African Journal of Biotechnology Vol. 8 (16), pp.

3887-3892, 18 August, 2009

Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2009 Academic Journals

Full Length Research Paper

Antioxidant activities of extracts from areca (Areca

catectu L.) flower, husk and seed
Wei-Min Zhang, Bin Li, Lin Han and Hai-De Zhang*
College of Food Science, Hainan University, Hainan P. R. China.
Accepted 22 May, 2009

The antioxidant activities of areca (Areca catectu L.) flower, husk and seed extracts were evaluated
using 3 complementary in vitro assays, inhibition of DPPH (1,1-diphenyl-2- picrylhydrazyl) radicals,
inhibition of hydroxyl radicals and reducing power system. The EC50 values were calculated for all the
methods in order to evaluate the antioxidant efficiency of areca extracts. The phenol and flavonoid
contents were also analyzed. Areca seed has the best antioxidant properties, presenting much lower
EC50 values for DPPH radicals scavenging activity, hydroxyl radicals scavenging activity and reducing
power. Furthermore, the highest polyphenols and flavonoids contents were found from areca seed
extracts. It is suggested that areca seed is an excellent food material with a potential nutrition and
antioxidation.

Key words: Areca catectu L. extracts, antioxidant activity.

INTRODUCTION

Free radicals were a major interest for early physicists ever, because of their toxic and carcinogenic effects, their
and radiologists and much later found to be a product of use is being restricted. Thereby, interest in finding natural
normal metabolism. Although oxygen is essential for antioxidants, without undesirable side effects, has increas-
aerobic forms of life, oxygen metabolites are highly ed greatly (Rechner et al., 2002).
toxic. As a conse quence, reactive oxygen species The number of antioxidant compounds synthesized by
(ROS) are known to be implicated in many cell plants as secondary products, mainly phenolics, serving in
disorders and in the development of many diseases plant defence mechanisms to counteract ROS in order to
including cardiovascular diseases, atherosclerosis, survive, is currently estimated to be between 4000 and
cataracts, chronic inflammation and neurodegenerative 6000 (Havsteen, 2002; Robards et al., 1999; Wollgast and
diseases (Gutteridge, 1993; Knight, 1995). ROS and Anklam, 2000). A direct relationship has been found bet-
free radicals are also considered as inducers of lipid ween the content of total phenolics and antioxidant capa-
peroxidation and cause the deterioration of foods city of plants (Ferreira et al., 2007; Robards et al., 1999). In
(Rechner et al., 2002). Although organisms have fact, to counteract deleterious action of ROS, phenolic
endogenous antioxidant defences produced during compounds, naturally distributed in plants are effective
normal cell aerobic respiration against ROS, other (Ferreira et al., 2007; Pereira et al., 2006).
antioxidants are taken from the diet, both from natural Because purified phenolic compounds are difficult to
and synthetic origin (Rechner et al., 2002). obtain and because extracts sometimes have better anti-
Antioxidants, which can inhibit or delay the oxidation of oxidant activities than those of pure molecules, there is a
an oxidizable substrate in a chain reaction, therefore, growing interest for the use of plant extracts (Calliste et al.,
appear to be very important in the prevention of many 2005). To find new natural sources of active compounds,
diseases (Halliwell et al., 1992). Thus, synthetic we studied the antioxidant potential of different extracts of
antioxidants are widely used in the food industry. How- Areca catectu L. The use of betel nut, as a masticatory by
th
humans has been known since the 4 century A. D. in
different parts of the world. It is estimated that over 600
million individuals consume areca nut (also called areca
*Corresponding author. E-mail: [email protected]. Tel.: nut) in one form or another world-wide. In old Indian
th
+086-898-23300681. Fax: +086-898-23305646. scripts, such as Vagbhata (4 century) and Bhavamista
3888 Afr. J. Biotechnol.

th
(13 century), betel nut has been described as a thera- and the results were expressed as mg of catechin equivalents/g of
peutic agent. Its use was recommended in many extract.
Flavonoid contents in the extracts were estimated by a colorimetric
diseases, such as leucoderma, leprosy, anaemia and assay based on procedures described by Hertog et al. (1992) with
obesity. It was also reported to have de-worming some modifications. Basically, 0.5 ml of the sample added to a test
properties. In China, it has been used as a vermifuge tube which contained 4.5 ml of distilled water and then added 0.3 ml
th
since the 6 century and is still employed as such in of 5% sodium nitrite solution and allowed to stand for 5 min. 0.6 ml of
some parts (Sharan, 1996). In the Philippines the 10% aluminium chloride was added, and after 6 min, 2.0 ml of 1 mol/l
flowers are sometimes added to salads. The nuts, sodium hydroxide was added and the mixture was diluted with
another 2.1 ml of distilled water. The absorbance of the mixture at
husks, young shoots, buds, leaves and roots are used 510 nm was measured immediately. Rutin was used for constructing
in various medicinal preparations (Staples and the standard curve (8.0 - 40 µg/ml; y = 0.022 x +0.0109, R2 = 0.9994)
Bevacqua, 2006). and flavonoid contents were calculated using a standard calibration
Although it has already been demonstrated that areca curve, prepared from rutin. The flavonoid content was expressed as
fruit contain total phenolics and tannin (Zhang et al., mg/g DW.
2008), little is known about the antioxidant potential of
areca fruit and other areca extracts, such as husk and DPPH radical scavenging activity
flower.
In this work, the antioxidant properties of areca To evaluate the free radical scavenging activity, the extracts were
extracts were evaluated through several biochemical allowed to react with a stable free radical, DPPH (Brand-Williams et
assays, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical– al., 1995). Various concentrations of areca extracts (0.3 ml) were
mixed with 2.7 ml of 70% ethanol solution containing DPPH radicals
scavenging activity, reducing power and inhibition of (40 µg/ml). The mixture was shaken vigorously and left to stand for
hydroxyl radicals. 60 min in the dark (until stable absorbance values were obtained).
The reduction of the DPPH radical was determined by reading the
absorbance at 517 nm. The radical-scavenging activity (RSA) was
MATERIALS AND METHODS calculated as a percentage of DPPH discoloration, using the equation
Materials
% RSA = [(ADPPH - AS)/ ADPPH]× 100
Areca flower, husk and seed were obtained from areca (Areca
catechu L.) plant grown in Hainan, China. Areca flower and fruit where AS is the absorbance of the solution when the sample extract is
were picked in April and June, 2008 respectively. The samples added at a particular level and ADPPH is the absorbance of the
were cleaned, washed with distilled water, cut into small pieces, DPPH˙solution (Barros et al., 2007). The extract concentration
dried overnight in an air dryer at 40°C, ground to a particle size of providing 50% of radical-scavenging activity (EC50) was calculated
25 mesh using a grinder. Rutin and DPPH (1, 1-diphenyl-2- from the graph of RSA percentage against extract concentration.
picrylhydrazyl) was purchased from Sigma Chemical Company Ascorbic acid was used as standards.
(St. Louis, MO) and vitamin C (ascorbic acid) was obtained from
Guangzhou chemical reagent factory, China. All other reagents
were analytical grade. Hydroxyl radical scavenging activity

The scavenging activity for the hydroxyl radical was evaluated using
Extraction of antioxidative compounds the method of Halliwell et al. (1987) at a wavelength of 510 nm with a
UV-visible spectrophotometer. The radical scavenging activity was
For antioxidant compounds extraction, a fine dried powder (25 calculated using the following equation,
mesh) of sample (areca flower, husk and seed, 5 g for all
extracts) was extracted using 50 ml of 70% ethanol at 75°C for Hydroxyl radical scavenging activity (%) = [(A0 - Ai)/A0] × 100
2.5 h by reflux. The extracts were filtered through Whatman No.4
paper under reduced pressure, frozen and then lyophilized (Ly-8- where A0 is the absorbance of the control at 510 nm and Ai is the
FMULE, Snijders). All the samples were redissolved in 70% absorbance of the sample at 510 nm.
ethanol at a concentration of 5.0 mg/ml and analysed for their
contents of polyphenols and flavonoids and DPPH radical-
scavenging activity, reducing power and inhibition of hydroxyl Reducing power
radicals.
The reducing power of the extracts was assessed by the method of
Oyaizu (1986). Various concentrations of the extracts (2.5 ml) were
Determination of antioxidant contents mixed with 2.5 ml of 200 mM sodium phosphate buffer (pH 6.6) and
2.5 ml of 1% potassium ferricyanide. The mixture was incubated at
Contents of total phenolics in the extracts were estimated by a 50°C for 30 min. After 2.5 ml of 10% trichloroacetic acid (w/v) was
colorimetric assay based on procedures described by Singleton added, the mixture was centrifuged at 1000 rpm for 10 min. The
and Rossi (1965) with some modifications. Basically, 1 ml of upper layer (5 ml) was mixed with 5 ml of deionised water and 1 ml of
sample was mixed with 1 ml of Folin and Ciocalteu’s phenol 0.1% of ferric chloride and the absorbance was measured
reagent. After 3 min, 1 ml of saturated sodium carbonate solution spectrophotometrically at 700 nm (Barros et al., 2007). Blank sample
was added to the mixture and it was adjusted to 10 ml with was prepared using distilled water instead of extract. The values are
distilled water. The reaction was kept in the dark for 90 min, after presented as the means of triplicate analyses. The extract
which the absorbance was read at 725 nm (UV-2450 spec- concentration providing 0.5 of absorbance (EC50) was calculated from
trophotometer). Catechin was used for constructing the standard the graph of absorbance at 700 nm against extract concentration.
curve (2.0 - 12.0 µg/ml; y = 0.0631 x -0.0611; R2 = 0.9992) and Ascorbic acid was used as standards.
Zhang et al. 3889

Table 1. Extraction yields and contents of total phenolics and flavonoids in areca extracts.

Fraction Extraction yield (%) Total phenolics (mg/g) Flavonoids (mg/g)

Flower 1.76 ± 0.19c 20.09 ± 1.21c 6.12 ± 0.24c
Husk 3.24 ± 0.11b 59.22 ± 1.48b 52.57 ± 3.02b
Seed 8.75 ± 0.95a 114.14 ± 3.41a 77.36 ± 5.06a
SD: standard deviation (means ± standard error of means of the 3 cultivars).
Data followed by different letters in the same column are significantly different at 0.05 probability
level.

76

Hydr oxyl radical scavenging

40
65
32
DPPH(%)

54

ability(%)
43 24

32 16
21 8
0.5 1 1.5 2 2.5
0
Concentraction(mg/ml)
0.5 1 1.5 2 2.5

Flower Husk Seed Concentration(mg/ml)

Figure 1. Radical-scavenging activity (RSA) of different Flower Husk Seed

concentrations areca flower, husk and seed extracts.
Figure 2. Radical-scavenging activity (hydroxyl radical) of
different concentrations areca flower, husk and seed extracts.

Statistical analysis

All experiments were conducted in triplicate and statistical

analyses was done according to the software DPS3.01user's
extraction yields, the antioxidant contents found were
guides. The data were presented as mean ± SD. Determination of good, indicating that the extraction was efficient.
significant differences of the means between various treatments Nevertheless, a relationship between the extracted mass
of areca flower, husk and seed were performed by t-test. and the corresponding total phenolics and flavonoids were
not observed in all cases. Most of the phenolic or poly-
phenolic compounds in nature have antioxidative activities,
RESULTS AND DISCUSSION e.g. tocopherols, flavonoids and derivatives of cinnamic
acid, phosphatidic and other organic acids. Flavonoids
Extraction yields and contents of antioxidative were the major components of the total phenolic content of
compounds areca flower, husk and seed (Table 1). The significant
difference was observed in the total phenolics and flavo-
Water with ethanol was selected as the extraction noids of 3 fractions of areca. And areca seed contained the
solvent since both are commonly used in the food most total phenolics and flavonoids, followed by husk and
industry in a variety of ways and are more highly stable flower.
in the human body than any other solvents. The
extraction yield is highly valued because a low
extraction yield means a lower productivity despite high Antioxidant activities in areca flower, husk and seed
antioxidation. Therefore, 70% ethanol was used as the
extraction solvent for this study. The extraction yields Figures 1 - 3 showed the antioxidant activity of areca
were expressed in terms of the solid content in the dried flower, husk and seed extracts examined as a function of
product per soluble solid content in areca husk, flower their concentration. Several biochemical assays were used
and seed used on a dry basis. Table 1 showed the to screen the antioxidant properties, scavenging activity on
extraction yields of the 70% ethanol extracts from areca DPPH radicals (measuring the decrease in DPPH radical
flower, husk and seed, the extraction yields of areca absorption after exposure to radical scavengers),
flower, husk and seed were 1.76, 8.75 and 3.24%, scavenging activity on hydroxyl radicals (measuring the
respectively. Despite the low values obtained for the decrease in hydroxyl radical absorption after exposure to
3890 Afr. J. Biotechnol.

Table 2. EC50 values (mg/ml) and anti-radical power obtained in the antioxidant
assays for areca extracts.

Assay Flower Husk Seed

DPPH radical scavenging activity EC50 1.838a 1.489b 0.409d
Hydroxyl radical scavenging activity EC50 6.754 a 5.380b 3.575c
Reducing power EC50 2.685a 1.466b 0.188d
Data followed by different letters in the same line are significantly different at 0.05
probability level.

2.1 can conclude that the scavenging effects of all extracts on

1.8 DPPH radicals increased with the concentration increase
Absorbance at 700nm

1.5 and were excellent, especially in the case of areca seed.

The RSA values were also good for husk, but areca flower
1.2 revealed a very low value.
0.9 The hydroxyl radical is an extremely reactive free radical
formed in biological systems and has been implicated as a
0.6
highly damaging species in free radical pathology, capable
0.3 of damaging the biomolecules of living cells. We investi-
0 gated the hydroxyl radical scavenging activity of ethanol
0.5 1 1.5 2 2.5 extracts using the Fenton reaction (Figure 2), the hydroxyl
radical scavenging activity also increased with concen-
Concentrat ion(mg/ml) tration. All extracts revealed a good scavenging activity on
hydroxyl radicals, especially in the case of areca seed. The
Flower Husk Seed scavenging activity was also good for husk, but areca
flower revealed a very low value (23.8% at 2.5 mg/ml).
Figure 3. Reducing power of different concentrations area The reducing capacity of a compound may serve as a
flower, husk and seed extracts concentration. significant indicator of its potential antioxidant activity. A
higher absorbance indicates a higher ferric reducing power
(Meir et al., 1995; Shimada et al., 1992; Duh et al., 1998).
radical scavengers) and reducing power (measuring the Figure 3 showed the reducing powers of the ethanol
3+
conversion of a Fe /ferricyanide complex to he ferrous extracts as a function of their concentration. The reducing
form). The assays were per-formed for each extract power also increased with concentration and the values
separately. Nevertheless, those assays were carried obtained for all the extracts were good. Areca seed had the
out using whole extracts instead of individual com- highest reducing power in 3 fractions of areca. With
pounds. Additive and synergistic effects of phyto- regards to reducing power, higher reducing activities can
chemicals in fruits and vegetables are responsible for be attributed to higher amounts of polyphenolics and the
their potent bioactive properties and the benefit of a reducing capacity of a compound may reflect its antioxidant
diet rich in fruits and vegetables is attributed to the potential (Lee et al., 2007). It has been reported that the
complex mixture of phytochemicals present in whole reducing properties are generally associated with the
foods (Liu, 2003). This explains why no single anti- presence of reductones, which have been shown to exert
oxidant can replace the combination of natural phyto- antioxidant action by breaking the free radical chain by
chemicals to achieve the health benefits. Analysis of donating a hydrogen atom (Shimada et al., 1992). Hence,
Figures 1 - 3 revealed that antioxidant activity increased areca seed may have the highest amounts of reductones
with the concentration, good results being obtained, and polyphenolics in 3 fractions of areca.
even at low extract concentrations. Table 2 showed antioxidant activity with EC50 values of
DPPH, a stable free radical with a characteristic areca flower, husk and seed measured by different
absorption at 517 nm, was used to study the radical biochemical assays. Areca seed revealed the best anti-
scavenging effects of ethanol extracts. The decrease in oxidant properties in 3 fractions of areca by the DPPH
absorption is taken as a measure of the extent of assay, hydroxyl radical assay and reducing power assay,
radical scavenging (Suja et al., 2005). The radical- followed by husk and flower. The obtained results are in
scavenging activity (RSA) values were expressed as agreement with the phenol and flavonoid contents
the ratio percentage of sample absorbance decrease determined for each sample and shown in Table 2. In
and the absorbance of DPPH solution in the absence of DPPH radicals scavenging activity, the EC50 of areca seed,
extract at 517 nm. From the analysis of Figure 1, we husk and flower were 0.409, 1.489 and 1.838 mg/ml,
Zhang et al. 3891

Table 3. Correlations established between the concentration of extracts with antioxidant activity EC50
values (df = 3).

DPPH radical Hydroxyl radical

Extract Reducing power
scavenging activity scavenging activity
Equation Y = 19.326x + 14.487 Y = 5.866x + 10.379 Y = 0.2242x - 0.1019
Flower 2 2 2
Linearity R = 0.9855 R = 9069 R = 0.9983
Equation Y = 20.078x + 20.111 Y = 8.754x + 2.901 Y = 0.3938x - 0.0775
Husk 2 2 2
Linearity R = 0.9363 R = 0.9654 R = 9846
Equation Y = 6.576x + 58.99 Y = 13.202x + 2.805 Y = 0.1742x + 1.2279
Seed 2 2 2
Linearity R = 0.9159 R = 9928 R = 0.9890

respectively. In hydroxyl radicals scavenging activity, radical scavenging activity and reducing power, while
the EC50 of areca seed, husk and flower was 3.575, areca flower showed the best radical-scavenging activity of
5.380 and 6.754 mg/ml, respectively. In reducing power hydroxyl radicals. It is interesting that the ethanol extracts
system, the EC50 of areca seed, husk and flower was of areca seed exhibited the highest antioxidative activity in
0.188, 1.466 and 2.685 mg/ml, respectively. The EC50 3 fractions of areca by the DPPH assay and reducing
values of areca seed obtained for reducing power were power, but it was the lowest antioxidative activity in 3
better than those for DPPH radicals, scavenging effects fractions of areca by hydroxyl radical assay.
on hydroxyl radicals. These results showed that areca The present study suggests that areca seed extracts are
seed exhibited significantly (p < 0.05) the highest useful nutritional antioxidants for the nutraceutical industry.
antioxidative activity than areca husk and flower. Total phenolics and flavonoids of areca extracts should be
In complex systems, such as food and food prepara- the most activity substances, but the components
tions, various different mechanisms may contribute to responsible for the antioxidative activity of areca seed
oxidative processes, such as in Fenton reactions, extracts are currently unclear. Therefore, further study is
where transition metal ions play a vital role, different required for isolation and identification of antioxidant active
reactive oxygen species might be generated and compounds from ethanol extracts of areca seed. After
various target structures such as lipids, proteins and adequate treatment they can, for
carbohydrates, can be affected. Therefore, it is impor- example, be included in foods with remarkable benefits for
tant to characterize the extracts by a variety of anti- human or animal health.
oxidant assays (Halliwell, 1997). In reducing power
assay, the general ability of the extracts to donate elec-
trons is tested whereas, in the DPPH assay, hydrogen ACKNOWLEDGEMENTS
atoms are involved as well. The results from the
antioxidant assays show that areca flower, husk and This work was supported by the part of grants from
seed can act as radical scavengers to a certain extent. National Key Technologies R and D Program of P. R.
Areca seed exhibited the highest antioxidative activities. China (2007BAD76B03). The authors would like to thank
In the present study, despite the high coefficient of the Faculty of College of Food Science, Hainan University,
2
correlation values (R ) obtained, proving the existence China, for the laboratory facilities.
of correlation, the only results that showed statistical
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