Vol. 64, No. 5 (2016)
Chem. Pharm. Bull.
505
+
AꢀportionꢀofꢀmethanolicꢀextractꢀSgꢀ2ꢀ(25.34ꢀg)ꢀwasꢀseparat- C29H35O15 [M+H]+ 623.1970), [−]ꢀ HR-ESI-MSꢀ m/z 667.1854
ed by sequential liquid–liquid partition to obtain ethyl acetate (Calcd for [C29H34O15+HCOO]−,ꢀ 667.1880).ꢀ Methylꢀ 2,3,4-tri-
(Sgꢀ2.1,ꢀ1.3ꢀg,ꢀ5.13%),ꢀn-BuOHꢀ(Sgꢀ2.2,ꢀ4.1ꢀg,ꢀ16.2%)ꢀandꢀaque- O-(trimethylsilyl)-α/β-L-rhamnopyranoside,ꢀ tR 6.39/6.599, RI
ousꢀ(Sgꢀ2.3,ꢀ18.0ꢀg,ꢀ71%)ꢀextracts.
1568/1581, MS m/zꢀ (%):ꢀ 73ꢀ (100),ꢀ 133ꢀ (18.31/24.31),ꢀ 147ꢀ
TheꢀbutanolicꢀextractꢀSgꢀ2.2ꢀ(4.0ꢀg)ꢀwasꢀseparatedꢀinꢀaꢀstep- (13.85/23.71), 204 (49.36/51.91), 217 (8.27/15.04). Methyl
wiseꢀgradientꢀofꢀwater–MeOHꢀ(10–100%ꢀMeOH,ꢀincrementꢀofꢀ 2,3,4,6-tetrakis-O-(trimethylsilyl)-α/β-D-glucopyranoside,ꢀ tR
20%ꢀinꢀeachꢀstep)ꢀonꢀaꢀDIAIONꢀHP-20ꢀcolumnꢀ(30ꢀcm×5cm 11.764/12.103, RI 1873/1891, MS m/zꢀ (%):ꢀ 73ꢀ (100),ꢀ 133ꢀ
i.d.)ꢀ toꢀ getꢀ fiveꢀ fractionsꢀ (recoveryꢀ 81%).ꢀ Flavonoid-richꢀ frac- (22.26/29.91), 147 (18.15/13.67), 204 (45.97/47.18), 217
tionꢀ Sgꢀ 2.2.3ꢀ (500ꢀmg)ꢀ wasꢀ chromatographedꢀ onꢀ MCI-Gelꢀ (12.85/12.74).
CHP20P (30cm×2ꢀcmꢀ i.d.)ꢀ withꢀ aꢀ gradientꢀ ofꢀ water–MeOHꢀ
Measurement of DPPH Radical Scavenging Activity
(30–100%ꢀ MeOH,ꢀ incrementsꢀ ofꢀ 5%ꢀ inꢀ eachꢀ step)ꢀ toꢀ ob- Theꢀ DPPHꢀ freeꢀ radicalꢀ scavengingꢀ ofꢀ pureꢀ compoundsꢀ wereꢀ
tainedꢀ fifteenꢀ fractionsꢀ (Recoveryꢀ 90.8%).ꢀ Fractionsꢀ 6,ꢀ 8,ꢀ 9,ꢀ measuredꢀinꢀaꢀ96-wellꢀmicroplatesꢀformat.24)ꢀBriefly,ꢀ100ꢀµLꢀofꢀ
10ꢀ andꢀ 15,ꢀ wereꢀ furtherꢀ purifiedꢀ byꢀ preparativeꢀ HPLCꢀ RP18ꢀ dilutedꢀsamplesꢀatꢀdifferentꢀconcentrationsꢀ(1–1024ꢀmg/L),ꢀwasꢀ
(H3PO4 (aq)ꢀ0.05%–isopropylꢀalcoholꢀ(IPA),ꢀ95ꢀ:ꢀ5,ꢀ92ꢀ:ꢀ8,ꢀ90ꢀ:ꢀ10,ꢀ mixedꢀ withꢀ 100ꢀµLꢀ ofꢀ 132ꢀmg/Lꢀ DPPHꢀ solutionꢀ inꢀ methanol,ꢀ
88ꢀ:ꢀ12ꢀ andꢀ 80ꢀ:ꢀ20).ꢀ Fractionꢀ 6ꢀ yieldedꢀ 4ꢀ (8.2ꢀmg);ꢀ fractionꢀ 8ꢀ the plate was incubated at room temperature, after 1h, mea-
yielded 3ꢀ (23.8ꢀmg);ꢀfractionꢀ 9ꢀ yieldedꢀ 3ꢀ (12.5ꢀmg),ꢀ 4ꢀ(7.4ꢀmg)ꢀ surements of absorbance were done at 520nm in a microplate
and 1ꢀ(12.8ꢀmg);ꢀfractionꢀ10ꢀyieldedꢀ4ꢀ(10.7ꢀmg)ꢀandꢀ6ꢀ(15.1ꢀmg)ꢀ reader (Metertech, AccuReader M 965+). Ascorbic acid and
and fraction 15 yielded 5ꢀ(13.8ꢀmg)ꢀandꢀ2ꢀ(21.3ꢀmg).
quercetin were used as positive controls and ran in parallel.
Methanolysis and Silylation of Flavonoid Glycosides Theꢀresultsꢀwereꢀexpressedꢀasꢀaꢀpercentageꢀofꢀradicalꢀscaveng-
Flavonoidꢀ glycosidesꢀ andꢀ authenticꢀ monosaccharidesꢀ (Sigma- ingꢀactivityꢀ(%FRS)ꢀaccordingꢀtoꢀtheꢀequation:ꢀ
Aldrich),ꢀ includingꢀ D-(−)-arabinose,ꢀ D-(−)-lyxose,ꢀ D-(−)-
fructose, L-(−)-fucose,ꢀL-rhamnose,ꢀD-(+)-xylose,ꢀD-(−)-ribose,ꢀ
%FRS=[(Ac − As) / Ac]×100
D-(+)-mannose,ꢀD-(+)-galactoseꢀandꢀD-(+)-glucoseꢀ(1ꢀmg)ꢀwereꢀ where Ac is the absrobance of DPPH radicals without sample
heated with 2Nꢀ methanolicꢀ HClꢀ (0.5ꢀmL)ꢀ forꢀ 3ꢀhꢀ inꢀ aꢀ boilingꢀ or positive controls and As is the absrobance of DPPH radicals
waterꢀ bathꢀ toꢀ giveꢀ 1-O-methylꢀ glycosides.ꢀ Afterꢀ removalꢀ ofꢀ withꢀsampleꢀorꢀpositiveꢀcontrol.ꢀTheꢀefficientꢀconcentrationꢀofꢀ
the solvent by evaporation in vacuo,ꢀ1ꢀmLꢀofꢀacetonitrileꢀwasꢀ samples and positive controls that inhibits FRS50 was calculed
addedꢀ andꢀ freeꢀ hydroxylꢀ groupsꢀ wereꢀ trimethylsilylatedꢀ withꢀ andꢀexpressedꢀasꢀmg/L.
100 µLꢀ ofꢀ N,O-bis(trimethylsilyl)ꢀtrifluoroacetamideꢀ (BSTFA)ꢀ
andꢀtrimethylchlorosilaneꢀ(TMCS)ꢀ(99ꢀ:ꢀ1)ꢀasꢀreagentꢀ(Supelco)ꢀ ity (ORAC)ꢀ ꢀORACꢀ assayꢀ wasꢀ carriedꢀ outꢀ accordingꢀ toꢀ
forꢀ15ꢀminꢀatꢀ70°C.
methodꢀ describedꢀ byꢀ Huangꢀ et al.25)ꢀ withꢀ someꢀ modifica-
Kaempferolꢀ 3-O-β-Xylopyranosyl-(1→2)-α-arabinofuranosideꢀ tions.ꢀ Theꢀ assayꢀ wasꢀ performedꢀ inꢀ aꢀ 96-wellꢀ plate.ꢀ Sodiumꢀ
Measurement of Oxygen Radical Absorbance Capac-
(1)
fluoresceinꢀ wasꢀ dissolvedꢀ inꢀ phosphateꢀ bufferꢀ solutionꢀ (PBS)ꢀ
Yellow powder; [α]D25 +73.3 (c=0.80), MeOH; UV (MeOH) (75 mM,ꢀpHꢀ7.4)ꢀtoꢀobtainꢀaꢀstockꢀsolutionꢀofꢀ4ꢀµM.ꢀTheꢀwork-
λmaxꢀ (logꢀε) 270 (3.91), 300 (3.67), 356 (3.70); +NaOMe 273 ingꢀ solutionꢀ (0.08ꢀµM) was prepared by subsequent dilution
(3.97), 315 (3.68), 388 (3.89); +NaOAc 271 (3.98), 300 (3.66), inꢀ PBS.ꢀ Aꢀ 10ꢀmLꢀ solutionꢀ ofꢀ 2,2-azobis(2-amidinopropane)-
357 (4.07); +NaOAc+H3BO3 264 (3.92), 344 (4.11); +AlCl3 dihydrochloride (AAPH) was prepared at a concentration of
271 (3.95), 297 (3.55), 336 (3.97), 397 (3.70); +AlCl3+HCl 250µMꢀ (67.8ꢀmg/mLꢀ inꢀ PBS).ꢀ Forꢀ eachꢀ sessionꢀ ofꢀ measure-
271 (3.94), 296 (3.56), 337 (4.00), 398 (3.07) nm; IR (ATR) νmax ments,ꢀ aꢀ standardꢀ curveꢀ ofꢀ Troloxꢀ wasꢀ plottedꢀ (0.625–5ꢀµM).
cm−1:ꢀ3383ꢀ(OH),ꢀ1652ꢀ(carbonyl),ꢀ1608,ꢀ1503,ꢀ1466,ꢀ1160,ꢀ1030;ꢀ Troloxꢀ solutionsꢀ wereꢀ preparedꢀ inꢀ PBS.ꢀ Aꢀ blankꢀ (PBS)ꢀ wasꢀ
1H-ꢀ andꢀ 13C-NMR,ꢀ seeꢀ Tableꢀ 1;ꢀ [+]ꢀ ESI-MSꢀ m/z 551 [M+H]+, run with each assay. Samples were diluted in PBS at dif-
287 [C15H11O6]+; [−]ꢀESI-MSꢀm/z 549 [M−H]−. [+]ꢀHR-ESI-MSꢀ ferent concentrations (0.625–5µM)ꢀ fromꢀ stockꢀ solutionsꢀ atꢀ
m/z 573.1188 (Calcd for C25H26NaO14+, 573.1215), [−]ꢀ HR-ESI- 1ꢀmg/mLꢀ inꢀ dimethylꢀ sulfoxideꢀ (DMSO).ꢀ Sampleꢀ (50ꢀµL)ꢀ wasꢀ
MS m/z 595.1278 (Calcd for [C25H26O14+HCOO]−, 595.1305). mixedꢀ withꢀ sodiumꢀ fluoresceinꢀ (100ꢀµL)ꢀ andꢀ incubatedꢀ forꢀ
Methylꢀ 2,3,4-tri-O-(trimethylsilyl)-α/β-D-xylopyranoside,ꢀ tR 10ꢀminꢀ atꢀ 37°C.ꢀ Theꢀ reactionꢀ wasꢀ startedꢀ byꢀ theꢀ additionꢀ ofꢀ
7.646/7.978, RI 1645/1666, MS m/zꢀ(%):ꢀ73ꢀ(100),ꢀ133ꢀ(33.12/30.31),ꢀ AAPH (50µL)ꢀ andꢀ fluorescenceꢀ wasꢀ measuredꢀ immediatelyꢀ
147 (32.81/25.29), 204 (58.25/66.00), 217 (43.93/47.27). Methyl duringꢀ 120ꢀminꢀ inꢀ anꢀ espectrofluorometerꢀ JASCOꢀ FP-8500ꢀ
2,3,5-tri-O-(trimethylsilyl)-α/β-arabinofuranoside,ꢀ tR 5.93/6.119, (λexc 490nm, λem 514nm). The area under the curve (AUC)
RI 1537/1550, MS m/zꢀ (%):ꢀ 73ꢀ (100),ꢀ 133ꢀ (21.25/23.64),ꢀ 147ꢀ of each sample was calculated relative to the initial value
(14.62/20.24), 204 (18.87/22.21), 217 (38.59/40.93).
(theꢀfluorescenceꢀintensityꢀatꢀ0ꢀmin),ꢀandꢀtheꢀblankꢀvalueꢀwasꢀ
Kaempferolꢀ3,7-Di-O-methyl-4′-O-α-rhamnopyranosyl-(1→2)- subtracted.ꢀTheꢀregressionꢀcoefficientꢀbetweenꢀAUC and anti-
β-glucopyranosideꢀ(2)
oxidantꢀconcentrationꢀwasꢀcalculatedꢀforꢀallꢀsamplesꢀ(r2>0.93).
Yellow powder; [α]D25 −70.6 (c=1.3), MeOH; UV (MeOH) Theꢀ resultsꢀ expressedꢀasꢀ µmolꢀ ofꢀTroloxꢀEquivalentꢀ (TE)ꢀperꢀ
λmaxꢀ (logꢀε) 268 (4.33), 338 (4.21); +NaOMe 268 (4.32), 338 µmol of sample. Further positive control measurements were
(4.18); +NaOAc 268 (4.26), 338 (4.17); +NaOAc+H3BO3 performed with quercetin.
268 (4.30), 338 (4.18); +AlCl3 280 (4.24), 300 (3.88), 342
Statistical Analysis All the reported values represent the
(4.28), 400 (3.72); +AlCl3+HCl 280 (4.24), 300 (3.88), 340 mean standardꢀ deviationꢀ (S.D.)ꢀ ofꢀ experimentsꢀ performedꢀ inꢀ
(4.17), 400 (3.53) nm; IR (ATR) νmax cm−1:ꢀ 3360ꢀ (OH),ꢀ 1653ꢀ triplicate and statistically processed in GraphPad Prism 6.0.
1
(carbonyl), 1597, 1498, 1348, 1165, 1041; H-ꢀ andꢀ 13C-NMR,ꢀ
seeꢀ Tableꢀ 1;ꢀ positiveꢀ ESI-MSꢀ m/z 623.15 [M+H]+, 477.10
Acknowledgments Authorsꢀ areꢀ gratefulꢀ toꢀ “Universi-
[(M+H)−146]+;ꢀ negativeꢀ ESI-MSꢀ 621.10ꢀ [M−H]−, 312.95 dadꢀ delꢀ Valle”ꢀ andꢀ COLCIENCIASꢀ forꢀ theꢀ financialꢀ supportꢀ
[(M−H)−146−162]−; [+]ꢀHR-ESI-MSꢀm/z 623.1945 (Calcd for throughꢀ theꢀ Grantꢀ CI7852,ꢀ CT-557-2011.ꢀ H.G.ꢀ Torresꢀ C.,ꢀ