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.