Antioxidant Effects of Herbal Tea Leaves from

Yacon (Smallanthus sonchifolius) on Multiple

Free Radical and Reducing Power Assays,
Especially on Different Superoxide Anion
C: Food Chemistry

Radical Generation Systems

Shintaro Sugahara, Yuto Ueda, Kumiko Fukuhara, Yuki Kamamuta, Yasushi Matsuda, Tatsuro Murata, Yasuhiro Kuroda,
Kiyotaka Kabata, Masateru Ono, Keiji Igoshi, and Shin Yasuda

Abstract: Yacon (Smallanthus sonchifolius), a native Andean plant, has been cultivated as a crop and locally used as a
traditional folk medicine for the people suffering from diabetes and digestive/renal disorders. However, the medicinal
properties of this plant and its processed foods have not been completely established. This study investigates the potent
antioxidative effects of herbal tea leaves from yacon in different free radical models and a ferric reducing model. A
hot-water extract exhibited the highest yield of total polyphenol and scavenging effect on 1,1-diphenyl-2-picryl hydrazyl
(DPPH) radical among four extracts prepared with hot water, methanol, ethanol, and ethylacetate. In addition, a higher
reducing power of the hot-water extract was similarly demonstrated among these extracts. Varying concentrations of
the hot-water extract resulted in different scavenging activities in four synthetic free radical models: DPPH radical
(EC50 28.1 μg/mL), 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (EC50 23.7 μg/mL), galvinoxyl
radical (EC50 3.06 μg/mL), and chlorpromazine cation radical (EC50 475 μg/mL). The yacon tea-leaf extract further
demonstrated superoxide anion (O2 − ) radical scavenging effects in the phenazine methosulfate-NADH-nitroblue
tetrazolium (EC50 64.5 μg/mL) and xanthine oxidase assay systems (EC50 20.7 μg/mL). Subsequently, incubating
human neutrophilic cells in the presence of the tea-leaf extract could suppress the cellular O2 − radical generation
(IC50 65.7 μg/mL) in a phorbol 12-myristate 13-acetate-activated cell model. These results support yacon tea leaves
may be a good source of natural antioxidants for preventing O2 − radical-mediated disorders.

Keywords: antioxidant, herbal tea, Smallanthus sonchifolius, superoxide anion radical, yacon

Practical Application: Yacon has been considered to be a potent alternative food source for patients who require a
dietary cure in regional area, while the leaf part has been provided and consumed as an herbal tea in local markets. We
demonstrated here potent antioxidative effects of the tea leaves from yacon in different free radical assays, reducing power
assay, and cellular superoxide anion radical generation assay. Results support yacon tea leaves may be a good source of
natural antioxidants for preventing O2 − radical-mediated disorders.

Introduction yacon has been provided and consumed in local markets. In recent
Yacon (Smallanthus sonchifolius) is a perennial plant originally evidences, yacon has been believed as a potent alternative food
cultivated in the Andean highlands of South America. Over the source for patients who require a dietary cure in regional area
past decades, yacon has been introduced into other places includ- (Delgado and others 2013). It is also used as a folk medicine for
ing Asia (Japan, South-Korea, Taiwan, Hainan, and Philippines), the people suffering from diabetes and digestive/renal disorders.
Oceania (New Zealand), and Europe (Czech Republic) (Ojansivu It is possibly due to its rich in indigestible fructo-oligosaccharides
and others 2011). The root part of this plant is sweet potato-like and beneficial polyphenols (Campos and others 2012). Studies of
shape and it has been historically taken as a fruit/vegetable, and the yacon root further disclose the antioxidant activity (Yan and
is currently also available as processed foods, such as syrup, juice others 1999), the anti-diabetic effect in rats (Oliveira and others
and marmalade in South America (Ojansivu and others 2011; 2013), the anti-obesity in humans (Genta and others 2009) and
Delgado and others 2013). As an herbal tea, the leaf part of the hypolipidemic effect on diabetic rats (Habib and others 2011).
In addition, the leaf part possesses antifungal activity (Lin and
others 2003), antioxidant activity (Valentova and others 2005) and
MS 20150511 Submitted 3/25/2015, Accepted 8/24/2015. Authors Sugahara, hypoglycemic effect in normal and diabetic rat models (Aybar and
Ueda, Fukuhara, Kamamuta, Matsuda, Murata, Kabata, Ono, Igoshi, and Yasuda others 2001). However, the mechanisms underlying the functional
are with School of Agriculture, Tokai Univ, Kawayo, Minamiaso, Aso, Kumamoto properties of this plant and even its processed foods remain to be
869–1404, Japan. Author Kuroda is with School of Engineering, Tokai Univ, 4-1-1
Kita-Kaname, Hiratsuka, Kanagawa 259–1292, Japan. Direct inquiries to author completely established.
Yasuda (E-mail: [email protected]). Since the effect of free radicals in biological systems was first
proposed (Harman 1956), there has been a considerable interests

C 2015 Institute of Food Technologists

 R

C2420 Journal of Food Science r Vol. 80, Nr. 11, 2015 doi: 10.1111/1750-3841.13092
Further reproduction without permission is prohibited
Yacon tea leaves suppress O2 − radical . . .

17503841, 2015, 11, Downloaded from https://ift.onlinelibrary.wiley.com/doi/10.1111/1750-3841.13092 by UEFS - Universidade Estadual de Feira de Santana, Wiley Online Library on [19/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
in examining whether antioxidant(s) from natural products and/or ide (DMSO), ABTS, DPPH, Folin-Ciocalteu phenol reagent,
sources can regulate oxidative stresses and reactions. Exaggerated iron(Ⅲ) chloride, NBT, β-nicotinamide adenine dinucleotide
oxidative stress and reactive oxygen species (ROS), for example, disodium salt (reduced form) (NADH), PMS, potassium hexa-
superoxide anion (O2 − ) radical, hydroxyl (OH) radical, perhy- cyanoferrate(Ⅲ), potassium peroxodisulfate, trichloroacetic acid
droxyl radical, hydrogen peroxide (H2 O2 ), and singlet oxygen, (TCA), xanthine, 12.5 U/mL XOD from buttermilk were pur-
have been implicated to exert a deleterious effect on normal tissue chased from Nacalai Tesque Inc. (Kyoto, Japan). Galvinoxyl and
functions, thereby leading to pathological conditions (Gutowski CPZ were products from Tokyo Chemical Industry Co., Ltd.

C: Food Chemistry
and Kowalczyk 2013) such as atherosclerosis (Droge 2002), cancer (Tokyo, Japan). Potassium dichromate (chromium(VI)), RPMI-
(Hail and others 2008), diabetes (Sabu and Kuttan 2002), neurode- 1640 medium, Hank’s balanced salt solution (HBSS), cytochrome
generation (Obata 2002; Przedborski and Ischiropoulos 2005) and c from horse heart prepared using TCA, and phorbol 12-myristate
liver cirrhosis (Bruck and others 2001), as well as aging process 13-acetate (PMA) were from Wako Pure Chemical Industries,
(Weisburger 2002). One of the important oxygen free radicals is Ltd. (Osaka, Japan). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-
the O2 − radical, which can easily form H2 O2 and highly reactive disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) was
OH radical in the presence of metal ions (Valko and others 2005). obtained from Dojindo Labs (Kumamoto, Japan). The HL-60 hu-
In the body, O2 − radical can be generated during mitochondrial man promyelocytic leukemia cells (JCRB00085) were obtained
respiration (Wiswedel and others 1998), and by NAD(P)H oxi- from Japanese Collection of Research Bioresources (Tokyo, Japan).
dase (Pagano and others 1995), cyclooxygenase and lipoxygenase Fetal bovine serum (FBS) was purchased from Biowest (Nuaille,
(Kukreja and others 1986), nitric oxide synthase (Cosentino and France). All other chemicals were of the highest grade commer-
others 1989), and cytochrome P450 (Fleming and others 2001). cially available.
Xanthine oxidase (XOD) is a key enzyme that catalyzes the oxi-
dation of hypoxanthine and xanthine to uric acid in concomitant Preparation of the tea-leaf extracts
with the generation of O2 − radical (Nishino and others 2008; Four extracts from yacon tea leaves were prepared by dif-
Cantu-Medellin and Kelley 2013). The excess amount of these ferent solvent extractions as described previously (Oliveira and
O2 − radical generated by this enzyme has been implicated in others 2009) with some modifications. Briefly, approximately 5 g
a wide variety of pathophysiological processes, such as diabetes, of crashed tea leaves (dried weight) was soaked in 250 mL of
ischemia-reperfusion injury and chronic heart failure (Pacher and MilliQ water in a conical beaker at 90 to 100 °C for 45 min.
others 2006). A previous study shows that free radical production The tea-leaf mixture was then centrifuged and filtrated. The hot-
through the higher activity of XOD is involved in type 1 dia- water extract was obtained from the filtrate after lyophilization,
betes (Desco and others 2002). Therefore, the suppression on the and reconstituted in water for following bioassays. For methanol,
XOD-mediated O2 − radical generation can be considered to be ethanol, and ethylacetae extracts, approximately 2.5 g of crashed
a strategy for screening compound(s) to prevent these disease risks tea leaves was soaked in 41.67 mL of individual solvent in a cen-
(Kim and others 2004; Pacher and others 2006; Connor 2009). trifuge tube at room temperature for 10 min with a continuous
In this study, we report potential antioxidative properties of shaking. After a brief centrifugation, the supernatant was placed
extracts prepared from commercial yacon tea leaves in different into another conical beaker. The tea leaves therein was totally ex-
free radical models. Four representative synthetic free radicals, tracted three times with the same amount of solvent, and filtrated
including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (Blois together. After vacuum evaporation, the each extract was obtained
1958), 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and reconstituted in DMSO for following bioassays.
cation (ABTS+ ) radical (Thaipong and others 2006), galvinoxyl
radical (Shi and others 2001), and chlorpromazine cation (CPZ+ )
radical (Nagaraja and others 2014), were used as valid and useful Determination of total polyphenol content
models for the discovery of antioxidative capacity of foodstuffs. In The amount of total polyphenol was determined by an es-
parallel, a reducing power assay using potassium hexacyanoferrate tablished procedure (Singleton and Rossi 1965). Briefly, the
was performed. This method has been developed to measure mixture containing test samples (25 μL) and 10-times diluted
the antioxidant capacity of various food materials with a ferric Folin-Ciocalteu phenol reagent solution (125 μL) was kept with
ion-mediated redox mechanism (Oyaizu 1986). A hot-water 10% sodium carbonate solution (125 μL) for 10 min at room
extract of yacon tea leaves was evaluated in both O2 − radical temperature. The assay mixture was allowed to a colorimetric
generation systems; phenazine methosulfate (PMS)-NADH- measurement at 600 nm using a grating microplate reader (SH-
nitroblue tetrazolium (NBT) and XOD enzymatic assays. To gain 1000Lab, Corona Electric, Ibaraki, Japan). Chlorogenic acid was
insight into the physiological relevance of the tea-leaf extract used as the standard sample for a calibration curve. In case the
with its potent O2 − radical suppressing effects, activated human photometric absorption of samples/reagents may interfere with
granulocytic neutrophil cells were incubated in the presence of the the data, a parallel experiment as background at each point was
extract. carried out.

Materials and Methods DPPH radical scavenging assay

The DPPH radical scavenging activity was measured based on
Materials the following method (Blois 1958). Briefly, the reaction was started
A yacon ‘Sarada otome (SY201)’ has been registrated as a by the addition of 0.5 mM DPPH dissolved in ethanol (50 μL)
cultivar in Japan (Sugiura and others 2007). A dried herbal into the assay mixture (200 μL) containing varying concentrations
tea leaves from this yacon, which were locally cultivated, pro- of test samples (10 μL), 70% ethanol (90 μL), and 0.1 M sodium
cessed, and marketed in Kikuchi area (Kumamoto, Japan), were acetate buffer (pH 5.5, 100 μL), then allowed to proceed for
obtained in March 2011 in a retail store. Dimethyl sulfox- 30 min at room temperature. The absorbance of the resulting

Vol. 80, Nr. 11, 2015 r Journal of Food Science C2421

 Yacon tea leaves suppress O2 − radical . . .

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solution was measured at 517 nm. Trolox was used as the standard 250 mM potassium phosphate buffer (pH 7.4, 40 μL) and water
sample. (90 μL), then allowed to proceed for 10 min at room temperature.
The absorbance of the resulting solution was measured at 570 nm.
ABTS+ radical scavenging assay Trolox was used as the standard sample.
+
The ABTS radical scavenging activity was measured based
on the following method (Thaipong and others 2006). Briefly, Determination of O2 − radical generated in XOD assay
ABTS-mixture solution was first prepared by mixing in equal The amount of O2 − radical enzymatically generated by XOD,
C: Food Chemistry

amount of 7.4 mM ABTS and 2.6 mM potassium peroxodisulfate was performed based on the measurement of formation of WST-1
solutions for 15-h rotation in the dark at room temperature. formazan during the enzymatic reaction (Ukeda and others 1999).
Thereafter, ABTS-working solution was prepared after dilution The standard assay mixture, in a final volume of 200 μL, contained
of the ABTS-mixture solution (150 μL) in methanol (2.9 mL) the sample solution (10 μL) at varying concentrations, 2.8 mM
before use. The reaction was started by the addition of the WST-1 (8 μL), 2.5 mM xanthine (10 μL), 280 mM potassium
ABTS-working solution (190 μL) into varying concentrations of phosphate buffer (pH 7.4, 40 μL), water (92 μL), and 0.05 U/mL
test samples (10 μL), then allowed to proceed at room temperature XOD (40 μL). The reaction was started by the addition of the
for 2 h in the dark. The absorbance of the resulting solution was enzyme, allowed to proceed at 25 °C for 0 min and 15 min in a 96-
measured at 734 nm. Trolox was used as the standard sample. well multiplate. The assay mixture was allowed to a colorimetric
measurement of O.D. at 450 nm. The amount of end product,
Galvinoxyl radical scavenging assay uric acid, enzymatically generated by XOD, was next monitored
The galvinoxyl radical scavenging activity was measured based for measurement of the enzyme activity. The enzymatic assays
on the following method (Shi and others 2001). Briefly, the were similarly performed under the replacement of WST-1 with
reaction was started by the addition of galvinoxyl assay mixture water. The assay mixture was allowed to a spectrophotometric
(380 μL) containing 0.0167 mM galvinoxyl in ethanol (228 μL) measurement of O.D. at 290 nm as following (Nguyen and
plus 0.01 M citrate buffer (pH 6.0, 152 μL) into the varying con- others 2004; Wang and others 2008). Trolox and allopurinol were
centrations of test samples (20 μL), then allowed to proceed for used as the standard antioxidant and XOD inhibitor, respectively.
10 min at room temperature. The absorbance of the resulting
solution was measured at 432 nm. Trolox was used as the Cellular O2 − radical generation assay
standard sample. In case the photometric absorption of samples or The HL-60 human promyelocytic leukemia cells were routinely
nonspecific residual color of the reagent interferes with the data, a maintained under a 5% CO2 atmosphere at 37 °C in RPMI-1640
parallel experiment as background at each point was carried out. medium supplemented with 5% FBS, 100 U/mL penicillin G, and
100 μg/mL streptomycin sulfate. For the cellular O2 − radical pro-
CPZ+ radical scavenging assay duction assay, DMSO-differentiated HL-60 human granulocyte-
The CPZ+ radical scavenging activity was measured based on like neutrophil cells were prepared as reported previously
the following method (Nagaraja and others 2014). Briefly, the (Nakamura and others 1998; Kim and others 2002). Cells seeded
reaction was started by the addition of 0.5 mM chromium(VI) so- in a culture dish at a density of 4×105 cells/mL in 10 mL were
lution (10 μL) into the assay mixture (240 μL) containing varying cultured in the presence of 1.25% DMSO for 6 d to undergo gran-
concentrations of test samples (12.5 μL), 10 mM CPZ dissolved ulocytic differentiation. After washing with HBSS, the viable cell
in ethanol (20 μL), 1:1 phosphoric acid plus ethanol mixture number was counted by trypan blue dye exclusion assay. There-
(200 μL), and ethanol (7.5 μL), then allowed to proceed for 10 after, the differentiated cells were prepared and suspended in HBSS
min at room temperature. The absorbance of the resulting solution at a density of 1×106 cells/mL. The cell suspension (250 μL) was
was measured at 530 nm. Trolox was used as the standard sample. preincubated in the presence of test sample (1.25 μL) in a 1.5 mL
test tube at 37 °C for 15 min. A mixture (13.75 μL) contained
Reducing power assay 20 μM PMA (1.25 μL) in DMSO to induce cellular O2 − radi-
Reducing power was measured by the following method (Oy- cal production and 20 mg/mL cytochrome c solution (12.5 μL)
−
aizu 1986), based on the reduction of ferrous ion from Fe3+ to in phosphate buffered saline to colorimetrically detect the O2
2+
Fe . Briefly, the reaction was started by the addition of 1% potas- radical. This mixture was added to the cell suspension and then
sium hexacyanoferrate(Ⅲ) (20.9 μL) into the assay mixture (29.1 incubated at 37 °C for another 15 min. The cells were immedi-
−
μL) containing varying concentrations of test samples (2.5 μL), ately placed on ice-cold water for 5 min to terminate O2 radical
MilliQ water (2.5 μL), and 0.2 M phosphate buffer (pH 6.6, 24.15 production, and centrifuged at 13000 xg for 1 to 2 min to collect
−
μL), then allowed to proceed for 20 min at 50 °C. Thereafter, 10% the supernatant. The level of O2 radical generated therein was
TCA (20.85 μL) and 0.05% iron(Ⅲ) chloride (141.7 μL) were determined colorimetric change of cytochrome c at 550 nm. In
added into the reaction mixture. The absorbance of the resulting parallel, cytotoxicity of the test sample was determined by trypan
solution was measured at 700 nm. Increased absorbance indicates blue dye exclusion assay.
the increase of reducing capability.
Miscellaneous method
O2 − radical scavenging assay in PMS-NADH-NBT system For the trypan blue dye exclusion assay, cell suspension (10 μL)
The O2 − radical scavenging activity was measured by us- was mixed with the equal amount of 0.4% trypan blue solution
ing PMS-NADH-NBT system according to previously described (Gibco-Invitrogen, Carlsbad, Calif., U.S.A.). After a 3-min incu-
methods (Gulcin 2006; Wang and others 2008). Briefly, the reac- bation at room temperature, the cells were mounted on a Burker-
tion was started by the addition of 2 mM NADH (20 μL) into the Turk-type hemocytometer and counted as trypan blue-penetrated
assay mixture (180 μL) containing varying concentrations of test (dead) cells or nonpenetrated (viable) cells using an inverted phase-
samples (10 μL), plus 1 mM NBT (20 μL), 0.1 mM PMS (20 μL), contrast microscope (AE-30, Shimadzu Co., Kyoto, Japan).

C2422 Journal of Food Science r Vol. 80, Nr. 11, 2015

Yacon tea leaves suppress O2 − radical . . .

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Table 1–Yield of four different extracts from yacon tea leaves, and total polyphenol content in four extracts and yacon tea leaves
by different extractions.

Total polyphenol Total polyphenol

Yield of extract (µg CAE/mg of (mg CAE/100 g of
a a
Extracts (%) extract) tea leaves)
b
Hot water 27.8 279 ± 11 7756 ± 306 (0.0776)
Methanol 6.60 80.0 ± 5.6 528 ± 37 (0.00528)

C: Food Chemistry
Ethanol 4.74 83.5 ± 8.8 396 ± 42 (0.00396)
Ethylacetate 3.91 70.5 ± 4.8 276 ± 19 (0.00276)
a
Data of total polyphenol shown represent mean ± S.D. from four experiments. CAE, chlorogenic acid equivalent.
b
Data in parentheses indicate calculated amount of total polyphenol expressed as gram order of CAE per gram of tea leaves.

Table 2–Antioxidative effect of four extracts from yacon tea leaves in DPPH radical scavenging assay.
∗
DPPH radical scavenging activity (%) at various concentrations
Extracts 0 µg/mL 50 µg/mL 100 µg/mL 250 µg/mL 500 µg/mL EC50 (µg/mL)
∗∗
Hot water 0.0 ± 1.5 75.3 ± 1.3 92.2 ± 0.2 93.0 ± 0.1 92.4 ± 0.1 <50 (28.1 ± 0.7a )
Methanol 0.0 ± 0.4 5.9 ± 1.8 11.3 ± 3.2 26.4 ± 1.7 52.0 ± 1.2 480 ± 12b
Ethanol 0.0 ± 0.4 10.9 ± 2.5 18.1 ± 2.4 43.8 ± 1.2 81.0 ± 2.3 292 ± 9c
Ethylacetate 0.0 ± 0.4 10.8 ± 4.8 16.8 ± 1.1 40.7 ± 1.1 72.6 ± 2.8 324 ± 12d
Trolox 0.0 ± 1.9 93.6 ± 0.1 93.4 ± 0.2 94.2 ± 0.1 94.5 ± 0.2 <50 (7.64 ± 0.06e )
∗
Data shown represent mean ± S.D. from four experiments. The final concentrations of each sample tested were 50, 100, 250, or 500 μg/mL in DPPH radical scavenging assay.
EC50 values not sharing a common superscript letter are considered significantly different at P < 0.05. Trolox was used as the standard sample.
∗∗
Data shown in parentheses indicate EC50 values calculated from the data obtained in Figure 2A.

Statistic analysis 4.0-times higher than the other three extracts. Thus, 0.0776 g of
For statistical analysis, the values are expressed as mean ± polyphenol can be effectively extracted from 1 g of tea leaves by
standard deviation derived from four parallel experiments. Data hot-water extraction, and the level was 14.7 to 28.1-times higher
in part were analyzed using statistical add-on software program than the other three extractions. It is to note that the values of
(Statcel, OMS Co., Saitama, Japan) for Excel 2004 (Microsoft yield and total polyphenol content determined in this study are
Co., Redmond, Wash., U.S.A.). Statistical differences were comparable with another report (Oliveira and others 2009).
considered significant at P < 0.01 or P < 0.001 using Student’s
t-test. With an one-factor analysis of variance (ANOVA), a Radical scavenging effects by yacon tea leaves
post-hoc Bonferroni-Dunn test was conducted for the multiple We attempted to investigate whether the hot-water, methanol,
comparison and significant difference at P < 0.05 was considered. ethanol, and ethylacetate extracts of the yacon tea leaves can
demonstrate potent antioxidant activities against representative
Results and Discussion free radicals. The DPPH is a stable free radical with the deep
Yacon is locally marketed and recently used as various processed violet color, and changes to the reduced form with the decol-
foods with an expectation for its health-promoting effects (Ojan- orization when any proton-donating antioxidant(s) is(are) present
sivu and others 2011; Delgado and others 2013). However, the (Blois 1958; Molyneux 2004). It is therefore an increasing of the
health-beneficial mechanisms of the yacon tea leaves remain to be interest to use this method for estimating the antioxidative effi-
completely established. This study investigates the antioxidative ciency in foodstuffs. A pilot study first revealed that the hot-water
properties of commercial yacon tea leaves in multiple free radical extract tested at 50 to 500 μg/mL showed 75.3% to 93.0% of
assays targeting to four representative artificial radical models and higher DPPH radical scavenging activity (Table 2). The methanol
O2 − radical, one of well-known reactive oxygen species. Reduc- extract, the ethanol extract, and the ethylacetate extract tested at
ing capability of these extracts were examined in parallel. To gain 500 μg/mL, respectively, resulted in showing 52.0%, 81.0%, and
insight into the relevance in the cellular physiology, suppressive 72.6% of the maximum activities. The EC50 value of the hot-water
effect of the tea-leaf extract on the cellular O2 − production was extract therefore estimated to be out of the range, indicating less
subsequently examined. than 50 μg/mL. On the ranging from 50 to 500 μg/mL tested,
the EC50 values of the methanol extract, the ethanol extract, and
Sample preparation and total polyphenol content in yacon the ethylacetate extract were 480, 292, and 324 μg/mL, respec-
tea leaves tively. Therefore, potent hydrophilic constituents present in the
We first prepared four yacon tea-leaf extracts by different ex- yacon tea leaves, for example, high water-soluble polyphenols,
tractions. A boiling hot-water extraction yielded 1.39 g of the may contribute to the DPPH radical scavenging capability rather
extract from 5.00 g of the crashed tea leaves. Yields of individual than the hydrophobic components. In parallel, the EC50 value of
extractions with methanol, ethanol, and ethylacetate, respectively, trolox, a standard sample, was also estimated to be out of the range,
were 0.165 g from 2.50 g, 0.119 g from 2.51 g, and 0.0981 g from <50 μg/mL.
2.51 g of yacon tea leaves. As compiled in Table 1, the hot-water It is an interesting issue whether the antioxidant capacity of these
extraction resulted in 27.8% of the yield of the extract and the yacon tea-leaf extracts shown in this scavenging assay is specific
value was 4.2 to 7.1-times higher than the other three extractions. to DPPH radical or not. To gain insight into the heterogenous
Amount of total polyphenol in 1 mg of the hot-water extract was antioxidant capability of these extracts, we next conducted a fer-
279 μg as the chlorogenic acid equivalent and the value was 3.3 to ric reducing power assay. Because the data of reducing power are

Vol. 80, Nr. 11, 2015 r Journal of Food Science C2423

 Yacon tea leaves suppress O2 − radical . . .

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generally shown as the measured absorbance at 700 nm (Oyaizu effectively contribute to the electron receiving reduction with
1986; Oktay and others 2003), increased absorbance indicates the its radical scavenging capacities. For determining ferric reducing
increase of reducing capability. In this study, we introduce and capability, at least three methods are currently available: (i) reduc-
define an arbitrary unit in the figure where the reducing power ing power assay using potassium hexacyanoferrate (Oyaizu 1986;
with 1.0 of absorbance is equal to 100% of reductivity as described Oktay and others 2003), (ii) ferric reducing antioxidant power
(Ferreira and others 2007). Thus, the effective concentration giv- (FRAP) assay using tripyridyltriazine (TPTZ) (Benzie and Strain
ing 0.5 of absorbance, or 50% of reductivity, is assumed to be EC50 1996; Pulido and others 2000), and (iii) ferric reducing capacity
C: Food Chemistry

values. As shown in Figure 1, the hot-water extract demonstrated (FRC) assay using phenanthroline instead of TPTZ (Lim and Lim
higher reducing capability than the other three extracts; EC50 2013). As investigated in a review paper, the reducing power as-
168 μg/mL of hot-water extract, compared with 566 μg/mL of say has been more frequently used rather than FRAP assay in the
methanol, 1009 μg/mL of ethanol, and 593 μg/mL of ethylac- in vitro antioxidant assays (Alam and others 2013). Therefore, we
etate extracts. Among four extracts, the hot-water extract may selected this reducing power assay in this study. It is to note that

Figure 1–Antioxidative effect of four extracts

from yacon tea leaves in reducing power assay.
Data shown represent mean ± S.D. from four
experiments. The final concentrations of each
sample tested in this assay were 10, 25, 50, 100,
250, 500, or 1000 µg/mL. An arbitrary unit is
defined and included in the figure where the
reducing power with 1.0 of absorbance is equal
to100% of reductivity. The effective
concentration giving 0.5 of absorbance, or 50%
reductivity, is assumed to be EC50 (µg/mL) as
described (Ferreira and others 2007). The inset
figure shows the whole region where the
experimental data were obtained. EC50 values
not sharing acommon superscript letter are
considered significantly different at P < 0.05.
Trolox was used as the standard sample.

Figure 2–Antioxidative effect of hot-water extract

from yacon tea leaves in DPPH radical (A),
ABTS+ radical (B), galvinoxyl radical (C), and
CPZ+ radical scavenging assays (D). Data
shown represent mean ± S.D. from four
experiments. Trolox was used as the standard
sample.

C2424 Journal of Food Science r Vol. 80, Nr. 11, 2015

Yacon tea leaves suppress O2 − radical . . .

17503841, 2015, 11, Downloaded from https://ift.onlinelibrary.wiley.com/doi/10.1111/1750-3841.13092 by UEFS - Universidade Estadual de Feira de Santana, Wiley Online Library on [19/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
there is a trend to obtain similar results by several different an- EC50 values were obtained. It is to note that the radical scaveng-
tioxidants between the reducing power assay and the FRAP assay ing activities of the tea leaf extract reached to the maximum level
in a recent report (Cakmakci and others 2015). Further studies at around 50 μg/mL against DPPH and ABTS+ radical, while
are needed in order to fully elucidate the effect of extractions and at 8.0 μg/mL against galvinoxyl radical. The CPZ, a neuroleptic
solvents in multiple antioxidant assays, for example, radical chain drug in the treatment of schizophrenia in psychopharmacology,
reaction-mediated liposome and/or β-carotene-linolate oxidation forms a stable cation radical in acidic condition under the oxidiza-
systems. It is to note that yacon leaves possess water-soluble phe- tion with chromium(IV) (Nagaraja and others 2014) or Fe(III)

C: Food Chemistry
nolic antioxidants such as protocatechuic acid, chlorogenic acid, (Miftode and others 2010), and can be used for analyzing antioxi-
and caffeic acid (Valentova and others 2005, 2006). It should be dation capability with electron transferring. At the concentrations
emphasized whether these polyphenols are stably present in the tested from 100 to 1000 μg/mL, the extract demonstrated an
tea leaves during food processing and remain to exert functional increase of CPZ+ radical scavenging activity (Figure 2D). The
effects will be another interesting issue. calculated EC50 values of the extract and trolox were 475 and
To quantitatively evaluate the antioxidative effect, the hot- 3.16 μg/mL, respectively. The tea-leaf extract showed only 63.0%
water tea extract was further tested at different concentrations of the scavenging activity as the maximum at 1000 μg/mL. It is
in DPPH radical and three other representative radical assays. suggested that the antioxidative capability of the tea extract may
As shown in Figure 2A, the hot-water tea extract and trolox be diminished by acidic environment because the CPZ+ radical
showed a concentration-dependent increase of DPPH radial scav- scavenging assay was carried out with phosphoric acid (see Sec-
enging activity. The calculated EC50 value of the tea extract was tion “Materials and Methods”). Therefore, it may be an important
28.1 μg/mL, while that of trolox was 7.64 μg/mL. In recent years, strategy to delineate potent antioxidative properties of foodstuffs
the importance of several comparative studies on antioxidants has by multiple free radical assay methods.
been raised because the activities of some antioxidants may vary The four free radicals tested here are valid and useful radical
with the assay methods (Takebayashi and others 2006; Tai and models for the screening of antioxidant capacity of foodstuffs;
others 2011, 2012; Nagaoka and others 2013). It is an interesting however, these chemicals do not exist in vivo. We next challenged
strategy to investigate whether the radical scavenging effect of the to investigate whether the hot-water extract can indeed demon-
yacon tea leaves shown was specific to DPPH or not. The ABTS, strate antioxidant capacity against O2 − radical, one of well-known
a stable cation radical, has been widely used to screen radical ROS. Generated O2 − radical can form further H2 O2 and highly
scavenging capacity of both lipophilic and hydrophobic antiox- reactive OH radical in the presence of metal ions, thereby leading
idant(s) responsible for electron and hydrogen-donating abilities to sequential oxidations (Valko and others 2005). As shown in
(Pellegrini and others 1999; Re and others 1999; Thaipong and Figure 3, the tea-leaf extract also demonstrated a concentration-
others 2006). As shown in Figure 2B, a concentration-dependent dependent increase of O2 − radical scavenging activity. The activity
increase of ABTS cation radical scavenging activity was similarly of the extract reached to the maximum level at 250 μg/mL con-
demonstrated in the tea extract. The calculated EC50 values of centration. The calculated EC50 values of the extract and trolox
the extract and trolox were 23.7 and 8.93 μg/mL, respectively. were 64.5 and 482 μg/mL, respectively. In contrast to the re-
The galvinoxyl, a stable radical utilized in electron spin resonance sults obtained in DPPH, ABTS+ , galvinoxyl, and CPZ+ radical
measurements (Havenith and others 2008), is available for mea- models (see Figure 2), the extract possesses 7.47-fold higher an-
surement of hydrogen-donating ability of phenolic compounds tioxidative capability than trolox in this O2 − radical scavenging
which can form resonance-stabilized phenoxyl radicals in their assay. It is suggested that the yacon tea leaves should be effective
active hydroxyl group (Shi and others 2001). The tea-leaf extract for the prevention of O2 − radical-mediated oxidation. In addi-
also showed a concentration-dependent increase of galvinoxyl rad- tion, oxygen radical absorbance capacity (ORAC) assay is one of
ical scavenging activity (Figure 2C). The calculated EC50 values the most widely used and also standardized methods for evalu-
of the tea extract and trolox were 3.06 and 0.571 μg/mL, re- ating the capacity of antioxidants in food and biological samples
spectively. Comparing to other two assays, the galvinoxyl radical (Prior and others 2005; Watanabe and others 2014). This method
scavenging assay was a relatively sensitive model because the lower enables us determining the antioxidant capacity of lipophilic and
hydrophilic antioxidants targeting to peroxyl radicals in lipid oxi-
dation systems. Due to the requirement of a high-spec fluorescent
microplate reader equipped with a heat control, however, there is
a technical limitation to perform this assay. Further investigation is
needed to clarify the oxygen radical specific antioxidant capacity
of the herbal yacon tea leaves.
Which bioactive compounds present in the hot-water extract
from the tea leaves were responsible for the O2 − radical scavenging
effect, remains to be entirely clarified. As described in previous re-
ports (Valentova and others 2005, 2006), chlorogenic acid, caffeic
acid, and feruric acid have been isolated and determined as the
major phenolic constituents from leaf part of yacon. To gain insight
into the active compounds, these three known chemicals were ex-
amined in O2 − radical scavenging assay. Among the different con-
centrations tested (ranging from 0, 100 to 1000 μM), chlorogenic
acid and caffeic acid, both contains a catecyl group, respectively,
Figure 3–Effect of concentration of hot-water extract from yacon tea leaves
− demonstrated concentration-dependent scavenging activities with
in O2 radical scavenging assay in PMS-NADH-NBT system. Data shown
represent mean ± S.D. from four experiments. Trolox was used as the 459 μM (167 μg/mL) and 620 μM (35.0 μg/mL) of EC50 values,
standard sample. while feruric acid showed approximately 10% activities (figure not

Vol. 80, Nr. 11, 2015 r Journal of Food Science C2425

 Yacon tea leaves suppress O2 − radical . . .

17503841, 2015, 11, Downloaded from https://ift.onlinelibrary.wiley.com/doi/10.1111/1750-3841.13092 by UEFS - Universidade Estadual de Feira de Santana, Wiley Online Library on [19/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Table 3–EC50 values and trolox equivalent antioxidant capacity of hot-water extract from yacon tea leaves in five different free
radical scavenging assays and ferric reducing power assay.

EC50 (µg/mL) EC50 TEAC TEAC

of hot-water (µg/mL) (mg TE/mg of (g TE/ 100 g
∗ ∗ ∗∗ ∗∗
Free radicals extract of trolox extract) of tea leaves)
DPPH radical 28.1 ± 0.7a 7.64 ± 0.06a 0.272 7.56
ABTS+ radical 23.7 ± 0.3a 8.93 ± 0.19a 0.377 10.5
C: Food Chemistry

Galvinoxyl radical 3.06 ± 0.15b 0.571 ± 0.014a 0.187 5.20

CPZ+ radical 475 ± 13c 3.16 ± 0.16a 0.00665 0.185
O2 − radical 64.5 ± 5.8d 482 ± 23b 7.47 208
Reducing power 168 ± 13e 47.5 ± 2.7c 0.283 7.87
∗
Data shown represent mean ± S.D. from four experiments and values not sharing a common superscript letter are considered significantly different at P < 0.05. Trolox was used as
the standard sample. In four radical scavenging assays, EC50 values indicate effective concentration at which the activity was 50%. In reducing power assay, EC50 values indicate
effective concentration at which the absorbance is 0.5, or the reducivity was 50% (Figure 1).
∗∗
Data shown represent mean value from four experiments. TEAC, trolox equivalent antioxidant capacity; TE, trolox equivalent.

shown). On the electron spin resonance trapping determination, leaves and can play a health beneficial role, is another interesting
these two catecholic chemicals are the excellent radical scavengers issue.
specific to O2 − radical in comparison with trolox rather than the The calculated EC50 values of the hot-water extract and
other ROS including OH radical, singlet oxygen, and also alkoxyl trolox among five radical scavenging assays and ferric reducing
radical (Sueishi and others 2014). Antioxidant capacity of these power assay (Figure 1 to 3) were compiled in Table 3. Based
chemicals are only 0.24 to 2.24-times of the activities obtained by on the ratio of EC50 values, the antioxidant capability of the
trolox on the various antioxidant assays, including DPPH radical tea extract can be defined as a “trolox equivalent antioxidant
and ABTS+ radical scavenging assays, reducing power, and linolate capacity (TEAC)”value. In DPPH radical scavenging assay, the tea
peroxidation assays (Olszewska and others 2012). These pheno- extract appeared to possess 0.272-fold extent of trolox-equivalent
lic compounds and/or possibly other candidate(s) may contribute antioxidant capacity, thereby calculating TEAC value of 1 mg
to exert as the O2 − radical-specific antioxidants. To investigate extract was 0.272 mg ( = 1.09 μmol) TE. According to 27.8%
whether these polyphenols are stably present in the processed tea yield of the hot-water extract (see Table 1), 100 g yacon tea leaves
is estimated to possess 7.56 g ( = 30.2 mmol) of TEAC. In ABTS
cation radical scavenging assay, the calculated TEAC values of mg
extract and 100 g tea leaves were 0.377 mg ( = 1.51 μmol) TE and
10.5 g ( = 42.0 mmol) TE, respectively. In the case of galvinoxyl
radical, the TEAC values of mg extract and 100 g tea leaves were
0.187 mg ( = 0.747 μmol) TE and 5.20 g ( = 20.8 mmol) TE,
respectively. The values obtained in CPZ cation radical assay were
0.00665 mg ( = 0.0266 μmol) TE per mg extract and 0.185
g ( = 0.739 mmol) TE per 100 g tea leaves. In O2 − radical
scavenging assay, the TEAC values determined per mg extract and
100 g tea leaves, respectively, were 7.47 mg ( = 29.8 μmol) TE
and 208 g ( = 831 mmol) TE. In addition, the values obtained
in reducing power assay were 0.283 mg ( = 1.13 μmol) TE per
mg extract and 7.87 mg ( = 31.4 mmol) TE per 100 g tea leaves.
It should be notable that the much higher TEAC values were
obtained in O2 − radical scavenging assay in contrast to the values
shown in DPPH, ABTS+ , galvinoxyl, and CPZ+ radical models,
as well as ferric reducing power model. It is therefore suggested
that the antioxidant capability of foodstuff(s) needs to be evaluated
in multiple assays as reported (Takebayashi and others 2006; Tai
and others 2011, 2012; Nagaoka and others 2013) and the TEAC
values are useful parameter to characterize its specific antioxidant
properties. Indeed, the excess amount of O2 − radical has been
implicated in a wide variety of pathophysiological processes,
such as diabetes, ischemia-reperfusion injury and chronic heart
failure (Pacher and others 2006). It is suggested that the intake
of the hot-water extract of commercial yacon tea leaves may
contribute to prevent exaggerated O2 − radical mediated disease
risks.
It will be a good strategy to delineate potent antioxidative prop-
erties of foodstuffs by multiple free radical assay methods as well
Figure 4–Effect of concentration of hot-water extract from yacon tea leaves
in XOD-mediated O2 − radical (A) and uric acid generation assays (B). as other antioxidant assay(s). We tried comparing the EC50 values
Data shown represent mean ± S.D. from four experiments. Trolox and obtained by hot-water extract and trolox among these five free rad-
allopurinol, respectively, were used as an antioxidant and an XOD-specific ical scavenging assays plus reducing power assay (Table 3). With a
enzyme inhibitor. XOD; xanthine oxidase. 1/20-folded volume of test sample, these assays were performed in

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Yacon tea leaves suppress O2 − radical . . .

17503841, 2015, 11, Downloaded from https://ift.onlinelibrary.wiley.com/doi/10.1111/1750-3841.13092 by UEFS - Universidade Estadual de Feira de Santana, Wiley Online Library on [19/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Table 4–IC50 values and O2 − radical scavenging index of hot-water extract from yacon tea leaves, trolox, and allopurinol obtained
in XOD-mediated O2 − radical and uric acid generation assays.
∗ ∗∗
IC50 value (µg/mL) O2 − Radical scavenging index
Ratio of IC50 values
O2 − radical Uric acid values(O2 − radical / Uric acid)
Yacon tea leaves 20.7 ± 1.4a 639 ± 50a 0.0324

C: Food Chemistry
Trolox 48.5 ± 0.9b 336 ± 7b 0.144
Allopurinol 0.306 ± 0.089c 0.449 ± 0.037c 0.682
∗
Data shown represent mean ± S.D. from four experiments and values not sharing a common superscript letter are considered significantly different at P < 0.05. Trolox and
allopurinol, respectively, were used as a standard antioxidant and an XOD-specific enzyme inhibitor. XOD, xanthine oxidase.
∗∗
Data shown represent mean value from four experiments. O2 − Radical scavenging index was defined as the ratio of IC50 values (O2 − radical / Uric acid).

reaction mixtures. Among 6 different assays, the lowest EC50 val- other antioxidant activities; for example DPPH free radical scav-
ues, for example 3.06 μg/mL by the extract and 0.571 μg/mL by enging, O2 − radical scavenging, hydrogen peroxide scavenging,
trolox, were given in galvinoxyl radical scavenging assay, implying and metal chelating activities (Oktay and others 2003). Therefore,
this method to be the superior method with high sensitivity. While multiple free radical assay methods may provide useful informa-
EC50 values in DPPH and ABTS+ radical assays, respectively, were tion to estimate the mode of action for their radical scavenging
comparable in both cases; 28.1 and 23.7 μg/mL by the extract, capabilities.
and 7.64 and 8.93 μg/mL by trolox. On the other side, CPZ+
and O2 − radical assays, respectively, were effective methods to de- Suppressive effect on enzymatic O2 − radical generation
lineate the characteristic differences of the yacon leaf extract (475 by yacon tea leaves
and 64.5 μg/mL EC50 values) compared with trolox (3.16 and XOD is a type of enzyme that can catalyze the oxidation of
482 μg/mL EC50 values), rather than the other assays. The EC50 xanthine to uric acid, thereby generating O2 − radical in our body
values of the extract and trolox in reducing power assay, which (Kim and others 2004; Pacher and others 2006; Connor 2009).
are positioned at the middle range of the values among these six We next performed XOD enzymatic assays to investigate whether
assays, may be changeable depending on the arbitrary unit flexibly the yacon tea-leaf extract was capable of inhibiting O2 − radi-
defined. However, the calculated TEAC values obtained in the cal generation. Upon this experimental setting, amount of O2 −
reducing power assay are closer to those values from DPPH radical radical generated by XOD was measured through the coloriza-
and ABTS+ radical assays, implying the comparable antioxidant tion of WST-1 formazan (Ukeda and others 1999). As shown in
characters of yacon-tea leaves among these three different analyses. Figure 4A, the extract suppressed the amount of O2 − radical in
Reducing power assay has been previously developed to measure a concentration dependent manner, and reached to the minimum
the antioxidant capacity of various food materials with a mecha- level at 200 μg/mL. The calculated IC50 value of the extract was
nism based on the ferric ion-based redox reaction (Oyaizu 1986). 20.7 μg/mL. The standard samples, trolox and allopurinol, used as
It is interesting to note that the reducing power is simultaneously the reference antioxidant and enzyme inhibitor, provided 48.5 and
demonstrated in a course of characterization for antioxidant ac- 0.306 μg/mL of IC50 values, respectively. Thus, the tea-leaf ex-
tivities of water and ethanol extracts from an herbal plant with tract was found to possess 2.34-times higher suppressive capability
compared with trolox in measuring O2 − radical, but 0.0148-fold
extent of suppression compared with allopurinol.
It is an interesting strategy to clarify whether the tea-leaf ex-
Yacon tea leaves tract can directly inhibit the XOD activity, thereby decreasing the
120 Trolox
amount of O2 − radical production or not. We next investigated the
Generated by Cells (%)
Amount of O2− Radical

100 effect of the extract on XOD activity. The enzymatic activity was
**
*** ** measured as the amount of uric acid generated as the end product
80 ***
*** *** *** through the reaction according to previous reports (Nguyen and
60 others 2004; Wang and others 2008). As shown in Figure 4B, the
40 *** *** extract weakly inhibited the XOD-catalyzed uric acid generation,
*** and the calculated IC50 value of the extract was 639 μg/mL. The
20 *** standard sample, allopurinol, provided 0.449 μg/mL of IC50 value,
0 and also trolox weakly inhibited the XOD with 336 μg/mL of
0 20 50 100 200 500 1,000 IC50 value. It is to note that the tea-leaf extract and trolox main-
Concentration (µg/ml) tain approximately 80% to 100% of the XOD activity at the con-
centrations ranging from 0 to 200 μg/mL, implying almost no
Figure 5–Effect of concentration of hot-water extract from yacon tea leaves inhibition. IC50 values obtained in these XOD-mediated assays
and trolox on the cellular O2 − radical generation in PMA-stimulated gran- are compiled in Table 4. Ratio of IC50 values from both assays can
ulocytic neutrophil cell model. HL-60 human leukemic cells were incubated
in the presence of 1.25% DMSO for 6 d to undergo neutrophilic differen- be defined as a parameter indicating a mode of suppressive action.
tiation (see Materials and Methods section). The differentiated cells were For instance, a low ratio value indicates a sample possesses strong
preincubated with the test sample for 15 min at 37°C in a test tube and O2 − radical scavenging effect, while a value close to 1.00 means
then exposed to PMA for another 15 min. The supernatants were collected a sample possesses insufficient scavenging effect due to its enzyme
and colorimetrically analyzed for determination of O2 − radical level by the
cytochrome c method. Data shown represent mean ± S.D. from four experi-
inhibition. The tea-leaf extract shows 0.0324 of lower ratio value,
ments. Significant differences from individual untreated cells (control), ∗∗ P indicating its dominant O2 − radical scavenging capacity. XOD-
<0.01 or ∗∗∗ P < 0.001. PMA, phorbol 12-myristate 13-acetate. mediated O2 − radical generation assay has been used as a useful

Vol. 80, Nr. 11, 2015 r Journal of Food Science C2427

 Yacon tea leaves suppress O2 − radical . . .

17503841, 2015, 11, Downloaded from https://ift.onlinelibrary.wiley.com/doi/10.1111/1750-3841.13092 by UEFS - Universidade Estadual de Feira de Santana, Wiley Online Library on [19/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
model for the discovery of both antioxidative capacity and candi- date the health beneficial contributions of this herbal tea leaves in
dates of inhibitors to prevent XOD-mediated disease risks (Kim suppressing the risk of O2 − free radical-mediated disorders.
and others 2004; Pacher and others 2006; Connor 2009). The
yacon tea leaves may contribute to prevent O2 − radical-mediated Acknowledgments
disease risks due to its O2 − radical scavenging mechanism. This work was supported in part by Grant-in-Aid for Young
Scientists (B) (JSPS KAKENHI Grant Numbers 23780143 and
Suppressive effect on cellular O2 − radical generation 15K18700), Research and Study Program/Project of Tokai Uni-
C: Food Chemistry

by yacon tea leaves versity Educational System General Research Organization (Kana-
Neutrophils originated from blood polymorphonuclear leuco- gawa, Japan), and “Center of Community (COC)” program (The
cytes are thought to be an important defense line against mi- Ministry of Education, Culture, Sports, Science, & Technology
croorganisms and inflammatory responses. When neutrophils are (MEXT), Japan). No potential conflicts of interest were disclosed.
activated by cytokines and/or stimulating agents, excess amount
of O2 − radical can be generated (Utsumi and others 1992; Shin- Author Contributions
tani 2013). To gain insight into the physiological relevance of S. Sugahara mainly executed experiments and drafted the
the yacon tea leaves with its potent antioxidative property, dif- manuscript. Y. Ueda, K. Fukuhara, and Y. Kamamuta col-
ferentiated HL-60 human granulocyte-like neutrophil cells were lected and analyzed the data. Y. Matsuda, T. Murata, Y. Kuroda,
preincubated in the presence of the extract for 15 min, and then K. Kabata, M. Ono, and K. Igoshi interpreted the results. S.
stimulated with PMA for 15 min in the presence of cytochrome Yasuda designed the study, interpreted the results, and wrote the
c. Amount of cellular O2 − radical generated was colorimetrically manuscript.
measured as the reduction of cytochrome c according to previ-
ous reports (Jiwajinda and others 2002; Kim and others 2002). References
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Yacon tea leaves suppress O2 − radical . . .

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