Microbial metabolism. Part 12. Isolation, characterization and bioactivity evaluation of eighteen microbial metabolites of 4′-hydroxyflavanone

Article abstract of DOI:10.1248/cpb.59.692

Fermentation of 4′-hydroxyflavanone (1) with fungal cultures, Beauveria bassiana (ATCC 13144 and ATCC 7159) yielded 6,3′,4′- trihydroxyflavanone (2), 3′,4′-dihydroxyflavanone 6-O-β -D-4-methoxyglucopyranoside (3), 4′-hydroxyflavanone 3′-sulfate (4), 6,4′-dihydroxyflavanone 3′-sulfate (5) and 4′- hydroxyflavanone 6-O-β-D-4-methoxyglucopyranoside (7). B. bassiana (ATCC 13144) and B. bassiana (ATCC 7159) in addition, gave one more metabolite each, namely, flavanone 4′-O-β -D-4-methoxyglucopyranoside (6) and 6,4′-dihydroxyflavanone (8) respectively. Cunninghamella echinulata (ATCC 9244) transformed 1 to 6,4′-dihydroxyflavanone (8), flavanone-4′-O- β -D-glucopyranoside (9), 3′-hydroxyflavanone 4′-sulfate (10), 3′,4′-dihydroxyflavanone (11) and 4′-hydroxyflavanone- 3′-O-β-D-glucopyranoside (12). Mucor ramannianus (ATCC 9628) metabolized 1 to 2,4-trans-4′-hydroxyflavan-4-ol (13), 2,4-cis-4′-hydroxyflavan-4-ol (14), 2,4-trans-3′,4′- dihydroxyflavan-4-ol (15), 2,4-cis-3′,4′-dihydroxyflavan-4-ol (16), 2,4-trans-3′-hydroxy-4′-methoxyflavan-4-ol (17), flavanone 4′-O-α-D-6-deoxyallopyranoside (18) and 2,4-cis-4-hydroxyflavanone 4′-O-α-D-6-deoxyallopyranoside (19). Metabolites 13 and 14 were also produced by Ramichloridium anceps (ATCC 15672). The former was also produced by C. echinulata. Structures of the metabolic products were elucidated by means of spectroscopic data. None of the metabolites tested showed antibacterial, antifungal and antiprotozoal activities against selected organisms.

Full text of DOI:10.1248/cpb.59.692

692
Regular Article
Chem. Pharm. Bull. 59(6) 692—697 (2011)
Vol. 59, No. 6
Microbial Metabolism. Part 12.1) Isolation, Characterization and
Bioactivity Evaluation of Eighteen Microbial Metabolites of
ꢀ
4 -Hydroxyflavanone
a
a
,a,b
*
Julie Rakel MIKELL, Wimal HERATH, and 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 November 13, 2010; accepted March 15, 2011; published online March 24, 2011
Fermentation of 4ꢀ-hydroxyflavanone (1) with fungal cultures, Beauveria bassiana (ATCC 13144 and ATCC
7159) yielded 6,3ꢀ,4ꢀ-trihydroxyflavanone (2), 3ꢀ,4ꢀ-dihydroxyflavanone 6-O-b-D-4-methoxyglucopyranoside (3),
4ꢀ-hydroxyflavanone 3ꢀ-sulfate (4), 6,4ꢀ-dihydroxyflavanone 3ꢀ-sulfate (5) and 4ꢀ-hydroxyflavanone 6-O-b-D-4-
methoxyglucopyranoside (7). B. bassiana (ATCC 13144) and B. bassiana (ATCC 7159) in addition, gave one more
metabolite each, namely, flavanone 4ꢀ-O-b-D-4-methoxyglucopyranoside (6) and 6,4ꢀ-dihydroxyflavanone (8) re-
spectively. Cunninghamella echinulata (ATCC 9244) transformed 1 to 6,4ꢀ-dihydroxyflavanone (8), flavanone-4ꢀ-
O-b-D-glucopyranoside (9), 3ꢀ-hydroxyflavanone 4ꢀ-sulfate (10), 3ꢀ,4ꢀ-dihydroxyflavanone (11) and 4ꢀ-hydroxyfla-
vanone-3ꢀ-O-b-D-glucopyranoside (12). Mucor ramannianus (ATCC 9628) metabolized 1 to 2,4-trans-4ꢀ-hydroxy-
flavan-4-ol (13), 2,4-cis-4ꢀ-hydroxyflavan-4-ol (14), 2,4-trans-3ꢀ,4ꢀ-dihydroxyflavan-4-ol (15), 2,4-cis-3ꢀ,4ꢀ-dihy-
droxyflavan-4-ol (16), 2,4-trans-3ꢀ-hydroxy-4ꢀ-methoxyflavan-4-ol (17), flavanone 4ꢀ-O-a-D-6-deoxyallopyranoside
(18) and 2,4-cis-4-hydroxyflavanone 4ꢀ-O-a-D-6-deoxyallopyranoside (19). Metabolites 13 and 14 were also pro-
duced by Ramichloridium anceps (ATCC 15672). The former was also produced by C. echinulata. Structures of
the metabolic products were elucidated by means of spectroscopic data. None of the metabolites tested showed
antibacterial, antifungal and antiprotozoal activities against selected organisms.
Key words flavonoid; microbial metabolism; Beauveria bassiana; Cunninghamella echinulata; Mucor ramannianus; Ramichlo-
ridium anceps
Flavonoids which constitute an important part of human (catechol) products. It was suggested that the semiquinones
diet are associated with many beneficial biological activi- or quinones that could result from the oxidation of catechol
ties,2) though some may exhibit mutagenic and/or prooxidant may be a source of mutagenic free radicals. However, it has
properties.3) They are known to interact with the cytochrome been demonstrated that partial detoxification takes place by
P450 drug-metabolizing enzymes found in liver and other O-methylation of the catechol moiety.5,6)
tissues including skin. The capability of flavonoids to induce
Flavanones, as compared to other flavonoids are less abun-
biosynthesis of Cytochromes P450 (CYPs) and to modulate dant in nature. Propolis, honey, citrus fruits and juices are
their activity could lead to loss of biological activity of a their main dietary sources.7) Flavanones are associated with
drug or to increase its concentration in the plasma resulting many biological properties. The major constituent of citrus
in an overdose.3) They may also activate or inhibit CYPs in- fruits, Naringenin for example was shown by in vitro experi-
volved in the process of chemical carcinogenesis. It has been ments to have antioxidant, antiproliferative and estrogenic
observed that Flavonoids with hydroxyl groups tend to in- activities.7) Since citrus fruits and juices are consumed by
hibit these enzymes, whereas those without seem to stimulate man in considerable amounts, investigations have been car-
them.4) The CYPs in turn can bring about transformation of ried out to evaluate their metabolic parthways. In a transfor-
flavonoids to metabolites which could interact with them and mation study with six flavanones by rat liver microsomes the
modify their activities. This underlines the importance of de- major metabolic pathway observed was aromatic hydroxyla-
termining the metabolic pathways of flavonoids which are tion. However, several previously unreported metabolic path-
being consumed in measurable amounts by man. There are ways including the reduction of the keto group were also dis-
two major sites of flavonoid metabolism.3) They are the liver covered.7) As many as 43 urinary metabolites of flavanone in
and the colon microflora. Several in vivo and in vitro experi- the rat were detected by a gas chromatographic mass spectro-
ments were conducted to identify the mammalian metabolites metric technique and the most prominent compounds among
of different classes of flavonoids. Based on these observa- them were identified.8) The main transformations observed
tions various suggestions were made with regards to their were hydroxylation at the 3- or 6-position and the reduction
metabolic pathways. The results of one such experiment in- of the keto group. However, only a little hydroxylation oc-
volving the metabolism of 16 naturally occurring flavonoids curred in B-ring.8)
by rat liver microsomes suggested that the B-ring was the
4ꢀ-Hydroxyflavanone, a synthetic analogue of flavanones
main structural moiety that was subjected to biotransforma- is associated with biological activities including, endo-
tion.5) The number and the position of the hydroxyl and thelium-independent full vasorelaxing efficacy on rat
methoxy groups in the B-ring seemed to determine the extent aortic rings,9) aromatase inhibitor properties,10) decreasing
of metabolism. The non polar flavonoids were metabolized collegen concentration in human dermal fibroblasts11) and
more extensively than the polar ones. The absence of hy- moderate binding ability to the rat uterine estrogen recep-
droxyl groups in the B-ring or the presence of one such tor.12) Its metabolic transformation by rat liver microsomes
group at C-4ꢀ position resulted in 3ꢀ and 4ꢀ dihydroxylated resulted in the formation of 3ꢀ,4ꢀ-dihydroxyflavanone, 4ꢀ-
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: ikhan@olemiss.edu

June 2011
693
hydroxyflavone, 6,4ꢀ-dihydroxyflavanone (tentative) and a
The known compounds, 8,15) 1116) and 1316) were identified
chromone.7) The formation of 3ꢀ,4ꢀ-dihydroxyflavanone as as 6,4ꢀ-dihydroxyflavanone, 3ꢀ,4ꢀ-dihydroxyflavanone and
the major metabolite was consistent with the observation that 2,4-trans-4ꢀ-hydroxyflavan-4-ol (13) respectively, by com-
the flavonoids with no hydroxyl groups in the B-ring or had parison with published data. Compound 11 was detected pre-
one at 4ꢀ position get converted to the corresponding cate- viously as a rat liver microsomal incubation product of 4ꢀ-hy-
chol by microsomal enzymes.5) Microorganisms are good droxyflavanone and 3ꢀ-dihydroxyflavanone7) as well as a mi-
predictors of mammalian drug metabolites with the ability to crobial metabolite of flavanone.16)
yield sufficient quantities of metabolites for structure analy-
Compound 2 (143.1 mg, 28.6% and 14.9 mg, 2.98% yield
sis and further pharmacological evaluation.13) In view of low by B. bassiana (ATCC 7159) and B. bassiana (ATCC
yields of metabolites produced in previous experiments pre- 13144), respectively), a yellowish solid with a molecular for-
venting complete structure elucidation as well as to obtain mula, C15H12O5 (m/z 273.0741 [MꢁH]ꢁ) corresponded to a
transformed products with enhanced biological properties, product of 1 with two more oxygen atoms present as a phe-
we subjected 4ꢀ-hydroxyflavanone to microbial transforma- nolic hydroxyl groups as determined by H- and 13C-NMR
1
tion and obtained 18 transformed products. Production, isola- data. The 1H-NMR spectrum of 2 was similar to that of 1 but
tion structure elucidation and some bioactivity studies of the showed a reduction of aromatic protons from 8 to 6 with A
metabolites are reported herein.
[dH 7.08 (1H, d, Jꢂ2.8 Hz), 7.00 dd (1H, Jꢂ9.2, 2.8 Hz) and
6.89 (1H, d, Jꢂ9.2 Hz)] and B [dH 6.88 (1H, br s) and 6.73
(2H, br s)] rings showing 1, 3, 4 aromatic substitution. The
Results and Discussion
4ꢀ-Hydroxyflavanone (1) was screened using the standard location of the hydroxyl group was determined by 2D-NMR
two stage procedure with forty fungal strains to identify cul- experiments. It was identified as 6,3ꢀ,4ꢀ-trihydroxyflavanone
tures that could give maximum yields of the metabolic prod- (2).
ucts.14) The results of the scale up studies with selected fun-
Metabolite 3 (38.4 mg, 7.08% and 9.4 mg, 1.88% yield by
gal cultures were summarized in Figs. 1 and 2. The total con- B. bassiana (ATCC 7159) and B. bassiana (ATCC 13144),
version of 1 to its metabolites was about 47%. Determination respectively) of 1 with a molecular mass of 448 was assigned
of the molecular formulae of the compounds was based on a molecular formula C22H24O10. Its spectroscopic data were
HR-ESI-MS data.
similar to those of 2. The difference observed was the pres-
Fig. 1. Microbial Metabolites of 4ꢀ-Hydroxyflavanone
B. bassiana (1): (ATCC 13144), B. bassiana (2): (7159).

694
Vol. 59, No. 6
Fig. 2. Microbial Metabolites of 4ꢀ-Hydroxyflavanone
ence of an O-methyl-D-glucopyranosyl moiety with 176 mass of a hydroxyl group in the molecule. 1H-NMR coupling con-
units. The coupling constant of the anomeric proton at d 4.77 stants, NMR correlation spectra along with MS (m/z
(1H, d, Jꢂ7.6 Hz, H-1ꢃ) indicated a b-configuration of the 255.1593; [MꢄSO4]ꢁ) and IR (1237, 1175, 827) data con-
glycosyl unit. The three bond correlation of the anomeric firmed that structure as 4ꢀ-hydroxyflavanone 3ꢀ-sulfate.
proton with the quaternary carbon at d 152.2 (C-6) indicated
Molecular formula C22H24O8, based on the molecular ion
O-glucosylation at C-6. The large coupling constants be- peak at m/z 439.1358 [MꢁNa]ꢁ was assigned to metabolite 6
tween H-2ꢃ/3ꢃ, H-3ꢃ/H-4ꢃ and H-4ꢃ/5ꢃ protons indicated (43.7 mg, 8.74% yield by B. bassiana (ATCC 13144). It was
trans-diaxial relationships. The downfield shift of C-4ꢃ (d found by spectroscopic data to be an O-methylated glucopy-
79.5) along with the HMBC correlations indicated that the ranosyl conjugate of 1. The large coupling constants ob-
O-methyl group was attached to C-4ꢃ. Thus, the metabolite 3 served between H-2ꢃ/3ꢃ, H-3ꢃ/H-4ꢃ and H-4ꢃ/5ꢃ protons of
was characterized as 3ꢀ,4ꢀ-dihydroxyflavanone 6-O-b-D-4- the sugar moiety established their trans-diaxial relationships.
methoxyglucopyranoside.
HMBC correlations and the downfield shift of C-4ꢃ (d 79.7)
Compound 5 (59.5 mg, 11.9% and 9.6 mg, 1.92% yield by detected in the 13C-NMR spectrum indicated that the O-
B. bassiana (ATCC 7159) and B. bassiana (ATCC 13144), methyl group was attached to C-4ꢃ carbon. O-glucosylation
respectively) showed a molecular ion peak at m/z 350.9835 was shown to be at C-4ꢀ by the three bond correlation of the
[MꢄH]ꢁ consistant with the molecular formula C15H12O8S. anomeric proton with the carbon at d 158.1 (C-4ꢀ) which in
Comparison of this compound with 2 revealed significant turn correlate similarly with H-2ꢀ and H-6ꢀ (d 7.44). The
similarity except for the presence of a sulfate moiety, the po- large coupling constant of the anomeric proton at d 4.89 (1H,
sition of which was determined as C-3ꢀ by correlation NMR d, Jꢂ8.0 Hz, H-1ꢃ) revealed the b-configuration of the gluco-
spectra. It was further supported by the shielding of the sig- syl unit. The structure of 6 was thus, elucidated as flavanone
nal due to the ipso carbon at C-3ꢀ position by 4.4 ppm and 4ꢀ-O-b-D-4-methoxyglucopyranoside.
the deshielding of C-2ꢀ and C-4ꢀ carbons ortho to and C-6ꢀ
Compound 7 (9.7 mg, 1.94% and 101.4 mg, 20.28% yield
carbon para to the sulfation site by values of 7.6, 3.9 and by B. bassiana (ATCC 7159) and B. bassiana (ATCC
5.7 ppm respectively, in the 13C-NMR spectrum when com- 13144), respectively) with a molecular formula C22H24O9
pared with that of 2.17) The loss of 80 mass units together (m/z 455.1277 [MꢁNa]ꢁ) was determined to be an O-methyl-
with 1250, 1019 and 883 absorptions observed in the MS ated glucopyranosyl conjugate of 1. The sugar moiety was
and IR spectra respectively, gave further evidence to the pres- identified as 4-O-methyl-D-glucopyranose by a detailed study
ence of a sulfate group in the molecule. The forgoing data of NMR data as in compound 6. Three bond correlation of
suggested 6,4ꢀ-dihydroxyflavanone 3ꢀ-sulfate as the structure anomeric proton with carbon at d 152.3 (C-6) and similar
of compound 5.
correlation of C-6 with H-8 (d 7.00) indicated O-glucosyla-
Compound 4 (40.5 mg, 8.1% and 87.8 mg, 17.56% yield tion at C-6. b-Configuration of the glucosyl unit was detr-
by B. bassiana (ATCC 7159) and B. bassiana (ATCC mined by the large coupling constant of the anomeric proton
13144), respectively) with the molecular formula C15H12O7S d 4.79 (1H, d, Jꢂ7.2 Hz, H-1ꢃ). The structure of 7 was thus,
(m/z 335.0165 [MꢄH]ꢁ) showed spectroscopic data in close established as 4ꢀ-hydroxyflavanone 6-O-b-D-4-methoxyglu-
agreement with those of 5 with the exception of the absence copyranoside.

June 2011
695
Metabolite 9 (129.3 mg, 25.86% yield) had a molecular d 64.7 in the 13C-NMR spectrum of 16 indicated the reduc-
formula C21H22O8 derived from molecular ion peak at m/z tion of the carbonyl group. The double doublet at d 5.01
425.1228 [MꢁNa]ꢁ. Its spectroscopic data revealed its close (Jꢂ12.0, 1.6 Hz) assigned to H-2 and the double doublet at d
resemblance to those of 1. The exception was the presence of 4.90 (Jꢂ11.2, 6.4 Hz) assigned to H-4 indicated a 2,4-cis
D-glucopyranose group. The structure and the position of the structure.18) 16 was thus, identified as 2,4-cis-3ꢀ, 4ꢀ-dihy-
sugar moiety were determined by correlation NMR data as in droxyflavan-4-ol.
compound 6. The upfield shift of the ipso and the downfield
Spectroscopic data of the metabolite 17 (2.8 mg, 0.56%
shift of the ortho carbon atoms together with the lowfield yield), with the molecular formula, C16H16O4 (m/z 271.1085
shift of the para carbon with a higher magnitude in the 13C- [MꢄH]ꢁ) were similar those of compound 11 with the ex-
NMR spectrum provided further evidence for the presence of ceptions of a methoxy group attached to C-4ꢀ as determined
the sugar unit at C-4ꢀ.17) Its structure was determined as fla- by NMR correlation data and the reduction of the carbonyl
vanone-4ꢀ-O-b-D-glucopyranoside (9).
group. The coupling constants of the signals of H-2 and H-4
A molecular ion of m/z 335.1140 [MꢄH]ꢁ displayed by in the 1H-NMR spectrum indicated that were in a trans
compound 10 (63.1 mg, 12.62% yield) was consistant with arrangement. It was assigned the structure 2,4-trans-3ꢀ-
the molecular formula, C15H12O7S. The presence of peaks at hydroxy-4ꢀ-methoxyflavan-4-ol.
1273, 1050 and 845 in the IR spectrum along with the loss of
Metabolite 18 (10 mg, 2% yield), was assigned the molec-
80 mass units in the MS spectrum indicated the presence of a ular formula C21H22O7 based on m/z 409.1473 [MꢁNa]ꢁ.
1
sulfate group in the molecule. The spectral data were similar The H-NMR spectral data of the compound was similar to
to those of metabolite 4 except for the position of the sul- those of 1, except for the presence of glycosyl unit shown by
phate group. NMR correlation data together with the shield- the additional five carbon resanances observed at d 67.9—
ing of the ipso carbon at C-4ꢀ position by 8.6 ppm and the 98.6 in the 13C-NMR spectrum. It was identified as D-6-de-
1
deshielding of C-3ꢀ and C-5ꢀ carbons ortho to and C-1ꢀ car- oxyallose by analysing the H-, 13C-NMR and their correla-
bon para to the sulfation site by values of 8.3, 5.7 and tion data. They were in total agreement with those of the fla-
5.5 ppm respectively, in the 13C-NMR spectrum compared to vanoid glycosides obtained during microbial transformation
that of 4 suggested structure 3ꢀ-hydroxyflavanone-4ꢀ-sulfate studies carried out by us using the same organism, M. raman-
for compound 10.
nianus.19) HMBC correlations permitted the assignment of
The NMR data of compound 12 (56.0 mg, 11.2% yield) the sugar unit at C-4ꢀ. The structure of the metabolite 18 was
with the molecular formula, C21H22O9 (417.0845 [MꢄH]ꢁ) determined as flavanone 4ꢀ-O-a-D-6-deoxyallopyranoside.
were in close resemblance to those of 1116) except for the
NMR data of flavanone 19 (30.1 mg, 6.02% yield,
presence of a D-glucopyranose moiety at C-3ꢀ. The structure C21H24O7, 411.1529 [MꢁNa]ꢁ) were in close agreement
and the position of the sugar moiety were determined by cor- with those of metabolite 13. However, there were additional
relation NMR data. b-Configuration of the glucosyl unit was resonances due to the presence of a sugar unit, which was es-
indicated by the large coupling constant of the anomeric pro- tablished as D-6-deoxyallose as in compound 18. In addition,
ton at d 4.70 (1H, d, Jꢂ7.2 Hz, H-1ꢃ). Its structure was eluci- the large J4,3ax value along with the downfield shift of the C-2
dated as 4ꢀ-hydroxyflavanone-3ꢀ-O-b-D-glucopyranoside.
compared to the trans-flavan-4-ols in the 13C-NMR spectrum
HR-ESI-MS (241.0566 [MꢄH]ꢁ) established the molecu- indicated a 2,4-cis structure.18) It was thus, identified as
lar formula of 13 (63.4 mg, 12.7% yield by R. anceps) as 2,4-cis-4-hydroxyflavanone 4ꢀ-O-a-D-6-deoxyallopyranoside
C15H14O3. It corresponded to a product of 1 with two addi- (19).
tional hydrogen atoms. The spectral data were in agreement
with those published for 2,4-cis-4ꢀ-hydroxyflavan-4-ol.16)
Metabolite 14 (56.51 mg, 11.3% yield) (HR-ESI-MS
Conclusion
In this study, extensive metabolism of 4ꢀ-hydroxyfla-
(241.0557 [MꢄH]ꢁ) was spectroscopically similar to com- vanone (1) by the fungal cultures, B. bassiana, C. echinulata,
pound 13, with the exception of the appearance of the C-4 M. ramannianus, and R. anceps was observed with the for-
signal d 4.92 as a quartet with Jꢂ12 Hz, as against a triplet mation of 18 compounds. Metabolites 2—5, 10—12 and
signal at d 4.62 (Jꢂ3.2 Hz) in the C-2, C-4 cis isomer (13) 15—17 suggested that the primarily conversion occurred in
indicating it to be 2,4-cis-4ꢀ-hydroxyflavan-4-ol.
the B-ring with the generation of catechol (2—3, 11 and 15,
Metabolite 15 (37.5 mg, 7.5% yield) with the molecular 16) and the conjugated products, 4, 5, 10, 12 and 17 showing
formula, C15H14O4 (257.0500 [MꢄH]ꢁ) showed spectro- metabolic pathways similar to those observed in vitro bio-
scopic data similar to those of compound 11 with the excep- transformations of flavonoids by mammalian microsomes.5)
tion of the reduction of the carbonyl group. A quartet signal Further, the formation of C-4 carbonyl group reduction com-
at d 5.02 (Jꢂ10.4 Hz) assigned to H-2 and a broad singlet at pounds, 13—17 and 19 along with the hydroxylation at 3ꢀ-
d 4.60 suggested a 2,4-trans structure. It was thus, identified position (2, 3, 10, 11 and 15—17), at 6-position (2, 5 and 8)
as, 2,4-trans-3ꢀ, 4ꢀ-dihydroxyflavan-4-ol.
and the conjugation products (3—5 and 7) also paralleled the
Compound 16 (47.0 mg, 9.4% yield) exhibited a molecular mammalian metabolism of flavanone.8) Present study under-
ion of m/z 257.0515 [MꢄH]ꢁ which was consistant with the lined the importance of using microbial models to mimic
molecular formula, C15H14O4 (257.0812 [MꢄH]ꢁ). The mammalian metabolism of xenobiotics. The completely char-
product whose spectroscopic data were similar to those of the acterized microbial metabolites of 1 could thus, be utilized to
metabolite 11 except for the reduction of the carbonyl group confirm tentatively identified mammalian metabolites5) due
in the former was identified by means of NMR data. The to lack of material and also to carry out further pharmacolog-
1
double doublet of 4-H at d 4.90 in the H-NMR and the re- ical studies. The formation of functional groups at carbon 6
placement of the C-4 signal of 11 at d 192.6 by the signal at position in this investigation is significant, as C-6 derivatives

696
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

June 2011
697
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