Food

Chemistry
Food Chemistry 95 (2006) 319327
www.elsevier.com/locate/foodchem

Antioxidant and antiproliferative activities of red pitaya

Li-chen Wu *, Hsiu-Wen Hsu, Yun-Chen Chen, Chih-Chung Chiu,
Yu-In Lin, Ja-an Annie Ho *
Department of Applied Chemistry, National Chi-Nan University, No. 1 University Road, Puli, Nantou, 545 Taiwan

Received 8 November 2004; received in revised form 4 January 2005; accepted 4 January 2005

Abstract

The red pitaya, rich in micronutrients, has recently generated a great deal of consumer interest, therefore, this paper was designed
to study the total phenolic content, antioxidant activity and antiproliferative activity of red pitaya on melanoma cells and to deter-
mine if it is a valuable source of antioxidants and an anticancer agent. The total phenolic contents of esh (42.4 0.04 mg of gallic
acid equivalents (GAE)/100 g of esh fresh weight) and peel (39.7 5.39 mg of GAE/100 g of peel fresh weight) were similar. The
avonoid contents of esh and peel did not vary much (7.21 0.02 mg vs. 8.33 0.11 mg of catechin equivalents/100 g of esh and
peel matters). The concentrations of betacyanins expressed as betanin equivalents per 100 g of fresh esh and peel were 10.3 0.22
and 13.8 0.85 mg, respectively. The antioxidant activity, measured by the DPPH method at EC50, was 22.4 0.29 and
118 4.12 lmol vitamin C equivalents/g of esh and peel dried extract; the values of EC50, determined by the ABTS+ approach,
were 28.3 0.83 and 175 15.7 lmol of trolox equivalents antioxidant capacity (TEAC)/g of esh and peel dried extract, respec-
tively. The antiproliferative study on B16F10 melanoma cells revealed that the peel (EC50 25.0 lg of peel matter) component
was a stronger inhibitor of the growth of B16F10 melanoma cancer cells than the esh. The results indicated that the esh and peel
were both rich in polyphenols and were good sources of antioxidants. The red pitaya peel fullled its promise to inhibit the growth
of melanoma cells.

2005 Elsevier Ltd. All rights reserved.

Keywords: Phenolics; Red pitaya; Polyphenols; Antioxidant activity; DPPH, ABTS+; B16F10 melanoma cells

1. Introduction by Willet (1994) that roughly 32% (range of 20%42%)

of deaths from cancer could be avoided by dietary mod-
The leading causes of death in the United States are ication. Epidemiological studies have strongly sug-
cardiovascular diseases and cancers. Similarly, in Tai- gested that diet plays an important role in the
wan, around 27% of deaths are from cancer and 18% prevention of chronic diseases (Bauman, 2004; Parillo
of deaths are from cardiovascular and heart diseases & Riccardi, 2004; Willet, 1995). Polyphenolics, thiols,
(Department of Health Web, 2004). It was estimated carotenoids, tocopherols, and glucosinolates commonly
found in fruits, vegetables and grains, provide chemo-
protective eects to combat oxidative stress in the body
Abbreviations: GAE, Gallic acid equivalents; TEAC, Trolox equiv- and maintain balance between oxidants and antioxi-
alent antioxidant capacity; CHD, Coronary heart disease; MTT, 3- dants to improve human health (Adom & Liu, 2002;
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Dragsted, Strube, & Larsen, 1993; Jia, Tang, & Wu,
*
Corresponding authors. Tel.: +886 49 2910960; fax: +886 49 291
7956.
1999; Wolfe, Wu, & Liu, 2003). An imbalance caused
E-mail addresses: lw25@ncnu.edu.tw (L.-c. Wu), jah@ncnu.edu.tw by excess oxidants leads to oxidative stress, resulting
(J.-a. Annie Ho). in damage to DNA and protein and increased risk of

0308-8146/$ - see front matter
2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2005.01.002
320 L.-c. Wu et al. / Food Chemistry 95 (2006) 319327

degenerative diseases such as cancer (Farombi, Hansen, torical documents, has a long lineage in the western
Ravn-Haren, Moller, & Dragsted, 2004; Llopiz et al., hemisphere. The skin is covered with bracts, or
2004; Moller & Loft, 2004; Sander, Chang, Hamm, Els- scales hence the name dragon fruit. They are cur-
ner, & Thiele, 2004; van Meeteren, Hendriks, Dijkstra, rently being grown commercially in Taiwan, Nicara-
& van Tol, 2004). gua, Colombia, Vietnam, Israel, Australia and the
Consumption of fresh fruits and vegetables, as well USA.
as grains, has been associated with reduced risk of In cacti, the most important fruit pigments are the
coronary heart disease (CHD) (Bazzano, Serdula, & betacyanins and betaxanthins (Wybraniec et al., 2001).
Liu, 2003; Joshipura et al., 2001; Polidori, 2003; Sri- Betalains, composed of red-violet betacyanin and yellow
nath Reddy & Katan, 2004), stroke (Gillman et al., betaxanthins, are water-soluble pigments that provide
1995; Voko, Hollander, Hofman, Koudstaal, & Bret- colours in owers and fruits. The known betacyanin
eler, 2003), symptoms of chronic obstructive pulmon- pigments of Hylocereus polyrhizus esh are betanin,
ary disease (Fabricius & Lange, 2003; Liu, Sobue, phyllocactin (6 0 -O-malonylbetanin), and a recently dis-
Otani, & Tsugane, 2004), and dierent types of can- covered betacyanin, hylocerenin (5-O-[6 0 -O-(300 -hydro-
cer, including breast and ovarian cancer (Duncan, xyl-300 -methyl-glutaryl)-b-D-glucopyranoside) (Wybraniec
2004; Hakimuddin, Paliyath, & Meckling, 2004; & Mizrahi, 2002; Wybraniec et al., 2001). Betacyanin,
Khanzode, Muddeshwar, Khanzode, & Dakhale, with 5-O-glycosides (betanin) or 6-O-glycosides, is com-
2004; Larsson, Holmberg, & Wolk, 2004) and colon monly detected in plants. More-complicated esterica-
cancer (Frydoonfar, McGrath, & Spigelman, 2003; tion of 5-O-glycosides with hydrocinnamic acids such
McCullough et al., 2003). Polyphenolic compounds, as ferulic or p-coumaric acids (Strack, Steglich, & Beta-
widely distributed in higher plants, have been found lains, 1993) and malonic acid (Kobayashi, Schmidt,
to have potential health benets that are believed to Nimtz, Wray, & Schliemann, 2000; Minale, Piattelli,
arise mainly from their antioxidant activity (Liu, De Stefano, & Nicolaus, 1996) have also been studied.
2003). There is considerable scientic and public inter- It has been reported that betalains from Amaranthus
est in the important role that antioxidants may play in (Cai, Sun, & Corke, 2003) and beet roots (Escribano,
health care, such as by acting as cancer chemopreven- Pedreo`o, Garcia-Carmona, & Munoz, 1998) demon-
tive and anti-inammatory agents and by reducing strated antiradical and antioxidant activity. Betanin,
risk of cardiovascular mortality (Cos et al., 2004; from red beet, eectively inhibited lipid peroxidation
Siddiqui, Afaq, Adhami, Ahmad, & Mukhtar, 2004). and heme decomposition, suggesting that these pig-
Numerous studies have reported the antioxidant and ments may provide protection against certain oxidative
antiproliferative activities of dierent fresh produces, stress-related disorders (Kanner, Harel, & Granit,
such as apples (Wolfe et al., 2003), grape seeds (Call- 2001). Polyphenolics, on the other hand, play an
iste, Trouillas, Allais, Simon, & Duroux, 2001; Chi- important role in antioxidant activity (Wu, Chen, Ho,
dambara Murthy, Singh, & Jayaprakasha, 2002), & Yang, 2003). They have evidently shown antiprolif-
strawberries (Meyers, Watkins, Pritts, & Liu, 2003), erative activity or cytotoxicity to human oral cancer
common vegetables (Chu, Sun, Wu, & Liu, 2002), cells (Seeram, Adams, Hardy, & Heber, 2004), mela-
cranberry fruits (Gunes, Liu, & Watkins, 2002), and noma cells (Rodriguez et al., 2002), and lung metastasis
raspberries (Liu et al., 2002). However, the antioxi- induced by B16F10 melanoma cells (Menon, Kuttan, &
dant activity of some plant foods has not been well Kuttan, 1995).
studied. Investigation of their antiradical characteris- Eorts have been made to study the chemistry of
tics is therefore of interest, primarily to discover betalains in H. polyrhizus (Stintzing, Conrad, Klaiber,
promising new sources of natural antioxidants, func- Beifuss, & Carle, 2004; Wybraniec et al., 2001;
tional foods, and neutraceuticals. Wybraniec & Mizrahi, 2002); however, there is little
The fruits of Hylocereus cacti, known as red pitaya information available on its phenolic content, antiox-
or pitahaya, have recently drawn much attention of idant activity, and antitumor activity. The objectives
growers worldwide, not only because of their red-pur- of this study were to evaluate the nutritional quality
ple colour and economic value as food products, but of red pitaya esh component and peel. Total pheno-
also for their antioxidative activity from the betacya- lic content, antioxidant capacity, and antiproliferative
nin contents (Wybraniec & Mizrahi, 2002). The Hyl- eect on B16F10 melanoma cells were quantied and
ocereus genus consists of climbing or epiphytic compared to those characteristics of betanin in rela-
tropical American cacti with angular stems and mostly tion to the same cancer cell. In addition, it could
white, fragrant, night-blooming owers. The pitaya, also be determined whether the peel of red pitaya,
the fruit of some strains of this cactus family, which the waste product from juice manufacture, could be
is native to the tropical forest regions of Mexico and utilized as a potential alternative for various sources
Central and South America (Mizrahi, Nerd, & Nobel, of nutrients or antioxidants to improve human
1997) and is mentioned as a popular Aztec fruit in his- health.
 L.-c. Wu et al. / Food Chemistry 95 (2006) 319327 321

2. Materials and methods on each sample; data shown later represent the means
of three measurements.
2.1. Chemicals
2.4. Determination of avonoid content (vanillin-HCl
FolinCiocalteu reagent, sodium nitrite, catechin, assay)
gallic acid, Sephadex G-25, MTT (3-[4,5-dimethylthia-
zol-2-yl]-2,5-diphenyltetrazolium bromide), and all other The amount of avonoid (condensed tannins) was
chemicals and organic solvents were purchased from measured by vanillin assay (Price, van Scoyoc, & Butler,
Sigma Chemical Co. (St. Louis, MO). LH-20 was ob- 1978). One millilitre aliquots of peel and esh extracts
tained from Amersham Biosciences (Uppsala, Sweden). were dispensed into test tubes. Tubes were incubated
Whatman No.1 lter paper was bought from Fisher Sci- in the water bath at 30 C. Five millilitres of the vanillin
entic (Fair Lawn, NY). HPLC-grade acetonitrile and reagent (1% vanillin in methanol) were added at 1.0 min
acetic acid came from Merck (Darmstadt, Germany). intervals to one set of samples, and 5.0 ml of the 4% HCl
The B16F10 melanoma cells were obtained from the solution were added at 1.0 min intervals to the second
American Type Culture Collection (ATCC) (Rockville, set of samples. After 20 min of incubation, the samples
MD). Fetal bovine serum (FBS) was purchased from were removed and the absorbance at 500 nm was read.
Gibco Life Technologies (Grand Island, NY), and beta- The condensed tannins content (avonoid) was ex-
nin standard was obtained from TCI (Tokyo, Japan). pressed as catechin equivalents in milligrammes. Tripli-
cate determinations were performed on each sample;
2.2. Sample preparation data shown later represent the means of three
measurements.
Red pitaya, H. polyrhizus, used in this study was ob-
tained locally from four lots of fruits (Puli, Nantou, Tai- 2.5. Quantitation of betacyanins
wan), which were washed and stored at
20 C before
analysis. The fruit was peeled prior to analysis. The phe- Red pitaya pigment content was measured in a simi-
nolic content, of dierent components of red pitaya, was lar way to that described by Wybraniec and Mizrahi
determined by extracting 50 g of the esh and peel com- (Wybraniec & Mizrahi, 2002). The betacyanin content
ponents with 200 g of chilled 80% acetone solution in a of dried extracts, determined by the spectrophotometric
Waring blender for 10 min (Wolfe et al., 2003). The slurry method, were calculated and expressed as betanin equiv-
was ltered through Whatman No. 1 lter paper in a alents (mg/100 g of fresh materials) by the following
Buchner funnel under vacuum. The solids were re- formulas: concentration of betacyanins (mg/g of dried
extracted with 150 g of 80% acetone and homogenized extracts weight) = A538 (MW)V (DF) 100/eLW. A538
again for an additional 5 min before reltering. The l- is the absorbance at 538 nm (kmax); L (path length)
trate was then evaporated using a rotorary evaporator = 1.0 cm; DF is the dilution factor; V is the pigment
at 45 C until less than 10% of the initial volume re- solution volume (ml); W is the dried pigment weight
mained. The extract was freeze-dried and frozen at (g). For betanin, e (molar extinction coecient) = 65,000

70 C prior to analysis. and MW = 550. Individual pigment content was ex-
pressed as the percentage of peak area determined by
2.3. Determination of total phenolic contents HPLC.

The total phenolic contents of esh and peel sam- 2.6. Determination of anthocyanin contents
ples of red pitaya were determined using a modied
Folin-Ciocalteu method (Wolfe et al., 2003). A 125 ll The monomeric anthocyanin content of red pitaya
aliquot of a known dilution of the extract was added esh and peel components was measured using a spec-
to the test tube and combined with 0.5 ml of Folin trophotometric pH dierential protocol (Boyles &
Ciocalteus reagent. The tubes were vortexed for 15 s Wrolstad, 1993). The red pitaya peel and esh extracts
and then allowed to stand for 6 min at 20 C. About were thoroughly mixed with 0.025 M potassium chloride
1.25 ml of 7% sodium carbonate solution was then (pH 1.0) in a known dilution. The absorbance of the
added to the test tubes, and the mixture was diluted mixture was measured at 515 and 700 nm using distilled
to 3 ml with distilled, de-ionized water. Colour devel- water to zero the spectrophotometer. The red pitaya
oped for 90 min, and absorbance was measured at peel and esh extracts were then combined with 0.4 M
760 nm using the Hewlett Packard UVVis spectro- sodium acetate buer (pH 4.5), and the absorbances
photometer (Palo Alto, CA). The measurement was were measured at the same wavelengths. The absor-
compared to a standard curve of prepared gallic acid bance of the diluted sample (A) was as follows:
solutions and expressed as gallic acid equivalents in A = (A515
A700)pH 1.0
(A515
A700)pH 4.5. The
milligrams. Triplicate determinations were performed anthocyanin content was calculated as the total of
322 L.-c. Wu et al. / Food Chemistry 95 (2006) 319327

monomeric anthocyanin pigment (mg/litre) = (A MW tested compounds should be reacted completely within
DF 1000)/(e 1), where A is the absorbance of the di- 180 min in this condition (Cai et al., 2003) and reaction
luted sample and DF is the dilution factor. MW and e in time for vitamin C is less than 1 min due to its fast oxi-
this formula correspond to the predominant anthocya- dation. Ethanol (80%) was used as a blank solution and
nin in the sample. Since the sample composition was DPPH solution without test samples (3.9 ml of
unknown, pigment content was calculated as cyanidin- DPPH + 0.1 ml of 80% ethanol) served as the control.
3-glucoside, where MW = 449.2 and e = 26,900. All tests were performed in triplicate. The antioxidant
activity of the test samples was expressed as (i) the med-
2.7. Chromatographic determination of betanin ian eective concentration for radical-scavenging activ-
ity (EC50): total phenolics (mg) of antioxidant (test
Standard betanin, containing betanin and isobetanin, sample) required for a 50% decrease in absorbance of
was obtained commercially and prepared in a two-step DPPH radicals, and (ii) inhibition (%) of DPPH absor-
procedure prior to HPLC analysis (Wybraniec & Miz- bance = (Acontrol
Atest) 100/Acontrol. A plot of absor-
rahi, 2002). Briey, the extracts were rst separated by bance of DPPH vs.concentration of antioxidant was
gel ltration on a Sephadex G-25 column (40 2.2 cm) made to establish the standard curves (doseresponse
pre-equilibrated with 1% acetic acid. The 1% acetic acid curves) and to calculate EC50. Acontrol is the absorbance
fractions, eluted at a ow rate of 1.0 ml/min, were col- of the control (DPPH solution without the test sample),
lected and examined spectrophotometrically at 538 nm and Atest is the absorbance of the test sample (DPPH
for the presence of betanin. Fractions with 538 nm solution plus 0.1 ml of 5 lM test compound). Vitamin
absorbance were collected and lyophilized for further C served as a standard, and the results of the assay were
purication. The residue was redissolved in water and expressed relative to vitamin C equivalent.
subsequently puried on an LH-20 column
(40 2.2 cm) with water as the eluent. Fractions show- 2.9. ABTS+ assay
ing 538 nm absorbance were collected and lyophilized
for HPLC analysis. For ABTS+ generation from ABTS salt, 2.45 mM of
A high-performance liquid chromatographic method potassium persulfate (K2S2O8) was reacted with 7 mM
was employed to analyze the betanin content for red ABTS salt in 0.01 M phosphate-buered saline, pH
pitaya peel and esh components (Wybraniec & Miz- 7.4, for 16 h at room temperature in the dark. The resul-
rahi, 2002). An Agilent 1100 liquid chromatographic tant ABTS+ radical cation was diluted with 0.01 M
system (Palo Alto, CA), equipped with a photodiode phosphate-buered saline, pH 7.4, to give an absor-
array detector and a Rheodyne (Cotati, CA) model bance of around 0.70 at 734 nm. The standard and sam-
7125 injector with a sample loop of 20 ll, was used, ple red pitaya peel and esh extracts were diluted 100 X
along with an Agilent ChemStation for LC. Final sig- with the ABTS+ solution to a total volume of 1 ml and
nal integration was performed using the Agilent Chem- allowed to react for 3 min (Re et al., 1999). Absorbance
Station for LC running on a Pentium-4 computer. A was measured spectrophotometrically at dierent time
Hypersil ODS (Supelco Inc., Bellefonte, PA), 5 lm, intervals. Control (without a standard or sample) was
250 4.6 mm I.D. column was used to determine the used as blank and 990 ll of PBS were added to these
betanin content, and the elution programme consisted control samples instead. Trolox, the water-soluble
of a 35 min isocratic elution of 90% solvent A (0.5% a-tocopherol (Vitamin E) analog, served as a standard,
aqueous TFA in water) with 10% solvent B (acetoni- and the results of the assay were expressed relative to
trile) with a ow of 0.5 ml/min. For each analysis, trolox in terms of TEAC (trolox equivalent antioxidant
15 ll of the ltered extracts was directly injected into capacity).
the chromatographic column. The identities of the dif-
ferent chromatographic peaks were conrmed by their 2.10. Determination of inhibition of B16F10 melanoma
visible spectral characteristics in comparison to stan- cell proliferation
dards and retention times.
The extracts of the esh and peel samples of red pi-
2.8. DPPH radical scavenging activity taya were used to measure their ability to inhibit
B16F10 melanoma cell proliferation (Nakano et al.,
A 0.1 ml aliquot of the acetone extract prepared 1998). The cell cultures were exposed to various concen-
above was mixed with 3.9 ml of an 80% ethanolic trations of the extracts during a 48 h growth period. Cell
0.6 mM DPPH solution. The tubes were vortexed for proliferation was measured by the ability of viable cells
15 s and allowed to stand for 180 min, as described by to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
Cai et al. (2003), after which the absorbance of the mix- razolium bromide (MTT) to formazan, whose absor-
ture was measured at 515 nm using the Hewlett Packard bance can be analyzed spectrophotometrically at
UVVis spectrophotometer (Palo Alto, CA). Most 570 nm. All measurements were performed in triplicate.
 L.-c. Wu et al. / Food Chemistry 95 (2006) 319327 323

The absorbance was measured using the Hewlett Pack- (a)

ard UVVis spectrophotometer (Palo Alto, CA). The
eective median dose (EC50) was determined and ex- 1
pressed as milligrammes of red pitaya esh and peel

Absorbance at 538 nm
samples SD.
2
2.11. Statistical analysis

Results are expressed as the means standard devia-

tion of three replicates. The Students t test was used for
statistical analysis of the dierence noted.
1

3. Results and discussion 0 10 20 30

Time (min)
3.1. Content of phenolic compounds
(b) 1
The total phenolic contents of the esh and peel of
the red pitaya were measured according to the Folin

Absorbance at 538 nm
Ciocalteu method. The FolinCiocalteu reagent deter- 2
mines total phenols (and other easily oxidized
substances), producing a blue colour by reducing yellow
heteropolyphosphomolybdate-tungstate anions. This
method gives a general measure of phenolic content,
as it is not completely specic for phenolic compounds
and not all phenolic compounds exhibit the same level
of activity in the assay. Phenolics were extracted from
red pitaya esh or peel with 80% acetone. The total 1
extractable phenolic contents were 42.4 0.04 and
39.7 5.39 mg of GAE/100 g of esh and peel, respec- 0 10 20 30

tively. The amounts of total phenolics per 100 g of esh Time (min)
or peel were similar, whereas the amounts of total phen- Fig. 1. HPLC chromatographs of betacyanins from esh of
olics per gramme of extract of esh and peel were H. polyrhizus (a) and peel (b). Peak 1 represents betanin (retention
4.55 0.03 and 25.4 2.10 mg of GAE, respectively. time: 6.5 min); Peak 1 0 is isobetanin (retention time: 8.3 min).
Although the total phenolic content per gramme of ex-
tract in peel was about six times more than that in esh,
the yield of the extracts from the same weight of raw (tR = 15.7 min) if compared with the results of Wybra-
materials (9.91 and 1.56 g from 100 g of raw materials) niec and Mizrahi (Wybraniec & Mizrahi, 2002), which
diminished the dierences in total phenolic compounds. represented the most abundant kind of betacyanin in
It is likely that some of the non-phenolic compounds esh and peel (51% and 58%, respectively). The percent-
were co-extracted from esh, which bulked up the age of isobetanins (1.3%) in peel was less than that in
extract. esh (4.4%).
Betacyanins, the pigments found in Hylocereus cacti, The avonoid contents of esh and peel were
also contributed to the total phenolics, due to a phenol 7.21 0.02 and 8.33 0.11 mg of catechin equivalents/
structure in the molecule. The concentrations of betacy- 100 g of esh and peel, respectively. The avonoid con-
anins, expressed as betanin equivalents in esh and peel, tents per gramme of extract in peel were about ve times
were 10.3 0.22 and 13.8 0.85 mg/ 100 g of esh and greater than those in esh (5.33 0.31 vs. 0.77
peel, respectively. The HPLC chromatogram of betacy- 0.02 mg catechin equivalents), which was similar to the
anins from fruit esh and peel after purication was set results of measuring total phenolic content. However,
at 538 nm in Fig. 1. At least four dierent kinds of beta- no anthocyanins were detected. The non-detectable level
cyanins were separated by the C18 column and detected of anthocyanins in red pitaya could be due to the ab-
by the photodiode array detector of the HPLC system. sence of monomeric anthocyanin in red pitaya. The
By comparing with commercially obtained betanin stan- red-violet colour of red pitaya might be attributed to
dards, peaks 1 and 1 0 were betanin and isobetanin with betacyanins, but not to anthrocyanin. Betacyanins are
retention times of 6.5 and 8.3 min, respectively. It was a class of water-soluble pigments that provide the col-
likely that peak 2 was contributed by phyllocactin ours in a wide variety of owers and fruits (Strack,
324 L.-c. Wu et al. / Food Chemistry 95 (2006) 319327

Vogt, & Schliemann, 2003). They cannot be found in 100

(a) y = 21.417x + 0.7107
plants containing anthocyanin pigments, and structur- 90 2
R = 0.9996
80
ally they are unrelated. Little is known about the role

Inhibition (%)
70
of betacyanin, and the gain and loss of anthocyanins 60
and betalains during plant evolution remain a mystery 50
(Clement & Mabry, 1996). 40
30
20
3.2. Assessment of antioxidant activity
10
0
Of the several approaches to the measurement of 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
antioxidative activity that could have been used, such Extract (mg)
as DPPH (Cai et al., 2003), TEAC (Miller, Rice-Evans,
100
Davies, Gopinathan, & Milner, 1993), TOSC (Winston, (b) y = 112.68x + 0.8443
90 2
R = 0.9993
Regoli, Dugas, Fong, & Blanchard, 1998), ORAC (Cao,

Inhibition (%)
80
Alessio, & Cutler, 1993), FRAP (Pulido, Bravo, & 70
Saura-Calixto, 2000), and TRAP (Ghiselli, Serani, 60
Maiani, Azzini, & Ferroluzzi, 1995), the DPPH ap- 50
proach and ABTS+ radical-scavenging assay were cho- 40
30
sen for this study. The DPPH approach is simple and 20
widely applied for the measurement of antioxidant 10
activity of polyphenolics and colourants (Cai et al., 0
0.1 0.2 0.3 0.4 0.5 0.6
2003; Shi & Le Maguer, 2000). The ABTS+ radical-
Extract (mg)
scavenging assay, which employs a specic absorbance
at a wavelength remote from the visible region and re- Fig. 2. Antioxidant activity of Hylocereus cacti determined by DPPH.
quires a short reaction time (Lee, Kim, Lee, & Lee, (a) esh; (b) peel.
2003), can be used in both organic and aqueous solvent
systems. It is suggested that the ABTS+ approach is bet-
ter for evaluating the antioxidant activity of phenolic of hydroxyl groups (OH) or other hydrogen-donating
phytochemicals than the DPPH radical-scavenging as- groups (@NH, SH) in the molecular structure led to
say (Awika, Rooney, Wu, Prior, & Cisneros-Zevallos, higher antioxidant activity (Cai et al., 2003). Betanins
2003). contain imino groups and hydroxyl groups and would
The relationship between phenolic content and anti- contribute antioxidant activity, which could explain, in
oxidant capacity of the pitaya esh and peel is shown part, that peel is a better antioxidant due to the higher
in Fig. 2. Similar patterns were found in the antioxidant level of betanin in the peel.
activity measured by ABTS+ assay. The eective con-
centrations (EC50) determined by DPPH radical-scav- 3.3. Inhibitory eect of extracts on B16F10 melanoma
enging activity for esh and peel were 22.4 0.29 and cell proliferation
118 4.12 lmol vitamin C equivalents/g dried extract,
respectively, and the values of EC50 for the ABTS ap- MTT assay was used to study the antiproliferative
proach were 28.3 0.83 and 175 15.7 lmol TEAC/g activity of red pitaya. MTT is reduced to an insoluble
dried extract, respectively. Peel extract was a better anti- purple formazan by mitochondrial dehydrogenase. Cell
oxidant than esh due to its slightly higher polyphenolic proliferative activity was measured by comparison of
content. The increase in phenolic content of the two ex- the purple colour formation. Dead cells, on the other
tracts from esh and peel was found to be linearly cor- hand, did not form the purple formazan due to their
related with antioxidant capacity. In the DPPH assay, lack of the enzyme. The eect of red pitaya esh and
as the total phenolic compounds increased from 0.7 to peel on the growth of B16F10 cells in vitro is shown
3.5 mg gallic acid equivalents of esh and from 0.12 to in Fig. 3(a). Cells were cultured for 48 h with several
0.6 mg gallic acid equivalents of peel, the percentage of dierent levels of esh or peel extracts. The curves
radical-scavenging activity of both increased linearly showed that the growth of melanoma cancer cells
about 4.8-fold (R2 = 0.99). was inhibited in a dose-dependent manner. The value
Phenolics also inuence antioxidant-activity measure- of EC50 of the antiproliferative activity of the peel ex-
ment. They interfere with the oxidation process by react- tract was 10.0 lg of GAE (25.0 mg of peel matter),
ing with free radicals, chelating catalytic metals, and whereas the EC50 value of esh could not be calculated
scavenging oxygen. The eect of esh and peel on anti- from the doseresponse curve. The dierence could be
oxidant activity could be a result of the types of poly- attributed to the dierent kinds of polyphenolics pres-
phenolics they contained. An increase in the number ent in the samples. Evidence indicated that hydroxyl-
 L.-c. Wu et al. / Food Chemistry 95 (2006) 319327 325

(a) 100 lue-added ingredient that may assist in the prevention

90 of chronic disease.
80
Cell growth (%)

70
60
50 Acknowledgments
40
30 The authors are grateful for the nancial support
20 from the Taiwan National Science Council, NSC 92-
10
2113-M-260-012 and the Taichung Veterans General
0
0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 HospitalNational Chi-Nan University Joint Research
Total phenolics (g GAE) Program under Grant TCVGH-NCNU-92-79-06.

(b) 100
90
80 References
Cell growth (%)

70
60 Adom, K. K., & Liu, R. H. (2002). Antioxidant Activity of Grains.
50 Journal of Agricultural and Food Chemistry, 50, 61826187.
40
Awika, J. M., Rooney, L. W., Wu, X., Prior, R. L., & Cisneros-
30
Zevallos, L. (2003). Screening methods to measure antioxidant
20
10
activity of sorghum (sorghum bicolour) and sorghum products.
0 Journal of Agricultural and Food Chemistry, 51, 66576662.
0 0.1 0.2 0.3 0.4 Bauman, A. E. (2004). Updating the evidence that physical activity is
Betanin (mg) good for health: an epidemiological review 20002003. Journal
Science and Medicine in Sport, 7, 619.
Fig. 3. Inhibition of B16F10 melanoma cell proliferation by phyto- Bazzano, L. A., Serdula, M. K., & Liu, S. (2003). Dietary intake of
chemical extracts of Hylocereus cacti esh (s) and peel (j) (a) and fruits and vegetables and risk of cardiovascular disease. Current
betanin (b). The cell growth of treated groups were standardized with Atherosclerosis Report, 5, 492499.
untreated control group as A/A0 (%), where A is the value of A570 Boyles, M. J., & Wrolstad, R. E. (1993). Anthrocyanin composition of
generated at a given concentration of extracts or betanin by MTT red raspberry juice: inuence of cultivar, processing, and environ-
assay, and A0 is from untreated control group. mental factors. Journal of Food Science, 58, 11351141.
Cai, Y., Sun, M., & Corke, H. (2003). Antioxidant activity of betalains
from plants of the Amaranthaceae. Journal of Agricultural and
ated avonoids without the C2C3 double bond could Food Chemistry, 51, 22882294.
not inhibit the growth of melanoma cell lines, such as Calliste, C. A., Trouillas, P., Allais, D.-P., Simon, A., & Duroux, J.-L.
B16F10, whereas the presence of at least three adjacent (2001). Free radical-scavenging activities measured by electron spin
methoxyl groups would confer a more potent antipro- resonance spectroscopy and b16 cell antiproliferative behaviors of
seven plants. Journal of Agricultural and Food Chemistry, 49,
liferative eect (Rodriguez et al., 2002). Moreover, 33213327.
avonoids such as myricetin, baicalein, and gallic acid Cao, G., Alessio, H. M., & Cutler, R. G. (1993). Oxygen-radical
signicantly inhibited the growth of B16F10 after 72 h absorbance capacity assay for antioxidants. Free Radical Biology
of exposure. It has been suggested that the presence and Medicine, 14, 303311.
of a C2C3 double bond and three adjacent hydroxyl Chidambara Murthy, K. N., Singh, R. P., & Jayaprakasha, G. K.
(2002). Antioxidant Activities of Grape Vitis vinifera Pomace
groups in the avonoid A- or B-rings confer greater Ectracts. Journal of Agricultural and Food Chemistry, 50,
antiproliferative activity to the avonoid (Martinez 59095914.
et al., 2003). Betanin would also have an inuence on Chu, Y., Sun, J., Wu, X., & Liu, R. H. (2002). Antioxidant and
the antiproliferative eect. As Fig. 3(b) indicates, with antiproliferative activities of common vegetables. Journal of
increase of betanin concentration, greater inhibitory Agricultural and Food Chemistry, 50, 69106916.
Clement, J. S., & Mabry, T. J. (1996). Pigment evolution in the
eect was observed. This could be due to the molecular Caryophyllales: a systematic overview. Botanica Acta, 109,
structural eects similar to those observed in 360367.
avonoids. Cos, P., De Bruyne, T., Hermans, N., Apers, S., Berghe, D. V., &
In summary, considerable amounts of phenolic com- Vlietinck, A. J. (2004). Proanthocyanidins in health care: current
pounds were found in red pitaya esh and peel. To our and new trends. Current Medicinal Chemistry, 11, 13451359.
Department of Health Web (Taiwan). <http://www.doh.gov.tw/statis-
knowledge, this is the rst time that the total phenolic tic/data/> (accessed June, 2004).
content and antioxidant activity of red pitaya esh Dragsted, L. O., Strube, M., & Larsen, J. C. (1993). Cancer-protective
and peel have been measured. Our results showed that, factors in fruits and vegetables: biochemical and biological
in addition to the grape seeds, the peel of red pitaya, background. Pharmacology and Toxicology, 72, 116135.
an inedible waste product of juice manufacture, might Duncan, A. M. (2004). The role of nutrition in the prevention of breast
cancer. AACN Clinical Issues, 15, 119135.
be another good source of antioxidants and an antimel- Escribano, J., Pedreo`o, M. A., Garcia-Carmona, F., & Munoz, R.
anoma agent. Waste pitaya peel should be regarded as a (1998). Characterization of the antiradical activity of betalains
valuable product and has potential as an economic va- from Beta vulgaris L. roots. Phytochemical Analysis, 9, 124127.
326 L.-c. Wu et al. / Food Chemistry 95 (2006) 319327

Fabricius, P., & Lange, P. (2003). Diet and lung cancer. Monaldi Menon, L. G., Kuttan, R., & Kuttan, G. (1995). Inhibition of lung
Archives of Chest Disease, 59, 207211. metastasis in mice induced by B16F10 melanoma cells by
Farombi, E. O., Hansen, M., Ravn-Haren, G., Moller, P., & Dragsted, polyphenolic compounds. Cancer Letters, 95, 221225.
L. O. (2004). Commonly consumed and naturally occurring dietary Meyers, K. J., Watkins, C. B., Pritts, M. P., & Liu, R. H. (2003).
substances aect biomarkers of oxidative stress and DNA damage Antioxidant and antiproliferative activities of strawberries. Journal
in healthy rats. Food and Chemical Toxicology, 42, 13151322. of Agricultural and Food Chemistry, 51, 68876892.
Frydoonfar, H. R., McGrath, D. R., & Spigelman, A. D. (2003). The McCullough, M. L., Robertson, A. S., Chao, A., Jacobs, E. J.,
variable eect on proliferation of a colon cancer cell line by the Stampfer, M. J., Jacobs, D. R., Diver, W. R., Calle, E. E., & Thun,
citrus fruit avonoid Naringenin. Colourectal Disease, 5, 149152. M. J. (2003). A prospective study of whole grains, fruits, vegetables
Gillman, M. W., Cupples, L. A., Gagnon, D., Posner, B. M., Ellison, and colon cancer risk. Cancer Causes and Control, 14, 959970.
R. C., & Castelli, W. P. (1995). A Protective eect of fruits and Miller, N. J., Rice-Evans, C. A., Davies, H. V., Gopinathan, V., &
vegetables on development of stroke in men. Journal of the Milner, A. (1993). A novel method for measuring antioxidant
American Medical Association, 273, 11131117. capacity and its application to monitoring the antioxidant status in
Ghiselli, A., Serani, M., Maiani, G., Azzini, E., & Ferroluzzi, A. premature neonates. Clininal Science, 84, 407412.
(1995). A uorescense-based method for measuring total plasma Minale, L., Piattelli, M., De Stefano, S., & Nicolaus, R. A. (1996).
anti-oxidant capability. Free Radical Biology and Medicine, 18, Pigments of centrospermae-VI. Acylated betacyanins. Phytochem-
2936. istry, 5, 10371052.
Gunes, G., Liu, R. H., & Watkins, C. B. (2002). Controlled- Mizrahi, Y., Nerd, A., & Nobel, P. S. (1997). Cacti as Crops.
atmosphere eects on postharvest quality and antioxidant activity Horticultural Reviews, 18, 291320.
of cranberry fruits. Journal of Agricultural and Food Chemistry, 50, Moller, P., & Loft, S. (2004). Interventions with antioxidants and
59325938. nutrients in relation to oxidative DNA damage and repair.
Hakimuddin, F., Paliyath, G., & Meckling, K. (2004). Selective Mutation Research, 551, 7989.
cytotoxicity of a red grape wine avonoid fraction against MCF-7 Nakano, Y., Matsunaga, H., Saita, T., Mori, M., Katano, M., &
cells. Breast Cancer Research and Treatment, 85, 6579. Okabe, H. (1998). Antiproliferative constituents in Umbelliferae
Jia, Z., Tang, M., & Wu, J. (1999). The determination of avonoid plants II. Screening for polyacetylenes in some Umbelliferae plants,
contents in mulberry and their scavenging eects on superoxide and isolation of panaxynol and falcarindiol from the root of
radicals. Food Chemistry, 64, 555559. Heracleum moellendori. Biological and Pharmaceutical Bulletin,
Joshipura, K. J., Hu, F. B., Manson, J. E., Stampfer, M. J., Rimm, E. 21, 257261.
B., Spiegelman, D., et al. (2001). The eect of fruit and vegetable Parillo, M., & Riccardi, G. (2004). Diet composition and the risk of
intake on risk for coronary heart disease. Annals of Internal type2 diabetes: epidemiological and clinical evidence. British
Medicine, 134, 11061114. Journal of Nutrition, 92, 719.
Kanner, K., Harel, S., & Granit, R. (2001). Betalains A new class of Polidori, M. C. (2003). Antioxidant micronutrients in the prevention of
dietary cationized antioxidants. Journal of Agricultural and Food age-related diseases. Journal of Postgraduate Medicine, 49,
Chemistry, 49, 51785185. 229235.
Khanzode, S. S., Muddeshwar, M. G., Khanzode, S. D., & Dakhale, Price, M. L., van Scoyoc, S., & Butler, L. G. (1978). A critical
G. N. (2004). Antioxidant enzymes and lipid peroxidation in evaluation of the vanillin reaction as an assay for tannin in
dierent stages of breast cancer. Free Radical Research, 38, 8185. sorghum grain. Journal of Agricultural and Food Chemistry, 26,
Kobayashi, N., Schmidt, J., Nimtz, M., Wray, V., & Schliemann, W. 12141218.
(2000). Betalains from Christmas cactus. Phytochemistry, 54, Pulido, R., Bravo, L., & Saura-Calixto, F. (2000). Antioxidant activity
419426. of dietary polyphenols as determined by a modied ferric reducing/
Larsson, S. C., Holmberg, L., & Wolk, A. (2004). Fruit and vegetable antioxidant power assay. Journal of Agricultural and Food Chem-
consumption in relation to ovarian cancer incidence: the Swedish istry, 48, 33963402.
Mammography Cohort. British Journal of Cancer, 90, 21672170. Re, R., Pellegrini, N. P., Proteggente, A., Pannala, A., Yang, M., &
Lee, K. W., Kim, Y. J., Lee, H. J., & Lee, C. Y. (2003). Cocoa has Rice-Evans, C. (1999). Antioxidant activity applying an improved
more phenolic phytochemicals and a higher antioxidant capacity ABTS radical cation decolourization assay. Free Radical Biology
than teas and red wine. Journal of Agricultural and Food Chemistry, and Medicine, 26, 12311237.
51, 72927295. Rodriguez, J., Yanez, J., Vicente, V., Alcaraz, M., Benavente-Garcia,
Liu, M., Li, X. Q., Weber, C., Lee, C. Y., Brown, J., & Liu, R. H. O., Castillo, J., Lorente, J., & Lozano, J. A. (2002). Eects of
(2002). Antioxidant and antiproliferative activities of raspberries. several avonoids on the growth of B16F10 and SK-MEL-1
Journal of Agricultural and Food Chemistry, 50, 29262930. melanoma cell lines: relationship between structure and activity.
Liu, R. H. (2003). Health benets of fruit and vegetables are from Melanoma Research, 12, 99107.
additive and synergistic combinations of phytochemicals. American Sander, C. S., Chang, H., Hamm, F., Elsner, P., & Thiele, J. J. (2004).
Journal of Clinical Nutrition, 78, 517S520S. Role of oxidative stress and the antioxidant network in cutaneous
Liu, Y., Sobue, T., Otani, T., & Tsugane, S. (2004). Vegetables, fruit carcinogenesis. International Journal of Dermatology, 43, 326335.
consumption and risk of lung cancer among middle-aged Japanese Seeram, N. P., Adams, L. S., Hardy, M. L., & Heber, D. (2004). Total
men and women: JPHC study. Cancer Causes and Control, 15, cranberry extract versus its phytochemical constituents: antiprolif-
349357. erative and synergistic eects against human tumor cell lines.
Llopiz, N., Puiggros, F., Cespedes, E., Arola, L., Ardevol, A., Blade, Journal of Agricultural and Food Chemistry, 52, 25122517.
C., & Salvado, M. J. (2004). Antigenotoxic eect of grape seed Shi, J., & Le Maguer, M. (2000). Lycopene in tomatoes: chemical and
procyanidin extract in Fao cells submitted to oxidative stress. physical properties aected by food processing. Critical Reviews in
Journal of Agricultural and Food Chemistry, 52, 10831087. Food Science and Nutrition, 40, 142.
Martinez, C., Yanez, J., Vicente, V., Alcaraz, M., Benavente-Garcia, Siddiqui, I. A., Afaq, F., Adhami, V. M., Ahmad, N., & Mukhtar, H.
O., Castillo, J., Lorente, J., & Lozano, J. A. (2003). Eects of (2004). Antioxidants of the beverage tea in promotion of human
several polyhydroxylated avonoids on the growth of B16F10 health. Antioxidation and Redox Signalling, 6, 571582.
melanoma and Melan-a melanocyte cell lines: inuence of the Srinath Reddy, K., & Katan, M. B. (2004). Diet, nutrition and the
sequential oxidation state of the avonoid skeleton. Melanoma prevention of hypertension and cardiovascular diseases. Public
Research, 13, 39. Health Nutrition, 7, 167186.
 L.-c. Wu et al. / Food Chemistry 95 (2006) 319327 327

Stintzing, F. C., Conrad, J., Klaiber, I., Beifuss, U., & Carle, R. (2004). Willet, W. C. (1995). Diet, Nutrition, and avoidable cancer. Environ-
Structural investigations on betacyanin pigments by LC NMR and mental Health Perspectives, 103, 165170.
2D NMR spectroscopy. Phytochemistry, 65, 415422. Winston, G. W., Regoli, F., Dugas, A. J., Fong, J. H., & Blanchard, K.
Strack, D., Vogt, T., & Schliemann, W. (2003). Recent advances in A. (1998). A rapid gas chromatographic assay for determining
betalain research. Phytochemistry, 62, 247269. oxyradical-scavenging capacity of antioxidants and biological
Strack, D., Steglich, W., & Betalains, Wray, V. (1993). In P. G. uids. Free Radical Biology and Medicine, 24, 480493.
Watermann (Ed.). Methods in Plant Biochemistry (vol. 8, Wolfe, K., Wu, X., & Liu, R. H. (2003). Antioxidant Activity of Apple
pp. 421450). London, UK: Academic Press. Peels. Journal of Agricultural and Food Chemistry, 51, 609614.
van Meeteren, M. E., Hendriks, J. J., Dijkstra, C. D., & van Tol, E. A. Wu, L. C., Chen, Y. C., Ho, J. A., & Yang, C. S. (2003). Inhibitory
(2004). Dietary compounds prevent oxidative damage and nitric Eect of red koji extracts on mushroom tyrosinase. Journal of
oxide production by cells involved in demyelinating disease. Agricultural and Food Chemistry, 51, 42404246.
Biochemical Pharmacology, 67, 967975. Wybraniec, S., Platzner, I., Geresh, S., Gottlieb, H. E., Haimberg, M.,
Voko, Z., Hollander, M., Hofman, A., Koudstaal, P. J., & Breteler, M. Mogilnitzki, M., et al. (2001). Betacyanins from vine cactus
M. (2003). Dietary antioxidants and the risk of ischemic stroke: the Hylocereus polyhizus. Phytochemistry, 58, 12091212.
Rotterdam Study. Neurology, 61, 12731275. Wybraniec, S., & Mizrahi, Y. (2002). Fruit esh betacyanin pigments
Willet, W. C. (1994). Diet and Health: what should we eat. Science, in Hylocereus Cacti. Journal of Agricultural and Food Chemistry,
254, 532537. 50, 60866089.