Evaluating the antihyperglycemic and antioxidative effects of ellagitannin and punicalagin from pomegranate fruit peel

Abstract

Background The vascular and metabolic health benefits of pomegranate fruit have been frequently mentioned. Despite being under-utilized, the peel of this fruit contains ellagitannins that can potentially increase the medicinal relevance of this fruit in managing metabolic diseases such as diabetes and oxidative complications. Unfortunately, the optimum recovery methods of these bioactive ellagitannins from pomegranate peel and their bioactivity profiling has been scarcely reported. Therefore, the aim of this study was to optimally recover ellagitannins and punicalagin with potent antioxidant and glycemic control properties from the fruit peel of “Wonderful” pomegranate. Methodology “Wonderful” pomegranate fruits (Class 1 or processing grade) were purchased from Sonlia Fruit Packhouse (Wellington, Western Cape Province, South Africa). The fresh peel was extracted using water at different temperatures (25, 37, 50, 65, 78 and 95 °C). The extracts were preliminary evaluated for phenol and flavonoid contents, as well as in vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, Fe3+ reducing and α-amylase and α-glucosidase inhibitory activities at a concentration of 45 μg/mL. An Amberlite® XAD16N resin was used to chromatographically recover tannins from the extracts. Ellagitannins in the tannins were quantified by Liquid chromatography – mass spectrometry (LC–MS). Punicalagin was purified from the tannin recovered from the 78 °C crude extract using Sephadex LH-20 gel. The dose dependent radical scavenging, oxygen radical absorbance capacity (ORAC), antiglycation, α-amylase inhibitory and anti-linoleic acid peroxidative potential of the purified punicalagin was evaluated in vitro and compared with that of the corresponding crude extract and tannin. In Chang liver cells induced with oxidative stress, the ability of the purified punicalagin and tannin, as well as the corresponding parent crude extract to inhibit lipid peroxidation and improve catalase and superoxide dismutase activity was measured. Also, their modulatory potential on glucose uptake/utilization was evaluated in L-6 myotubes. Results The extract obtained at 78 °C had the highest phenolic, flavonoid, tannin and punicalagin contents, which potentiated a stronger DPPH and ABTS radical scavenging and enzyme inhibitory action compared to the other extracts. Dose-dependently, the purified ellagitannins showed stronger DPPH (IC50 = 4.80 versus 25.0 and 32.7 μg/mL, respectively) and ABTS (IC50 = 1.63 versus 27.5 and 37.5 μg/mL, respectively) radical scavenging, ORAC (IC50 = 9.64 versus 15.6 and 25.6 μg/mL, respectively), anti-linoleic acid peroxidative (IC50 = 9.97 versus 19.9 and 27.3 μg/mL, respectively) and antiglycation (IC50 = 29.1 versus 34.2 and 79.1 μg/mL, respectively) activities than the extract obtained at 78 °C and the purified punicalagin. The anti-lipid peroxidative effect in Chang liver cells, as well as the glucose uptake modulatory activity in L-6 myotubes also followed similar trend, with the recovered tannin (IC50 = 3.67 μg/mL; EC50 = 14.8 μg/mL) outperforming the extract (IC50 = 24.3 μg/mL; EC50 = 55.0 μg/mL) and the purified punicalagin (IC50 = 59.0 μg/mL; EC50 = 112 μg/mL), which suggests antioxidant and glycemic control potentials of the recovered tannin. The ORAC, anti-linoleic acid peroxidative and cellular anti-lipid peroxidative effects of the purified tannin was comparable to and/or more potent than that of ascorbic acid (IC50 = 8.10, 7.18, 22.4 μg/mL, respectively), suggesting its potential relevance in managing oxidative stress. The potent antioxidant and glycemic control effect of the recovered tannin may be influenced by the presence of LC-MS identified ellagitannins and/or phenolics (punicalagin, ellagic acid, granatin A, corilagin, casuarinin, gallic acid and quercetin hexoside). The α-amylase inhibitory data however showed the purified punicalagin as the most potent, suggesting its potential in influencing postprandial glycemic control. Conclusion While punicalagin appears to be a major ellagitannin influencing the α-amylase inhibitory action of pomegranate peel, it works in concert with other ellagitannin and polyphenol constituents, including ellagic acid and gallic acid in exerting potent radical scavenging, anti-lipid peroxidative and glucose uptake modulatory activities. The data of the present suggest that tannin/ellagitannins purified from pomegranate peel aqueous extract obtained at 78 °C may be a promising dietary supplement for exerting glycemic control and mitigating oxidative stress.