ISOLATION OF BRADYOXETIN


 


Preparation of CDF


 

1.                  Bacteria cultures were grown up in Minimal medium to
late log phase (OD600 = 2.0).

2.                  Equal volume of ethyl acetate (Optima Grade, Fisher
Scientific) was then added and incubated, with shaking at 200 rpm, for 1
h.  

3.                  The flasks were then allowed to sit for 1 h, or as
long as it takes to separate the aqueous phase from the ethyl acetate
phase.  Should the layers fail to separate, additional ethyl acetate is
added and step 3 is repeated.

4.                  Remove the top layer containing the ethyl acetate
using a glass pipette, and store in glass container.  The ethyl acetate
is removed by blow-drying, using the house air-line.

5.                  The concentrated extract, containing CDF, is then
stored at -20 C.

 


HPLC analyses of CDF


 

 

1.                  For analyses of CDF, the extracts are first
reconstituted in water, and then applied to a Sep-pak column (C18-M,
phenomenex).  The sep-pak columns are prepared using the following
steps:

 

a.       wash in Methanol

b.      wash in Water

2.                  The sample is then applied and pass through the
column with the pressure provided by a disposable syringe.

3.                  Reapply flow through of sample 4 times.

4.                  Wash column with water, and elute with 100%
methanol.

5.                  Eluted sample is then concentrated via blow-drying
(as above), and then resuspended in water before being applied to the
HPLC (C18 jupiter, phenomenex; 250 x 4.6 mm; 5 micron).

6.                  The following gradient is used for CDF isolation:

 

1 min 95% water/5% MeOH (inject sample)

1 min to 10 min (gradient to 40% MeOH)

hold 10 min

gradient to 100% MeOH  in 30 min

return solutions to  95% water/5% MeOH

 

7.                  CDF should elute at about 20 min after start of
gradient (ie. During the 10 min hold at 40% MeOH.