Isolation and structural determination of steroidal glycosides from the bulbs of Easter lily (Lilium longiflorum Thunb.)

Article abstract of DOI:10.1021/jf101410d

The bulbs of the Easter lily (Lilium longiflorum Thunb.) are used as a food and medicine in several Asian cultures, and they are cultivated as an ornamental plant throughout the world. A new steroidal glycoalkaloid and two new furostanol saponins, along with two known steroidal glycosides, were isolated from the bulbs of L. longiflorum. The new steroidal glycoalkaloid was identified as (22R,25R)-spirosol-5-en-3β-yl O-α-l-rhamnopyranosyl-(1→2)- [6-O-acetyl-β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside. The new furostanol saponins were identified as (25R)-26-O-(β-d-glucopyranosyl)- furost-5-en-3β,22α,26-triol 3-O-α-l-rhamnopyranosyl-(1→2)- α-l-arabinopyranosyl-(1→3)-β-d-glucopyranoside and (25R)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-α-l-rhamnopyranosyl-(1→2)-α-l-xylopyranosyl-(1→3) -β-d-glucopyranoside. The previously known steroidal glycosides, (22R,25R)-spirosol-5-en-3β-yl O-α-l-rhamnopyranosyl-(1→2)- β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside and (25R)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4) -β-d-glucopyranoside were identified in L. longiflorum for the first time. These new compounds from L. longiflorum and the isolation methodologies employed can be used for studies on the biological role of steroidal glycosides in plant development and plantpathogen interactions, as well as for studies in food and human health, for which little is known.

Full text of DOI:10.1021/jf101410d

8806 J. Agric. Food Chem. 2010, 58, 8806–8813
DOI:10.1021/jf101410d
Isolation and Structural Determination of Steroidal Glycosides
from the Bulbs of Easter Lily (Lilium longiflorum Thunb.)
‡
,
J
OHN P. MUNAFO
J
R
.,† AHALYA
R
AMANATHAN,‡ LESLIE S. JIMENEZ
,†
AND
T
HOMAS J. GIANFAGNA*
†Department of Plant Biology and Pathology, Rutgers—The State University of New Jersey, 59 Dudley
Road, New Brunswick, New Jersey 08901, and ‡Department of Chemistry and Chemical Biology,
Rutgers—The State University of New Jersey, 610 Taylor Road, Piscataway, New Jersey 08854
The bulbs of the Easter lily (Lilium longiflorum Thunb.) are used as a food and medicine in several
Asian cultures, and they are cultivated as an ornamental plant throughout the world. A new steroidal
glycoalkaloid and two new furostanol saponins, along with two known steroidal glycosides, were
isolated from the bulbs of L. longiflorum. The new steroidal glycoalkaloid was identified as (22R,25R)-
spirosol-5-en-3β-yl O-R-
pyranoside. The new furostanol saponins were identified as (25R)-26-O-(β-
5-en-3β,22R,26-triol 3-O-R- -rhamnopyranosyl-(1f2)-R- -arabinopyranosyl-(1f3)-β-
side and (25R)-26-O-(β- -glucopyranosyl)-furost-5-en-3β,22R,26-triol 3-O-R- -rhamnopyranosyl-(1f2)-
R- -xylopyranosyl-(1f3)-β- -glucopyranoside. The previously known steroidal glycosides, (22R,25R)-
spirosol-5-en-3β-yl O-R- -rhamnopyranosyl-(1f2)-β- -glucopyranosyl-(1f4)-β- -glucopyranoside and
(25R)-26-O-(β- -glucopyranosyl)-furost-5-en-3β,22R,26-triol 3-O-R- -rhamnopyranosyl-(1f2)-β-
glucopyranosyl-(1f4)-β- -glucopyranoside were identified in L. longiflorum for the first time. These
L
-rhamnopyranosyl-(1f2)-[6-O-acetyl-β-
D
-glucopyranosyl-(1f4)]-β-
-glucopyranosyl)-furost-
-glucopyrano-
D-gluco-
D
L
L
D
D
L
L
D
L
D
D
D
L
D-
D
new compounds from L. longiflorum and the isolation methodologies employed can be used for studies
on the biological role of steroidal glycosides in plant development and plant-pathogen interactions,
as well as for studies in food and human health, for which little is known.
KEYWORDS: Lilium longiflorum Thunb.; Liliaceae; Easter lily; steroidal glycoside; steroidal glycoalkaloid;
furostanol saponin
INTRODUCTION
Extensive work has been done on the isolation and characteriza-
tion of steroidal saponins in Lilium. Steroidal saponins have been
reported in L. auratum Lindl. (6), L. brownii (2,7), L. brownii var.
viridulum (5, 8), L. candidum (9-11), L. hansonii (12), L. henryi
Baker (13), L. longiflorum (14), L. martagon L. (15), L. pardalinum
Kellogg (16), L. pensylvanicum (1), L. pumilum (6), L. regale E. H.
Wilson (13, 17, 18), L. speciosum var. speciosum (19), and
L. speciosum ꢀ L. nobilissimum (Makino) Makino (20). Steroidal
saponins have been reported to exhibit a wide range of biological
activities including antifungal (21), platelet aggregation inhibi-
tion (22, 23), antidiabetic (24), cholesterol lowering (25), anti-
inflammatory (26), antiviral (27), and anticancer (28-30).
Although the putative biological activities of steroidal saponins
are well documented, the biological role of these compounds
within the plant is poorly understood.
The occurrence of steroidal glycoalkaloids in the Liliaceae
family is also well documented (31). Steroidal alkaloids isolated
from Veratrum and Fritillaria species show various biological
activities including antihypertensive (32), anticholinergic (33),
antifungal (34), and anticancer (35). In Lilium, steroidal glycoalk-
aloids have been identified in L. philippinense Baker (36),
L. candidum L. (37), and L. brownii var. viridulum (5); however,
no steroidal glycoalkaloids have been previously reported from
L. longiflorum.
The Easter lily (Lilium longiflorum Thunb., family Liliaceae)
has beautiful white flowers and a delicate aroma and is appre-
ciated worldwide as an attractive ornamental. In addition to its
economic importance and popularity in horticulture, lily bulbs are
regularly consumed in Asia, as both food and medicine. The bulbs
of several Lilium species, including L. longiflorum, L. brownii F. E.
Br. ex Miellez var. viridulum Baker, L. pensylvanicum Ker Gawl.,
and L. pumilum DC, have been used traditionally in China as
sedatives, anti-inflammatory and antitussive agents, and a
general tonic (1, 2). The crude drug “Bai-he”, used in tradi-
tional Chinese medicine, is prepared from the bulbs of Lilium
sp. and is regularly used for lung ailments in China today.
Although the medicinal use of L. longiflorum is well documen-
ted, the compounds responsible for the reported properties are
not known.
Bulbs of the genus Lilium are a rich source of secondary
metabolites, including bitter phenylpropanoid glycosides identi-
fied in the bulbs of L. speciosum Thunb. (3), antitumor alkaloids
identified in L. hansonii Leichtlin ex D. D. T. Moore (4), and
steroidal glycoalkaloids identified in L. brownii var. viridulum (5).
*Author to whom correspondence should be addressed [e-mail
gianfagna@aesop.rutgers.edu; phone (731) 932-9711, ext. 252].
pubs.acs.org/JAFC
Published on Web 07/20/2010
© 2010 American Chemical Society

Article
With regard to the steroidal glycosides of L. longiflorum bulbs,
J. Agric. Food Chem., Vol. 58, No. 15, 2010 8807
six spirostanol saponins and three furostanol saponins with
antitumor activity have been reported (14). To set the stage for
biological activity studies on the role of steroidal glycosides in
plant development and plant-pathogen interactions, as well as
for studies in food and human health, this paper reports the
isolation and structural determination of several new steroidal
glycosides from the bulbs of L. longiflorum. The structures of the
steroidal glycosides were elucidated by a combination of spectro-
scopic and chemical analysis.
MATERIALS AND METHODS
Plant Material. L. longiflorum, cultivar 7-4, bulbs were provided from
the Rutgers University Easter lily breeding program. Bulbs were treated
with Captan (Bayer CropScience AG, Monheim am Rhein, Germany)
fungicide prior to planting. Bulbs were planted in beds containing Pro-Mix
(Premier Horticulture Inc., Quakertown, PA) soil mix and were grown to
mature plants under greenhouse conditions for 9 months prior to harvest.
The greenhouse temperatures were set to provide a minimum day temp-
erature of 24 °C and a minimum night temperature of 18 °C. Plants were
fertilized biweekly with a 100 mg/L solution of NPK 15-15-15 fertilizer
(J. R. Peters Inc., Allentown, PA). Each plant produced three to five new
bulbs, which we used for extraction. The new bulbs were full-sized, fresh,
and mature at harvest. Each plant was harvested by hand, and the bulbs
were manually separated, immediately frozen under liquid nitrogen,
lyophilized on a VirTis AdVantage laboratory freeze-dryer (SP Industries
Inc.,Warminster, PA), and stored at -80 °C until analysis.
Figure 1. RP-HPLC chromatogram (λ = 210 nm) of 1-5 separated from
L. longiflorum n-butanol extract fractionated by gel permeation chroma-
tography (GPC).
solvent mixture overnight. Chromatography was performed with isocratic
ethanol/water (70:30, v/v) at a flow rate of 3.5 mL min-1. The first 200 mL
of effluent was discarded, and 30 fractions (25 mL each) were collected and
analyzed by LC-MS. LC-MS analyses were performed on a HP 1100 series
HPLC (Agilent Technologies Inc., Santa Clara, CA) equipped with an
autoinjector, a quaternary pump, a column heater, and a diode array
detector and interfaced to a Bruker 6300 series ion-trap mass spectrometer
equipped with an electrospray ionization chamber. HP ChemStation and
BrukerData Analysis software were used for data acquisition and data
analysis. Reverse phase separations were performed using a Prodigy C18
column (250 mm ꢀ 4.6 mm i.d.; 5.0 μm particle size) (Phenomenex,
Torrance, CA). The flow rate was set to 1.0 mL min-1, and the column
temperature was at 23 ( 2 °C. The binary mobile phase composition
consisted of (A) 0.1% formic acid in deionized water and (B) 0.1% formic
acid in acetonitrile. Chromatography was performed using a linear
gradient of 15-43% B over 40 min and then to 95% B over 5 min;
thereafter, elution with 95% B was performed for 10 min. The re-
equilibration time was 10 min. All mass spectra were acquired in positive
ion mode over a scan range of m/z 100-2000. Ionization parameters
included capillary voltage, 3.5 kV; end plate offset, -500 V; nebulizer
pressure, 50 psi; drying gas flow, 10 mL min-1; and drying gas temperature,
360 °C. Trap parameters included ion current control, 30000; maximum
accumulation time, 200 ms; trap drive, 61.2; and averages, 12 spectra. On
the basis of the LC-MS profile, GPC fractions 6-17 were combined,
evaporated under reduced pressure (30 °C; 1.0 ꢀ 10-3 bar), and lyoph-
ilized, yielding GPC fraction A (180 mg).
Chemicals. The following compounds were obtained commercially:
(25R)-spirost-5-en-3β-ol, Sephadex LH-20, N,O-bis(trimethylsiyl)trifluo-
roacetamide with trimethylchlorosilane (99:1) silylating reagent, Dragendorff
reageant, p-(dimethylamino)benzaldehyde, hydrochloric acid, sodium hydro-
xide, pyridine-d5 (0.3% v/v TMS),
rhamnose, -(-)-rhamnose,
-(þ)-arabinose,
lose, and -(-)-xylose (Sigma-Aldrich, St. Louis, MO); and (22R,25R)-
D
-(þ)-glucose,
L
-(-)-glucose,
D
-(þ)-
L
D
L
-(-)-arabinose,
D
-(þ)-xy-
L
spirosol-5-ene-3β-ol (Glycomix Ltd., Reading, U.K.). All solvents
(acetonitrile, chloroform, ethanol, ethyl acetate, formic acid, n-butanol,
and n-pentane) were of chromatographic grade (Thermo Fisher Scientific
Inc., Fair Lawn, NJ). Water was deionized (18 MΩ cm) using a Milli-Q-
water purification system (Millipore, Bedford, MA).
Isolation and Purification of Steroidal Glycosides 1-5 from
L. longiflorum. Sequential Solvent Extraction of Lyophilized L. longi-
florum Bulbs. Lyophilized lily bulbs (100 g) were frozen in liquid nitrogen,
ground into a fine powder with a laboratory mill (IKA Labortechnik,
Staufen, Germany), and extracted with n-pentane (3 ꢀ 100 mL) on a wrist-
action autoshaker (Burrell Scientific, Pittsburgh, PA) at room temperature
for 30 min. After centrifugation (5000 rpm for 10 min) (Sorvall RC-3C
Plus, Thermo Fisher Scientific Inc.), the organic layers were discarded and
the pellet was freed from residual solvent in a fume hood overnight. The
residual defatted material was then extracted with a mixture of ethanol and
water (7:3, v/v; 2 ꢀ 150 mL) on an autoshaker for 45 min at room
temperature (25 °C). After centrifugation (5000 rpm for 10 min) and
vacuum filtration through a Whatman 114 filter paper (Whatman Inter-
national Ltd., Maidstone, U.K.), the supernatant was collected and the
residue discarded. The supernatant was then evaporated under reduced
pressure (30 °C; 1.0 ꢀ 10-3 bar) using a Laborota 4003 rotary evaporator
(Heidolph Brinkman LLC, Elk Grove Village, IL) and lyophilized,
yielding a crude bulb extract (13.7 g). The lyophilized crude bulb extract
was then dissolved in deionized water (100 mL) and washed with ethyl
acetate (5 ꢀ 100 mL), and the organic phase was discarded. The aqueous
phase was then extracted with n-butanol (5 ꢀ 100 mL) and the aqueous
phase discarded. The organic phase was then evaporated under reduced
pressure (30 °C; 1.0 ꢀ 10-3 bar) and lyophilized, yielding a crude glycoside
extract (2.42 g).
Semipreparative Reverse-Phase High-Performance Liquid Chromato-
graphy (RP-HPLC). Fractionation of GPC fraction A was achieved by
semipreparative RP-HPLC performed on a Luna C18 column (250 mm ꢀ
21.2 mm i.d.; 10 μm particle size) (Phenomenex) to afford 1-5 (Figure 1).
Chromatography was performed on a Shimadzu LC-6AD liquid chroma-
tograph (Shimadzu Scientific Instruments Inc., Columbia, MD) using a
UV-vis detector and a 2 mL injection loop. Mixtures of (A) 0.1% formic
acid in deionized water and (B) 0.1% formic acid in acetonitrile were used
as the mobile phase. The flow rate was set to 20 mL min-1, the column
temperature was 23( 2 °C, and UV detection was recorded at λ = 210 nm.
GPC fraction A was dissolved in a mixture of mobile phase A and mobile
phase B (75:25, v/v) and filtered through a 0.45 μm PTFE syringe filter
prior to injection. Chromatography was performed using a linear gradient
of 5-30% B over 45 min and then to 90% B over 10 min; thereafter,
elution with 90% B was performed for 10 min. The re-equilibration time
was 10 min. The target compounds were collected, freed from solvent
under reduced pressure (30 °C; 1.0 ꢀ 10-3 bar), and lyophilized. Final
purification of 1 and 2 was performed with an isocratic separation using a
mixture of 0.1% formic acid in deionized water and 0.1% formic acid in
acetonitrile (80:20, v/v). The target compounds were collected, freed from
solvent under reduced pressure (30 °C; 1.0 ꢀ 10-3 bar), and lyophilized,
yielding 1 (15 mg) and 2 (7 mg) as white amorphous powders in high purity
(>98%), as determined by LC-MS (Figure 2) and NMR. Final purification
of 3-5 was performed with an isocratic separation using a mixture of 0.1%
Gel Permeation Chromatography (GPC). Crude glycoside extract
(1.0 g) was dissolved in a solution of ethanol and water (7:3, v/v;
5.0 mL), filtered with a 0.45 μm PTFE syringe filter (Thermo Fisher
Scientific Inc.), and then applied onto a standard threaded 4.8 cm ꢀ 100
cm glass column (Kimble Chase Life Science and Research Products LLC,
Vineland, NJ) packed with Sephadex LH-20 (Amersham Pharmacia
Biotech, Uppsala, Sweden) that was washed and conditioned in the same

8808 J. Agric. Food Chem., Vol. 58, No. 15, 2010
Munafo et al.
helium as the carrier gas with a constant flow of 1 mL min-1. MS para-
meters were as follows: operatedin electron impact (EI) ionizationmode at
70 eV; scan range, m/z 50-550. The transfer line was maintained at 250 °C.
Comparisons were madewith retention times and mass spectra of aglycone
reference standards prepared according to the same procedure.
Sugar Composition Analysis. The aqueous fraction obtained after
hydrolysis was evaporated to dryness under reduced pressure (30 °C;
1.0 ꢀ 10-3 bar), dissolved in a mixture of pyridine and BSTFA with TMCS
(99:1) silylating reagent (1:1, v/v, 100 μL), and refluxed in a sealed tube
(60 °C for 1 h). After cooling to room temperature, the solution was
analyzed by GC-MS. The temperature program was as follows: 50 °C for
6 min, then increased at 4 °C/min to 160 °C, and held for 5 min. The other
parameters used were a splitless injector heated to 200 °C and helium as the
carrier gas with a constant flow of 1 mL min-1. MS parameters were the
same as reported above. Identifications were made based on retention
times and mass spectra of sugar standards prepared according to the same
procedure.
Determination of Sugar Absolute Configurations. Absolute configura-
tion of sugars was determined by enantioselective GC-FID. A chiral RT-
BetaDEXsm capillary column (30 m ꢀ 0.25 mm ꢀ 0.25 μm; Restek Corp.,
State College, PA) was used for chromatographic separation. The tem-
perature program was as follows: 60 °C for 0 min, then increased at 4 °C/
min to 160 °C, then increased at 15 °C/min to a final temperature 230 °C,
and maintained for 15 min. The other parameters used were as follows:
FID detector heated to 230 °C; split injector with a 10:1 split ratio
maintained at 230 °C; helium as the carrier gas with a constant flow of
1 mL min-1. Comparisons were made with retention times of optically
pure sugar standards prepared following the same procedure.
Figure 2. (A) Total ion chromatogram (TIC) of crude L. longiflorum
n-butanol extract. (B-F) Total ion chromatograms (TIC) of 1-5 isolated
by RP-HPLC.
formic acid in DI water and 0.1% formic acid in acetonitrile (75:25, v/v).
The target compounds were collected, freed from solvent under reduced
pressure (30 °C; 1.0 ꢀ 10-3 bar), and lyophilized, yielding 3 (25 mg), 4
(7 mg), and 5 (5 mg) as white amorphous powders in high purity (>98%),
as determined by LC-MS (Figure 2) and NMR.
Thin Layer Chromatography (TLC). Each compound (1 mg) was
dissolved in methanol (0.5 mL), spotted on a 20 cm ꢀ20 cm silica gel
60 F254 TLC plate (Merck & Co., Inc., Whitehouse Station, NJ), and
developed with chloroform/methanol/water (8:4:1, v/v/v). To detect
furostanols, TLC plates were developed with Ehrlich’s reagent [3.2 g of
p-(dimethylamino)benzaldehydein 60 mL of 95%ethanol and 60 mL of 12
N HCl] and heated to 110 °C for 5 min. Bright red spots were indicative of
a positive reaction. To detect alkaloids, the TLC plates were developed
with Dragendorff’s reagent and heated to 110 °C for 5 min. Orange spots
were indicative of a positive reaction.
Identification of Compounds 1-5. Compounds 1-5 were identified
by a combination of spectroscopic data (1H NMR, 13C NMR, HMBC,
HMQC, MS, IR), chromatographic data, and chemical analysis. Melting
points were obtained using a Thomas-Hoover Capillary Melting Point
Apparatus (Arthur H. Thomas Co., Philadelphia, PA) and by differential
scanning calorimetry using a Perkin-Elmer Diamond DSC (Perkin-Elmer,
Waltham, MA). IR spectra were recorded on a Nexus 670 FT-IR spectro-
photometer. Observed rotations were obtained on a Perkin-Elmer model
341LC polarimeter. High -resolution mass spectra (HRMS) were recorded
on a BioTOF II ESI under the following conditions: source temperature,
150 °C; acceleration voltage, 8500; mass resolution, 10000 fwhm; scan
range, m/z 100-1000; drying gas, N2. ESIþ-MS spectra were recorded on a
Bruker 6300 series ion-trap mass spectrometer under the conditions
reported above. 1D 1H NMR and 13C NMR spectra and 2D heteronuclear
multiple bond coherence (HMBC) and heteronuclear multiple quantum
coherence (HMQC) spectra were acquired on an AMX-400 spectrometer
and an AMX-500 spectrometer (Bruker, Rheinstetten, Germany). Sam-
ples for NMR analysis were dissolved in pyridine-d5, and chemical shifts
are given as δ values with reference to tetramethylsilane (TMS).
Compound 1, (22R,25R)-spirosol-5-en-3β-yl O-R-
(1f2)-β- -glucopyranosyl-(1f4)-β- -glucopyranoside: amorphous solid;
L-rhamnopyranosyl-
D
D
[R]28 D -76.9 (c 0.02; MeOH); mp 303 °C (dec); IR νmax (film) cm-1 3336
(OH), 2930 (CH), 1585, 1450, 1347, 1255, 1029, 984, 897, 811; HRESI-
TOFMS, m/z 884.5028 [M þ H]þ (calculated for C45H74NO16, 884.5002);
ESIþ-MS, m/z 884.7 (100, [M þ H]þ), 738.5 (3, [M - Rha þ H]þ), 576.4
(19, [M - Glu - Rha þ H]þ), 453.8 (1.6, [M þ H þ Na]þ2), 414.6 (15,
[M - 2Glu - Rha þ H]þ) (Figure 3); 1H NMR (400 MHz) δH 0.82 [d, 3H,
J = 5.2 Hz, 27-H], 0.88 [s, 3H, 18-H], 1.06 [s, 3H, 19-H], 1.09 [d, 3H, J =
6.8 Hz, 21-H], 1.78 [d, 3H, J = 6.4 Hz, 600-H], 2.75 [m, 2H, 26-H], 3.88 [m,
1H, 3-H], 3.90 [m, 1H, 20-H], 4.33 [1H, 6b000-H], 4.56-4.40 [1H, 6a000-H],
4.48 [m, 2H, 60-H], 4.61 [dd, 1H, J = 9.2, 3.2, 300-H], 4.97 [d, 1H, J = 7.2
Hz, 10-H], 5.15 [d, 1H, J = 7.6 Hz, 1000-H], 5.30 [d, 1H, J = 4.8 Hz, 6-H],
6.27 [s, 1H, 100-H]; 13C NMR (400 MHz, pyridine-d5) δ 37.5 [C-1], 30.2
[C-2], 78.1 [C-3], 38.9 [C-4], 140.8 [C-5], 121.8 [C-6], 32.4 [C-7], 31.6 [C-8],
50.3 [C-9], 37.2 [C-10], 21.2 [C-11], 40.1 [C-12], 40.7 [C-13], 56.7 [C-14],
32.6 [C-15], 78.8 [C-16], 63.6 [C-17], 16.5 [C-18], 19.4 [C-19], 41.6 [C-20],
15.7 [C-21], 98.4 [C-22], 34.7 [C-23], 31.1 [C-24], 31.7 [C-25], 48.1 [C-26],
19.90[C-27], 100.0 [C-10], 77.8 [C-20], 76.2 [C-30], 82.1 [C-40], 77.3 [C-50], 62.0
[C-6 ], 101.8 [C-100], 72.5 [C-200], 72.8 [C-300], 74.2 [C-400], 69.5 [C-500], 18.7
[C-600], 105.3 [C-1000], 75.0 [C-2000], 78.3 [C-3000], 71.2 [C-4000], 78.5 [C-5000],
62.1 [C-6000]; 1H NMR and 13C NMR are consistent with the literature (5).
Acid Hydrolysis of Compounds 1-5. A solution of each compound
(1 mg) in 1 N HCl in methanol (0.5 mL) was refluxed at 80 °C for 2 h. After
hydrolysis, the solution was adjusted to pH 7 with NaOH (4 N) and
evaporated to dryness under reduced pressure (30 °C; 1.0 ꢀ 10-3 bar). The
residue was dissolved in water (1 mL) and extracted withn-pentane (2 mL).
The n-pentane phase was used for aglycone analysis, and the aqueous
phase was used for sugar analysis.
Aglycone Analysis. The n-pentane phase obtained after hydrolysis was
evaporated to dryness under reduced pressure (30 °C; 1.0 ꢀ 10-3 bar),
dissolved in a mixture of pyridine and BSTFA with TMCS (99:1) silylating
reagent (1:1, v/v, 100 μL), and refluxed in a sealed tube (60 °C for 1 h).
After cooling to room temperature, the solution was analyzed by GC-MS.
An Agilent 6890 series GC system coupled to an Agilent 5973 mass spec
detector (Santa Clara, CA) was used for GC-MS analysis. A capillary
column (HP-5, 5% phenyl, 95% dimethyl polysiloxane stationary phase,
30 m ꢀ 0.25 mm i.d. ꢀ 0.25 mm film thickness) was used for the
chromatographic separation. The temperature program was as follows:
70 °C for 2 min, then increased 8 °C/min to 240 °C, and held for 10 min.
The other parameters used were splitless injector heated at 250 °C and
Compound 2, (22R,25R)-spirosol-5-en-3β-yl O-R-
(1f2)-[6-O-acetyl-β- -glucopyranosyl-(1f4)]-β- -glucopyranoside: amor-
L-rhamnopyranosyl-
D
D
phous solid; [R] D28 -32.0° (MeOH; c 0.05); mp 285 °C (dec); IRνmax (film)
cm-1 3353 (OH), 2932 (CH), 1732 (CdO), 1588, 1451, 1370, 1252, 1033,
983, 897, 813; HRESI-TOFMS m/z 926.5085 [M þ H]þ (calculated for
C47H76NO17, 926.5108); ESIþ-MS, m/z 926.6 (100, [M þ H]þ), 780.5 (14,
[M - Rha þ H]þ), 576.4 (15, [M - Glu - Ac - Rha þ H]þ), 474.8 (3,
[M þ H þ Na]2þ), 414.3 (30, [M - 2Glu - Ac - Rha þ H]þ) (Figure 3); 1H
NMR (400 MHz) δH 0.82 [d, 3H, J = 4.8 Hz, 27-H], 0.88 [s, 3H, 18-H],
1.06 [s, 3H, 19-H], 1.11 [d, 3H, J = 6.4 Hz, 21-H], 1.78 [d, 3H, J = 6 Hz,
600-H], 2.06 [s, 3H, CO-CH3], 4.35 [t, 1H, J = 9.4, 400-H], 4.60 [dd, 1H,

Article
J. Agric. Food Chem., Vol. 58, No. 15, 2010 8809
Figure 3. ESIþ-MS mass spectra of 1-5.
J = 8.8, 3.2, 300-H], 4.64 [d, 1H, J = 8.4 Hz, 6b000-H], 4.92 [dd, 1H, J =
11.6, 2, 6a000-H], 4.98 [d, 1H, J = 7.2 Hz, 10-H], 5.09 [d, 1H, J = 8.1000-H],
5.29 [d, 1H, J = 4.4 Hz, 6-H], 6.24 [br s, 1H, 100-H]; for 13C NMR (500
MHz, pyridine-d5) spectroscopic data, see Table 1.
32.5 [C-7], 31.7 [C-8], 50.4 [C-9], 37.2 [C-10], 21.2 [C-11], 39.9 [C-12], 40.8
[C-13], 56.6 [C-14], 32.4 [C-15], 81.1 [C-16], 63.9 [C-17], 16.5 [C-18], 19.4
[C-19], 40.7 [C-20], 16.5 [C-21], 110.7 [C-22], 37.1 [C-23], 28.4 [C-24], 34.3
[C-25], 75.3 [C-26], 17.5 [C-27], 100.0 [C-10], 77.8 [C-20], 76.2 [C-30], 82.1
[C-40], 77.3 [C-50], 62.1 [C-60], 101.8 [C-10], 72.5 [C-200], 72.8 [C-300], 74.2
[C-400], 69.5 [C-500], 18.7 [C-600], 105.2 [C-1000], 75.0 [C-2000], 78.5 [C-3000],
71.2 [C-4000], 78.3 [C-5000], 61.9 [C-6000], 105.0 [C-10000], 75.2 [C-20000], 78.6
[C-30000], 71.7 [C-40000], 78.2 [C-50000], 62.8 [C-60000]; 1H NMR and 13C NMR
are consistent with the literature (12).
Compound 3,(25R)-26-O-(β-
D
-glucopyranosyl)-furost-5-en-3β,22R,26-triol
3-O-R- -rhamnopyranosyl-(1f2)-β-
L
D
-glucopyranosyl-(1f4)-β- -gluco-
D
pyranoside: amorphous solid; [R]28 D -60.0 (c 0.05; MeOH); mp 207 °C
(dec); IR νmax (film) cm-1 3347 (OH), 2893 (CH), 1662, 1377, 1256, 1027,
909, 839, 812; HRESI-TOFMS m/z 1087.5296 [M þ Na]þ (calculated for
C51H84O23Na, 1087.5307); ESIþ-MS, m/z 1087.7 (20, [M þ Na]þ), 1047.7
(100, [M - 18 þ H]þ), 901.5 (9, [M - 18 - Rha þ H]þ), 739.4 (17, [M -
18 - Glu - Rha þ H]þ), 577.4 (11, [M - 18 - 2Glu - Rha þ H]þ), 415.3
(9, [M - 18 - 3Glu - Rha þ H]þ) (Figure 3); 1H NMR (500 MHz) δH 0.91
[s, 3H, 18-H], 1.00 [d, 3H, J = 6.4 Hz, 27-H], 1.07 [s, 3H, 19-H], 1.35 [d,
3H, J = 6.8 Hz, 21-H], 1.76 [d, 3H, J = 6.5 Hz, 600-H], 4.56 [dd, 1H, J =
12, 2.5, 60000-H], 4.60 [dd, 1H, J = 9.5, 3.5, 300-H], 4.83 [d, 1H, J = 8 Hz,
Compound 4, (25R)-26-O-(β-
triol 3-O-R- -rhamnopyranosyl-(1f2)-R-
glucopyranoside: amorphous solid; [R]
D-glucopyranosyl)-furost-5-en-3β,22R,26-
L
L
-arabinopyranosyl-(1f3)-β-
-48.6° (MeOH; c 0.07); mp
D-
28
D
200 °C (dec); IR νmax (film) cm-1 3367 (OH), 2899 (CH), 1699, 1377, 1256,
1040, 912, 840, 811, 780; HRESI-TOFMS m/z 1057.5211 [M þ Na]þ
(calculated for C50H82O22Na, 1057.5190); ESIþ-MS, m/z 1057.7 (5, [M þ
Na]þ), 1017.7 (100, [M - 18 þ H]þ), 871.6 (7, [M - 18 - Rha þ H]þ),
739.4 (13, [M - 18 - Ara - Rha þ H]þ), 577.2 (7, [M - 18 - Ara - Rha -
Glu þ H]þ), 415.2 (4, [M - 18 - 2Glu - Ara - Rha þ H]þ) (Figure 3); 1H
NMR (400 MHz) δH 0.91 [s, 3H, 18-H], 1.00 [d, 3H, J = 6.4 Hz, 27-H],
1
0000-H], 4.96 [d, 1H, J = 6.5 Hz, 10-H], 5.14 [d, 1H, J = 8, 1000-H], 5.29 [d,
1H, J = 4 Hz, 6-H], 6.26 [br s, 1H, 100-H]; 13C NMR (400 MHz, pyridine-
d5) δ 37.5 [C-1], 30.2 [C-2], 78.2 [C-3], 39.0 [C-4], 140.8 [C-5], 121.9 [C-6],

8810 J. Agric. Food Chem., Vol. 58, No. 15, 2010
Munafo et al.
Table 1. 13C NMR Chemical Shift Assignments for 2, 4, and 5 in Pyridine-d5
(dec); IR νmax (film) cm-1 3362 (OH), 2898 (CH), 1636, 1377, 1256, 1035,
912, 838, 811; HRESI-TOFMS m/z 1057.5242 [M þ Na]þ (calculated for
C50H82O22Na, 1057.5190); ESIþ-MS, m/z 1057.7 (2, [M þ Na]þ), 1017.7
(100, [M - 18 þ H]þ), 871.6 (7, [M - 18 - Rha þ H]þ), 739.4 (9, [M - 18 -
Xyl - Rha þ H]þ), 577.2 (3, [M - 18 - Xyl - Rha - Glu þ H]þ), 415.2 (2,
[M - 18 - 2Glu - Xyl - Rha þ H]þ) (Figure 3); 1H NMR (500 MHz) δH
0.91 [s, 3H, 18-H], 1.00 [d, 3H, J = 6.5 Hz, 27-H], 1.08 [s, 3H, 19-H], 1.35
[d, 3H, J = 7 Hz, 21-H], 1.77 [d, 3H, J = 6.5 Hz, 600-H], 4.83 [d, 1H, J = 8
Hz, 10000-H], 4.89-4.88 [m, 1H, 16-H], 4.99 [d, 1H, J = 7 Hz, 10-H], 5.01 [d,
1H, J = 5.5, 1000-H], 5.33 [d, 1H, J = 4 Hz, 6-H], 6.34 [br s, 1H, 100-H]; for
13C NMR (400 MHz, pyridine-d5) spectroscopic data, see Table 1.
compound
carbon
C-1
2
4
5
37.5
30.2
78.1a
39.0
140.8
121.9
32.4
31.6b
50.4
37.2
21.2
40.1
40.7
56.7
32.6
79.0
63.5
16.5
19.4
41.7
15.7
98.4
34.7
31.0
31.7b
48.0
19.8
99.9
77.6
76.1
83.3
77.4
62.0
102.0
72.5
72.8
74.2
69.6
18.7
105.6
75.1
78.2a
71.9
74.9
64.8
37.5
30.1
77.7a
38.7
140.7
121.9
32.4
31.7
50.3
37.2
21.1
39.9
40.8b
56.6
32.5
81.1
63.9
16.5
19.4
40.7b
16.5
110.7
37.2
28.4
34.3
75.3c
17.5
99.9
78.0
88.0
69.7
77.6a
62.4
102.5
72.5
72.9
74.1
69.5
18.7
105.6
72.3
74.6
69.7
67.8
37.5
30.1
77.7a
38.7
140.7
121.9
32.4
31.7
50.3
37.2
21.1
39.9
40.8b
56.6
32.5
81.1
63.9
16.5
19.4
40.7b
16.5
110.7
37.2
28.4
34.3
75.3c
17.5
100.0
78.0
88.2
69.7
77.7a
62.4
102.4
72.5
72.9
74.1
69.5
18.7
105.5
74.7
78.4
70.7
67.3
C-2
C-3
C-4
C-5
C-6
C-7
RESULTS AND DISCUSSION
C-8
C-9
Structure Elucidation. Lyophilized lily bulbs were washed with
n-pentane and extracted with ethanol and water. After the
removal of the solvent, the extract was dissolved in deionized
water, washed with ethyl acetate, and extracted with n-butanol.
The organic phase was evaporated under reduced pressure and
lyophilized, yielding a crude steroidal glycoside extract. The crude
glycoside extract was fractionated by GPC (Sephadex LH-20)
and repeated semipreparative RP-HPLC to yield compounds
1-5 (Figure 2). On the basis of 1H NMR, 13C NMR, 2D NMR
(HMQC and HMBC), HRESI-TOFMS, and chemical analysis,
including GC-MS analysis of the sugar and aglycone TMSi
derivatives after acid hydrolysis, 1 and 3 were identified as
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-10
(22R,25R)-spirosol-5-en-3β-yl O-R-
β- -glucopyranosyl-(1f4)-β- -glucopyranoside, previously iso-
lated from L. brownii var. viridulum (5), and (25R)-26-O-(β-
glucopyranosyl)-furost-5-en-3β,22R,26-triol 3-O-R- -rhamnopy-
ranosyl-(1f2)-β- -glucopyranosyl-(1f4)-β- -glucopyranoside,
L-rhamnopyranosyl-(1f2)-
D
D
D-
L
D
D
previously isolated from Dioscorea deltoidea Wall. ex Griseb.,
Ophiopogon planiscapus Nakai, L. hansonii, and Allium nutans
L. (38, 39, 12, 40) (Figure 5). This is the first report of these
compounds isolated from the bulbs of L. longiflorum.
C-20
C-30
C-40
C-500
Compound 2 was obtained as a white amorphous powder. The
compound was positive to the Dragendorff reaction, indicative of
an alkaloid (5). The IR spectrum showed absorption at 1732 and
3353 cm-1 due to the presence of an acetyl group and hydroxyl
groups. HRESI-TOFMS showed a [M þ H]þ ion at m/z 926.5085
(calculated for C47H76NO17, 926.5108). Additionally, a [M þ H þ
Na]2þ ion was observed at m/z 474.7520 (calculated for C47H76-
NO17Na, 474.7500). Thus, the molecular formula was calculated
as C47H75NO17, suggestive of an acetylated steroidal glycoalk-
C-600
C-100
C-200
C-300
C-400
C-500
C-6000
C-1
C-2000
C-3000
C-4000
C-5000
C-6000
C-10000
C-20000
C-30000
C-40000
C-50000
C-60000
Ac—CH3
Ac—CdO
1
aloid. The aglycone was readily deduced from H NMR, 13C
NMR, ESIþ-MS, and chemical analysis. The 1H NMR spectrum
showed two singlets at δH 1.06 and 0.88, which are indicative of
tertiary methyl groups of the spirosolane skeleton. Furthermore,
two doublet signals at δH 1.11 and 0.82 were assignable to secondary
methyl groups. A quaternary carbon signal at δH 98.4 and olefinic
carbon signals at δC 140.8 and 121.9 in the 13C NMR spectrum
are consistent with a Δ5 spirosolane aglycone. The unsaturation
between C-5 and C-6 was further substantiated by a doublet at δH
105.0
75.2c
78.6d
71.7
78.5d
62.8
105.0
75.2c
78.6d
71.7
78.5d
62.8
1
20.8
170.9
5.29 in the H NMR spectrum for the H-6 signal. Upon acid
hydrolysis, derivatization, and GC-MS analysis, the retention
time and mass spectrum of the TMSi derivative of the aglycone
were consistent with those of (22R,25R)-spirosol-5-en-3β-ol,
which was prepared following the same procedure. The structure
of the oligosaccharide moiety was readily deduced from 1H
NMR, 13C NMR, HMBC, ESIþ-MS, and chemical analysis.
Three anomeric protons were observed at δH 4.98, 6.24, and 5.09,
which implied the presence of three saccharide residues. Coupling
constants for the anomeric proton resonances suggested β-inter-
glycosidic linkages. Additionally, the 13C NMR spectrum con-
tained three anomeric carbon signals observed at δC 105.6, 102.0,
and 99.9, consistent with the presence of three saccharide residues.
The HMBC experiment showed long-range correlations between
a Assignments may be interchanged in each column. b Assignments may be
interchanged in each column. c Assignments may be interchanged in each column.
d Assignments may be interchanged in each column.
1.07 [s, 3H, 19-H], 1.35 [d, 3H, J = 6.8 Hz, 21-H], 1.76 [d, 3H, J = 6 Hz,
600-H], 3.08 [d, 1H, J = 11.6, 5000-H], 4.05 [m, 1H, Hz, 20000-H], 3.90-3.85
[m, 1H, 20-H], 4.84 [d, 1H, J = 7.6 Hz, 10000-H], 4.91 [m, 1H, 16-H], 4.92 [d,
1H, J = 7.6, 1000-H], 4.99 [d, 1H, J = 7.2 Hz, 10-H], 5.33 [d, 1H, J = 4 Hz,
6-H], 6.30 [br s, 1H, 100-H]; for 13C NMR (400 MHz, pyridine-d5)
spectroscopic data, see Table 1.
Compound 5, (25R)-26-O-(β-
triol 3-O-R- -rhamnopyranosyl-(1f2)-R-
copyranoside: amorphous solid; [R] D28 -46.4° (MeOH; c 0.03); mp 200 °C
D
-glucopyranosyl)-furost-5-en-3β,22R,26-
L
L
-xylopyranosyl-(1f3)-β- -glu-
D

Article
J. Agric. Food Chem., Vol. 58, No. 15, 2010 8811
Figure 4. Partial HMBC spectrum of 4, showing the correlation between
H-21 and the carbon signals of C-20, C17, and C-22.
the anomeric proton signal at δH 4.98 [H-10] and the carbon signal
at δC 78.1 [C-3], between the anomeric proton signal at δH 5.09
[H-1000] and the carbon signal at δC 83.3 [C-40], and the between
anomeric proton signal at δH 6.24 [H-100] and the carbon signal at
δC 77.6 [C-20]. Upon acid hydrolysis, derivatization, and GC-MS/
enantioselective GC-FID analysis, the sugars of the trisaccharide
Figure 5. Structures of steroidal glycosides prepared from L. longiflorum
bulbs.
C50H82O22, consistent with a furostanol saponin. The aglycone
1
moiety were identified as
D
-(þ)-glucose and
L
-(-)-rhamnose in a
was readily deduced from H NMR, 13C NMR, HMBC, ESI-
2:1 ratio. The 1H NMR spectrum showed a doublet integrating
for three protons at δH 1.78, indicative of the methyl group of
rhamnose. The ESIþ-MS mass spectrum showed the protonated
molecular ion 926.6 [M þ H]þ and the protonateddoublecharged
sodium adduct at 474.8 [M þ H þ Na]2þ. Additionally, ion frag-
ments at m/z 780.5 [M - Rha þ H]þ, 576.4 [M - Glu - Ac -
Rha þ H]þ, and 414.3 [M - 2Glu - Ac - Rha þ H]þ were
observed and were consistent with a trisaccharide moiety contain-
MS, and chemical analysis. The 1H NMR spectrum showed two
singlets at δH 1.07 and 0.91, which are indicative of the tertiary
methyl groups of the furostane skeleton. Furthermore, two
doublet signals at δH 1.35 and 1.0 were assignable to secondary
methyl groups. (25R)-26-O-β-
furost-5-en-3β,26-diol 3-O-R-
arabinopyranosyl-(1f3)-β- -glucopyranoside, previously identi-
D-glucopyranosyl-22ξ-methoxy-
L
-rhamnopyranosyl-(1f2)-R-
L-
D
fied in L. longiflorum possessing antitumor activity, contains a
methoxy group at the C-22 position with a -OCH3 signal at δC
47.73, which is missing in 4. Also, the quaternary carbon signal
for C-22 had a minor upfield shift to δC 110.7 instead of the
reported value of δC 112.7 for the C-22 methoxy derivative (14).
Long-range coupling was observed between the methyl proton
signal at δH 1.35 [H-21] and the carbon signals at δC 40.7 [C-20],
63.9 [C-17], and 110.7 [C-22], supporting that the shift seen in this
region is consistent with a C-22 hydroxy compared to the C-22
methoxy derivative with the reported values of δC 40.5 [C-20],
64.2 [C-17], and 112.7 [C-22] (14) (Figure 4). The carbon peaks
from C-5 to C-27 were similar to those reported for (25R,S)-26-O-
ing
D
-(þ)-glucose, an acetylated
D
-(þ)-glucose, and a
L
-(-)-
rhamnose moiety (Figure 3). The presence of an acetyl group
was shown by the IR absorption at 1732 cm-1, 1H NMR δH 2.06
(s, 3H, CO-CH3), and 13C NMR δC 170.9 and 20.8. The carbon
signals corresponding to the saccharide moiety of 2 were similar
to those reported for (25R,26R)-26-methoxyspirost-5-en-3β-yl
O-R-
L
-rhamnopyranosyl-(1f2)-[6-O-acetyl-β-
D-glucopyranosyl-
(1f4)]-β-
D
-glucopyranoside, isolated from L. speciosum ꢀ L.
nobilissimum (20). Alkaline hydrolysis of 2 with 1 N sodium
hydroxide yielded 1. Upon comparison of the 13C NMR spectra
of 1 and 2, the C-6000 and C-4000 signals were shifted from δC 62.1
and 71.2 to δC 64.8 and 71.9, respectively. The signal for C-5000
was shifted from δC 78.5 to 74.9, and all other carbon signals were
similar. Upon comparison of the 1H NMR spectra of 1 and 2, the
signals assignable to H2-6000 methylene protons of the terminal
glucose were shifted to a lower field as compared to those of 2.
Thus, the acetyl moiety was linked to the C-6 hydroxy position of
the terminal glucose unit. Accordingly, the structure of 2 was
β-
D
-glucopyranosyl-furost-5-en-3β,22R,26-triol 3-O-β-
D-galacto-
-glucopyranosyl-(1f4)-β- -galactopyra-
pyranosyl-(1f2)-O-β-
D
D
noside, isolated from Tribulus terrestris L., which suggests the
presence of an R-hydroxy group at the C-22 position of the
furostane skeleton (41). Consistent with (25R)-26-O-β-
pyranosyl-furost-5-en-3β,22R-triol 3-O-β- -glucopyranosyl-(1f2)-
β- -glucopyranosyl-(1f4)-β- -glucopyranoside, isolated from
D-gluco-
D
D
D
determined to be (22R,25R)-spirosol-5-en-3β-yl O-R-
L
-rhamnop-
D
the rhizomes of Tupistra chinensis Bak., the peak of C-25 at δC
34.3 as compared to δC 34.42 suggests an R configuration (42).
Olefinic carbon signals at δC 140.7 and 121.9 in the 13C NMR
spectrum were consistent with a Δ5 furostane skeleton. The
unsaturation between C-5 and C-6 is further substantiated by
an olefinic proton signal at δH 5.33 in the 1H NMR spectrum for
H-6. Upon acid hydrolysis, derivatization, and GC-MS analysis,
the retention time and mass spectrum of the TMSi derivative of
the aglycone were consistent with that of (25R)-spirost-5-en-3β-ol,
which was prepared following the same procedure. The structure
yranosyl-(1f2)-[6-O-acetyl-β-
D
-glucopyranosyl-(1f4)]-β- -gluco-
pyranoside (Figure 5).
Compound 4 was obtained as a white amorphous powder. The
compound was positive to Ehrlich’s reaction, indicative of a furo-
stanol saponin (39). The IR spectrum showed absorption at 3367
cm-1 due to the presence of hydroxyl groups. HRESI-TOFMS
showed a [M þ Na]þ ion at m/z 1057.5211 (calculated for C50H82-
O22Na, 1057.5190). Additionally, a [M - H]- ion was observed at
m/z 1033.6. Thus, the molecular formula was calculated as

8812 J. Agric. Food Chem., Vol. 58, No. 15, 2010
Munafo et al.
of the oligosaccharide moiety was readily deduced from 1H
NMR, 13C NMR, HMBC, ESIþ-MS, and chemical analysis.
All four sugars of 4 have carbon NMR values similar to those
glycoalkaloids that contain naturally occurring acetylated sac-
charides. Interestingly, the presence or absence of the acetyl
moiety of the S. chacoense glycoalkaloids markedly affected resi-
stance to foliar feeding of both adults and larvae of L. decemlineata.
Differences in acetylation of the terminal glucose of the trisac-
charide moiety of 1 and 2 may also play a biological role in
L. longiflorum. Work is currently underway to compare the
antifungal activity of these compounds against common lily
pathogens by quantifying the ability of each pathogen to cleave
the sugar residues of 1 and 2.
The furostanol saponins 3-5 are similar in structure except for
the terminal monosaccharide residues and interglycosidic lin-
kages. 3 has a hexose as the terminal sugar linked via the C-40
carbon of the inner glucose, whereas both 4 and 5 contain a
pentose as the terminal sugar linked via the C-30 carbon of the
inner glucose. In fact, differences in oligosaccharide composition
and interglycosidic linkages have been shown to affect the
biological activity of steroidal saponins possessing the same
aglycone moiety (46). Differences in oligosaccharide composition
and interglycosidic linkages in 3-5 may also play a role in the
biology of these compounds in L. longiflorum.
reported for (25R)-26-O-β-
5-en-3β,26-diol 3-O-R-
binopyranosyl-(1f3)-β-
-glucopyranoside (14). The 1H NMR
D-glucopyranosyl-22ξ-methoxy-furost-
L
-rhamnopyranosyl-(1f2)-R- -ara-
L
D
spectrum contained four anomeric proton signals observed at δH
6.30, 4.99, 4.92, and 4.84. The coupling constants of the anomeric
proton resonances suggested β-interglycosidic linkages. The car-
bon signals were at δC 105.6, 105.0, 102.5, and 99.9, which is
consistent with the presence of four saccharide residues. The
HMBC experiment showed long-range correlations between the
anomeric proton signal at δH 4.99 [H-10] and the carbon signal at
δC 77.7 [C-3], between the anomeric proton signal at δH 4.92
[H-1000] and the carbon signal at δC 88 [C-30], and between the
anomeric proton signal at δH 6.30 [H-100] and the carbon signal at
δ
C 78 [C-20]. Upon acid hydrolysis, derivatization, and GC-MS/
enantioselective GC-FID analysis, the sugars were identified as
-(-)-rhamnose, and -(-)-arabinose in a 2:1:1
D
-(þ)-glucose,
L
L
ratio. The 1H NMR spectrum showed a doublet integrating for
three protons at δH 1.76, indicative of the methyl group of
rhamnose. The ESIþ-MS mass spectrum showed a base ion peak
at 1017.7 [M - 18 þ H]þ and the sodium adduct at 1057.7 [M þ
Na]þ. Additionally, ion fragments at m/z 871.6 [M - 18 - Rha þ
H]þ, 739.4 [M - 18 - Ara - Rha þ H]þ, 577.2 [M - 18 - Ara -
Rha - Glu þ H]þ, and 415.2 [M - 18 - 2Glu - Ara - Rha þ
H]þ were observed and were consistent with 4 being bidesmodic
with the trisaccharide moiety at the C-3 position containing
In the present work, a new acetylated steroidal glycoalkaloid,
(22R,25R)-spirosol-5-en-3β-yl O-R-
[6-O-acetyl-β- -glucopyranosyl-(1f4)]-β-
two new furostanol saponins, (25R)-26-O-(β-
furost-5-en-3β,22R,26-triol 3-O-R- -rhamnopyranosyl-(1f2)-R-
-arabinopyranosyl-(1f3)-β- -glucopyranoside and (25R)-26-
O-(β- -glucopyranosyl)-furost-5-en-3β,22R,26-triol 3-O-R-
-rhamnopyranosyl-(1f2)-R- -xylopyranosyl-(1f3)-β- -gluco-
L
-rhamnopyranosyl-(1f2)-
-glucopyranoside, and
-glucopyranosyl)-
D
D
D
L
L
D
D
D
-(þ)-glucose,
L
-(-)-arabinose, and
L
-(-)-rhamnose, and a
D
-(þ)-
L
L
D
glucose moiety at the C-26 position, indicative of a furostanol
pyranoside, from the bulbs of L. longiflorum have been isolated
saponin (Figure 3). Accordingly, the structure of 4 was deter-
and their structures elucidated. Additionally, a known steroidal
mined to be (25R)-26-O-(β-
22R,26-triol 3-O-R- -rhamnopyranosyl-(1f2)-R-
nosyl-(1f3)-β- -glucopyranoside (Figure 5).
D
-glucopyranosyl)-furost-5-en-3β,
glycoalkaloid, (22R,25R)-spirosol-5-en-3β-yl O-R-
anosyl-(1f2)-β- -glucopyranosyl-(1f4)-β- -glucopyranoside,
and a known furostanol saponin, (25R)-26-O-(β- -glucopyran-
osyl)-furost-5-en-3β,22R,26-triol 3-O-R- -rhamnopyranosyl-
(1f2)-β- -glucopyranosyl-(1f4)-β- -glucopyranoside, were
isolated for the first time from the bulbs of L. longiflorum. On the
basis of our identification of several new steroidal glycosides of
L. longiflorum, we are studying the distribution of these com-
pounds in all of the organs of the plant and their potential roles in
plant-pathogen interactions. The extraction and purification
techniques reported above are also currently being used for the
production of sufficient quantities of pure compounds to study
the effects of steroidal glycosides on human health and nutrition.
L-rhamnopyr-
L
L-arabinopyra-
D
D
D
D
Compound 5 was determined to be an isomer of compound 4.
The structure of the oligosaccharide moiety was readily deduced
from 1H NMR, 13C NMR, HMBC, ESIþ-MS, and chemical ana-
lysis. 5 showed carbon NMR peaks similar to those of com-
L
D
D
1
pounds 3 and 4, except for the 3000 sugar peaks. The H NMR
spectrum contained four anomeric proton signals observed at δH
6.34, 5.01, 4.99, and 4.89. The 13C NMR spectrum contained four
anomeric carbon signals observed at δC 105.4, 105.0, 102.4, and
100.0. The HMBC experiment showed long-range correlations
between the anomeric proton signal at δH 4.99 [H-10] and the
carbon signal at δC 77.4 [C-3], between the anomeric proton
signal at δH 5.01 [H-1000] and the carbon signal at δC 88.2 [C-30],
and between the anomeric proton signal at δH 6.34 [H-100] and the
carbon signal at δC 78 [C-20]. Upon acid hydrolysis, derivatiza-
tion, and GC-MS/enantioselective GC-FID analysis, the sugars
ACKNOWLEDGMENT
We thank Robert Carhart (Mars, Inc., Hackettstown, NJ) for
his assistance in acquiring FT-IR spectra; Dr. Richard Merritt
and the Rutgers University Easter lily breeding program for
providing lily bulbs, and Jacob Schmidt and the University of
Minnesota DepartmentofChemistry MassSpectrometryFacility
for HRMS acquisition.
were identified as
xylose in a 2:1:1 ratio. Accordingly, the structure of 5 was
determined to be (25R)-26-O-(β- -glucopyranosyl)-furost-5-en-
3β,22R,26-triol 3-O-R- -rhamnopyranosyl-(1f2)-R- -xylopyra-
nosyl-(1f3)-β- -glucopyranoside (Figure 5).
D
-(þ)-glucose,
L-(-)-rhamnose, and
L-(-)-
D
L
L
D
LITERATURE CITED
Although saponins are widely distributed secondary metabo-
lites and have been identified in over 100 plant families and in
marine organisms such asstarfishand sea cucumber(43), thus far,
steroidal glycoalkaloids are limited to members of the families
Solanaceae and Liliaceae (44). Steroidal saponins with acetyla-
tion of saccharide residues have been identified; however, the
biological significance of acetylation of these compounds is
unclear. Some genotypes of Solanum chacoense, a wild potato
species that is resistant to Leptinotarsa decemlineata, contain
glycoalkaloids that are acetylated at the C-23 position of the
steroid aglycone (45), but we are unaware of any steroidal
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Received for review April 14, 2010. Revised manuscript received July 2,
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