Extraction from Osage orange fruit
Osage orange fruits were collected from Tazewell and Peoria counties, Illinois, USA in 2018 and stored in a freezer at -20oC until processed. Fruits were sliced into 1-cm thick pieces and frozen for 4 h at -80 oC then immediately freeze-dried for 96 h using a Labconco freeZone 12L -50oC Console Freeze dryer (Labconco Corp., Kansas City, MO, USA). Freeze-dried fruit lost approximately 75-80% of their original weight. Freeze-dried fruits were sealed in vacuum bags until extracted with a retail food vacuum sealer (Model 4800, Foodsaver, Sunbeam Inc., Boca Raton, FL, USA). Freeze-dried fruits were ground using a food processor (Model BFP-703, Cuisinart, East Windsor, NJ, USA) to a powder. Approximately 230 g of powder was then transferred to a 90 mm diameter × 200 mm height × 1 mm thickness Cellulose Extraction thimble (LabExact, I.W. Tremont Co., Inc., Hawthorne, NJ, USA) and inserted in Soxhlet extractor system consisting of 8,000 ml of hexane in a 12,000 ml capacity round bottom boiling flask fitted into a Soxhlet extractor body (12.7 cm diameter × 48.2 cm height, Size 25, Kimble Chase, Vineland, NJ, USA) and a condenser. Heating and stirring were conducted with a Stirmantle equipped with a motor drive set at 70oC and 100 rpm (Model 100D EMS116, Glas-Col, Terre Haute, IN, USA). Condenser cooling was provided 8 oC antifreeze using a refrigerated recirculating chiller (Model 6000, PolyScience, Niles, IL, USA). After 72 hours, the heat was turned off while the stirring continued. Once room temperature was achieved, the condenser was disconnected, the thimble was removed, and another thimble containing freeze-dried fruit was inserted. Additional hexane was added in order to maintain the volume of hexane at 8,000 ml. Five thimbles of freeze-dried fruit (230 g) were extracted in the same hexane solvent. Yellowish precipitates accumulated in the solution and adhered to the glass walls. The extract solution was filtered through coffee filters fitted into Büchner funnel/flask filtration system to separate a yellow precipitate. 34 g (3.0% yield from dry fruit) of the precipitate, which is referred to as Osage orange fruit extract (OOE), was collected after drying under a fume hood. The filtrate was saved for further studies. Thin layer chromatography (TLC) was employed to initially identify the composition of OOE on silica glass plates using hexane: ethyl acetate: formic acid (60:20:3 ratio). At the conclusion of the TLC run, the plate was sprayed with a solution of 30% sulfuric acid in methanol. Spots were revealed by heating the plate in an oven at 100 oC for 3-5 min. Standards were run to identify the spots based on their Rf values. OOE contained two distinct spots at Rf = 0.19 and 0.31 for pomiferin and osajin, respectively.