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282, 321, 384; ꢁNaOAc: 270, 329; EI-MS m/z (rel. int.): 254
(Mꢃ80)ꢁ(100), 226 (26), 152 (25), 124 (29), 102 (9), 96 (15), 81 (4), 82
(5), 80 (1); HR-ESI-MS: 333.00612 (MꢃH) (Calcd for C15H9O7S:
333.00690, Dꢂ0.77); IR (nmax) (KBr) cmꢃ1: 1640 (CꢂO), 1610, 1250
(SꢂO), 1050 (C–O–S), 790 (S–O). 1H-NMR (DMSO-d6) d: 6.59 (1H, s, H-
6), 7.04 (1H, s, H-8), 6.99 (1H, s, H-3), 7.58 (3H, m, H-3ꢀ, 4ꢀ, 5ꢀ), 8.08 (2H,
d, Jꢂ7.2 Hz, H-2ꢀ, 6ꢀ).
Apigenin 7,4ꢀ-Disulfate (4): Rf values: 0.05, 0.61, 0.20, 0.12 in solvent
systems A, B, C, D, respectively; HR-ESI-MS: 427.94768 (Mꢃ2H) (Calcd
for C15H8O11S2: 427.95080, Dꢂ3.12); 347.99416 (Mꢃ2Hꢃsulfate) (Calcd
for C15H8O8S: 347.99399, Dꢂ0.18).
Fermentation of Apigenin (2) with C. elegans Apigenin [2, 40 mg]
was incubated with stage 2 cultures of C. elegans as explained previously.
After 8 d, the fermentation was stopped, cells were removed by filtration and
the broth was extracted with an equal volume of n-BuOH. The butanolic
residue (30 mg) was chromatographed on a short Sephadex LH20 column to
give 5 mg of apigenin 7,4ꢀ-disulfate (4) in addition to 3 mg of apigenin 7-
sulfate (5).
Acid Hydrolysis of 3, 4 and 531) One milligram samples of 3, 4 and 5
were separately dissolved in 2 ml MeOH and mixed with 5 ml HCl at room
temperature. After evaporation of MeOH, under reduced pressure, the agly-
cone (1) or (2) was extracted with CHCl3 and ether, respectively. They were
analyzed by TLC using solvent systems CHCl3–MeOH (6 : 1) and
AcOEt–toluene–formic acid (45 : 55 : 5) as well as UV spectroscopy. On the
other hand, sulfate was detected in the concentrated aqueous layer based on
the white precipitate produced with BaCl2.
Enzyme Hydrolysis of 3, 4 and 532) One milligram of 3, 4 or 5 was
separately dissolved in 1 ml 0.1 M acetate buffer (pHꢂ5) and incubated with
aryl sulfatase from Helix pomatia (Sigma) at 37 °C. Complete hydrolysis
was observed to take place after 3—5 h of incubation. The mixture was then
extracted with CHCl3 or ether and the concentrated organic phase was ana-
lyzed by TLC using the same solvent systems as indicated under acid hy-
drolysis in addition to UV spectroscopy, which showed that the aglycone of
3 is chrysin, while that of 4 and 5 was identical to apigenin. Furthermore,
the addition of BaCl2 to the concentrated aqueous layer gave a white precipi-
tate presumably due to BaSO4.
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Acknowledgements The author is grateful to professor Ragai Ibrahim,
Concordia University, Biological Sciences, Montreal, Canada for the gener-
ous gifts of apigenin 7-sulfate and apigenin 7,4ꢀ-disulfate. The author also
appreciates the help of Dr. Meselhy Ragab, Egyptian cultural attaché, Tokyo,
Japan for the HRESIMS data.
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