ANTIFUNGAL SAPONINS FROM THE MAYA MEDICINE CESTRUM SCHLECHTENDAHLII
441
633 [M + Na]+ (100), 611 [M + H]+ (40), 449
[M À C6O5H11]+ (25), 431 [MÀC6H11O5 À H2O]+ (25),
413 [M ÀC6H11O5 À 2H2O]+ (70).
with 90% B for 3min and changed back to the initial con-
dition in 1min. The flow rate was set at 0.8mL/min with
the column temperature set at 50°C. The photodiode ar-
ray detector was set to monitoring wavelengths from 190
to 400nm. The mass spectrometer with ESI interface
was operating in positive and negative scan modes; the
nebulizing gas flow was set at 1.5L/min, and drying gas
flow was at 10L/min. The desolvation line temperature
and heat block temperature were set at 300 and 450°C,
respectively. The m/z range of both positive and negative
scan is from 150 to 600 with 938u/s scan speed.
Isolated saponins were analyzed using a Waters Xevo
G2 UPLC–QTOF–MS/MS system to confirm their
masses. UPLC conditions: Acquity BEH C18 1.7μm
2.1×100mm column connected with a VanGuard Pre-
column 2.1×5mm. Mobile phases were water+0.1%
formic acid for A and acetonitrile+ 0.1% formic acid for
B (Fisher Optima LC–MS); the flow rate was 0.5 mL/min,
column temperature set at 50°C, and sample temperature
at 10°C. Mobile phase composition was as follows:
0–1min 5% A isocratic, 1–6 min linear gradient 5–50% B,
6–8 min 50–95% B, 8.01–10min 5% A isocratic (total
run time 10min). Sample injection conditions: 1-μL injec-
tion followed by a strong wash 200μL (90% acetonitrile
+10% water) and weak wash 600μL (10% acetonitrile
+90% water). QTOF analysis conditions: MassLynx soft-
ware, MSe ESI+ mode, lock mass Leucine Enkephalin
12C 556.2615, source temperature 120°C, desolvation
304, temperature 400°C, cone gas (N2) flow 50L/h,
desolvation gas (N2) flow 1195L/h. MSe conditions: mass
range 100–1500Da, F1 CE, 6V, F2 CER 10–30V, cone
voltage 20V, scan time 1s. Calibration was done using
50–1000Da sodium formate.
Acid hydrolysis of compounds 1 and 2. Samples of 1
(10 mg) and 2 (1 mg) were dissolved separately in 5 mL
of 2 M HCl (dioxane–H2O 1:1). The solutions were
refluxed for 4 h at 100°C. The reaction mixture was
diluted with H2O and extracted three times with dichlo-
romethane (10 mL each). The organic layer from com-
pound 1 hydrolysis was passed through anhydrous
magnesium sulfate and dried in vacuo to give sapogenin
1
1A (6 mg). The structure of 1A was confirmed by H-
NMR and ESI–MS (see Supporting Information). The
aqueous phases from the hydrolysis of compounds 1
and 2 were neutralized by passing through an Amberlite
IRA-93ZU column (Organo, Japan) and concentrated
by lyophilization. The sugar identities were confirmed
by TLC methods [EtOAc:MeOH:CH3COOH:H2O
(11:2:2:2)] using authentic monosaccharide standards
(Sigma-Aldrich, St. Louis, MO, USA). TLC plates were
visualized with an anisaldehyde solution. This confirma-
tion of sugar identity by TLC has been used by previous
phytochemical studies involving saponins (Ahmad et al.,
1995; González et al., 2004; Lu et al., 2009). In support of
the TLC results, optical rotational measurements con-
firmed the presence of L-rhamnose ([αD] À13.6°] on 1
and D-galactose on both 1 and 2 ([αD] +18.5° and [αD]
+17.9°, respectively).
Phytochemical analyses. Chromatographic analyses of
the crude extract and fractions were performed on an
Agilent 1100 series HPLC system comprising a quater-
nary pump, a degasser, an autosampler with 100 μL
loop, a column thermostat, and a diode array detector
(DAD). The identification of the phenolics was corrob-
orated by comparing the retention time and maximum
UV absorption values with authentic commercial stan-
dards (Sigma-Aldrich, St. Louis, MO, USA). The
analyses were performed using a Luna C18 column
(250 mm × 4.6mm, 5 μm particle size) with column tem-
perature set at 55°C and a flow rate of 1.5 mL/min.
The mobile phase A was acetonitrile containing 0.05%
trifluoroacetic acid, and B was water containing 0.05%
trifluoroacetic acid. Optimized separation was achieved
with the following method: initial conditions of 5% A
and 95% B with an increasing gradient to 100% A in
25 min; the column was flushed with 100% A for 5 min
and then set back to the initial conditions. DAD was
set to monitor wavelengths 254, 280, and 330nm.
The active fractions were analyzed using a Shimadzu
UPLC–PDA–MS system (Mandel Scientific Company
Inc, Guelph, ON, Canada), which consisted of
LC30AD pumps, a CTO20A column oven, a SIL-
30 AC autosampler, and an LCMS-2020 mass spectrom-
eter with an electrospray ionization source. Briefly, 1 μL
of each fraction was injected through the autosampler to
an Acquity CSH C18 column (100 × 2.1 mm, 1.7 μm
particle size; Waters, Mississauga, ON, Canada) with an
Acquity CSH C18 VanGuard Pre-column (5×2.1mm).
The mobile phases were H2O (A) and acetonitrile (B).
The gradient elution method initiated with 5% B then in-
creased to 95% B in 5min. The column was then washed
Fungal strains. Three yeast-like fungal strains, Saccharo-
myces cerevisiae S288C, Cryptococcus neoformans, and
C. albicans D10, were used for disk diffusion assays. The
strains used to determine minimum inhibitory concentra-
tions (MICs) in liquid culture were as follows: S. cerevisiae
BY4741 (haploid), S. cerevisiae BY4743 (diploid), and
Fusarium graminearum [teleomorph Gibberella zeae
(Schwein.) Petch]. F. graminearum ZTE-2A (DAOM
227650), a gift from Robert Proctor (USDA), constitu-
tively expresses green fluorescent protein (Skadsen and
Hohn, 2004).
Antifungal disk diffusion assay. Saccharomyces cerevisiae
S288C, C. neoformans, and C. albicans D10 were cultured
in Sabouraud dextrose broth (Difco) at 30°C. Berberine
(95%, Sigma-Aldrich, St. Louis, MO, USA) and ketoco-
nazole (>98%, Sigma-Aldrich, St. Louis, MO, USA)
were used as antifungal positive controls and methanol
as a negative control. Overnight cultures were grown to
an optical density of 1.0 (~1.0×107 CFU/mL) at 600nm
and diluted 1:100. Aliquots (100μL) of this inoculum
were spread over the surface of Sabouraud agar plates.
Paper disks (7.0mm diameter) were loaded with crude
extract (2mg/disk), berberine (0.5mg/disk), ketoconazole
(90μg/disk), fractions (0.5mg/disk), saponin (0.5mg/
disk), or methanol (carrier solvent) and allowed to air
dry. The amended disks were placed treated side down
on the prepared medium and incubated in the dark at
30°C for 48h, prior to measurement of inhibition zones
Copyright © 2015 John Wiley & Sons, Ltd.
Phytother. Res. 30: 439–446 (2016)