopy and spectrophotometric methods. Evaluation of antioxidant activity was performed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′- Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and metal chelating activity. Anti glycation activity of the extract was evaluated in bovine serum albumin (BSA)/glucose or BSA/methylglyoxal (MGO) bioassay systems in presence or absence of samples. Antiglycation property was measured by determination of the level of formation of fructosamine, protein carbonyl and AGE or loss of thiol group. Also, effect of extract on glucose consumption and lipid formation in 3T3-L1 cell line in media containing MGO was investigated in vitro. The result showed that eight hour extraction with methanol using Soxhlet extraction (SM8) was the best extraction process in term of total polyphenolic compounds (59.69 ± 2.913 mg gallic acid equivalent (GAE)/g dry weight (DW), DPPH scavenging capacity [half maximal effective concentration (EC50) 14.99 ± 1.18 mg/L], ABTS●+ radical scavenging equal to 2.636 mM trolox equivalent antioxidant capacity (TEAC)/100 g DW and metal chelation activity (EC50 888.1±48.38). Standardization of SM8 extract by HPLC-PAD showed gallic acid as 0.19% and catechin 0.03% of the extract. SM8 extract reduced formation of AGE significantly (p<0.01) by 77% in concentration of 250 μg /ml. Moreover, it reduced protein carbonyl (60.2%) and fructosamine formation (33.99 %) and simultaneously inhibited thiol group loss (by 1.84 folds). The SM8 extract increased glucose consumption (by 1.95 folds) in 3T3-L1 cells in glycemic condition. In conclusion, it is recommended that pomegranate stem bark extract as a potential source of raw material to be further investigated for the development of health supplement with AGEs inhibitory properties.