PaPer Ital. J. Food Sci., vol. 28 - 2016 25 - Keywords: Polygonum cuspidatum, antioxidant, polyphenol, neochlorogenic acid - IdentIfIcatIon of neochlorogenIc acId as the predomInant antIoxIdant In Polygonum cusPidatum leaves serIka kurIta, takehIro kashIwagI*, tomoyo ebIsu, tomoko shImamura and hIroyukI ukeda Faculty of Agriculture, Kochi University, B 200 Monobe, Nankoku City, Kochi prefecture, Japan *Corresponding author: Tel. +81 88 864 5184, Fax +81 88 864- 5189, email: tkashi@kochi-u.ac.jp AbstrAct to identify the predominant antioxidant compound in Polygonum cuspidatum leaves, the meth- anol extract of fresh samples were separated by liquid–liquid partitioning, octadecylsilyl sep-pak® cartridge and high-performance liquid chromatography. the main active compound was identified as (1R,3R,4S,5R)-3-{[(2E)-3-(3,4-dihydroxyphenyl)-2-propenoyl]oxy}-1,4,5-trihydroxycyclohexane- carboxylic acid (neochlorogenic acid) by nuclear magnetic resonance and liquid chromatography- mass spectroscopic analysis. Its content was found to be 2.31 mg/g of fresh leaves. As shown by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide anion scavenging assays, the con- tributions of neochlorogenic acid as an antioxidant were 16.5% and 36.5%, respectively, suggest- ing that neochlorogenic acid is the predominant antioxidant in P. cuspidatum leaves. mailto:tkashi%40kochi-u.ac.jp?subject= 26 Ital. J. Food Sci., vol. 28 - 2016 IntroDuctIon Polygonum cuspidatum, commonly known as Japanese knotweed, originated in East Asia and has spread widely to European and Ameri- can countries where it has been listed as one of the most invasive plants. In some invaded are- as it has become a severe environmental prob- lem and governmental actions have been tak- en to thwart its spread (GrEVstAD et al., 2013). However, the chemical and mechanical methods that have been used have not been successful in eliminating this plant, owing to its viability. con- trastingly, in other areas, P. cuspidatum has been used as medicine and consumed as a food. For example, in china its dried rhizomes are used in traditional chinese medicine to treat inflam- matory diseases, hepatitis, tumors, and diarrhea (cHEn et al., 2013). It is also reported that the young stems of P. cuspidatum were consumed by native people of north America (cHEn et al., 2013). In some areas of Japan, such as Kochi prefecture, the edible portions of young stems are pickled and cooked to be served as tradition- al dishes even today. the young leaves have also been recognized as edible (HAsHIMoto, 2003). over the past few decades the health-pro- moting effects of P. cuspidatum have attracted the attention of researchers and several bioac- tive compounds, particularly those with antiox- idant activity, have been identified. resveratrol, or trans-3,5,4′-trihydroxystilbene, also found in grape skins and wine, is abundant in the rhi- zomes of P. cuspidatum. numerous health-pro- moting effects of resveratrol, including antican- cer, anti-inflammatory, antiviral, and antifun- gal activities have been described (PEnG et al., 2013). Polydatin, a glycoside precursor to resver- atrol, is also found in abundance in the rhizomes of P. cuspidatum. Polydatin has been linked with beneficial lipid-regulating, melanogenesis-inhib- itory and hepatoprotective effects (PEnG et al., 2013; cHu et al., 2005). besides stilbene com- pounds, other antioxidants including anthraqui- nones, such as emodin and physcion, and flavo- noids, such as catechin and quercetin, that pos- sess health-promoting properties have also been found in the rhizomes of P. cuspidatum (cHEn et al. 2013; PEnG et al., 2003; cHu et al., 2005). Less research has been performed on the different parts of the plant. the rhizomes have been the most studied however the health-pro- moting effects of other parts of P. cuspidatum have not been studied. Although the stems and leaves are not as commonly used as the rhi- zomes, in a previous study we observed the an- tioxidant effect of the leaves was comparable to that of the rhizomes (KurItA et al., 2014). De- spite their high antioxidant capacity, only a few studies have been performed to identify antioxi- dant compounds in the leaves. In this study, we isolated and identified the predominant antioxi- dant compounds in the leaves of P. cuspidatum. MAtErIALs AnD MEtHoDs Instruments to determine 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, a tEcAn cts-r r-10 microplate reader (tEcAn, Manne- dorf, switzerland) was used. High-performance liquid chromatography (HPLc) was performed with an Lc-7100 pump, L-2300 column oven, and L-2420 uV VIs detector (Hitachi, tokyo, Ja- pan). Liquid chromatography-mass spectroscopy (Lc-Ms) was performed with a Waters AcQuItY uPLc system (Waters, Milford, usA) with a cos- mosil® 5c 18 Ar-II column (150 × 4.6 mm i.d., particle size 5 µm, pore size 12 nm), (nacalai tesque Inc., Kyoto, Japan). the mobile phase of Lc-Ms included 20% MeoH, 1% acetic acid and 79% H 2 o at a flow rate of 0.5 mL. Positive ion EsI with the capillary voltage at 3 kV was used. the source and desolvation temperatures were 150°c and 400°c, respectively, and the eluted compounds were detected at 254 nm. 1H- and 13c-nMr data for compound 1 were measured using a JEoL JnM-EcX500 (JEoL resonance Inc., tokyo, Japan) at 500 MHz. the letters (br.) s, d, t, q and m represent (broad)singlet, doublet, triplet, quartet, and multiplet, respectively, and coupling constants are expressed in Hz. specif- ic rotation was determined by Horiba sEPA-500 (HorIbA Ltd., Kyoto, Japan), and the uV spec- trum was measured with a Pharmacia biotech ultraspec 3000 uV/Visible spectrophotometer (GE Healthcare uK Ltd., buckinghamshire, uK). For the Folin–ciocalteu method, uVmini-1240 uV-Vis spectrophotometer (shimadzu, Kyoto, Japan) was used for measurement. Chemicals and reagents All reagents used were of analytical grade or better. DPPH and HPLc-grade methanol were purchased from Wako Pure chemical Indus- tries (osaka, Japan). neochlorogenic acid was obtained from sigma chemical co. (st. Louis, usA), and chlorogenic acid was from MP bio- medicals, Lcc (santa Ana, usA). Phenol re- agent solution for Folin–ciocalteu assay was purchased from nacalai tesque Inc. (Kyoto, Ja- pan). superoxide dismutase (soD) Assay Kit- Wst was purchased from Dojindo Laboratories (Kumamoto, Japan). Isolation of antioxidants from P. cuspidatum sample materials were collected in Muroto- shi, Kochi prefecture, Japan, in May 2013. the roots, stems, and leaves of P. cuspidatum were separated and extracted in an aqueous solu- tion containing 80% methanol (MeoH) for 24 h, and the extraction was repeated twice. the extract was filtered using Minisart® rc 15 sy- ringe Filters made from regenerated cellulose Ital. J. Food Sci., vol. 28 - 2016 27 with a pore size of 0.45 µm (sartorius stedium, Göttingen, Germany). twenty grams equiva- lents of fresh leaf weight (f.w.) were evaporat- ed until dry under reduced pressure (1110 mg) and subjected to liquid–liquid partitioning. the residue of the MeoH extract was dissolved in 27.7 mL of water, and the solution was parti- tioned between hexane (19.6 mL × 3) and wa- ter and then between ethyl acetate (19.6 mL × 3) and water. the hexane (53.3 mg), ethyl ace- tate (93.2 mg), and water (960 mg) layers were collected. the water layer (1 g f.w. equivalent) was applied to a sep-Pak® Plus c18 cartridge (Waters, Milford, usA), containing 360 mg of octadecylsilyl (oDs), and eluted with increas- ing concentrations of MeoH to obtain four frac- tions: 0% MeoH (25.6 mg), 20% MeoH (8 mg), 40% MeoH (3.6 mg), and 100% MeoH (trace amount) fractions. the oDs 20% MeoH frac- tion was further separated into six fractions by reverse-phase semipreparative HPLc (cosmos- il® 5c 18 Ar-II column, 250 × 10 mm i.d., parti- cle size 5 µm, pore size 12 nm, nacalai tesque Inc.) and eluting with 20% MeoH containing 1% acetic acid at a flow rate of 3 mL/min and detected at 254 nm. compound 1 was isolated from fraction 2, and its structure is (1r,3r,4s,5r)-3-{[(2E)-3-(3,4- dihydroxyphenyl)-2-propenoyl]oxy}-1,4,5-trihy- droxycyclohexanecarboxylic acid, neochlorogen- ic acid. [α] D 20 + 12.00° (c = 0.02, MeoH). uV λ max (MeoH) nm (ε): 238.5 (5383), 324.2 (8729). Pos- itive-ion EsI-Ms: m/z 355 [M+H]+, 163 [M-quin- ic acid]+. nMr spectral data were as follows. 1H- nMr (500 MHz, DMso-d 6 ) δ: 7.44 (d, 1H, J = 16.0 Hz, H c -3), 7.00 (d, 1H, J = 2.5 Hz, H c -2′), 6.93 (dd, 1H, J = 8.0, 2.5, Hz, H c -6′), 6.75 (d, 1H, J = 8.0 Hz, H c -5′), 6.21 (d, 1H, J = 16.0 Hz, H c - 2), 5.16 (dt, 1H, J = 3.5, 8.5 Hz, H Q -3), 3.84 (dt, 1H, J = 7.5, 4.0 Hz, H Q -5), 3.15 (m, 1H, H Q -4), 2.00 (dd, 1H, J = 15.0, 4.0 Hz, H Q -2′), 1.89 (dd, 1H, J = 15.0, 7.5Hz, H Q -2), 1.83 (m, 2H, H Q -6). 13c-nMr (125 MHz, DMso-d 6 ) δ: 176.1 (c Q -7, s), 166.2 (c c -1, s), 148.2 (c c -4’, s), 145.6 (c c -3’, s), 144.5 (c c -3, d), 125.9 (c c -1’, s), 121.2 (c c -6’, d), 115.9 (c c -5’, d), 115.2 (c c -2, d), 114.6 (c c - 2’, d), 73.1 (c Q -1, s), 71.6 (c Q -5, d), 71.1 (c Q -3, d), 67.2 (c Q -4, d), 39.5 (c Q -2, t), δ 35.2 (c Q -6, t). Structural determination of compound 1 the structure of compound 1 was estab- lished by independent injection and co-injec- tion of fraction 2 with an authentic prepara- tion in HPLc to confirm the retention times. the following conditions were used to identi- fy the compound found in fraction 2: a cos- mosil® 5c 18 Ar-II column (150 × 4.6 mm i.d., particle size 5 µm, pore size 12 nm, nacalai tesque Inc.) was used with a mobile phase of 20% MeoH containing 1% acetic acid at a flow rate of 0.5 mL/min, and uV detection was set at 254 nm. Determination of total phenolic content the polyphenol content of P. cuspidatum leaves was determined by the Folin-ciocalteu method as described by singleton et al. with some modi- fications (sInGLEton et al., 1999). In a test tube, 0.25 mL of sample solution, 0.1 mL of phenol re- agent (1.8 n), and 0.25 mL of saturated sodium carbonate were added within 15 s and mixed. then, 2.15 mL of water was added and mixed, followed by 1 h of incubation at room tempera- ture. After incubation, the sample was measured at 725 nm. the measured value for the crude extract was expressed as gallic acid equivalent (GAE) per gram of the sample material. DPPH radical scavenging activity assay Antioxidant activity was measured using the DPPH method as described in our previous study (KurItA et al., 2014). In a 96-well plate, 20 µL of sample solution, 80 µL of 0.1 M tris-Hcl buffer (pH 7.4), and 0.2 mM DPPH in ethanol solution were added and mixed. the mixture was incu- bated in the dark at room temperature for exact- ly 30 min. the radical scavenging rates of each sample and a control solution were measured at 517 nm. All experiments were performed in triplicate. the radical scavenging rate was cal- culated using following equation: scavenging rate (%) = = (A control – A sample ) / A control × 100 where A control is the absorbance of the control and A sample is that of the sample. sc 50 , which is the sam- ple concentration at 50% of the scavenging ratio, was used to express the antioxidant capacity of each sample. to determine the contribution rate, sc 50 was then converted to 6-hydroxy-2,5,7,8-te- tramethylchroman-2-carboxylic acid (trolox) equiv- alent (tE) antioxidant capacity, tEAc, using the following equation (sHIMAMurA et al., 2014): tEAc (mg tE/mg) = trolox sc 50 (mg/mL)/ /sample sc 50 (mg /mL) the contribution rate of the active compound was calculated using the following equation: contribution rate (%) = (tEAc of active com- pound × concentration of active compound in P. cuspidatum) / (tEAc of crude extract) × 100 Superoxide anion scavenging assay A soD Assay Kit-Wst was used to determine the superoxide scavenging activity (sosA) of each sample. the assay was performed accord- ing to the manufacturer’s procedure. the result- ing 50% inhibitory concentration (Ic 50 ) was used to determine the sosA, which was further used to evaluate the contribution of compound 1 to 28 Ital. J. Food Sci., vol. 28 - 2016 the total antioxidative capacity. sosA was de- fined using following equation: sosA (unit/g) = [1/Ic 50 (mg/mL)] × 0.02 mL × × 1000 mg/g the contribution rate from sosA was calcu- lated using the following equation: contribution rate (%) = (sosA of active com- pound × concentration of active compound in P. cuspidatum) / (sosA of crude extract) × 100 rEsuLts Antioxidant capacities of different parts of P. cuspidatum All results of DPPH radical scavenging activity assays had relative standard deviations (rsD) of < 5%. Among the MeoH extracts of the different parts of P. cuspidatum, the strongest activity was observed in the leaves (sc 50 : 1.24 mg f.w./mL), followed by the rhizomes (sc 50 : 1.63 mg f.w./mL) and stems (sc 50 : 14.1 mg f.w./mL). this is con- sistent with our previous study which also found that the leaves and rhizomes showed almost equiv- alent antioxidant capacities (KurItA et al. 2014). Fractionation and antioxidant activity of the leaf extract the fractionated leaf extracts and antioxidant activities are shown in Fig. 1. Among all the lay- ers, the water layer showed the highest activity (sc 50 : 1.90 mg f.w./mL), followed by the ethyl ac- etate layer (sc 50 : 13.2 mg f.w./mL). the separat- ed hexane, ethyl acetate, and water layers were further combined for measurement. the com- bined sample yielded an sc 50 of 1.8 mg f.w./mL, although some activity had been lost in the sep- aration process. the combinations of the hexane and water layers (sc 50 : 1.93 mg f.w./mL), and the ethyl acetate and water layers (sc 50 : 1.69 mg f.w./mL) were measured and compared with the water layer only. the results suggest that the antioxidants were present mainly in the water layer because the activities of these combina- tions were close to that of the water layer only. Antioxidant activities were observed in the oDs water and in the 20% and 40% MeoH fractions (Fig. 1). the oDs 20% MeoH fraction showed the highest activity, yielding an sc 50 of 4.3 mg f.w./ mL. All the fractions combined had an sc 50 of 1.55 mg f.w./mL. When the oDs 20% MeoH fraction was combined with the second highest fraction, the oDs water fraction (sc 50 : 5.56 mg f.w./mL), the sc 50 of the combined sample was 1.68 mg f.w./mL. this suggests that the oDs water and the 20% MeoH fractions account for the majori- ty of the antioxidant capacity of the water layer. the oDs 20% MeoH fraction was further fractionated by reversed phase semipreparative HPLc, and the chromatogram is shown in Fig. 2. the highest antioxidant capacity was seen in fraction 6 (sc 50 : 22.4 mg f.w./mL), followed by fraction 1 (sc 50 : 32.4 mg f.w./mL) and fraction 2 (sc 50 : 36.1 mg f.w./mL). A further HPLc anal- ysis with multiple-wavelength detection using a sPD-M10A photodiode array detector (shimadzu, Kyoto, Japan) detected no other distinct peaks in fraction 1 or 6. Fractions 1 and 6 were further separated to isolate and identify the compound; however, the antioxidant activity was dispersed during the process. In contrast fraction 2, which exhibited relatively high antioxidant activity, con- tained a major single peak at the retention time of 10.07 min. this major peak was assigned as com- pound 1, which was further purified. compound Fig. 1 - the separation process of the leaf extract and the antioxidant capacity of each fraction. Ital. J. Food Sci., vol. 28 - 2016 29 1 was present not only in the oDs 20% MeoH fraction but also in the oDs water fraction, which showed the second highest antioxidative activi- ty among the oDs fractions. In the oDs water fraction, compound 1 was also found abundant- ly and accordingly was inferred to be the major compound in the water layer of the leaf extract. Identification of compound 1 compound 1 was found to have sixteen car- bon atoms consisting of two methylene, eight methine, and six quaternary carbon atoms in- cluding two carbonyl groups (c Q -7, δ 176.1 and c c -1, δ 166.2) as a result of 13c-nMr. this result was consistent with 1H-nMr, which showed the presence of twelve hydrogen atoms in the spec- trum. this compound contains a trans-form double bond (c c -2, δ 115.2 and c c -3, δ 144.5) signified by two hydrogen signals (δ 6.21 and δ 7.44) corresponding to a double bond where dou- blet with 16 Hz coupling constant. this double bond and a carbonyl group (c c -1, δ176.1) were observed to be conjugated, consistent with these chemical shifts and the result from Heteronu- clear Multiple bond correlation (HMbc). be- cause the observed six aromatic carbons in the 13c-nMr spectrum corresponded to an AbX sys- tem at δ 6.75 (Hc-5′, d, J = 8 Hz), δ 6.93 (Hc-6′, dd, J = 2, 8 Hz), and δ 7.00 (Hc-2′, d, J = 2 Hz) in 1H-nMr, compound 1 was found to contain a 1,2,4-trisubstituted benzene ring. For the above- mentioned reasons, compound 1 was inferred to contain a caffeic acid moiety. In the rest of the structure, three methine car- bon atoms with oxygen atoms, one quaternary carbon, two methylene carbon atoms, and one carbonyl carbon were found. two-dimensional nMr spectral data imply a six-membered ring substituted with four oxygen atoms. the meth- ylene proton at δ 1.85 and the carbonyl carbon (c Q -7, δ 176.1) were interrelated in HMbc spec- troscopy. the other moiety was thus determined to be a quinic acid derivative. the proton corresponding to the carbon of quinic acid (c Q -3, δ 71.1) showed a downfield shift at 5.16 ppm, suggesting that this com- pound formed a caffeate ester. the molecular formula of a caffeoylquinic acid is c 16 H 18 o 9 and its molecular weight is calculated to be 354. based on the EsI mass data (m/z 355 [M+H]+), the molecular weight of compound 1 was found to be 354; therefore, compound 1 was assigned the molecular formula c 16 H 18 o 9. the data in the literature from 13c-nMr and 1H-nMr studies on chlorogenic acid (5-caffeoylquinic acid), cryp- tochlorogenic acid (4-caffeoylquinic acid) and neochlorogenic acid (3-caffeoylquinic acid) were compared with our observed data and most of the values for compound 1 matched with those of neochlorogenic acid (Fig. 3) (QIn et al., 2006; HYun et al., 2010). the specific rotation value of compound 1 was also consistent with that of a neochlorogenic acid standard. to determine the structure, fraction 2 was fur- ther analyzed using HPLc under the conditions described in Determination of structure of com- pound 1, and the result is shown in Fig. 4. the peak of compound 1 was observed at 8.1 min (Fig. 4a). the retention time of neochlorogenic acid was clearly different from that of chlorogenic acid; the neochlorogenic acid peak appeared at 8.04 min, whereas the peak of chlorogenic acid appeared at 16.67 min (Fig. 4b and 4c). co-injection analysis showed that the peak of compound 1 was iden- tical to that of neochlorogenic acid. Accordingly, compound 1 was assigned as neochlorogenic acid. Quantification of neochlorogenic acid and its contribution to the whole leaf extract the leaves of P. cuspidatum were freshly collect- ed in otoyo-cho in May 2014 to determine neo- chlorogenic acid content. one gram of fresh P. cus- Fig. 2 - the chromatogram of oDs 20% MeoH fraction of the leaf extract. the sc 50 of Fr. 1, 2, 3, and 6 were 32.4, 36.1, 62.4 and 22.4 mg f.w./mL, respectively. Fr. 4 and 5 was not determined since their sc 50 were over 200 mg f.w./mL. 30 Ital. J. Food Sci., vol. 28 - 2016 pidatum leaves contained 2.31 mg of neochloro- genic acid. by the Folin–ciocalteu method, 17.9 mg GAE of phenolic compounds were found to be present in the fresh leaves; thus, neochlorogen- ic acid comprises 12.8% of the total polyphenol content. to evaluate the antioxidant capacity of neochlorogenic acid in P. cuspidatum two differ- ent assays, each measuring the sample’s ability to quench reactive oxygen species in a different way, were performed. Antioxidant capacity can- not be evaluated by a single method because reac- tive oxygen species in the body do not always op- erate through the same mechanisms. the assays we used in this study were the DPPH radical scav- enging (tEAc) and superoxide anion scavenging assays (sosA). the tEAc values of the crude ex- tract and neochlorogenic acid were 59.7 mg tE/g f.w. and 4.25 mg tE/mg, respectively, indicating the neochlorogenic acid contribution is 16.5%. Fig. 3 - the structures of neochlorogenic acid and chloro- genic acid. However, by the superoxide anion scavenging as- say, the sosA values of the crude extract and ne- ochlorogenic acid were 22.7 unit/g f.w. and 3.57 unit/mg, respectively, suggesting 36.5% of the an- tioxidant activity is by neochlorogenic acid. the disparate results may be explained by the differ- ent mechanisms of the two antioxidant activities (sHIMAMurA et al., 2007). In the DPPH method, free radical scavenging activity is achieved by sin- gle electron transfer, and the assay simply meas- ures the rate of free radical quenching. the super- oxide anion scavenging assay, however, measures the sample’s ability to scavenge superoxide ani- ons produced by xanthine oxidase, thus evaluat- ing the soD-like activity of the sample. the su- peroxide anion further reduces 2-(4-iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetra- zolium (Wst-1) to produce formazan, which is de- tectable at a wavelength of 450 nm. thus, the su- peroxide anion scavenging assay involves compe- tition by the sample antioxidants with Wst-1 in addition to the enzymatic reactions of xanthine oxidase. taken together our results indicate that neochlorogenic acid in P. cuspidatum contributes a large part of its antioxidant activity, particular- ly as a superoxide anion scavenger. In a study by Kirino et al. (2012) chlorogenic acid was reported as one of the major polyphe- nols in the leaves of P. cuspidatum (KIrIno et al., 2012). the amount of chlorogenic acid was re- ported to be 0.36 mg/g of fresh leaves, which is only 1/6th of the neochlorogenic acid content observed in this study. In the chromatogram in Fig. 4, chlorogenic acid appeared to be a small peak in the water layer of the leaf extract. How- ever, according to the data from Kirino et al., the peak of chlorogenic acid was much more distinct than in our study. the contents of such antioxi- dants in P. cuspidatum may differ depending on its origin and harvest season, as we mentioned in a previous report (KurItA et al., 2014). stress factors such as sunlight and insects can influ- ence antioxidant production levels as well. Comparison of neochlorogenic acid contents in other food sources and its possible effects on human health to the best of our knowledge, this is the first study to report the presence of neochlorogen- ic acid in P. cuspidatum leaves. neochlorogen- ic acid is also found in rosaceae fruits such as plums, cherries, and apples and brassica veg- etables such as broccoli and kale (bALLIstrErI et al., 2013; KIM et al., 2003; KAuLMAnn et al., 2014). Among different kinds of sweet cherries, its content varied between 6.27–71.5 mg/100 g f.w. (bALLIstrErI et al., 2013). Plums contain even higher amounts of up to 179 mg/100 g f.w., un- surprisingly neochlorogenic acid has been recog- nized as the predominant polyphenol in plums (KIM et al., 2003). brassica vegetables are also rich in the compound. Green vegetables such Fig. 4 - the chromatogram of the water layer of the leaf ex- tract, neochlorogenic acid and chlorogenic acid. In the water layer of leaf extract (A), compound 1 was ob- served at 8.11 min. neochlorogenic acid (b) was found at 8.04 min whereas chlorogenic acid (c) was at 16.67 min. Ital. J. Food Sci., vol. 28 - 2016 31 as kale, broccoli, and brussels sprouts contain 7.06, 5.61, and 4.59 mg/100 g f.w., respectively, of neochlorogenic acid (KAuLMAnn et al., 2014). In comparison with these neochlorogenic-rich fruits and vegetables, the content was much higher in the leaves of P. cuspidatum, which yielded 231 mg of neochlorogenic acid per 100 g of fresh materi- al. our study suggests that the leaves of P. cusp- idatum are a rich source of neochlorogenic acid. besides its antioxidant activity, neochlorogen- ic acid has been shown to exert health-promot- ing effects. As an antitumor agent, neochlorogen- ic acid has been found to suppress the growth of estrogen-independent MDA-Mb-435 breast cancer cells (norAtto et al., 2009). this sup- pressive effect is selective for cancer cells and is more pronounced than that of chlorogenic acid. the compound has also been investigated in a weight-control study (sHIMoDA et al., 2006). In the study performed by shimoda et al. (2006), experimental mice were fed a diet containing ne- ochlorogenic acid (0.028% and 0.055%, respec- tively) extracted from green coffee beans for 6 days. the hepatic carnitine palmitoyltransferase activity of the experimental mice increased, indi- cating they had improved fat metabolism. these studies suggest that neochlorogenic acid could play a role in preventing chronic diseases and preserving healthy body weight when consumed in the diet. As a natural source of neochlorogen- ic acid, the leaves of P. cuspidatum may be used to improve human health in modern society. concLusIons For their medicinal effects the antioxidants in P. cuspidatum have been of interest to research- ers, but other than the rhizomes the plant has not been extensively studied. the leaves possess high antioxidant activity and can be consumed in the diet as they currently are in Japan. Given the re- ports of health-promoting effects of neochlorogenic acid, our result that neochlorogenic acid is a main antioxidant in the leaves of P. cuspidatum may in- crease the utility of this hardy and prolific plant. AcKnoWLEDGEMEnts We thank soichiro ueta from nPo sakihama Genki project for providing the samples for our study. rEFErEncEs ballistreri G., continella A., Gentile A., Amenta M., Fabro- ni s. and rapisarda P. 2013. Fruit quality and bioactive compounds relevant to human health of sweet cherry (Prunus avium L.) cultivars grown in Italy. Food chem. 140(4): 630-8. chen H., tuck t., Ji X., Zhou X., Kelly G., cuerrier A. and Zhang J. 2013. Quality assessment of Japanese knot- weed (Fallopia japonica) grown on Prince Edward Island as a source of resveratrol. J. Agric. Food chem. 61(26): 6383-92. chu X., sun A. and Liu r. 2005. Preparative isolation and purification of five compounds from the chinese medic- inal herb Polygonum cuspidatum sieb. et Zucc by high- speed counter-current chromatography. J. chromatogr. A. 1097(1-2): 33-9. Grevstad F., shaw r., bourchier r., sanguankeo P., cortat G. and reardon r.c. 2013. Efficacy and host specificity compared between two populations of the psyllid Aphalara itadori, candidates for biological control of invasive knot- weeds in north America. biol. control. 65(1): 53-62. Hashimoto I. 2003. “Wild food lexicon, Japan: a unique photographic guide to finding cooking and eating wild plants ferns and lichen” 1st ed. p. 149. Kashiwashobo co. Ltd., tokyo. Hyun s.K., Jung H. A., Min b-s., Jung J.H. and choi Js. 2010. Isolation of Phenolics, nucleosides, saccharides and an Alkaloid from the root of Aralia cordata. nat. Prod. sci. 16(1): 20-25. Kim D.o., chun o.K., Kim Y.J., Moon H.Y. and Lee c.Y. 2003. Quantification of polyphenolics and their anti- oxidant capacity in fresh plums. J. Agric. Food chem. 51(22): 6509-15. Kaulmann A., Jonville M.c., schneider Y.J., Hoffmann L. and bohn t. 2014. carotenoids, polyphenols and micro- nutrient profiles of brassica oleraceae and plum varie- ties and their contribution to measures of total antioxi- dant capacity. Food chem. 155: 240-50. Kirino A., takasuka Y., nishi A., Kawabe s., Yamashita H., Kimoto M., Ito H. and tsuji H. 2012. Analysis and func- tionality of major polyphenolic components of Polygo- num cuspidatum (itadori). J. nutr. sci. Vitaminol. 58(4): 278-86. Kurita s., Kashiwagi t., Ebisu t., shimamura t. and uke- da H. 2014. content of resveratrol and glycoside and its contribution to the antioxidative capacity of Polygonum cuspidatum (Itadori) harvested in Kochi. biosci. biotech. bioch. 78: 499-502. noratto G., Porte W., byrne D. and cisneros-Zevallo L. 2009. Identifying peach and plum polyphenols with chemopre- ventive potential against estrogen-independent breast cancer cells. J. Agric. Food chem. 57(12): 5219-26. Peng W., Qin r., Li X. and Zhou H. 2013. botany, phyto- chemistry, pharmacology, and potential application of Polygonum cuspidatum sieb.et Zucc.: A review. J. Eth- nopharmacol. 148(3): 729-745. Qin L., Han t., Li H., Zhang Q. and Zheng H. 2006. A new thiazinedione from Xanthium strumarium. Fitoterapia. 77(3): 245-6. shimamura t., Matsuura r., tokuda t., sugimoto n., Yamazaki t., Matsufuji H., Matsui t., Matsumoto K. and ukeda H. 2007. comparison of conventional anti- oxidants assays for evaluatingpotencies of natural an- tioxidants as food additives by collaborative study. nip- pon shokuhin Kagaku Kogaku Kaishi (in Japanese). 54: 482-487. shimamura t., sumikura Y., Yamazaki t., tada A., Kashi- wagi t., Ishikawa H., Matsui t., sugimoto n., Akiyama H. and ukeda H. 2014. Applicability of the DPPH assay for evaluating the antioxidant capacity of food additives – inter-laboratory evaluation study –. Analytical scienc- es. 30(7): 717-721. shimoda H., seki E. and Aitani M. 2006. Inhibitory effect of green coffee bean extract on fat accumulation and body weight gain in mice. bMc complement. Altern. Med. 6: 9. singleton V.L., orthofer r. and Lamuela-raventons rM. 1999. Analysis of total phenols and other oxidation sub- strates and antioxidants by means of Folin-ciocalteu re- agent. Methods Enzymol. 299: 152-178. Paper Received November 5, 2014 Accepted February 19, 2015