CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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(CHCl3/MeOH 9.3 :0.7), and Fr. EE (CHCl3/MeOH 9.0 :1.0). The Frs. EA and EB could not be worked up
due to paucity of material. The Fr. EC contained a major compound with lingering traces of impurities,
which could be removed through HPLC on JAIGEL ODS-M 80 column with H2O/MeOH 1:1 at a flow
rate of 3 ml/min. The pure compound 1 was obtained as an off-white amorphous solid (tR 40 min; 14 mg).
The Fr. ED was also subjected to HPLC on same column, mobile phase, and flow rate to furnish
compound 2 as light-yellow amorphous solid (tR 31 min; 11 mg). The Fr. EE contained a major
compound, which crystallized from MeOH, m.p. 2388, and could be identified as bergenin (3) by
comparison of physical and spectral data with those reported in the literature [10].
Bergecin A (¼4-O-(3,5-Di-O-methylgalloyl)bergenin; 1). Off-white amorphous solid. [a]2D5 ¼ ꢀ7.5
(c ¼ 0.056, MeOH). UV (MeOH): 220 (4.39), 274 (3.95). IR (nujol): 3320, 1710, 1600, 1530, 1510, 1220.
1H- and 13C-NMR: see Tables 2 and 1, resp. EI-MS: 508 (100), 327 (37), 208 (77), 181 (52). HR-EI-MS:
508.1220 (Mþ, C23H24O1þ3 ; calc. 508.1216).
Bergecin B (¼4-O-[(E)-4-Hydroxy-3,5-dimethoxycinnamoyl]bergenin; 2). Light-yellow amorphous
solid. [a]2D5 ¼ ꢀ48.1 (c¼0.042, MeOH). UV (MeOH): 218 (4.41), 275 (3.98). IR (nujol): 3330, 1710,
1630, 1600, 1525, 1500, 1220. 1H- and 13C-NMR: see Tables 2 and 1, resp. EI-MS: 534 (100), 327 (40), 208
(81), 207 (40). HR-EI-MS: 534.1369 (Mþ, C25H26O1þ3 ; calc. 534.1373).
Acid Hydrolysis of 1 and 2. Controlled acid hydrolysis was carried out according to a previously
reported protocol [15]. To 1 (10 mg) in H2O (1 ml) was added CF3CO2H (0.1 ml), and the mixture was
refluxed for 40 h. The residue obtained after evaporation was methylated with CH2N2/Et2O at 48 for 3 h.
Prep. TLC (C6H6/Me2CO 1:1) afforded dimethyl bergenin (2.1 mg), m.p. 2068, and methyl tri-O-
methylgallate (2 mg), m.p. 79–818. Analogus hydrolysis of 2 gave dimethylbergenin (2.2 mg) and methyl
(E)-3,4,5-trimethoxycinnamate (2.3 mg), m.p. 99–1008. All the hydrolysis products were identified by
comparison with authentic samples (m.p., IR, and 1H-NMR).
In vitro Lipoxygenase Inhibitory Assay. Lipoxygenase inhibitory activity was determined by slightly
modifying the spectrometric method developed by Tappel [16]. Lipoxygenase (1.13.11.12) type I-B (from
soybean) and linoleic acid were purchased from Sigma Chemicals. A mixture of 160 ml of 100 mm
phosphate buffer, pH 5.0, 10 ml of test compound, and 20 ml of lipoxygenase soln. was incubated for
10 min at 258. The reaction was then initiated by the addition of 10 ml of linoleic acid (substrate) soln.
[17], resulting in the formation of (9Z,11E,13S)-13-hydroperoxyoctadeca-9,11-dienoate. The change in
absorbance was followed for 6 min at 234 nm. Test compounds and the control were dissolved in MeOH
or 50% EtOH. All the reactions were performed in triplicate on a 96-well plate reader Spectromax 384
plus (Molecular Devices, USA). The IC50 values were calculated using the EZ-Fit Enzyme Kinetics
Program (Perrella Scientific Inc., Amherst, USA). The percentage [%] inhibition was calculated by the
formula (EꢀS)/Eꢂ100, where E is the activity of the enzyme without test compound, and S is the
activity of enzyme with test compound.
Dissociation constant/inhibition constant (K1) was determined by the interpretation of Dixon and
LineweaverꢀBurk plots and their secondary replots using initial velocities obtained over a substrate
(linoleic acid) concentration range between 0.06–0.5 mm.
Antioxidant Assay. The DPPH (¼1,1-diphenyl-2-picrylhydrazyl) assay was performed essentially
according to the protocol in [18]: 95 ml of 3.2 mm DPPH soln. in abs. EtOH and 5 ml of sample soln. in
DMSO were mixed in a 96-well plate. The optical density was measured at 515 nm after incubation of the
plate for 1 h at 378. The DPPH control contained no sample but was otherwise identical.
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