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Vol. 59, No. 6
(59.5 mg, 11.9% and 9.6 mg, 1.92% yield by B. bassiana (ATCC 7159) and
B. bassiana (ATCC 13144), respectively). Rf 0.17 [MeOH–CHCl3 (3 : 17)];
[a]D27 ꢄ14.4 (cꢂ0.06, MeOH).
Flavanone 4ꢀ-O-b-D-methylglucopyranoside (6), obtained as a yellowish
solid (43.7 mg, 8.74% yield by B. bassiana (ATCC 13144) showed a Rf
value of 0.61 [MeOH–CHCl3 (1 : 9)]; [a]D27 ꢄ17.6 (cꢂ0.06, MeOH).
4ꢀ-Hydroxyflavanone 6-O-b-D-4-methoxyglucopyranoside (7) was iso-
lated as a white solid (9.7 mg, 1.94% and 101.4 mg, 20.28% yield by B.
bassiana (ATCC 7159) and B. bassiana (ATCC 13144), respectively). Rf
0.31 [MeOH–CHCl3 (1 : 9)]; [a]D27 ꢄ16 (cꢂ0.06, MeOH).
Microbial transformation of 1 by C. echinulata (ATCC 9244) resulted in
the formation of six metabolites, 8—13. Compound 8 was also given by B.
bassiana (ATCC 7159).
6,4ꢀ-Dihydroxyflavanone (8), a yellowish solid (38.7 mg, 7.74% and
1.0 mg, 0.2% yield by C. echinulata (ATCC 9244) and B. bassiana (ATCC
7159), respectively) with a Rf value of 0.26 [MeOH–CHCl3 (1 : 19)]; [a]D27
0.0 (cꢂ0.43, MeOH) was identified by comparison with published data.15)
Flavanone-4ꢀ-O-b-D-glucopyranoside (9) was obtained as a white solid
(129.3 mg, 25.80% yield). Rf 0.29 [MeOH–CHCl3 (1 : 9)]; [a]D27 0.00
(cꢂ0.10, MeOH).
of flavonoids are somewhat rare. This method therefore,
could be of immense help in generating C-6 derivatives of
flavanoids in synthetic medicinal and organic chemistry. Fur-
ther, although the organisms showed similar patterns of
phase I conversions, certain specificities were observed in the
conjugation products. B. bassiana strains whilst introduced a
4-methoxyglucopyranosyl moiety, C. echinulata coupled the
molecule with a glucopyranosyl unit. M. ramannianus on the
other hand was able to convert some of the phase I metabo-
lites to the respective 6-deoxyallopyranosides. In addition,
only M. ramannianus was able to bring about reduction of
the carbonyl group. Investigators have characterized ring
cleavage products during the microbial transformation stud-
ies of flavanone.16) However, several attempts made during
the present investigation to detect and isolate cleavage prod-
ucts of 1 using available microbial cultures were unsuccess-
ful indicating perhaps the necessity of specific microbial
strains for conversion. Flavanones are associated with a vari-
ety of biological activities. However, none of the metabolites
subjected to in vitro investigations carried out with the avail-
able tests, namely, antibacterial, antiviral and antihelmenthis,
showed any activity.
3ꢀ-Hydroxyflavanone 4ꢀ-sulfate (10) was isolated as a brownish solid
(63.1 mg, 12.62% yield). Rf 0.31 [MeOH–CHCl3 (1 : 4)]; [a]D27 ꢄ3.9
(cꢂ0.25, MeOH).
3ꢀ,4ꢀ-Dihydroxyflavanone (11), purified as
a yellowish white solid
(8.3 mg, 1.66% yield). Rf 0.28 [MeOH–CHCl3 (1 : 19)]; [a]D27 ꢄ11.7 (cꢂ
0.54, MeOH) was identified with the aid of literature data.15)
4ꢀ-Hydroxyflavanone-3ꢀ-O-b-D-glucopyranoside (12). (56.0 mg, 11.2%
yield). Rf 0.27 [MeOH–CHCl3 (1 : 9)]; [a]D27 2.3 (cꢂ0.18, MeOH).
Microbial transformation of 1 by Mucor ramannianus (ATCC 9628)
yielded seven compounds, 13—19. Metabolites 13 and 14 were also pro-
duced by Ramichloridium anceps (ATCC 15672). They were isolated as
white solids.
Experimental
General Experimental Procedures UV spectra were measured on a
Hewlett Packard 8452A diode array spectrometer. An ATI Mattson Genesis
series FT-IR spectrophotometer was used to run IR spectra in CHCl3. 1H-
and 13C-NMR were acquired in CDCl3 and DMSO-d6 on a Varian Unity
Inova 600 spectrometer unless otherwise stated. HR-ESI-MS data were ob-
tained using a Bruker GioApex 3.0. Jasco DIP-370 digital polarimeter was
used to measure optical rotations.
Substrate 4ꢀ-Hydroxyflavanone (1) was purchased from Aldrich Co.
(Milwaukee, Wisconsin, U.S.A.) and its authenticity was confirmed by NMR
data.
Organisms and Metabolism Organisms capable of converting the
flavonoid, 1 to their metabolites were selected by screening forty culture
samples from the microbial collection of The National Center for Natural
Products Research of The University of Mississippi. Screening experiments
were carried out by the usual two-stage procedure in 125 ml Erlenmeyer
flasks containing 25 ml medium a.20) Each compound was added 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. Precoated Si gel 60 F254 TLC plates (E. Merck) and p-anisalde-
hyde spray reagent were used to monitor the progress of conversion. In
preparative scale fermentations 500 mg of each substrate in dimethylfor-
mamide were distributed equally among five 2 l flasks, each containing
500 ml medium a. The combined culture filtrates were extracted with
(Me)2CHCH2OH–EtOAc (2 : 9). The residues obtained by the evaporation of
solvents were flash chromatographed using silica gel with CHCl3 enriched
with MeOH as the mobile phase. The fractions thus obtained were separated
over sephadex LX-20 using MeOH as the eluent to isolate the metabolites.
Culture and substrate controls were run along with the above experiments.21)
Microbial transformation of 1 by B. bassiana (ATCC 7159) and B.
bassiana (ATCC 13144) yielded metabolites, 2—5 and 7. The latter in addi-
tion gave compound 6.
2,4-trans-4ꢀ-Hydroxyflavan-4-ol (13): (63.4 mg, 12.7% yield by R.
anceps). Rf 0.27 [MeOH–CHCl3 (1 : 19)]; [a]D27 18.4 (cꢂ0.31, MeOH).
2,4-cis-4ꢀ-Hydroxyflavan-4-ol (14): (56.51 mg, 11.3% yield by R. an-
ceps). Rf 0.27 [MeOH–CHCl3 (1 : 19)]; [a]D27 1.1 (cꢂ0.28, MeOH).
2,4-trans-3ꢀ,4ꢀ-Dihydroxyflavan-4-ol (15): (37.5 mg, 7.5% yield). Rf 0.40
[MeOH–CHCl3 (1 : 9)]; [a]D27 ꢄ11.4 (cꢂ0.31, MeOH).
2,4-cis-3ꢀ,4ꢀ-Dihydroxyflavan-4-ol (16): (47.0 mg, 9.4% yield). Rf 0.40
[MeOH–CHCl3 (1 : 9)]; [a]D27 6.5 (cꢂ0.31, MeOH).
2,4-cis-3ꢀ-hydroxy-4ꢀ-Methoxyflavan-4-ol (17): (2.8 mg, 0.56% yield). Rf
0.60 [MeOH–CHCl3 (1 : 19)]; [a]D27 ꢄ1.5 (cꢂ0.33, MeOH).
Flavanone 4ꢀ-O-a-D-6-deoxyallopyranoside (18): (10 mg, 2% yield). Rf
0.36 [MeOH–CHCl3 (1 : 9)]; [a]D27 ꢄ24.0 (cꢂ0.06, MeOH).
2,4-cis-4-Hydroxyflavanone 4ꢀ-O-a-D-6-deoxyallopyranoside (19):
(30.1 mg, 6.02% yield). Rf 0.24 [MeOH–CHCl3 (1 : 9)]; [a]D27 ꢄ87.3
(cꢂ0.22, MeOH).
Structures of the metabolic products were elucidated by means of spectro-
scopic data.
Biological Activity Evaluation of biological activities of selected
metabolites was carried out at NCNPR in the School of Pharmacy of The
University of Mississippi.22) Metabolites, 3, 8—12 and 15—19 showed no
antibacterial activity against Staphylococcus aureus, methicillin-rsistant S.
aureus (MRSA), Escherichia coli, Pseudomonas aeroginosa and Micobac-
terium intracellulare. The positive control used was Ciprofloxacin. The same
metabolites when tested against the fungal strains, Candida albicans, C.
glabra, C. kusei, C. neoformans and Aspergillus fumigatus also gave nega-
tive results. Amphotericin B was used as the positive control. Similar results
were obtained when compounds 1, 4—7 and 14 were screened against C. al-
bicans, A. fumigatus, C. neoformans, E. coli, P. aeroginosa, MRSA and M.
intracellulare. No antileishmanial activity was observed with compounds 3,
8—12 and 15—19 when tested against Leishmania donovani. The standard
drug, pentamidine was the positive control used. In vitro antimalarial activ-
ity was conducted using chloroquine-sensitive and chloroquine-resistant
strains of Plasmodium falciparum. The compounds used were 1—19.
Supplementary Material Spectroscopic data of metabolites 2—19 are
available as supplementary material.
6,3ꢀ,4ꢀ-Trihydroxyflavanone (2) was obtained as
a yellowish solid
(143.1 mg, 28.6% and 14.9 mg, 2.98% yield by B. bassiana (ATCC 7159)
and B. bassiana (ATCC 13144), respectively). Rf 0.64 [MeOH–CHCl3
(1 : 9)]; [a]D27 0.0 (cꢂ0.06, MeOH). It was identified by means of spectro-
scopic data.
3ꢀ,4ꢀ-Dihydroxyflavanone 6-O-b-D-4-methoxyglucopyranoside (3) was a
white solid (38.4 mg, 7.08% and 9.4 mg, 1.88% yield by B. bassiana (ATCC
7159) and B. bassiana (ATCC 13144), respectively) with a Rf 0.21 [MeOH–
CHCl3 (1 : 9)]; [a]D27 ꢄ9.5 (cꢂ0.32, MeOH).
Acknowledgements The authors thank Mr. Frank Wiggers for assis-
tance in obtaining 2D-NMR spectra and Dr. 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.
4ꢀ-Hydroxyflavanone 3ꢀ-sulfate (4) was isolated as a brownish solid
(40.5 mg, 8.1% and 87.8 mg, 17.56% yield by B. bassiana (ATCC 7159) and
B. bassiana (ATCC 13144), respectively). Rf 0.24 [MeOH–CHCl3 (3 : 17)];
[a]D27 3.5 (cꢂ0.63, MeOH).
6,4ꢀ-Dihydroxyflavanone 3ꢀ-sulfate (5) was purified as a brownish solid