Research Article

1812
proposed that destabilization of cell membranes by saponins
requires their deglycosylation in the immediate vicinity of the
membrane. In agreement, we observed that purified steroidal
sapogenin had no effect on ruminal cellulolytic bacteria (Wang
et al., unpublished data). Similarly, others have found that once
deglycosylated, saponins no longer exhibit their antifungal
properties. 16,17 Thus, further definition of the mechanisms of
microbial metabolism of saponins in the rumen could provide
valuable information with regard to their potential to favorably
manipulate ruminal fermentation.
This study was conducted with the objective of modifying
the spectrophotometric method of Baccou et al. 12 to enable
measurement, in ruminal fluid, of steroidal saponin and of
sapogenin, its deglycosylated, insoluble form, and to use the
modified procedure to assess the deglycosylase activity of ruminal
microbes by comparing the activities in cell-associated and extracellular
enzyme fractions.
MATERIALS AND METHODS
This study used smilagenin (minimum 98%; Sigma Chemical Co.,
St Louis, MO, USA) and steroidal saponin extracted from Yucca
schidigera (Desert King International, San Diego, CA USA) as
model steroidal sapogenins and steroidal saponins, respectively.
Smilagenin was dissolved in absolute ethanol (50 mg 100 mL −1 )
for use in all assays. Baccou et al. 12 reported that all steroidal
sapogenins and/or saponins tested including smilagenin and
saponins containing smilagenin, produced similar chromophores
(max. absorption at 430 nm) upon reaction with p-anisaldehyde
and sulfuric acid in ethyl acetate. Thus, steroidal saponin and/or
sapogenin measured in this study were expressed as smilagenin
equivalents (SE).
Extraction of steroidal saponins from Yucca schidigera
Powdered Yucca schidigera (YS) plant material was used as a
source of saponins in this study. To remove fat and endogenous
smilagenin from the dry powder, 60 g were combined with 200 mL
of petroleum ether (Sigma Chemical Co.) and mixed continuously
for 2 h in a sealed container at room temperature (21 ◦ C), then
filtered through Waterman No. 1 filter paper. The residue was
washed with 100 mL of petroleum ether, filtered again and ether
in the residue was allowed to volatilize under continuous air flow in
a fume hood. The ether-extracted residue was mixed with 200 mL
of dH2O at room temperature for 60 min prior to being filtered
through Waterman No. 1 filter paper. The residue was washed
with 100 mL of dH2O and filtered two more times and filtrates
were combined. To remove tannins and phenolic compounds, the
combined filtrate was mixed with 5 g of polyvinyl-polypyrolidone
(GAF Materials Corporation, Wayne, NJ, USA). After centrifugation
at 5000 × g (10 min, 4 ◦ C), the supernatant was lyophilized and the
dried residue was dissolved in 100 mL of dH2O and centrifuged
(10 000 × g,20min,4 ◦ C). To capture saponins from the resulting
supernatant (100 mL), it was mixed with 200 mL of n-butanol
(Sigma Chemical Co.) and 0.2 mL of concentrated HCl and held at
room temperature for 30 min, then centrifuged (10 000×g,10min,
4 ◦ C). The butanol fraction was collected and the aqueous fraction
was subjected to a second n-butanol extraction. Butanol fractions
were combined and rotary evaporated at 50 ◦ C to dryness. The
residue was dissolved in 100 mL of dH2O, centrifuged (12 000 × g,
20 min, 4 ◦ C), and the supernatant was freeze-dried. The dried YS
saponin extract was stored in a sealed amber container at 0 ◦ C.
www.soci.org Y Wang, TA McAllister
High-performance thin-layer chromatography (HPTLC) of the
extract prepared as described above confirmed that it produced
no band and that the acid-hydrolysed (deglycosylated) extract
produced a band corresponding to steroidal sapogenin (Wang
et al., unpublished data). Using the modified spectrophotometric
method described below, the steroidal saponin concentration in
the YS extract was determined as 242.5 mg SE 100 g −1 .
Experiment1:Modificationofthespectrophotometricmethod
for application in ruminal fluid
This study was based on the spectrophotometric method as
described by Baccou et al. 12 In that method, a dry sample
containing saponin or sapogenin is dissolved in 2 mL of ethyl
acetate, to which is added 1 mL of 0.5% (v/v) anisaldehyde in
ethyl acetate and then, after mixing, 1 mL of 50% (v/v) H2SO4
in ethyl acetate. Reaction mixtures are then incubated at 60 ◦ C
for 20 min for color development. Saponin present in samples
is deglycosylated via acid hydrolysis, such that chromophore
development arises from total saponin+sapogenin in a sample.
Preliminary studies indicated to us that the Baccou et al. 12 assay
was unsuitable for analysis of steroidal saponin and sapogenin in
ruminal fluid because of interference from an intense background
chromophore. Further investigation showed that reducing the
reaction time at 60 ◦ C from 20 min to 10 min did not affect
the density of chromophores formed, as judged by the optical
density of the solution at 425 nm (OD425 values), and also that
adding 0.5 mL of dH2O to the post-reaction solution (i.e. after a
10minincubationat60 ◦ C)significantlyincreased the constancyof
the reaction mixture OD425. These conditions (10 min incubation;
post-reaction addition of dH2O) were used subsequently in testing
saponin analysis in defined solvent vs. ruminal fluid.
Preparation of samples for analysis from solution in dH2Oorruminal
content
Ruminal fluids collected from two steers fed a 40 : 60 barley
grain : barley silage diet were strained through four layers of
cheesecloth, combined in equal portions and then centrifuged
(10 000 × g,20min,4 ◦ C), and the supernatant (denoted ‘partially
clarified ruminal fluid’, pcRF) was used as a test solvent. The ruminal
fluid donors used in this study were cared for according to the
standards of the Canadian Council on Animal Care. 18
Smilagenin (as a model sapogenin) and YS saponin extracted
as described above were each assayed following suspension in
pcRF to assess the usefulness of the protocol improvements in a
ruminal application. Aqueous solutions of these compounds were
included for comparison. Smilagenin–ethanol solution (50 mg SE
100 mL −1 ) was combined with four volumes of either pcRF or
dH2O. In 10-mL plastic tubes, 5-mL quantities of the suspension
were frozen and lyophilised, and the residues were re-suspended
into 2.0 mL of methanol. Extracted YS saponins were dissolved
(125 µg SEmL −1 )indH2O or pcRF, and 4.0-mL quantities were
frozen, lyophilised and the residues were re-suspended in 2.0 mL of
methanol. Four replicates of each solution were prepared. Samples
were centrifuged (1000 × g; 10 min) to remove particulates prior
to assay.
Assay and test of post-reaction addition of dH2O. Aliquots of the
clear methanol solutions as prepared above were transferred
into 20-mL glass test tubes to produce duplicate series of 0,
5, 10, 20, 30 and 40 µg of steroidal saponin or smilagenin (to
model sapogenin) per tube. Tubes were placed in a 60 ◦ Cwater
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