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Antioxidantactivityandfreeradicalscavengingcapacityofextractsfromguava
(PsidiumguajavaL.)leaves
ArticleinFoodChemistry·January2007
DOI:10.1016/j.foodchem.2006.02.047
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Antioxidant activity and free radical-scavenging capacity of
extracts from guava (Psidium guajava L.) leaves
Hui-Yin Chen
a
a,*
, Gow-Chin Yen
b
Department of Food Science, Central Taiwan University of Science and Technology, 11, Putzu Lane, Peitun District, Taichung 40601, Taiwan, ROC
b
Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan, ROC
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 scavenging effects were decreased when the extract concentration was greater than 10 lg/ml. The extracts from leaves of various guava cultivars 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 radical-scavenging ability and the content of phenolic compounds of guava leaf extracts.
2006 Published by Elsevier Ltd.
Keywords: Guava leaves; Antioxidant activity; Radical scavenging; Phenolic compound
1. Introduction
The involvement of active oxygen and free radicals in
the pathogenesis of certain human diseases, including cancer, aging and atherosclerosis is increasingly being recognized (Moskovitz, Yim, & Chock, 2002). Active oxygen
and free radicals, such as superoxide anion ðO2 Þ, hydrogen peroxide (H2O2) and hydroxyl radical (OH), are constantly formed in the human body by normal metabolic
action. Their action is opposed by a balanced system of
antioxidant defences, including antioxidant compounds
and enzymes. Upsetting this balance causes oxidative
stress, which can lead to cell injury and death (Halliwell
& Gutteridge, 1999). Therefore, much attention has been
*
Corresponding author. Tel.: +886 4 22391647 7501 19; fax: +886 4
22396771.
E-mail address: [email protected] (H.-Y. Chen).
0308-8146/$ - see front matter 2006 Published by Elsevier Ltd.
doi:10.1016/j.foodchem.2006.02.047
focussed on the use of antioxidants, especially natural antioxidants, to inhibit lipid peroxidation, or to protect against
the damage of free radicals (Vendemiale, Grattagliano, &
Altomare, 1999). Current research into free radicals has
confirmed that foods rich in antioxidants play an essential
role in the prevention of cardiovascular diseases and cancers (Kris-Etherton et al., 2002) and neurodegenerative diseases (Di Matteo & Esposito, 2003).
Recently, there is strong evidence to show that diabetes
is associated with increased oxidative stress (Abou-Seif &
Youssef, 2004; Freitas, Filipe, & Rodrigo, 1997). Moreover, the generation of oxidative stress may play an important role in the etiology of diabetic complications, such as
vascular complications (Cooper, Bonnet, Oldfield, &
Jandeleit-Dahm, 2001), diabetic cataract (Ozmen, Mutaf,
Ozmen, Mentes, & Bayindir, 1997) and diabetic nephropathy (Ha & Kim, 1999). Administration of streptozotocin
induces diabetes in experimental animals. The development
of diabetes induced by streptozotocin was related to the
production of radicals, including superoxide anion and
hydroxyl radicals (Chang et al., 1993; Ohkuwa, Sato, &
Naoi, 1995). Kakkar, Mantha, Radhi, Prasad, and Kalra
(1997) also reported that the lipid peroxide level in the kidney of streptozotocin-induced diabetic rats was significantly higher than that of the control (P < 0.05).
Evidence indicates that many biochemical pathways associated with hyperglycemia can increase the production of
free radicals (Lee & Chung, 1999). Diabetic patients have
reduced antioxidant defences and suffer from an increased
risk of free radical-mediated damage (Maxwell et al., 1997).
The levels of plasma lipid peroxide products, including
malondialdehyde in diabetic patients, were increased when
compared with control subjects (Freitas et al., 1997). Fortunately, supplementation of vitamin E can lower lipid peroxidation in diabetic patients (Jain et al., 1996). Noda,
Mori, and Packer (1997) indicated that gliclazide, commonly used in the treatment of diabetes, was not only effective in reducing blood sugar, but also might be useful in the
scavenging of free radicals. Armstrong, Chestnutt, Gormley, and Young (1996) suggested that an improved antioxidant status and reduced lipid peroxidation might be one
mechanism by which dietary treatment contributes to the
prevention of diabetic complications.
Recently, research on phytochemicals and their effects
on human health have been intensively studied. In particular, research has focussed on a search for antioxidants,
hypoglycemic agents, and anticancer agents from vegetables, fruit, tea, spices and medicinal herbs. Prince and
Menon (1999) showed that oral administration of aqueous
Tinospora cordifolia root extract, an indigenous plant used
as medicine in India, resulted in a decreased level of
TBARS and an increase in the levels of glutathione and
vitamin C in alloxan diabetes. The extracts from neem
(Azadirachta indica A. Juss Meliaceae) seed also expressed
significant protection against the oxidative stress damage
induced by streptozotocin in the heart and erythrocytes
of rats (Gupta, Kataria, Gupta, Murganandan, & Yashroy, 2004). Guava (Psidium guajava L.) is widely cultivated
and its fruit is popular in Taiwan. Guava was also used as a
hypoglycemic agent in folk medicine. The leaves and skin
of the fruit have greater effects. Guava tea, the infusion
of dried guava fruit and leaves, has recently become popular as a drink in Taiwan. Cheng and Yang (1983) proved
that guava juice exhibited hypoglycemic effects in mice.
Interestingly, the decreased serum glucose level of infusions
from the African mistletoe (Loranthus bengwensis L.) parasite on guava trees was more affected than that prepared
from mistletoe parasitic on other trees (Obatomi, Bikomo,
& Temple, 1994). In other studies, the anti-diarrhoeal
(Lutterodt, 1989) antipyretic (Olajide, Awe, & Makinde,
1999), antimicrobial (Jaiarj et al., 1999) and bio-antimutagenic (Matsuo, Hanamure, Shimoi, Nakamura, & Tomita,
1994) properties of guava leaf extract have been demonstrated. There is an important role of oxidative stress in
the development of cancer and diabetes. As noted above,
the infusion of guava leaves has potential as a functional
drink. However, information concerning the antioxidant
activity of guava leaves is unavailable. The objectives of
this study were to study the antioxidant activity and free
radical-scavenging effects of guava leaf and dried fruit
extracts.
2. Materials and methods
2.1. Chemicals
2,2 0 -Azino-bis-(3-ethylbenzthiazoline-6-sulfonic
acid)
(ABTS), ferrozine, linoleic acid, nitro blue tetrazolium
(NBT), peroxidase, b-phycoerythrin and polyphenon 60
were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). 2,2-Azobis(2-amidinopropane) dihydrochloride (AAPH) was purchased from Wako Pure Chemical
Co. (Tokyo, Japan). Ammonium thiocyanate, dihydro-nicotin-amidadenin-dinucleotide (NADH), Folin–Ciocalteau
reagent and phenazine methosulphate (PMS) were purchased from E. Merck Co. (Darmstadt, Germany).
2.2. Plant material and extraction
Fresh leaves of various guava cultivars, including Hong
Ba, Shi Ji Ba, Shui Jing Ba and Tu Ba, were provided by
Fengshan Tropical Horticultural Experiment Branch of
the Agricultural Research Institute, Taiwan. The commercial products, guava tea A (dried leaves), guava tea B (dried
leaves:dried fruit = 1:1; w/w) and dried fruit were purchased at a local market in Taichung, Taiwan. The fresh
guava leaves were dried at 40 C for 12 h in an oven and
then broken into pieces of about 0.5 cm2. Samples (20 g)
were extracted with boiling water (500 ml) for 5 min. The
extracts were filtered through Whatman No. 2 filter paper
and the filtrates were freeze-dried.
2.3. Antioxidant activity in a linoleic acid system
The antioxidant activities of extracts from guava were
determined by the thiocyanate method (Mitsuda, Yasumoto, & Iwami, 1966). Each sample, in 0.5 ml of distilled
water, was mixed with linoleic acid emulsion (2.5 ml
0.02 M, pH 7.0) and phosphate buffer (2 ml, 0.2 M, pH
7.0) in a test tube and stood, in darkness, at 37 C, to
accelerate oxidation. The linoleic acid emulsion was prepared by mixing an equivalent weight of linoleic acid
and Tween 20 in phosphate buffer (0.2 M, pH 7.0). The
peroxide value was determined by reading the absorption
at 500 nm with a spectrophotometer (Hitachi U-2000)
after colour development with FeCl2 and thiocyanate at
various intervals during incubation. The peroxidation of
linoleic acid was calculated as peroxidation (%) = (A1/
A0) · 100, where A0 was the absorption of the control
reaction and A1 was the absorption in the presence of
sample. All analyses were run in triplicate and mean values were calculated.
2.4. Radical-scavenging assays
2.6. HPLC analysis of phenolic compounds
2.4.1. Scavenging effects on ABTS+ radicals
This decoloration method consists of an enzymatic system containing a peroxidase, hydrogen peroxide and
ABTS. A radical is generated from ABTS and has a characteristic absorption spectrum with a maximum of
414 nm. The ability of extracts from guava to scavenge
ABTS+ radicals was determined by the method described
in the work of Okamoto, Hayase, and Kato (1992) with
some modifications. In brief, 30 lM H2O2 and extracts
were added to 0.1 M phosphate buffer (pH 6.0) medium
containing 0.02% ABTS and 6 units of peroxidase. Absorbance at 414 nm was measured after incubation for
15 min. All analyses were run in triplicate and mean values were calculated.
The contents of phenolic compounds in extracts from
guava were determined by HPLC, performed with a Hitachi liquid chromatograph (Hitachi, Ltd., Tokyo, Japan)
consisting of a model L-6200 pump, and a model L-4200
UV–Vis detector set at 285 nm. The analyses were carried
out on a LiChrospher RP-18 column (250 mm · 4 mm
i.d., 5 lm, E. Merck Co., Darmstadt, Germany). Extracts
were filtered through a 0.45 lm filter before use. The elution solvents were (A) 0.2 M NaH2PO4 adjusted to pH
3.0 by phosphoric acid, and then diluted with distilled
water to 50 mM NaH2PO4 and (B) 50 mM NaH2PO4/
methanol/acetonitrile (30/20/50, v/v/v). The solvent gradient elution programme used was as follows: initial 100% A,
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 gradient to 62% A in 10 min and hold for 15 min; linear gradient
to 0% A in 10 min and hold for 10 min. The flow rate was
1 ml/min. Phenolic compounds were identified by comparison of their retention time (Rt) values and UV spectra with
those of known standards and determined by peak areas
from the chromatograms. All analyses were run in triplicate and mean values were calculated.
2.4.2. Scavenging effects on superoxide anion
The influence of extracts from guava on the generation
of superoxide anion was measured according to the method
described in previously work (Yen & Chen, 1995). Superoxide anion was generated in a non-enzymic system and
determined by a spectrophotometric measurement for
reduction of nitro blue tetrazolium. The reaction mixture,
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
of NADH (468 lM) in phosphite buffer and 1 ml of NBT
(150 lM) in phosphate buffer, was incubated at ambient
temperature for 5 min, and the colour was read at
560 nm against blank samples. All analyses were run in
triplicate and mean values were calculated.
2.4.3. Scavenging effects on peroxyl radicals
The ability of extracts from guava to scavenge peroxyl
radicals was measured by monitoring the loss of b-phycoerythrin fluorescence induced by AAPH. The Fluorescent
degradation of b-phycoerythrin was measured at 540 nm
excitation and 575 nm emission (Glazer, 1990). The reaction mixture contained 5 · 1010 M b-phycoerythrin,
25 mM AAPH and extracts in 75 mM phosphate buffer
at pH 7.4. b-Phycoerythrin fluorescence was almost
lost by incubation with 25 mM AAPH for 30 min. All
analyses were run in triplicate and mean values were
calculated.
2.5. Determination of total phenolic compounds
Total phenolic compounds in the extracts from guava
were determined with Folin–Ciocalteu reagent using gallic
acid and (+)-catechin as the standards (Taga, Miller, &
Pratt, 1984). Extracts (100 ll) were added to 2 ml of 2%
Na2CO3. After 2 min, 50% Folin–Ciocalteau reagent
(100 ll) was added to the mixture which was then left to
stand for 30 min. Absorbance was measured at 750 nm
on a spectrophotometer and compared to gallic acid and
(+)-catechin calibration curves. All analyses were run in
triplicate and mean values were calculated.
2.7. Statistical analysis
Data were analyzed using the Statistical Analysis System
software package. Analyses of variance were performed
using ANOVA procedures. Significant differences between
means were determined using Duncan’s multiple range test.
3. Results and discussion
3.1. Antioxidant activity in a linoleic acid system
The antioxidant activities of extracts from guava, determined using the thiocyanate method, were compared with
that of polyphenon 60, which is a commercial polyphenol
product extracted from green tea. Comparison of antioxidant activity of extracts from guava at various concentrations is shown in Table 1. In all samples, the extracts
from Shi Ji Ba showed a stronger antioxidant activity than
did other samples at 50 lg/ml. However, the extracts from
guava leaves and dried fruit all exhibited over 95% inhibition at a concentration of 200 lg/ml. Except for the dried
fruit extracts, the other samples were found to have weaker
antioxidant activity when the concentration was over
200 lg/ml. The polyphenon 60 showed more significantly
prooxidant effects and the maximum antioxidant activity
was weaker than those of extracts from guava leaves and
guava tea at a concentration of 100 lg/ml. The extracts
from dried fruit of guava displayed weaker antioxidant
activity than did other samples at 50 lg/ml. However, there
was no significant difference in antioxidant activity
(P > 0.05) between dried fruit of guava and other guava
extracts at a concentration of 200 lg/ml.
Table 1
Antioxidant activities of extracts from leaves and dried fruit of guava
Sample concentration (lg/ml)
50
100
150
200
500
Peroxidation (%)a
Shi Ji Ba
Shui Jing Ba
Tu Ba
Hong Ba
Guava tea A
Guava tea B
Dried fruit
Tea polyphenon 60
9.3 ± 0.4
3.8 ± 0.1
4.5 ± 0.1
4.7 ± 0.2
9.0 ± 0.3
14.3 ± 1.9
4.0 ± 0.2
4.1 ± 0.1
4.8 ± 0.4
8.0 ± 0.1
13.0 ± 2.6
4.4 ± 0.4
4.6 ± 0.1
4.8 ± 0.1
8.7 ± 0.3
15.6 ± 1.1
5.0 ± 0.3
4.9 ± 0.2
4.5 ± 0.1
8.9 ± 0.1
20.6 ± 2.9
5.7 ± 1.3
3.8 ± 0.1
3.4 ± 0.2
6.3 ± 0.2
17.8 ± 2.6
4.7 ± 0.2
3.7 ± 0.2
4.5 ± 0.1
6.2 ± 0.2
49.9 ± 2.7
11.6 ± 0.8
7.3 ± 0.3
4.8 ± 0.2
0.7 ± 0.1
17.8 ± 1.8
10.1 ± 1.1
11.3 ± 0.4
13.1 ± 0.4
19.5 ± 1.2
a
Peroxidation (%) = [(absorbance of sample at 500 nm)/(absorbance of control at 500 nm)] · 100. A low peroxidation (%) indicated a high antioxidant
activity.
Recently, there has been considerable interest in preventive medicine through the quest for natural antioxidants
from plant material. Various phytochemical components,
such as flavonoids, phenolic acids and carotenoids, are
known to be responsible for the antioxidant capacity of
plants. However, the effectiveness of flavonoids as effective
antioxidants is dependent upon the environment. A number of factors may influence the behaviour of flavonoids
and may result in alterations to their efficacy as antioxidants. The antioxidant activity of flavonoids may be
reduced by the autoxidation of flavonoids, catalyzed by
transition metals to produce superoxide anion. The latter
dismutates to generate hydrogen peroxide and form hydroxyl radicals via a Fenton reaction in the presence of transition metals (Canada, Giannella, Nguyen, & Mason, 1990).
Most plant polyphenol compounds possess both antioxidant and prooxidant properties, depending on concentration and environmental factors (Cao, Sofic, & Prior,
1997). A possible mechanism of polyphenol cytotoxicity
may be related to their prooxidant properties. In our previous work, tea extracts showed both antioxidant and prooxidant activities in oxidative damage of biomolecules (Yen,
Chen, & Peng, 1997). Azam, Hadi, Khan, and Hadi
(2004) proposed that the prooxidant action of tea polyphenolics may be an important mechanism of their anticancer and apoptosis properties. In addition to
polyphenolics, the prooxidant activity of green tea extracts
may be caused by their chlorophyll components (Wanasundara & Shahidi, 1998). In the above results, the extracts
from guava leaf and fruit exhibited strong potential antioxidant activity. The true prooxidant effects that guava
extract has on cells remains as a matter to be studied
further.
3.2. Radical-scavenging effects
In this study, three free radicals were used to assess the
potential free radical-scavenging activities of guava
extracts, namely ABTS+ radical, superoxide anion and
peroxy radicals. ABTS is a peroxidase substrate which,
when oxidized in the presence of H2O2 in a typical peroxidative reaction, generates a metastable radical with a
characteristic absorption spectrum and an absorption
maximum of 414 nm (Arnao, Cano, Hernandez-Ruiz, Garcia-Canovas, & Acosta, 1996). The ABTS+ radicals are
scavenged by antioxidants via the mechanism of electron-/hydrogen-donation and are assessed by measuring
the decrease in absorption at 414 nm. In the superoxide
anion-scavenging test, superoxide anion, that is derived
from dissolved oxygen through the PMS/NADH coupling
reaction, reduces NBT and increases absorption at 560 nm.
The decrease in absorption at 560 nm with antioxidants
thus indicates the consumption of superoxide anion. In
the peroxyl radical-scavenging assay, thermal decomposition of AAPH leads to the formation of carbon-centred
radicals which, under aerobic conditions, yield alkylperoxyl radicals. These radical species can be detected by assay
of fluorescent decomposition of b-phycoerthrin, a major
pigment protein of sea algae. The absorption assay for
antioxidants was based on oxidation of b-phycoerthrin
molecules by peroxyl radicals (Cao, Alessio, & Cutler,
1993).
The abilities of extracts from guava, assayed to be scavenging the ABTS+ radical in comparison with polyphenon
60, are shown in Fig. 1. The scavenging effect of polyphenon 60 was observed to be higher than that of extracts from
guava. The polyphenon 60 showed a linear increase in
ABTS+ radical-scavenging activity with increasing concentration, reaching 97.1 ± 0.9% scavenging activity at a concentration of 5 lg/ml. In various samples of guava extracts,
the scavenging activities of Shi Ji Ba, Hong Ba, and Tu Ba
extracts on ABTS+ radicals were stronger than those of
Shui Jing Ba, guava tea A and guava tea B extracts. However, the extracts from four guava cultivars expressed over
95% scavenging activity at a concentration of 20 lg/ml. In
all samples, extracts from dried guava fruit had the weakest
scavenging ability on ABTS+ radicals. The decolorization
assay, using free blue-green ABTS+ radicals, was shown to
be a very useful tool in expeditiously measuring the antioxidant activity of individual chemical compounds or complex extracts. This method can express total antioxidant
activity as vitamin C equivalent antioxidant capacity
(VCEAC) or as trolox equivalent antioxidant capacity
(TEAC) value (Kim, Lee, Lee, & Lee, 2002; Re et al.,
1999).
As shown in Fig. 2, the scavenging effects of polyphenon
60 and extracts from guava on the superoxide anion were
similar to the results of the scavenging effects on ABTS+
radicals. The abilities of all samples to scavenge superoxide
anion decreased in the order: polyphenon 60 > Shi Ji Ba,
100
Scavenging effects (%)
80
Shi Ji Ba
Shui Jing Ba
Tu Ba
Hong Ba
Guava tea A
Guava tea B
Dried fruit
Tea polyphenon 60
60
40
20
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
Scavenging effects (%)
80
Shi Ji Ba
Shui Jing Ba
Tu Ba
Hong Ba
Guava tea A
Guava tea B
Dried fruit
Tea polyphenon 60
60
40
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.
100
β -phycoerythrin
Relative fluorescence (%)
Hong Ba and Tu Ba > Shui Jing Ba, guava tea A and
guava tea B > dried fruit.
The influence of Shi Ji Ba extracts on the decrease in the
fluorescence of b-phycoerthrin induced by AAPH is shown
in Fig. 3. When compared to a time of 0 min, the relative
fluorescence of b-phycoerthrin had decreased to 87.7%
after incubation for 10 min, indicating that auto-composition of b-phycoerthrin had occurred in the solution. The
rapid decomposition of b-phycoerthrin was induced by
addition of AAPH to the solution, and a resulting 2.4%
fluorescence remained after incubation for 10 min, when
compared with the control. The extracts from Shi Ji Ba
exhibited a concentration-dependent biphasic effect on
the fluorescent decomposition of b-phycoerthrin induced
by AAPH. The inhibitions of guava leaf extracts on the
fluorescent decomposition of b-phycoerthrin, induced by
AAPH, increased with concentration (2.5–10 lg/ml) up
to a maximum, and then decreased at the concentration
C
80
D
B
E
60
A
40
20
β -phycoerythrin + AAPH
0
0
2
4
6
8
10
Time (min)
Fig. 3. The effects of extracts from leaves of Shi Ji Ba on b-phycoerythrin
fluorescence decay induced by AAPH. The concentrations of extracts were
(A) 2.5 lg/ml; (B) 5 lg/ml; (C) 10 lg/ml; (D) 25 lg/ml; and (E) 50 lg/ml.
25–50 lg/ml. This finding was similar to the results of the
antioxidant activity assay in a linoleic acid system. The
prooxidant effects of guava extracts at high concentrations
may be correlated with phenoxy radical formed by the
change of phenolic compounds with phenoxy radical
reacted with b-phycoerythrin to participate in radical chain
propagation. Bowry, Ingold, and Stocker (1992) also indicated that a-tocopherol could form a-tocopherol radical
and be a strong prooxidant for low-density lipoprotein at
high concentration of a-tocopherol and low fluxes of
AAPH. The most interesting point from the results
(Fig. 3) was the decomposition rate of b-phycoerythrin
induced by AAPH in the presence of various concentrations of guava leaf extracts. When b-phycoerythrin and
AAPH was incubated with guava leaf extracts at 2.5–
50 lg/ml (Fig. 3, curves A–E), the fluorescent decomposition of b-phycoerythrin decreased more rapidly than that
of control (b-phycoerythrin + AAPH) after incubation
for 1–2 min. However, with the exception of 5 lg/ml guava
leaf extracts (Fig. 3, curve B), the fluorescent decomposition of b-phycoerythrin, induced by AAPH in the presence
of other concentrations of guava leaf extracts, tended to
moderate at an incubation of 2–10 min. In contrast to
other concentrations, curve B showed a linear decrease
with an increase of the incubation time. The reaction rate
was made possible in a different way and resulted a change
in expression of the antioxidant activity of samples.
Table 2 summarizes that extracts from guava that inhibited the fluorescent decomposition of b-phycoerythrin by
AAPH-derived peroxyl radical. With the exception of
polyphenon 60 and dried fruit, the extracts from four cultivars of guava leaves and two kinds of guava tea all
showed over 85% scavenging ability on peroxyl radicals
at a concentration of 10 lg/ml. The extracts from Shi Ji
Ba, Hong Ba and Tu Ba expressed a stronger inhibition
than that of Shui Jing Ba, guava tea A and guava tea B
at a concentration of 2.5 lg/ml. However, there was no significant difference in inhibition (P > 0.05) between those six
extracts at a concentration of 10 lg/ml. The inhibitory
effect of dried fruit extracts was increased with increase
of the concentration. On the contrary, 90.6% fluorescent
decomposition of b-phycoerythrin was inhibited by
2.5 lg/ml polyphenon 60, and then decreased with increasing concentration. The prooxidant effects of guava leaf
extracts and polyphenon 60 extracted from green tea leaves
may be attributed to the plant phenolic compounds. However, the components of guava fruit which provided inhibitory effects on peroxidation remain a matter to be studied
further.
As shown above, polyphenon 60 and guava leaf extracts
exhibited a concentration-dependent increase in their scavenging effects on ABTS+ radicals and superoxide anion.
However, the antioxidant activity and radical-scavenging
ability decreased at high concentrations in the linoleic acid
peroxidation and peroxyl radical systems. Comparison
between the four methods, ABTS+ radicals and superoxide
anion-scavenging assays was obtained by the absorption
change induced by the formation of radicals. Nevertheless,
antioxidant activity and peroxyl radicals are determined by
the oxidation damage of biomolecular matrices, such as
linoleic acid and protein. The biomolecular matrices may
be attacked by derivatives from sample components, especially the phenolic compounds, resulting in secondary oxidation damage. Hanasaki, Ogawa, and Fukui (1994)
reported that multiple hydroxyflavonoids, especially with
OH in the B-ring, significantly increased production of
hydroxyl radicals in a Fenton system. Liu, Han, Lin, and
Luo (2002) demonstrated that 4-hydroxyquinoline derivatives could inhibit the free radical-induced peroxidation,
but also play a prooxidative role in the vesicle of dipalmitoyl phosphatidylcholine. This could be due to the electron-attracting group at the ortho position to hydroxyl
group in the phenoxy radical of quinoline derivatives. At
high concentrations, the phenoxy radical initiated additional propagation of lipid peroxidation. The polyphenon
60 and guava leaf extracts showed weaker effects, at high
concentrations, in antioxidant activity and peroxyl radical
scavenging assays. Therefore, the methods and concentrations used to measure the antioxidant activity of natural
material, especially polyphenol-rich samples, should take
into consideration the influence of prooxidant effects.
3.3. Determination of phenolic compounds
Phenolic compounds, such as quercetin, rutin, narigin,
catechins, caffeic acid, gallic acid and chlorogenic acid,
are very important plant constituents because of their antioxidant activities (Croft, 1998; Paganga, Miller, & RiceEvans, 1999). The analysis of phenolic compounds in the
guava extracts is shown in Table 3 and Fig. 4. As shown
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
5.0
10.0
25.0
50.0
83.3 ± 6.0
87.6 ± 8.5
88.6 ± 7.9
74.2 ± 2.4
58.7 ± 6.9
77.3 ± 3.1
84.9 ± 4.5
88.6 ± 4.4
51.8 ± 3.7
23.5 ± 2.9
82.0 ± 5.4
85.2 ± 2.2
92.5 ± 1.2
68.1 ± 2.8
48.5 ± 4.2
86.1 ± 4.1
87.7 ± 3.6
94.2 ± 4.0
77.5 ± 5.7
58.9 ± 8.7
60.2 ± 5.1
86.0 ± 6.5
90.2 ± 2.5
82.2 ± 8.2
64.9 ± 7.1
37.9 ± 2.4
66.8 ± 1.7
90.5 ± 3.3
89.0 ± 4.9
78.7 ± 8.0
16.6 ± 7.3
44.0 ± 4.3
79.3 ± 6.6
89.2 ± 2.3
91.2 ± 4.2
90.6 ± 6.7
89.2 ± 4.0
79.0 ± 4.6
62.9 ± 5.7
39.8 ± 5.7
a
Scavenging effects (%) = [(fluorescence of b-phycoerythrin with AAPH and sample-fluorescence of b-phycoerythrin with AAPH)/(fluorescence of bphycoerythrin 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
Shi Ji Ba
Shui Jing Ba
Tu Ba
Hong Ba
Guava tea A
Guava tea B
Dried fruit
Tea polyphenon 60
Total phenolic compounds
Phenolic acid
Expressed as (+)-catechin (mg/g)
Expressed as gallic acid (mg/g)
Gallic acid (lg/g)
Ferulic acid (lg/g)
296 ± 5.4
267 ± 5.4
313 ± 4.7
294 ± 4.0
103 ± 9.3
177 ± 9.2
69.6 ± 2.8
643 ± 8.5
458 ± 8.1
414 ± 8.2
483 ± 7.1
455 ± 6.1
166 ± 14.1
279 ± 14.0
115 ± 4.2
985 ± 12.8
1621 ± 87.4
793 ± 52.3
1022 ± 62.4
1137 ± 79.6
725 ± 54.1
1278 ± 92.7
266 ± 15.4
–
672 ± 65.2
108 ± 15.7
355 ± 45.4
296 ± 25.9
147 ± 16.8
234 ± 27.5
–
–
Fig. 4. HPLC chromatographs of (A) phenolic compound standards and
(B) water extracts from Shyj Jih Pa leaves: (1) gallic acid; (2) 3,4dihydroxybenzoic acid; (3) 4-hydroxybenzoic acid; (4) chlorogenic acid;
(5) (+)-catechin; (6) caffeic acid; (7) syringic acid; (8) 2-hydroxybenzoic
acid; (9) epicatechin; (10) ferulic acid; (11) naringin; (12) morin; (13)
quercetin.
in Table 3, the contents of total phenolic compounds,
shown as (+)-catechin equivalents, were less than those
as gallic acid equivalents. This may be affected by the
molecular weight of standards. The contents of total phenolic compounds in the extracts were in the order: tea
polyphenon 60 > four guava cultivars > guava tea > dried
fruit. Tea polyphenon 60, which is a commercial polyphenol product extracted from green tea, contained about
60% polyphenolic compounds, such as catechins. However,
the result from the chromatogram indicated that guava
extracts contained phenolic acids. Fig. 4 shows the chromatogram of mixed standards (A) and Shi Ji Ba extracts
(B). A good resolution, with sharp peaks, was achieved
for all phenolic compound standards within 90 min. The
results of HPLC analyses show that three main peaks were
found in the Shi Ji Ba extracts at the absorbance of 285 nm.
Gallic acid (Rt = 18.60 min) and ferulic acid (Rt =
68.41 min) were identified by comparison of their retention
time values and UV spectra with those of known standards.
The contents of two phenolic acids in guava extracts are
shown in Table 3. The highest amounts of gallic acid and
ferulic acid were found in Shi Ji Ba extracts, while the dried
fruit extracts contained the lowest amounts of these compounds. In addition to phenolic acids, the other complex
phenolic compounds in guava extracts may also provide
the antioxidant acitiviy. However, further studies on the
effective components in guava extracts, which contribute
to the antioxidant ability, are required.
Ferulic acid and its precursors, p-coumaric acid and caffeic acid are synthesized in plants. Ferulic acid occurs in
cereals and vegetables, such as rice, wheat, oats, tomatoes,
asparagus, olives and many other plants. Recently, a great
deal of focus has been placed on the antioxidant potentialities of ferulic acid and its n-alkyl esters (Anselmi et al., 2004;
Kanski, Aksenova, Stoyanova, & Butterfield, 2002). Sánchez-Moreno, Larrauri, and Saura-Calixto (1999) indicated
that the inhibition of lipid oxidation of the phenolic compounds and antioxidant standards followed the order: rutin,
ferulic acid > tannic acid, gallic acid, resveratrol > BHA,
quercetin > tocopherol > caffeic acid, in a linoleic acid system. Meanwhile, the free radical-scavenging activity was
in the order: gallic acid > tannic acid, caffeic acid, quercetin,
BHA, rutin > ferulic acid, tocopherol, resveratrol. In contrast, several researches have indicated that ferulic acid
was ineffective, and even promoted the oxidation of lowdensity lipoprotein induced by copper (Chalas et al., 2001;
Cirico & Omaye, 2006). In our results, gallic acid and ferulic
acid may have important roles in the antioxidant activity
and free radical scavenging ability of guava extracts.
3.4. Correlation analysis
Calculated coefficients of correlations between antioxidant activity, scavenging effects on radicals and contents
of phenolic compounds of guava extracts are shown in
Table 4. The antioxidant activity of guava extracts was sig-
Table 4
Correlation between the antioxidant properties and phenolic compounds of extracts from leaves and dried fruit of guava
Antioxidant activitya
Scavenging effectsa
Phenolic compounds
+
Superoxide anion
ABTS
0.876*
0.880*
0.853**
1.000
0.991*
0.921*
1.000
0.944*
1.000
0.797**
0.810**
0.685
0.927*
0.759**
0.763**
0.909*
0.698
0.729
0.875*
0.585
0.631
Antioxidant activity
1.000
Scavenging effects
Superoxide anion
ABTS+ radicals
Peroxyl radicals
Phenolic compounds
Total
Gallic acid
Ferulic acid
radicals
Peroxyl radicals
Total
Gallic acid
Ferulic acid
1.000
0.688
0.682
1.000
0.895*
1.000
a
The concentrations of guava extracts in the antioxidant activity, scavenging effects on superoxide anion, ABTS+ radicals and peroxyl radicals used for
correlation analysis were 50, 200,10, 2.5 lg/ml, respectively.
*
P < 0.01.
**
P < 0.05.
nificantly correlated with their scavenging effects on superoxide anion (P < 0.01), ABTS+ radicals (P < 0.01) and
peroxyl radicals (P < 0.05). Therefore, the antioxidant
activities of guava extracts may be due to their scavenging
effects on radicals and blocking of the chain reaction in the
peroxidation of linoleic acid. For scavenging effects on radicals, high correlations (R = 0.921–0.991) were observed
between various radicals, indicating that these three methods have satisfactory correlations for the examination of
antioxidants. The antioxidant activity (P < 0.05) and scavenging effects on superoxide anion, ABTS+ radicals and
peroxyl radicals (P < 0.01) of guava extracts was also well
correlated with their contents of total phenolic compounds.
The same trends were observed in the correlation of the
content of gallic acid and the antioxidant activity and scavenging effects on superoxide anion. According to recent
reports, a highly positive relationship existed between total
phenolics and antioxidant activity in many plant species
(Dasgupta & De, 2004; Dorman & Hiltunen, 2004).
4. Conclusions
In the present study, the extracts from guava were found
to possess strong antioxidant activity. The antioxidant
mechanisms of guava leaf extracts may be attributed to
their free radical-scavenging ability. In addition, phenolic
compounds appear to be responsible for the antioxidant
activity of guava extracts. On the basis of the results
obtained, guava extracts from either the leaf or dried fruit
can be used for a variety of beneficial chemo-preventive
effects. However, further studies on the antioxidative components of guava extracts and more in vivo evidence from
diabetic patients are required.
Acknowledgement
This work is part of a Research Project, NSC 88-2313-B166-001, supported by the National Science Council,
Republic of China.
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