full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 5 (3) 1282-1297 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)
1285
equivalent basis. After selection of the best
source, different concentrations 2.5, 3, 3.76, 4,
4.5 g/l were investigated.
Mineral salts : Different concentrations (0-4 %)
of NaCl were used in the cultivations to
determine the best for antifungal production.
Different concentrations (0-1.6 %) of K 2
HPO 4
in the culture medium were tested.
Different pH and shaking rate : Varied initial
pH values were tested using different buffering
solutions. A control flask was done by adjusting
the pH at 7 by 1M HCl and 1M NaOH. The buffer
solutions used were: Citrate phosphate buffer at
pH 6, 6.5 and 7, Phosphate buffer at pH 7, 7.5
and 8 and Tris-maleate buffer at pH 8 and 8.5.
Different rpm were investigated (100, 150, 200)
as well as static conditions were maintained.
Extraction of the antifungal bioactive
compound (s) : After fermentation of the media,
the culture broth was filtered in vacuo and the
filtrate was extracted with equal volume of ethyl
acetate. The latter was dried under reduced
pressure using a rotary evaporator at a
temperature not exceeding 50 °C, yielding an
orange-yellow residue (270 mg).
Thin Layer chromatography (TLC) : Ethyl
acetate extract of the culture filtrate was
developed on TLC plate using solvent system
consisting of Benzene: Ethyl acetate: Acetic acid
(1: 3.5: 0.5 v/v) to evaluate the secondary
metabolite constituents of Nocardioides luteus.
The chromatogram was detected under a
UV lamp and each spot was scratched from the
TLC plate and dissolved in ethyl acetate. Silica
gel was removed by centrifugation at 2000 rpm
for 10 min. The supernatant was air-dried and
the constituents were tested by paper disc assay
method (12).
Isolation of the active compound : The residue
(270 mg) was dissolved in 5 ml ethyl acetate and
adsorbed on silica gel G for column
chromatography. The mixture was added at the
top of a column chromatography (45 cm long ×
4 cm wide) using silica gel G as an adsorbent,
benzene/ ethyl acetate (1:1) was used as an
eluent. Fractions of each equal to 15 ml, were
collected individually. Then, the polarity was
increased after 53 fractions to benzene/ ethyl
acetate (1:2) for 97 fractions followed by ethyl
acetate for 25 fractions then, 5 % methanol in
ethyl acetate for 11 fractions and finally methanol
for 39 fractions.
Fractions were grouped in accordance to
their TLC profiles and tested for their
antimicrobial activity (Bioassay- guided
fractionation). The TLC plates revealed that the
fractions were to be pooled into 8 fractions, only
5 of which were active against C.albicans. The
most active was subjected to further purification
by re-fractionation using column
chromatography.
The elution with benzene/ ethyl acetate
(1:2) afforded 97 fractions. The fractions from
45 to 70 were collected together to give rise to
80 mg of an orange - yellow mixture. It was refractionated
on flash silica gel column (45 cm
long × 4 cm wide) using chloroform/ methanol
(9:1) as eluent. Fractions, each equal to 10 ml,
were collected individually. The fractions from
27 to 34 were collected together once again and
purified on RP-18 column (30 cm long × 4 cm
wide) using 20 % water in methanol to afford 10
mg of semi-pure compound. Further purification
failed due to the instability of the compound.
The partially purified active material
obtained was subjected to various examinations
by ultraviolet, infrared, 1 H-NMR 500MHz and
13
C-NMR.
Cytotoxicity assay to human tumor cell lines:
Sulphorhodamine-B (SRB) assay of cytotoxic
activity (13):
El-Refai et al