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Received 8 November 2004; received in revised form 7 February 2006; accepted 7 February 2006
Abstract
The objectives of this study were to study the antioxidant activity and free radical-scavenging effects of extracts from guava leaves and
dried fruit. The results indicated that 94.4–96.2% of linoleic acid oxidation was inhibited by the addition of guava leaf and guava tea
extracts at a concentration of 100 lg/ml. The guava dried fruit extracts exhibited weaker antioxidant effects than did the leaf extracts.
The results also demonstrated that the scavenging effects of guava leaf extracts on ABTS+ radicals and superoxide anion increased with
increasing concentrations. The guava leaf extracts displayed a significant scavenging ability on the peroxyl radicals. However, the scav-
enging effects were decreased when the extract concentration was greater than 10 lg/ml. The extracts from leaves of various guava cul-
tivars exhibited more scavenging effects on free radicals than did commercial guava tea extracts and dried fruit extracts. The
chromatogram data indicated that guava extracts contained phenolic acids, such as ferulic acid, which appeared to be responsible for
their antioxidant activity. Correlation analysis indicated that there was a linear relationship between antioxidant potency, free radi-
cal-scavenging ability and the content of phenolic compounds of guava leaf extracts.
2006 Published by Elsevier Ltd.
2.4.1. Scavenging effects on ABTS+ radicals The contents of phenolic compounds in extracts from
This decoloration method consists of an enzymatic sys- guava were determined by HPLC, performed with a Hit-
tem containing a peroxidase, hydrogen peroxide and achi liquid chromatograph (Hitachi, Ltd., Tokyo, Japan)
ABTS. A radical is generated from ABTS and has a char- consisting of a model L-6200 pump, and a model L-4200
acteristic absorption spectrum with a maximum of UV–Vis detector set at 285 nm. The analyses were carried
414 nm. The ability of extracts from guava to scavenge out on a LiChrospher RP-18 column (250 mm · 4 mm
ABTS+ radicals was determined by the method described i.d., 5 lm, E. Merck Co., Darmstadt, Germany). Extracts
in the work of Okamoto, Hayase, and Kato (1992) with were filtered through a 0.45 lm filter before use. The elu-
some modifications. In brief, 30 lM H2O2 and extracts tion solvents were (A) 0.2 M NaH2PO4 adjusted to pH
were added to 0.1 M phosphate buffer (pH 6.0) medium 3.0 by phosphoric acid, and then diluted with distilled
containing 0.02% ABTS and 6 units of peroxidase. Absor- water to 50 mM NaH2PO4 and (B) 50 mM NaH2PO4/
bance at 414 nm was measured after incubation for methanol/acetonitrile (30/20/50, v/v/v). The solvent gradi-
15 min. All analyses were run in triplicate and mean val- ent elution programme used was as follows: initial 100% A,
ues were calculated. hold for 6 min; linear gradient to 92% A in 8 min and hold
for 6 min; linear gradient to 82% A in 35 min; linear gradi-
2.4.2. Scavenging effects on superoxide anion ent to 62% A in 10 min and hold for 15 min; linear gradient
The influence of extracts from guava on the generation to 0% A in 10 min and hold for 10 min. The flow rate was
of superoxide anion was measured according to the method 1 ml/min. Phenolic compounds were identified by compar-
described in previously work (Yen & Chen, 1995). Super- ison of their retention time (Rt) values and UV spectra with
oxide anion was generated in a non-enzymic system and those of known standards and determined by peak areas
determined by a spectrophotometric measurement for from the chromatograms. All analyses were run in tripli-
reduction of nitro blue tetrazolium. The reaction mixture, cate and mean values were calculated.
which contained 1 ml of extract in distilled water, 1 ml of
PMS (60 lM) in phosphate buffer (0.1 M, pH 7.4), 1 ml 2.7. Statistical analysis
of NADH (468 lM) in phosphite buffer and 1 ml of NBT
(150 lM) in phosphate buffer, was incubated at ambient Data were analyzed using the Statistical Analysis System
temperature for 5 min, and the colour was read at software package. Analyses of variance were performed
560 nm against blank samples. All analyses were run in using ANOVA procedures. Significant differences between
triplicate and mean values were calculated. means were determined using Duncan’s multiple range test.
Recently, there has been considerable interest in preven- scavenged by antioxidants via the mechanism of elec-
tive medicine through the quest for natural antioxidants tron-/hydrogen-donation and are assessed by measuring
from plant material. Various phytochemical components, the decrease in absorption at 414 nm. In the superoxide
such as flavonoids, phenolic acids and carotenoids, are anion-scavenging test, superoxide anion, that is derived
known to be responsible for the antioxidant capacity of from dissolved oxygen through the PMS/NADH coupling
plants. However, the effectiveness of flavonoids as effective reaction, reduces NBT and increases absorption at 560 nm.
antioxidants is dependent upon the environment. A num- The decrease in absorption at 560 nm with antioxidants
ber of factors may influence the behaviour of flavonoids thus indicates the consumption of superoxide anion. In
and may result in alterations to their efficacy as antioxi- the peroxyl radical-scavenging assay, thermal decomposi-
dants. The antioxidant activity of flavonoids may be tion of AAPH leads to the formation of carbon-centred
reduced by the autoxidation of flavonoids, catalyzed by radicals which, under aerobic conditions, yield alkylper-
transition metals to produce superoxide anion. The latter oxyl radicals. These radical species can be detected by assay
dismutates to generate hydrogen peroxide and form hydro- of fluorescent decomposition of b-phycoerthrin, a major
xyl radicals via a Fenton reaction in the presence of transi- pigment protein of sea algae. The absorption assay for
tion metals (Canada, Giannella, Nguyen, & Mason, 1990). antioxidants was based on oxidation of b-phycoerthrin
Most plant polyphenol compounds possess both antioxi- molecules by peroxyl radicals (Cao, Alessio, & Cutler,
dant and prooxidant properties, depending on concentra- 1993).
tion and environmental factors (Cao, Sofic, & Prior, The abilities of extracts from guava, assayed to be scav-
1997). A possible mechanism of polyphenol cytotoxicity enging the ABTS+ radical in comparison with polyphenon
may be related to their prooxidant properties. In our previ- 60, are shown in Fig. 1. The scavenging effect of polyphe-
ous work, tea extracts showed both antioxidant and proox- non 60 was observed to be higher than that of extracts from
idant activities in oxidative damage of biomolecules (Yen, guava. The polyphenon 60 showed a linear increase in
Chen, & Peng, 1997). Azam, Hadi, Khan, and Hadi ABTS+ radical-scavenging activity with increasing concen-
(2004) proposed that the prooxidant action of tea poly- tration, reaching 97.1 ± 0.9% scavenging activity at a con-
phenolics may be an important mechanism of their anti- centration of 5 lg/ml. In various samples of guava extracts,
cancer and apoptosis properties. In addition to the scavenging activities of Shi Ji Ba, Hong Ba, and Tu Ba
polyphenolics, the prooxidant activity of green tea extracts extracts on ABTS+ radicals were stronger than those of
may be caused by their chlorophyll components (Wanas- Shui Jing Ba, guava tea A and guava tea B extracts. How-
undara & Shahidi, 1998). In the above results, the extracts ever, the extracts from four guava cultivars expressed over
from guava leaf and fruit exhibited strong potential antiox- 95% scavenging activity at a concentration of 20 lg/ml. In
idant activity. The true prooxidant effects that guava all samples, extracts from dried guava fruit had the weakest
extract has on cells remains as a matter to be studied scavenging ability on ABTS+ radicals. The decolorization
further. assay, using free blue-green ABTS+ radicals, was shown to
be a very useful tool in expeditiously measuring the antiox-
3.2. Radical-scavenging effects idant activity of individual chemical compounds or com-
plex extracts. This method can express total antioxidant
In this study, three free radicals were used to assess the activity as vitamin C equivalent antioxidant capacity
potential free radical-scavenging activities of guava (VCEAC) or as trolox equivalent antioxidant capacity
extracts, namely ABTS+ radical, superoxide anion and (TEAC) value (Kim, Lee, Lee, & Lee, 2002; Re et al.,
peroxy radicals. ABTS is a peroxidase substrate which, 1999).
when oxidized in the presence of H2O2 in a typical peroxi- As shown in Fig. 2, the scavenging effects of polyphenon
dative reaction, generates a metastable radical with a 60 and extracts from guava on the superoxide anion were
characteristic absorption spectrum and an absorption similar to the results of the scavenging effects on ABTS+
maximum of 414 nm (Arnao, Cano, Hernandez-Ruiz, Gar- radicals. The abilities of all samples to scavenge superoxide
cia-Canovas, & Acosta, 1996). The ABTS+ radicals are anion decreased in the order: polyphenon 60 > Shi Ji Ba,
100
80
0
0 5 10 15 20
Concentration (μg /ml)
Fig. 1. Scavenging effects of extracts from leaves and dried fruit of guava on ABTS+ radicals.
100
80
Scavenging effects (%)
Shi Ji Ba
Shui Jing Ba
60 Tu Ba
Hong Ba
Guava tea A
40 Guava tea B
Dried fruit
Tea polyphenon 60
20
0
0 100 200 300 400 500
Concentration (μg /ml)
Fig. 2. Scavenging effects of extracts from leaves and dried fruit of guava on superoxide anion.
Table 2
Scavenging effects of extracts from leaves and dried fruit of guava on the peroxyl radicals
Sample concentration (lg/ml) Scavenging effects (%)a
Shi Ji Ba Shui Jing Ba Tu Ba Hong Ba Guava tea A Guava tea B Dried fruit Tea polyphenon 60
2.5 83.3 ± 6.0 77.3 ± 3.1 82.0 ± 5.4 86.1 ± 4.1 60.2 ± 5.1 37.9 ± 2.4 16.6 ± 7.3 90.6 ± 6.7
5.0 87.6 ± 8.5 84.9 ± 4.5 85.2 ± 2.2 87.7 ± 3.6 86.0 ± 6.5 66.8 ± 1.7 44.0 ± 4.3 89.2 ± 4.0
10.0 88.6 ± 7.9 88.6 ± 4.4 92.5 ± 1.2 94.2 ± 4.0 90.2 ± 2.5 90.5 ± 3.3 79.3 ± 6.6 79.0 ± 4.6
25.0 74.2 ± 2.4 51.8 ± 3.7 68.1 ± 2.8 77.5 ± 5.7 82.2 ± 8.2 89.0 ± 4.9 89.2 ± 2.3 62.9 ± 5.7
50.0 58.7 ± 6.9 23.5 ± 2.9 48.5 ± 4.2 58.9 ± 8.7 64.9 ± 7.1 78.7 ± 8.0 91.2 ± 4.2 39.8 ± 5.7
a
Scavenging effects (%) = [(fluorescence of b-phycoerythrin with AAPH and sample-fluorescence of b-phycoerythrin with AAPH)/(fluorescence of b-
phycoerythrin fluorescence of b-phycoerythrin with AAPH)] · 100.
Table 3
The contents of total phenolic compounds and phenolic acids of extracts from leaves and dried fruit of guava
Samples Total phenolic compounds Phenolic acid
Expressed as (+)-catechin (mg/g) Expressed as gallic acid (mg/g) Gallic acid (lg/g) Ferulic acid (lg/g)
Shi Ji Ba 296 ± 5.4 458 ± 8.1 1621 ± 87.4 672 ± 65.2
Shui Jing Ba 267 ± 5.4 414 ± 8.2 793 ± 52.3 108 ± 15.7
Tu Ba 313 ± 4.7 483 ± 7.1 1022 ± 62.4 355 ± 45.4
Hong Ba 294 ± 4.0 455 ± 6.1 1137 ± 79.6 296 ± 25.9
Guava tea A 103 ± 9.3 166 ± 14.1 725 ± 54.1 147 ± 16.8
Guava tea B 177 ± 9.2 279 ± 14.0 1278 ± 92.7 234 ± 27.5
Dried fruit 69.6 ± 2.8 115 ± 4.2 266 ± 15.4 –
Tea polyphenon 60 643 ± 8.5 985 ± 12.8 – –