Microbial metabolism. Part 6. Metabolites of 3- and 7-hydroxyflavones

Article abstract of DOI:10.1248/cpb.54.320

Fermentation of 3-hydroxyflavone (1) with Beauveria bassiana (ATCC 13144) yielded 3,4′-dihdroxyflavone (3), flavone 3-O-β-D-4-O- methylglucopyranoside (4) and two minor metabolites. 7-Hydroxyflavone (2) was transformed by Nocardia species (NRRL 5646) to 7-methoxyflavone (5) whilst Aspergillus alliaceus (ATCC 10060) converted it to 4′,7-dihydroxyflavone (6). Flavone 7-O-β-D-4-O-metylglucopyranoside (7) and 4′- hydroxyflavone 7-O-β-D-4-O-methylglucopyranoside (8) were the metabolic products of 7-hydroxyflavone (2) when fermented with Beauveria bassiana (ATCC 7159). One of the minor metabolites of 3-hydroxyflavone (1) was tentatively assigned a β′-chalcanol structure (9). Compounds 4, 7 and 8 are reported as new compounds. Structure elucidation of the metabolites was based on spectroscopic data.

Full text of DOI:10.1248/cpb.54.320

320
Chem. Pharm. Bull. 54(3) 320—324 (2006)
Vol. 54, No. 3
Microbial Metabolism. Part 6.1) Metabolites of 3- and 7-Hydroxyflavones
Wimal HERATH,a Julie Rakel MIKELL,a Amber Lynn HALE,a Daneel FERREIRA,a,b and
Ikhlas Ahmad KHAN*,a,b
a National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, The University of
Mississippi: and b Department of Pharmacognosy, School of Pharmacy, The University of Mississippi; University, MS
38677, U.S.A. Received September 8, 2005; accepted November 29, 2005
Fermentation of 3-hydroxyflavone (1) with Beauveria bassiana (ATCC 13144) yielded 3,4ꢀ-dihdroxyflavone
(3), flavone 3-O-b-D-4-O-methylglucopyranoside (4) and two minor metabolites. 7-Hydroxyflavone (2) was trans-
formed by Nocardia species (NRRL 5646) to 7-methoxyflavone (5) whilst Aspergillus alliaceus (ATCC 10060) con-
verted it to 4ꢀ,7-dihydroxyflavone (6). Flavone 7-O-b-D-4-O-metylglucopyranoside (7) and 4ꢀ-hydroxyflavone 7-O-
b-D-4-O-methylglucopyranoside (8) were the metabolic products of 7-hydroxyflavone (2) when fermented with
Beauveria bassiana (ATCC 7159). One of the minor metabolites of 3-hydroxyflavone (1) was tentatively assigned
a bꢀ-chalcanol structure (9). Compounds 4, 7 and 8 are reported as new compounds. Structure elucidation of the
metabolites was based on spectroscopic data.
Key words flavonoid; microbial metabolism; Beauveria bassiana; Aspergillus alliaceus; Nocardia
Flavonoids and their glycosides form a large group of zymes in the colon.21) Using animal models, human trials and
polyphenolic compounds which are widely distributed in in vitro fermentation experiments, it was shown that the in-
plants.2,3) Animals depend on plants for flavonoids as they testinal microorganisms are greatly responsible for catabo-
are unable to biosynthesize them.4) The exception was the de- lism and scission of the flavonoid. Scission of the flavonoid
tection of flavonoids in butterfly wings with the likely source structure depends on their hydroxylation pattern.21) Absence
being the plant food of the larva.5) Flavonoids add colour,6) of hydroxyl groups in the B-ring for example, prevents ring
flavour and processing characteristics to many foods (fruits scission. In vitro metabolism studies of flavonoids using rat
and vegetables) and drinks (tea, wine).7) So far, over 4000 liver microsomes showed that the B-ring is the main struc-
flavonoid derivatives have been identified and numerous ben- tural moiety that undergoes biotransformation. B-rings with
eficial health effects including, anti-inflammatory,8—10) anti- a single hydroxyl group at C-4ꢀ or none at all get hydroxy-
viral11) and anti-cancer8,12,13) properties have been reported. lated by microsomal cytP450 enzymes.21) It has been demon-
Some epidemiological studies have also demonstrated that strated that the bioavailability of flavonoids in general is low
the intake of flavonoids reduced the risk of cardiovascular due to limited absorption and rapid elimination. For example,
diseases.8,14—16) Most of the pharmacological activities could the peak plasma concentrations of citrus flavonoids was less
be attributed to their ability to inhibit certain enzymes and to than 1 mM/l.20) It is also known that they are rapidly and ex-
their oxygen free radical scavenging and iron chelating capa- tensively bio-transformed into metabolites which do not al-
bilities.17) The antioxidant properties are suggested to be due ways have the same biological activities of the parent com-
to the number and arrangement of their phenolic hydroxyl pounds.22) As seen in in vitro antioxidant experiments, most
groups.18) It has been shown that 3-hydroxy-, 7-hydroxy- and circulating flavonoids are actually the metabolites rather than
5-hydroxyflavones have hypochlorite scavenging activity the original compounds indicating that the active compounds
with 3-hydroxyflavone showing the greatest effect.19) De- may not be the dietary flavonoids.22,23) Thus, to ascertain the
pending on the concentration and the reaction conditions, role of flavonoids as biologically active compounds it is es-
flavonoids could act as antioxidants as well as prooxidants.17) sential to know the chemical nature and the bioavailability of
Toxicity of some flavonoids has been attributed to their the absorbed forms. Being predictive models for mammalian
prooxidant behavior. Hence, careful examination of flavonoids drug metabolism, microorganisms are used to establish the
for their behavior in varying reaction conditions has to be metabolic fate of biologically active compounds.24—26) This
carried out before being considered for therapeutic uses.17) procedure gives significant amounts of metabolites for com-
Although in vitro experiments have shown that flavonoids plete structure elucidations and for further pharmacological
possess a wide range of biological activities, their overall evaluations. Reports on the microbial transformation of
function in vivo has to be clarified. It has been observed that flavonoids are relatively few27) and thus, we initiated a pro-
only aglycones and flavonoid glucosides are absorbed in the gram to investigate the microbial metabolism of some se-
small intestine.18) They are then metabolized rapidly to lected flavonoids. Here we report the transformation of 3-hy-
methylated, glucuronidated or sulphated metabolites in the droxyflavone (1) by Beauveria bassiana (ATCC 13144) and
jejunal and ileal parts of the small intestine. After metabo- 7-hydroxyflavone (2) by Nocardia species, Aspergillus alli-
lism in the intestine, the flavonoids are further metabolized in aceus (ATCC 10060) and Beauveria bassiana (ATCC 7159).
the liver to yield various conjugated forms. The reactions in-
clude methylation, sulphation and glucuronidation. Conjuga- Results and Discussion
tion is essentially a detoxification process in which the com-
Initial screening of compounds 1 and 2 was carried out
pounds are made to eliminate by way of bile and urine by in- with forty organisms using the standard two stage
creasing their hydrophilic character.20) The unabsorbed procedure.24) Several organisms showed the capability of
flavonoids undergo further metabolism by the bacterial en- transforming the flavonoids. However, B. bassiana (ATCC
∗ To whom correspondence should be addressed. e-mail: ikhan@olemiss.edu
© 2006 Pharmaceutical Society of Japan

March 2006
321
13144) was selected for scale-up studies of compound 1 as it
gave a number of metabolites compared to other organisms.
Flavonol (1) was converted to 3,4ꢀ-dihdroxyflavone (3),
flavone 3-O-b-D-4-O-methylglucopyranoside (4) and two
other minor metabolites, whilst 7-hydroxyflavone (2) was
metabolized to 7-methoxyflavone (5) and 4ꢀ,7-dihydroxy-
flavone (6) by Nocardia species (NRRL 5646) and A. alli-
aceus (ATCC 10060), respectively. Transformation of flavone
(2) to flavone 7-O-b-D-4-O-methylglucopyranoside (7) and
4ꢀ-hydroxyflavone 7-O-b-D-4-O-methylglucopyranoside (8)
was observed when fermented with B. bassiana (ATCC
7159). None of the extracts showed detectable amounts of
substrates 1 and 2.
Metabolite 3, (5 mg, 1.25% yield) isolated as a faint yel-
low solid, was shown by high resolution electrospray ioniza-
tion mass spectrometric (HR-ESI-MS) data to have a molec-
ular formula C15H10O4 corresponding to a monooxygenated
product of flavonol (1). IR absorption bands at 3308, 2924,
1602 and 1653 cmꢁ1 suggested the presence of –OH, –C–H,
1
–CꢂC and –CꢂO groups, respectively. The H-NMR spec-
trum differed from that of starting compound (1) in the re-
duction of aromatic protons from nine to eight resulting in
the appearance of two doublets at d 8.09 and 6.93 in an
AAꢀBBꢀ system characteristic of a 1,4-disubstituted benzene
moiety showing p-hydroxylation. The 13C-NMR spectrum re-
vealed a highly deshielded carbon at d 159.9 corresponding
to C-4ꢀ. Long-range heteronuclear multiple correlation
(HMBC) confirmed this assignment. The metabolite 3 was
thus characterized as the known 3,4ꢀ-dihdroxyflavone.
Compound 4 (20 mg, 5% yield) was obtained as an amor- pound, flavone 3-O-b-D-4ꢀ-O-methylglucopyranoside.
phous white solid with a molecular formula, C22H22O8, as de-
One of the minor metabolites (9) (3 mg, 0.75% yield) of
termined by HR-ESI-MS data. The IR spectrum showed flavonol (1) had a molecular formula of C22H28O8 by HR-
strong absorption bands at 3374 (OH), 2924 (C–H), 1615 ESI-MS. This was an increase of six mass units as compared
1
(br) cmꢁ1 (CꢂC) and (CꢂO). Distortionless enhancement by to 4. It was reflected in the H-NMR spectrum, where addi-
polarization transfer (DEPT) spectra indicated that the 19 tional peaks were observed at d 4.17 (1H), 3.50 (1H), 3.44
signals observed in the 13C-NMR comprised one primary, (1H) and 2.20 (2H). The IR spectrum showed a broad ab-
one secondary, eleven tertiary and six quaternary carbons. sorption at 3319 cmꢁ1 indicating the presence of hydroxyl
1H- and 13C-NMR spectra showed the characteristic pattern group(s). The absence of a peak due to carbonyl in the 13C-
of flavonol (1) with a 4-O-methyl-b-D-glucopyranosyl moiety NMR spectrum indicated the absence of a carbonyl group in
attached to the 3-hydroxyl. Resonances at d 4.68 (1H, d, the molecule. The NMR spectra of compound 9 however,
Jꢂ8.4 Hz, H-1ꢃ), 3.51 (1H, dd, Jꢂ8.4 Hz, H-2ꢃ), 3.62 (1H, showed close resemblance to those of 4 with respect to the A
dd, Jꢂ9.0 Hz, H-3ꢃ), 3.02 (1H, dd, Jꢂ9.0 Hz, H-4ꢃ), 3.46 and B rings and the sugar moiety. NMR data also suggested
(3H, s, OMe-4ꢃ), 3.08 (1H, br, m, H-5ꢃ) 3.34, 3.45 (2H, m, 3-O-glucosylation as in 4. However, the disappearance of
1
H-6ꢃ) in the H-NMR spectrum revealed the presence of the peaks due to C-2 (d 159.1), C-3 (d 138.0) and C-4 (d 175.9)
4-O-methyl-b-D-glucopyranosyl moiety. Further evidence in 4 and the appearance of peaks due to a methylene group (d
came from the 13C-NMR spectrum which showed signals at d 30.0) and carbons linked to oxygen at d 76.7 and 69.7 in the
74.1, 75.8, 77.2 and 78.9 for hydroxymethine carbons, sig- 13C-NMR spectrum together with the observation of proton
1
nals at d 60.8 for an O-methyl carbon, at d 62.3 for a hydroxy- signals at d 4.17, 3.50, 3.44, 2.2 in the H-NMR spectrum,
methylene carbon and at d 105.7 for the anomeric carbon. suggested a new bꢀ-chalcanol structure for 9.
The 3-O-glucosylation was deduced from the chemical shifts
Metabolite 5 (57.2 3% yield) with a molecular formula
of C-3 (d 138.0) and C-2 (d 159.1). The b-configuration of of C16H12O3 as determined by HR-ESI-MS, 13C-NMR and
the sugar unit was consistent with the coupling constant of DEPT data, was isolated as a white solid. Strong absorption
the anomeric proton (d 4.68, d, Jꢂ8.4 Hz).28) The sugar part peaks at 2925 (C–H), 1633 (br) cmꢁ1 (CꢂC) and (CꢂO)
was further confirmed by comparing the 13C resonances with were observed in the IR spectrum. The NMR spectra of 5
those reported for the corresponding carbons in a similar 4- resembled those of 2 in all aspects except that the hydroxyl
O-methylglucopyranoside.29) The remaining signals of the group at C-7 has been methylated. Analysis of high-resolu-
NMR spectra confirmed the structure of the unchanged agly- tion NMR spectra and a comparison with reported data31)
cone moiety. The assignment of signals of compound 4 was confirmed that 5 was 7-methoxyflavone.
based on 1H–1H correlation spectroscopy (COSY), heteronu-
HR-ESI-MS, 13C-NMR and DEPT data suggested a mo-
clear multiple-quantum coherence (HMQC), HMBC spectra. lecular formula C15H10O4 for metabolite 6 (123 mg, 10%
Consequently, metabolite 4 was identified as the new com- yield). The additional 16 mass units indicated that it was a

322
Vol. 54, No. 3
monohydroxylated derivative of flavone 2. The IR spectrum cient for full structure elucidations. However, one was as-
showed absorption bands at 3190 (OH) and 1630 (br) (CꢂC, signed a tentative structure (9) using available spectroscopic
CꢂO) cmꢁ1. The NMR data of 6 closely resembled those of data. The transformations observed in this study are the re-
2. The metabolite however, displayed a para-substituted B sults of functionalization (Phase I) and conjugation (Phase II)
ring [d: 7.87 (2H, d, Jꢂ8.4 Hz), 6.90 (2H, d, Jꢂ8.4 Hz)]. reactions. As detected in previous transformation experi-
Comparison with literature data31) confirmed that compound ments with most microorganisms,27) we observed hydroxyla-
6 was 4ꢀ,7-dihydroxyflavone.
tion of the B-ring at C-4ꢀ in metabolites, 3, 6 and 8. Rela-
Metabolite 7 (186.9 mg, 21% yield) showed a molecular tively poor yields of the three metabolites could be due to de-
mass of 414 comprising of a 7-hydroxyflavone (m/z 238) and creased binding of flavones 1 and 2 to the enzymes responsi-
an O-methylglucopyranosyl moiety (m/z 178). The exact mo- ble for hydroxlation.27) Both flavones yielded the conjugated
lecular mass was consistent with a molecular formula of products, 4, 5, 7, 8 and 9. The formation of hydroxylation
1
C22H22O8. The H-NMR spectrum showed that the aromatic and conjugation products in this study are paralleled in mam-
protons had nearly the same chemical shifts and J values as mals32—34) indicating the use of microbes to mimic mam-
recorded for 7-hydroxyflavone (2). Similarly, the 13C-NMR malian metabolism. Microbes usually form glucosides rather
data were nearly identical to that of flavone 2, confirming than glucuronides as occurs in mammals.35) As reported, con-
that the flavone nucleus had not been transformed. Five of jugation are the most common final step reactions in mam-
the resonances in the 13C-NMR spectrum were in the region malian metabolism of intact flavonoids.20) Metabolite 9 is a
for aliphatic oxygenated carbons (d 60—80) and were con- cleavage product of flavone 1.36) Previous work on microbial
sistent with the presence of a glucosyl moiety. The methine transformation of flavonoids also reported some cleavage
carbon resonating at d 100.3 showed a one-bond correlation products.32) Cleavage of flavonoids in mammals has been
(HMQC) with the anomeric proton at d 5.14 which in turn shown to be brought about by the colonic microflora.20) The
had a three-bond correlation (HMBC) with the methine car- metabolites obtained are in sufficient quantities for biological
bon at d 162.3 (C-7). The latter showed two-bond correlation as well as further chemical studies. Compounds 4, 7 and 8
to H-6 (d 7.12). These data suggested O-glucosylation at C-7 are described as new compounds
of the flavone. HMBC correlations together with an observed
Experimental
downfield shift for C-4ꢃ (d 79.5) in the 13C-NMR spectrum
General Experimental Procedures UV spectra were run on a Hewlett
indicated the presence of an O-methyl group at C-4ꢃ. The O-
Packard 8452A diode array spectrometer. IR spectra were recorded in CHCl3
methyl group resonated at d 60.4 in the 13C-NMR spectrum
using an ATI Mattson Genesis series FTIR spectrophotometer. H- and 13C-
1
1
NMR were recorded in CDCl3 on Varian Mercury 400 and Varian Unity
Inova 600 spectrometers. HR-ESI-MS data were obtained using a Bruker
GioApex 3.0.
Substrates 3-Hydroxyflavone (1) and 7-hydroxyflavone (2) were ob-
tained from Aldrich Co. (Milwaukee, Wisconsin).
and at d 3.46 in the H-NMR spectrum. The large coupling
constant (Jꢂ7.6 Hz) between the (anomeric) proton H-1ꢃ and
H-2ꢃ indicated a trans-diaxial relationship establishing a b-
glycosidic linkage. Large coupling constants, J2ꢃ/3ꢃ, J3ꢃ/4ꢃ
,
Organisms and Metabolism Microorganisms (37) from the collection
of the National Center for Natural Products Research, University of Missis-
sippi, were used in the preliminary screening experiments to identify organ-
isms capable of metabolizing 1 and 2. Medium a,24) consisting of dextrose,
20 g; NaCl, 5 g; K2HPO4, 5 g; bacto-peptone (Difco Labs, Detroit, MI,
U.S.A.), 5 g and yeast extract (Difco Labs), 5 g per liter of distilled water
was used in all fermentation experiments based on a two-stage procedure.24)
Initial screening was performed in 125 ml Erlenmeyer flasks (125 ml) con-
taining 25 ml medium-a. Compounds 1 and 2 were added separately in di-
methylformamide (0.5 mg/ml) solution to 24 h old stage II cultures. Incuba-
tion was carried out at room temperature for a period of 14 d on a rotary
J4ꢃ/5ꢃ, indicated the trans-diaxial relationships between each
of the consecutive pairs of protons. The collective spectro-
scopic data suggested that compound 7 represent a methyl-
glucoside of 2. Thus, 7 was identified as the new compound,
flavone 7-O-b-D-4-O-methylglucopyranoside.
Compound 8 (64.8 mg, 7% yield), the most polar metabo-
lite of 2 was assigned the molecular formula, C22H22O9, as
determined from HR-ESI-MS. NMR data of 7 were similar
to those of 2, except in the region of B-ring protons which
showed para-substitution [d 7.97 (2H, d, Jꢂ8.8 Hz), 6.92 shaker (New Brunswick Model G10-21) at 100 rpm. Monitoring of the sam-
ples was carried out at 7-d intervals, using precoated Si gel 60 F254 TLC
plates (E. Merck) with EtOAc–hexane (3 : 2) as the solvent system. Spots on
TLC were visualized by UV light (254, 365 nm) and also with the help of
the spray reagent, p-anisaldehyde. Five 2 l flasks, each containing 100 mg of
(2H, d, Jꢂ8.8 Hz)]. The mass spectrum showed an additional
oxygen indicating that compound 8 was a monohydroxy de-
rivative of metabolite 7. Correlations observed in the HMBC
spectrum permitted the assignment of the hydroxyl group at
C-4ꢀ. Thus, the structure of compound 8 was established as
the new compound, 4ꢀ-hydroxyflavone 7-O-b-D-4-O-methyl-
glucopyranoside.
It is unclear whether the 4-O-methylglucopyranosyl moi-
ety in the metabolites, 4, 7 and 8 originated from the medium
or the fungus.
substrate dispersed in 500 ml of medium-a were used for preparative scale
fermentation of 3-hydroxyflavone (1) with B. bassiana. Similarly, 100 mg of
7-hydroxyflavone (2) in each of five 2 l flasks containing 500 ml of the
same medium were used with each of Nocardia species, A. alliaceous and
B. bassiana. The combined culture filtrates were extracted with EtOAc/
(Me)2CHOH–EtOAc (2 : 9) and the solvents were evaporated. The residues
obtained were subjected to partition or column chromatography. Wherever
needed, further purification was carried out by preparative thin layer (Silica
gel 60 F254) chromatography. Substrate and culture controls were run simul-
taneously with the above experiments.24)
Microbial Transformation of 3-Hydroxyflavone (1) by B. bassiana
The combined culture filtrates were extracted with EtOAc and the solvent
was evaporated to dryness. The resulting brown solid was column chro-
matographed over silica gel (Si gel 230—400 mesh: E. Merck, 30 g, column
diameter: 20 mm.) with CHCl3 gradually enriched with MeOH. Purification
of the metabolites was by repeated column and preparative layer chromatog-
raphy (EtOAc–hexane, 3 : 2). Four compounds were isolated. Only structures
of metabolites 3 (5 mg) and 4 (20 mg) were fully characterized as the other
Conclusion
In the present study, four metabolites of 3-hydroxyflavone
(1) produced by B. bassiana (ATCC 13144) and four of 7-hy-
droxyflavone (2), converted by Nocardia species (NRRL
5646), A. alliaceus (ATCC 10060) and B. bassiana (ATCC
7159) were isolated. Whilst all the products of flavone 2 were
characterized, only two metabolites of compound 1 were
identified. The other two were isolated in quantities insuffi- two compounds were in quantities insufficient for full structure elucidation.

March 2006
323
(4.38). IR nmax (KBr) cmꢁ1: 3190, 2360, 2336, 1630, 1602, 1554, 1450,
1383, 1273, 1231, 1178, 827, 665. HR-ESI-MS [MꢄH]ꢄ: (m/z) 255.0619
(Calcd for [C15H10O4ꢄH]: 255.0658).
Microbial Transformation of 7-Hydroxyflavone (2) by B. bassiana
The EtOAc/(Me)2CHOH–EtOAc (2 : 9) extract of the combined fermenta-
tion broth when evaporated yielded a brown solid. It was subjected to col-
umn chromatography (Alumina-neutral: 50—200 mÅ: Acrose Organic)
using CHCl3 gradually enriched with MeOH. One compound (7) (186.9 mg)
was isolated and identified.
Flavone 7-O-b-D-4-O-methylglucopyranoside (7) was isolated as a white
solid (186.9 mg, 21% yield). Rf 0.34 [MeOH–CHCl3 (1 : 9)]; 1H-NMR
400 MHz (DMSO-d6) d: 8.05 (2H, d, Jꢂ7.2 Hz, H-2ꢀ, 6ꢀ), 7.95 (1H, d,
Jꢂ8.4 Hz, H-5), 7.55 (3H, m, H-3ꢀ—H-5ꢀ), 7.37 (1H, s, H-8), 7.12 (1H,
br d, Jꢂ8.4 Hz, H-6), 6.94 (1H, s, H-3), 5.55 (1H, d, Jꢂ8.4 Hz, OH-2ꢃ), 5.34
(1H, d, Jꢂ4.8 Hz, OH-3ꢃ), 5.14 (1H, d, Jꢂ7.6 Hz, H-1ꢃ), 4.78 (1H, m, OH-
6ꢃ), 3.67 (2H, m, H-6ꢃ), 3.52 (1H, br d, Jꢂ8.4 Hz, H-5ꢃ), 3.46 (3H, s, OMe-
4ꢃ), 3.44 (1H, m, H-3ꢃ) 3.32 (1H, dd, Jꢂ8.4 Hz, H-2ꢃ), 3.07 (1H, dd,
Jꢂ8.4 Hz, H-4ꢃ). 13C-NMR 150 MHz (DMSO-d6): d 177.2 (C-4), 163.1 (C-
2), 162.3 (C-7), 157.8 (C-9), 132.4 (C-4ꢀ), 131.8 (C-1ꢀ), 129.8 (C-5ꢀ), 126.9
(C-2ꢀ), 118.7 (C-10), 116.2 (C-6), 107.5 (C-3), 104.4 (C-8), 100.3 (C-1ꢃ),
79.5 (C-4ꢃ), 76.8 (C-3ꢃ), 76.4 (C-5ꢃ), 74.0 (C-2ꢃ), 60.8 (C-6ꢃ), 60.4 (OMe).
UV lmax (MeOH) nm (log e): 214 (4.27), 252 (4.24), 308 (4.35). IR nmax
(CHCl3) cmꢁ1: 3429, 29051447, 1372, 1086, 773, 650. HR-ESI-MS
[MꢁH]ꢄ: (m/z) 413.1267 (Calcd for C22H22O8ꢁH: 413.1236).
However, one of the minor compounds was assigned a tentative chalcanol
structure (9) (2 mg).
3,4ꢀ-Dihdroxyflavone (3) was obtained as a pale yellow solid (5 mg,
1.25% yield). Rf 0.72 [MeOH–CH2Cl2 (1 : 19)]; 1H-NMR 600 MHz (CDCl3)
d: 8.09 (2H, d, Jꢂ8.4 Hz, H-2ꢀ, 6ꢀ), 8.08 (d, Jꢂ7.2 Hz, H-5), 7.76 (br dd,
Jꢂ7.8, 7.2 Hz, H-7), 7.71 (br d, Jꢂ7.8 Hz, H-8), 7.43 (1H, br dd, Jꢂ7.8,
7.2 Hz, H-6), 6.93 (2H, d, Jꢂ8.4 Hz, H-3ꢀ—H-5ꢀ). 13C-NMR 150 MHz
(CDCl3, CD3OD): d 173.2 (C-4), 159.9 (C-4ꢀ), 155.1 (C-9), 146.8 (C-2),
138.5 (C-3), 134.1 (C-7), 130.5 (C-1ꢀ), 130.2 (C-2ꢀ, -6ꢀ), 125.4 (C-5), 125.1
(C-6), 118.9 (C-8), 116.1 (C-3ꢀ). UV lmax (MeOH) nm (log e): 208 (3.67),
232 (3.49), 250 (3.44), 314 (3.19), 358 (3.49); IR nmax (CHCl3) cmꢁ1: 3308,
2924, 1653, 1602, 1558, 1484, 1280, 1178, 750. HR-ESI-MS [MꢄH]ꢄ:
(m/z) 255.0596 (Calcd for C15H10O4ꢄH: 255.0659).
Flavone 3-O-b-D-4-O-methylglucopyranoside (4) was isolated as a light
yellow solid (20 mg, 5% yield). Rf 0.34 [MeOH–CHCl3 (1 : 9)]; 1H-NMR
600 MHz (CDCl3) d: 8.24 (br d, Jꢂ8.4 Hz, H-5), 8.08 (2H, d, Jꢂ7.8 Hz, H-
2ꢀ, 6ꢀ), 7.73 (br dd, Jꢂ8.4, 7.2 Hz, H-7), 7.56 (br d, Jꢂ8.4 Hz, H-8), 7.52
(3H, m, H-3ꢀ—H-5ꢀ), 7.44 (1H, br d, Jꢂ8.2 Hz, H-6), 4.68 (1H, d,
Jꢂ8.4 Hz, H-1ꢃ), 3.62 (1H, dd, Jꢂ9.0 Hz, H-3ꢃ) 3.51 (1H, dd, Jꢂ8.4 Hz, H-
2ꢃ), 3.46 (3H, s, OMe-4ꢃ), 3.45 (1H, m, H-6ꢃ), 3.34 (1H, m, H-6ꢃ), 3.08 (1H,
br m, H-5ꢃ), 3.02 (1H, dd, Jꢂ9.0 Hz, H-4ꢃ). 13C-NMR 150 MHz (CDCl3,
CD3OD): d 175.9 (C-4), 159.1 (C-2), 155.8 (C-9), 138.0 (C-3), 134.6 (C-7),
131.5 (C-1ꢀ), 129.6 (C-2ꢀ), 128.3 (C-3ꢀ, -4ꢀ, -5ꢀ), 126.2 (C-5), 125.6 (C-6),
123.7 (C-10), 118.4 (C-7), 105.7 (C-1ꢃ), 78.9 (C-4ꢃ), 77.2 (C-3ꢃ), 75.8 (C-
5ꢃ), 74.1 (C-2ꢀ), 62.3 (C-6ꢃ), 60.8 (OMe). UV lmax (MeOH) nm (log e): 214
(2.15), 244.0 (4.23), 300 (4.19); IR nmax (CHCl3) cmꢁ1: 3374, 2924, 1615,
1469, 1397, 1240, 1077, 762. HR-ESI-MS [MꢄH]ꢄ: (m/z) 415.1364 (Calcd
for C22H22O8ꢄH: 415.1394).
A fraction of the above extract when subjected to flash chromatography
(Biotage Horizon) over silica gel (KP-silTM 60 Å cartidge) with
MeOH–CHCl3 (1 : 9) as the solvent system yielded the metabolite 8
(64.8 mg).
Chalcane 3-O-b-D-4-O-methylglucopyranoside (9) was obtained as a
white solid (3 mg, 0.75% yield). Rf 0.12 [MeOH–CHCl3 (1 : 9)]; 1H-NMR
600 MHz (CDCl3) d: 8.17 (br d, Jꢂ8.4 Hz, H-2ꢀ), 8.04 (2H, d, Jꢂ7.8 Hz, H-
2, 6), 7.69 (br dd, Jꢂ8.4 Hz, H-4ꢀ), 7.53 (br d, Jꢂ8.4 Hz, H-5ꢀ), 7.45 (3H, m,
H-3, -4, -5), 7.40 (1H, m, H-3ꢀ), 4.69 (1H, d, Jꢂ7.8 Hz, H-1ꢃ), 4.17 (1H, m,
H-bꢀ), 3.56 (1H, m, H-3ꢃ), 3.50 (1H, m, H-2ꢃ), 3.44 (1H, m, H-6ꢃ), 3.04
(1H, br m, H-5ꢃ), 2.20 (2H, m, H, b). 13C-NMR 150 MHz (CDCl3, CD3OD):
d 159.1 (C-6ꢀ), 134.6 (C-4ꢀ), 131.5 (C-1), 129.5 (C-2), 128.3 (C-3, -4, -5),
126.0 (C-2ꢀ), 125.6 (C-3ꢀ), 123.6 (C-1ꢀ), 118.4 (C-5ꢀ), 105.2 (C-1ꢃ), 76.7
(C-3ꢃ, 4ꢃ), 76.3 (C-5ꢃ), 74.0, 69.7 (C-2ꢃ), 62.0 (C-6ꢃ), 59.0 (OMe), 30.0. UV
lmax (MeOH) nm (log e): 210 (4.07), 228 (4.05), 278 (3.33); IR nmax
(CHCl3) cmꢁ1: 3319, 2926, 1629, 1451, 1385, 1092, 771, 649. HR-ESI-MS
[MꢄH]ꢄ: (m/z) 421.2331 (Calcd for C22H28O8ꢄH: 421.1863).
Microbial Transformation of 7-Hydroxyflavone (2) by Nocardia Species
(NRRL 5646) The combined culture medium including the mycelium was
extracted with EtOAc/(Me)2CHOH–EtOAc (2 : 9). The solvents were evapo-
rated to dryness and the resulting brown solid was subjected to flash chro-
matography (Biotage Horizon) over silica gel (KP-silTM 60 Å cartidge) with
CHCl3 enriched with MeOH as the eluent. A single metabolite (5) (57.2 mg)
was obtained.
7-Methoxyflavone (5) was isolated as a white solid (57.2 mg, 3% yield).
Rf 0.64 [MeOH–CHCl3 (1 : 19)]; 1H-NMR 400 MHz (DMSO-d6) d: 8.05
(2H, d, Jꢂ7.2 Hz, H-2ꢀ, 6ꢀ), 7.90 (1H, d, Jꢂ8.4 Hz, H-5), 7.55 (3H, m, H-
3ꢀ—H-5ꢀ), 7.27 (1H, d, Jꢂ2.4 Hz, H-8), 7.03 (1H, dd, Jꢂ8.4, 2.4 Hz, H-6),
6.91 (1H, s, H-3), 3.89 (3H, s, –OMe), 13C-NMR 150 MHz (DMSO-d6): d
177.1 (C-4), 164.5 (C-7), 163.0 (C-2), 158.1 (C-9), 132.3 (C-4ꢀ), 131.8 (C-
1ꢀ), 129.8 (C-3ꢀ), 126.9 (5), 126.9 (C-2ꢀ), 117.8 (C-10), 115.4 (C-6), 107.4
(C-3), 101.6 (C-8), 56.8 (OMe). UV lmax (MeOH) nm (log e): 212 (4.11),
252 (3.95), 306 (4.03). IR nmax (KBr) cmꢁ1: 3386, 2925, 2853, 1633, 1451,
1439, 1376, 1357, 1248, 772. HR-ESI-MS [MꢄH]ꢄ: (m/z) 253.0864 (Calcd
for C16H12O3ꢄH: 253.0865).
Microbial Transformation of 7-Hydroxyflavone (2) by A. alliaceus
(ATCC 10060) The EtOAc/(Me)2CHOH–EtOAc (2 : 9) extract of the com-
bined culture media and the EtOAc extract of the MeOH extract of the
mycelium showed a similar TLC profile. Partition chromatography (Saki
High HPCPC: LLB-M) however, was effected on the extracts using hexane–
EtOAc–(Me)2CꢂO–MeOH–H2O (3 : 2 : 2 : 2 : 2) as the solvent system. One
metabolite (6) (123.5 mg) was isolated and characterized.
4ꢀ-Hydroxyflavone 7-O-b-D-4-O-methylglucopyranoside (8) was isolated
as a white solid (64.8 mg, 7% yield). Rf 0.16 [MeOH–CHCl3 (3 : 2)]; 1H-
NMR 400 MHz (DMSO-d6) d: 7.97 (2H, d, Jꢂ8.8 Hz, H-2ꢀ, 6ꢀ), 7.92 (1H, d,
Jꢂ8.8 Hz, H-5), 7.33 (1H, d, Jꢂ2.4 Hz, H-8), 7.10 (1H, dd, Jꢂ8.8, 2.4 Hz,
H-6), 6.92 (2H, d, Jꢂ8.8 Hz, H-3ꢀ, H-5ꢀ), 6.77 (1H, s, H-3), 5.50 (1H, d,
Jꢂ4.8 Hz, OH-2ꢃ), 5.30 (1H, d, Jꢂ5.2 Hz, OH-3ꢃ), 5.12 (1H, d, Jꢂ8.0 Hz,
H-1ꢃ), 4.75 (1H, m, OH-6ꢃ), 3.65 (2H, m, H-6ꢃ), 3.50 (1H, Jꢂ7.6 Hz, H-5ꢃ),
3.45 (3H, s, OMe-4ꢃ), 3.43 (1H, m, H-3ꢃ), 3.28 (1H, Jꢂ8.0 Hz, H-2ꢃ), 3.05
(1H, Jꢂ8.8 Hz, H-4ꢃ). 13C-NMR 150 MHz (DMSO-d6): d 177.0 (C-4),
163.6 (C-2), 162.1 (C-7), 161.5 (C-4ꢀ), 157.7 (C-9), 128.9 (C-2ꢀ, 6ꢀ), 126.9
(C-5), 122.3 (C-1ꢀ), 118.7 (C-10), 116.6 (C-3ꢀ, 5ꢀ), 116.0 (C-6), 105.3 (C-3),
104.3 (C-8), 100.3 (C-1ꢃ), 79.5 (C-4ꢃ), 76.8 (C-3ꢃ), 76.4 (C-5ꢃ), 74.0 (C-2ꢃ),
60.8 (C-6ꢃ), 60.4 (OMe). UV lmax (MeOH) nm (log e): 212 (4.11), 230
(4.02), 254 (3.79), 326 (4.08). IR nmax (KBr) cmꢁ1: 3426, 2921, 1628, 1564,
1509, 1447, 1383, 1251, 1138, 639. HR-ESI-MS [MꢁH]ꢄ: (m/z) 429.1212
(Calc for C22H22O9ꢁH: 429.1185).
Acknowledgements The authors thank Mr. Frank Wiggers for assis-
tance in obtaining 2D NMR spectra and Drs. Chuck Dunbar and Bharathi
Avula for conducting HR-ESI-MS analysis. This work was supported, in
part, by the United States Department of Agriculture, Agricultural Research
Specific Cooperative Agreement No. 58-6408-2-00009.
References and Notes
1) Part 5: Herath W. H. M. W., Ferreira D., Mikell J. R., Khan I. A.,
Chem. Pharm. Bull., 52, 1372—1374 (2004).
2) Geissman T. A., Crout D. H. G., “Organic Chemistry of Secondary
Plant Metabolism,” Vol. 52, Freeman, Cooper and Company, San Fran-
cisco, 1969, pp. 183—230.
3) Beecher G. R., J. Nutr., 133, 3248S—3254S (2003).
4) Venkataraman K., “Progress in the Chemistry of Organic Natural
Products,” Vol. 17, Chap. 1, ed. by Zechmeister L., Springer-Verlag,
Vienna, 1959, pp. 1—69.
5) Harbone J. B., Nat. Prod. Rep., 18, 361—379 (2001).
6) Brouillard R., Dangles O., “The Flavonoids: Advances in Research
Since 1986,” ed. by Harbone J. B., Chapman and Hall, London, 1993,
p. 565.
7) Yang C. S., Landau J. M., Huang M., Newmark L., Ann. Rev. Nutr., 21,
381—406 (2001).
4ꢀ,7-Dihydroxyflavone (6) was obtained as a light yellow solid (123.5 mg,
1
10% yield). Rf 0.2 [EtOAc–Hexane (3 : 2)]; H-NMR 400 MHz (DMSO-d6)
8) Middleton E., Kandaswami C., Theoharides T. C., Pharmacol. Rev.,
52, 673—751 (2000).
d: 7.87 (2H, d, Jꢂ8.4 Hz, H-2ꢀ, 6ꢀ), 7.84 (1H, d, Jꢂ8.8 Hz, H-5), 6.94 (1H,
br s, H-8), 6.90 (2H, d, Jꢂ8.4 Hz, H-3ꢀ—H-5ꢀ), 6.88 (1H, d, Jꢂ8.8 Hz, H-6),
6.67 (1H, s, H-3), 13C-NMR 150 MHz (DMSO-d6): d 177.0 (C-4), 163.4 (C-
7), 163.2 (C-2), 161.4 (C-4ꢀ), 158.1 (C-9), 128.8 (C-2ꢀ, 6ꢀ), 127.1 (C-5),
122.5 (C-1ꢀ), 116.8 (C-10), 116.6 (C-3ꢀ, 5ꢀ), 115.5 (C-6), 105.1 (C-3), 103.1
(C-8). UV lmax (MeOH) nm (log e): 216 (4.31), 232 (4.28), 254 (4.05), 3.08
9) Middleton E., Jr., Adv. Exp. Med. Biol., 439, 175—182 (1998).
10) Gabor M., Prog. Clin. Biol. Res., 213, 471—480 (1986).
11) Selway J. W., Prog. Clin. Biol. Res., 213, 521—536 (1986).
12) Block G. A., Nur. Rev., 50, 207—213 (1992).
13) Elangovan V., Sekar N., Govindasamy S., Cancer Lett., 50, 278—284

324
Vol. 54, No. 3
(1994).
1986, pp. 391—392.
14) Facino R. M., Carini M., Aldini G., Berti F., Rossoni G., Bombardelli
26) Clark A. M., McChesney J. D., Hufford C. D., Med. Res. Rev., 5,
E., Morazzoni P., Life Sci., 64, 943—949 (1999).
231—253 (1985).
15) Herteg M. G. L., Feskens E. J. M., Hollman P. C. H., Katan M. B., 27) Ibrahim A., Abdul-Hajj Y. J., J. Nat. Prod., 53, 1471—1478 (1990)
Kromhout D., Lancet, 342, 1007—1011 (1993).
16) Mazur A., Bayle D., Atherosclerorsis, 149, 421—422 (1999).
17) Kessler M., Ubeaud G., Jung L., J. Pharm. Pharmacol., 55, 131—142
(2003).
and references therein.
28) Markham K. R., Geiger H., “The Flavonoids: Advances in Research
Since 1986,” Chap. 10, ed. by Harbone J. B., Chapman & Hall, Lon-
don, 1993, pp. 441—473.
18) Farks O., Jakus J., Héberger K., Molecules, 9, 1079—1088 (2004).
19) Firuzi O., Mlade˘nka P. M., Petrucci R., Marrosu G., Saso L., J. Pharm.
Pharmacol., 56, 801—807 (2004).
29) Vigne B., Archelas A., Fourneron J. D., Furstoss R., Tetrahedron, 42,
2451—2456 (1986).
30) Kingsbury C. A., Looker J. H., J. Org. Chem., 40, 1120—1124 (1975).
20) Manach C., Scalbert A., Morand C., Rémésy C., Jiménez L., Am. J. 31) Yoo H. S., Lee J. S., Kim C. Y., Kim J., Arch. Pharm. Res., 27, 544—
Clin. Nutr., 79, 727—747 (2004).
546 (2004).
21) Rice-Evens C., Curr. Med. Chem., 8, 797—807 (2001).
32) Griffiths L. A., Smith S. E., Biochem. J., 128, 901—911 (1972).
22) Williams R. J., Spencer J. P. E., Rice-Evans C., Free. Radic. Biol. 33) Ibrahim A., Abdul-Hajj Y. J., J. Nat. Prod., 53, 644—656 (1990).
Med., 36, 838—849 (2004).
34) Hackett A. M., Griffiths L. A., Xenobiotica, 15, 907—914 (1985).
35) Davis P. J., “Antibiotics and Microbial Transformations,” Chap. 3, ed.
by Lamber S. S., Walker C. A., CRC Press, Florida, 1987, pp. 47—68.
36) Karoon P. A., Clifford M. N., Crozier A., Day A. J., Donavan J. L.,
Manach C., Williamson G., Am. J. Clin. Nutr., 80, 15—21 (2004).
23) Frei B., Higdon J. V., J. Nutr., 133, 3275S—3284S (2003).
24) Abourashed E. A., Khan I. A., Chem. Pharm. Bull., 48, 1996—1998
(2000).
25) Rosazza J. P. N., Duffel M. W., “Alkaloids: Chemistry and Pharmacol-
ogy,” Vol. 27, Chap. 4, ed. by Brossi A., Acadamic Press, New York,