A.-R.S. Ibrahim / Phytochemistry 53 (2000) 209±212
211
1H- and 13C-NMR spectra were obtained in DMSO-d6
on a Bruker DRX-500 NMR spectrometer operating
at 500 and 125 MHz, respectively. The chemical shift
values are reported as ppm using tetramethylsilane
plates F254 using CHCl ±MeOH (4:1) or EtOAc±
3
MeCOEt±HCO H±H O (5:3:1:1) as solvent systems.
2
2
Flavonoid spots were examined in UV light (254 nm),
then sprayed with P-anisaldehyde, AlCl or NH sol-
utions.
3
3
(
TMS) as internal standard and coupling constants are
expressed in Hz. Electron ionization (EI) mass spectra
were taken on Shimadzu QP5000 mass spectrometer.
3
.5. Fermentation of naringenin (1) with C. elegans
3.2. Microorganisms and culture conditions
Naringenin [1, 600 mg], dissolved in 15 ml dimethyl-
Microorganisms were obtained from either
formamide, was evenly distributed among 60 ¯asks
containing stage II cultures. Fermentation was stopped
after 3 weeks, the cells were removed by ®ltration and
the fermentation broth was extracted with an equal
volume of n-BuOH. Repeated sephadex LH20 column
chromatography of the butanolic residue (1.8 g), using
American Type Culture Collection (ATCC) or
Northern Regional Research Laboratories (NRRL).
Organisms were maintained on Sabouraud dextrose
agar (Oxoid) slants at 48 and were used to inoculate
the autoclaved culture medium. Twenty microorgan-
isms were used for the preliminary screening as fol-
lows.
Aspergillus alliaceous NRRL 315, A. ¯avips ATCC
1013, A. niger NRRL 599, A. niger NRRL 2295, A.
ochraceous NRRL 398, A. ochraceous NRRL 405,
Candida albicans, Lab isolate, Cunninghamella blacke-
sleeana MR 198, C. echinulata NRRL 1382 (ATCC
MeOH as eluent, gave 140 mg of pure 2; mp > 3008;
MeOH
aD+21. UV lmax nm: 228, 281, 334 (sh ); +AlCl
+
:
223, 306, 390; + NaOAc: 280, 330 (sh ); +NaOMe:
3
1
244, 324; EIMS m/z (rel. int.): 272 (M-80) (12), 166
�
1
(7), 153 (19), 83 (27); IR ꢀnmax (KBr)cm : 1640
1
(C1O), 1250 (S1O), 1030 (C±O±S), 800 (S±O); H-
13
and C-NMR (DMSO-d )d (see Table 1).
6
42616), C. elegans NRRL 1392 (ATCC 10028a),
Gymnascella citrina NRRL 6050 (ATCC 16956),
Lindera pinnespora NRRL 2237, Penicillium chryso-
genum ATCC 10002, P. chrysogenum ATCC 10002-K,
P. purpureus UI 193, P. vermiculatum NRRL 1009,
Rhizopus nigricans NRRL 1477, Rhodotorula rubra
NRRL y1592, Saccharomyces cerevisae (Baker's yeast)
and Streptomyces fulvissimus NRRL 1453B.
3
1
.6. Acid hydrolysis (Mann, Tofern, Kaloga & Eich,
999)
5
mg of 2 was dissolved in 10 ml MeOH and mixed
with 25 ml 3% HCl at room temperature. After evap-
oration of MeOH, the aglycone (1) was extracted with
Et O and analyzed by TLC on silica gel with CH Cl ±
2
2
2
Me CO±HCO H (76:16:8). Sulfate was detected in the
2
concentrated aqueous layer by giving white precipitate
with BaCl2.
3
1
.3. Components of culture medium (Ibrahim et al.,
997)
2
All fermentation experiments were carried out in a
medium of the following composition: 10 g dextrose,
0 ml glycerol, 5 g yeast extract, 5 g peptone, 5 g
K HPO , 5 g NaCl and 1000 ml distilled water. The
3
.7. Enzyme hydrolysis (Hackett & Grith, 1982)
mg of 2 was incubated with aryl sulfatase from the
1
2
4
5
pH was adjusted to 6.0 before autoclaving at 1218 for
snail, Helix pomatia (Sigma) in 5 ml of 0.1 M acetate
buer (pH = 5) at 378. Complete hydrolysis was
observed after 3 h of incubation. The mixture was
then extracted with Et O and the concentrated organic
2
phase was analyzed by TLC, which showed a com-
pound identical with naringenin (mp, R and NMR
15 min.
3
1
.4. Cultivation of microorganisms (Ibrahim et al.,
997)
f
Cells of microorganisms were transferred from 2-
signals). Again, addition of BaCl to concentrated aqu-
2
eous layer resulted in formation of white precipitate.
week old slants into sterile culture medium and kept
on a gyratory shaker for 72 h to give stage I culture. 5
ml of stage I cultures were used as inoculum for stage
II cultures (50 ml per 250 ml ¯ask). After 24 h incu-
bation of stage II cultures, naringenin (1) was added
�
1
as a solution in dimethylformamide (10 mg 0.25 ml ).
Both substrate and organism controls were made.
Fermentations were sampled by extracting 5 ml culture
with 5 ml EtOAc or n-BuOH. After evaporation of the
solvent, the residue was chromatographed on silica gel
Acknowledgements
The author is grateful to Dr. Charles, D. Huord,
School of Pharmacy, University of Mississippi, USA,
for the NMR spectra.