Vol. 26, No. 10 (2014)
Stigmasteryl Arachidate Constituent from the Straw of Oryza sativa 3019
13
26 x CH2, 7 x CH); C NMR (150 MHz; CDCl3): δ 37.19
using tetramethylsilane (TMS) as an internal standard, with
chemical shifts expressed in parts per million (δ) and coupling
constants (J) in hertz. Highresolution ESI/FT mass spectra were
recorded on a Thermo-Finnigan LTQ-Orbitrap instrument
(Thermo Scientific, USA) equipped with a Dionex U 3000
HPLC system (NICEM, Seoul National University).All chemicals
were of analytical grade. n-Hexane, ethyl acetate, methanol,
ethanol, sulfuric acid and vanillin were purchased from
Daejung Chemicals and Metals (Seoul, South Korea). Thin-
layer chromatography was performed on precoated silica gel
60 F254 plates (Merck). Visualization of the TLC plates was
performed using 5 % H2SO4 in ethanol spray reagent. Column
chromatography was performed using silica gel (70-230 mesh)
and LiChroprep RP-18 [40-63 µm; octadecyl silica (ODS) gel]
from Merck.Authentic standards of stigmasterol and arachidic
acid was purchased from Sigma-Aldrich (St. Louis, MO,
USA).
(C-1), 31.47 (C-2), 71.65 (C-3), 42.23 (C-4), 140.67 (C-5),
121.58 (C-6), 31.82 (C-7), 33.86 (C-8), 50.07 (C-9), 36.29
(C-10), 21.01 (C-11), 39.54 (C-12), 43.13 (C-13), 56.69
(C-14), 24.23 (C-15), 29.07 (C-16), 55.93 (C-17), 11.91
(C-18), 19.32 (C-19), 36.07 (C-20), 18.64 (C-21), 138.25
(C-22), 129.18 (C-23), 45.74 (C-24), 29.24 (C-25), 18.92
(C-26), 18.89 (C-27), 22.98 (C-28), 11.78 (C-29), 171. 99
(C-1'), 51.16 (C-2'), 40.44 (CH2), 39.54 (CH2), 39.61 (CH2),
29.65 (6 x CH2), 28.85 (CH2), 28.67 (CH2), 28.13 (CH2), 26.01
(CH2), 25.34 (CH2), 24.76 (CH2), 22.63 (CH2), 14.06 (C-20');
ESIMS m/z rel. int. 707 [M + H]+ (C49H87O2) (2.2), 411 (45.3),
394 (32.8), 311 (6.7), 295 (35.1), 139 (37.6) (Fig. 2).
29
28
21
27
25
26
22
20
24
23
18
The rice (O. sativa L.) straw used in the present study
was collected after harvesting of rice cereal at the Konkuk
University experimental farm,Yeoju, South Korea, in October
2010. The collected samples were dried in the laboratory in
the temperature range of 25-30 °C for 3 weeks with some
modifications of a previous study15. A voucher specimen
(reference code ILPUM variety) has been dried and deposited
in the herbarium of the Department of Applied Life Science,
Konkuk University.
12
H
17 16
11
13
19
10
H
9
1
4
15
14
2
8
m/z 311
H
H
3
7
5
O
m/z 394
6
m/z 411
CO CH2 (CH2)17 CH3
20'
1'
2'
m/z 295
Extraction of rice straw: Dried O. sativa straw (10 kg)
was immersed in methanol (55 L) for 1 week at room tempe-
rature and then the supernatant was concentrated under vacuum
to yield 78 g of extract. This material was suspended in water
and extracted with hexane, ethyl acetate and n-butanol succe-
ssively and evaporated to produce hexane (9.4 g), ethyl acetate
(11.2 g) and n-butanol (14.2 g) extracts.
Fig. 2. Fragmentation patterns of stigmasteryl-3β-arachidate (1)
Acid hydrolysis of compound 1: Compound 1 (5 mg)
was refluxed with 2 mL of 1M hydrochloric acid/dioxane (1:1)
in a water bath for 4 h. The reaction mixture was evaporated
to dryness and column chromatographed over silica gel with
hexane and ethyl acetate to obtain the stigmasterol and arachidic
acid and confirmed with authentic standards.
Isolation of compounds from hexane extract: The
hexane extract (9.4 g) was column chromatographed (CC) over
silica gel (70-230 mesh, 200 g, 800 × 25 mm) with hexane
and ethyl acetate solvents and yielding 25 fractions (each
frcation 250 mL): fractions 1-2 in hexane, fractions 3-4 in
hexane/EtOAc (9.5:0.5), fractions 5-6 in hexane/EtOAc (9:1),
fractions 7-8 in hexane/EtOAc (8:2), fractions 9-10 in hexane/
EtOAc (7:3), fractions 11-12 in hexane/EtOAc (6:4), fractions
13-14 in hexane/EtOAc (1:1); fractions 15-16 in hexane/EtOAc
(4:6) fractions 17-18 in hexane/EtOAc (3:7); fractions 19-20
in hexane/EtOAc (2:8), fractions 21-22 in hexane/EtOAc (1:9);
fractions 23-25 in EtOAc. Fractions 19-20 combined and rechro-
matographed over silica gel with chloroform and methanol to
afford compound 1 in crystalline form with eluants chloroform-
methanol (97:3).
RESULTS AND DISCUSSION
Compound 1, stigmasteryl-3β-arachidate, was obtained
as a yellow crystals from hexane-ethyl acetate (2:8) eluants.
Its IR spectrum showed characteristic absorption bands at 2932,
2845 and ester function (1723 cm-1), double bonds (1637 cm-1)
On the basis of ESI mass and 13C NMR spectra, the molecular
ion peak of 1 was determined at m/z 706 consistent to the
molecular formula of sterol attached with some long chain
acid C49H86O2.
1
The H NMR spectrum of 1 indicated steroidal moiety
attached arachidic acid moiety at C-3 position, as displayed
one-proton multiplet at δ 5.33 and two double doublets at δ
5.01 (J = 9.0, 8.4 Hz), 5.13 (J = 9.0, 9.0) were assigned to
H-6, H-22 and H-23 protons, respectively. A one-proton broad
multiplet at δ 3.51 with half-width of 15.6 Hz was ascribed to
oxygenated methane H-3α proton. Two proton triplet at δ 2.32
(J = 7.2 Hz) was due to methylene H2-protons adjacent to ester
group. Two three protons broad signals at δ 0.67 and 1.02 and
three doublets at δ 1.02 (J = 6.3), 0.87 (J = 6.2) and 0.85 (J =
6.6) intergrating for three protons each were accounted to
terteiary C-18 and C-19 and secondary C-21, C-26 and C-27
Stigmasteryl-3β-arachidate (1):Yellow crystals; Rf 0.34
(hexane:ethyl acetate; 2:8); m.p. 112-114°; IR (KBr, νmax, cm-1):
2932, 2845, 1723, 1637, 1401, 1379, 1240, 1192, 1064, 966,
930, 801, ; 1H NMR (600 MHz, CDCl3): δ 5.33 (1H, m, H-6),
5.13 (1H, dd, J = 9.0, 9.0 Hz, H-22), 5.01 (1H, dd, J = 9.0, 8.4
Hz, H-23), 3.51 (1H, br m, W1/2 = 15.6 Hz, H-3a), 2.32 (2H, t,
J = 7.2 Hz, H2-2), 1.02 (3H, br s, Me-19), 0.92 (3H, d, J = 6.3
Hz, Me-21), 0.87 (3H, d J = 6.2 Hz, Me-26), 0.85 (3H, d, J =
6.2 Hz, Me-27), 0.83 (3H, t, J = 6.5 Hz, Me-20'), 0.79 (3H, t,
6.1 Hz, Me-29), 0.67 (3H, br s, Me-18), 2.80-1.04 (59 H, m,