Comparison of extraction methods
A Soxhlet extractor with hexane as solvent was used to extract
freeze-dried and ground fruit. This method conveniently provided an
extract that contained 42.9% osajin and 30.0% pomiferin, which was
collected by filtration of the precipitates from the hexane solution
without further purification.
Previously, Schall and Quackenbush
(1956) reported that extractions of Osage orange fruit with petroleum
ether, acetone, carbon tetrachloride, ethyl acetate, ethyl ether, and
methanol produced extracts in 12.8, 22.8, 20.4, 23.9, 25.5, 18.2, 21.8,
and 51.8% yields, respectively. However, yields were unreasonably high
(e.g. 51.8% with methanol), and detailed information (e.g. contents of
osajin and pomiferin) was not provided. Therefore, a literature search
was conducted for more recent studies to compare the extraction method
in the current study with other previous methods. Table 1 summarizes
comparisons of the extraction yields and the contents of osajin and
pomiferin, when reported. Among the solvents, 95% ethanol had the
highest extraction yields ranging between 13.3 and 14.8% (Mahmoud,
1981; Su et al., 2017). However, Su et al.
(2017) reported low contents of
osajin and pomiferin (0.09 and 0.03%, respectively) in the extract
while Mahmoud (1981) reported high
contents of osajin (10.0%) and pomiferin (5.0%) in the extract. In
another study using ethanol
extraction of wet fruit, the
extract (yield: 0.43%) contained 6.3% osajin and 6.3% pomiferin
(Orazbekov et al., 2018). Methanol extraction produced an extract with
relatively high contents of osajin (26.4%) and pomiferin (24.0%) in
5.6% yield from dry fruit (Abourashed et al., 2015). Methanol was also
used in the study by Darji et al. (2013), which determined contents of
osajin and pomiferin in dry fruit to be 0.6-2.1% and 1.6-5.0%,
respectively, yet the extraction yield was not determined. The ethyl
acetate gave an extract with higher contents of osajin (25.7%) and
pomiferin (36.2%) compared to ethanol and methanol providing a 1.53%
yield from wet fruit (Tsao et al., 2003). Similarly, Orhan et al. (2016)
reported that their ethyl acetate extract of Osage orange fruit had
higher total phenolic and flavonoid contents than ethanol, water and
butyl alcohol extracts. To the best of our knowledge, no study has been
conducted with hexane for Osage orange fruit. The hexane extraction of
dried fruit in this study gave the extract containing 42.9% osajin and
30.0% pomiferin in a 3.0% yield. The total osajin and pomiferin
content (72.9%) contained in this extract was the highest among the
extraction methods reported so far.
Other solvents and such as supercritical CO2 (Filip et
al., 2015), chloroforms (Kupeli et al., 2006), and water (Kupeli et al.,
2006) were used to give the yield of 5.3-7.9% from partially dried
fruit, 4.03% from wet fruit, and 7.17% from wet fruit, respectively.
However, the contents of osajin and pomiferin were not reported in these
studies. Extraction methods using petroleum ether (Wolfrom & Mahan,
1942) and diethyl ether (Ribaudo et al., 2018) followed by the formation
of lead complexes, separation and removal of lead were also developed to
produce pure osajin and pomiferin.