Food Chemistry 121 (2010) 480–486

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Inhibitory effects of sweet potato leaves on nitric oxide production

and protein nitration
Ming-Hsing Huang a, Heuy-Ling Chu b, Lih-Jeng Juang c, Bor-Sen Wang b,*
a
Department of Cosmetic Science, Chia Nan University of Pharmacy and Science, 60 Erh-Jen Rd., Sec. 1, Tainan, Taiwan, ROC
b
Department of Food Science and Technology, Chia Nan University of Pharmacy and Science, 60 Erh-Jen Rd., Sec. 1, Tainan, Taiwan, ROC
c
Department of Applied Cosmetic Science, Ching Kuo Institute of Management and Health, 336, Fu-Hsing Rd., Keelung, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: The inhibitory effects of a water extract of sweet potato leaves (WSPL) on nitric oxide (NO) production
Received 16 February 2009 and protein tyrosine residue nitration were investigated. The results showed that WSPL inhibited NO pro-
Received in revised form 14 December 2009 duction in a concentration-dependent manner. In the range of 0–1.0 mg/ml, the inhibitory effect on NO
Accepted 22 December 2009
generation in macrophages increased with increasing concentration of WSPL. Meanwhile, the protein
tyrosine residue nitration in mouse heart homogenates was inhibited by 1 mg/ml WSPL. In addition,
WSPL, in the range of 0–0.4 mg/ml, also exhibited radical scavenging, reducing and chelating activities
Keywords:
and protected liposomes against oxidative damage. A high performance liquid chromatography analysis
Sweet potato leaves
Nitric oxide
revealed that phenolic acids and flavonols such as chlorogenic acid, caffeic acid, and quercetin were pres-
Macrophages ent in the WSPL, which could contribute to the protective effect against oxidative damage. Thus, WSPL
Protein nitration might be useful in preventing protein nitration and oxidative stress.
High performance liquid chromatography Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction been given to studies investigating the protective effects provided

Previous studies have indicated that the intracellular oxidative
stresses induced by non-essential reactive oxygen species (ROS)
and reactive nitrogen species (RNS) are capable of damaging bio-
logical molecules, such as proteins, resulting in protein oxidation,
which is regarded as a marker of tissue injury and aging (Valko
et al., 2007). For example, ROS and RNS, including hydroxyl radicals
and excessively produced nitric oxide (NO), have significant effects
in the development of degenerative diseases, especially increasing
the progression of chronic heart failure (Ungvari, Gupte, Recchia,
Batkai, & Pacher, 2005). It is widely recognized that human low-
density lipoprotein (LDL) could be oxidized by transition-metal
ions, myeloperoxidase, and ROS in plasma (Steffen, Wiswedel, Pe-
ter, Schewe, & Sies, 2006). In fact, oxidation of LDL plays a key role
to promote the development of cardiovascular diseases in humans
(Stocker & Keaney, 2004). On the other hand, peroxidase, in the
presence of nitrite and hydrogen peroxide, could result in the
nitration of the apo B-100 protein tyrosine residues and transform
LDL into an atherogenic particle (Steffen et al., 2006). However,
epidemiological reports have revealed that natural dietary antiox-
idants could exhibit protective effects against LDL lipid oxidation
and decrease the crises of coronary artery diseases (Campbell,
Efendy, Smith, & Campbell, 2001). Therefore, much attention has
* Corresponding author. Tel./fax: +886 6 2668618.
E-mail address: wangbs@mail.chna.edu.tw (B.-S. Wang).
by natural dietary ingredients against lipid and protein modifica-
tion in LDL oxidation.
Further, under physiological conditions, massive amounts of
superoxide are produced by different enzymatic metabolism path-
ways (e.g., xanthine oxidase and NADPH oxidase) (Lampe, 1999). It
is well known that intracellular NO is generated by a family of iso-
forms of NO synthase in different cells. For example, macrophages
and neutrophils could generate much NO in response to extracellu-
lar stimulants (e.g. lipopolysaccharide) by expressing an inducible
NO synthase (iNOS) and executing massive NO production. At the
same time, the high concentration of NO could overcome the
endogenous antioxidant system and react with the surrounding
superoxide to yield the more toxic peroxynitrite oxidant. Peroxyni-
trite can then cause extensive damage to proteins, lipids, and DNA
biomolecules (Uppu et al., 2007). Therefore, decreasing NO produc-
tion under physiological conditions is an important goal for inhib-
iting the production of oxidative stresses and preventing the
oxidation of biomolecules.
Phytochemicals-rich vegetables, which have a complex array of
antioxidants, could exhibit protective effects against oxidative
stress in different models. The anti-oxidation mechanisms of these
phytochemicals can be illustrated by four crucial properties,
including the scavenging activity against free radicals, the reducing
activity, the metal ion chelating activity, and the inhibition of oxi-
dative enzymes. As mentioned above, inhibiting the oxidative
stress derived from free radicals is the first line defense to prevent

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.12.068
 M.-H. Huang et al. / Food Chemistry 121 (2010) 480–486
oxidation in biomolecules (e.g. lipids, proteins, and DNA) and to
protect cells against the progression of aging. Further, much atten-
tion has also been paid to the search for natural dietary products
that may inhibit protein nitration or decrease NO production in
activated macrophages (Olmos, Manez, Giner, Redo, & Rios, 2007).
The sweet potato (Ipomoea batatas [L.] Lam.) containing a large
amount of b-carotene and anthocyanin showed the activities of
stabilizing blood sugar and decreasing insulin resistance (Ludvik,
Neuffer, & Pacini, 2004). The sweet potato leaves commonly called
Digua-Yeh is as an important dark green leafy vegetable in Asian
dishes. The sweet potato leaves exhibited radical scavenging activ-
ity, antimutagenicity, anticarcinogenic, antidiabetic activity, and
antibacterial activity (Matsui et al., 2002). Further, Huang, Sheu,
Chen, Chang, and Lin (2007) have indicated that a trypsin inhibitor,
isolated from the sweet potato storage roots, had an efficient abil-
ity to scavenge RNS. However, in spite of the large number of stud-
ies on sweet potato roots, there have been relatively limited
studies focusing on the bioactive constituents in sweet potato
leaves on protein nitration and activated macrophage NO produc-
tion. Consequently, the aim of this study was to investigate the
modulation capacity of sweet potato leaves on NO production
activity, and the protective effects provided against protein nitra-
tion damage in vitro.
2. Materials and methods
2.1. Materials
Lipopolysaccharide (LPS, Escherichia coli 0127:B8), N-(1-naph-
thyl) ethylenediamine dihydrochloride, sulphanilamide, 2,20 -azi-
no-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS),
potassium ferricyanide, ferrozine, 2-thiobarbituric acid (TBA) and
Trolox were purchased from Sigma–Aldrich Chemical Co. (St. Louis,
MO, USA). Lecithin was purchased from TCI AMERICA Co. (Portland,
OR, USA). Ten fresh batches of mature sweet potato leave (Ipomoea
batatas [L.] Lam ‘Tainon 71’) were obtained from five local markets
in Tainan, Taiwan. Sixty grams of sweet potato leaves were sampled
from each batch (60  10 g), and then used for the preparation of
water extract.
2.2. Samples preparation
The sweet potato leaves (600 g) were frozen at 80 °C and then
lyophilized for 48 h. These freeze-dry leaves (70.2 g) were then
ground by the CemotecTM 1090 Sample Mill (FOSS Analytical, Hille-
roed, Denmark). The leave powder (50 g) was extracted with boil-
ing water (1000 ml) for 5 min, and the filtrate was freeze-dried.
The final dehydrated powder (4.3 g) was then dissolved in phos-
phate buffer saline (138 mM NaCl, 2.7 mM KCl, 10 mM phosphate
buffer, pH 7.4). This sample was named as the water extracts of
sweet potato leaves (WSPL).
2.3. Determination of the trolox equivalent antioxidant capacity
(TEAC)
This assay determined the capacity of WSPL to scavenge the
ABTS+ and then compared with the calibration curve of trolox.
The ABTS+ scavenging activity was measured as previously de-
scribed (Arnao, Cano, & Acosta, 2001). The ABTS+ was generated
by reacting 1 mM ABTS with 0.5 mM hydrogen peroxide and
10 units/ml horseradish peroxidase in the dark at 30 °C for 2 h.
After 1 ml ABTS+ was added to WSPL or trolox standards, the
absorbance at 734 nm was recorded after 10 min. The radical scav-
enging capacity was plotted as a function of concentration and the
481
trolox equivalent antioxidant capacity (TEAC) of WSPL was calcu-
lated against a trolox calibration curve.
2.4. Determination of liposome oxidation
A solution containing the lecithin (580 mg) and phosphate buf-
fer (58 ml, 10 mM, pH 7.4) was sonicated by an ultrasonic cleaner
(Branson 8210, Branson ultrasonic Corporation, Danbury, CT, USA)
in an ice-cold water bath for 2 h. The sonicated solution, FeCl3,
ascorbic acid and samples of WSPL (0.2 ml) were mixed to produce
a final mixture with a concentration of 3.12 lM FeCl3 and 125 lM
ascorbic acid. The mixture was incubated at 37 °C for 1 h by the
thiobarbituric acid (TBA) method (Tamura & Shibamoto, 1991).
The absorbance of the sample was read at 532 nm against a blank,
which contained all reagents except lecithin. A lower level of
absorbance indicated a stronger protective activity.
2.5. Determination of reducing activity
The reducing power of WSPL was determined as previously de-
scribed (Oyaizu, 1986). Potassium ferricyanide (2.5 ml, 10 mg/ml)
was added to WSPL in phosphate buffer (2.5 ml, 200 mM, pH 6.6)
and the mixture was incubated at 50 °C for 20 min. Trichloroacetic
acid (2.5 ml, 100 mg/ml) was added to the mixture, which was
then centrifuged at 1000g for 10 min. The supernatant (2.5 ml)
was mixed with distilled water (2.5 ml) and ferric chloride
(0.5 ml, 1.0 mg/ml), and then the absorbance at 700 nm was read.
Higher absorbance of the reaction mixture indicated greater reduc-
ing activity. The reducing activity was plotted as a function of con-
centration and the ascorbic acid equivalent reducing activity was
calculated against an ascorbic acid calibration curve.
2.6. Determination of chelating activity
The chelating activity of WSPL on Fe2+ was measured as previ-
ously described (Carter, 1971). The WSPL thus prepared (0.6 ml)
were reacted with FeCl2 (2 mM, 0.2 ml) and ferrozine (5 mM,
0.2 ml) for 10 min, and the absorbance at 562 nm was determined.
A lower level of absorbance indicated a higher chelating activity.
2.7. Determination of total flavonoid contents
Different concentrations of WSPL solutions were added with
0.1 ml (2-aminoethyl) diphenyl borate (0.2% in ethanol) individu-
ally. After 20 min of incubation, the absorbance at 405 nm was
measured and compared to a rutin calibration curve (Hairi, Salle,
& Andary, 1991).
2.8. Determination of total polyphenolic contents
Total polyphenolics were determined as gallic acid equivalents
(Taga, Miller, & Pratt, 1984). Different concentrations of WSPL solu-
tions were added with 2 ml sodium carbonate (20% (w/v)) individ-
ually to a 10 ml volumetric flask. After 5 min, 0.1 ml Folin–
Ciocalteu reagent (50% (v/v)) was added and the volume were
made up to 10 ml with H2O. After incubation at 30 °C for 1 h, the
absorbance at 750 nm was measured and compared to a gallic acid
calibration curve.
2.9. Determination of nitric oxide (NO) production
The RAW 264.7 cells (ATCC number: TIB-71) were purchased
from Bioresources Collection and Research Center (Shin-chu, Tai-
wan) and cultured in the RPMI-1640 medium containing 10%
heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM pyru-
vate, and maintained in humidified 5% CO2/95% air at 37 °C. Nitrite
482 M.-H. Huang et al. / Food Chemistry 121 (2010) 480–486
levels, which reflect the nitric oxide synthase activity in the cul-
tured media, were determined by Griess reaction. Briefly, cells
were cultured with WSPL with or without LPS (200 ng/ml) for
24 h. Then, the culture medium was mixed with the same volume
of Griess reagent (1% sulphanilamide in 5% phosphoric acid and
0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in water);
absorbance of the mixture at 550 nm was determined by using a
microplate reader (Wang et al., 2006).
2.10. Determination of inducible NO synthase (iNOS) levels
After the RAW 264.7 cells were cultured with WSPL in the pres-
ence of LPS (200 ng/ml) for 24 h, the cells were washed with ice-
cold PBS, and then treated with lysis buffer. Cellular lysates were
centrifuged at 10,000g at 4 °C for 20 min. The supernatants were
collected and the protein contents were determined by using the
bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Each
sample, contained 50 lg protein, was separated on 8% SDS poly-
acrylamide gels. After electrophoresis, the gels were transferred
to nitrocellulose paper. After washing with distilled water, the
membrane was incubated with 5% albumin in PBS (containing
0.1% Tween-20), and then immunoblotted with mouse monoclonal
anti-iNOS antibody (Santa Cruz, CA, USA) and mouse monoclonal
anti-Tubulin antibody (Sigma–Aldrich, St. Louis, MO, USA). The
blots were then incubated with anti-mouse or anti-rabbit IgG anti-
body conjugated to horseradish peroxidase (Santa Cruz, CA, USA)
and visualized using an enhanced chemiluminescence (ECL) kit
(Amersham Bioscience, Piscataway, NJ, USA).
2.11. Determination of protein tyrosine residue nitration levels
The mouse heart homogenates were prepared as previously de-
scribed (Wang et al., 2006). Then, aliquots of mouse heart homog-
enate supernatants (100 ll) were incubated with or without WSPL,
in the presence of horseradish peroxidase (HRP, 500 units/ml), so-
dium nitrite (0.5 mM), and H2O2 (0.2 mM) at 37 °C for 6 h. Reac-
tions were stopped by the addition of SDS sample buffer and the
mixture was heated at 90 °C for 10 min (Sampson, Ye, Rosen, &
Beckman, 1998). Each sample was separated on 10% SDS polyacryl-
amide minigels. After electrophoresis, gels were transferred to
nitrocellulose paper and then immunoblotted with rabbit poly-
clonal anti-nitrotyrosine (Cayman Chemical, Ann Arbor, Michigan,
USA) and mouse monoclonal anti-Tubulin antibody (Sigma–Al-
drich, St. Louis, MO, USA). The blots were then incubated with a
secondary antibody as described above. Then, proteins were de-
tected with an enhanced chemiluminescence (ECL) kit.
2.12. High performance liquid chromatography (HPLC) analysis
HPLC was performed with a Hitachi Liquid Chromatograph (Hit-
achi Ltd., Tokyo, Japan), consisting of two model L-7100 pumps,
and one model L-7455 photodiode array detector (254 nm). Sam-
ple (50 mg/ml) was filtered through a 0.45 lm filter and injected
into the HPLC column. The injection volume was 20 ll and the flow
rate was 0.8 ml/min. The separation temperature was 25 °C. The
column was a Mightysil RP-18 GP (5 lm, 250  4.6 mm I.D.; Kanto
Corporation, Portland, OR, USA). The method involved the use of a
binary gradient with mobile phases containing: (A) phosphoric
acid in water (0.1%, v/v) and (B) H2O/CH3CN (3:7, v/v). The solvent
gradient elution program was as follows: 0–10 min, 100–80% A, 0–
20% B; 10–20 min, 80–70% A, 20–30% B; 20–30 min, 70–55% A, 30–
45% B; 30–40 min, 55–45% A, 45–55% B; 40–45 min, 45–20% A, 55–
80% B; and finally 45–60 min, 20–0% A, 80–100% B.
Table 1
TEAC, total flavonoids, and total polyphenolics of WSPL.
WSPL (mg/ TEAC (lg/ Total flavonoids (lg/ Total polyphenolics
ml) ml) ml) (lg/ml)
0.01 1.19 ± 0.03A 0.25 ± 0.01A 0.27 ± 0.01A
0.02 2.42 ± 0.04B 0.41 ± 0.01B 0.59 ± 0.02B
0.04 4.91 ± 0.11C 0.91 ± 0.02C 1.15 ± 0.02C
TEAC is the mg/ml solution of the sample having the antioxidant activity equivalent
to the lg/ml concentration of a Trolox solution under investigation. Results are
mean ± SD for n = 3. Values with different superscripts in a column are significantly
different (p < 0.05).
2.13. Statistical analysis
Statistical analysis adopted in this study involved the use of the
StatView statistical package (SAS institute Inc.). Analysis of vari-
ance was performed by ANOVA procedures. Significant differences
between means were determined by Duncan’s multiple range tests
at a level of P < 0.05.
3. Results
3.1. Free radical scavenging effect
Table 1 shows TEAC, total flavonoids, and total polyphenolics of
the WSPL. In the range of 0.01–0.04 mg/ml, the TEAC of the WSPL
against ABTS radicals was equal to that of trolox in the range of
1.19–4.91 lg/ml. The WSPL exhibited a concentration-dependent
increase in the effect of free radical scavenging. The total flavo-
noids and total polyphenolics of natural products were regular in-
dexes of their antioxidant activity. Thus we determine the total
flavonoids and total polyphenolics of WSPL. For the other columns
of Table 1, the contents of total flavonoids and total polyphenolics
in the WSPL were expressed in rutin and gallic acid equivalents,
respectively. The results showed that the WSPL at 0.04 mg dry ex-
tract/ml contained the amounts of total flavonoids and total poly-
phenolics, respectively equal to those of 0.91 lg rutin/ml and
1.15 lg gallic acid/ml. In other words, the levels of total flavonoids
and total polyphenolics of WSPL were 22.75 and 28.75 mg/g ex-
tract, respectively. Further, the correlation between the TEAC and
total flavonoids or total polyphenolics was determined by linear
regression analysis. While a positive correlation (r2 = 0.991) be-
tween the TEAC and the total flavonoids of WSPL was found, the
TEAC correlated even better (r2 = 0.998) with the total polypheno-
lics of the WSPL. This implied that the radical scavenging effects of
the WSPL might be attributed to the total flavonoids and total poly-
phenolic constituents of the WSPL.
3.2. HPLC chromatograph of WSPL
The free radical scavenging activities of sweet potato leaves were
contributed to various natural polyphenolics including chlorogenic
acid, caffeic acid, and quercetin (Chu, Chang, & Hsu, 2000; Islam,
2006). Thus chlorogenic acid, caffeic acid, and quercetin were se-
lected as three marker compounds for HPLC chromatographic finger-
print analysis of WSPL, and the chromatogram was shown as in Fig. 1.
Based on the plots of the peak-area (y) vs. concentration (x, lg/ml),
the regression equations of the three phenolic constituents and their
correlation coefficients (r2) were as follows: chlorogenic acid,
y = 0.0066x + 0.0209 (r2 = 0.998); caffeic acid, y = 0.0118x + 0.0307
(r2 = 0.996); and quercetin, y = 0.0245x + 0.0292 (r2 = 0.998). In a
comparison of the amounts of the three polyphenolic compounds
present in the WSPL, chlorogenic acid (11.5 mg/g extract) > caffeic
acid (5.1 mg/g extract) > quercetin (0.3 mg/g extract). On the other
 M.-H. Huang et al. / Food Chemistry 121 (2010) 480–486
Fig. 1. HPLC chromatogram of WSPL.
hand, in Fig. 1, three main peaks from unknown compounds A, B and
C with retention time at 32.8 min, 33.2 min and 34.7 min, respec-
tively, were observed. By ultraviolet–visible spectra analysis, com-
pounds A, B and C showed the absorbance maximum wavelength
at 217, 242 and 323 nm; 219, 242 and 325 nm; 217, 242 and
325 nm, respectively. Peak A, B and C could be recognized as charac-
teristic peaks of WSPL on the basis of their retention time and UV
spectra. However, the HPLC analyses showed that chlorogenic acid,
caffeic acid, and quercetin, which are known with bioactive actions,
were found in WSPL.
3.3. Protective effect of WSPL on liposome oxidation
Table 2 shows the liposome protection, chelating activity, and
reducing power of the WSPL. Lipid peroxidation is a harmful
process, producing toxic aldehyde and promoting cellular patho-
logical metabolism. In this study, liposome protection was used
as an index to assay the protective action of the WSPL on lipid
oxidation. WSPL in the range of 0.1–0.4 mg/ml exhibited a con-
centration-dependent inhibitory effect, 42.22–83.36%, on the
liposome oxidation induced by the Fe3+/H2O2 system. Mean-
while, the WSPL at 0.4 mg/ml inhibited liposome oxidation by
83.36%, which indicated the WSPL, could be an effective protec-
tor in preventing lipid peroxidation in vitro. The chelating activ-
ity of natural products could also serve as an important
inhibition factor in oxidation processes; thus the metal ions che-
lating capacity of WSPL was determined. As shown in Table 2,
WSPL in the range of 0.1–0.4 mg/ml exhibited a 4.66–14.52%
chelating activity on ferrous ions.
Table 2
The liposome protection, chelating activity, and reducing power of WSPL.
WSPL (mg/ Liposome Chelating Reducing power
ml) protection (%) activity (%) (lg/ml)
0.1 42.22 ± 0.97A 4.66 ± 0.21A 16.19 ± 0.51A
0.2 58.66 ± 1.57B 6.72 ± 0.34B 31.26 ± 1.28B
0.4 83.36 ± 2.75C 14.52 ± 0.72C 55.39 ± 1.82C
Reducing power is the mg/ml solution of the sample having the reducing activity
equivalent to the lg/ml concentration of an ascorbic acid solution under investi-
gation. Results are mean ± SD for n = 3. Values with different superscripts in a
column are significantly different (p < 0.05).
483
Reducing activity of natural products, which can be regarded
as a hydrogen donating capacity, can usually achieved by termi-
nating the radicals’ chain reaction and thus minimizing their
damage. The results on WSPL on this effect were compared to
that of ascorbic acid. As shown in Table 2, the reducing capacity
of WSPL was also concentration-dependent and at 0.4 mg/ml was
as high as 55.39 lg/ml of ascorbic acid. Overall speaking, there
was a positive linear correlation (r2 = 0.999) between liposome
protection and the reducing activity of the WSPL. Meanwhile
the metal ion chelating activity also correlated well (r2 = 0.959)
with the liposome protection of WSPL.
3.4. Inhibitory effect on NO production and protein nitration
The NO scavenging activity of the WSPL was detected by using
the LPS activated macrophage system to produce NO radicals, and
then measured as nitrite in a cell culture medium by Griess reac-
tion. As shown in Fig. 2, the WSPL, in the range of 0.1–1.0 mg/ml,
reduced the NO production of activated macrophages in a dose-
dependent manner. In the presence of 0.4 and 1.0 mg/ml WSPL,
the levels of NO in medium were decreased to 42.81% and 8.11%,
respectively, of that observed in LPS (200 ng/ml) alone. This sug-
gests that WSPL could be a potential NO scavenger. In addition,
no cell toxicity was observed in the presence of the WSPL, as mea-
sured by the MTT cell viability test. The results unambiguously
indicated that the WSPL could reduce RNS generation in LPS stim-
ulated macrophages.
On the other hand, the effects of the WSPL on iNOS protein lev-
els of macrophages were examined by immunoblot. As shown in
Fig. 3, with the WSPL at 0.4 and 1.0 mg/ml, the level of 200 ng/
ml LPS induced iNOS production was reduced by 33.31% and
98.17%, respectively, as compared with LPS alone. This result im-
plied that the WSPL could play an inhibitory role on RNS produc-
LPS (200 ng/ml)
100
80
NO production (%)
60
40
20
0
0.0 0.1 0.2 0.4 1.0
WSPL (mg/ml)
Fig. 2. Effects of WSPL on nitric oxide production in lipopolysaccharide stimulated
macrophages. The data were displayed with mean ± S.D. of three individual
experiments (p < 0.05).
484 M.-H. Huang et al. / Food Chemistry 121 (2010) 480–486

WSPL (mg/ml) 0 0 0.1 0.2 0.4 1 WSPL (mg/ml) 0 0 0 0.1 0.2 0.4 1
LPS (200ng/ml) - + + + + +
Trolox (mg/ml) - - 1 - - - -
-
iNOS peroxidase+NO 2+H2O2 - + + + + + +
130
Tubulin
100

C 70
C
100 C

55
(% of LPS group)

80 B
iNOS level

60 MW 35
(kDa)
40

20 E
A A
100
0

Protein nitration level (%)

1 2 3 4 5 6
Lane
80
Fig. 3. Effects of WSPL on levels of iNOS and tubulin protein in lipopolysaccharide
stimulated macrophages. Each bar in chart was displayed with mean ± SD (n = 3),
60 D
and marked by different letters with levels of significant difference (p < 0.05). D

tion in a cellular model by decreasing iNOS production in stimu- 40

lated macrophages.
Furthermore, the protective effect of the WSPL against protein
tyrosine residue nitration was performed by the model of HRP/
hydrogen peroxide/nitrite system. As shown in Fig. 4, the band sig-
nal for the nitrotyrosine levels of the positive group (lane 2) was
obviously increased at 35–130 kDa protein when compared with
the control group (lane 1). In particular, in the presence of 0.2,
0.4 and 1.0 mg/ml WSPL (lanes 5–7), this protective effect showed
a strong concentration-dependence, with inhibition rate of 51.62%,
85.34% and 95.41%, respectively. When compared to the reference
sample of trolox (a water soluble derivative of vitamin E, at 1 mg/
ml to show an 86.89% inhibitory effect, lane 3), the effect of the
WSPL obviously overwhelmed that of trolox.
4. Discussion
Previous studies revealed that a methanolic extract of sweet po-
tato leaves exhibited scavenging effects against radicals (Chu et al.,
2000). The levels of polyphenolics such as caffeic and chlorogenic
acids in sweet potato leaves are superior to other vegetables (Is-
lam, 2006). In this study, WSPL also exhibited the scavenging activ-
ity against ABTS radicals in a concentration-dependent manner
(Table 1). These results indicated that the bioactive constituents
of the WSPL could scavenge various species of harmful radicals.
Therefore, the WSPL could be used as a natural protector against
oxidative stress in different biological systems.
Three polyphenolics including chlorogenic acid, caffeic acid and
quercetin were detected in WSPL. Other three unknown com-
pounds A, B and C which might contribute to the antioxidant activ-
ity of WSPL (Fig. 1) have to be identified in our future study.
However, the concentrations of chlorogenic acid (11.5 mg/g ex-
tract), caffeic acid (5.1 mg/g extract), quercetin (0.3 mg/g extract),
total flavonoids (22.75 mg/g extract) and total polyphenolics
(28.75 mg/g extract) in the WSPL were determined. And, WSPL also
exhibited the inhibitory effects on NO production and protein
nitration. Therefore, the antioxidant effects of the WSPL could be
attributed to these bioactive polyphenolic constituents present.
When lipid peroxidation occurs in cell membranes, it releases
arachidonic acid, which is responsible for long term oxidative
20 BC C
A AB
0
1 2 3 4 5 6 7
Lane
Fig. 4. Effects of WSPL on levels of protein tyrosine residue nitration in mouse heart
homogenate. Each bar in chart was displayed with mean ± SD of (n = 3), and marked
by different letters with levels of significant difference (p < 0.05).
stress to cells. In this study, WSPL showed a protective activity
against the lipid damage caused by the hydroxyl radicals produced
from a Fenton-like reaction (Table 2). In fact, 4-hydroxy-2-nonenal
(HNE), a major lipid peroxidation product, can bind covalently to
cellular DNA to form the exocyclin etheno-DNA-base adducts
(Nair, De Flora, Izzotti, & Bartsch, 2007), whereas chlorogenic acid
prevented the loss of total GSH and the accumulation of malondi-
aldehyde (MDA) in hepatocytes cultured with glucose oxidase
(Miccadei et al., 2008). In the current study, the WSPL showed an
obvious inhibitory activity on thiobarbituric acid reactive sub-
stances (TBARS) production, indicating that WSPL could protect
lipids against oxidative damage in tissues.
It has been suggested that an antioxidant capacity is due to the
development of a reducing ability when reacting with free radicals
and terminating the radical chain reaction (Huang, Ou, & Prior,
2005). Previous reports have revealed that a methanolic extract
of sweet potato leaves exhibited a reducing ability (Chu et al.,
2000). As shown in Table 2, we also found that the WSPL exhibited
a remarkable reducing ability. In fact, multifarious evidence has
indicated that the reducing effects of polyphenolic compounds
could play a critical role not only in radical inhibition but also in
the prevention of biofilm formation (Palikova et al., 2008). For
example, the phenolic fraction of blue honeysuckle inhibited rat li-
ver microsome peroxidation and microorganisms adhesion to the
artificial surface (Palikova et al., 2008). On the other hand, other
studies have supported the critical role of metal ion chelation in
protecting against oxidation. As expected, the WSPL showed a che-
lating effect on ferrous ions (Table 2). Iron ions not only take part in
 M.-H. Huang et al. / Food Chemistry 121 (2010) 480–486
the electron transfer processes of superoxide production, but also
lead to the formation of hydroxyl radicals by the Fenton reaction.
Yet, polyphenolic compounds, such as flavonoids, could form com-
plexes with metal ions. The antioxidant activities of flavonoids
have been reported to mainly depend on the number and configu-
ration of hydroxyl groups in the B-ring for inhibiting the produc-
tion of free radicals (Heim, Tagliaferro, & Bobilya, 2002).
Chelating may be formed between the 5-OH and 4-oxo groups,
or between the 30 -OH and 40 -OH groups of flavonoids and the metal
ions (Heim et al., 2002). Therefore, the ability of the flavonoids pre-
sented in the WSPL to inhibit hydroxyl radicals and metal ions
could also involve these mechanisms. Meanwhile, it was found
that lipid protection effects of the WSPL had a better correlation
with its reducing activity (r2 = 0.999) than its chelating activity
(r2 = 0.959). This implies that the reducing activity of the WSPL
might play a more important role than its chelating action in its li-
pid protection effects.
A burst of NO would interact with cellular molecules and accel-
erate the progression of illness. The overproduction of NO could in-
duce DNA damage, as well as cytotoxicity. Besides, a direct
suppressive activity of chlorogenic and caffeic acids on nitrosamine
formation has been demonstrated in a previous study (Kono, Shi-
bata, Kodama, & Sawa, 1995). Further, the markedly increased pro-
duction of nitrites by stimulation of J774 cells with LPS was
inhibited by the anthocyanin fraction of a blackberry extract (Per-
gola, Rossi, Dugo, Cuzzocrea, & Sautebin, 2006). The results shown
in Fig. 2, demonstrated that the WSPL inhibited NO generation in
LPS stimulated macrophages. Moreover, as shown in Fig. 3, the
WSPL clearly showed inhibitory effects on iNOS protein production
in LPS stimulated macrophages as revealed by immunoblotting.
Therefore, the inhibitory effect of the WSPL on NO production
could have contributed to its inhibition to iNOS protein expression.
Hence, the WSPL, which contained different polyphenolic compo-
nents, could be a considerable NO scavenger to protect cells from
RNS toxicity.
The mechanisms of these protective actions including: attenu-
ating iNOS protein expression and decreasing iNOS activity. The
polyphenolics present in the WSPL might form a complex with
proteins through hydrogen and covalent bonds causing enzyme
inhibition (Van Sumere, Albrecht, Denonder, DePooter, & Pe,
1975). Thus, the affinity or complex between the WSPL and iNOS
may lead to the inhibitory effect on iNOS activity by the WSPL.
Consequently, WSPL decreased NO production in stimulated mac-
rophages could result from modulating the expression of iNOS pro-
tein. However, the fact that the WSPL could scavenge superoxide
and nitric oxide is significantly important for consumer health.
To decrease production of both superoxide and NO, leads to the
prevention of peroxynitrite formation, which has been proposed
as a protection for tissue from a variety of oxidative damage.
Nitrotyrosine is a specific marker for protein nitration and tis-
sue injury by RNS in vivo. Previous studies have indicated that ni-
trite, a major end-product of NO metabolism, readily promotes
tyrosine nitration through the formation of nitryl chloride (NO2Cl)
and nitrogen dioxide (NO2) by a reaction with the inflammatory
mediators such as hydrogen peroxide and myeloperoxidase (Samp-
son et al., 1998). Therefore, the peroxidase/H2O2/nitrite pathway
could be an important source of RNS to produce the nitration of
tyrosine residue in a complex biological system. However, in the
pathogenesis of numerous diseases, including neurodegenerative
diseases, inflammation and cardiovascular pathologies, protein
nitration has been found to play a critical role (Uppu et al.,
2007). In the current study, the WSPL showed remarkable inhibi-
tory effects on the peroxidase/H2O2/nitrite induced nitration of
mouse heart homogenate. In fact, chlorogenic acid and caffeic acid
could exhibit powerful prevention to inhibit the peroxynitrite-dri-
ven oxidation reaction (Boveris, Valdez, & Alvarez, 2002). These
485
data implied that the WSPL and its active constituents could be
excellent natural scavengers of RNS and could protect cells against
the nitration of protein tyrosine residues. The results implied that
the WSPL also could have a protective effect on tissues because it
reduced RNS formation.
In conclusion, WSPL, with its flavonoids and polyphenolics,
which were good oxidative inhibitors, could exert significant anti-
oxidative effects and anti-NO production activity in a cellular sys-
tem. Further, the WSPL also exhibited inhibitory effects on protein
tyrosine residue nitration in mouse heart homogenate. Based on
the results of these experiments, the WSPL had the potential of
scavenging free radicals and inhibiting lipid oxidation. The protec-
tion effects provided by the WSPL against protein oxidation may be
attributed to its free radical scavenging and reducing activities.
These activities were closely correlated with its flavonoid and poly-
phenolic constituents, though other unknown components (e.g.
compounds A, B and C) in the WSPL could also play important roles
in its protective effects. However, these results revealed the possi-
ble protective roles of the WSPL against oxidative damage and
aging diseases, further investigations on the nutritional and phys-
iological effects of sweet potato leaves are still intensively
required.
Acknowledgement
This work was supported by a research grant from the National
Science Council of the Republic of China (NSC 97-2313-B-041-003).
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