Research Article

1814
Calculations and statistical analyses
In Experiment 1, OD425 readings, which reflected chromophores
formed during reaction among sapogenin, p-anisaldehyde and
sulfuric acid, were compared over a 24-h period between dH2Oand
RF-based suspensions, and with and without dH2O added to
the post-reaction mixture. In Experiments 2 and 3, recoveries of
steroidal saponin were calculated on the basis of the amounts of
saponin/sapogenin detected in various fractions as a proportion
of the amount of saponin present initially. All data were analyzed
statistically by analysis of variance using the MIXED procedure
of SAS, 21 with individual vial or tube as a random factor. The
significance of differences among treatments was tested using
LSMEANS with the PDIFF option. 21
RESULTS
Experiment 1
Addition of dH2O to the post-reaction mixture after a 10-min
incubation in the water bath reduced (P < 0.01) interference from
background color and improved (P < 0.01) the stability of the
OD425 readings over 24-h period (Fig. 1). With no water added,
OD425 readings increased (P < 0.01) between 0.5 and 24 h postreaction,
whereas if 0.5 mL of dH2O had been added, the OD425
readings remained essentially unchanged over this period. This
response was observed both with smilagenin (Fig. 1A) and with
OD 425
OD 425
A 2.4
2.0
1.6
1.2
0.8
0.4
0.0
2.8
2.4
2.0
1.6
1.2
0.8
0.4
Recovered from dH 2 O
Recovered from pcRF
No H 2O added
0.0
0 8 16 24 32 40
Smilagenin added (µg mL-1 )
www.soci.org Y Wang, TA McAllister
YS saponin (Fig. 1B), irrespective of their having been recovered
from suspension in dH2OorinpcRF.
The utility of centrifugation as a means of separating sapogenin
from saponin was confirmed. After saponin or sapogenin
suspensions in cRF were centrifuged, essentially all saponin measured
in the whole (uncentrifuged) mixture remained detectable
in the supernatant, whereas virtually no sapogenin (smilagenin
equivalents) was present in the supernatant (Table 1). Nearly all
(>99%) of the saponin and sapogenin added to cRF was recoverable
from the non-centrifuged mixture. Rates of recovery from
cRF were similar across steroidal sapogenin concentrations of
250–1000 µgmL −1 , and between saponin concentrations of 450
and 500 µgSEmL −1 .
Experiment 2
Patterns of recovery of YS saponins added to dRF or cRF were
unaffected by the ruminal fluid donor heifers having been fed
powdered Y. schidigera (0 vs. 20 or 60 g day −1 ; Table 2). When
the mixtures were assayed without centrifugation, all of the YS
saponin (99.1 to 100.6%) added to dRF or cRF was detected
as steroidal saponin+sapogenin. In contrast, measurement of
steroidal saponin in the supernatant fraction of the incubations
was greatly reduced (P < 0.01) in dRF (58.5% recovered), as
compared with cRF (98.7% recovered).
Recovered from dH 2 O
Recovered from pcRF
H 2 O added post-reaction
0.5 h
2.0 h
6.0 h
24 h
0 8 16 24 32 40
Smilagenin added (µg mL-1 )
Figure 1. Stabilization of chromophore development (OD425) during colorimetric determination of (A) smilagenin or (B) saponin from Y. schidigera that
had been recovered from solution in dH2O or in partially clarified ruminal fluid (pcRF). Values shown are means of quadruplicate determinations. Bars
representing standard error fell within symbols.
www.interscience.wiley.com/jsfa Copyright c○ 2010 Crown in the right of Canada. J Sci Food Agric 2010; 90: 1811–1818
Published by John Wiley & Sons, Ltd