40
A. Begum et al. / Steroids 107 (2016) 37–44
(870 mg, 94%); m.p. 131–135 °C (from EtOH) (lit. [15], 131–
132 °C); IR, 1H and 13C NMR data were found to be consistent with
literature values [15]. MS (m/z): 613 [M+Na]+. Anal. calcd. for:
(m/z): 637 [M+Na]+. Anal. calcd. for: C40H54O5: C, 78.18; H, 8.79.
Found: C, 78.16; H, 8.77.
Ergosteryl ferulate, viz., 3-O-(trans-4-feruloyl)-ergosterol
(17b): 1 g of 3-O-(trans-4-O-acetylferuloyl)-ergosterol (12a) was
hydrolyzed under basic condition as per procedure described
above to get pure ergosteryl ferulate (17b) as white solid
C39H58O4: C, 79.32; H, 9.83. Found: C, 79.30; H, 9.81.
3-O-(trans-4-O-acetylferuloyl)-b-campesterol (15a): 1 g of b-
campesterol (15) was esterified with trans-4-O-acetylferulic acid
(9) under MW irradiation as per procedure described above to
get pure 3-O-(trans-4-O-acetylferuloyl)-b-campesterol (15a) as
(875 mg, 94%); m.p. 166–169 °C (from EtOH); IR:
m
max/cmꢀ1
3395, 2957, 2871, 1703, 1633, 1594, 1514, 1460, 1269, 1159,
758; NMR: dH (300 MHz; CDCl3; Me4Si) 0.7–1.2 (18H, C(18), C
(19), C(21), C(25), C(27) and C(28)Me), 3.9 (3H, s, OMe), 4.7 (1H,
m, C(3)H), 5.2 (2H, m, C(5)H, C(6)H), 5.3 (1H, d, J = 2.4 Hz, C(23)
H), 5.6 (1H, d, J = 2.5 Hz, C(22)H), 6.39 (1H, d, J = 16 Hz, C(80)H),
7.6 (1H, d, J = 8.8 Hz, C(70)H) and 6.8–7.1 (3H, m, Ph); dC
(75 MHz; CDCl3; Me4Si) 166.6, 147.8, 144.6, 144.6, 141.5, 138.6,
135.5, 131.9, 127.0, 123.0, 120.2, 120.2, 116.3, 114.7, 109.2, 72.7,
55.9, 55.7, 54.5, 46.0, 42.8, 40.4, 39.0, 37.9, 37.1, 36.8, 33.0, 28.3,
28.2, 23.0, 21.1, 21.0, 20.5, 20.5, 19.9, 19.9, 19.6, 17.6; MS (m/z):
595 [M+Na]+. Anal. calcd. for: C38H52O4: C, 79.72; H, 9.09. Found:
C, 79.70; H, 9.08.
white solid (1.46 g, 95%); m.p. 160–163 °C (from EtOH); IR: mmax
/
cmꢀ1 3010, 2862, 1765, 1710, 1640, 1510, 1464, 1080, 758;
NMR: dH (300 MHz; CDCl3; Me4Si) 0.7–1.3 (18H, C(18), C(19), C
(21), C(25), C(27) and C(28)Me), 2.3 (3H, s, COMe), 3.8 (3H, s,
OMe), 4.8 (1H, m, C(3)H), 5.4 (1H, m, C(5)H), 6.36 (1H, d,
J = 16 Hz, C(80)H), 7.6 (1H, d, J = 8.8 Hz, C(70)H) and 6.8–7.2 (3H,
m, Ph); dC (75 MHz; CDCl3; Me4Si) 168.4, 165.4, 152.4, 144.2,
141.9, 140.3, 133.7, 123.7, 122.5, 121.3, 118.7, 111.3, 74.3, 56.8,
56.2, 55.8, 50.4, 45.8, 42.4, 39.9, 38.5, 37.4, 36.9, 36.4, 34.2, 31.9,
31.6, 29.4, 28.5, 28.1, 24.5, 23.6, 21.5, 20.6, 20.6, 19.4, 19.4, 18.8,
17.4, 17.1; MS (m/z): 641 [M+Na]+. Anal. calcd. for: C40H58O5: C,
77.67; H, 9.39. Found: C, 77.65; H, 9.40.
b-Campesteryl ferulate, viz., 3-O-(trans-4-feruloyl)-b-cam-
pesterol (15b): 1 g of 3-O-(trans-4-O-acetylferuloyl)-b-cam-
pesteryl (15a) was hydrolyzed under basic condition as per
procedure described above to get pure b-campesteryl ferulate
(15b) as white solid (870 mg, 94%); m.p. 168–171 °C (from EtOH);
2.6. Antioxidant activity assays
2.6.1. Radical scavenging activity
The free radical scavenging activity of the samples was mea-
sured using the stable 2,20-diphenyl-picrylhydrazyl radical (DPPHÅ)
as described originally by Blois [24] with slight modifications.
Briefly, various concentrations of each of the synthesized steryl fer-
ulates (10b–17b) and their mixture (equimolar mixture of 10b–
IR:
m
max/cmꢀ1 3020, 2865, 1712, 1645, 1515, 1470, 1085, 760;
NMR: dH (300 MHz; CDCl3; Me4Si) 0.7–1.4 (18H, C(18), C(19), C
(21), C(25), C(27) and C(28)Me), 3.9 (3H, s, OMe), 4.8 (1H, m, C
(3)H), 5.3 (1H, m, C(5)H), 6.36 (1H, d, J = 16 Hz, C(80)H), 7.7 (1H,
d, J = 8.8 Hz, C(70)H) and 6.6–7.1 (3H, m, Ph); dC (75 MHz; CDCl3;
Me4Si) 165.3, 152.4, 144.4, 141.8, 140.3, 133.5, 123.7, 122.5,
121.4, 118.8, 111.7, 74.3, 56.8, 56.2, 55.9, 50.4, 45.8, 42.5, 39.9,
38.4, 37.2, 36.9, 36.3, 34.0, 31.9, 31.6, 29.2, 28.3, 28.0, 24.3, 23.6,
21.8, 20.4, 20.4, 19.5, 19.5, 18.6, 17.4; MS (m/z): 599 [M+Na]+. Anal.
calcd. for: C38H56O4: C, 79.17; H, 9.72. Found: C, 79.18; H, 9.73.
3-O-(trans-4-O-acetylferuloyl)-b-campestanol (16a): 1 g of b-
campestanol (16) was esterified with trans-4-O-acetylferulic acid
(9) under MW irradiation as per procedure described above to
get pure 3-O-(trans-4-O-acetylferuloyl)-b-campestanol (16a) as
white solid (1.48 g, 96%); m.p. 158–162 °C (from EtOH) (lit. [18],
161–162 °C); IR, 1H and 13C NMR data were found to be consistent
with literature values [18]. MS (m/z): 643 [M+Na]+. Anal. calcd. for:
C40H60O5: C, 77.41; H, 9.68. Found: C, 77.42; H, 9.70.
b-Campestanyl ferulate, viz., 3-O-(trans-4-feruloyl)-b-cam-
pestanol (16b): 1 g of 3-O-(trans-4-O-acetylferuloyl)-campesterol
(16a) was hydrolyzed under basic condition as per procedure
described above to get pure b-campestanyl ferulate (16b) as white
solid (872 mg, 94%); m.p. 155–158 °C (from EtOH) (lit. [18],
157 °C); IR, 1H and 13C NMR data were found to be consistent with
literature values [18]. MS (m/z): 601 [M+Na]+. Anal. calcd. for:
17b), and
c-oryzanol in methanol were taken in different test-
tubes. The volume was adjusted to 2 ml with methanol. 0.5 ml of
freshly prepared methanolic DPPH solution (0.05 mM) was added
to the tubes and shaken vigorously. The tubes were kept in the
dark for 30 min at room temperature and then the absorbance of
the samples was measured at 517 nm using a UV–Vis spectropho-
tometer (Lambda 35, Perkin-Elmer, Singapore). The free radical
scavenging activity of all the compounds was calculated as per-
centage of DPPH discoloration using the following formula:
% Radical scavenging activity ¼ ½ðAo ꢀ AÞ=Aoꢁ ꢂ 100
where Ao is the absorbance of the solution without the compound,
and A is the absorbance of the solution with the compound.
The scavenging activity of each of the compound was expressed
as 50% effective concentration, EC50 (mg/ml), which refers to the
concentration of the compound that produces 50% scavenging of
the DPPHÅ radical.
2.6.2. Total antioxidant capacity
The total antioxidant capacity of the compounds, viz., synthe-
sized steryl ferulates (10b–17b) and their mixture (equimolar mix-
ture of 10b–17b), and c-oryzanol, was determined by the Cupric
C
38H58O4: C, 78.89; H, 10.03. Found: C, 78.87; H, 10.10.
3-O-(trans-4-O-acetylferuloyl)-ergosterol (17a): 1 g of ergos-
Reducing Antioxidant Capacity (CUPRAC) assay, as described in lit-
erature [25]. Briefly, 1 ml each of CuCl2 solution (1.0 ꢂ 10ꢀ2 M),
neocuproine solution (7.5 ꢂ 10ꢀ3 M) and ammonium acetate buf-
fer (pH 7.0, 1.0 M) were added to a test- tube. After that 0.4 ml
sample solution in methanol and 0.7 ml of deionized water were
added and mixed. After 30 min, the absorbance was measured at
450 nm using a UV–Vis Spectrophotometer (Lambda 35, Perkin-
Elmer, Singapore). The antioxidant capacity was determined
terol (17) was esterified with trans-4-O-acetylferulic acid (9) under
MW irradiation as per procedure described above to get pure 3-O-
(trans-4-O-acetylferuloyl)-ergosterol (17a) as white solid (1.46 g,
94%); m.p. 166–169 °C (from EtOH); IR:
m
max/cmꢀ1 2955, 2872,
1766, 1706, 1644, 1509, 1464, 1261, 1221, 1184, 758; NMR: dH
(300 MHz; CDCl3; Me4Si) 0.7–1.2 (18H, C(18), C(19), C(21), C(25),
C(27) and C(28)Me), 2.2 (3H, s, COMe), 3.9 (3H, s, OMe), 4.7 (1H,
m, C(3)H), 5.2 (2H, m, C(5)H, C(6)H), 5.4 (1H, d, J = 2.5 Hz, C(23)
H), 5.7 (2H, m, C(5) and C(6)H), 6.4 (1H, d, J = 16 Hz, C(80)H), 7.7
(1H, d, J = 8.8 Hz, C(70)H) and 6.8–7.1 (3H, m, Ph); dC (75 MHz;
CDCl3; Me4Si) 168.8, 166.2, 151.3, 143.8, 143.8, 141.3, 138.5,
135.5, 133.4, 131.9, 123.2, 121.2, 121.2, 118.8, 116.3, 111.1, 73.0,
55.8, 55.7, 54.5, 46.0, 42.8, 40.4, 39.0, 37.9, 37.1, 36.7, 33.1, 28.2,
28.2, 23.0, 21.1, 21.0, 20.6, 20.6, 19.9, 19.9, 19.6, 17.6, 16.2; MS
against Trolox standard and the results were expressed as
Trolox equivalent (TE) per g of compound.
lmol
2.6.3. Reducing power assay
The reducing power of the compounds, viz., synthesized steryl
ferulates (10b–17b) and their mixture (equimolar mixture of
10b–17b), and
c-oryzanol, was determined according to the
method of Oyaizu [26]. Briefly, different concentrations of the