Antioxidant Effects of Herbal Tea Leaves from Yacon (Smallanthus sonchifolius) on Multiple Free Radical and Reducing Power Assays,
Especially on Different Superoxide Anion Radical Generation Systems
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 (Smallanthus sonchifolius) is a perennial plant originally cultivated in the Andean highlands of South America. Over the past decades, yacon has been introduced into other places includ- ing Asia (Japan, South-Korea, Taiwan, Hainan, and Philippines), Oceania (New Zealand), and Europe (Czech Republic) (Ojansivu and others 2011). The root part of this plant is sweet potato-like shape and it has been historically taken as a fruit/vegetable, and is currently also available as processed foods, such as syrup, juice and marmalade in South America (Ojansivu and others 2011; Delgado and others 2013). As an herbal tea, the leaf part of MS 20150511 Submitted 3/25/2015, Accepted 8/24/2015. Authors Sugahara, Ueda, Fukuhara, Kamamuta, Matsuda, Murata, Kabata, Ono, Igoshi, and Yasuda are with School of Agriculture, Tokai Univ, Kawayo, Minamiaso, Aso, Kumamoto 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 Yasuda (E-mail: [email protected]).
yacon has been provided and consumed in local markets. In recent evidences, yacon has been believed as a potent alternative food source for patients who require a dietary cure in regional area (Delgado and others 2013). It is also used as a folk medicine for the people suffering from diabetes and digestive/renal disorders. It is possibly due to its rich in indigestible fructo-oligosaccharides and beneficial polyphenols (Campos and others 2012). Studies of the yacon root further disclose the antioxidant activity (Yan and others 1999), the anti-diabetic effect in rats (Oliveira and others 2013), the anti-obesity in humans (Genta and others 2009) and 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 hypoglycemic effect in normal and diabetic rat models (Aybar and others 2001). However, the mechanisms underlying the functional properties of this plant and even its processed foods remain to be completely established.
Since the effect of free radicals in biological systems was first proposed (Harman 1956), there has been a considerable interests in examining whether antioxidant(s) from natural products and/or sources can regulate oxidative stresses and reactions. Exaggerated oxidative stress and reactive oxygen species (ROS), for example, superoxide anion (O2−) radical, hydroxyl (OH) radical, perhy- droxyl radical, hydrogen peroxide (H2O2), and singlet oxygen, have been implicated to exert a deleterious effect on normal tissue functions, thereby leading to pathological conditions (Gutowski and Kowalczyk 2013) such as atherosclerosis (Droge 2002), cancer (Hail and others 2008), diabetes (Sabu and Kuttan 2002), neurode- generation (Obata 2002; Przedborski and Ischiropoulos 2005) and liver cirrhosis (Bruck and others 2001), as well as aging process (Weisburger 2002). One of the important oxygen free radicals is the O2− radical, which can easily form H2O2 and highly reactive OH radical in the presence of metal ions (Valko and others 2005). In the body, O2− radical can be generated during mitochondrial respiration (Wiswedel and others 1998), and by NAD(P)H oxidase (Pagano and others 1995), cyclooxygenase and lipoxygenase (Kukreja and others 1986), nitric oxide synthase (Cosentino and others 1989), and cytochrome P450 (Fleming and others 2001). Xanthine oxidase (XOD) is a key enzyme that catalyzes the oxi- dation of hypoxanthine and xanthine to uric acid in concomitant with the generation of O2− radical (Nishino and others 2008; Cantu-Medellin and Kelley 2013). The excess amount of these O2− radical generated by this enzyme has been implicated in a wide variety of pathophysiological processes, such as diabetes, ischemia-reperfusion injury and chronic heart failure (Pacher and others 2006). A previous study shows that free radical production through the higher activity of XOD is involved in type 1 dia- betes (Desco and others 2002). Therefore, the suppression on the XOD-mediated O2− radical generation can be considered to be
a strategy for screening compound(s) to prevent these disease risks (Kim and others 2004; Pacher and others 2006; Connor 2009).
In this study, we report potential antioxidative properties of extracts prepared from commercial yacon tea leaves in different free radical models. Four representative synthetic free radicals, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (Blois 1958), 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) 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 models for the discovery of antioxidative capacity of foodstuffs. In parallel, a reducing power assay using potassium hexacyanoferrate was performed. This method has been developed to measure the antioxidant capacity of various food materials with a ferric ion-mediated redox mechanism (Oyaizu 1986). A hot-water
extract of yacon tea leaves was evaluated in both O2− radical generation systems; phenazine methosulfate (PMS)-NADH- nitroblue tetrazolium (NBT) and XOD enzymatic assays. To gain insight into the physiological relevance of the tea-leaf extract with its potent O2− radical suppressing effects, activated human granulocytic neutrophil cells were incubated in the presence of the extract.
Materials and Methods
Materials
A yacon ‘Sarada otome (SY201)’ has been registrated as a cultivar in Japan (Sugiura and others 2007). A dried herbal tea leaves from this yacon, which were locally cultivated, pro- cessed, and marketed in Kikuchi area (Kumamoto, Japan), were obtained in March 2011 in a retail store. Dimethyl sulfoxide (DMSO), ABTS, DPPH, Folin-Ciocalteu phenol reagent, iron(Ⅲ) chloride, NBT, β-nicotinamide adenine dinucleotide disodium salt (reduced form) (NADH), PMS, potassium hexa- cyanoferrate(Ⅲ), potassium peroxodisulfate, trichloroacetic acid (TCA), xanthine, 12.5 U/mL XOD from buttermilk were pur- chased from Nacalai Tesque Inc. (Kyoto, Japan). Galvinoxyl and CPZ were products from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Potassium dichromate (chromium(VI)), RPMI- 1640 medium, Hank’s balanced salt solution (HBSS), cytochrome c from horse heart prepared using TCA, and phorbol 12-myristate 13-acetate (PMA) were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) was obtained from Dojindo Labs (Kumamoto, Japan). The HL-60 hu- man promyelocytic leukemia cells (JCRB00085) were obtained from Japanese Collection of Research Bioresources (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Biowest (Nuaille, France). All other chemicals were of the highest grade commer- cially available.
Preparation of the tea-leaf extracts
Four extracts from yacon tea leaves were prepared by dif- ferent solvent extractions as described previously (Oliveira and others 2009) with some modifications. Briefly, approximately 5 g of crashed tea leaves (dried weight) was soaked in 250 mL of MilliQ water in a conical beaker at 90 to 100 °C for 45 min. The tea-leaf mixture was then centrifuged and filtrated. The hot- water extract was obtained from the filtrate after lyophilization, and reconstituted in water for following bioassays. For methanol, ethanol, and ethylacetae extracts, approximately 2.5 g of crashed tea leaves was soaked in 41.67 mL of individual solvent in a cen- trifuge tube at room temperature for 10 min with a continuous shaking. After a brief centrifugation, the supernatant was placed into another conical beaker. The tea leaves therein was totally ex- tracted three times with the same amount of solvent, and filtrated together. After vacuum evaporation, the each extract was obtained and reconstituted in DMSO for following bioassays.
Determination of total polyphenol content
The amount of total polyphenol was determined by an es- tablished procedure (Singleton and Rossi 1965). Briefly, the mixture containing test samples (25 μL) and 10-times diluted Folin-Ciocalteu phenol reagent solution (125 μL) was kept with 10% sodium carbonate solution (125 μL) for 10 min at room temperature. The assay mixture was allowed to a colorimetric measurement at 600 nm using a grating microplate reader (SH- 1000Lab, Corona Electric, Ibaraki, Japan). Chlorogenic acid was used as the standard sample for a calibration curve. In case the photometric absorption of samples/reagents may interfere with the data, a parallel experiment as background at each point was carried out.
DPPH radical scavenging assay
The DPPH radical scavenging activity was measured based on the following method (Blois 1958). Briefly, the reaction was started by the addition of 0.5 mM DPPH dissolved in ethanol (50 μL) into the assay mixture (200 μL) containing varying concentrations of test samples (10 μL), 70% ethanol (90 μL), and 0.1 M sodium acetate buffer (pH 5.5, 100 μL), then allowed to proceed for 30 min at room temperature. The absorbance of the resulting solution was measured at 517 nm. Trolox was used as the standard sample.
ABTS+ radical scavenging assay
The ABTS+ radical scavenging activity was measured based on the following method (Thaipong and others 2006). Briefly, ABTS-mixture solution was first prepared by mixing in equal amount of 7.4 mM ABTS and 2.6 mM potassium peroxodisulfate solutions for 15-h rotation in the dark at room temperature. Thereafter, ABTS-working solution was prepared after dilution of the ABTS-mixture solution (150 μL) in methanol (2.9 mL) before use. The reaction was started by the addition of the ABTS-working solution (190 μL) into varying concentrations of test samples (10 μL), then allowed to proceed at room temperature for 2 h in the dark. The absorbance of the resulting solution was measured at 734 nm. Trolox was used as the standard sample.
Galvinoxyl radical scavenging assay
The galvinoxyl radical scavenging activity was measured based on the following method (Shi and others 2001). Briefly, the reaction was started by the addition of galvinoxyl assay mixture (380 μL) containing 0.0167 mM galvinoxyl in ethanol (228 μL) plus 0.01 M citrate buffer (pH 6.0, 152 μL) into the varying con- centrations of test samples (20 μL), then allowed to proceed for 10 min at room temperature. The absorbance of the resulting solution was measured at 432 nm. Trolox was used as the standard sample. In case the photometric absorption of samples or nonspecific residual color of the reagent interferes with the data, a parallel experiment as background at each point was carried out.
CPZ+ radical scavenging assay
The CPZ+ radical scavenging activity was measured based on the following method (Nagaraja and others 2014). Briefly, the
reaction was started by the addition of 0.5 mM chromium(VI) so- lution (10 μL) into the assay mixture (240 μL) containing varying concentrations of test samples (12.5 μL), 10 mM CPZ dissolved in ethanol (20 μL), 1:1 phosphoric acid plus ethanol mixture (200 μL), and ethanol (7.5 μL), then allowed to proceed for 10 min at room temperature. The absorbance of the resulting solution was measured at 530 nm. Trolox was used as the standard sample.
Reducing power assay
Reducing power was measured by the following method (Oy- aizu 1986), based on the reduction of ferrous ion from Fe3+ to Fe2+. Briefly, the reaction was started by the addition of 1% potas- sium hexacyanoferrate(Ⅲ) (20.9 μL) into the assay mixture (29.1 μL) containing varying concentrations of test samples (2.5 μL), MilliQ water (2.5 μL), and 0.2 M phosphate buffer (pH 6.6, 24.15 μL), then allowed to proceed for 20 min at 50 °C. Thereafter, 10% TCA (20.85 μL) and 0.05% iron(Ⅲ) chloride (141.7 μL) were added into the reaction mixture. The absorbance of the resulting solution was measured at 700 nm. Increased absorbance indicates the increase of reducing capability.
O2− radical scavenging assay in PMS-NADH-NBT system
The O2− radical scavenging activity was measured by us- ing PMS-NADH-NBT system according to previously described methods (Gulcin 2006; Wang and others 2008). Briefly, the reac- tion was started by the addition of 2 mM NADH (20 μL) into the assay mixture (180 μL) containing varying concentrations of test samples (10 μL), plus 1 mM NBT (20 μL), 0.1 mM PMS (20 μL), 250 mM potassium phosphate buffer (pH 7.4, 40 μL) and water (90 μL), then allowed to proceed for 10 min at room temperature. The absorbance of the resulting solution was measured at 570 nm. Trolox was used as the standard sample.
Determination of O2− radical generated in XOD assay
The amount of O2− radical enzymatically generated by XOD, was performed based on the measurement of formation of WST-1 formazan during the enzymatic reaction (Ukeda and others 1999). The standard assay mixture, in a final volume of 200 μL, contained the sample solution (10 μL) at varying concentrations, 2.8 mM WST-1 (8 μL), 2.5 mM xanthine (10 μL), 280 mM potassium phosphate buffer (pH 7.4, 40 μL), water (92 μL), and 0.05 U/mL XOD (40 μL). The reaction was started by the addition of the enzyme, allowed to proceed at 25 °C for 0 min and 15 min in a 96- well multiplate. The assay mixture was allowed to a colorimetric measurement of ∆O.D. at 450 nm. The amount of end product, uric acid, enzymatically generated by XOD, was next monitored for measurement of the enzyme activity. The enzymatic assays were similarly performed under the replacement of WST-1 with water. The assay mixture was allowed to a spectrophotometric measurement of ∆O.D. at 290 nm as following (Nguyen and others 2004; Wang and others 2008). Trolox and allopurinol were used as the standard antioxidant and XOD inhibitor, respectively.
Cellular O2− radical generation assay
The HL-60 human promyelocytic leukemia cells were routinely maintained under a 5% CO2 atmosphere at 37 °C in RPMI-1640 medium supplemented with 5% FBS, 100 U/mL penicillin G, and 100 μg/mL streptomycin sulfate. For the cellular O2− radical pro- duction assay, DMSO-differentiated HL-60 human granulocyte-like neutrophil cells were prepared as reported previously (Nakamura and others 1998; Kim and others 2002). Cells seeded in a culture dish at a density of 4×105 cells/mL in 10 mL were cultured in the presence of 1.25% DMSO for 6 d to undergo gran- ulocytic differentiation. After washing with HBSS, the viable cell number was counted by trypan blue dye exclusion assay. There- after, the differentiated cells were prepared and suspended in HBSS at a density of 1×106 cells/mL. The cell suspension (250 μL) was
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 20 μM PMA (1.25 μL) in DMSO to induce cellular O2− radi- cal production and 20 mg/mL cytochrome c solution (12.5 μL) in phosphate buffered saline to colorimetrically detect the O2− radical. This mixture was added to the cell suspension and then incubated at 37 °C for another 15 min. The cells were immedi- ately placed on ice-cold water for 5 min to terminate O2− radical production, and centrifuged at 13000 xg for 1 to 2 min to collect the supernatant. The level of O2− radical generated therein was determined colorimetric change of cytochrome c at 550 nm. In
parallel, cytotoxicity of the test sample was determined by trypan blue dye exclusion assay.
Miscellaneous method
For the trypan blue dye exclusion assay, cell suspension (10 μL) was mixed with the equal amount of 0.4% trypan blue solution (Gibco-Invitrogen, Carlsbad, Calif., U.S.A.). After a 3-min incu- bation at room temperature, the cells were mounted on a Burker- Turk-type hemocytometer and counted as trypan blue-penetrated (dead) cells or nonpenetrated (viable) cells using an inverted phase- contrast microscope (AE-30, Shimadzu Co., Kyoto, Japan).
Statistic analysis
For statistical analysis, the values are expressed as mean ± standard deviation derived from four parallel experiments. Data in part were analyzed using statistical add-on software program (Statcel, OMS Co., Saitama, Japan) for Excel 2004 (Microsoft Co., Redmond, Wash., U.S.A.). Statistical differences were considered significant at P < 0.01 or P < 0.001 using Student’s t-test. With an one-factor analysis of variance (ANOVA), a post-hoc Bonferroni-Dunn test was conducted for the multiple comparison and significant difference at P < 0.05 was considered. Results and Discussion Yacon is locally marketed and recently used as various processed foods with an expectation for its health-promoting effects (Ojan- sivu and others 2011; Delgado and others 2013). However, the health-beneficial mechanisms of the yacon tea leaves remain to be completely established. This study investigates the antioxidative properties of commercial yacon tea leaves in multiple free radical assays targeting to four representative artificial radical models and O2− radical, one of well-known reactive oxygen species. Reduc- ing capability of these extracts were examined in parallel. To gain insight into the relevance in the cellular physiology, suppressive effect of the tea-leaf extract on the cellular O2− production was subsequently examined. Sample preparation and total polyphenol content in yacon tea leaves We first prepared four yacon tea-leaf extracts by different ex- tractions. A boiling hot-water extraction yielded 1.39 g of the extract from 5.00 g of the crashed tea leaves. Yields of individual extractions with methanol, ethanol, and ethylacetate, respectively, were 0.165 g from 2.50 g, 0.119 g from 2.51 g, and 0.0981 g from 2.51 g of yacon tea leaves. As compiled in Table 1, the hot-water extraction resulted in 27.8% of the yield of the extract and the value was 4.2 to 7.1-times higher than the other three extractions. Amount of total polyphenol in 1 mg of the hot-water extract was 279 μg as the chlorogenic acid equivalent and the value was 3.3 to 4.0-times higher than the other three extracts. Thus, 0.0776 g of polyphenol can be effectively extracted from 1 g of tea leaves by hot-water extraction, and the level was 14.7 to 28.1-times higher than the other three extractions. It is to note that the values of yield and total polyphenol content determined in this study are comparable with another report (Oliveira and others 2009). Radical scavenging effects by yacon tea leaves We attempted to investigate whether the hot-water, methanol, ethanol, and ethylacetate extracts of the yacon tea leaves can demonstrate potent antioxidant activities against representative free radicals. The DPPH is a stable free radical with the deep violet color, and changes to the reduced form with the decol- orization when any proton-donating antioxidant(s) is(are) present (Blois 1958; Molyneux 2004). It is therefore an increasing of the interest to use this method for estimating the antioxidative effi- ciency in foodstuffs. A pilot study first revealed that the hot-water extract tested at 50 to 500 μg/mL showed 75.3% to 93.0% of higher DPPH radical scavenging activity (Table 2). The methanol extract, the ethanol extract, and the ethylacetate extract tested at 500 μg/mL, respectively, resulted in showing 52.0%, 81.0%, and 72.6% of the maximum activities. The EC50 value of the hot-water extract therefore estimated to be out of the range, indicating less 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 the ethylacetate extract were 480, 292, and 324 μg/mL, respec- tively. Therefore, potent hydrophilic constituents present in the yacon tea leaves, for example, high water-soluble polyphenols, may contribute to the DPPH radical scavenging capability rather than the hydrophobic components. In parallel, the EC50 value of trolox, a standard sample, was also estimated to be out of the range, <50 μg/mL. It is an interesting issue whether the antioxidant capacity of these yacon tea-leaf extracts shown in this scavenging assay is specific to DPPH radical or not. To gain insight into the heterogenous antioxidant capability of these extracts, we next conducted a fer- ric reducing power assay. Because the data of reducing power are generally shown as the measured absorbance at 700 nm (Oyaizu 1986; Oktay and others 2003), increased absorbance indicates the increase of reducing capability. In this study, we introduce and define an arbitrary unit in the figure where the reducing power with 1.0 of absorbance is equal to 100% of reductivity as described (Ferreira and others 2007). Thus, the effective concentration giv- ing 0.5 of absorbance, or 50% of reductivity, is assumed to be EC50 values. As shown in Figure 1, the hot-water extract demonstrated higher reducing capability than the other three extracts; EC50 168 μg/mL of hot-water extract, compared with 566 μg/mL of methanol, 1009 μg/mL of ethanol, and 593 μg/mL of ethylac- etate extracts. Among four extracts, the hot-water extract may effectively contribute to the electron receiving reduction with its radical scavenging capacities. For determining ferric reducing capability, at least three methods are currently available: (i) reduc- ing power assay using potassium hexacyanoferrate (Oyaizu 1986; Oktay and others 2003), (ii) ferric reducing antioxidant power (FRAP) assay using tripyridyltriazine (TPTZ) (Benzie and Strain 1996; Pulido and others 2000), and (iii) ferric reducing capacity (FRC) assay using phenanthroline instead of TPTZ (Lim and Lim 2013). As investigated in a review paper, the reducing power as- say has been more frequently used rather than FRAP assay in the in vitro antioxidant assays (Alam and others 2013). Therefore, we selected this reducing power assay in this study. It is to note that there is a trend to obtain similar results by several different an- tioxidants between the reducing power assay and the FRAP assay in a recent report (Cakmakci and others 2015). Further studies are needed in order to fully elucidate the effect of extractions and solvents in multiple antioxidant assays, for example, radical chain reaction-mediated liposome and/or β-carotene-linolate oxidation systems. It is to note that yacon leaves possess water-soluble phe- nolic antioxidants such as protocatechuic acid, chlorogenic acid, and caffeic acid (Valentova and others 2005, 2006). It should be emphasized whether these polyphenols are stably present in the tea leaves during food processing and remain to exert functional effects will be another interesting issue. To quantitatively evaluate the antioxidative effect, the hot- water tea extract was further tested at different concentrations in DPPH radical and three other representative radical assays. As shown in Figure 2A, the hot-water tea extract and trolox showed a concentration-dependent increase of DPPH radial scav- enging activity. The calculated EC50 value of the tea extract was 28.1 μg/mL, while that of trolox was 7.64 μg/mL. In recent years, the importance of several comparative studies on antioxidants has been raised because the activities of some antioxidants may vary with the assay methods (Takebayashi and others 2006; Tai and others 2011, 2012; Nagaoka and others 2013). It is an interesting strategy to investigate whether the radical scavenging effect of the yacon tea leaves shown was specific to DPPH or not. The ABTS, a stable cation radical, has been widely used to screen radical scavenging capacity of both lipophilic and hydrophobic antiox- idant(s) responsible for electron and hydrogen-donating abilities (Pellegrini and others 1999; Re and others 1999; Thaipong and others 2006). As shown in Figure 2B, a concentration-dependent increase of ABTS cation radical scavenging activity was similarly demonstrated in the tea extract. The calculated EC50 values of the extract and trolox were 23.7 and 8.93 μg/mL, respectively. The galvinoxyl, a stable radical utilized in electron spin resonance measurements (Havenith and others 2008), is available for mea- surement of hydrogen-donating ability of phenolic compounds which can form resonance-stabilized phenoxyl radicals in their active hydroxyl group (Shi and others 2001). The tea-leaf extract also showed a concentration-dependent increase of galvinoxyl rad- ical scavenging activity (Figure 2C). The calculated EC50 values of the tea extract and trolox were 3.06 and 0.571 μg/mL, re- spectively. Comparing to other two assays, the galvinoxyl radical scavenging assay was a relatively sensitive model because the lower EC50 values were obtained. It is to note that the radical scaveng- ing activities of the tea leaf extract reached to the maximum level at around 50 μg/mL against DPPH and ABTS+ radical, while at 8.0 μg/mL against galvinoxyl radical. The CPZ, a neuroleptic drug in the treatment of schizophrenia in psychopharmacology, forms a stable cation radical in acidic condition under the oxidiza- tion with chromium(IV) (Nagaraja and others 2014) or Fe(III) (Miftode and others 2010), and can be used for analyzing antioxi- dation capability with electron transferring. At the concentrations tested from 100 to 1000 μg/mL, the extract demonstrated an increase of CPZ+ radical scavenging activity (Figure 2D). The calculated EC50 values of the extract and trolox were 475 and 3.16 μg/mL, respectively. The tea-leaf extract showed only 63.0% of the scavenging activity as the maximum at 1000 μg/mL. It is suggested that the antioxidative capability of the tea extract may be diminished by acidic environment because the CPZ+ radical scavenging assay was carried out with phosphoric acid (see Section “Materials and Methods”). Therefore, it may be an important strategy to delineate potent antioxidative properties of foodstuffs by multiple free radical assay methods. The calculated EC50 values of the hot-water extract and trolox among five radical scavenging assays and ferric reducing power assay (Figure 1 to 3) were compiled in Table 3. Based on the ratio of EC50 values, the antioxidant capability of the tea extract can be defined as a “trolox equivalent antioxidant capacity (TEAC)”value. In DPPH radical scavenging assay, the tea extract appeared to possess 0.272-fold extent of trolox-equivalent antioxidant capacity, thereby calculating TEAC value of 1 mg extract was 0.272 mg ( = 1.09 μmol) TE. According to 27.8% 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 as other antioxidant assay(s). We tried comparing the EC50 values obtained by hot-water extract and trolox among these five free rad- ical scavenging assays plus reducing power assay (Table 3). With a 1/20-folded volume of test sample, these assays were performed in reaction mixtures. Among 6 different assays, the lowest EC50 val- ues, for example 3.06 μg/mL by the extract and 0.571 μg/mL by trolox, were given in galvinoxyl radical scavenging assay, implying this method to be the superior method with high sensitivity. While EC50 values in DPPH and ABTS+ radical assays, respectively, were comparable in both cases; 28.1 and 23.7 μg/mL by the extract, 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- lineate the characteristic differences of the yacon leaf extract (475 and 64.5 μg/mL EC50 values) compared with trolox (3.16 and 482 μg/mL EC50 values), rather than the other assays. The EC50 values of the extract and trolox in reducing power assay, which are positioned at the middle range of the values among these six assays, may be changeable depending on the arbitrary unit flexibly defined. However, the calculated TEAC values obtained in the reducing power assay are closer to those values from DPPH radical and ABTS+ radical assays, implying the comparable antioxidant characters of yacon-tea leaves among these three different analyses. Reducing power assay has been previously developed to measure the antioxidant capacity of various food materials with a mecha- nism based on the ferric ion-based redox reaction (Oyaizu 1986). It is interesting to note that the reducing power is simultaneously demonstrated in a course of characterization for antioxidant ac- tivities of water and ethanol extracts from an herbal plant with other antioxidant activities; for example DPPH free radical scav- enging, O2− radical scavenging, hydrogen peroxide scavenging, and metal chelating activities (Oktay and others 2003). Therefore, multiple free radical assay methods may provide useful informa- tion to estimate the mode of action for their radical scavenging capabilities. Suppressive effect on enzymatic O2− radical generation by yacon tea leaves XOD is a type of enzyme that can catalyze the oxidation of xanthine to uric acid, thereby generating O2− radical in our body (Kim and others 2004; Pacher and others 2006; Connor 2009). We next performed XOD enzymatic assays to investigate whether the yacon tea-leaf extract was capable of inhibiting O2− radical generation. Upon this experimental setting, amount of O2− radical generated by XOD was measured through the colorization of WST-1 formazan (Ukeda and others 1999). As shown in Figure 4A, the extract suppressed the amount of O2− radical in a concentration dependent manner, and reached to the minimum level at 200 μg/mL. The calculated IC50 value of the extract was 20.7 μg/mL. The standard samples, trolox and allopurinol, used as the reference antioxidant and enzyme inhibitor, provided 48.5 and 0.306 μg/mL of IC50 values, respectively. Thus, the tea-leaf extract 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- tract can directly inhibit the XOD activity, thereby decreasing the amount of O2− radical production or not. We next investigated the effect of the extract on XOD activity. The enzymatic activity was measured as the amount of uric acid generated as the end product through the reaction according to previous reports (Nguyen and others 2004; Wang and others 2008). As shown in Figure 4B, the extract weakly inhibited the XOD-catalyzed uric acid generation, and the calculated IC50 value of the extract was 639 μg/mL. The standard sample, allopurinol, provided 0.449 μg/mL of IC50 value, and also trolox weakly inhibited the XOD with 336 μg/mL of IC50 value. It is to note that the tea-leaf extract and trolox main- tain approximately 80% to 100% of the XOD activity at the con- centrations ranging from 0 to 200 μg/mL, implying almost no inhibition. IC50 values obtained in these XOD-mediated assays are compiled in Table 4. Ratio of IC50 values from both assays can be defined as a parameter indicating a mode of suppressive action. For instance, a low ratio value indicates a sample possesses strong O2− radical scavenging effect, while a value close to 1.00 means a sample possesses insufficient scavenging effect due to its enzyme inhibition. The tea-leaf extract shows 0.0324 of lower ratio value, indicating its dominant O2− radical scavenging capacity. XOD- mediated O2− radical generation assay has been used as a useful model for the discovery of both antioxidative capacity and candi- dates of inhibitors to prevent XOD-mediated disease risks (Kim and others 2004; Pacher and others 2006; Connor 2009). The yacon tea leaves may contribute to prevent O2− radical-mediated disease risks due to its O2− radical scavenging mechanism. Conclusion In this report, we demonstrated potential radical scavenging ef- fects of herbal tea leaves from yacon in different O2− radical gen- eration systems. A hot water extract found to exhibit the highest yield of total polyphenol and radical scavenging effect with its higher reducing capability. Incubating cells in the presence of the tea-leaf extract displayed suppressive effect in the O2− radical gen- eration of PMA-stimulated human neutrophilic cell model. These results may indicate herbal tea leaves from yacon are the good source of natural antioxidants effective to free radicals for health-beneficial use. In view of traditional usage of this herbal tea leaves, hydrophilic phenolic constituents, such as chlorogenic acid, caffeic acid, and feruric acid, and also possibly other chemicals may be involved in its O2− radical regulation mechanism(s). As a part of our overall study, we focused here on the functional effect of yacon tea leaves. Extensions of this work are warranted to fully elucidate the health beneficial contributions of this herbal tea leaves in suppressing the risk of O2− free radical-mediated disorders.