Column chromatographySilica (230-40

Column chromatography
Silica (230-400 mesh) gel slurry was prepared using methanol.
The column was packed with silica gel. After washing
the column with same solvents, the sample (3 to 5 ml) was
poured and then eluted with methanol: water on different
percentages (10 to 100% methanol). The active fractions
were collected and used for NMR analysis.
Antibacterial activity
Disc diffusion method was employed to test the active
fractions of H. atra obtained from the column chromatography
Bauer et al. (1966). Antibacterial activity was determined
using Muller Hinton agar (Hi Media).The bacterial
cultures were obtained from the Microbial type culture
collection and gene bank (MTCC), Institute for Microbial
Technology, Chandigarh, India. They were Staphylococcus
aureus MTCC 737, E.coli MTCC 443, Klebsiella pneumonia
MTCC 109, Listeria monocytogenes MTCC 1143, and
Serratia liquefaciens MTCC 3039. The plates were aseptically
streaked with the test microorganism using a sterile
swab and allowed to dry for a few minutes. Sterilized
filter paper discs (Whatman no.1; 6 mm diameter) were
used. The fractions were collected from 10 to 100% levels
of methanolic extracts and were evaluated at 100 μl
concentration. The plates were then incubated for 24 h at
37°C. Controls were blank discs impregnated with solvent.
The diameter of the inhibition zone formed around the
disc was measured.
GC-MS analysis
The methanol extract of the sea cucumber H. atra was
analyzed by GC-MS (Make: Fisons GC8000 series and MS:
md800). The GC column dimension was: 30 mm, 0.25 mm,
0.5 mm AB-35MS fused silica capillary column. The GC
conditions were as follows: injector temperature 250°C
column temp isothermal at 100°C then programmed to
rise up to 250°C at 6°C/min and held at this temperature
for 10 minutes. The ion source temperature was 200°C
and the interface temperature was 250°C. Helium gas was