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IC50
µg/mL µM
pelargonidin 130 420
cyanidin 7.0 22
delphinidin 0.8 2.4
Figure 4. Relationship between the signal intensity of the DMPO−OOH spin adducts and concentration of pelargonidin, cyanidin, or delphinidin (concentration
of DMPO, 0.45 M or 0.045 M).
Antioxidant Activities of Pomegranate Anthocyanidins J. Agric. Food Chem., Vol. 50, No. 1, 2002 169
DISCUSSION
Pomegranate extract showed potent scavenging activity for
superoxide radicals. A filtered sample from the extract only
slightly decreased the activity when compared to that of the
nontreated sample. These results suggest that the scavenging
activity of pomegranate extract is due to its relatively smaller
molecular weight components (MW < 10,000). Anthocyanidins
showed potent activity in the order of delphinidin, cyanidin,
and pelargonidin. Anthocyanidins, aglycons of anthocyanins,
are known to exist in red fruits (19). Delphinidin-3,5-diglucoside
Figure 5. Hydroxyl radical scavenging activity of pomegranate extract. is a major anthocyanin in pomegranate juice (1). From the
H2O2/UV system was compared to the DMPO-OH signals from spectrophotometric observation, the estimated value of del-
the H2O2/UV system and the delphinidin (cyanidin or pelar- phinidin content in fresh juice was approximately 0.06% if it
gonidin)/UV system (Figure 8). These anthocyanidins did not was calculated on the basis of delphinidin. The yield of freeze-
show direct scavenging from the H2O2/UV system. dried sample was about 11% of the fresh juice and seed coats.
Inhibitory Effect of Pomegranate Extract and Anthocya- Meanwhile, the activity of pomegranate extract and delphinidin
nidins on H2O2-Induced Lipid Peroxidation. Pomegranate were 17 ( 1 and 1360 ( 214 SOD-equivalent units/mg.
extract lowered the levels of malonaldehyde plus 4-hydroxy- Therefore, the activity would most likely be due to delphinidin
alkenals produced by H2O2-induced lipid peroxidation in rat which is a major component contained as delphinidin-3,5-
brain homogenates during the incubation (Figure 9a). The ID50 diglucoside in pomegranate juice (1), and may be in part due
value of the inhibition was 10 mg/mL. Anthocyanidins inhibited to cyanidin which is also contained in the seed coats (2). In
lipid peroxidation in a dose-dependent manner (Figure 9b). The this study, a hypoxanthine-xanthine oxidase system was used
ID50 values were 0.7, 3.5, and 85 µM for delphinidin, cyanidin, for superoxide radical generation. It has been reported that
and pelargonidin, respectively. flavonoids, such as flavones, flavonols, and isoflavones, inhib-
Nitric Oxide Radical Scavenging Activity. Pomegranate ited xanthine oxidase (20). Meanwhile, it was mentioned that
extract did not show NO scavenging activity up to 2.5 mg/mL. flavonoids inhibited various enzymes such as alkaline phos-
Figure 6. Relationship between the signal intensity of the DMPO-OH spin adducts and concentration of pelargonidin, cyanidin, or delphinidin (concentration
of DMPO. 45 mM or 4.5 mM).
in the B-ring at neutral pH (33, 34), although the mechanism (15) Mori, A.; Edamatsu, R.; Kohno, M.; Ohmori, A. A new hydroxyl
in this case needs clarification. radical scavenger: EPC-K1. Neurosciences 1989, 15, 371-376.
Delphinidin, cyanidin, and pelargonidin inhibited H2O2- (16) Noda, Y.; Mori, A.; Ogata, K.; Packer, L. Hydroxyl and nitric
induced lipid peroxidation in rat brain homogenates in the order oxide radical scavenging activities of new synthetic derivatives
of delphinidin > cyanidin > pelargonidin. It was suggested that of R-tocopherol and glutathione; 2-S-[2-(N-carbonylaminoeth-
delphinidin, the main component in pomegranate juice, is the ylsulfonic acid)-1-(R-tocopheryl-6-yloxy-carbonyl)ethyl] glu-
most contributing to the inhibitory effect of pomegranate on tathione and 2-S-[2-(N-carbonyl-3-β-aminoethyl-5- hydroxyindol)-
H2O2-induced lipid peroxidation. 1-(R-tocopheryl-6-yloxy-carbonyl)ethyl] glutathione. Res. Commun.
Biochem. Cell & Mol. Biol. 1999, 3, 131-143.
Under in vitro experimental conditions, pomegranate extract
(17) Lowry, O. J.; Rosebrough, N. J.; Farr, A. L.; Randall, R. L.
and anthocyanidins did not show nitric oxide scavenging
Protein measurement with folin-phenol reagent. J. Biol. Chem.
activity.
1951, 193, 265-275.
ABBREVIATIONS USED (18) Kimura, K. Mechanism of active oxygen species reduction by
non-steroidal anti-inflammatory drugs. Int. J. Biochem. Cell Biol.
ESR, electron spin resonance; DMPO, 5,5-dimethyl-1-pyr- 1997, 29, 437-446.
roline-N-oxide; MGD, N-methyl-D-glucamine-dithiocarbamate; (19) Harborne, J. B.; Grayer, R. J. The anthocyanins. In The
[(MGD)2Fe2+], complex Fe2+ with N-methyl-D-glucamine dithio- FlaVonoids: AdVances in Research Since 1980; Harborne, J. B.,
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mutase; EPC-K1, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8- (20) Hayashi, T.; Sawa, K.; Kawasaki, M.; Arisawa, M.; Shimizu,
tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl- M.; Morita, N. Inhibition of cow’s milk xanthine oxidase by
hydrogen phosphate] potassium salt. flavonoids. J. Nat. Prod. 1988, 51, 345-348.
(21) Havsteen, B. Flavonoids, a class of natural products of high phar-
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