Microbial metabolism part 9.1 Structure and antioxidant significance of the metabolites of 5,7-dihydroxyflavone (chrysin), and 5- and 6-hydroxyflavones

Article abstract of DOI:10.1248/cpb.56.418

5,7-Dihydroxyflavone (chrysin) (1) when fermented with fungal cultures, Aspergillus alliaceous (ATCC 10060), Beauveria bassiana (ATCC 13144) and Absidia glauco (ATCC 22752) gave mainly 4′-hydroxychrysin (4), chrysin 7-O-β-D-4-O-methylglucopyranoside (5) and chrysin 7-sulfate (6), respectively. Mucore ramannianus (ATCC 9628), however, transformed chrysin into six metabolites: 4′-hydroxy-3′-methoxychrysin (chrysoeriol) (7), 4′-hydroxychrysin (apigenin) (4) 3′,4′-dihydroxychrysin (luteolin) (8), 3′-methoxychrysin 4′-O-α-D-6- deoxyallopyranoside (9), chrysin 4′-O-α-D-6-deoxyallopyranoside (10), and luteolin 3′-sulfate (11). Cultures of A. alliaceous (ATCC 10060) and B. bassiana (ATCC 13144) metabolized 5-hydroxyflavone (2) into 5,4′-dihydroxyflavone (12) and 4′-hydroxyflavone 5-O-β-D-4-O-methylglucopyranoside (13), respectively. 6-Hydroxyflavone (3) was transformed into 6-hydroxyflavanone (14), flavone 3-O-β-D-4-O- methylglucopyranoside (15) and (±)-flavanone 6-O-β-D-4-O- methylglucopyranoside (16) by cultures of Beauveria bassiana (ATCC 13144). The structures of the metabolic products were elucidated by means of spectroscopic data. The significance of the metabolites as antioxidants in relation to their structure is briefly discussed.

Full text of DOI:10.1248/cpb.56.418

418
Chem. Pharm. Bull. 56(4) 418—422 (2008)
Vol. 56, No. 4
Microbial Metabolism Part 9.1) Structure and Antioxidant Significance
of the Metabolites of 5,7-Dihydroxyflavone (Chrysin), and 5- and
6-Hydroxyflavones
Wimal HERATH,a Julie Rakel MIKELL,a Amber Lynn HALE,a Daneel FERREIRA,a,b and
,a,b
*
Ikhlas Ahmad KHAN
a National Center for Natural Products Research, The University of Mississippi; and b Department of Pharmacognosy,
Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi; University, MS 38677,
U.S.A. Received August 16, 2007; accepted January 18, 2008; published online January 25, 2008
5,7-Dihydroxyflavone (chrysin) (1) when fermented with fungal cultures, Aspergillus alliaceous (ATCC
10060), Beauveria bassiana (ATCC 13144) and Absidia glauco (ATCC 22752) gave mainly 4ꢀ-hydroxychrysin (4),
chrysin 7-O-b-D-4-O-methylglucopyranoside (5) and chrysin 7-sulfate (6), respectively. Mucore ramannianus
(ATCC 9628), however, transformed chrysin into six metabolites: 4ꢀ-hydroxy-3ꢀ-methoxychrysin (chrysoeriol)
(7), 4ꢀ-hydroxychrysin (apigenin) (4) 3ꢀ,4ꢀ-dihydroxychrysin (luteolin) (8), 3ꢀ-methoxychrysin 4ꢀ-O-a-D-6-de-
oxyallopyranoside (9), chrysin 4ꢀ-O-a-D-6-deoxyallopyranoside (10), and luteolin 3ꢀ-sulfate (11). Cultures of A.
alliaceous (ATCC 10060) and B. bassiana (ATCC 13144) metabolized 5-hydroxyflavone (2) into 5,4ꢀ-dihydroxy-
flavone (12) and 4ꢀ-hydroxyflavone 5-O-b-D-4-O-methylglucopyranoside (13), respectively. 6-Hydroxyflavone (3)
was transformed into 6-hydroxyflavanone (14), flavone 3-O-b-D-4-O-methylglucopyranoside (15) and (ꢁ)-fla-
vanone 6-O-b-D-4-O-methylglucopyranoside (16) by cultures of Beauveria bassiana (ATCC 13144). The struc-
tures of the metabolic products were elucidated by means of spectroscopic data. The significance of the metabo-
lites as antioxidants in relation to their structure is briefly discussed.
Key words flavonoid; microbial metabolism; Beauveria bassiana; Aspergillus alliaceus; Absidia glauco; Mucore ramannianus
Flavonoids are an important class of polyphenolic natural isms which effected transformation, scale up studies was car-
products that are biosynthesized from phenylalanine.2) They ried out with selected cultures to isolate the maximum num-
are widely distributed in the plant kingdom but confined ber of metabolites in reasonable yields. Structure elucidation
mainly to higher plants. Mosses and liverworts are some of of the metabolites of the three flavonoids, 1—3 and their pos-
the lower plant groups that contain flavonoids.3) Rare detec- sible impact on the antioxidant activity in relation to struc-
tions of these compounds have been made in fungi.4) Ani- ture are discussed.
mals are not able to biosynthesize flavonoids.5) Besides im-
parting vivid colors to various parts of plants, flavonoids play Results and Discussion
many roles in the development and growth of plants.6) Sev-
Forty fungal strains were used in the initial screening of
eral biological activities including anticarcinogenic,7—9) anti- the flavonoids 1—3. The two-stage standard procedure was
inflammatory7,10,11) and antioxidant12,13) have been attributed used in all the screening experiments.18) Selection of fungal
to this class of compounds. Flavonoids are widely consumed cultures for scale up studies was made in such a manner as to
in the form of herbal preparations or dietary supplements by obtain all the transformed products with maximum yield.
humans. Although most of the flavonoids are considered to Chrysin (1) was converted to eight derivatives whilst 5-hy-
be safe, their efficacy is yet to be determined by means of droxyflavone (2) and 6-hydroxyflavone (3) were transformed
clinical trials.14) Moreover, potential toxicities of flavonoids into two and three metabolites respectively (Table 1).
as well as their drug interactions have not been well stud-
Metabolites, 4, 6—8, 11, 12, 14 and 15 being known com-
ied.14) With the publicity given to the beneficial effects, the pounds were identified by comparison with published data
consumption of dietary supplements containing flavonoids as, 4ꢀ-hydroxychrysin (apigenin),19,20) chrysin 7-sulfate,21) 4ꢀ-
has increased significantly. It has been pointed out that the hydroxy-3ꢀ-methoxychrysin (chrysoeriol),19,20) 3ꢀ,4ꢀ-dihy-
excess use of these compounds could have drastic effects, as droxychrysin (luteolin),19,20) luteolin 3ꢀ-sulfate, 5,4ꢀ-dihy-
high concentrations of flavonoids may act as mutagens, pro- droxyflavone,19,20) (ꢁ)-6-hydroxyflavanone,22) and flavone 3-
oxidants and inhibitors of hormone metabolizing enzymes.15) O-b-D-4-O-methylglucopyranoside.23) respectively. (ꢁ)-6-hy-
Microorganisms may be utilized as models of drug metab- droxyflavanone (14) indicated a non-stereoselective process
olism to predict the fate of xenobiotics in mammalian sys- of hydroxylation of the olefinic bond in the starting material
tems.16,17) Since this method often gives sufficient quantities (3). However, due to the non availability of spectral informa-
of metabolites, complete chemical structure and pharmaco- tion, the structure of compound 11 was elucidated by a de-
logical activities could be determined. Also, they can be tailed study of its high resolution spectroscopic data.
used as analytical standards to detect and characterize
Metabolite 5 (221.0 mg, 44.2% yield), a white solid, was
particular flavonoid metabolites which are present in very shown to have a molecular formula C22H22O9 (HR-ESI-MS).
small quantities in biological fluids.17) In our ongoing re- IR absorption bands at 3408, 2925, 1620 and 1665 cmꢂ1 sug-
search on microbial transformation of flavonoids, 5,7-dihy- gested the presence of –OH, –CH, –CꢃC and –CꢃO func-
droxyflavone (chrysin), and 5- and 6-hydroxyflavones were tional groups, respectively. The chemical shifts and the J
1
screened using 40 different microorganisms. Of the organ- values for aromatic protons observed in the H-NMR spec-
∗ To whom correspondence should be addressed. e-mail: ikhan@olemiss.edu
© 2008 Pharmaceutical Society of Japan

April 2008
419
Table 1. Microbial Transformation of Chrysin and 5- and 6-Hydroxyflavones
Antioxidant
activity IC50
Yield
(%)
Substrate
Microorganism
Metabolite
(mM)
Chrysin (1)
Aspergillus alliaceous (ATCC 10060) 4ꢀ-Hydroxychrysin (apigenin) (4)
4.1
44.2
5.74
1.4
3.0
10.2
3.2
18.7
10.2
0.46
N/A
N/A
N/A
N/A
N/A
11.0
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Beauveria bassiana (ATCC 13144)
Absidia glauco (ATCC 22752)
Chrysin 7-O-b-D-4-O-methylglucopyranoside (5)
Chrysin 7-sulfate (6)
Mucore ramannianus (ATCC 9628)
4ꢀ-Hydroxy-3ꢀ-methoxychrysin (chrysoeriol) (7),
4ꢀ-Hydroxychrysin (apigenin) (4),
3ꢀ,4ꢀ-Dihydroxychrysin (luteolin) (8),
3ꢀ-Methoxychrysin 4ꢀ-O-a-D-6-deoxyallopyranoside (9),
Chrysin 4ꢀ-O-a-D-6-deoxyallopyranoside (10),
Luteolin 3ꢀ-sulfate (11)
5-Hydroxyflavone (2)
6-Hydroxyflavone (3)
Aspergillus alliaceous (ATCC 10060) 5,4ꢀ-Dihydroxyflavone (12)
Beauveria bassiana (ATCC 13144)
Beauveria bassiana (ATCC 13144)
4ꢀ-Hydroxyflavone 5-O-b-D-4-O-methylglucopyranoside (13) 98
(ꢁ)-6-Hydroxyflavanone (14),
8.3
1.6
0.8
Flavone 3-O-b-D-4-O-methylglucopyranoside (15),
(ꢁ)-Flavanone 6-O-b-D-4-O-methylglucopyranoside (16)
N/A: No noticeable activity.
bond correlations to H-6 (6.47) and H-8 (6.87) suggesting O-
glucosylation at C-7. A trans-diaxial relationship was in-
ferred by the large coupling constant (8.4 Hz) between the
anomeric proton and H-2ꢄ establishing a b-glycosidic link-
age. trans-Diaxial relationships were suggested between H-
2ꢄ/3ꢄ, H-3ꢄ/4ꢄ, H-4ꢄ/5ꢄ protons, as large coupling constants,
(J2ꢄ/3ꢄ, J3ꢄ/4ꢄ, J4ꢄ/5ꢄ) were observed between the respective pairs
of protons. The downfield shift of C-4ꢄ (d 78.8) in the 13C-
NMR spectrum together with HMBC correlations showed
the presence of an O-methyl group at C-4ꢄ. Compound 5 was
thus identified as chrysin 7-O-b-D-4-O-methylglucopyra-
noside.
Compound 10 (93.6 mg, 18.7% yield), was assigned the
1
molecular formula, C22H22O9, (HR-ESI-MS). The H-NMR
data of 10 were similar to those of 1, except for the B-ring
protons which showed para-substitution [d 8.00 (2H, d,
Jꢃ8.0 Hz), 7.18 (2H, d, Jꢃ8.0 Hz)]. The additional five car-
bon resonances (d 60—80) observed in the aliphatic region
of the 13C-NMR spectrum indicated the presences of a glyco-
syl moiety. Correlations observed in the HMBC spectrum
permitted the assignment of the glycosyl group at C-4ꢀ. Iden-
tification of the sugar moiety was by the analysis of 1H-, 13C-
NMR and their correlation spectra. The anomeric proton at d
5.55 was coupled (HMQC) to the methine carbon at d 98.4
and to the proton at d 3.78 (H-2ꢄ) with a small coupling con-
stant (Jꢅ1 Hz). This suggested that the anomeric proton was
equatorially disposed. H-2ꢄ was coupled to H-3ꢄ with a small
coupling constant which in turn was similarly coupled to H-
4ꢄ. Therefore, H-2ꢄ and H-3ꢄ were required to be equatorial.
The large coupling constant (7.2 Hz) between H-4ꢄ and H-5ꢄ
suggested that they were trans-diaxial oriented. H-5ꢄ was
coupled to the methyl doublet implying that the sugar was a
6-deoxy sugar. On acid hydrolysis, 10 afforded deoxyallose
trum, as well as the 13C-NMR data were similar to those whose structure was supported by the 1H-NMR coupling pat-
of chrysin. These indicated that the chrysin architecture tern and optical rotation24,25) and hence, the sugar was identi-
remained largely unchanged during transformation. The 13C- fied as D-6-deoxyallose. The chemical shift of the anomeric
NMR spectrum additionally showed five carbon resonances carbon (d 98.5) and the coupling constant of the anomeric
(d 60—80) in the aliphatic region indicative of the presence proton suggested the presence of an a-O-glycosidic bond.
of a glycosyl moiety. The anomeric proton at d 5.11 showed The spectroscopic data of the aglycon formed were similar in
a one bond correlation (HMQC) with the methine carbon at d all respects to those of 4ꢀ-hydroxychrysin (4). The structure
99.5 and three bond correlations (HMBC) with the quater- of compound 10 was determined as chrysin 4ꢀ-O-a-D-6-de-
nary carbon at d 163.1 (C-7), which in turn exhibited two- oxyallopyranoside.

420
Vol. 56, No. 4
Metabolite 9 (16.0 mg, 3.2% yield) was obtained as a metabolites of flavonoids reported, are paralleled in mam-
white solid with a molecular formula, C22H22O10 as deter- mals27—29) indicating the use of microbes to mimic mam-
mined by HR-ESI-MS. Comparison of this compound with malian metabolism. Conjugation is the most common final
metabolite 10 revealed significant similarity except for the step in mammalian metabolism of intact flavonoids.30)
presence of an O-methyl group, the position of which was
Flavonoids are associated with many biological properties,
determined as C-3ꢀ by correlation NMR spectra. Compound among which the antioxidant activity is well covered. Most
9 was therefore identified as 3ꢀ-methoxychrysin 4ꢀ-O-a-D-6- of the results have been based on in vitro experiments. The
deoxyallopyranoside.
antioxidant nature of these compounds has been defined by
Compound 13 (490 mg, 98% yield) the second metabolic the ability to donate electrons to free radicals, due to the
product of 2 had a molecular formula, C22H22O9 (HR-ESI- presence of a catechol group in the B-ring.31) This feature is
MS data). Very similar chemical shifts and J-values of aro- considered to be highly important in addition to the presence
matic protons together with similar 13C-NMR data, suggested of a double bond between C-2 and C-3 in conjugation with
that this compound and metabolite 12 had the same flavonoid the 4-oxo-group, and the ability to chelate ions like copper
framework. However, the presence of resonances between d and iron.31) The flavonoid, quercetin meets all the criteria to
3.05 and 5.05 in the 1H-NMR and five carbon resonances be- be a good antioxidant in vitro. It is reflected in experiments
tween d 100.2 and 60.9 in the 13C-NMR spectra revealed the such as the DPPH assay which indicated a 2.2 mM IC50
presence of a glycosyl moiety. The coupling constant of the value.32) Examination of the structures of the metabolites of
anomeric proton at d 5.05 (1H, d, Jꢃ7.8 Hz, H-1ꢄ) was con- compounds 1—3, which have been O-methylated, O-gluco-
sistent with a b-configuration of the glycosyl unit. The three sylated or sulfated, showed that their effectiveness as antioxi-
bond correlation of the anomeric proton with the quaternary dants, compared to the starting compounds (1—3), should
carbon at d 160.5 (C-5) together with its three-bond correla- decrease due to their reduced ability to donate electrons. Al-
tion to H-7 (d 7.64) suggested O-glucosylation at C-5. The though the metabolite 3ꢀ,4ꢀ-dihydroxychrysin (luteolin) (8)
large coupling constants observed between H-2ꢄ/3ꢄ, H-3ꢄ/4ꢄ, has the necessary structural features to show the ability to
H-4ꢄ/5ꢄ protons showed trans-diaxial relationships. HMBC donate electrons,32) the substantial formation of conjugated
correlations and the downfield shift of C-4ꢄ (d 79.6) in the products implied its rapid inactivation. This was supported
13C-NMR spectrum revealed the presence of an O-methyl by the DPPH assay conducted on the metabolites which
group at C-4ꢄ. Metabolite 13 was thus, characterized as 4ꢀ- showed significant antioxidant activity only by 3ꢀ,4ꢀ-dihy-
hydroxy-5-O-b-D-4-O-methylglucopyranoside.
droxychrysin (luteolin) (8).26) These results tend to support
Metabolite 16 (4 mg, 0.8% yield) of 3 with a molecular the view that although flavonoids exhibit powerful antioxi-
mass of 416 was assigned a molecular formula C22H24O8 dant properties in vitro, such an activity has yet to be clari-
(HR-ESI-MS data). Its spectroscopic data were in close fied in in vivo.33) Thus, as has been pointed out,11) it is essen-
agreement with those of metabolite 14 with the exception of tial to establish how flavonoids are absorbed in the intestine,
additional NMR resonances due to an O-methylated glucopy- their bioavalability, and the properties of the circulating
ranoside unit which was indicated by the excess of 176 mass metabolites to consider them as effective antioxidants. Fur-
units. 1H-NMR coupling constants together with NMR corre- ther, the formation of conjugates facilitates rapid elimination
lation data confirmed the structure of 16 as (ꢁ)-flavanone 6- of flavonoids, resulting in the observed lower concentrations
O-b-D-4-O-methylglucopyranoside, again indicating a non- of the parent compounds and their metabolites in the plasma
stereoselective hydrogenation process.
and in organs as compared to antioxidants like ascorbic acid
The DPPH assay conducted on the metabolites showed and a-tocopherol which are present in high concentrations.
that only 3ꢀ,4ꢀ-dihydroxychrysin (luteolin) (8) had significant Hence, in competition, the former is expected to play an in-
antioxidant activity as compared to the literature value.26) All significant role as far as antioxidant activity is concerned.29)
the other metabolites showed no noticeable activity.
However, in the light of new data it has been pointed out that
even though flavonoids and their in vivo metabolites may not
act as antioxidants, they are present in sufficient amounts in
Conclusion
In this study, Beauveria bassiana (ATCC 13144) trans- vivo to have pharmacological activity at receptors, transcrip-
formed 6-hydroxyflavone (3) to 6-hydroxyflavanone (14), tion factors and enzymes.29) The formation of conjugated
flavone 3-O-b-D-4-O-methylglucopyranoside (15), and (ꢁ)- products may be viewed as a safety mechanism which pre-
flavanone 6-O-b-D-4-O-methylglucopyranoside (16). The vents the accumulation of flavonoids to levels harmful to the
starting compound (3) was reduced during functionalization body15) by aiding in rapid elimination and retaining the
(Phase I) to yield compound 14 followed by conjugation in amounts necessary to have beneficial effects.
Phase II conversion to yield metabolite 16. It was also sus-
Experimental
ceptible to several conversion steps, namely, deoxygenation,
General Experimental Procedures IR spectra were measured in
CHCl3 on an ATI Mattson Genesis series FT-IR spectrophotometer. UV
oxygenation and conjugation to form metabolite 15. In con-
trast, 5-hydroxyflavanone (2) and chrysin (3) did not undergo
spectra were run on a Hewlett Packard 8452A diode array spectrometer. Op-
1
reduction but yielded the oxygenated products during the
functionalization phase. Similar results were obtained with
our previous microbial conversion of the flavonoids 3-hy-
droxyflavone and 7-hydroxyflavone.23) However, conjugation
seems to be the preferred reaction in most of these conver-
sions. Oxygenation and conjugation products obtained with
compounds, 1—3 together with many other similar microbial
tical rotations were measured with a Jasco DIP-370 digital polarimeter. H-
and 13C-NMR were obtained in CDCl3 and DMSO-d6 on a Varian Unity
Inova 600 spectrometer unless otherwise stated. HR-ESI-MS data were ac-
quired using a Bruker GioApex 3.0.
Substrates Chrysin (1), 5-hydroxyflavone (2) and 6-hydroxyflavone (3)
were purchased from Aldrich Co. (Milwaukee, Wisconsin) and their authen-
ticity was confirmed by NMR data.
Organisms and Metabolism Forty culture samples from the microbial

April 2008
421
collection of The National Center for Natural Products Research of The Uni-
(ATCC 10060) The combined (Me)2CHOH–EtOAc (2 : 9) extracts of the
versity of Mississippi were used in the initial screening stage of the experi- culture filtrates were flash chromatographed with hexane enriched with
ment to identify organisms capable of converting the flavonoids, 1—3 to
EtOAc, to give the metabolite 12.
their respective metabolites in good yield. Initial screening was carried out
5,4ꢀ-Dihydroxyflavone (12) was isolated as a light yellow solid (2.3 mg,
in 125 ml Erlenmeyer flasks containing 25 ml medium a.18) A two-stage pro- 0.46% yield). Rf 0.28 [EtOAc–hexane (3 : 7)]. It was identified by compari-
cedure was used in all experiments.18) Each compound was administered son with published data.19,20)
separately in dimethylformamide (0.5 mg/ml) solution to 24 h old stage II
cultures and incubated for 14 d on a rotary shaker (New Brunswick Model
G10-21) at 100 rpm. The progress of each reaction was monitored at 7-d
intervals using precoated Si gel 60 F254 TLC plates (E. Merck) with p-
Microbial Transformation of 5-Hydroxyflavone (2) by B. bassiana
(ATCC 13144) Recrystalization of the MeOH soluble portion of the com-
bined (Me)2CHOH–EtOAc (2 : 9) extracts yielded 13.
4ꢀ-Hydroxyflavone 5-O-b-D-4-O-methylglucopyranoside (13) was iso-
anisaldehyde as the spray reagent. Preparative scale fermentations were car- lated as a white solid (490 mg, 98% yield). Rf 0.34 [MeOH–CHCl3 (3 : 17)];
ried out in five 2 l flasks, each containing 100 mg of substrate in 500 ml [a]D23 ꢂ49.4° (cꢃ0.08, MeOH). Spectroscopic analysis confirmed the struc-
medium a. EtOAc or EtOAc/(Me)2CHOH–EtOAc (2 : 9) was used to extract ture of 11.
the combined culture filtrates. The residues obtained by the evaporation of
solvents were column chromatographed (Silica gel 60 F254) to isolate the
Microbial Transformation of 6-Hydroxyflavone (3) by B. bassiana
(ATCC 13144) The brown solid obtained from the EtOAc extract of the
metabolites. Purification of the compounds was by repeated column and combined culture filtrates was subjected to silica gel column chromatogra-
preparative thin layer (Silica gel 60 F254) chromatography, where appropri- phy (Si gel 230—400 mesh: E. Merck, 30 g, column diameter: 20 mm) with
ate. Culture and substrate controls were run along with the above experi- CH2Cl2 and increasing amounts of MeOH. The fractions obtained were
ments.18)
purified by repeated column and preparative layer chromatography (CH2Cl2–
Microbial Transformation of Chrysin (1) by A. alliaceous (ATCC MeOH, 3 : 2) to obtain three compounds, 14—16 which were characterized
10060) The combined (Me)2CHOH–EtOAc (2 : 9) extract of the culture fil- by spectroscopic data.
trates when flash chromatographed with CHCl3 enriched with MeOH, af-
6-Hydroxyflavanone (14) was a white solid (41.4 mg, 8.3% yield). Rf 0.52
forded metabolite 4. The structure was elucidated by spectroscopic data.
[MeOH–CH2Cl2 (1 : 19)]; [a]D27 0° (cꢃ0.2, MeOH). Identification of the
4ꢀ-Hydroxychrysin (4) was obtained as a pale yellow solid (20.6 mg, 4.1% metabolite was by comparison with published data.22)
yield). Rf 0.38 [MeOH–CHCl3 (1 : 9)]. It was identified by comparison with
Flavone 3-O-b-D-4-O-methylglucopyranoside (15) was separated as a
white solid (8 mg, 1.6% yield). Rf 0.18 [MeOH–CHCl3 (1 : 19)]. It was iden-
literature data.19,20)
Microbial Transformation of Chrysin (1) by B. bassiana (ATCC tified by comparing the spectral data with of the literature values.23)
13144) The methanol soluble fraction of the combined (Me)2CHOH–
EtOAc (2 : 9) extract of the culture filtrates gave compound 5 on recrystal-
lization. The structure was determined by spectroscopic data.
(ꢁ)-Flavanone 6-O-b-D-4-O-methylglucopyranoside (16) was purified as
a white solid (4 mg, 0.8% yield). Rf 0.18 [MeOH–CH2Cl2 (1 : 19)]; [a]D27 0°
(cꢃ0.2, MeOH). Identification was by detailed study of spectroscopic data.
The free radical scavenging activity of the metabolites was analyzed by
the DPPH assay as described by S. Burda et al.34) 1 ml of 10ꢂ4 M solution in
methanol of each metabolite was added to 2 ml of DPPH solution (10 mg/l in
5-Hydroxyflavone 7-O-b-D-4-O-methylglucopyranoside (5) was a pale
yellow solid (221.0 mg, 44.2% yield) with a Rf 0.34 [MeOH–CH2Cl2 (3 :
17)]; [a]D23 ꢂ13.9° (cꢃ0.11, MeOH).
Microbial Transformation of Chrysin (1) by A. glauco (ATCC 22750) MeOH), vortexed and kept for 10 min in the dark at room temperature. Ab-
Flash chromatographic separation of the combined (Me)2CHOH–EtOAc sorbance of each solution was measured at 517 nm and the scavenging per-
(2 : 9) extract of the culture media resulted in the isolation of the metabolite
centage of activity was calculated. Quercetin was used as a positive con-
6. CHCl3 enriched with MeOH was used as the eluent.
trol.26) Only 3ꢀ,4ꢀ-dihydroxychrysin (luteolin) (8) showed significant antioxi-
Chrysin 7-sulphate (6) was isolated as a pale yellow solid (28.7 mg, dant activity as compared to the literature value (IC50 (mM) 11.0).26) All other
5.74% yield). Rf 0.24 [MeOH–CHCl3 (3 : 17)]. The compound was identi-
metabolites showed no noticeable activity.
Supplementary Material Spectroscopic data of metabolites 4—16 are
fied by comparison with published data.21)
Microbial Transformation of Chrysin (1) by M. ramannianus (ATCC available as supplementary material.
9628) Compounds 4 (14.8 mg, 3.0% yield) and 7—9 were isolated by
Flash Chromatography of the combined (Me)2CHOH–EtOAc (2 : 9) extracts
Acknowledgements The authors thank Mr. Frank Wiggers for assis-
of the culture media using CHCl3 enriched with MeOH, followed by re- tance in obtaining 2D NMR spectra and Dr. Bharathi Avula for conducting
peated column chromatography. The structures were elucidated by spectro- HR-ESI-MS analysis. This work was supported, in part, by the United States
scopic data.
Department of Agriculture, Agricultural Research Specific Cooperative
4ꢀ-Hydroxy-3ꢀ-methoxychrysin (7) (6.9 mg, 1.4% yield), a white solid Agreement No. 58-6408-2-00009.
with a Rf 0.53 [MeOH–CHCl3 (1 : 9)]. The metabolite was identified by
comparison with published data.19,20)
References
4ꢀ-Hydroxychrysin (4), isolated as a pale yellow solid (14.8 mg, 3.0%
yield); Rf 0.38 [MeOH–CHCl3 (1 : 9)] was identified with the aid of litera-
ture data.19,20)
3ꢀ,4ꢀ-Dihydroxychrysin (8) (51.0 mg, 10.2% yield), a white solid with an
Rf 0.24 [MeOH–CHCl3 (1 : 9)] was identified by comparison with literature
data.19,20)
3ꢀ-Methoxychrysin 4ꢀ-O-a-D-6-deoxyallopyranoside (9) (16.0 mg, 3.2%
yield), was obtained as a white solid. Rf 0.14 [MeOH–CHCl3 (1 : 9)]; [a]D23
ꢂ45.5° (cꢃ0.08, MeOH). Spectroscopic analysis confirmed the structure of
9.
1) Herath W., Reddy N., Khan I. A., Chem. Pharm. Bull., 55, 1512—
1513 (2007).
2) Winkel-Shirley B., Curr. Opin. Plant. Biol., 5, 375—381 (2002).
3) Basile A., Sorbo S., López-Sáez J. A., Cobianchi R. C., Phytochem-
istry, 62, 1145—1151 (2003).
4) “Flavonoids: Chemistry, Biochemistry and Applications,” ed. by An-
dersen Ø. M., Markham K. R., CRC Press, Boca Raton, 2006.
5) 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.
Chrysin 4ꢀ-O-a-D-6-deoxyallopyranoside (10) (93.6 mg, 18.7% yield),
with a Rf 0.12 [MeOH–CHCl3 (1 : 9)] was obtained as a white solid. [a]D23
ꢂ76.3° (cꢃ0.12, MeOH). Identification was by means of spectroscopic
data.
Acid Hydrolysis of Chrysin 4ꢀ-O-a-D-6-Deoxyallopyranoside (10): A so-
lution of 10 (8 mg) in 1.2 ml 2 N HCl–MeOH was refluxed for 4 h. On cool-
6) Havesteen B. H., Pharmacol. Ther., 96, 67—202 (2002).
7) Middleton E., Kandaswami C., Theoharides T. C., Pharmacol. Rev.,
52, 673—751 (2000).
8) Block G. A., Nur. Rev., 50, 207—213 (1992).
9) Elangovan V., Sekar N., Govindasamy S., Cancer Lett., 50, 278—284
(1994).
ing an off-white precipitate was formed which was identified as 4ꢀ-hydroxy- 10) Middleton E., Jr., Adv. Exp. Med. Biol., 439, 175—182 (1998).
chrysin (4) by means of spectroscopic data. The supernatant was purified by 11) Gabor M., Prog. Clin. Biol. Res., 213, 471—480 (1986).
preparative layer chromatography (CH2Cl2–MeOH, 9 : 1) to give a white 12) Kessler M., Ubeaud G., Jung L., J. Pharm. Pharmacol., 55, 131—142
compound which was identified by spectroscopic data as D-6-deoxyallose
(2003).
sugar. Rf 0.37 [MeOH–CHCl3 (1 : 9)]; [a]D27 ꢂ3.9° (cꢃ0.25, MeOH).24,25)
13) Farks O., Jakus J., Héberger K., Molecules, 9, 1079—1088 (2004).
Luteolin 3ꢀ-sulfate (11) (51.0 mg, 10.2% yield), a white solid with an Rf 14) Galati G., O’Brien P. J., Free Radic. Biol. Med., 37, 287—303 (2004).
0.26 [MeOH–CHCl3 (1 : 3)] was identified by a detailed study of its spectro-
15) Skibola C. F., Smith M. T., Free Radic. Biol. Med., 29, 375—383
(2000).
scopic data.19,20)
Microbial Transformation of 5-Hydroxyflavone (2) by A. alliaceous
16) Rosazza P. N., Duffel M. W., “Alkaloids: Chemistry and Pharmacol-

422
Vol. 56, No. 4
ogy,” Vol. 27, Chap. 4, ed. by Brossi A., Academic Press, New York, 24) Olafasdottir E. S., Jaroszewski J. W., Seigler D. S., Phytochemistry, 30,
1986, pp. 391—392.
867—869 (1991).
17) Clark A. M., McChesney J. D., Hufford C. D., Med. Res. Rev., 5,
25) Hunger A., Reichstein T., Helv. Chim. Acta, 35, 1073—1103 (1952).
26) Seyoum A., Asres K., El-Fiky F. K., Phytochemistry, 67, 2058—2070
(2006).
231—253 (1985).
18) Abourashed E. A., Khan I. A., Chem. Pharm. Bull., 48, 1996—1998
(2000).
27) Griffiths L. A., Smith S. E., Biochem. J., 128, 901—911 (1972).
19) Markham K. R., Geiger H., “The Flavonoids: Advances in Research 28) Ibrahim A., Abdul-Hajj Y. J., J. Nat. Prod., 53, 644—656 (1990).
Since 1986,” Chap. 10, ed. by Harbone J. B., Chapman & Hall, Lon-
don, 1994, pp. 448—450.
20) Agrawal P. K., Thakur R. S., Bansal M. C., “Carbon-13 NMR of
29) Hackett A. M., Griffiths L. A., Xenobiotica, 15, 907—914 (1985).
30) Manach C., Scalbert A., Morand C., Remesy C., Jimenez L., Am. J.
Clin. Nutr., 79, 727—747 (2004).
Flavonoids,” Chap. 3, ed. by Agrawal P. K., Elsevier, Amsterdam, 31) Williams R. J., Spencer J. P. E., Rice-Evans C., Free. Radic. Biol.
1989.
Med., 36, 838—849 (2004).
21) Ibrahim A.-R. S., Chem. Pharm. Bull., 53, 671—673 (2005).
22) Kagawa H., Shigematsu A., Ohta S., Harigaya Y., Chem. Pharm. Bull.,
53, 547—554 (2005).
32) Furusawa M., Tanaka T., Ito T., Nishikawa A., Yamazaki N., Nakaya
K., Matsuura N., Tsuchiya H., Nagayama M., Iinuma M., J. Health
Sci., 51, 376—378 (2005).
23) Herath W., Mikell J. R., Hale A. L., Ferreira D., Khan I. A., Chem.
Pharm. Bull., 54, 320—324 (2006).
33) Rice-Evans C., Curr. Med. Chem., 8, 797—807 (2001).
34) Burda S., Oleszek W., J. Agric. Food Chem., 49, 2774—2779 (2001).