Journal of Natural Products
ARTICLE
reaction mixtures were acidified to pH 4.0 with 2 N HCl and extracted
with CHCl3 (2 mL ꢀ 2) and n-BuOH (3 mL ꢀ 2). The organic layer was
washed with H2O, dried over anhydrous Na2SO4, concentrated in
vacuo, and analyzed by GC-MS on a model 6890 GC interfaced with
a model 5975 MS (Agilent) at 70 eV under the following conditions (30
m ꢀ 0.32 mm ꢀ 0.25 μm, DB-5 MS column; He, 0.8 mL/min; 50 °C, 3
min; 50-300 °C, Δ 10 °C/min). From the GC-MS spectrum and by
comparison with authentic samples of trans-cinnamic acid (tR 10.6 min):
m/z 148 [M]þ (76), 147 (100), 131 (22), 120 (6), 103 (49), 102 (24),
91 (23), 77 (35), 74(7), 63 (6), 51 (36), 50 (10), 45 (15); n-dodecanoic
acid (tR 11.4 min): m/z 200 [M]þ (8), 183 (1), 171 (8), 157 (27), 143
(10), 129 (36), 115 (17), 101 (12), 87 (15), 85 (26), 83 (14), 73 (90),
71 (26), 60 (100), 57 (55), 55 (60), 43 (77), 41 (66), 29 (27), 27 (14);
n-decanoic acid (tR 9.9 min): m/z 172 [M]þ (4), 155 (1), 143 (9), 129
(49), 115 (12), 101 (6), 87 (15), 73 (79), 60 (100), 57 (48), 55 (45), 43
(52), 41 (50), 29 (20), 27 (13); 2-methylbutanoic acid (tR 4.0 min): m/z
87 (24), 74 (100), 73 (15), 57 (71), 55 (11), 45 (21), 41 (60), 39 (38),
29 (45), 27 (23); 2-methylpropanoic acid (tR 3.2 min): m/z 88 [M]þ
(7), 73 (26), 71 (2), 60 (1), 55 (6), 45 (13), 43 (100), 41 (47), 39 (14),
29 (5), 27 (20); n-hexanoic acid (tR 5.9 min): m/z 99 (1), 87 (12), 73
(40), 60 (100), 55 (14), 45 (14), 43 (33), 41 (26), 29 (10), 27 (13); and
n-octanoic acid (tR 8.1 min): m/z 144 [M]þ (1), 115 (9), 101 (22), 85
(17), 73 (58), 69 (10), 60 (100), 55 (32), 45 (12), 43 (47), 41 (36), 39
(13), 29 (16), 27 (13) were identified. The n-BuOH layer was subjected
to an open ODS column (MeOH-H2O, 70:30, v/v) to obtain the
glycosidic acid simonic aicd B (11).12,13 It gave key fragments at m/z
1001 [M - H]-, 855 [M - H - C6H10O6]-, 709 [855 - C6H10O6]-,
563 [709 - C6H10O6]-, 417 [563 - C6H10O6]-, and 271 [417 -
C6H10O6]- in the negative ESIMS. The organic fraction (3.2 mg) of the
alkaline hydrolysis of 1 was purified on ODS column chromatography
eluting with MeOH-H2O (25:75, v/v) to give 2-methylbutanoic acid
(0.5 mg). This was proved to be S-configured by comparing the specific
rotation ([R]2D5 þ18.9) with that of authentic 2S-methylbutanoic
acid.12,13
Acid Hydrolysis and Sugar Analysis. The glycosidic acid (15
mg, from alkaline hydrolysis), which was methylated with MeOH,
catalyzed with 0.5 N H2SO4 gave simonic acid B methyl ester (12).
Compound 12 was hydrolyzed with 1 N H2SO4 and extracted with Et2O
to yield 11-hydroxyhexadecanoic acid methyl ester (13).12b The aqu-
eous layer of acidic hydrolysis was concentrated under reduced pressure
to yield a residue of the sugars fraction. The protocols applied to
determine the configuration of sugars were the same as our previous
research, which permitted the identification of the mixture sugars of
L-rhamnose and D-fucose by comparison of their derivatives with those
of authentic samples.15
Preparation of Mosher’s Esters. The procedures for the pre-
paration of Mosher’s esters to determine the absolute configuration of
the aglycone were the same as described previously for resin glycosides
from Ipomoea batatas.12b The selected ΔδH values [ΔδH = δ(S) -
δ(R)] (ΔδH = -0.06, H-10; ΔδH = þ0.14, H-12; ΔδH = þ0.08, H-16)
of 11-(R-MPA)-hexadecanoic acid methyl ester (14) and 11-(S-MPA)-
hexadecanoic acid methyl ester (15) (Scheme 1) facilitated assignment
of the 11S absolute configuration.
1.66 (2H, m, H-10), 1.40 (2H, m, H-12), 0.76 (3H, t, J = 7.0 Hz, H-16);
ESIMS m/z 457 [M þ Na]þ.
11-(S-MPA)-Hexadecanoic acid methyl ester (15): colorless oil
(CHCl3), [R]2D5 þ1.5 (c 0.21, CHCl3); IR (KBr) νmax 3451, 2960,
2927, 2854, 1741, 1260 cm-1; 1H NMR (500 MHz, CDCl3) δH 7.45
(2H, m, C6H2), 7.35 (3H, m, C6H3), 4.74 (1H, s, OCH), 4.90 (1H, m,
H-11), 3.68 (3H, s, OCH3), 3.41 (3H, s, OCH3), 2.30 (2H, t, J = 7.5 Hz,
H-2), 1.60 (2H, m, H-10), 1.54 (2H, m, H-12), 0.84 (3H, t, J = 7.2 Hz,
H-16); ESIMS m/z 457 [M þ Na]þ.
Cytotoxicity Assays. MCF-7/ADR cells were maintained in
DMEM medium supplemented with 10% fetal bovine serum (Clark,
Australia), harvested with trypsin, and resuspended in a final concentra-
tion of 4.5 ꢀ 104 cells/mL. Aliquots (0.1 mL) of cell suspension were
seeded evenly into 96-well culture multiplates and incubated in a 37 °C
incubator containing 5% CO2 for 24 h. A series of concentrations for
pure compounds ranging from 400 to 1 μg/mL in DMSO were added to
designated wells. After 48 h, an MTT assay was performed as described
previously.21
MDR Reversal Assays. MCF-7/ADR cells were distributed into
96-well culture plates at 4.5 ꢀ 103 cells per well. Serial dilutions ranging
from 0.008 to 25 μg/mL of the known antitumor agent doxorubicin
(Zhejiang Haizheng Pharmaceutical Co., Ltd., China) with or without 5
μg/mL samples were added to the cells. Verapamil (5 μg/mL) was used
as positive control. After 48 h, the MTT assay was performed as
described above. IC50 values of doxorubicin were calculated from plotted
results using untreated cells as 100%. The reversal fold (RF) as potency
of reversal was obtained from fitting the data to RF = IC50 of doxorubicin
alone/IC50 of doxorubicin in the presence of sample.22 All assays were
peformed in triplicate.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR, ESIMS, and
S
b
HRESIMS spectra of compounds 1-10. This material is avail-
’ AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: þ86-25-83271405. E-mail: cpu_lykong@126.com.
’ ACKNOWLEDGMENT
This research was financially supported by the National Key
Scientific and Technological Special Projects (2009ZX09502-
011) and a grant from Key Laboratory of Bioactive Substances
and Resources Utilization of Chinese Herbal Medicine (Peking
Union Medical College); the Ministry of Education of China
(2010JZ-W-02) is also acknowledged.
’ REFERENCES
(1) Kinghorn, A. D.; Falk, H.; Kobayashi, J. In Progress in the
Chemistry of Organic Natural Products; Budzikiewicz, H.; Pereda-Mir-
anda, R.; Rosas-Ramírez, D.; Castanꢀeda-Gꢀomez, J., Eds.; Springerwien:
New York, 2010; pp 77-153.
(2) (a) Pereda-Miranda, R.; Mata, R. J. Nat. Prod. 1993, 56, 571–582.
(b) Bah, M.; Pereda-Miranda, R. Tetrahedron 1997, 53, 9007–9022.
(3) (a) Leꢀon, I.; Mirꢀon, G.; Alonso, D. J. Nat. Prod. 2006, 69, 896–
902. (b) Leꢀon, I.; Enríquez, R. G.; Gnecco, D.; Villarreal, M. L.; Cortꢀes,
D. A.; Reynolds, W. F.; Yu, M. J. Nat. Prod. 2004, 67, 1552–1556. (c)
Cao, S.; Guza, R. C.; Wisse, J. H.; Miller, J. S.; Evans, R.; Kingston,
D. G. I. J. Nat. Prod. 2005, 68, 487–492. (d) ChꢀeRigo, L.; Pereda-
Miranda, R. J. Nat. Prod. 2006, 69, 595–599.
11S-Hydroxyhexadecanoic acid methyl ester (13): colorless oil
(CHCl3), [R]2D5 þ1.3 (c 0.21, CHCl3); IR (KBr) νmax 3335, 2923,
2852, 1205 cm-1; 1H NMR (500 MHz, CDCl3) δH 3.65 (3H, s, OCH3),
3.57 (1H, m, H-11), 2.28 (2H, t, J = 7.5 Hz, H-2), 1.60 (2H, t, J = 7.0 Hz,
H-10), 1.42 (2H, m, H-12), 0.88 (3H, t, J = 7.0 Hz, H-16); HRESIMS m/
z 309 [M þ Na]þ.
11-(R-MPA)-Hexadecanoic acid methyl ester (14): colorless oil
(CHCl3), [R]2D5 -2.1 (c 0.11, CHCl3); IR (KBr) νmax 3443, 2925,
2857, 1742, 1263 cm-1; 1H NMR (500 MHz, CDCl3) δH 7.43 (2H, m,
C6H2), 7.33 (3H, m, C6H3), 4.72 (1H, s, OCH), 4.89 (1H, m, H-11),
3.66 (3H, s, OCH3), 3.40 (3H, s, OCH3), 2.29 (2H, t, J = 7.5 Hz, H-2),
627
dx.doi.org/10.1021/np100640f |J. Nat. Prod. 2011, 74, 620–628