Optimization of Ultrasound-Assisted Extraction of Phloretin and Other Phenolic Compounds from Apple Tree Leaves (Malus domestica Borkh.) and Comparison of Different Cultivars from Estonia

Abstract

Polyphenolic compounds, plant secondary metabolites essential for plant survival, are known for their high antioxidant and anti-inflammatory activity. In addition, several polyphenols, such as phloretin, also have potential antiviral effects, making these compounds potential ingredients of biofunctional foods. A promising source for the extraction of phloretin is a by-product of apple production—apple tree leaves. Focusing on green technologies, the first aim of the present study was to optimize the direct ultrasound-assisted extraction conditions to gain the maximum yield of phloretin from air-dried apple leaves. For the optimization of process parameters, we applied the response surface method with Box–Behnken design. The optimal extraction conditions were extraction time 14.4 min, sonication amplitude 10% and 10 g of sample per 100 mL solvent (70% ethanol, w/w). Using these conditions, we assessed the content of individual and total polyphenolic compounds along with antioxidant activity in the leaves of different autumn and winter apple cultivars grown in Estonia. The analyses were carried out with chromatographic (HPLC-DAD-MS/MS) and spectrophotometric methods. The phloretin concentration ranged from 292 to 726 µg/g and antioxidant activity from 6.06 to 11.42 mg GA eq./g, these being the highest in the local winter cultivars ‘Paide taliõun’ and ‘Tellissaare’, respectively.

Optimization of Phlorizin Extraction from Annurca Apple Tree Leaves Using Response Surface Methodology

“…As reported in Table 1 , for the optimization of phlorizin extraction protocol from AALs, different extraction times (30, 60, 120, 240 min), solvent compositions (80% aqueous methanol solution containing 0.1, 1, or 5% formic acid), incubation temperatures (30, 35, and 45 °C), with and without a sonication stage of 30 min, were opportunely combined. According to the general extraction protocol applied, 250 mg of AALs were treated with 2 mL of extraction solvent, as previously optimized by Othman et al [ 12 ] the mixture obtained was left in incubation at selected temperatures and times on an orbital shaker. At the end of the extraction time, where expected, 30 min of sonication (continuous operative mode, 150 W Power, 40 kHz Frequency; Branson Fisher Scientific 150E Sonic Dismembrator) was performed.…”

“…The main natural sources of phlorizin are the plants of the Malus genus, although it also reaches a valuable concentration in other plant species, such as Punica granatum (pulp) [ 7 ], Polygonum cuspidatum (flower), Prunus persica (pulp) [ 8 ], Rosa canina (flesh) [ 9 ], Vaccinium vitis-idaea (flesh) [ 10 ], and Vaccinium macrocarpon [ 11 ]. Specifically, phlorizin is not equally distributed in all parts of the apple tree, however, it reaches its maximum concentration in the non-edible parts of the plant, e.g., leaves [ 12 ], twigs [ 13 ], root bark, seeds [ 14 ], and unripe fruits [ 15 ]. Phloretin, and its glucoside phlorizin, are found to be the major phenolic compounds in apple leaves, reaching a concentration ranging from 5.4% to 14% of leaf dry weight (DW) [ 12 ] Moreover, the phlorizin content of leaves seems to be less affected by some variables, such as apple cultivar or harvesting period, than its aglycone, making phlorizin concentration stable over time and in the type of apple cultivar analyzed.…”

“…Specifically, phlorizin is not equally distributed in all parts of the apple tree, however, it reaches its maximum concentration in the non-edible parts of the plant, e.g., leaves [ 12 ], twigs [ 13 ], root bark, seeds [ 14 ], and unripe fruits [ 15 ]. Phloretin, and its glucoside phlorizin, are found to be the major phenolic compounds in apple leaves, reaching a concentration ranging from 5.4% to 14% of leaf dry weight (DW) [ 12 ] Moreover, the phlorizin content of leaves seems to be less affected by some variables, such as apple cultivar or harvesting period, than its aglycone, making phlorizin concentration stable over time and in the type of apple cultivar analyzed.…”

“…Considering the conventional method of cultivation, during the pruning period in summer, unripe fruits and leaves are harvested to improve the quality of fruit production, resulting in a high amount of non-utilized apple leaves that would be classified as agro-food waste materials [ 12 ]. As widely reported, the interest of the nutraceutical industry in the reutilization of agro-food waste products is progressively increasing, since they represent still rich sources of biologically active compounds that can be conveniently used for the formulation of food supplements [ 16 ].…”

Intensification Technologies to Efficiently Extract Antioxidants from Agro-Food Residues

“…Ben-Othman and collaborators [16] focused on the production of extracts from apple tree leaves rich in phloretin, a phenolic compound with antiviral properties. They optimized the ultrasound assisted (USAE) extraction parameters using hydroalcoholic mixtures as solvent.…”