Allimacrosides A–E, new steroidal glycosides from Allium macrostemon Bunge

Article abstract of DOI:10.1016/j.steroids.2016.12.002

A new pregnane-type steroidal glycoside (1), two new spirostane-type steroidal glycosides (2, 3), and two new furostane-type steroidal glycosides (4, 5), named allimacrosides A–E, together with four known compounds (6–9) were isolated from a 80% MeOH extract of Allium macrostemon Bunge. The identification and structural elucidation of these compounds were based on their 1D- and 2D-NMR spectra, and HR-FAB-MS data analysis. The isolated compounds were tested for cytotoxicity against four human tumor cell lines in vitro using the sulforhodamine B bioassay.

Full text of DOI:10.1016/j.steroids.2016.12.002

Accepted Manuscript
Allimacrosides A-E, new steroidal glycosides from Allium macrostemon Bunge
Yun Sik Kim, Won Se Suh, Kyoung Jin Park, Sang Un Choi, Kang Ro Lee
PII:
S0039-128X(16)30181-7
http://dx.doi.org/10.1016/j.steroids.2016.12.002
STE 8058
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Reference:
Steroids
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Accepted Date:
11 July 2016
3 December 2016
5 December 2016
Please cite this article as: Sik Kim, Y., Se Suh, W., Jin Park, K., Un Choi, S., Ro Lee, K., Allimacrosides A-E, new
steroidal glycosides from Allium macrostemon Bunge, Steroids (2016), doi: http://dx.doi.org/10.1016/j.steroids.
2016.12.002
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Steroids
Original Research Article
Allimacrosides A-E, new steroidal glycosides from Allium macrostemon
Bunge
Yun Sik Kim a, Won Se Suh a, Kyoung Jin Park a, Sang Un Choi b, Kang Ro Lee*,a
a
Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon
16419, Republic of Korea
b Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
*Correspondence to:
Prof. Dr. KANG RO LEE
Natural Products Laboratory, School of Pharmacy,
Sungkyunkwan University, 2066 Seobu-ro,
Jangan-gu, Suwon 16419, Republic of Korea.
TEL: 82-31-290-7710; FAX: 82-31-290-7730; E-mail: krlee@skku.edu
1

A new pregnane-type steroidal glycoside (1), two new spirostane-type steroidal glycosides (2,
3), and two new furostane-type steroidal glycosides (4, 5), named allimacrosides A-E,
together with four known compounds (6-9) were isolated from a 80% MeOH extract of
Allium macrostemon Bunge. The identification and structural elucidation of these compounds
were based on their 1D- and 2D-NMR spectra, and HR-FAB-MS data analysis. The isolated
compounds were tested for cytotoxicity against four human tumor cell lines in vitro using the
sulforhodamine B bioassay.
Key words Allium macrostemon; Liliaceae; steroidal glucosides; cytotoxicity
2

1. Introduction
Allium macrostemon Bunge (Liliaceae) is a perennial herb that grows in the fields and
mountains of Southeast Asia. Its dried bulbs are popularly known as traditional Chinese
medicine “Xiebai” and are used as treatments for several heart diseases such as thoracic pain,
stenocardia, heart asthma, etc [1]. Through our search for structurally interesting compounds
from Allium sp., we have reported the isolation of flavonoid glycosides and phenolic
compounds from A. victorialis var. platyphyllum [2,3] and A. tuberosum [4]. A number of
steroidal saponins with various biological activities, such as macrostemonosides A-S, have
been isolated from the dried bulbs of A. macrostemon [5-9].
Continuing investigation of the steroidal glycoside constituents of the n-BuOH fraction of
the MeOH extract has resulted in the isolation of a new pregnane-type steroidal glycoside (1),
two new spirostane-type steroidal glycosides (2, 3), and two new furostane-type steroidal
glycosides (4, 5), named allimacrosides A-E, together with a known pregnane-type steroidal
glycoside (6) and three known furostane-type steroidal glycosides (7-9). The structure of the
new compounds were elucidated by analysis of 1D- and 2D-NMR (1H- and 13C-NMR,
1
distortionless enhancement by polarization transfer (DEPT), H-1H correlated spectroscopy
(COSY), heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond
correlation (HMBC) and nuclear overhauser effect spectroscopy (NOESY)) data, acid
hydrolysis and GC-MS. All isolated compounds (1-9) were evaluated for their cytotoxic
activities against four human cancer cell lines.
2. Experimental
2.1 General experimental procedures
Optical rotations were measured on a Jasco P-1020 polarimeter in MeOH. IR spectra were
recorded on a Bruker IFS-66/S FT-IR spectrometer. UV spectra were recorded using a
Schimadzu UV-1601 UV-visible spectrophotometer. HR-FAB mass spectra were obtained on
1
a JEOL JMS700 mass spectrometer. NMR spectra, including H-1H COSY, DEPT, HSQC,
HMBC and NOESY experiments, were recorded on a Varian UNITY INOVA 700 NMR
spectrometer operating at 700 MHz (1H) and 175 MHz (13C) with chemical shifts given in
3

ppm (δ). Preparative HPLC was conducted using a Gilson 306 pump with Shodex refractive
index detector and Econosil RP-C18 10 µm column (250 × 10 mm). Silica gel 60 (Merck, 70-
230 mesh and 230-400 mesh) and RP-C18 silica gel (YMG GEL ODS-A, 12 nm, S-75 µm)
were used for column chromatography. TLC was performed using percolated Silica gel F254
plates and RP-18 F254s plates (Merck). Spots were detected by TLC under UV light or by
heating after spraying with 10% H2SO4 in C2H5OH (v/v). A Hewlett-Packard (HP) GC
system 6890 Series equipped with a 5973 Mass Selective Detector (MSD) system was
controlled by the Enhanced ChemStation Version B.01.00 software. The capillary column
used for GC was an Agilent J&W HP-5MS UI (30.0 m × 0.25 mm i.d., 0.25 µm film
thickness coated 5% diphenyl 95% dimethylpolysiloxane).
2.2 Plant material
A. mactostemon was collected in Taeback, Gangwon province, Korea in April, 2010, and
the plant was identified by one of the authors (K. R. Lee). A voucher specimen (SKKU-NPL
1202) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University,
Suwon, Korea.
2.3 Extraction and isolation
Dried whole plants of A. mactostemon (1.5 kg) were extracted with 80% MeOH three
times at room temperature and evaporated under reduced pressure to give a residue (210.0 g),
which was dissolved in water (800 ml) and partitioned with solvent to give n-hexane (10.0 g),
CHCl3 (5.5 g), EtOAc (1.9 g), and n-BuOH (12.2 g) soluble layers. The n-BuOH-soluble
layer (12.2 g) was chromatographed on a silica gel column (diameter × height: 5.5×35.0 cm,
500.0 g) with a CHCl3-MeOH-H2O (20:10:1 to 2:3:1) to give 10 fractions (B1-B10) based on
a TLC analysis. Fraction B5 (1.3 g) was separated on a RP-C18 silica gel open column
(2.5×33.0 cm, 80.0 g), eluting with 60% aqueous MeOH to give five subfractions (B51-B55).
Subfraction B54 (140 mg) was separated on Lobar A®-column eluted with CHCl3-MeOH-
H2O (30:10:1) to give two subfractions (B541-B542). Subfraction B542 (120 mg) was
separated on RP-C18 Lobar A®-column (40% aqueous MeCN) to give two subfractions
(B5421-B5422). Subfraction B5422 (40 mg) was purified by RP-C18 semi-prep. HPLC (35%
4

aqueous MeCN) to afford compounds 1 (13 mg, tR = 18.8 min) and 6 (9 mg, tR = 21.8 min).
Subfraction B5421 (70 mg) was purified by RP-C18 semi-prep. HPLC (25% aqueous MeCN)
to afford compounds 7 (18 mg, tR = 21.5 min) and 8 (36 mg, tR = 25.0 min). Subfraction
B541 (20 mg) was purified by RP-C18 semi-prep. HPLC (25% MeCN) to afford compound 9
(14 mg, tR = 13.2 min). Fraction B7 (2.6 g) was separately chromatographed on a Diaion HP-
20 column (3.0×30.0 cm, 100.0 g) eluting with a gradient solvent system of 100% H2O and
100% MeOH, yielding subfractions B71 and B72. Subfraction B72 (1.5 g) was separated on
an RP-C18 silica gel open column (2.5×30.0 cm, 80 g), eluting with 40% aqueous MeOH to
give six subfractions (B721-B726). Subfraction 724 (95 mg) was purified by RP-C18 semi-
prep. HPLC (23% aqueous MeCN) to afford compound 4 (10 mg, tR = 16.2 min). Subfraction
725 (40 mg) was purified by RP-C18 semi-prep. HPLC (30% aqueous MeCN) to afford
compounds 3 (10 mg, tR = 11.8 min), 2 (7 mg, tR = 13.7 min), and 5 (3 mg, tR = 15.7 min).
2.3.1 Allimacroside A (1)
White amorphous powder; [α]D25 -48.4 (MeOH); UV (MeOH) λmax: 239 nm; IR (KBr) νmax
:
1
3385, 2934, 1664, 1371, 1070 cm-1; H-NMR (C5D5N, 700 MHz) data for aglycone moiety,
see Table 1; 1H-NMR data for sugar moiety: H-1′ [4.87, d (7.7)], H-2′ (4.38, m), H-3′ (4.08,
m), H-4′ [4.57, d (3.2)], H-5′ [3.95, dd, (9.0, 5.6)], H-6′ (4.65, m; 4.15, m), H-1′′ [5.14, d
(8.0)], H-2′′ (4.38, m), H-3′′ (4.20, m), H-4′′ (3.83, m), H-5′′ (3.84, m), H-6′′ [4.48, d (10.5);
3.99, m], H-1′′′ [5.58, d (7.7)], H-2′′′ (4.05, m), H-3′′′ (4.13, m), H-4′′′ (4.14, m), H-5′′′ (4.03,
m), H-6′′′ (4.56, m; 4.26, m), H-1′′′′ [5.29, d (8.0)], H-2′′′′ (4.04, m), H-3′′′′ (4.18, m), H-4′′′′
(4.23, m), H-5′′′′ (3.86, m), H-6′′′′ (4.53, m; 4.36, m); 13C-NMR (C5D5N, 175 MHz) see Table
2; HR-FAB-MS m/z 985.4251 [M + Na]+ (calcd. for C45H70NaO22 : 985.4251).
Pregna-5,16-dien-3β-ol-20-one (1a): Colorless gum; [α]2D5 -27.3 (MeOH); 1H-NMR (CDCl3,
700 MHz): δH 6.64 (1H, t, J = 3.0 Hz, H-16), 5.32 (1H, d, J = 5.2 Hz, H-6), 3.47 (1H, m, H-
3), 2.19 (3H, s, H-21), 0.92 (3H, s H-19), 0.87 (3H, s, H-18). HR-FAB-MS m/z 315.2319 [M
+ H]+ (calcd. for C21H31O2: 315.2319).
2.3.2 Allimacroside B (2)
Colorless gum; [α]2D5 -32.0 (MeOH); IR (KBr) νmax: 3385, 2927, 1563, 1512, 1451, 1371,
1075, 895 cm-1; 1H-NMR (C5D5N, 700 MHz) data for aglycone moiety, see Table 1; 1H-NMR
data for sugar moiety: H-1′ [4.86, d (7.5)], H-2′ (4.42, m), H-3′ (4.10, m), H-4′ [4.58, d (2.8)],
5

H-5′ [3.95, dd (8.7, 5.7)], H-6′ (4.68, m; 4.20, m), H-1′′ [5.13, d (8.0)], H-2′′ (4.37, m), H-3′′
(4.19, m), H-4′′ (3.77, m), H-5′′ (3.82, m), H-6′′ (4.45, m; 3.98, m), H-1′′′ [5.56, d (7.7)], H-2′′′
(4.05, m), H-3′′′ (4.12, m), H-4′′′ (4.13, m), H-5′′′ (4.02, m), H-6′′′ (4.52, m; 4.25, m), H-1′′′′
[5.29, d (8.0)], H-2′′′′ (4.05, m), H-3′′′′ (3.85, m), H-4′′′′ (4.23, m), H-5′′′′ (3.85, m), H-6′′′′
(4.52, m; 4.37, m), H-1′′′′′ [4.90, d (7.7)], H-2′′′′′ (4.05, m), H-3′′′′′ (4.18, m), H-4′′′′′ (4.26, m),
H-5′′′′′ (3.84, m), H-6′′′′′ (4.51, m; 4.35, m); 13C-NMR (C5D5N, 175 MHz) see Table 2; HR-
FAB-MS m/z 1265.5778 [M + Na]+ (calcd. for C57H94NaO29 : 1265.5773).
2.3.2 Allimacroside C (3)
Colorless gum; [α]2D5 -28.0 (MeOH); IR (KBr) νmax: 3385, 2928, 1549, 1452, 1434, 1371,
1
1305, 1074, 896 cm-1; H-NMR (C5D5N, 700 MHz) data for aglycone moiety, see Table 1;
1H-NMR data for sugar moiety: H-1′ [4.87, d (7.7)], H-2′ [4.43, t (8.4)], H-3′ (4.07, m), H-4′
[4.57, d (2.6)], H-5′ [3.95, dd (8.7, 5.7)], H-6′ (4.67, m; 4.17, m), H-1′′ [5.14, d (8.0)], H-2′′
(4.35, m), H-3′′ (4.21, m), H-4′′ (3.81, m), H-5′′ (3.83, m), H-6′′ (4.47, m; 3.99, m), H-1′′′
[5.57, d (8.0)], H-2′′′ (4.05, m), H-3′′′ (4.13, m), H-4′′′ (4.14, m), H-5′′′ (4.03, m), H-6′′′ (4.53,
m; 4.26, m), H-1′′′′ [5.29, d (8.0)], H-2′′′′ (4.06, m), H-3′′′′ (3.86, m), H-4′′′′ (4.24, m), H-5′′′′
(3.86, m), H-6′′′′ (4.52, m; 4.37, m), H-1′′′′′ [4.92, d (8.0)], H-2′′′′′ (4.06, m), H-3′′′′′ (4.19, m),
13
H-4′′′′′ (4.27, m), H-5′′′′′ (3.85, m), H-6′′′′′ (4.52, m; 4.36, m); C-NMR (C5D5N, 175 MHz)
see Table 2; HR-FAB-MS m/z 1263.5621 [M + Na]+ (calcd. for C57H92NaO29 : 1263.5622).
2.3.4 Allimacroside D (4)
Colorless gum; [α]2D5 -23.3 (MeOH); IR (KBr) νmax: 3385, 2931, 1549, 1452, 1377, 1161,
1077 cm-1; 1H-NMR (C5D5N, 700 MHz) data for aglycone moiety, see Table 1; 1H-NMR data
for sugar moiety: H-1′ [4.92, d (7.5)], H-2′ (4.53, m), H-3′ (4.11, m), H-4′ (4.58, m), H-5′
(4.02, m), H-6′ (4.61, m; 4.16, m), H-1′′ [5.17, d (8.0)], H-2′′ (4.32, m), H-3′′ (4.17, m), H-4′′
(3.82, m), H-5′′ (3.82, m), H-6′′ (4.47, m; 4.00, m), H-1′′′ [5.57, d (8.0)], H-2′′′ (4.03, m), H-
3′′′ (4.15, m), H-4′′′ (4.13, m), H-5′′′ (3.87, m), H-6′′′ (4.54, m; 4.40, m), H-1′′′′ [5.29, d (7.7)],
H-2′′′′ (4.03, m), H-3′′′′ (4.18, m), H-4′′′′ (4.23, m), H-5′′′′ (3.93, m), H-6′′′′ (4.53, m; 4.26, m),
H-1′′′′′ [4.81, d (7.7)], H-2′′′′′ (4.02, m), H-3′′′′′ (4.23, m), H-4′′′′′ (4.14, m), H-5′′′′′ (4.03, m),
H-6′′′′′ (4.54, m; 4.38, m); 13C-NMR (C5D5N, 175 MHz) see Table 2; HR-FAB-MS m/z
1281.5728 [M + Na]+ (calcd. for C57H94NaO30 : 1281.5722).
6

2.3.5 Allimacroside E (5)
Colorless gum; [α]2D5 -14.0 (MeOH); IR (KBr) νmax: 3385, 2930, 1549, 1450, 1369, 1306,
1
1
1161, 1076 cm-1; H-NMR (C5D5N, 700 MHz) data for aglycone moiety, see Table 1; H-
NMR data for sugar moiety: H-1′ [4.87, d (7.5)], H-2′ (4.43, m), H-3′ (4.09, m), H-4′ [4.57, d
(2.8)], H-5′ (3.95, m), H-6′ (4.67, m; 4.16, m), H-1′′ [5.14, d (8.0)], H-2′′ (4.37, m), H-3′′
(4.20, m), H-4′′ (3.83, m), H-5′′ (3.84, m), H-6′′ (4.48, m; 4.00, m), H-1′′′ [5.57, d (7.7)], H-2′′′
(4.06, m), H-3′′′ (4.12, m), H-4′′′ (4.14, m), H-5′′′ (3.86, m), H-6′′′ (4.53, m; 4.37, m), H-1′′′′
[5.29, d (8.0)], H-2′′′′ (4.05, m), H-3′′′′ (4.19, m), H-4′′′′ (4.23, m), H-5′′′′ (3.95, m), H-6′′′′
(4.53, m; 4.26, m), H-1′′′′′ [4.86, d (7.5)], H-2′′′′′ (4.05, m), H-3′′′′′ (4.25, m), H-4′′′′′ (4.25, m),
H-5′′′′′ (4.03, m), H-6′′′′′ (4.58, m; 4.40, m); 13C-NMR (C5D5N, 175 MHz) see Table 2; HR-
FAB-MS m/z 1277.5778 [M + Na]+ (calcd. for C58H94NaO29 : 1277.5773).
2.4 Acid hydrolysis of compounds 1-5 and sugar determination
Each compound (2.5 mg) was dissolved in 2 mL of 15% HCl. The solution was heated at
80°C for 2 hrs. The hydrolysate was extracted with CH2Cl2, and the aqueous layer was
neutralized using an Amberlite IRA-67 column to yield the sugar. The sugar acquired from
the hydrolysis was dissolved in anhydrous pyridine (0.1 mL), and 2.0 mg of L-cysteine
methyl ester hydrochloride was added. The mixture was stirred at 60 °C for 1.5 h and
trimethylsilylated through adding 0.1 mL of 1-trimethylsilylimidazole for 2 hrs. The mixture
was partitioned with n-hexane and H2O (0.3mL each), and the n-hexane layer (1.0 µL) was
analyzed through GC/MS. Identification of D-galactose (20.093 min) and D-glucose (22.103
min) were detected in each case by co-injection of the hydrolysate with standard silylated
sugars.
2.5 Cytotoxicity test
A sulforhodamine B bioassay (SRB) was used to determine the cytotoxicity of the
compounds. The cytotoxic activity of each compound against four cultured human tumor
cells was examined in vitro at the Korea Research Institute of Chemical Technology. The
tumor cell lines were A549 (non small cell lung adenocarcinoma), SK-OV-3 (ovarian cancer
cells), SK-MEL-2 (skin melanoma), and HCT15 (colon cancer cells) [10]. Etoposide was
7

used as the positive control. IC50 values for the cytotoxicity of etoposide were 1.02, 1.74,
0.21, and 1.93 µM to A549, SK-OV-3, SK-MEL-2, and HCT15 cells, respectively.
3. Results and discussion
Compound 1 was isolated as a white amorphous powder. The molecular formula was
determined to be C45H70O22 from the molecular ion peak [M + Na]+ at m/z 985.4251 (calcd.
for C45H70NaO22 : 985.4251) in the positive-ion high resolution (HR)-fast atom bombardment
(FAB)-MS. The IR spectrum showed characteristic absorptions for α,β-unsaturated ketone
1
(1664 cm-1), hydroxyl (3385 cm-1), and glycosidic linkage (1000-1160 cm-1) [11]. The H-
NMR spectrum of 1 (Table 1) displayed the signals of two olefinic protons at δH 6.59 (dd, J =
3.1, 1.8 Hz, H-16) and 5.30 (overlap, H-6), an oxymethine proton at δH 3.86 (m, H-3), and
three methyl singlet signals at δH 2.23 (s, H-21), 0.91 (s, H-18), and 0.87 (s, H-19) of
aglycone, and four anomeric protons at 4.87 (d, J = 7.7 Hz, H-1′), 5.14 (d, J = 8.0 Hz, H-1′′),
5.58 (d, J = 7.7 Hz, H-1′′′), and 5.29 (d, J = 8.0 Hz, H-1′′′′) of four sugar moieties. The 13C-
NMR spectrum (Table 2) showed a total of 45 carbon signals, of which 21 carbons were
assigned to the aglycone and the remaining 24 carbons to four sugar moieties.
A
comparison of the NMR spectral findings of 1 with literature data revealed that the aglycone
pair of 1 was identical to that of pregna-5,16-dien-3β-ol-20-one [12]. The remaining 24
carbon signals were supposed to be the sugar moiety (R1), which was identical to that of
solanigroside B (6) [13]. Starting from these four anomeric protons, the exact identity of the
monosaccharides and the sequence of the tetrasaccharide chain were also determined by
1
analysis of a combination of DEPT, H-1H COSY, HSQC, and HMBC spectra. The
connectivity of the four sugars was mainly based on the HMBC correlations: H-1′ (δH 4.87, d,
J = 7.7 Hz,) with C-3 (δC 79.4) of the aglycone, H-1′′ (δH 5.14, d, J = 8.0 Hz) with C-4′ (δC
81.5), H-1′′′ (δH 5.58, d, J = 7.7 Hz) with C-2′′ (δC 82.7), and H-1′′′′ (5.29, d, J = 8.0 Hz) with
C-3′′ (δC 89.8). Acid hydrolysis of 1 afforded the aglycone pregna-5,16-dien-3β-ol-20-one
(1a) and two kinds of sugars, D-glucose and D-galactose, which were identified by GC
1
analysis after derivatizion [14]. The identification of the aglycone was confirmed by H-
NMR, optical rotation, and HR-FAB-MS analyses [15]. The relative stereochemistry of the
aglycone was corroborated by the NOESY cross-peaks of H-1ax/H-3, H-8/H3-18 and H3-19,
H-9/H-14. The anomeric configuration of D-glucose and D-galactose was determined to be β
8

form, respectively, on the basis of the J value of the anomeric proton in D-glucose (8.0 Hz)
and D-galactose (7.7 Hz). Thus, the structure of 1 was established as pregna-5,16-dien-3β-ol-
20-one-3-O-β-D-glucopyranosyl(1→2)-[β-D-glucopyranosyl(1→3)]-β-D-
glucopyranosyl(1→4)-β-D-galactopyranoside, named allimacroside A.
Compound 2 was isolated as a colorless gum. The molecular formula was determined to be
C57H94O29 from the molecular ion peak [M + Na]+ at m/z 1265.5778 (calcd. for C57H94NaO29 :
1265.5773) in the positive-ion HR-FAB-MS. The 1H-NMR spectrum of 2 showed signals for
four steroidal methyl groups at δH 1.13 (d, J = 6.5 Hz, H-27), 1.03 (d, J = 6.9 Hz, H-21), 0.72
(s, H-18), and 0.60 (s, H-19). The 13C-NMR spectrum showed a total of 57 carbon signals, of
which 27 carbons were assigned to the aglycone and the remaining 30 carbons were assigned
to the sugar moieties. A comparison of the NMR spectral data of 2 with literature data
revealed that the aglycone pair of 2 was identical to that of (24S,25S)-5α-spirostane-3β,24-
diol [16]. The remaining 30 carbon signals were supposed to be 5 sugars, one D-galactose and
four D-glucose moieties. The anomeric configuration of D-glucose was determined by J
values at δH 5.56 (d, J = 7.7 Hz, H-1′′′), 5.29 (d, J = 8.0 Hz, H-1′′′′), 5.13 (d, J = 8.0 Hz, H-
1′′), 4.90 (d, J = 8.0 Hz, H-1′′′′′) as all β forms, D-galatose by J values at δH 4.86 (d, J = 8.0
Hz, H-1′) as β form, and the sequence of sugars in the oligosaccharide chain was deduced by
the HMBC correlations between H-1′′′′′/C-24, and H-1′/C-3. Acid hydrolysis of 2 gave D-
galactose and D-glucose in a ratio of 1:4, while the aglycone was decomposed under acidic
conditions. Identification of D-galactose and D-glucose was performed by GC analysis. The
relative stereochemistry of the aglycone was corroborated by cross-peaks of H-14/H-5, H-9,
H-16, and H-17 and H-21/H-17 in the NOESY spectrum. The chemical shifts of C-5 (δC 44.4),
C-9 (δC 54.1) and C-19 (δC 12.0) were consistent with those of the 5αH configuration [17-19].
The proton multiplicity of H-24, with J values of 10.7 Hz (H-24/H-23ax), 10.7 Hz (H-24/H-
25), 4.7 Hz (H-24/H-23eq), and NOE correlations between H-23ax and H-20/H-25, and
between H-26ax and H-24/H-16 in the phase-sensitive NOESY spectrum were consistent
with the 22α, 24S, and 25S configurations (Fig. 2). Thus, the structure of 2 was established as
(24S,25S)-24-[(β-D-glucopyranosyl)oxy]-5α-spirostan-3β-yl-O-β-D-glucopyranosyl(1→2)-[β-
D-glucopyranosyl(1→3)]-β-D-glucopyranosyl(1→4)-β-D-galactopyranoside,
named
allimacroside B.
Compound 3 was isolated as a colorless gum. The molecular formula was determined to be
C57H92O29 from the molecular ion peak [M + Na]+ at m/z 1263.5621 (calcd. for C57H92NaO29 :
9

1263.5622) in the positive-ion HR-FAB-MS. The 1H-NMR spectrum of 3 showed signals for
four steroidal methyl groups at δH 1.14 (d, J = 6.5 Hz, H-27), 1.05 (d, J = 6.9 Hz, H-21), 0.85
(s, H-19), and 0.73 (s, H-18), as well as signals for five anomeric protons at δH 5.57 (d, J =
8.0 Hz, H-1′′′), 5.29 (d, J = 8.0 Hz, H-1′′′′), 5.14 (d, J = 8.0 Hz, H-1′′), 4.92 (d, J = 8.0 Hz, H-
1′′′′′), and 4.87 (d, J = 7.7 Hz, H-1′), and an olefinic proton signal at δH 5.26 (br d, J = 4.1 Hz,
H-6) (Table 1). The 13C-NMR spectrum showed a total of 57 carbon signals, of which 27
carbons were assigned to the aglycone and the remaining 30 carbons were assigned to the
1
sugar moieties. The H-NMR spectra of 3 were very similar to those of compound 2 except
for an additional double bond at δH 5.26 (br d, J = 4.1 Hz, H-6) and δC 142.2 (C-5) and 122.9
(C-6). A comparison of the NMR spectral data of 3 with those in the literature revealed that
the aglycone of 3 was identical to that of (3β,24S,25S)-spirost-5-ene-3,24-diol [20]. The
relative stereochemistry of the aglycone was confirmed by cross-peaks of H-14/H-9, H-16,
and H-17 and H-21/H-17 in the NOESY spectrum. The sugar units were the same with those
in the compound 2. Acid hydrolysis of 3 gave D-galactose and D-glucose in a ratio of 1:4,
while the aglycone was decomposed under acidic conditions. Thus, the structure of 3 was
established as (24S,25S)-24-[(β-D-glucopyranosyl)oxy]-spirost-5-ene-3β,24-diol-3-O-β-D-
glucopyranosyl(1→2)-[β-D-glucopyranosyl(1→3)]-β-D-glucopyranosyl(1→4)-β-D-
galactopyranoside, named allimacroside C.
Compound 4 was isolated as a colorless gum. The molecular formula was determined to be
C57H94O30 from the molecular ion peak [M + Na]+ at m/z 1281.5728 (calcd. for C57H94NaO30 :
1281.5722) in the positive-ion HR-FAB-MS. The 1H-NMR spectrum of 4 showed signals for
four steroidal methyl groups at δH 1.31 (d, J = 6.9 Hz, H-21), 0.97 (d, J = 6.7 Hz, H-27), 0.93
(s, H-19), and 0.86 (s, H-18), as well as signals for five anomeric protons at δH 5.57 (d, J =
7.7 Hz, H-1′′′), 5.29 (d, J = 8.0 Hz, H-1′′′′), 5.17 (d, J = 8.0 Hz, H-1′′), 4.92 (d, J = 7.5 Hz, H-
1′), and 4.86 (d, J = 7.5 Hz, H-1′′′′′), and an olefinic proton signal at δH 5.28 (brs, H-6) (Table
13
1). The C-NMR spectrum of 4 had signals at δ 141.3 and 123.2, indicating the presence of
an olefinic bond. A comparison of the NMR spectral findings of 4 with literature data
revealed that the aglycone pair of 4 was identical to that of fistulosaponin F [21]. The
position of OH group at C-2 was confirmed by the HMBC correlations between C-2 and H-
1b (δH 1.29, m) and H-4a (δH 2.69, dd, J = 14.0, 5.2 Hz), and α-form of OH group by the
NOESY cross-peak of H-2 with H-19 [21]. The stereochemistry of the OH group at C-22 was
assigned as α-orientation by comparison of NMR data with other same skeleton derivatives
10

[21, 22]. The 25R form was determined by the chemical shifts of C-23 (δC 38.5), C-24 (δC
29.6), C-25 (δC 35.5), C-26 (δC 76.6) and C-27 (δC 18.7) [7, 21- 23]. This was also confirmed
by the difference between the chemical shifts of two proton signals of H-26 [23]. The relative
stereochemistry of the aglycone was corroborated by the NOESY cross-peaks of H-14/H-9,
13
H-16, and H-17 and H-21/H-17. Among the 57 carbon signals in the C-NMR spectrum, 27
signals were assigned to the aglycone and the remaining 30 signals to five hexose units. In
the HMBC experiment, long-range correlations between the anomeric proton H-1′′′′′ and C-
26 of the aglycone, and between the anomeric proton H-1′ and C-3 of the aglycone, allowed
us to identify C-26 and C-3 as the glucosidic linkage sites. Acid hydrolysis of 4 gave D-
galactose and D-glucose in a ratio of 1:4, while the aglycone was decomposed under acidic
conditions. Thus, the structure of 4 was established as (25R)-26-O-β-D-glucopyranosyl-5-
enefurostan-2α,3β,22α,26-tetraol-3-O-β-D-glucopyranosyl(1→2)-[β-D-
glucopyranosyl(1→3)]-β-D-glucopyranosyl(1→4)-β-D-galactopyranoside,
named
allimacroside D.
Compound 5 was isolated as a colorless gum. The molecular formula was determined to be
C58H94O29 from the molecular ion peak [M + Na]+ at m/z 1277.5778 (calcd. for C57H94NaO30 :
1277.5773) in the positive-ion HR-FAB-MS. The 1H-NMR spectrum of 5 showed signals for
five steroidal methyl groups at δH 1.38 (s, H-21), 1.08 (d, J = 6.7 Hz, H-27), 0.85 (s, H-19),
0.83 (s, H-18), and 3.15 (s, OMe) as well as signals for five anomeric protons at δH 5.57 (d, J
= 7.7 Hz, H-1′′′), 5.29 (d, J = 8.0 Hz, H-1′′′′), 5.14 (d, J = 8.0 Hz, H-1′′), 4.97 (d, J = 7.5 Hz,
H-1′), and 4.86 (d, J = 8.0 Hz, H-1′′′′′). The 13C-NMR spectrum (Table 2) showed a total of 58
carbon signals, of which 28 carbons were assigned to the aglycone and the remaining 30
carbons to the sugar moieties. Comparison of the 13C-NMR spectroscopic signals of the
aglycone moiety of 5 with literature values and an extensive HMQC and HMBC data analysis,
showed that the aglycone of 5 was 20-methoxyl-furosta-5,22-dien-3β,26-diol [24]. The
relative stereochemistry of the aglycone was corroborated by the cross-peaks of H-14/H-9, H-
16, and H-17 in the NOESY spectrum. The β-configuration of methoxy group at C-20 was
determined by cross-peak between H-21 (δH 1.38) and H-17 (δH 2.06) in the NOESY
1
spectrum. Comparison of H-NMR, 13C-NMR, COSY, and HMBC spectrum for the sugar
moieties of 5 with those of 4 suggested the same sugar chains. Acid hydrolysis of 5 gave D-
galactose and D-glucose in a ratio of 1:4, while the aglycone was decomposed under acidic
conditions. Thus, the structure of 5 was established as 26-O-β-D-glucopyranosyl-20β-
11

methoxyl-25(R)-furostan-5,22(23)-dien-3β,26-diol-3-O-β-D-glucopyranosyl(1→2)-[β-D-
glucopyranosyl(1→3)]-β-D-glucopyranosyl(1→4)-β-D-galactopyranoside,
allimacroside E.
named
Additionally, four known compounds were isolated. By comparing their NMR
spectroscopic data with those in the literature, their structures were determined to be
solanigroside B (6) [13], macrostemonoside O (7) [8], (25R)-26-O-β-D-glucopyranosyl-22-
→2')
hydroxy-5β-furost-3β-26-diol-3-O-β-D-glucopyranosyl-(1''
-β-D-galactopyranoside (8)
[25], and ceparoside B (9) [26].
The cytotoxic activities of the isolated compounds (1-9) were evaluated by determining
their inhibitory effects on human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT15)
in vitro using the sulforhodamine B (SRB) assay. Compound 3 showed moderate cytotoxicity
against SK-MEL-2 cell with IC50 values of 23.43 µM. Also, Compound 6 showed moderate
cytotoxicity against A549 and SK-MEL-2 cells with IC50 values of 23.68 and 14.27 µM,
respectively. But the other compounds were inactive (IC50: >30.0 µM).
In summary, five new steroidal saponins, allimacrosides A-E (1-5), together with four
known compounds (6-9) were isolated from the MeOH extract of A. macrostemon. Their
structures were determined by the spectral data, including 2D NMR spectra. All the isolated
compounds were evaluated their cytotoxic activities against A549, SK-OV-3, SK-MEL-2,
and HCT-15 cell lines. Compounds 3 and 6 showed moderate cytotoxic activity against the
SK-MEL-2 cells, with IC50 values of 23.43 and 14.27 µM, respectively. However, the other
compounds were inactive against the four human tumor cell lines.
Acknowledgements
This research was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
Technology (2012R1A5A2A28671860). We thank the Korea Basic Science Institute (KBSI)
for the MS spectral measurements.
Supporting Information
The spectral data of compounds 1-5 are available on request from the corresponding author.
12

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13

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14

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15

Table legends
1
Table 1. H-NMR data (700 MHz) for the aglycone moieties of compounds 1-5a (δ in ppm,
C5D5N, J in Hz).
Table 2. 13C-NMR data (175 MHz) for compounds 1-5a (δ in ppm, C5D5N).
Figure legends
Fig. 1. Structures of compounds 1-5.
Fig. 2. Important NOE correlations of compounds 2.
16

21
21
20
26
O
27
O
O
18
20
18
OR2
OR2
23
17
11
11
17
19
13
19
13
O
O
H
1
9
9 H
1
H
H
H
15
10
H
H
15
10
H
H
5 H
H
H
H
3
7
5
3
7
R1O
R1O
R1O
H
3
1
2
R2O
21
R2O
OMe
26
OH
18
27
20
17
23
11
19
13
O
1
9 H
H
O
HO
H
H
H
15
10
H
H
H
3
7
H
H
5
R1O
R1O
4
5
OH
O
OH
OH
O
OH
O
O
1''
HO
1''''
O
O
OH
1'''''
R2 :
R1 :
O
1'
HO
HO
HO
HO
HO
O
OH
1'''
OH
OH
OH
HO
HO
Fig. 1. Structures of compounds 1-5.
17

H
H
H
O
H
Me
Me
Me
O
H
H
H
O
H
H
Fig. 2. Important NOE correlations of compound 2.
18

Table 1. 1H-NMR data (700 MHz) for the aglycone moieties of compounds 1-5a (δ in ppm, C5D5N, J in Hz).
Position
1
2
3
4
δH
5
δH
δH
J (Hz)
δH
J (Hz)
δH
J (Hz)
J (Hz)
J (Hz)
1
2
a
b
a
b
1.65 m
0.90 m
2.07 m
1.67 m
3.86 m
2.65 dd
2.41 t-like
-
1.47 m
0.75 m
2.01 m
1.57 m
3.90 m
1.75 m
1.75 m
0.85 m
1.06 m
1.06 m
1.45 m
0.73 m
1.37 m
0.45 m
-
1.64 m
0.93 m
2.08 m
1.68 m
3.85 m
2.63 dd
2.40 t-like
-
2.30 dd
1.29 m
4.06 m
-
12.8, 4.4
1.64 m
0.93 m
2.08 m
1.68 m
3.88 m
2.63 dd
2.39 t-like
-
3
4
3.82 m
2.69 dd
2.54 t-like
-
a
13.3, 2.4
12.2
13.2, 2.1
12.4
14.0, 5.2
12.4
13.6, 2.4
12.4
b
5
6
a
b
a
b
5.30 ob
5.26 br d
-
4.1
5.28 br s
-
5.26 br d
-
4.1
-
7
1.84 m
1.52 m
1.50 m
0.90 m
-
1.79 m
1.42 m
1.45 m
0.83 m
-
1.80 m
1.45 m
1.47 m
0.93 m
-
1.77 m
1.41 m
1.42 m
0.79 m
-
8
9
10
11
a
b
a
b
1.46 m
1.46 m
2.59 m
1.35 m
-
1.33 m
1.12 m
1.57 m
0.97 m
-
1.38 m
1.32 m
1.61 m
1.03 m
-
1.46 m
1.38 m
1.69 m
1.07 m
-
1.33 m
1.33 m
1.79 m
1.08 m
-
12
13
14
15
1.28 td
2.12 ddd
1.87 m
6.59 dd
-
11.5, 6.6
0.96 m
2.08 m
1.31 m
4.49 m
1.72 m
0.72 s
0.60 s
1.90 m
1.03 d
-
0.99 m
1.94 m
1.35 m
4.50 m
1.72 m
0.73 s
0.85 s
1.91 m
1.05 d
-
1.01 m
1.97 m
1.41 m
4.93 m
1.91 m
0.86 s
0.93 s
2.20 m
1.31 d
-
0.87 m
1.97 m
1.37 m
4.94 m
2.06 m
0.83 s
0.85 s
-
a
16.9, 6.6, 3.4
b
16
17
18
19
20
21
22
23
3.1, 1.8
0.91 s
0.87 s
-
2.23 s
6.9
6.9
6.9
1.38 s
-
a
b
a
b
2.66 dd
1.95 dd
4.02 ddd
-
12.9, 4.7
12.9, 10.7
2.67 dd
1.96 dd
4.03 ddd
-
13.0, 4.8
13.0, 10.7
2.04 m
2.00 m
2.04 m
1.67 m
1.90 m
3.93 m
3.60 m
0.97 d
4.32 m
-
24
10.7, 10.7, 4.7
10.7, 10.7, 4.8
2.46 dt
2.22 dt
2.10 m
4.00 m
3.70 dd
1.08 d
3.15 s
14.0, 6.2
14.3, 7.9
25
26
1.88 m
3.62 dd
3.56 dd
1.13 d
1.90 m
3.63 dd
3.57 dd
1.14 d
a
11.3, 4.7
11.4, 11.3
6.5
11.3, 4.9
11.4, 11.3
6.5
b
9.5, 6.0
6.7
27
6.7
OMe
a Assignments are based on DEPT, HSQC, COSY, HMBC, and NOESY experiments.
b Signal pattern unclear due to overlapping.
19

Table 2. 13C-NMR data (175 MHz) for compounds 1-5a (δ in ppm, C5D5N).
Position
Position
Gal 1′
2′
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
6
7
8
9
38.6
31.4
79.4
40.5
142.7
122.6
33.0
31.6
52.0
38.3
22.2
36.4
47.5
57.7
33.6
146.0
156.5
17.2
20.5
197.6
28.4
36.8
29.7
77.1
34.6
44.4
28.7
32.1
34.9
54.1
35.4
21.0
39.8
40.5
56.1
31.7
81.3
62.3
16.4
12.0
41.9
14.6
113.7
40.6
81.2
38.0
64.9
13.3
38.7
31.4
79.3
40.5
142.2
122.9
33.5
32.8
51.5
38.3
22.3
41.0
41.7
57.9
33.3
82.7
63.7
17.6
20.6
43.4
16.4
112.9
42.1
82.8
39.5
66.4
14.8
47.0
71.3
85.7
39.0
141.3
123.2
33.5
32.3
51.5
39.2
22.4
41.1
42.0
57.7
33.7
82.4
65.1
17.7
21.7
42.0
17.7
111.9
38.5
29.6
35.5
76.6
18.7
37.2
29.9
77.9
39.0
140.8
121.3
31.7
30.7
49.7
36.7
20.3
38.9
40.0
56.4
33.2
83.7
66.4
13.4
19.1
82.1
15.0
157.0
96.0
29.4
34.7
75.0
17.3
48.6
104.0
74.5
76.8
81.5
76.5
61.8
106.4
82.7
89.8
72.1
78.8
64.3
106.2
77.4
79.1
72.8
79.9
63.6
105.8
76.6
79.9
72.2
79.9
63.6
102.2
73.0
75.4
80.0
75.1
60.4
104.9
81.3
88.3
70.6
77.3
62.8
104.7
75.9
77.7
71.3
78.4
62.1
104.3
75.1
78.4
70.7
77.8
62.1
106.2
75.3
78.4
71.5
78.4
62.6
104.0
74.5
77.0
81.5
76.6
61.8
106.4
82.8
89.7
72.1
78.8
64.3
106.2
77.4
79.1
72.9
80.0
63.6
105.9
76.5
80.0
72.2
79.9
63.6
107.7
76.8
80.0
73.0
79.3
64.1
104.6
73.9
76.8
80.9
76.6
61.8
105.9
82.6
89.9
72.1
78.8
64.3
106.1
77.3
79.5
72.5
79.7
63.8
105.8
76.9
79.9
73.0
79.8
63.6
106.2
76.5
80.0
72.9
79.9
64.1
102.5
72.9
75.3
80.0
75.0
60.3
104.9
81.2
88.8
70.6
77.3
62.8
104.7
75.9
77.6
71.4
78.3
62.1
104.3
75.0
78.4
70.7
78.4
62.1
104.7
75.0
78.4
71.5
78.4
62.6
3′
4′
5′
6′
Glc 1′′
2′′
3′′
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
OMe
4′′
5′′
6′′
Glc 1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
Glc 1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
Glc 1′′′′′
2′′′′′
3′′′′′
4′′′′′
5′′′′′
6′′′′′
a Assignments are based on DEPT, HSQC, and HMBC experiments.
20

Highlights
► Nine steroidal compounds including five new compounds were isolated from Allium
macrostemon.
► Their chemical structures were determined based on spectroscopic methods.
► The isolated compounds were tested for cytotoxicity against four human tumor cell lines
in vitro using the sulforhodamine B bioassay.
21

Graphical Abstracts
Allimacrosides A-E, new steroidal glycosides from Allium macrostemon
Bunge
R2O
OH
R2O
OMe
O
O
O
OR2
OR2
O
O
O
O
HO
R1O
R1O
R1O
R1O
R1O
H
2
4
1
3
5
OH
O
OH
O
OH
OH
O
1''
HO
1''''
O
O
OH
1'''''
O
R2
:
R1
:
O
1'
HO
HO
HO
HO
HO
O
OH
1'''
OH
OH
OH
HO
HO
22