Journal of Natural Products
Article
Extraction and Isolation. Silybin was isolated from silymarin by
suspending silymarin in MeOH and filtration. 2,3-Dehydrosilybin was
prepared as described before.14
S24, S25, S26−S32); ESIMS m/z 479.1 (100%) [M − H]−, 375.2 (7),
309.2 (15), 301.0 (39), 293.2 (84); (−)-HRESIMS m/z 497.0976
(calcd for C25H19O10 479.0984).
Silychristin (3). 3 (natural mixture of diastereomers A and B; total
content of 3 is more than 96%) was isolated from S. marianum using
LH-20 column chromatography as published previously9 as a pale
yellow amorphous solid, and it was used for further reactions and
separations.
2,3-Dehydroanhydrosilychristin (5). To a solution of 2,3-
dehydrosilychristin (4, 200 mg, 0.42 mmol) in EtOH−H2O (15 mL,
9:1) was added concentrated HCl (5 mL), and the mixture was stirred
at 80 °C for 1 h in a sealed glass vessel. The mixture was cooled and
filtered through a pad of silica gel, which was then washed with EtOH,
and the combined filtrates were evaporated to yield compound 5 (80
mg, 41%) as a brown, amorphous solid, which was then lyophilized
from t-BuOH. Reddish-brown lyophilizate; UV (MeOH) λmax 267,
Silychristin A (3a) and Silychristin B (3b). A 1.0 g aliquot of natural
silychristin (3) was injected onto an HPLC column (25 × 250 mm,
silica gel, 10 μm particle size) in 10 injections (3 mL total volume
injected in acetone, mobile phase CHCl3−acetone−HCO2H, 9:1:0.01)
to obtain silychristin A (3a, 332 mg, purity 95.2%) and silychristin B,
which was reinjected twice to obtain pure compound (3b, 13 mg,
purity >98%).
1
303, 367, 425 nm; H, 13C NMR, MS, and UV data, see Supporting
Information (Table S36, Figures S34, S35, S37−S42); ESIMS m/z
461.1 [M − H]− (100%), 301.0 (7); (−)-HRESIMS m/z 461.0868
(calcd for C25H17O9 461.0878).
3a: white, amorphous solid; [α]D +6.1 (c 0.014, MeOH), lit.7 +112
(c 0.3, MeOH, 26 °C); UV (MeOH) λmax 205, 230, 238, 288 nm; 1H,
S5, Figures S3, S4, S6−S11); ESIMS m/z 481 (100%) [M − H]−, 451
(1), 385 (1), 309 (3), 293 (9), 249 (5), 216 (2); (−)-HRESIMS m/z
481.1131 (calcd for C25H21O10 479.1140).
Anhydrosilychristin (6). An improved procedure5 was used. To a
solution of silychristin (3, 100 mg, 0.21 mmol) in EtOH (5 mL, 90%)
was added concentrated HCl (2.5 mL), and the mixture was stirred at
70 °C overnight. The mixture was then evaporated, re-evaporated
twice with water, and purified by preparative HPLC to obtain 6 (23
mg, 24%), which was lyophilized from t-BuOH, yielding a pale yellow
lyophilizate: [α]D −4.3 (c 0.0067, MeOH); UV (MeOH) λmax 204,
3b: white, amorphous solid; [α]D −10.0 (c 0.0063, MeOH), lit.8
1
290, 317 nm; H, 13C NMR, MS, ECD, and UV data, see Supporting
1
+47 (c 0.03, MeOH); UV (MeOH) λmax 204, 231, 239, 288 nm; H,
S15, Figures S13, S14, S16−S21); ESIMS m/z 481 (100%) [M − H]−,
451 (2), 375 (4), 337 (1), 309 (14), 293 (49); (−)-HRESIMS m/z
481.1131 (calcd for C25H21O10 479.1140).
Information (Table S46, Figures S44, S45, S47−S52); ESIMS m/z
463.1 [M − H]− (88), 411.2 (2), 375.2 (9), 337.1 (3), 309.2 (19),
293.2 (100); (−)-HRESIMS m/z 463.1025 (calcd for C25H19O9
463.1035).
Antiradical and Antioxidant Assays. The compounds were
dissolved in DMSO; stock solutions (100 mM) were stored at −20 °C
and before each assay further diluted with the respective solvent.
DMSO content in the reaction mixtures was always kept below 0.1%.
Folin−Ciocalteau Reduction Assay. Folin−Ciocalteau reduction
capacity was determined as described previously,15 with some
modifications. Briefly, 5 μL of samples (1 mM) or standard (gallic
acid, 0.25−4 mM) in phosphate-buffered saline (PBS, pH 7.4) was
mixed with 100 μL of Folin−Ciocalteau reagent (Merck) diluted 10-
fold with distilled water. After 5 min at room temperature, 100 μL of
Na2CO3 solution (75 g/L) was added, and the reaction mixture was
further incubated for 90 min at room temperature. The absorbance
was measured at 725 nm using a Tecan Sunrise plate reader (Tecan
Silyhermin (7). A crude extract containing 7 was obtained by
extraction of the crushed fruits of S. marianum with acetone−H2O
(95:5). The crude extract was concentrated and defatted by extraction
with hexane, and the solvents were evaporated. A red-brown residue
containing about 15% (HPLC) of 7 was separated on preparative
HPLC (see General Experimental Procedures). Then 908 mg of the
mixture was injected in three portions (1 mL total volume), with 7
eluting at 31 min. The recovery was 83%. The combined fractions
were evaporated and lyophilized from t-BuOH to obtain 7 (90 mg,
purity >95%). The structure of 7 was inferred from its spectroscopic
data, which were in accordance with previously characterized 7-
pentaacetate.10 White lyophilizate; [α]D +23.5 (c 0.0068, MeOH); UV
1
(MeOH) λmax 203, 230, 287 nm; H, 13C NMR, MS, ECD, and UV
Group Ltd., Mannedorf, Switzerland), and the reducing capacity was
̈
S62); ESIMS m/z 465.1 [M − H]− (100), 435.1 (15), 293.2 (13);
(−)-HRESIMS m/z 465.1183 (calcd for C25H21O9 465.1191).
expressed as gallic acid equivalents.
DPPH Assay. The antiradical activity was evaluated using the
DPPH (Sigma-Aldrich) radical as previously described16,17 with minor
modifications. A 15 μL aliquot of each test substance (final
concentration 0−150 μM in MeOH) was mixed with 285 μL of
methanolic DPPH solution (final concentration 20 μM). After 30 min,
the absorbance was read at 517 nm. The percentage inhibition was
calculated using the negative control, and IC50 values were obtained
from the inhibition curves.
Isosilychristin (8). A crude extract containing 8 was obtained by
extraction of the crushed fruits of S. marianum with acetone−H2O
(95:5). The crude extract was concentrated and defatted with hexane,
and solvents were evaporated. A red-brown residue containing about
50% (HPLC) of 8 was separated by preparative HPLC. Then, 835 mg
of the mixture was injected in four portions, with 8 eluting at 21 min.
Recovery was 70%. The combined fractions were evaporated and
lyophilized from t-BuOH to obtain 8 (213 mg, purity over 95%),
which was identified by comparison with literature data.11 White
lyophilizate; [α]D +212.0 (c 0.012, MeOH), lit.11 +245 (c 0.1,
ABTS+• Scavenging. The capacity to scavenge ABTS+• was
evaluated using an antioxidant assay kit (CS0790, Sigma-Aldrich),
according to the manufacturer’s instructions. Briefly, 10 μL of the
sample (final concentration 100 μM), blank, or standard in an assay
buffer (pH 7.48) was mixed with 20 μL of myoglobin (3.5 mg/L of the
assay buffer), and 150 μL of a working solution containing 0.2 mM
ABTS and 2.45 mM H2O2 in a phosphate-citrate buffer, pH 5, was
added. The reaction mixture (pH 5.09) was incubated at room
temperature for ≥5 min, and then 100 μL of a stop solution was
added. Absorbance was measured at 405 nm, and the scavenging
activity was expressed as trolox (6-hydroxy-2,5,7,8-tetramethylchro-
man-2-carboxylic acid) equivalents (TE) from the trolox calibration
curve.
Measurement of logP. The hydrophobicity of the compounds
was determined by the measurement of partition coefficient P in a
mixture of two immiscible phases, octan-1-ol and 6.6 mM phosphate
buffer (pH 7.4), to simulate physiological conditions. Before use,
octan-1-ol was stirred with the buffer for 16 h at 25 °C to achieve
saturation of both phases, which were then separated. Stock solutions
(0.5 mM) of the compounds tested were prepared in octan-1-ol; 300
1
pyridine); UV (MeOH) λmax 203, 230, 288 nm; H, 13C NMR, MS,
S64, S65, S67−S72); ESIMS m/z 481.1 [M − H]− (100);
(−)-HRESIMS m/z 483.1284 (calcd for C25H23O10 483.1286).
Synthetic Procedures. 2,3-Dehydrosilychristin (4). A new
optimized procedure was used. (For optimization conditions, see
100 mg, 0.21 mmol in 5 mL of DMSO) was added 40 μL of
triethylamine, and the mixture was stirred at 60 °C under an O2
atmosphere overnight. Then, the mixture was diluted with water and
extracted with ethyl acetate. The organic phase was dried over Na2SO4
and concentrated in vacuo. The residue was separated by preparative
HPLC (see General Experimental Procedures) and lyophilized (t-
BuOH) to obtain the desired compound (27 mg, 27%). Yellow
lyophilizate; [α]D +72.1 (c 0.039, MeOH), lit.6 +71.2 (solvent and c
1
not given); UV (MeOH) λmax 230, 256, 373 nm; H, 13C NMR, MS,
E
J. Nat. Prod. XXXX, XXX, XXX−XXX
Biedermann, David
