O-demethylation and sulfation of 7-methoxylated flavanones by Cunninghamella elegans

Article abstract of DOI:10.1248/cpb.51.203

Metabolism of 7-O-methylnaringenin (sakuranetin) by Cunninghamella elegans NRRL 1392 yielded naringenin and naringenin-4′-sulfate. C. elegans also converted 5,3′,4′-trihydroxy-7-methoxyflavanone into eriodictyol-4′-sulfate. Furthermore, incubation of 5,4′

Full text of DOI:10.1248/cpb.51.203

February 2003
Notes
Chem. Pharm. Bull. 51(2) 203—206 (2003)
203
O-Demethylation and Sulfation of 7-Methoxylated Flavanones by
Cunninghamella elegans
,
a
b
c
Abdel-Rahim Sayed IBRAHIM,* Ahmed Mohamed GALAL, Mohammed Shamim AHMED, and
Gabir Salem MOSSA
c
a
b
Pharmacognosy Departments, College of Pharmacy, Tanta University; Tanta 8130, Egypt: College of Pharmacy, Cairo
c
University; Cairo 11562, Egypt: and College of Pharmacy, King Saud University; Riyadh 11451, KSA.
Received September 19, 2002; accepted November 11, 2002
Metabolism of 7-O-methylnaringenin (sakuranetin) by Cunninghamella elegans NRRL 1392 yielded narin-
genin and naringenin-4؅-sulfate. C. elegans also converted 5,3؅,4؅-trihydroxy-7-methoxyflavanone into eriodic-
tyol-4؅-sulfate. Furthermore, incubation of 5,4؅-dihydroxy-7,3؅-dimethoxyflavanone with the same fungus gave
homoeriodictyol (5,7,4؅-trihydroxy-3؅-methoxyflavanone) and homoeriodicytol-7-sulfate. The structures of the
new metabolites were established by spectral analysis including 2D-NMR, HR-ESI-FT-MS beside hydrolysis by
acid.
Key words 7-methoxyflavanone; sulfation; Cunninghamella elegans; NMR; ESI-MS
Interest to study microbial transformation of flavanones, tous fungus Cunninghamella elegans is able to sulfate narin-
4
)
beside the availability of relatively large quantities of 7- genin (2), specifically at C-7 hydroxyl group. Experiments
1
,2)
methoxylated flavanones (1, 4, 6) from a previous work
encouraged us to initiate the present study. Previous micro- a support for these results.
bial transformation research indicated the capability of the The isolation of large amounts of flavanone aglycones,
filamentous fungus Cunninghamella elegans to specifically which were not commercially available, has given us an ini-
with other flavonoids such as chrysin and apigenin provided
5
)
3
)
sulfate naringenin, a flavanone with cytotoxic potential, at tiative to pursue such a reaction with other flavonoids. Initial
C-7 hydroxyl group. The sulfation at the same position in screening experiments have unequivocally shown that C. ele-
the flavones chrysin and apigenin has also been documented, gans was the sole organism which converted compounds 1,
4
)
5
)
but longer incubation produced also 4Ј-sulfate conjugates.
4, 6 to polar metabolites. Trials to extract these metabolites
Thus, it was deemed interesting to explore the nature of the with AcOEt, without decomposition, failed. Thus n-BuOH
microbial reaction when flavanones, methoxylatyed at C-7 was successfully used for the extraction. The acid liability of
are used as substrates for this microorganism. Phytochemical these metabolites to decomposition to the corresponding
1
13
investigation of several plant families culminated in charac- aglycones, as evidenced from the H- and C-NMR spectral
terization of numerous naturally occurring flavone and data, suggested the sulfation of the substrates by the microor-
6
—8)
11)
flavonol sulfates.
Neverthless, sulfates of other minor ganism. Thus, the polar metabolites 3, 5, 8 were subjected
9
)
8)
flavonoid classes have not been described, except for a re- to acid hydrolysis to provide the aglycones 2, 9 and 7, re-
cent report on the the isolation and characterization of tor- spectively. Furthermore, the aqueous layer left after extrac-
vanol, an antiviral C-4 sulfated isoflavonoid from the fruits tion of the aglycones, gave positive test for sulfate with
of Solanum torvum. However, there seems no reason why BaCl2.
such compounds should not be found in nature. Flavanones
exhibited cytotoxic, inhibition of xanthine Oxidase enzyme,
and superoxide scavenging activities. The use of C. ele-
gans to study the metabolism of these plant flavanones would
give rise to a series of novel and potentially useful flavonoid
sulfate derviatives, which can also be used as reference stan-
dards to check their production by synthetic means. Sulfates
of numerous natural flavones showed antioxidant activity
and inhibited lens aldose reductase. While a number of the
microbially-produced flavanone sulfates showed antioxidant
1
0)
1
1)
1
2)
1
3)
1
4)
1
5)
activity, this particular class of compounds is yet to be ex-
plored for other bioactivities.
Results and Discussion
Sulfation of xenobiotics by mammalian systems is a well-
1
6)
established finding in the literature. However, there are
only a few reports on sulfation reactions carried out by mi-
1
7)
croorganisms. Demonstration of parallelism between mam-
malian and microbial systems, in phase II conjugation reac-
tions is of considerable importance for metabolic drug inves-
tigations. Recent experiments established, that the filamen-
*
To whom correspondence should be addressed. e-mail: arsib16@hotmail.com
© 2003 Pharmaceutical Society of Japan

2
04
Vol. 51, No. 2
4
)
1
The HR-ESI-MS of 3 displayed a molecular ion peak at identical to those of naringenin (2). Inspection of H- and
ϩ
13
m/z 351.0203 [MϪH] corresponding to C H O S, which
C-NMR spectra of 3 revealed that all its proton and carbon
1
5
12
8
supported the presence of sulfur as being the unusual het- signals except those belonging to ring B were identical to the
eroatom. The presence of a sulfate grouping at 1257, 1053 corresponding signals of NMR spectra of naringenin (Tables
Ϫ1
and 860 cm (SϭO stretching and deformation) in the IR 1, 2). The shift in proton and carbon signals of ring B was in-
4
,20)
spectrum of 3 substantiated the sulfation reaction. Inspection dicative of C-4Ј sulfation of 3.
of the UV spectral data indicated the presence of 5,7-dihy-
The foregoing data suggested that substrate 1 underwent
droxyflavanone. This was demonstrated by a bathochromic C-7 demethylation followed by sulfation at C-4Ј. Thus struc-
1
8)
shift of band II with NaOAc (38 nm) and a bathochromic ture 3 was established as naringenin-4Ј-sulfate. In addition to
1
9)
shift of band II with NaOMe (39 nm). Also, in the AlCl3 naringenin-4Ј-sulfate (3), the fermentation of 7-O-methyl-
UV spectrum, a significant bathochromic shift indicated the naringenin (1) with C. elegans gave a metabolite 2, which
1
8)
presence of free C-5 hydroxyl group. Observation of only a lacked the methyl group signal, indicating 7-O-demethyla-
weak shoulder at 419 nm indicated the presence of an esteri- tion. The identification of 2 as naringenin was confirmed by
1
13
fied hydroxyl group at C-4Ј. The foregoing results suggested
H-, C-NMR, EI-MS spectral analysis and by direct com-
that substrate 1 underwent C-7 demethylation followed by parison with reference compound. It is noteworthy to men-
sulfation at C-4Ј position. EI-MS exhibited mass fragments tion that fermentation of naringenin with C. elegans pro-
ϩ
4)
at m/z 272 [MϪsulfate] and at m/z 120 and 153 which are duced naringenin-7-sulfate. The difference in Rf values be-
1
Table 1. H-NMR (500 MHz, d, DMSO-d ) Data for Compounds 1—8
6
a)
a)
Position
1
2
3
4
5
6
7
8
2
3
5.46 dd
5.41 dd
5.52 dd
5.30 dd
5.46 dd
5.33 dd
5.40 dd
5.49 dd
(
2.6, 12.0)
2.70 cis dd
2.8, 18.0)
.3 trans m
(2.7, 12.7)
2.67 cis dd
(2.86, 17.1)
3.2 trans m
(2.4, 12.7)
2.75 cis dd
(2.7, 17.1)
3.29 trans m
(3.2, 12.2)
2.70 cis dd
(2.8, 18)
(2.8, 11.0)
2.74 cis dd
(2.8, 17.1)
3.2 trans m
(3.0, 12.5)
2.78 cis dd
(2.4, 17)
(2.8, 12.8)
2.66 cis dd
(2.8, 17.1)
3.22 trans m
(2.5, 12.9)
2.76 cis dd
(2.71, 17.1)
3.38 trans dd
(13.0, 17.1)
(
3
3.20 trans m
3.1 trans dd
(
12.4, 19.2)
4
5
6
7
8
9
6.08 d (2.0)
6.06 d (2.0)
5.89 s
5.89 s
5.92 s
5.93 s
6.10 d (2.0)
6.10 d (2.0)
5.90 s
5.92 s
6.06 d (2.2)
6.06 d (2.2)
5.87 s
5.87 s
6.34 d (1.9)
6.38 d (1.9)
1
0
1
2
3
4
5
6
Ј
Ј
Ј
Ј
Ј
Ј
7.31 d (8.4)
6.78 d (8.4)
7.30 d (8.4)
6.80 d (8.4)
7.44 d (8.3)
7.24 d (8.3)
6.80 s
6.99 br s
6.94 br s
7.07 d (1.7)
6.77 d (8.1)
7.12 br s
6.78 d (8.4)
7.31 d (8.4)
6.80 d (8.4)
7.30 d (8.4)
7.24 d (8.3)
7.44 d (8.3)
6.80 d (8.0)
7.22 d (8.2)
6.95 d (8.8)
6.82 d (8.1)
6.93 dd
(1.4, 8.1)
3.80 s
6.91 br d (8.0) 6.89 br d (8.2) 6.96 br d (8.8) 6.88 dd
8.1, 1.7)
3.87 s
(
OCH3
3.76 s
3.79 s
3.93 s, 3.80 s
The numbers in parentheses are J values in Hz. a) Taken in DMSO-d at 300 MHz.
6
13
Table 2.
C-NMR (125 MHz, d, DMSO-d ) for Compounds 1—8
6
a)
a)
Position
1
2
3
4
5
6
7
8
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
79.0
42.4
197.3
163.6
95.0
78.8
42.3
196.7
163.8
96.2
78.6
42.4
196.4
163.8
96.6
78.6
42.1
198.2
163.4
94.5
78.5
42.4
196.3
163.8
96.3
79.5
42.8
197.6
163.8
95.3
78.6
41.9
196.3
163.3
94.8
79.1
42.7
197.7
162.7
99.6
167.8
94.1
167.0
95.4
167.1
95.4
167.3
93.7
167.0
95.4
168.2
94.4
166.6
94.8
162.5
98.7
163.2
102.9
129.1
128.7
115.5
158.1
115.5
128.7
56.2
163.3
102.1
129.2
128.6
115.6
158.1
115.6
128.6
163.2
102.1
133.1
128.6
120.8
154.0
120.8
128.6
162.8
102.5
129.2
114.3
145.7
145.1
115.2
117.9
55.8
163.1
102.1
135.6
115.8
149.4
141.3
123.1
118.0
163.5
103.2
129.9
111.8
148.2
147.6
115.8
120.4
56.3, 56.5
162.8
101.6
129.2
111.0
147.4
146.8
115.0
119.5
55.5
162.2
104.0
129.6
111.5
147.9
147.9
115.6
120.0
56.1
1
Ј
Ј
Ј
Ј
Ј
Ј
OCH3
a) Taken in DMSO-d at 75 MHz.
6

February 2003
205
tween C-4Ј and C-7 sulfate conjugates of naringenin was flavanones, the fungus carries out a demethylation reaction as
very small and the differentiation between sulfation at C-4Ј a prelude for incorporation of a sulfate grouping. On the
1
13
and C-7 was accomplished by inspection of the H-, C- other hand, incubation for longer time resulted in sulfation of
NMR and UV spectra.
the hydroxyl group at C-4Ј position. Much less frequently,
Incubation of 7-O-Methyleriodictyol (4) with C. elegans only the C-4Ј sulfated flavanones could be characterized,
produced the polar metabolite 5, in a yield approaching 11%, which would be explained on the basis of the unstability of
in addition to a minor unidentified metabolite. Metabolite 5 sulfate conjugates of the C-7 hydroxyl groups.
was found to be a sulfate conjugate, based on acid hydrolysis
Sulfation is rarely encountered as metabolic pathway in
and spectral data. EI-MS exhibited an ion peak at m/z 288 the biosynthesis of plant secondary metabolites. However,
21)
ϩ
(
MϪSO ) , which indicated loss of the methyl group. HR- flavone, flavonol and more recently cardenolide sulfates have
4
2
1)
ESI-MS showed a molecular ion peak at m/z 369.0278 been isolated from higher plants. Isolation of flavanone
ϩ
[
MϩH] consistent with a molecular formula C H O S. sulfates from angiospermae has not been reported yet, and
The loss of C-7 methyl group was evident from the H- and utilization of the fungus C. elegans seems to be indespensi-
15 12 9
1
1
3
C-NMR spectral data, where signals at d 3.79 and d 55.8 ble in preparation of such derivatives.
corresponding to OMe-7 in the substrate are lacking (Tables
, 2). The NMR data were also useful in determining the lo-
cation of sulfate moiety, where the deshielding of C-3Ј and
Experimental
-Methoxyflavanones (1, 4, 6) were purified from Rhus retinorrhea. In-
frared spectra were recorded with an ATI mattson genesis series fourier
C-5Ј carbons, which are ortho to sulfation site compared to transform (FT-IR) spectrophotometer. Ultraviolet spectra were obtained on a
1
1,2)
7
1
3
Shimadzu 1601 PC and Hewlett Packard 8452A diode array spectropho-
tometers. Optical rotations were recorded at ambient temperature using
JASCO, DIP 370 digital polarimeter. 1D- and 2D-NMR spectra were ob-
tained on a Bruker Avance DRX500 spectrometer. EI-MS was measured on
those of the substrate 4 was observed in the C-NMR of 5 by
.7 and 7.9 ppm, respectively. Furthermore, the pararelated
carbon at C-1Ј in 5 was deshielded by about 6 ppm, while the
3
ipso carbon at C-4Ј position was shielded by 3.8 ppm. The Shimadzu QP5000 mass spectrometer, while HR-ESI-FT-MS were taken on
data mentioned herein supported sulfation of C-4Ј hydroxyl a Bruker Bioapex FT-MS in ESI mode. TLC was performed using glass sup-
2
0)
ported silica gel plates (0.25-mm layer, F254, Merck). For column chro-
matography, Sephadex LH 20 columns, Pharmacia were used.
Microorganisms and Culture, Conditions The microorganisms, used
in this study, were purchased from two sources: American Type Culture Col-
group. Further support was deriven from deshielding of H-
5
1
Ј proton signal in the H-NMR of 5 by 0.42 ppm compared
to H-5Ј signal in the substrate (4) spectrum. Concerning UV
spectra, both NaOAc and AlCl bathochromic shifts of bands lection (ATCC) and National Center for Agricultural Utilization Research
3
II and I, respectively indicated that 5 was a 5,7-dihydroxy- (NCAUR). Sabouraud dextrose agar (Oxoid) slants were used to subculture
1
8,19)
the battery of microorganisms every three months and kept at 4 °C. Two-
week old slants were used to inoculate sterilized culture medium and initiate
stage I culture. Several microorganisms were used in the initial screening as
lated flavanone.
On the other hand, the C-4Ј hydroxyl
group is not free due to lack of shift with NaOMe.
The metabolism of 5,4Ј-dihydroxy-7,3Ј-dimethoxyfla-
vanone (persicogenin, 6), gave a major metabolite 8 in a 27%
yield, in addition to 1.5% of a minor metabolite 7. Examina-
tion of the H-NMR spectral data of the minor metabolite ex-
hibited the presence of a single methoxyl group, which res-
onated at d 3.87. These data suggested that one methyl group
was lost. A closer look at the C-NMR signals of 7 showed used to inoculate the autoclaved medium and kept on a gyratory shaker for
that they are identical to those reported for homoeriodictyol
5,7,4Ј-trihydroxy-3Ј-methoxyflavanone)
22)
previously reported.
Components of the Culture Medium
2
3)
The Components of the cul-
ture medium, used in all fermentation experiments, are 10 g dextrose, 10 ml
glycerol, 5 g yeast extract, 5 g peptone, 5 g K HPO , 5 g NaCl and 1000 ml
1
2
4
distilled water. The pH was adjusted to 6.0 before sterilization at 121 °C for
1
5 min.
24)
Cultivation of Microorganisms
Cells from two week-old slants were
1
3
7
2 h to give stage I culture. An inoculum of 5 ml of stage I culture was used
to inoculate stage II cultures (50 ml per 250-ml flasks). After incubation for
4 h, substrates 1, 4 or 6 were added separately as a solution in dimethyl-
2
0)
(
(Table 2). The
2
foregoing data are consistent with the tendency of C. elegans
to carry out C-7 demethylation, which was also observed in made simultaneously. Fermentations were analyzed periodically by extrac-
Ϫ1
formamide (10 mg, 0.25 ml ). The substrate and organism controls were
case of substrates 1, 4. The positive ion HR-ESI-MS spec- tion of culture samples with an equal volume of AcOEt or n-BuOH. After
ϩ
evaporation of the solvent, the residue was chromatographed on silica gel
trum of 8 displayed a pseudomolecular ion peak [MϩH] at
m/z 383.0442 consistent with the molecular formula
plates (E. Merck, F254) using CHCl –MeOH (4 : 1) beside other solvent sys-
tems. Flavonoid spots were inspected under the UV light (254, 365 nm) be-
3
C H O S. Mild acid hydrolysis of 8 gave the aglycone 7 in
16
14
9
fore and after spraying with AlCl , NH or p-anisaldehyde reagents.
3
3
addition to a sulfate (BaCl precipitation reaction). These
Fermentation of Sakuranetin (1) with C. elegans 7-Methylnaringenin
2
data suggested that 8 is a sulfate conjugate of homoeriodic- [1, 700 mg], dissolved in 17.5 ml of dimetylformamide, was equally distrib-
uted among 70 flasks containing stage II cultures of the fungus. After 10 d,
tyol, which was also displayed by the presence of SϭO ab-
Ϫ1
the fermentation was stopped and the cells were removed by filtration. The
fermentation broth, Which contained the metabolite, was extracted with an
equal volume of n-butanol. The n-BuOH extract was concentrated at 50 °C,
sorption bands at n
1272, 1063, 801 cm in the IR spec-
max
1
3
trum. Comparison of C-NMR spectrum of 8 with that of
homoeriodictyol, showed that while C-7 signal is shielded by under reduced pressure to give 1.9 g of a brownish residue, which was re-
peatedly chromatographed on sephadex LH 20 columns using MeOH as an
eluent to give 60.0 mg of naringenin (2) and 110 mg of narigenin-4Ј-sulfate
about 4 ppm, C-6 and C-8 signals are deshielded by about 4.8
and 3.9 ppm, respectively (Table 2). This was consistent with
homoeriodictyol-7-sulfate structure. Moreover, the UV spec-
tral data provided additional support to this conclusion, as it
(
3).
Naringenin (2): EI-MS (rel. int.) 272 (M ) (25), 254 (4), 153 (49), 120
ϩ
1
13
(
30), H- and C-NMR (DMSO-d ) d (see Tables 1, 2, respectively). All
6
1
9,20)
showed free 5, 4Ј positions (AlCl and NaOMe shifts), while these data are identical to those reported for naringenin.
The metabolite
3
was also directly compared with an authentic sample of naringenin (TLC,
mp).
position 7 was shown to be blocked (lack of shift with
NaOAc).
In conclusion, the fungus C. elegans NRRL1392 exhibited
2
5
Naringenin-4Ј-sulfate (3): mp Ͼ300 °C; [a]D Ϫ45° (cϭ0.020; MeOH);
UV lmax (MeOH) nm: 289, 325 (sh), ϩNaOMe: 290 (sh), 325; ϩNaOAc:
preference towards sulfation of the hydroxyl group located at 287 (sh), 325; ϩNaOAc/H BO : 288, 322; ϩAlCl : 310, 380; EI-MS m/z
3
3
3
4
,5)
ϩ
C-7 of the flavonoid nucleus. In case of 7-methoxylated (%rel. int): 272 (50) (MϪ80) , 179 (32), 153 (76), 120 (45); HR-ESI-MS:

2
3
06
Vol. 51, No. 2
51.0203 (MϪH), using the negative mode (Calcd: 351.0180); IR (nmax
)
References
Ϫ1
(
film) cm : 1644 (CϭO), 1505, 1257 (SϭO), 1053 (C–O–S), 1017, 860;
1) Mossa J. S., Abdel Sattar E., Abou-Shoer M., Galal A. M., Int. J.
Pharmacognosy, 34, 198—201 (1996).
1
13
H- and C-NMR: See Tables 1 and 2.
Fermentation of 5,3؅,4؅-Trihydroxy-7-methoxyflavanone (4) 7-
2) Ahmed M. S., Galal A. M., Ross S. A., Ferreira D., El-Sohly M. A.,
Ibrahim A.-R. S., Mossa J. S., El-Feraly F. S., Phytochemistry, 58,
599—602 (2001).
3) Habtemariam S., J. Nat. Prod., 60, 775—778 (1997).
4) Ibrahim A.-R. S., Phytochemistry, 53, 209—212 (2000).
5) Ibrahim A.-R. S., Manuscript in preparation.
6) Harborne J. B., Phytochemistry, 14, 1331—1337 (1975).
7) Barron D., Ibrahim R. K., Phytochemistry, 26, 1181—1184 (1987).
8) Mann P., Tofern B., Kaloga M., Eich E., Phytochemistry, 50, 267—271
(1999).
9) Barron D., Varin L., Ibrahim R. K., Harborne J. B., Williams C. A.,
Phytochemistry, 27, 2375—2395 (1988).
Methyleriodictyol (4, 305 mg) was dissolved in 7.5 ml DMF and equally dis-
tributed among 30 flasks containing stage II cultures of C. elegans. After an
incubation period of 7 d, cells were removed by filtration over cheese cloth
and the broth which contained the metabolite was extracted with an equal
volume of n-BuOH and processed as described previously to give 900 mg of
an amber colored semisolid residue. Sephadex LH 20 column chromatogra-
phy of the butanolic residue gave 34 mg of eriodictyol-4Ј-sulfate (5) beside
1
4 mg of an unidentified minor product.
2
5
Eriodictyol-4Ј-sulfate: mp Ͼ300 °C; [a]D Ϫ23° (cϭ0.024, MeOH); UV
(MeOH) nm: 216, 290, 328 (sh); ϩAlCl : 218, 308, 378; ϩNaOMe:
l
2
(
3
(
1
max
3
16, 232, 284, 327 (sh) decomposition; ϩNaOAc: 302, 390. EI-MS m/z
ϩ
%rel. int.): 288 (MϪ80) (9), 166 (12), 153 (21), 136 (10); HR-ESI-MS: 10) Arthan D., Suasti J., Kittakoop P., Pittayakhachonwut D., Tanticharoen
ϩ
ϩ
69.0278 (MϩH) (Calcd: 369.0275), 289.0716 (MϪ80ϩH) ; IR (nmax)
M, Thebtaranonth Y., Phytochemistry, 59, 459—463 (2002).
11) Harborne J. B., Phytochemistry, 14, 1147—1155 (1975).
12) Cos P., Ying L., Calomme M., Hu J. P., Cimanga K., Poel B. V., Pieters
L., Vlietinck A. J., Berghe D. V., J. Nat. Prod., 61, 71—76 (1998).
13) Yagi A., Uemura T., Okamura N., Haraguchi H., Imoto T., Hashimoto
R., Phytochemistry, 35, 885—887 (1994).
Ϫ1
KBr) cm : 1640 (CϭO), 1507, 1340, 1343, 1272 (SϭO), 1185, 1162,
048 (C–O–S), 804 (S–O); H- and C-NMR (DMSO-d ): See Tables 1, 2.
1
13
6
Fermentation of 5,4؅-Dihydroxy-7,3؅-dimethoxyflavanone with C. ele-
gans 7-Methylhomoeriodictyol [6, 550 mg], dissolved in 14 ml DMF, was
equally distributed among 56 Flasks containing stage II culture of C. ele-
gans. After incubation on a gyratory shaker for two weeks, the fermentation 14) Haraguchi H., Ohmi I., Sakai S., Fukuda A., J. Nat. Prod., 59, 443—
was stopped, cells filtered and the broth extracted with an equal volume of n-
445 (1996).
BuOH. Sephadex LH 20 column chromatography of the extract (2 g) gave 15) Takamatsu S., Galal A. M., Ross S. A., Ferreira D., Elsohly M. A.,
1
50 mg of homoeriodictyol-7-sulfate (8) and 8 mg of homoeriodictyol (7).
Ibrahim A.-R. S., El-Feraly F. S., 42nd Annual Meeting of the Ameri-
can Society of Pharmacognosy, Oaxaca, Mexico, Abstract book, 2001,
p. 207.
2
5
Homoeriodictyol-7-sulfate (8): mp Ͼ300 °C; [a]D Ϫ2.7° (cϭ0.046,
MeOH); UV lmax (MeOH) nm: 218, 308, 378; ϩNaOMe: 214, 282, 337;
ϩAlCl : 302, 390; ϩNaOAc: 218, 308, 378; EI-MS m/z (%rel. int.): 302 16) Williams R. T., “Biochemistry of Phenolic Compounds,” ed. by Har-
3
ϩ
(
(
(
MϪ80) (55), 166 (22), 153 (61), 150 (56); HR-ESI-MS: 383.0442
MϩH) (Calcd: 383.0431); IR (n ) (KBr) cm : 1639 (CϭO), 1272
SϭO), 1063 (CϭO), 801 (S–O); H- and C-NMR: See Tables 1, 2.
borne J. B., Academic Press, London, 1964, p. 205.
17) Cerniglia C. E., Freeman J. B., Mitchum R. K. Appl. Environ. Micro-
biol., 43, 1070—1075 (1982).
18) Harborne J. B., Mabry T. J., Mabry H. (ed.), “The Flavonoids,” Acade-
mic Press, New York, 1975, p. 75.
ϩ
Ϫ1
max
1
13
1
13
Homoeriodictyol (7): H- and C-NMR data: See Tables 1, 2, respec-
tively. All the NMR data were identical to those reported for homoeriodic-
tyol.
Acid Hydrolysis of 3, 5 and 8
19,20)
19) Mabry T. J., Markham K. R., Thomas M. B. (ed.), “The Systematic
Identification of Flavonoids,” Springer, New York, 1970.
8
)
5 Mg of 3, 5, 8 were separately dis-
solved in 10 ml MeOH and mixed with 25 ml 3% HCl at room temperature. 20) Agrawal P. K., Bansal M. C. (ed.), “Carbon-13 NMR of Flavonoids,”
After evaporation of MeOH, under reduced pressure, CHCl was used to ex-
Elsevier, Berlin, 1989.
tract the aglycones 2, 9, 7, respectively. After evaporation of the solvent, the 21) Pauli G. F., Matthiesen U., Fronezek F. R., Phytochemistry, 52, 1075—
residue was analyzed by direct comparison with reference samples by TLC
1084 (1999).
and spectroscopic measurements. The sulfate remaining in the aqueous layer 22) Ibrahim A-R. S., Galal A. M., Mossa J. S., El-Feraly F. S., Phytochem-
3
was detected by giving white precipitate with BaCl2.
istry, 46, 1193—1195 (1997).
2
3) Galal A. M., Ibrahim A.-R. S., Mossa J. S., El-Feraly F. S., Phy-
tochimstry, 51, 761—765 (1999).
Acknowledgments The authors are grateful to Dr. Charles D. Hufford,
School of Pharmacy, University of Mississippi for collecting the NMR data 24) Orabi K. Y., Galal A. M., Ibrahim A.-R. S., El-Feraly F. S., Khalifa S.
and Dr. Chuck Dunbar, Natural Products Center, School of Pharmacy, Uni-
versity of Mississippi, MS, U.S.A. for the HR-ESI-FT-MS analysis.
I., El-Sohly H. N., Phytochemistry, 51, 257—261 (1999).