Allelochemical Extraction from Plant Exudates
To detect whether the phenolic allelochemicals were
released into water environment by the two plants, solid
phase extraction (SPE) and subsequent LLE were performed
to extract phenolic allelochemicals exuded into the
culture
solution. Before SPE, the culture solution of plants
was
filtered through GF/F filters to remove particles and
acidified
to pH 2 to enhance the binding of phenolic compounds.
Oasis HLB cartridges (500 mg, 6 cc, Waters,
Milford,
Massachusetts, USA) were used to adsorb exuded
phenolic
compounds. Each cartridge was preconditioned
with
15 ml methanol and equilibrated with 15 ml Milli-Q
ultra-pure
water.
Two
litres of acidified culture solution
were
passed over one cartridge at a rate of 3 ml/min, totally
10 L
on 5 cartridges for one plant species. Then,
the
adsorbed
cartridges were washed with ultra-pure water,
dried
under reduced pressure, and finally eluted with 18 ml
methanol
per one cartridge. The
methanol eluates were
combined
separately for the two plants, evaporated to dryness,
and re-dissolved in ultra-pure water.
The
same LLE
procedure
and sequent treatment as plant extracts were used
for
further separation and purification. The
control culture
solution
was extracted likewise.