Food Chemistry 129 (2011) 804–809

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Food Chemistry

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Antioxidant properties of Lactobacillus-fermented and non-fermentedGraptopetalum paraguayense E. Walther at different stages of maturity

She-Ching Wu ⇑, Yi-Shun Su, Huang-Yu ChengDepartment of Food Science, National Chiayi University, No. 300 Syuefu Rd., Chiayi City 60004, Taiwan, ROC

a r t i c l e i n f o

Article history:Received 29 August 2010Received in revised form 22 March 2011Accepted 4 May 2011Available online 17 May 2011

Keywords:Graptopetalum paraguayense E. WaltherMaturityGallic acidQuercetinLactobacillus plantarum

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.05.025

⇑ Corresponding author. Tel.: +886 5 2717622; fax:E-mail address: scwu@mail.ncyu.edu.tw (S.-C. Wu

a b s t r a c t

Accumulation of bioactive compounds, during developmental stages of Graptopetalum paraguayenseE. Walther, was investigated between 30 and 90 days as a function of physiological maturity. Three dis-tinct phases were defined: immature phase (30 days), intermediate developmental phase (30–60 days),and maturation phase (60–90 days). Gallic acid and quercetin, antioxidative bioactive compounds, wereidentified as biomarkers for determining the optimum physiological maturity stage in G. paraguayenseE. Walther. With regard to the antioxidant activity of G. paraguayense E. Walther at different developmen-tal stages, the results indicated that the leaves of immature G. paraguayense E. Walther had the highest2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS-), superoxide radical-, and 1,1-diphenyl-2-picrylhydrazyl (DPPH�)-scavenging activities. Fermentation of G. paraguayense E. Walther withLactobacillus plantarum BCRC 10357 significantly increased the level of flavonoids and total phenolics,including quercetin and gallic acid. Total phenols were the major naturally occurring antioxidant compo-nents in lactic acid bacteria-fermented G. paraguayense E. Walther.

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1. Introduction

During fermentation of foods, desirable biochemical changesand significant modifications in flavour and texture are broughtabout through the activity of microorganisms or enzymes. Lacticacid bacteria are important starter cultures for food fermentation;the characteristic feature of fermentation by lactic acid bacteria isthe production of different organic acids due to the degradation ofsome components in the raw material and the resultant decreasein pH. The beneficial health effects of probiotic bacteria in food in-clude protection against gastrointestinal infections, reduction inserum cholesterol levels, and stimulation of the immune system(Leroy & De-Vuyst, 2004; Vinderola & Reinheimer, 2003). Tradi-tionally, probiotic products are usually marketed in the form offermented milks and yogurts; however, with an increase in vege-tarianism amongst consumers throughout developed countries,there is also a demand for vegetarian probiotic products. Moreover,lactose intolerance and cholesterol content are the 2 drawbacksassociated with fermented dairy products. Hence, there is increas-ing interest in a wide variety of non-dairy probiotic beverages;many are non-alcoholic beverages manufactured with fruits andvegetables as the principal raw material (Ouwehand, Derrien,De-Vos, Tiihonen, & Rautonen, 2005).

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+886 5 2717590.).

A study on lactic acid bacteria and bifidobacteria has shownthat implementation of probiotic strains (in fruit- and vegetable-based media) may also increase health benefits (Parker, 1974). Inthe Korean fermented vegetable, kimchi, which showed strongantipathogenic activity during fermentation, the number of lacticacid bacteria increased from 105 to 108. Studies have demonstratedthe ability of Lactobacillus pentosus and Leuconostoc mesenteroidesto improve mineral availability during carrot juice fermentation,and have observed an increase in free radical-scavenging activityduring fermentation of carrot juice by Lactobacillus bulgaricus andLactobacillus rhamnosus, for increasing 1,1-diphenyl-2-pic-rylhydrazyl (DPPH) radical-scavenging activity (Bergqvist,Sandberg, Carlsson, & Andlid, 2005; Nazzaro, Fratinni, Sada, & Or-lando, 2008). In addition, Chen, Ng, Wang, and Chang (2009) foundthat the level of antioxidative activity varied, depending on thestarter organism used. In their case study, they reported that theantioxidative activities of ginger juice fermented withBifidobacterium longum were better than those of ginger juicesfermented with lactobacilli.

Graptopetalum paraguayense E. Walther is a tropical and sub-tropical plant that was originally cultivated in Mexico. Accordingto an ancient Chinese prescription, G. paraguayense has severalhealth benefits: it reduces blood pressure, alleviates hepatic disor-ders, and acts as a diuretic. Recently, Chung, Chen, Hsu, Chang, andChou (2005) found that G. paraguayense contains various potentialantioxidants, which may help reduce oxidative damages that occurin the human body and prevent lipid peroxidation in foods. In

S.-C. Wu et al. / Food Chemistry 129 (2011) 804–809 805

addition to the antioxidative activity of G. paraguayense, severalstudies have also demonstrated that G. paraguayense has variousbiological properties, including inhibition of mushroom tyrosinaseand angiotensin-converting enzyme activity, and production of anantimutagenic effect (Chen, Chang, Chung, & Chou, 2007; Chunget al., 2005; Huang, Chen, Chang, & Chou, 2005). However, thereis no information in the literature on the fermentation ofG. paraguayense juice with probiotic bacteria.

Therefore, the aim of this study is to investigate the use ofG. paraguayense plants, at different stages of maturity, as a rawmaterial for the production of antioxidants by fermentation withlactic acid bacteria.

Fig. 1. Different stages of growth and development of G. paraguayense E. Walther.

2. Materials and methods

2.1. Chemicals and reagents

2,20-Azino-bis (3-ethylbenzthiasoline-6-sulphonic acid) (ABTS+�),1,1-diphenyl-2-picryl hydrazyl (DPPH�), aluminium nitrate, butyl-ated hydroxyl anisole (BHA), ferrous chloride, Folin–Ciocalteu’s re-agent, caffeic acid, chlorogenic acid, rutin, quercetin, kaempferol,gallic acid, peroxidase, nitro bluetetrazolium (NBT), phenazinemethosulphate (PMS), dihydronicotinamide-adenine dinucleotide(NADH), sodium n-itroprusside (SNP), sulphanilamide, H3PO4,and N-(1-naphthyl)-ethylenediamine dihydrochloride were pur-chased from Sigma Corporation (St. Louis, MO, USA). Potassiumdihydrogen phosphate (KH2PO4) and dipotassium hydrogen phos-phate (K2HPO4) were purchased from Merck Corporation (Darms-tadt, Germany); 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) was purchased from Aldrich Corporation(Milwaukee, USA).

2.2. Plant material

Graptopetalum paraguayense E. Walther was purchased from theWeisen Organic Farm, Chayi, Taiwan. The different maturity stagesof G. paraguayense E. Walther were according to leaf size (diame-ter); immature (53 cm; 30 days), intermediate mature (3–4 cm;30–60 days) and mature (=4 cm; 60–90 days) (Fig. 1). After arrivalat the laboratory, fresh leaves from the plants were detached andwashed with distilled water. Afterwards, G. paraguayenseE. Walther juice was obtained, using a commercial food processor(CookPot JF-102; Taipei, ROC) at room temperature.

2.3. Microbial material

Strains of Lactobacillus acidophilus BCRC 10695, Lactobacillusplantarum BCRC 10357, and Lactobacillus paracasei BCRC 14023were purchased from the Bioresources Collection and ResearchCenter (BCRC), Taiwan.

2.4. Inoculation and fermentation

The non-fermented G. paraguayense E. Walther juice was fil-tered and freeze-dried. L. acidophilus BCRC 10695, L. plantarumBCRC 10357 and L. paracasei BCRC 14023 were used as fermenta-tion starters; they were subcultured twice on a MRS plate (BD Dif-co, NJ, USA) of broth prior to inoculation at 37 �C according to theinstructions in the user manual. The starters were cultured in MRSbroth for 18 h at 37 �C and reached an OD600 of 0.8, equivalent to105 CFU/ml. They were used in quantity, inoculated into 100 mlof G. paraguayense E. Walther juice. The fermentation processwas performed at 37 �C for 72 h. The G. paraguayense E. Walther-fermented juice was filtered and centrifuged (8000g) to remove

bacteria, and then the fermented-supernatant was freeze-driedand stored at �20 �C.

2.5. Total antioxidant activity

The antioxidant capacity of G. paraguayense E. Walther wasmeasured, using the method of Miller and Rice-Evans (1997) andArnao, Cano, and Acosta (2001). Peroxidase, H2O2, ABTS, and dis-tilled water were mixed and stored in the dark for 1 h at 25 �C. Asample was subsequently added and the absorbance at 734 nmwas determined. The antioxidant capacity was calculated by thefollowing formula:

Total antioxidant activity ð%Þ¼ ½1� ðA734 nm sample=A734 nm controlÞ� � 100%

2.6. Reducing power

The reducing power was measured using the approach of Duhand Yen (1997). Sample, phosphate buffer, and potassium hexacy-anoferrate solution were mixed and heated at 50 �C for 20 min andtrichloroacetic acid was added. Following centrifugation at 3000gfor 10 min, the supernatant was mixed with distilled water andferric chloride, and the reaction was then maintained for 10 min.The absorbance at 700 nm was measured.

2.7. DPPH free radical-scavenging activity

The DPPH� removal activity was measured by the method ofShimada, Fujikawa, Yahara, and Nakamura (1992). Briefly, sampleand DPPH� methanolic solution were mixed and kept in the darkfor 60 min. The absorbance of the reaction mixture at 517 nmwas measured. The percentage of free radical-scavenging activitywas calculated as follows:

Scavenging effect ð%Þ ¼ 1� ðA517 nm sample=A517 nm blankÞ½ �� 100%

2.8. Scavenging effect on superoxide anion radicals

The method used was that of Robak and Gryglewski (1998).Phenazine methosulphate, a-nicotinamide-adenine-dinucleotideand nitro blue tetrazolium (NBT) solution were mixed with sampleand reacted for 5 min. Absorbance of 560 nm was measured andthe percentage of free radical-scavenging activity was calculatedas follows:

806 S.-C. Wu et al. / Food Chemistry 129 (2011) 804–809

Scavenging effect ð%Þ ¼ 1� ðA560 nm sample=A560 nm blankÞ½ �� 100%

Table 1The total phenolic acid and flavonoid levels of non-fermented and Lactobacillus-fermented G. paraguayense E. Walther water extracts.

Maturity stage lg/mg of extract

Flavonoid Total phenolics

Non-fermentationImmature 17.2 ± 0.14aC 92.2 ± 0.17aB

Intermediate mature 13.3 ± 0.18b 44.2 ± 0.22b

Mature 11.9 ± 0.29b 33.2 ± 0.28c

L. acidophilus BCRC 10695Immature 19.0 ± 0.22aB 99.5 ± 0.38aB

Intermediate mature 14.6 ± 0.35b 51.5 ± 0.26b

Mature 13.5 ± 0.32b 38.9 ± 0.15c

L. plantarum BCRC 10357Immature 22.9 ± 0.28aA 111 ± 0.28aA

Intermediate mature 17.6 ± 0.24b 58.8 ± 0.31b

Mature 15.6 ± 0.26b 39.2 ± 0.22c

L. paracasei BCRC 14023Immature 18.6 ± 0.21aB 94.2 ± 0.31aC

Intermediate mature 14.3 ± 0.25b 47.7 ± 0.30b

Mature 12.2 ± 0.17b 32.3 ± 0.21c

Each value represents the mean ± SD (n = 3). Different letters are significantlydifferent at P < 0.05. a–c: Significant difference in each column. A–C: significantdifference in immature of each group.

2.9. Assay for total phenolic acid and flavonoids

The flavonoid contents were determined according to the mod-ified method of Jia, Tang, and Wu (1999). Sample was mixed withwater, Al(NO3)3 and CH3COOK. The mixture was left in the darkroom for 40 min, the absorbance was measured against blank at415 nm, using a spectrophotometer (Analytikijena 200–2004 spec-trophotometer). Quercetin was used for the standard calibrationcurve and the flavonoid contents in the samples were calculatedusing the linear equation based on the calibration curve. Phenoliccompounds were estimated using the method described bySingleton and Rossi (1965), with modifications, and gallic acid asa standard phenolic compound. Briefly, the sample was mixed withFolin and Ciocalteu’s phenol reagent and sodium carbonatesolution (7.5%) was added and left for 90 min, the absorbancewas measured at 760 nm. The results were expressed as milligramsof gallic acid equivalents per gram of extract.

2.10. High-performance liquid chromatography (HPLC) assay

HPLC performed with a Hitachi liquid chromatograph (Hitachi,Ltd., Tokyo, Japan) consisting of a model L-6200 pump, and a modelL-4200 UV–Vis detector set at 320 nm. The analyses were carriedout on a LiChrospher RP-18 column (250, 4.6 mm i.d., 5 lm, E.Merck Co., Darmstadt, Germany). Extracts were filtered through a0.45 lm filter before use. The mobile phase A was 2% acetic acid,and the mobile phase B was 0.5% acetic acid/water (1:1; v/v).Caffeic acid, chlorogenic acid, rutin, quercetin, kaempferol, gallicacid, oxalic acid and hydroxybutanedioic acid were determinedby ultraviolet detector (Hitachi L-7455 diode array detector).

2.11. Cell culture

The mouse FL83B normal liver cell line was obtained from theBioresource Collection and Research Center (BCRC) in Taiwan(Hsinchu, Taiwan). Cells were maintained in F12-K media supple-mented with 10% FBS, 1.5 g sodium bicarbonate, and 1% antibi-otic/antimitotic solution in 1 litre, and incubated in 5% CO2, 95%humidified atmosphere at 37 �C. After treatment with fermented-or non-fermented G. paraguayense E. Walther for 3 h, cells werewashed with PBS and suspended in 250 ll cell lysis buffer to incu-bate on ice for 10 min, centrifuged (10,000g) for 10 min, and super-natant transferred (cytosolic extract) to a fresh tube and put on icefor immediate assay.

2.12. Assay for antioxidant enzymes

Glutathione peroxidase (GPx) activity was determined as previ-ously described (Mohandas, Marshall, Duggin, Horvath, & Tiller,1984). Briefly, supernatant was mixed with potassium phosphatebuffer for 5 min. After adding hydrogen peroxide (H2O2), GPx activ-ity was calculated by the change of the absorbance at 340 nm for5 min. Another reaction mixture containing phosphate buffer wasadded to the supernatant for glutathione reductase (GR) activitydetermination (Bellomo et al., 1987). The catalase (CAT) activitywas determined by the method of Aebi (1984). SOD activity wasdetermined by the method of Marklund and Marklund (1974).

2.13. Statistical analysis

Experimental results were averages of triplicate analyses. Thedata were recorded as means ± standard deviation and analysis

was by a statistical analysis system (SAS Inc., NC, USA). One-wayanalysis of variance was performed by ANOVA procedures. Signif-icant differences between means were determined by Duncan’smultiple range tests. Results were considered statistically signifi-cant at p < 0.05.

3. Results and discussion

3.1. Yield from G. paraguayense E. Walther at different stages ofmaturity

There were no significant differences in the yields of waterextracts of immature, intermediate and mature G. paraguayenseE. Walther plants, which were 8.4%, 8.7%, and 8.5%, respectively.In the present study, the water content obtained from G.paraguayense, at all stages of maturity was greater than 96%.

3.2. Flavonoids and total phenolic acid

The flavonoid and phenolic contents in G. paraguayense E.Walther were quantified at different developmental phases of theplant. As shown in Table 1, the levels of flavonoids in water ex-tracts of immature, intermediate and mature G. paraguayenseE. Walther were 17.2, 13.3 and 11.9 lg/mg, respectively, whereasthe total phenolic contents were 92.2, 44.2 and 33.2 lg/mg, respec-tively. A decrease in the phenolic content may be attributed to thestrengthening of plant cell walls into lignans and lignins bypolymerisation (Randhir & Shetty, 2005). Moreover, water extractsof immature G. paraguayense E. Walther were fermented byL. acidophilus BCRC 10695, L. plantarum BCRC 10357 andL. paracasei BCRC 14023. Results indicated that fermentation byL. plantarum BCRC 10357 increased both the flavonoid and pheno-lic contents up to 22.9 and 111 lg/mg from 17.2 and 92.2 lg/mg,respectively; this was greater than that observed during fermenta-tion by L. acidophilus BCRC 10695 and L. paracasei BCRC 14023.Several studies have reported that the levels of many compounds,such as beta-carotene, polyphenols and flavonoids, are increasedbecause of fermentation by lactic acid bacteria (Figueiredo,Campos, de Freitas, Hogg, & Couto, 2008; Hernandez et al., 2007;Panda & Ray, 2007). Therefore, immature G. paraguayenseE. Walther, fermented by L. plantarum BCRC 10357, may induce

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Fig. 2. Antioxidant properties of non-fermented and fermented G. paraguayenseE. Walther. (n = 3). (A–D) water extracts of G. paraguayense E. Walther at differentmaturity stages, sample was filtered and freeze-dried and dissolved with distilledwater to prepare different concentrations, (E–H) G. paraguayense E. Walther(immature) was fermented by L. plantarum, L. acidophilus, and L. paracasei for72 h; sample was filtered and centrifuged to remove bacteria, and then thefermented-supernatant was freeze-dried and dissolved with distilled water toprepare different concentrations.

S.-C. Wu et al. / Food Chemistry 129 (2011) 804–809 807

the expression of flavonoid precursors and polyphenol degrada-tion, which in turn leads to the accumulation of phenolics.

3.3. HPLC assays for organic acid and phenolic compounds

A study has demonstrated that herbal plants contain a largeamount of antioxidants, and these components may reduce oxida-tive damage via free radical-scavenging activity (Zheng & Wang,2001). It has been reported that G. paraguayense E. Walther con-tains various antioxidants, such as gallic acid, quercetin, genistinand daidzin (Kao et al., 2010). However, changes in the major anti-oxidative component in G. paraguayense E. Walther during devel-opment are unknown; hence, the level of several organic acids(oxalic acid and hydroxybutanedioic acid) and phenolic com-pounds (caffeic acid, chlorogenic acid, rutin, quercetin and gallicacid) were investigated in immature, intermediate, and matureG. paraguayense E. Walther plants in the present study (Table 2).The results showed that no caffeic acid was present in G.paraguayense E. Walther at different stages of maturity (data notshown). However, chlorogenic acid, rutin, quercetin and gallic aciddecreased with ageing. Thus, immature G. paraguayense E. Waltherhad a higher content of phytochemicals than had intermediate andmature G. paraguayense E. Walther. However, high levels of oxalicacid and hydroxybutanedioic acid were observed in immature G.paraguayense E. Walther. Calcium ethanedioate is formed by abond between calcium and oxalic acid, which leads to lithogenesisin the kidney. Further, the acidity of G. paraguayense E. Walther isattributable to hydroxybutanedioic acid, which destroys its edibleflavour.

In the present study, it was found that fermentation by lacticacid bacteria increased antioxidant levels in immature G.paraguayense E. Walther. Both quercetin and gallic acid levels weresignificantly higher in immature G. paraguayense E. Walther plantsfermented by L. plantarum BCRC 10357 (increasing 25.6% and 40%,respectively) than in those fermented by L. acidophilus BCRC 10695and L. paracasei BCRC 14023. The levels of caffeic acid and chloro-genic acid were not significantly increased by lactic acid bacteriafermentation; however, fermentation by L. plantarum BCRC10357, L. acidophilus BCRC 10695 and L. paracasei BCRC 14023 re-duced the oxalic acid level in immature G. paraguayense E. Walther.These results suggested that the level of phytochemicals can be in-creased by fermenting G. paraguayense E. Walther at differentdevelopmental stages with lactic acid bacteria to obtain food withsuitable nutritional value.

3.4. Antioxidant activity of G. paraguayense E. Walther in vitro

Fig. 2 shows the antioxidation activity of non-fermented(Fig. 2A–D) and Lactobacillus-fermented immature G. paraguayenseE. Walther (Fig. 2E–H). G. paraguayense E. Walther exhibited grad-ual decreases in DPPH�, 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS+�), and superoxide radical-scavenging activitiesat different levels of maturity because the levels of flavonoids

Table 2The total phenolic acid and flavonoid levels of non-fermented and Lactobacillus-fermented

Maturity stage mg/g of extract

Chlorogenic acid Rutin

Immature (non-fermentation) 1.8 ± 0.2a 0.8 ± 0Intermediate mature (non-fermentation) 1.7 ± 0.2a 0.5 ± 0Mature (non-fermentation) 1.2 ± 0.1b 0.4 ± 0Immature (L. acidophilus BCRC 10695 fermentation) 1.9 ± 0.3a 0.8 ± 0Immature (L. plantarum BCRC 10357 fermentation) 2.0 ± 0.3a 0.9 ± 0Immature (L. paracasei BCRC 14023 fermentation) 1.8 ± 0.4a 0.8 ± 0

Each value represents the mean ± SD (n = 3). Different letters are significantly different

and phenolics in immature G. paraguayense E. Walther plants werehigher than those in intermediate and mature G. paraguayenseE. Walther plants. These phenomena have been attributed to theantioxidant activity of G. paraguayense E. Walther (Chung et al.,

G. paraguayense E. Walther water extracts.

Quercetin Gallic acid Oxalic acid Hydroxybutanedioic acid

.1a 7.8 ± 0.1b 11.0 ± 1.5b 27.2 ± 1.8a 17.6 ± 0.6a

.1b 4.3 ± 0.1c 8.4 ± 1.1c 22.3 ± 2.1c 15.5 ± 0.7b

.1b 2.8 ± 0.1c 6.8 ± 0.8c 20.4 ± 1.8c 14.3 ± 0.5b

.2a 8.7 ± 0.3a 12.5 ± 1.3b 24.8 ± 2.2b 15.7 ± 0.6b

.1a 9.8 ± 0.3a 15.4 ± 1.8a 24.1 ± 2.1b 14.5 ± 0.6b

.1a 8.0 ± 0.2a 11.6 ± 0.9b 24.6 ± 2.3b 15.8 ± 0.3b

at P < 0.05.

Table 3The stimulating-antioxidase activity of fermented or non-fermented G. paraguayense E. Walther on FL83B liver cell line.

Groupsa lmol NADPH/min/mg protein U/mg protein

GPx GR CAT SOD

Normal 2.4 ± 0.4c 3.4 ± 0.2c 1.8 ± 0.2b 0.7 ± 0.2c

Immature (non-fermentation) 4.1 ± 0.3b 4.7 ± 0.1a 2.2 ± 0.2b 1.4 ± 0.1b

Intermediate mature (non-fermentation) 3.9 ± 0.3b 4.0 ± 0.2b 2.0 ± 0.2b 1.3 ± 0.1b

Mature (non-fermentation) 2.7 ± 0.4c 4.2 ± 0.3b 2.1 ± 0.3b 1.0 ± 0.1c

Immature (L. acidophilus BCRC 10695 fermentation) 4.5 ± 0.1a 5.1 ± 0.2a 2.3 ± 0.2b 1.9 ± 0.1a

Immature (L. plantarum BCRC 10357 fermentation) 5.0 ± 0.2a 5.3 ± 0.1a 2.9 ± 0.2a 2.1 ± 0.1a

Immature (L. paracasei BCRC 14023 fermentation) 4.1 ± 0.2b 4.9 ± 0.3a 2.3 ± 0.1b 1.7 ± 0.1a

Normal group means that cells were not treated and is a comparing group. Each value represents the mean ± SD (n = 3). Different letters are significantly different at P < 0.05.GPx: glutathione peroxidase, GR: glutathione reductase, CAT: catalase, SOD: superoxide dismutase.

Table 4The correlations between the antioxidant materials and antioxidant activity in fermented G. paraguayense E. Walther extracts.

Correlation* Flavonoids Total phenolics Quercetin Gallic acid Scavenge activity Stimulatory activity

DPPH Superoxide anion ABTS Reducing power GR GPx SOD CAT

Flavonoids 0.814 0.325 0.824 0.722 0.785 0.863 0.774 0.728 0.586 0.604Total phenolics 0.423 0.901 0.985 0.941 0.969 0.958 0.943 0.929 0.721 0.871Quercetin 0.764 0.625 0.613 0.774 0.586 0.541 0.423 0.402Gallic acid 0.912 0.902 0.910 0.925 0.914 0.908 0.682 0.733

⁄Correlation values were performed with statistical software (SPSS, version 11, SPSS Inc.).

808 S.-C. Wu et al. / Food Chemistry 129 (2011) 804–809

2005). A previous study has reported the antioxidant activity ofgallic acid and phenolics in G. paraguayense E. Walther(Kao et al., 2010). An increase in the antioxidant activity associatedwith accumulation of bioactive compounds such as phenolics andflavonoids in G. paraguayense E. Walther, could be a better methodfor determining the optimum stage of physiological maturity in or-der to harvest G. paraguayense E. Walther rather than following theconventional method of harvesting between 30 and 90 days afterplanting. In addition, the capacity of the samples to reduce the fer-ricyanide complex to the ferrous form may serve as a significantindicator of their antioxidant capacity (Meir, Kanner, Akiri, &Hadas, 1995). The reducing power of immature G. paraguayenseE. Walther was higher than that of intermediate and matureG. paraguayense E. Walther. This may be attributed to the presenceof a high concentration of gallic acid and phenolics in G.paraguayense E. Walther, as reported in a previous study(Chung et al., 2005).

On the other hand, the level of antioxidants was significantly in-creased in immature G. paraguayense E. Walther fermented byL. acidophilus BCRC 10695, L. plantarum BCRC 10357 and L. paraca-sei BCRC 14023. Therefore, the antioxidant activity of Lactobacillus-fermented immature G. paraguayense E. Walther was further eval-uated in the present study. The DPPH�-, ABTS+�-, and superoxideanion-scavenging activities and reducing power were optimal insamples fermented with L. plantarum BCRC 10357 as comparedto those fermented with L. acidophilus BCRC 10695 and L. paracaseiBCRC 14023. However, fermentation of immature G. paraguayenseE. Walther by L. plantarum BCRC 10357 produced good antioxidantactivity, as compared to fermentation of intermediate and matureG. paraguayense E. Walther, because of significantly elevated levelsof total phenolics and flavonoids in the immature plants.

3.5. Antioxidant activity of G. paraguayense E. Walther in vivo

There is considerable evidence indicating that excessive levelsof reactive oxygen species (ROS) and reactive nitrogen species(RNS) may lead to oxidative stress and loss of cell function, andthus increased risk of various diseases and certain forms of cancers.Under physiological conditions, non-enzymatic antioxidants (e.g.,

vitamin E and glutathione) and antioxidant enzymes, such as glu-tathione peroxidase (GPx), glutathione reductase (GR), glutathioneS-transferase (GST) and catalase (CAT), can scavenge these reactiveoxygen species and thus protect cells from oxidative damage(Hung, Fu, Shih, Lee, & Yen, 2006). Several biological compoundsin plants may stimulate antioxidant enzyme transcription anddetoxification defence systems through antioxidant response ele-ments (AREs) (Masella, Benedetto, Vari, Filesi, & Govannini,2005), and increase c-glutamylcysteine (c-GCS) synthesis(Kim et al., 2007). Notable changes in CAT, GPx, GR and superoxidedismutase (SOD) were observed when the mouse normal liver cellline FL83B was treated with G. paraguayense E. Walther (Table 3).The results indicated that the intracellular antioxidase activitywas significantly more stimulated by treatment with immatureG. paraguayense E. Walther than with intermediate or matureG. paraguayense E. Walther. Moreover, immature G. paraguayenseE. Walther, fermented with L. plantarum BCRC 10357, significantlyincreased CAT, GPx, GR and SOD activities; this result suggests thatfermentation with L. plantarum BCRC 10357 increased antioxidantsin G. paraguayense E. Walther and stimulated antioxidase activity.

4. Conclusions

For the first time, a new set of optimum maturity indices for theharvesting of G. paraguayense E. Walther has been established.Interestingly, the water extracts of immature G. paraguayense E.Walther had greater total phenolic and flavonoid content thanhad intermediate and mature G. paraguayense E. Walther. Fermen-tation by L. plantarum BCRC 10357 could elevate the total phenolicand flavonoid contents in G. paraguayense E. Walther at differentstages of maturity; the increase in content was more than that pro-duced by other lactic acid bacteria. Further, we found that imma-ture G. paraguayense E. Walther had the highest levels ofchlorogenic acid, rutin, quercetin and gallic acid, and that the levelsof these phytochemicals decreased with ageing. The levels of thesephytochemicals increased, and oxalic acid and hydroxybutanedioicacid levels decreased in immature G. paraguayense E. Walther fer-mented with L. plantarum BCRC 10357. Immature G. paraguayenseE. Walther, fermented with L. plantarum BCRC 10357, was found to

S.-C. Wu et al. / Food Chemistry 129 (2011) 804–809 809

possess strong antioxidant ability in vitro and produced strongantioxidase activity in FL83B cells. In addition, the association be-tween antioxidants and the radical-scavenging activity was inves-tigated and the results are shown in Table 4. Compared to the levelof flavonoid compounds, the level of total phenolic compounds inG. paraguayense E. Walther extracts significantly correlated withthe radical-scavenging ability and antioxidase activity, and gallicacid appears to be responsible for the antioxidant activity ofG. paraguayense E. Walther extracts.

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