Determining the influence of the extraction method on the yield and chemical composition of ellagitannin and punicalagin from pomegranate fruit peel

Master of Health Sciences in Biomedical Technology

Background Pomegranate fruit has oxidative, metabolic and vascular health benefits. This has been attributed to its phenolic constituents, including bioactive ellagitannins. Although underutilized, the peel of pomegranate also contains these beneficial ellagitannins, which could be of medicinal relevance in managing oxidative stress. However, the extraction conditions for optimum recovery of antioxidant ellagitannins from pomegranate fruit peel remains elusive. To ascertain extraction the possible extraction conditions for optimum recovery of antioxidant ellagitannins from pomegranate peel, this study investigated the influence of aqueous extraction temperature or type of organic solvent extraction on the antioxidant and ellagitannins profile of pomegranate peel using advanced chemometrics models. Methodology Class 1 (processing grade) “Wonderful” pomegranate fruits were procured from a local fruit producing and packaging company. The fruits were washed, and peel was removed. First, 1 kg freshly removed peel was separately soaked in 2.5 L of distilled water at different temperatures (25 °C, 37 °C, 50 °C, 65 °C, 78 °C and 95 °C) for 1 hour. There after the soaked peel was blended with the water, filtered and the extracts were recovered by freeze drying. On the other hand, equivalent dry mass (250 g) of the dried peel was extracted with 1 L of different solvents (methanol, ethanol, acetone and water). The extracts were recovered using a rotary evaporator (for the organic solvent extracts) or by freeze drying (for the water extract). The extracts were evaluated for total phenol (TPC) and flavonoid (TFC) contents, 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radicals scavenging activity, Fe3+ reducing activity (FRAP), oxygen radical absorbance capacity (ORAC), as well as inhibitory capacity on FeSO4-induced linoleic acid peroxidation and lipid peroxidation in Chang liver cells. Tannins were recovered from the extracts and ellagitannins were quantified using liquid chromatography–mass spectrometry (LC-MS). Advanced chemometric models, including principal component analysis (PCA) and orthogonal projections to latent structures (OPLS) were used to correlate the influence of the extraction methods on the ellagitannins and antioxidant profiles of the extracts. Results and discussion In the dry peel extraction, acetone extraction had the highest (p˂0.05) TFC and TFC and strongest (p˂0.05) Fe3+ reducing effect, while methanol extraction had the strongest (p˂0.05) radical scavenging activity. The ORAC and anti-lipid peroxidative activities of both solvent extractions outperformed that of the ethanol and water extractions. Tannins were highest for acetone and methanol extractions. LCMS-quantified ellagitannins in the tannins varied for the different extractions. OPLS bi-plot showed that the high ellagic acid constituents and total tannins yield of the methanol extraction influenced its potent radical scavenging activity, while the ellagitannin constituents including punicalagins, granatin A, geraniin and casuarinin influenced the high TFC, as well as the potent Fe3+ reducing, ORAC and anti-lipid peroxidative activities of acetone extraction. On the other hand, extraction at 78 °C had the most potent radical scavenging potency, oxygen ORAC and anti-lipid peroxidative activity, which was consistent with its highest ellagitannins constituents. OPLS biplot showed distinct clustering pattern of the antioxidant and ellagitannins variables around 78 °C extraction temperature, suggesting the observable influence of the ellagitannins on the potent antioxidant profile of the 78 °C extraction. The variable contribution plots, as well as the obtained antioxidant data suggest punicalagins, granatin A and ellagic acid as notable influencing ellagitannins. Also, more ellagitannins were recovered from fresh peel extraction at 78 °C that dry peel organic solvent extraction. Conclusion While acetone and/or methanol extraction may be promising for appreciable recovery of antioxidant ellagitannins from dry pomegranate peel, it is safe to speculate that water extraction at 78 °C optimally influenced the ellagitannins and antioxidant profiles of freshly removed pomegranate fruit peel than other extraction temperatures. Thus, may be promising for optimally recovering the ellagitannins from pomegranate peel, without sacrificing the antioxidant capacity.

Supervisor: Dr Chika Ifeanyi Chukwuma (PhD: Biochemistry) Co-supervisor: Prof. Samson S. Mashele (PhD: Medicinal Chemistry)