Food and Chemical Toxicology 45 (2007) 328–336

www.elsevier.com/locate/foodchemtox

Evaluation of free radical scavenging and antityrosinase activities

of standardized longan fruit extract
Nuchanart Rangkadilok a, Somkid Sitthimonchai b, Luksamee Worasuttayangkurn a,
Chulabhorn Mahidol c, Mathuros Ruchirawat b, Jutamaad Satayavivad a,d,*
a
Laboratory of Pharmacology, Chulabhorn Research Institute (CRI), 54 Moo 4, Vipavadee-Rangsit Highway, Laksi, Bangkok 10210, Thailand
b
Laboratory of Chemical Carcinogenesis, Chulabhorn Research Institute (CRI), 54 Moo 4, Vipavadee-Rangsit Highway, Laksi, Bangkok 10210, Thailand
c
Laboratory of Natural Products, Chulabhorn Research Institute (CRI), 54 Moo 4, Vipavadee-Rangsit Highway, Laksi, Bangkok 10210, Thailand
d
Department of Pharmacology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand

Received 2 February 2006; accepted 23 August 2006

Abstract

The protective effects of fruits and vegetables against chronic diseases have been attributed to the antioxidant properties of some
secondary metabolites present in these foods. Plant polyphenols have been reported to exhibit bioactive properties, and in particular
antioxidant activities. Longan seeds are found to contain high levels of some beneficial polyphenolic compounds such as corilagin, gallic
acid and ellagic acid. The present study examined the free radical scavenging activity of longan seed extract by using three different assay
methods. Longan extracts contained corilagin ranging from zero to 50.64 mg/g DW, gallic acid from 9.18 to 23.04 mg/g DW, and ellagic
acid from 8.13 to 12.65 mg/g DW depending on the cultivars. Dried longan seed extracts of cultivar Edor contained high levels of gallic
acid and ellagic acid and also exhibited the highest radical scavenging activities when comparing fresh seed and dried pulp extracts. For
scavenging activity of DPPH and superoxide radicals, longan seed extract was found to be as effective as Japanese green tea extract while
dried longan pulp and mulberry green tea extracts showed the least scavenging activities. In the ORAC assay, both fresh and dried
longan seed also had higher activity than dried pulp and whole fruit. However, the results demonstrate that three polyphenolics may
not be the major contributors of the high antioxidant activity of longan water extracts but this high activity may be due to other
phenolic/flavonoid glycosides and ellagitannins present in longan fruit. In addition, longan seed also showed tyrosinase inhibitory
activity with IC50 values of 2.9–3.2 mg/ml. Therefore, the preliminary observations suggest that longan seed extract could be another
potential source of potent natural dietary antioxidants and also in an application as a new natural skin-whitening agent.
 2006 Elsevier Ltd. All rights reserved.

Keywords: Longan seed; Ellagic acid; Gallic acid; Corilagin; Free radical scavenging; Antityrosinase

1. Introduction

Abbreviations: AEAC, L-ascorbic acid-equivalent antioxidant capacity;
FRAP, ferric-reducing antioxidant power; HPLC, High Performance
Liquid Chromatography; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DMSO,
dimethyl sulfoxide; AAPH, 2,2 0 -Azobis(2-amidinopropane) dihydrochlo-
ride; XO, xanthine oxidase; X/XO, xanthine/xanthine oxidase assay; XTT,
2, 3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide;
ORAC, oxygen radical absorbance capacity.
*
Corresponding author. Address: Laboratory of Pharmacology, Chu-
labhorn Research Institute (CRI), 54 Moo 4, Vipavadee-Rangsit High-
way, Laksi, Bangkok 10210, Thailand. Tel.: +66 2 5740622x3917; fax: +66
2 5742027.
E-mail address: jutamaad@cri.or.th (J. Satayavivad).
Fruits and vegetables contain many antioxidant com-
pounds, including carotenoids, thiols, vitamins such as
ascorbic acid and tocopherols, flavonoids, and phenolics
(Prior, 2003; Heber, 2004). An increase in the consumption
of antioxidants from these foods can reduce oxidative
stress and prevent chronic diseases. Longan fruit (Euphoria
longana Lam.) is a subtropical fruit, which belongs to the
Sapindaceae family. Longan, known as ‘Lumyai’ in Thai-
land, is widely grown in China, Taiwan, and South East
Asia including Thailand and Vietnam. However, it can also

0278-6915/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2006.08.022
 N. Rangkadilok et al. / Food and Chemical Toxicology 45 (2007) 328–336
be grown in other areas such as Queensland (NSW, Austra-
lia), California, and Florida (USA) (Menzel et al., 1995).
The flesh is sweet and juicy, therefore it can be consumed
in both fresh and processed products such as canned lon-
gan in syrup or dried fruits, and fruit drink for refreshment
(Mortin, 1987).
A previous study demonstrated that aqueous extract of
longan contained high levels of polyphenolic compounds
such as corilagin, gallic acid, and ellagic acid (Rangkadilok
et al., 2005). The content of these polyphenolics was depen-
dent upon cultivars and plant tissues. Dried seed extract
was found to contain the highest levels of these three com-
pounds while dried pulp contained the lowest. Lin et al.
(1993) and Hsu et al. (1994) also indicated that longan
seeds contained corilagin and acetonylgeraniin as the active
compounds. In addition, three unusual amino acids were
identified in dried longan seeds (Minakata et al., 1985).
Okuyama et al. (1999) found high levels of adenosine,
which exhibited the anxiolytic-like effect, in pulp or flesh.
Longan fruit also contained the lowest total dietary fiber
and the lowest phytate contents (Nititham et al., 2004).
Corilagin possessed the ability to lower the blood pressure
of spontaneously hypertensive rats (SHR) through the
blockade of noradrenaline release and (or) by direct vaso-
relaxation (Lin et al., 1993). In addition, corilagin had anti-
fungal activity against Candida glabrata strains (Latte and
Kolodziej, 2000), potently inhibited HIV-1 replication in
HeLa CD4+ cells (Notka et al., 2003), inhibited the tumor
necrosis factor-a (TNF-a) release (Okabe et al., 2001), and
also remarkably reduced the resistance level of beta-
lactams in methicillin-resistance Staphylococcus aureus
(MRSA) (Shiota et al., 2004). Gallic acid and ellagic acid
are reported to be potent antioxidants and anticarcinogenic
agents (Constantinou et al., 1995; Festa et al., 2001; Kaw-
ada et al., 2001; Yilmaz and Toledo, 2004). Gallic acid
reduced cell viability of mouse lung cancer cells in vitro
dose dependently (IC50  200 lM) and the combination
of gallic acid with an anti-cancer drug such as cisplatin
may be an effective treatment for lung cancer (Kawada
et al., 2001). Gallic acid could also prevent oxidative
damage to cellular DNA at low concentration (Fan and
Lou, 2004) and exerted stronger antiproliferative activity
than quercetin on both Caco-2 human colon cancer cells
(Caco-2 cells) and WB-F344 normal rat liver epithelial cells
(WB cells) in DMEM media (Lee et al., 2005). Ellagic acid
expressed a selective cytotoxicity and antiproliferative
activity, and induced apoptosis in Caco-2, MCF-7,
Hs578T, and DU 145 cancer cells without any toxic effect
on the viability of normal human lung fibroblast cells
(Losso et al., 2004). In addition, ellagic acid also induced
G1 arrest within 48 h, inhibited overall cell growth, and
induced apoptosis in cervical carcinoma cell line (CaSki)
after 72 h of treatment (Saleem et al., 2002). This may sup-
port the role of ellagic acid as a chemopreventive agent.
Tyrosinase (EC1.14.18.1) is an enzyme in the undesir-
able browning of fruits and vegetables, and is involved in
the natural development of skin, hair and eye coloring
329
(Roh et al., 2004). This enzyme is also involved in the initial
step in melanin synthesis. Many tyrosinase inhibitors are
used as skin-whitening agents including linoleic acid, arbu-
tin, and kojic acid. Recently, there has been much attention
focused on the application of natural plant extracts in the
cosmetics industry. Methanol and ethyl acetate extracts
of safflower seeds possessed potent tyrosinase inhibitory
activity and the active compounds were identified as N-
feruloylserotonin, N-(p-coumaroy)serotonin, and acacetin
(Roh et al., 2004). Mulberroside F isolated from Morus
alba leaves also inhibited tyrosinase activity, 4.5 fold more
potent than kojic acid, and also showed superoxide scav-
enging activity including inhibition of melanin production
in cultured melan-a cells (Lee et al., 2002).
All these data indicate that longan fruit extract may be a
new source of dietary antioxidants for use in supplements,
as a natural chemopreventive agent, or for use as skin-
whitening agents because of the high levels of the three
beneficial polyphenolic compounds as well as other poly-
phenolics. However, there is limited information on the
pharmacological studies of longan and especially for the
commonly prepared extracts used in herbal medicine.
Water extracts of litchi, longan or dried longan (regarded
as ‘heating food’) showed a dose-dependent enhancing
effect on PGE2 production in the absence of lipopolysac-
charide (LPS), which was accompanied by a significant
induction of COX-2 protein expression (Huang and Wu,
2002). There are a few studies on antioxidant activity of
longan extracts. Dimocarpus longan Lour exhibited the
highest hydroxyl radical scavenging activity determined
by GC/MS method (Wang and Smythe, 2003) and freeze-
dried longan seed also showed antioxidant activity higher
than longan flesh (using AEAC and FRAP assay) (Soong
and Barlow, 2004). Therefore, the present study evaluates
the antioxidant activities of standardized water extracts
of longan (seed and pulp) which contained different levels
of the three polyphenolic compounds by using different
assay methods: (1) determination of radical scavenging
(DPPH assay), (2) inhibition of superoxide anion radical
formation (X/XO assay), and (3) measurement of oxygen
radical absorbance capacity (ORAC). The study also
investigates tyrosinase inhibitory activity of longan seed
extracts.
2. Materials and methods
2.1. Chemicals and apparatus
Dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA),
absolute ethanol, and methanol (HPLC grade) were obtained from Merck
(Darmstadt, FR Germany). 2,2 0 -Azobis(2-amidinopropane) dihydrochlo-
ride (AAPH) was purchased from Cayman (Ann Arbor, MI). Gallic acid,
ellagic acid, sodium bicarbonate (NaHCO3), L-tyrosine, kojic acid, and all
other chemicals were purchased from Sigma-Aldrich Chemicals (Stein-
heim, Germany). Mushroom tyrosinase was obtained from Fluka. Milli-Q
deionized water (Branstead, Newton, USA) was used in all of the
experiments.
The UV–VIS and fluorescence measurements were performed using
Spectramax 384 Plus with Softmax Pro 4.0 software (Molecular Devices;
330 N. Rangkadilok et al. / Food and Chemical Toxicology 45 (2007) 328–336
Sunnyvale, CA) and Spectramax GeminiXS with Softmax Pro 4.3.1 LS
software (Molecular Devices; Sunnyvale, CA), respectively. Ninety-six-
well microplates were purchased from Greiner (Frickenhausen, Germany).
2.2. Plant materials
Longan fruit (cultivars Edor, Baidam, Biewkiew) were freshly har-
vested from the field in August, 2004 (Department of Horticulture, Fac-
ulty of Agriculture, Maejo University, Chiang Mai) and transported to
Laboratory of Pharmacology (Chulabhorn Research Institute), Bangkok.
A selection of the fruits was washed and separated into 2 groups, one was
dried in the oven at 75 C for 48 h and the other group was used as fresh
fruit. After drying, all seeds were separated from peels and pulps and
ground into fine powder. For fresh fruit, seeds were ground into powder
after peels and pulps were removed. Baidam and Biewkiew cultivars were
selected as they contained high levels of corilagin while fresh seed of Edor
contained the lowest. Edor cultivar is a commercial cultivar, which is
widely grown in the North of Thailand, therefore it was included in this
study. Dried seeds of Edor were also collected from fruit processor, Malee
Sampran Public Co., Ltd. (Nakornpratom province) in 2004. Japanese
green tea (Camellia sinensis) ‘Ban-Cha’ product was from Yokohama,
Japan and Mulberry green tea (Morus alba) was produced in Nakorn-
ratshasrima province, in the North-East of Thailand.
2.3. Preparation of plant extracts
Five hundred grams of dried seed powders and pulps of longan were
extracted with 2 L of hot water (70–75 C) for 1 h. The extract was then
filtered and collected. The residue was re-extracted twice with 2 L of hot
water each time. The three water extracts were combined, and concen-
trated and lyophilized using a freeze-dryer. For the fresh sample, one
kilogram of seeds was used for extraction using the procedure described
above. The whole longan fruits (peel, pulp, seed) were also boiled in water
at an extraction ratio of 1:5 for 3 h, filtered, and then lyophilized; these
fruits were only extracted once with water. The percentage yield of extracts
was between 7.5% and 12% depending on cultivars, plant tissues, and fresh
or dried samples. Hot water was used to extract three polyphenolic
compounds since this is a common traditional longan preparation method
used in China for alternative medicine i.e., boiling the whole longan fruit
in water. Plant extracts, 22–25 mg, were dissolved in 3.0 ml of hot water
(70–75 C) (2 replicates per sample) and vigorously shaken. The extracts
were left at room temperature until they cooled down and then filtered
through a 0.45 lm Nylon membrane (13 mm, Orange Scientific, Belgium)
prior to HPLC analysis. All extracts were analysed for the contents of
three polyphenolic compounds, corilagin, gallic acid, and ellagic acid by
HPLC analysis using a previously described method (Rangkadilok et al.,
2005). For the preparation of green tea and mulberry green tea extracts,
dried leaves were extracted with hot water using the same procedure as
used for longan extraction.
For the antioxidant studies, all samples were dissolved in DMSO
(100%) at a final concentration of 4 mg/ml prior to chemical-based assay.
2.4. Scavenging of diphenyl-picrylhydrazyl (DPPH) radicals
This assay detects scavenging of free radicals by samples through the
scavenging activity of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH)
free radical. The reduced DPPH formazan form was determined using a
spectrophotometer.
This assay was performed using a previously described method (van
Amsterdam et al., 1992) with some minor modifications. Five microliters
of each sample or 100% DMSO (as a negative control) or 10 mM ascorbic
acid (as a positive control as well as a blank for background subtraction)
were allowed to react with 195 ll of 100 lM DPPH ethanolic solution in a
96-well microplate. The plate was then incubated at 37 C for 30 min after
which the absorbance was measured at 515 nm using a UV–VIS micro-
plate reader. Scavenging capacity of the sample was compared to that of
DMSO (0% radical scavenging) and ascorbic acid as positive control
(100% radical scavenging). The results expressed as the concentration of
the extracts or pure compounds which scavenged free radicals by 50%
(SC50).
2.5. Inhibition of superoxide radical formation by
xanthine/xanthine oxidase (X/XO assay)
The ROS-generating enzyme, xanthine oxidase (XO) converts xanthine
to uric acid and superoxide anion radicals. The radical formation was
detected indirectly by measuring the rate of reduced XTT (2, 3-bis [2-
methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) (orange
formazan dye) production using methods previously described (Ukeda
et al., 1997). The mixture contained 115 ll of 50 mM NaHCO3 buffer (pH
9.4), 20 ll of 0.5 mM hypoxanthine, 20 ll of 1 mM EDTA, and 20 ll of
0.25 mM XTT. To this mixture was added 10 ll of either sample or 100%
DMSO (as a negative control) or 600 U/ml SOD (as a positive control).
The reaction was initiated by addition of 15 ll of 200 mU/ml XO after
which the production of reduced XTT was kinetically determined at
480 nm every 20 s for 5 min. The results expressed as the concentration of
the extracts or pure compounds which scavenged free radicals by 50%
(SC50).
2.6. Inhibition of xanthine oxidase
The major product of xanthine/xanthine oxidase system is uric acid.
An inhibition of XO activity was examined directly by measuring the rate
of uric acid production as previously described (Gerhauser et al., 2003).
The reaction mixture comprised 135 ll of 50 mM NaHCO3 buffer (pH
9.4), 20 ll of 1 mM xanthine, and 20 ll of 1 mM EDTA. This reaction
mixture was added to 10 ll of either sample or 100% DMSO (as a negative
control) or 10 mM of allopurinol, an XO inhibitor (as a positive control).
The reaction was initiated by addition of 15 ll of 200 mU/ml XO, and the
rate of uric acid production was immediately measured at 295 nm every
20 s for 5 min.
2.7. Measurement of oxygen radical absorbance capacity (ORAC)
Peroxyl radical (ROO) absorbance capacity was determined using
previously described methods (Huang et al., 2002) with some minor mod-
ifications. Briefly, the reaction mixture containing 175 ll of 7 · 105 mM
fluorescein solution in 75 mM phosphate buffer (pH 7.4) was added to 10 ll
of either sample or 10 ll DMSO diluted in 75 mM phosphate buffer (pH
7.4) (as a blank) and pre-incubated at 37 C for 10 min. To start the reac-
tion, 15 ll of 255 mM AAPH, a peroxyl radical generator, was added to the
pre-incubated mixture. A change in intensity of the fluorescent probe
caused by free radicals was then monitored at 37 C every 2 min for 45 min
by using the fluorescent microplate reader at the excitation and emission
wavelengths of 485 and 530 nm, respectively. In parallel, 10 ll of 20 lM
Trolox, a water soluble vitamin E analogue, was used as a standard with
75 mM phosphate buffer (pH 7.4) as a blank. The area under the curve
(AUC) was analyzed by the Pro 4.3.1LS software and subtracted by the
AUC of the blank to obtain the net AUC. The relative ORAC value was
calculated using the equation shown below. The unit was expressed as lmol
Trolox equivalents per gram sample.
Relative ORAC value ¼ ½ðAUCsample  AUCblank Þ=ðAUCTrolox  AUCblank Þ
2.8. Inhibition of tyrosinase activity
Tyrosinase-inhibition activity was determined by using L-tyrosine as a
substrate. Forty microliters of 200 units/ml of mushroom tyrosinase
solution, 100 ll of phosphate buffer (pH 6.8), and 20 ll of sample with or
without enzyme were mixed. The assay mixture was pre-incubated at
37 C for 10 min and then 40 ll of 10 mM L-tyrosine was added. The
reaction was then further incubated at 37 C for 15 min. The amount of
dopachrome was measured at 475 nm in a microplate reader. The data
 N. Rangkadilok et al. / Food and Chemical Toxicology 45 (2007) 328–336 331

were expressed as a percentage of inhibition of tyrosinase activity. Kojic
acid was used as a standard tyrosinase inhibitor control.
3. Results
The contents of three polyphenolics in the concentrated
longan extracts are shown in Table 1. The contents of these
compounds were dependent upon plant tissues (ontogeny),
cultivars, and fruit drying processes. Fresh and dried seed
contained higher levels of three compounds than dried pulp
and whole fruit. The corilagin content varied from zero to
50.64 mg/g DW (Table 1), gallic acid from 0.29 to
23.04 mg/g DW, and ellagic acid from 0.20 to 12.65 mg/g
DW. Dried seed of Baidam and Biewkiew cultivars
contained the highest contents of corilagin, 50.64 and
38.65 mg/g DW, respectively, while Edor (a commercial
cultivar) contained the highest content of gallic acid
(23.04 mg/g DW) and ellagic acid (12.65 mg/g DW). Lon-
gan pulp contained the lowest contents of all polyphenolic
compounds.
3.1. Free radical scavenging ability of longan extracts
(DPPH assay)
HPLC analysis determined that longan extracts con-
tained high levels of phenolic compounds. These com-
pounds have been reported to be good scavengers of free
radicals. In the present study longan extracts were evalu-
ated for their abilities to neutralize the stable free radicals
such as DPPH radicals. Longan extracts, both fresh and
dried seeds, exhibited free radical scavenging activities that
were as good as green tea extract while the whole fruit and
dried pulp extracts had less activity (Table 2). The SC50 val-
ues (the concentration that scavenges 50% of the DPPH
radical) for these longan extracts were between 10.8 and
77.3 lg/ml. The highest scavenging activity was found in
Japanese green tea, and both fresh and dried seed of longan
cultivar Edor (SC50 = 10.2, 10.8, and 11.6 lg/ml, respec-
tively). Longan cultivars Baidam and Biewkiew, containing
high levels of corilagin, had lower scavenging activities
than Edor. Dried longan pulp and mulberry green tea
extracts exhibited very weak scavenging activities (only
7% and 12%, respectively). In addition, pure gallic acid
and ellagic acid showed very high activity towards DPPH
radicals with SC50 2.3 and 2.2 lg/ml, respectively.
3.2. Inhibition of superoxide radical formation
(X/XO assay)
These studies determined the superoxide scavenging abil-
ity of longan extracts using the xanthine/xanthine oxidase
assay. Superoxide radicals were produced by oxidation of

Table 1
The contents of corilagin, gallic acid, and ellagic acid in different longan extracts
Extract Corilagin Gallic acid Ellagic acid
(mg/g DW) (mg/g DW) (mg/g DW)
Fresh seed Edor 3.01 ± 0.03 9.75 ± 0.23 9.24 ± 0.38
Dried seed Edor No. 1 26.12 ± 0.22 23.04 ± 0.03 12.65 ± 0.40
Dried seed Edor No. 2 26.30 ± 0.46 21.30 ± 0.41 5.36 ± 0.34
Dried pulp Edor ND 0.29 ± 0.01 0.20 ± 0.01
Dried fruit Edor 2.11 ± 0.12 2.39 ± 0.10 1.21 ± 0.01
Dried seed Baidam 50.64 ± 0.34 17.06 ± 0.28 10.52 ± 0.26
Dried seed Biewkiew 38.65 ± 0.10 9.18 ± 0.06 8.13 ± 0.34
ND = Not detectable.

Table 2
Free radical scavenging activities (DPPH and X/XO assay) of longan extracts and some reference standards
Extracts and pure compounds DPPH assay SC50 X/XO assay SC50
(lg/ml) (lg/ml)
Fresh seed Edor 10.8 ± 0.1 7.6 ± 0.3
Dried seed Edor-1 11.6 ± 0.3 7.2 ± 0.2
Dried seed Edor-2 13.3 ± 0.4 7.4 ± 0.2
Dried pulp Edor >100 (7%)a >200 (13%)b
Dried fruit Edor 77.3 ± 3.4 47.8 ± 0.5
Dried seed Baidam 12.9 ± 0.7 11.6 ± 0.2
Dried seed Biewkiew 16.2 ± 0.3 13.3 ± 0.2
Japanese green tea 10.2 ± 0.7 5.3 ± 1.2
Mulberry green tea >100 (12%)a >200 (29%)b
Gallic acid 2.3 ± 0.1 0.7 ± 0.0
Ellagic acid 2.2 ± 0.1 6.9 ± 0.2
Ascorbic acid 4.7 ± 0.1 –
SC50 = The concentration of extract that scavenges 50% of DPPH and superoxide radicals.
a
% scavenging of DPPH radical.
b
% scavenging of superoxide radical.
332 N. Rangkadilok et al. / Food and Chemical Toxicology 45 (2007) 328–336

hypoxanthine using xanthine oxidase to uric acid. Superox- Table 3

ide radical scavenging activities of plant extracts in decreas- Oxygen radical absorbance capacity (ORAC) values of longan extracts
and some reference standards
ing order were Japanese green tea = dried and fresh seed
of Edor > dried seed of Baidam > dried seed of Biewkiew > Extracts and reference standards ORAC unita
whole fruit of Edor > mulberry green tea and dried pulp of Fresh seed Edor (1.4 ± 0.1) · 103
Edor (Table 2). In this study, gallic acid showed the highest Dried seed Edor-1 (1.4 ± 0.2) · 103
Dried seed Edor-2 (1.2 ± 0.2) · 103
activity with SC50 = 0.7 lg/ml. Although, the gallic acid Dried pulp Edor (1.3 ± 0.1) · 102
content in fresh seed of Edor (9.75 mg/g DW) was lower Dried fruit Edor (7.4 ± 0.5) · 102
than Baidam cultivar (17.06 mg/g DW), the activity of fresh Dried seed Baidam (1.3 ± 0.1) · 103
seed of Edor was greater than Baidam. This may be due Dried seed Biewkiew (1.2 ± 0.1) · 103
to other compounds, such as conjugated polyphenolics, Japanese green tea (3.6 ± 0.2) · 103
Mulberry green tea (5.1 ± 0.2) · 102
present in fresh seed, which also exhibit high scavenging Gallic acid (5.3 ± 0.2) · 103
activity against the superoxide radical. In addition, we also Ellagic acid (4.9 ± 0.5) · 103
examined plant extracts for the inhibition of xanthine a
Expressed as lmol Trolox equivalents per gram sample (of extracts or
oxidase by measuring the production of uric acid. However, pure compounds).
the results indicated that all the plant extracts exhibited
weak inhibition of xanthine oxidase enzyme at the maxi-
mum concentration used in this study (200 lg/ml) (data
not shown). The percentage inhibition of the extracts
ranged from 2% to 33%. Gallic acid and ellagic acid also 80
inhibited xanthine oxidase enzyme by only 21% and 26%, Dried seed
respectively. Therefore, it can be concluded that longan Fresh seed
60
extracts are strong scavengers of superoxide radicals but
% Inhibition

they are weak inhibitors of xanthine oxidase enzyme. Since

corilagin standard is not commercially available, it was not
40
possible to test the antioxidant activity of this compound.
However, the results from Baidam and Biewkiew cultivars,
containing high levels of corilagin, showed less scavenging 20
activity than Edor cultivar. Therefore, corilagin may exhibit
only weak scavenging activity.
0
3.3. Measurement of oxygen radical absorbance capacity 0.6 1.3 2.5 5
(ORAC) Conc. (mg/ml)

Fig. 1. Tyrosinase inhibitory activity of fresh and dried longan seed.

Antioxidants can be generally classified into two mech-
anistic classes; firstly, preventive antioxidants and sec-
ondly, chain-breaking antioxidants (Ou et al., 2002).
Preventive antioxidants, such as superoxide dismutase, cat-
alase, and transferrin, inhibit formation of reactive oxygen
species while chain-breaking antioxidants scavenge oxygen
radicals and thereby break radical chain sequences. The
ORAC assay directly measures the antioxidant activities
of chain-breaking antioxidants against peroxyl radicals.
On the basis of ORAC values, the strength of peroxyl
radical scavenging activity was in the order of Japanese
green tea > all fresh and dried seed of longan (all culti-
vars) > whole longan fruit = mulberry green tea > dried
longan pulp (Table 3). Gallic acid had a high peroxyl scav-
enging activity as did ellagic acid. Our present results indi-
cated that longan seed extract also had a potent peroxyl
radical scavenging capacity.
3.4. Inhibition of tyrosinase activity
Both fresh and dried longan seed clearly showed tyrosi-
nase inhibitory activity in a concentration-dependent man-
ner (Fig. 1). The IC50 values for fresh and dried seed
extracts were 2.9 and 3.2 mg/ml, respectively. However,
the inhibitory activity of these extracts was weaker than a
reference inhibitor, kojic acid (IC50 = 8.9 lg/ml).
4. Discussion
The reactive oxygen species (ROS) include superoxide
anions (O2 ), hydrogen peroxide (H2O2), and hydroxyl
radicals (OH) and accumulation of these ROS can result
in oxidative stress that has been related to human diseases
such as cardiovascular diseases, cancers, aging, diabetes,
and atherosclerosis (Willcox et al., 2004). Many polyphe-
nolic compounds including gallic acid and ellagic acid have
been shown to possess strong scavenging activity of free
radicals (Yilmaz and Toledo, 2004; Festa et al., 2001).
Therefore, plant extracts containing high levels of free
gallic acid and ellagic acid may be able to scavenge
excessive free radicals such as superoxide anion radical

(O
2 ) and peroxyl radical (ROO ) in the body and protect
human cells or tissues against oxidative stress.
 N. Rangkadilok et al. / Food and Chemical Toxicology 45 (2007) 328–336
The results showed that longan seed extracts contained
three major polyphenolic compounds, gallic acid, ellagic
acid, and corilagin at higher levels than pulp and whole
fruit (Fig. 2). These results were similar to those of a previ-
ous study where a 70% methanol extraction of longan fruit
was analyzed (Rangkadilok et al., 2005). However, the con-
tents of three compounds in longan water extracts in the
present study were higher than those of methanol extracts.
In the previous study, a rapid method was developed to
extract and determine the contents of these polyphenolics
in different longan tissues whereas in the present study,
the concentrated water extracts of longan were prepared
using multiple extraction and mechanical process (stirring
1 h each time, 3 times) to increase the efficiency of the
extraction method.
The content of ellagic acid in longan seed, cultivar Edor,
No. 1 was higher than Edor seed No. 2 while there was no
difference in the contents of corilagin and gallic acid. These
two seed materials were obtained from the same source in
2004 but the drying process of these two batches was differ-
ent. Edor seed No. 1 was dried immediately after washing
Fig. 2. HPLC chromatograms (detected at 270 nm) of gallic acid (GA), corilagin (CG), and ellagic acid (EA) in longan extracts (cultivar Edor). A = Fresh
seed extract; B = Dried seed extract; C = Dried pulp extract.
333
while seed No. 2 was kept at 4 C one night before drying.
A decrease in ellagic acid in seed No. 2 may be due to enzy-
matic browning during the storage period i.e., peroxidases
and polyphenoloxidases catalyzing oxidation and polymer-
ization of simple phenolics during storage, before the
drying process. The enzymatic browning reaction can be
controlled using heat inactivation of enzymes. Blanching
of apple peels for 10 s was found to be much more effective
in the inactivation of the enzymes than citric acid or ascor-
bic acid treatment (Wolfe and Liu, 2003). Therefore,
blanching fresh longan seed for 10 s before drying in the
oven may be a simple method to inactivate the browning
enzymes and preserve the polyphenol contents. In addition,
less water activity (drying immediately after washing) may
increase chemical stability and shelf life of longan seed.
The data presented in this study shows that longan seed
extract is a potent scavenger of ROS. In the DPPH and
X/XO assays, dried seed of longan exhibited scavenging
activity as strong as Japanese green tea extract while pulp
and whole fruit extracts had less activity. These results were
similar to the previous study by Soong and Barlow (2004)
334 N. Rangkadilok et al. / Food and Chemical Toxicology 45 (2007) 328–336
who reported the antioxidant activity of longan seed was
higher than longan flesh. Longan extracts contain high
levels of polyphenolic compounds which are composed of
one (or more) aromatic rings bearing one or more hydroxyl
groups, thus, they are capable of direct free radical scaveng-
ing and inactivation. From the XO inhibition studies on
these extracts it was found that longan seed extracts pos-
sessed weak inhibitory activity of XO (<30%). Therefore,
the antioxidant activity of these extracts is mainly due to
the direct scavenging of free radicals. Both gallic acid and
ellagic acid were more potent scavengers than ascorbic acid
in DPPH assay while gallic acid had much higher scaveng-
ing activity than ellagic acid in the X/XO assay. These two
compounds were also reported to be responsible for the
scavenging activity of Terminalia chebula extract in the
DPPH assay (Naik et al., 2004). However, longan seed
extract containing high levels of both gallic acid and ellagic
acid (cultivar Baidam) showed less scavenging activity than
longan fresh seed (cultivar Edor) that contained lower levels
of these compounds. In the DPPH assay, the SC50 values of
fresh seed and dried seed extracts (cultivar Edor) were
10.8 lg/ml (gallic acid = 0.11 lg/ml, ellagic acid = 0.10
lg/ml) and 11.6 lg/ml (gallic acid = 0.27 lg/ml, ellagic
acid = 0.15 lg/ml), respectively. The contents of both gallic
acid and ellagic acid present in these concentrated longan
extracts were much lower than the SC50 of individual pure
compound (2.3 and 2.2 lg/ml, respectively). Therefore,
both gallic acid and ellagic acid may not be the major con-
tributors to the high antioxidant effects of these longan
extracts. In the X/XO assay, the SC50 values of fresh seed
and dried seed extracts (Edor) were 7.6 lg/ml (gallic acid
and ellagic acid = 0.07 lg/ml) and 7.2 lg/ml (gallic
acid = 0.17 lg/ml, ellagic acid = 0.09 lg/ml), respectively.
The contents of both gallic acid and ellagic acid present in
these concentrated extracts were close to the SC50 of gallic
acid (0.7 lg/ml) but not ellagic acid (6.9 lg/ml). Gallic acid
may partially contribute to the scavenging activity of super-
oxide radical by longan extract. However, the present
results indicated that other polyphenolic/flavonoid glyco-
sides or ellagitannins in fresh and dried seed, which can
be easily extracted in hot water, may account for the potent
antioxidant activity of these longan extracts rather than
these two compounds. From HPLC fingerprints, there are
other peaks present in both fresh and dried seed (Fig. 2).
In a previous study, there were at least two unknown com-
pounds present in longan seed that were ellagic acid deriv-
atives and one of them (at 12.67 min) was present at a
high concentration in fresh seed (Rangkadilok et al.,
2005). A recent study on the isolation of the phenolic com-
pounds from longan seed also indicated the presence of an
ellagic acid-pentose conjugate (m/z 433), flavonoids, and
proanthocyanidins (Soong and Barlow, 2005). In addition,
longan cultivars Baidam and Biewkiew containing high lev-
els of corilagin also had less activity than others; therefore,
it clearly showed that the high scavenging activity of DPPH
and superoxide radicals of these longan extracts may not be
due to corilagin.
In the ORAC assay, the comparison of ORAC values
from the present study with those from a previous study
(Huang et al., 2002) showed that longan seed extracts
had lower peroxyl scavenging activity (1.4 · 103) than
strawberry extract (5.4 · 104) and grape juice (3.3 · 104).
Those fruits may contain more phytochemical compounds
that exhibit potent antioxidant activity and there may also
be more complex synergistic effects in these species. In
strawberry, the most abundant phytochemical compounds
are ellagic acid, various anthocyanin glycosides with
pelargonidin 3-O-glucoside predominant, catechin, and
various quercetin, and kaempferol glycosides (Hunnum,
2004) while grape juice or red wines contains gallic acid,
ellagic acid, epicatechin, catechin, resveratrol, and procy-
anidins (Blanco et al., 1998; Tedesco et al., 2000).
The results in the present study show that longan seed
water extract scavenged DPPH, superoxide radical (O 2 ),
and peroxyl radical (ROO). The O 
2 and ROO radicals
are among the most important free radicals which can be
generated as harmful byproducts and have been implicated
in lipid peroxidation and some diseases (Gyamfi and Aniya,
2002). The ROO radical is generated in normal metabolic
reactions by all aerobic organisms and also the source of
the highly biologically reactive, hydroxy radical (OH).
Therefore, scavenging of these free radicals by longan seed
extract can be an effective prevention for a living organism
against oxidative stress. In addition, longan seed extract
had significant antityrosinase activity, although, its activity
was lower than kojic acid. These results suggest that longan
seed extract may be developed further as a natural source of
free radical scavenging phytochemicals and also has appli-
cations as a natural skin-whitening agent in cosmetic prod-
ucts. However, the toxicity of these longan seed extracts
should be evaluated in more details before long term
consumption or use in formulated products. Therefore,
studies are currently underway to investigate the toxicity
of the longan extract in animal models.
In conclusion, longan extracts of both fresh and dried
seed exhibited strong antioxidant activities similar to those
of Japanese green tea extract while dried longan pulp had
the lowest activity in all tests. The present results indicate
that longan seed extract is an efficient scavenger of free rad-
icals such as superoxide and peroxyl radicals. Most antiox-
idant capacities of longan extracts are attributable to the
polyphenolic compounds in this plant. Free gallic acid,
ellagic acid, and a major ellagic acid conjugate, corilagin,
may not be the major free radical scavenging compounds
in longan seed extracts, but the activities may be due to
as yet uncharacterised polyphenolic/flavonoid glycosides
and ellagitannins. Interestingly, the longan seed extract
not only showed high scavenging activity, but also signifi-
cantly inhibited tyrosinase activity. Our preliminary studies
suggest that longan seed extract can be used to produce a
natural dietary antioxidant supplement or added into
healthy food products such as cereals, fruit bars or drinks,
to prevent some chronic diseases. In addition, they may be
used as an anti-cancer agent, anti-inflammatory drug, and
 N. Rangkadilok et al. / Food and Chemical Toxicology 45 (2007) 328–336
even anti-aging agent as they may help to protect cells/
DNA damages. In cosmetic products, longan extract could
be developed for use as an active ingredient for antioxidant
properties or a natural skin-whitening agent. The develop-
ment of a value-added ingredient from the longan seed to
use in food and cosmetic products will be beneficial for
human health rather than just being waste products from
canned longan manufacturers and dried fruit small
enterprises.
Acknowledgements
The authors would like to thank Supachai Ritruechai
and researchers in laboratory of Pharmacology, CRI, for
preparing of longan extracts. We gratefully acknowledge
Assoc. Prof. Pawin Manochai, Faculty of Agricultural
Production, Maejo University, and Malee Sampran Public
Co., Ltd. for supplying longan fruit and seed. We sincerely
thank Dr. Richard N. Bennett for his valuable suggestions
on the manuscript. This study was supported by a grant
from the Chulabhorn Research Institute.
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