A.-R.S. Ibrahim, A.E. Ragab
PhytochemistryLetters25(2018)86–89
3.3. Microorganism strain and culture conditions
Table 2
Antimicrobial activity testing of fusidic acid and the isolated metabolites.
Biotransformation studies were performed following previously re-
twenty-five), obtained from either the American Type Culture Collec-
tion (ATCC) or National Center for Agricultural Utilization Research
(NCAUR), were used in the preliminary screening. The strains were
subcultured quarterly and stored at 4° C on Sabouraud dextrose agar
slants.
Microorganism
MIC (μg/mL)
Fusidic acid 1
2
3
Streptomyces faecalis
Staphyllococcus aureus
Bacillus subtlis
Escherichia coli
Pseudomonas aeruginosa
Mycobacterium smegmatis
1.50
0.38
0.38
−ve
12.5
50
2.5
100
−ve
−ve
−ve
25
2.5
50
−ve
−ve
100
3.4. Culture media
* −ve at the highest tested concentration (100 μg/mL).
The fermentation medium is composed of 10 ml of glycerol, 10 g of
glucose, 5 g of peptone, 5 g of yeast extract, 5 g of NaCl, and 5 g of
K2HPO4 in 1 l of distilled water. The pH was adjusted to 6.0, and the
medium was autoclaved at 121 ° C for 15 min.
of the existing functionalities (Al-Footy, 2008). The hydroxylation site
is commonly at least four to five atoms away from the binding site (Al-
Footy, 2008). The proposed binding sites of steroids are C-3 and C-16
functionalities (Al-Footy, 2008). In fusidic acid (1), the hydroxylation
of ring B is directed by the hydroxyl group at C-3 and the ester group at
C-16. The C-7 position is four carbons away from both C-3 and C-16,
while C-6 is five carbons away from C-16, justifying our results. The
average distance between the hydroxyl groups at C-6 and C-3 is 4.73 Å,
while the average distance between the C-6 hydroxyl and C-16 ester is
7.359 Å. The average distance between the hydroxyl groups at C-7 and
C-3 is 6.18 Å, while the average distance between the C-7 hydroxyl and
C-16 ester is 5.379 Å. Although it was proposed that oxidation occurs at
approximately 5.5 Å away from an electron-rich functionality, some
hydroxylation reactions do not follow this rule, indicating that the
electron-rich centre is not the sole directing element (Holland, 1982).
The antimicrobial activity testing of compounds 2 and 3 revealed
that the hydroxylation of fusidic acid (1) at C-6 and C-7 diminishes the
antimicrobial activity (Table 2) and showed the same activity profile as
27-hydroxy and 26-formylfusidic acid, respectively, as our previous
crease the binding affinity at the binding pocket of the elongation factor
EF-G with a consequent reduction in antimicrobial activity.
3.5. Biotransformation initial screening experiments
Stage I cultures of the tested microorganisms were prepared by in-
oculating cells from two-week-old slants of the respective strains into
sterile liquid medium (50 ml/250 ml flasks). These cultures were kept
on a gyratory shaker for 72 h at 28 ° C, 200 rpm. Stage I culture (5 ml)
was inoculated into fresh liquid medium (50 ml/250 ml flask), and in-
cubation was continued at 28 ° C, 200 rpm to obtain stage II culture.
One day later, a solution of sodium fusidate or fusidic acid in absolute
ethanol (10 mg/250 μl) was added to each flask. On the third and sixth
days of incubation, samples (5 ml) were withdrawn and acidified with a
few drops of 10% HCl. The acidified samples were extracted with an
equal volume of chloroform. The chloroform extracts were evaporated
under vacuum, and the obtained residues were examined by precoated
silica gel plates using chloroform-methanol (5:1) or benzene:ethyl
acetate:formic acid (3 ml:7 ml:1 drop) as the mobile phase. Detection of
compounds was achieved by UV light visualization and p-anisaldehyde
spray reagent. Both substrate and organism-free cultures were pro-
cessed in the same way. The results of the preliminary screening using
fusidic acid were identical to those of sodium fusidate. Out of the tested
strains, C. echinulata NRRL 1382 and C. elegans 1392 displayed the best
transformations. This paper discusses the metabolites obtained by using
C. elegans NRRL 1392.
3. Experimental section
3.6. Large-scale fermentation
3.1. General procedures
A solution of sodium fusidate in absolute ethanol (1080 mg/27 ml)
was equally distributed among 108 flasks each containing 50 ml of
stage II culture of C. elegans NRRL 1392. Incubation was continued for
six days at 28 °C, 200 rpm. Substrate and organism-free control cultures
were prepared alongside the experiment. The cultures were collected
and acidified with 10% HCl (1 ml/30 ml culture). The cells were filtered
and extracted with chloroform. The filtrate was also extracted with
chloroform (1:1). Chloroform extracts were combined and dried over
anhydrous sodium sulfate and evaporated under vacuum to give
translucent residue (1.37 g). TLC was carried out using benzene:ethyl
acetate:formic acid (3 ml:7 ml:1 drop) or chloroform-methanol (5:1) as
the mobile phase, and detection was carried out by UV light visuali-
zation and p-anisaldehyde spray reagent.
Sodium fucidate was obtained from Leo Pharmaceutical Company
(Ballerup, Denmark). PerkinElmer IR and Shimadzu 60/PC ultraviolet
spectrophotometers were used to record the IR and UV spectra, re-
spectively. A Bruker Avance 500 spectrophotometer, using methanol-d4
as a solvent and tetramethyl silane (TMS) as an internal standard, was
used for NMR analysis at 500 MHz for 1H NMR and 125 MHz for 13C
NMR. Varian Pulse Sequences at 125 and 500 MHz were used for DEPT,
COSY and HETCOR analyses. A Bruker Bioapex FT-mass spectrometer
in ESI mode was used for HRESI-FTMS analysis. Precoated silica gel
60 F254 plates (0.25 mm layer, E. Merck) were used, and visualization
was completed by heating at 110 ° C after spraying with p-anisaldehyde
reagent for the detection of compounds.
3.2. Preparation of fusidic acid
3.7. Isolation of metabolites
A clear solution of sodium fusidate in distilled water was made
distinctly acidic to litmus paper with acetic acid. Fusidic acid pre-
cipitate was filtered and washed thoroughly with distilled water until
free from acidity. After drying to constant weight, the prepared fusidic
acid was analysed by NMR and MS. The resulting data were identical to
The residue obtained from the chloroform extract after evaporation
(1.37 g) was partially purified by a Sephadex LH20 column using me-
thanol as the eluent to yield 600 mg of a mixture of two compounds.
The last mixture was loaded onto a silica gel column (100 g) and eluted
with a gradient of ethyl acetate in benzene (40–55%) containing 0.2%
formic acid. Fractions of 100 ml were collected. Fractions 47–59 and
69–86 eluted with 0.2% formic acid in ethyl acetate:benzene (5.5:4.5)
88