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.