Microbial metabolism. Part 10: Metabolites of 7,8-dimethoxyflavone and 5-methoxyflavone

Article abstract of DOI:10.1080/14786410902829797

Microbial transformation of 7,8-dimethoxyflavone (1) by Mucor ramannianus produced five metabolites: 7,8-dimethoxy-4′-hydroxyflavone (2), 3′,4′-dihydroxy-7,8-dimethoxyflavone (3), 7,3′-dihydroxy-8- methoxyflavone (4), 7,4′-dihydroxy-8-methoxyflavone (5) a

Full text of DOI:10.1080/14786410902829797

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Microbial metabolism. Part 10:
Metabolites of 7,8-dimethoxyflavone
and 5-methoxyflavone
Wimal Herath a , Julie Rakel Mikell a & Ikhlas Ahmad Khan a b
a National Center for Natural Products Research, University of
Mississippi , University, MS 38677, USA
b Department of Pharmacognosy , Research Institute of
Pharmaceutical Sciences, School of Pharmacy, University of
Mississippi , University, MS 38677, USA
Published online: 05 Nov 2010.
To cite this article: Wimal Herath , Julie Rakel Mikell & Ikhlas Ahmad Khan (2009) Microbial
metabolism. Part 10: Metabolites of 7,8-dimethoxyflavone and 5-methoxyflavone, Natural Product
Research: Formerly Natural Product Letters, 23:13, 1231-1239, DOI: 10.1080/14786410902829797
To link to this article: http://dx.doi.org/10.1080/14786410902829797
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Natural Product Research
Vol. 23, No. 13, 10 September 2009, 1231–1239
Microbial metabolism. Part 10: Metabolites of 7,8-dimethoxyflavone and
5-methoxyflavone
Wimal Heratha, Julie Rakel Mikella and Ikhlas Ahmad Khanab
*
aNational Center for Natural Products Research, University of Mississippi, University, MS 38677,
USA; Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of
Pharmacy, University of Mississippi, University, MS 38677, USA
b
(Received 18 December 2008; final version received 16 February 2009)
Microbial transformation of 7,8-dimethoxyflavone (1) by Mucor ramannianus
produced five metabolites: 7,8-dimethoxy-40-hydroxyflavone (2), 30,40-dihy-
droxy-7,8-dimethoxyflavone (3), 7,30-dihydroxy-8-methoxyflavone (4), 7,40-
dihydroxy-8-methoxyflavone (5) and 8-methoxy-7,30,40-trihydroxyflavone (6).
It was, however, completely converted to a single metabolite, 7-hydroxy-8-
methoxyflavone (7) by Aspergillus flavus. 5-Methoxyflavone (8), when
fermented with Beauveria bassiana, gave a single product, 5-methoxyflavanone
(9). Conversion of 8 with Aspergillus alliaceus yielded the metabolite 40-
hydroxy-5-methoxyflavone (10). The structures of the compounds 2–7, 9 and
10 were established by spectroscopic methods.
Keywords: flavonoids; microbial metabolism; Mucor ramannianus; Aspergillus
flavus
1. Introduction
Flavonoids are polyphenolic compounds found in vegetables, fruits and beverages
(Herath, Mikell, Hale, Ferreira, and Khan 2008; Nikolic & van Breemen, 2004;
Tasdemir et al., 2006). Cell culture experiments on these compounds show potential use
as chemopreventive agents against cardiovascular disease, cancer and other diseases
(Middleton, Kandaswami, & Theoharides, 2000). These activities, however, are not
often observed in vivo in studies of animals and humans due to their very low oral
bioavailability (Tsuji, Winn, & Walle, 2006; Walle, Walle, & Halushka, 2001; Walle,
Otake, Brubaker, Walle, & Halushka, 2001a; Wen, & Walle, 2006). Excretion
facilitated by rapid presystemic hepatic and intestinal glucuronidation and sulphation
of the free hydroxyl groups of the flavonoids is responsible for their poor
bioavailability (Walle, 2007; Walle et al., 2001a). The observation that methylation
of the free hydroxyl groups greatly improves their intestinal absorption and hepatic
metabolic stability, limiting metabolism to less efficient CYP-mediated oxidation
(Walle, 2007; Wen & Walle, 2006) is the suggested reason for the improvements in
some of their biological properties, including the inhibition of cancer cell proliferation
(Walle et al., 2007). The early assumption that free hydroxyl groups are necessary for
*Corresponding author. Email: ikhan@olemiss.edu
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2009 Taylor & Francis
DOI: 10.1080/14786410902829797
http://www.informaworld.com

1232
W. Herath et al.
bioactivities of flavonoids (Walle, 2007; Walle et al., 2007) stems from the belief that
the main beneficial property of these compounds is their antioxidant activity (Takano
et al., 2007; Walle et al., 2007). Lack of such activity in methylated flavonoids has
attracted little attention as potent chemopreventive agents, even though many of them
occur naturally in
a variety of plant species (Wen & Walle, 2006). Some
methoxyflavones exhibit multidrug-resistant reversing effects in cancer cells. Their
potency is believed to be related to the number and positions of the methoxyl groups
(Ohtani et al., 2007). In selecting methoxyflavones as potential chemopreventive agents,
it is important to determine how susceptible they are towards metabolism (Walle &
Walle 2007). Since microorganisms can be used as predictive models for mammalian
drug metabolism, we investigated prospectively the microbial metabolism of 7,8-
dimethoxyflavone (1) and 5-methoxyflavone (8) using 40 microorganisms. Cultures
which gave the maximum number of metabolites in good yields were selected for scale-
up studies. The structures of the eight metabolites formed (2–7,9, 10) were established
by detailed study of their high resolution spectroscopic data.
4
R
5
R
1
3'
B
R
4'
1'
2
R
O
8
A
1
C
7
3
5
3
R
O
R1
R2
R3
R4
R5
C-2,3
dihydro
1
2
3
4
5
6
7
8
9
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OH
OH
OH
OH
H
H
H
H
H
H
H
H
OMe
OMe
H
H
OH
OH
H
OH
H
H
-
-
-
-
-
-
-
-
OH
OH
H
OH
OH
H
H
H
H
H
H
H
C-2,3
dihydro
-
10
H
H
OMe
H
OH
2. Results and discussion
Microorganisms capable of metabolising
1
and
8
were selected by screening
40 organisms using the standard two stage procedure (Abourashed & Khan, 2000).
Scale-up studies of 1 with Mucor ramannianus yielded five metabolites: 7,8-dimethoxy-
40-hydroxyflavone (2), 30,40-dihydroxy-7,8-dimethoxyflavone (3) (Menichincheri et al.,
2004), 7,30-dihydroxy-8-methoxyflavone (4), 7,40-dihydroxy-8-methoxyflavone (5) and 8-
methoxy-7,30,40-trihydroxyflavone (6). Aspergillus flavus converted 1 to 7-hydroxy-8-
methoxyflavone (7). 5-Methoxyflavone (8) yielded a single metabolite each when

Natural Product Research
1233
fermented with Beauveria bassiana and Aspergillus alliaceus. They were characterised as
5-methoxyflavanone (9) (Lee, Jung, & Jung, 2007) and 40-hydroxy-5-methoxyflavone
(10), respectively. The molecular formulae of all the metabolites were determined by
HR-ESI-MS.
Metabolite 2 (10 mg, 2.5%) was a white solid with a molecular formula of C17H14O5,
corresponding to a monoxygenated product of 1. Compound 2 showed a hydroxyl band at
1
3411 cmꢀ1 in the IR spectrum. The H-NMR data of 2 were similar to those of 1, except
for the B-ring protons, which showed para-substitution [ꢀ 7.89 (2H, d, J ¼ 9.0 Hz), 6.92
(2H, d, J ¼ 9.0 Hz)]. The hydroxyl group was assigned at C-40 by a detailed NMR study,
and the compound was identified as 7,8-dimethoxy-40-hydroxyflavone.
Compound 3 was obtained as a white solid (30 mg, 7.5%), which gave a molecular ion
peak at m/z 313.0750 [MꢀH]þ in its HR-ESI-MS, corresponding to the elemental formula
1
C17H14O6. The H-NMR data of 3 showed close resemblance to those of 2. The B-ring,
however, showed 1, 3, 4 aromatic substitution [ꢀH 6.98 (1H, d, J ¼ 8.5 Hz), 7.55
(1H, dd, J ¼ 8.5,2.0 Hz), 7.56 (1H, d, J ¼ 2.0 Hz)]. It was identified as 30,40-dihydroxy-7,8-
dimethoxyflavone by correlation spectra. Although 3 had been synthesised previously,
limited published data prevented a direct comparison (Menichincheri et al., 2004).
Compound 4 (10 mg, 2.5%) was isolated as a light yellow solid with a molecular
1
formula of C16H12O5. The H-NMR data of 4 were also similar to those of 1, with the
exception of the B-ring protons showing meta-substitution (Table 1). The 13C-NMR
spectrum revealed a highly deshielded singlet carbon at ꢀ 158.4. It was assigned to C-30 by
HMBC correlations. Further, the 1H-NMR of 4 showed that one of the methoxy groups of
1 was demethylated during transformation. HMBC correlations permitted the assignment
of the hydroxyl group at C-7. Thus, the structure of compound 4 was established as 7,30-
dihydroxy-8-methoxyflavone.
The NMR data of 5, a pale yellow solid (37 mg, 9.25%) having a molecular formula
C16H12O5, were similar to those of 4 except for the position of the hydroxyl group in the B-
ring. It was determined to be at C-40 by correlation NMR spectra. Compound 5 was thus
identified as 7,40-dihydroxy-8-methoxyflavone.
Compound 6 (7 mg, 1.75%), isolated as a faint yellow solid, was shown to have
a molecular formula C16H12O6. Except for the absence of a methyl group, all other NMR
resonances of 6 were similar to those of the metabolite 3. Correlation of the remaining O-
methyl resonance with the carbon resonance at ꢀ 135.8 (C-8) by HMBC enabled the
assignment of structure 6 as 8-methoxy-7,30,40-trihydroxyflavone.
Compound 7, obtained as a white solid (90 mg, 36%), was formulated as C16H12O4.
The NMR spectral data of 7 were similar to those of the metabolite 4 except that its B-ring
was free of hydroxyl groups. NMR correlation data were used to characterise the
compound as 7-hydroxy-8-methoxyflavone.
The white solid 9 (65.4 mg, 7.47%) with a molecular formula C16H14O3 was identified
as 5-methoxyflavanone by comparison with published data (Lee et al., 2007).
Metabolite 10,
formula C16H12O4, corresponded to a monoxygenated product of 8. A hydroxyl band
a
white solid (89.9 mg, 71.92% yield) with the molecular
1
was observed at 3428 cmꢀ1 in the IR spectrum of 10. The H-NMR spectrum of 10 was
similar to that of 8 except for the presence of two doublets (AA0 BB0 system, ꢀ 6.93,7.90),
indicating hydroxylation at the para position of the B-ring. The singlet carbon at ꢀ 161.1 in
13
the C-NMR spectrum was assigned to C-40 by correlation spectra of 10. Thus,
compound 10 was characterised as 40-hydroxy-5-methoxyflavone.

1234
W. Herath et al.

Natural Product Research
1235
3. Conclusion
Experiments with human liver microsomes had shown that the fully methylated flavanoids
resist oxidative metabolism compared to their partially methylated and unmethylated
analogues (Walle & Walle, 2007). However, a considerable variation of resistance had been
observed among them (Tsuji et al., 2006; Walle & Walle, 2007). Stability of 7-
methoxyflavone, for example, was increased or decreased by additional methoxy
groups. The variation was from highly susceptible 7,30-dimethoxyflavone to the more
stable 5,7-dimethoxyflavone (Walle & Walle, 2007). The study, which had been conducted
with 15 flavones, indicated the importance of positions rather than the number of methoxy
groups towards oxidation (Walle & Walle, 2007). This investigation was an attempt to
determine the susceptibility of 7,8-dimethoxyflavone towards metabolism and to identify
its metabolites prospectively with microorganisms as predictive models for mammalian
drug metabolism. The microbial model concept is based on the fundamental grounds that
the same Phase I paths of metabolism of xenobiotics observed in mammals are paralleled
in microorganisms (Davis, 1987). Of the 40 microbial cultures screened, many were
capable of transforming 1 to a limited number of metabolites with each organism. The
TLC comparisons of the EtOAc extracts of the culture filtrates showed relatively higher
biotransformational efficiency with M. ramannianus yielding all metabolites generated by
other organisms. The exception was compound 7, formed with A. flavus. About 25%
conversion of 1 to the respective metabolites was observed, indicating moderate
susceptibility towards oxidative metabolism. It yielded five compounds (2–6).
In compounds 4–7, demethylation occurred at position 7. The 8-position methoxy
group, however, remained unchanged during all the transformations. 5-Methoxyflavone,
which was highly resistant to human microsomal oxidation (Walle & Walle, 2007)
underwent transformation to metabolites 9 (7.47%) and 10 (71.92%) when fermented with
B. bassiana and A. alliaceus, respectively.
4. Experimental section
4.1. General experimental procedures
1
Unless otherwise stated, the H- and 13C-NMR were obtained in DMSO-d6 on a Varian
Unity Inova 600 spectrometer. Optical rotations were determined using a Jasco DIP-370
digital polarimeter. The UV spectra were measured on a Hewlett Packard 8452A diode
array spectrometer. IR spectra were run in CHCl3 on an ATI Mattson Genesis series
FTIR spectrophotometer. HR-ESI-MS data were obtained using a Bruker GioApex 3.0.
4.2. Substrates
7,8-Dimethoxyflavone (1) and 5-methoxyflavone (8) were purchased from Sigma Aldrich
Chemical Co. (Milwaukee, Wisconsin) and their authenticity was confirmed by NMR
data.
4.3. Organisms and metabolism
Initial screening experiments to select microorganisms capable of converting compounds 1
and 7 to their metabolites in good yields were carried out with 40 microbes from the

1236
W. Herath et al.
collection of the National Center for Natural Products Research of the University of
Mississippi. A two-stage procedure was used in all experiments, including the preparative
scale fermentations, as described in the literature (Abourashed & Khan, 2000).
Four 2-L flasks, each containing 100 mg of substrate dispersed in 500 mL of
medium-ꢁ (Herath, Ferreira, & Khan, 2003), were used for preparative scale
fermentation of 7,8-dimethoxyflavone (1) by M. ramannianus. Similarly, 200 mg of 1
in two 2-L flasks were used with A. flavus. Six such flasks were used to ferment 250 mg
of 5-methoxyflavone (8) with B. bassiana. The EtOAc extracts of the combined culture
filtrates were column chromatographed (silica gel 60 F254) to isolate the metabolites.
Repeated column and preparative thin layer (silica gel 60 F254) chromatography were
performed to purify the metabolites. Substrate and culture controls were run alongside
the above experiments (Jurgens, Hufford, & Clark, 1992).
4.4. Microbial transformation of 7,8-dimethoxyflavone (1) by M. ramannianus (ATCC
9628)
The ethyl acetate extract of the combined fermentation broth was column chromato-
graphed (Si gel 230–400 mesh: E. Merck, 30 g, column diameter: 20 mm) with CH2Cl2 by
increasing amounts of MeOH. The fractions were further purified by repeated column and
preparative layer chromatography (CH2Cl2 : MeOH, 16 : 1) to obtain five compounds (2–
6) that were identified by spectroscopic data.
7,8-Dimethoxy-40-hydroxyflavone (2) was isolated as a white solid (10 mg, 2.5%). Rf
0.42 [MeOH : CH2Cl2 (1 : 13)]; UV ꢂmax (MeOH) nm (log " ): 330 (4.31), 306 sh (4.19), 259
(4.16), 251 sh (4.09), 233 sh (4.18), 2.15 sh (4.34), 215 (4.34), 204 (4.42); IR
1
ꢃ
max(CHCl3) cmꢀ1: 3411, 2939, 1667, 1614, 1512, 1444, 1388, 1290, 1021, 817; H-NMR
600 MHz (DMSO-d6) and 13C-NMR 150 MHz (DMSO-d6): see Tables 1 and 2; HR-ESI-
MS [M þ Na]þ: (m/z) 321.0722 (Calcd for C17H14O5 þ Naþ: 321.0738).
30,40-Dihydroxy-7,8-dimethoxyflavone (3) was obtained as a white solid (30 mg, 7.5%).
Rf 0.32 [MeOH : CH2Cl2 (1 : 13)]; UV ꢂmax (MeOH) nm (log " ): 340 (3.89), 307 sh (4.70),
2.61 sh (3.71), 241 (3.83), 209 (4.12); IR ꢃmax(CHCl3) cmꢀ1: 3444, 2926, 1624, 1582, 1513,
1436, 1377, 1281, 1206, 1033, 810; 1H-NMR 600 MHz (DMSO-d6) and 13C-NMR
150 MHz (DMSO-d6): see Tables 1 and 2; HR-ESI-MS [MꢀH]þ: (m/z) 313.0750 (Calcd for
C17H14O6 ꢀ Hþ: 313.0721).
7,30-Dihydroxy-8-methoxyflavone (4) was obtained as a white solid (10 mg, 2.5%). Rf
0.26 [MeOH : CH2Cl2 (1 : 13)]; UV ꢂmax (MeOH) nm (log "): 309 (2.70), 284 (2.62), 259
(2.78), 2.39 sh (2.75), 207 (3.04); IR ꢃmax(CHCl3) cmꢀ1: 3428, 2921, 1625, 1582, 1448, 1385,
1348, 1203, 1078, 1040, 1004, 959; 1H-NMR 600 MHz (DMSO-d6) and 13C-NMR
150 MHz (DMSO-d6): see Tables 1 and 2; HR-ESI-MS [M þ H]þ: (m/z) 285.0745 (Calcd
for C16H12O5 þ Hþ: 285.0764).
7,40-Dihydroxy-8-methoxyflavone (5) was a pale yellow solid (37 mg 9.25%) with a Rf
0.20 [MeOH : CH2Cl2 (1 : 13)]; UV ꢂmax (MeOH) nm (log " ): 329 (3.89), 260 (3.73), 2.15
(3.96), 202 (4.09); IR ꢃmax(CHCl3) cmꢀ1: 3352, 2952, 1613, 1572, 1510, 1447, 1383, 1293,
1245, 1208, 1079, 826; H-NMR 600 MHz (DMSO-d6) and 13C-NMR 150 MHz (DMSO-
1
d6): see Tables 1 and 2; HR-ESI-MS [M þ H]þ: (m/z) 285.0779 (Calcd for C16H12O5 þ Hþ:
285.0764).
8-Methoxy-7,30,40-trihydroxyflavone (6) was purified as
(7 mg, 1.75%). Rf 0.10[MeOH : CH2Cl2 (1 : 13)]; UV ꢂmax (MeOH) nm (log "): 330
a
faint yellow solid

Natural Product Research
1237
(4.40), 308 sh (4.31), 260 (4.23), 2.51 sh (4.18), 234 sh (4.27), 214 sh (4.49), 202 (4.66); IR
1
ꢃmax (CHCl3) cmꢀ1: 3121, 2918, 1630, 1507, 1443, 1381, 1290, 1242, 1174, 1076, 824; H-
NMR 600 MHz (DMSO-d6) and 13C-NMR 150 MHz (DMSO-d6): see Tables 1 and 2; HR-
ESI-MS [M þ H]þ: (m/z) 301.0850 (Calcd for C16H12O6 þ Hþ: 301.0713).
4.5. Microbial transformation of 7,8-dimethoxyflavone (1) by A. flavus (ATCC 9170)
The combined culture medium was extracted with EtOAc and the solvent was evaporated.
The light brown gummy solid obtained was column chromatographed over silica gel with
CHCl3 gradually enriched with MeOH as the eluent.
7-Hydroxy-8-methoxyflavone (7) was obtained as a white solid (90 mg, 36%). Rf 0.65
[MeOH : CH2Cl2 (1 : 13)]; UV ꢂmax (MeOH) nm (log "): 308 (3.34), 259 (3.50), 2.12 (3.62),
201 (3.65); IR ꢃmax(CHCl3) cmꢀ1: 3453, 3087, 1635, 1618, 1589, 1568, 1451, 1388, 1363,
1
1247, 1212, 1073, 1039, 940, 768, 680; H-NMR 600 MHz (DMSO-d6) and 13C-NMR
150 MHz (DMSO-d6): see Tables 1 and 2; HR-ESI-MS [M-H]þ: (m/z) 267.0693 (Calcd for
C16H12O4 ꢀ Hþ: 267.0657).
4.6. Microbial transformation of 5-methoxyflavone (8) by B. bassiana (ATCC 7159)
The EtOAc extract of the combined culture filtrates yielded 9 when column
chromatographed over Si gel with CH2Cl2 enriched with MeOH.
The white solid, 5-metoxyflavanone (9) (65.4 mg, 7.47% yield), showed a Rf 0.85
27
[MeOH : CH2Cl2 (1 : 16)]; ½ꢁꢁD 0ꢂ (c ¼ 0.51, MeOH); UV ꢂmax (MeOH) nm (log "): 330
(3.87), 269 (4.12), 213 sh (4.49), 202 (4.60); IR ꢃmax(CHCl3) cmꢀ1: 2976, 1677, 1601, 1573,
1472, 1103,1090, 786, 734; HR-ESI-MS [M þ H]þ: (m/z) HR-ESI-MS [M þ H]þ: (m/z)
Table 2. 13C-NMR data for 2–7 and 10.
2
3
4
5
6
7
10
C-2
3
4
5
6
163.4
104.8
177.2
120.8
111.2
157.0
137.0
150.4
116.6
120.8
128.8
116.8
161.0
116.8
128.8
–
162.4
105.0
176.9
120.4
115.4
148.5
135.5
150.9
116.8
122.7
110.3
148.5
150.9
116.4
128.2
–
162.2
106.7
177.0
120.5
116.1
151.1
135.6
151.1
116.1
133.2
113.0
158.4
119.1
130.8
117.4
–
162.7
104.6
176.8
120.4
115.4
155.7
135.5
150.9
117.1
122.3
128.4
116.5
161.3
116.5
128.4
–
162.7
103.8
176.7
120.2
116.8
160.0
135.8
151.1
115.0
121.0
113.0
147.0
150.0
116.6
118.7
–
162.4
107.1
177.2
120.8
115.8
155.9
135.7
151.3
117.6
132.1
126.7
129.8
132.2
129.8
126.7
–
161.0
106.8
176.8
159.5
110.4
134.5
107.6
158.0
114.1
121.8
128.5
116.4
161.1
116.4
128.5
56.5
7
8
9
10
10
20
30
40
50
60
5-OMe
7-OMe
8-OMe
61.8
57.1
61.3
56.3
–
61.4
–
61.4
–
60.9
–
61.7
–
–
Note: Measured in DMSO-d6 at 150 MHz.

1238
W. Herath et al.
255.1109 (Calcd for C16H14O3 þ H: 255.10219). Identification of 9 was by comparison with
published data (Lee et al., 2007).
4.7. Microbial transformation of 5-methoxyflavone (8) by A. alliaceus (ATCC 10060)
40-Hydroxy-5-methoxyflavone (10) formed was purified as a white solid by column
chromatography (89.9 mg, 71.92% yield). Rf 0.31 [MeOH : CH2Cl2 (1 : 16)]; UV ꢂmax
(MeOH) nm (log "): 329 (4.42), 265(4.25), 219 (4.38), 202 (4.45); IR ꢃmax(CHCl3) cmꢀ1
:
3428, 2988, 1594, 1580, 1475, 1395,1285, 1131, 834; HR-ESI-MS [M þ H]þ: (m/z) 269.0811
(Calcd for C16H12O4 þ H: 269.08146).
Acknowledgements
The authors thank Mr. Frank Wiggers for assistance in obtaining 2D-NMR spectra and Dr Bharathi
Avula for conducting HRESIMS analysis. This work was supported, in part, by the United States
Department of Agriculture, Agricultural Research Specific Cooperative Agreement No. 58-6408-2-
00009.
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