Antioxidant capacity of leafy vegetables as affected by high tunnel environment, fertilisation and growth stage

Journal of the Science of Food and Agriculture J Sci Food Agric 87:2692–2699 (2007)

Antioxidant capacity of leafy vegetablesas affected by high tunnel environment,fertilisation and growth stageXin Zhao,1† Takeo Iwamoto2 and Edward E Carey3∗1Department of Horticulture, Forestry, and Recreation Resources, Kansas State University, 2021 Throckmorton Hall, Manhattan, KS66506-5506, USA2Department of Biochemistry, Kansas State University, 214 Burt Hall, Manhattan, KS 66506-5506, USA3K-State Horticulture Research and Extension Center, 35230 W 135th Street, Olathe, KS 66061-9423, USA

Abstract

BACKGROUND: This study was conducted to evaluate the influences of protected environment, organicfertilisation and growth stage on the oxygen radical absorbance capacity (ORAC) values of leafy vegetables.

RESULTS: In a first experiment, pac choi grown in high tunnels had significantly lower ORAC than field-grownplants. Organic fertiliser markedly increased the antioxidant capacity of pac choi compared with conventionaltreatment, especially in the open field. However, both open field and organic production resulted in significantlylower yield and more severe leaf damage due to insect attack. In a second study, spinach showed the highestORAC, followed by pac choi, red leaf lettuce and romaine lettuce. A significant decline in ORAC under hightunnel production was observed only in spinach. In contrast to the first trial, organic fertilisation did not cause anincrease in antioxidant capacity of the leafy vegetables. The ORAC values of spinach from the open field and pacchoi from high tunnels were significantly higher when harvested at the mature head stage than at the baby sizestage.

CONCLUSION: Further studies will help to elucidate effects of genotype, growth stage, and productionenvironment on antioxidant capacities of vegetables and may lead to recommended practices to maximiseantioxidant capacity of vegetable crops. 2007 Society of Chemical Industry

Keywords: pac choi; lettuce; spinach; oxygen radical absorbance capacity (ORAC); organic; conventional

INTRODUCTIONOxidative damage to DNA, lipids and proteins inthe human body can be induced by reactive oxygenand nitrogen species from endogenous and exogenoussources. Such oxidative stress is believed to beassociated with the aging process and the developmentof chronic diseases, including cancer, cardiovasculardisease, diabetes, Alzheimer’s disease, immune systemdecline and cataracts.1–4 The value of fruits andvegetables in reducing the risk of degenerativehuman diseases has been largely attributed to thediversified antioxidants that abound in them.5,6 Majorantioxidants in fruits and vegetables include vitaminsC and E, carotenoids and polyphenols. Additive andsynergistic effects of phytochemicals account for thepotent antioxidant activities of plant foods.7

Among a variety of chemical methods used todetermine antioxidant capacity in foods, the oxygenradical absorbance capacity (ORAC) assay has becomewidely adopted and fairly standardised.8,9 The ORAC

method measures scavenging capacity against theperoxyl radical, which is the predominant free radicalinvolved in lipid oxidation in foods and biologicalsystems. The ORAC assay uses the ‘area under thecurve’ technique to measure both inhibition timeand inhibition percentage in the reaction. Hydrophilicand lipophilic antioxidant capacities can be analysedusing the ORAC method, although their sum may notprecisely reflect total antioxidant capacity owing tolimitations of employing a single radical source.9 Givenits effectiveness for comparing the relative antioxidantcapacity of different food samples, ORAC has beenused extensively for fruits and vegetables.10–13

Genotype is the primary factor contributing tovariation in antioxidant capacity of fruits andvegetables, as demonstrated in numerous studies.10–15

However, the influences of crop developmentalstage and cultivation conditions are also of highinterest, since these may be manipulated to maximiseantioxidant capacity of food crops at harvest. Patterns

∗ Correspondence to: Edward E Carey, K-State Horticulture Research and Extension Center, 35230 W 135th Street, Olathe, KS 66061-9423, USAE-mail: [email protected]†Present address: Department of Horticultural Sciences, University of Florida, Gainesville, FL 32611-0690, USAContract/grant sponsor: US Organic Farming Research Foundation(Received 17 October 2006; revised version received 21 May 2007; accepted 23 May 2007)Published online 19 September 2007; DOI: 10.1002/jsfa.3032

2007 Society of Chemical Industry. J Sci Food Agric 0022–5142/2007/$30.00

Antioxidant capacity of leafy vegetables

of change in antioxidant activity during fruit ripeningdiffer among crops, with red raspberries15 andtomatoes16 harvested at the red-ripe stage having peakantioxidant activity. Peppers exhibit a similar increasein antioxidant activity with maturation.17 In contrast,strawberry and blackberry antioxidant capacities peakduring the immature green stages.15 Few studies haveevaluated antioxidant levels at different growth stagesof leafy vegetables, many of which are consumed atvariable plant ages. Differences in cultural practicesmay also result in significant changes in antioxidantproperties of plants. The antioxidant capacity ofstrawberries grown on raised beds covered with blackplastic mulch was considerably higher than that ofstrawberries grown in matted row beds without plasticmulch.18 Addition of compost to root media increasedthe antioxidant activity of well-fertilised strawberriesgrown in a greenhouse.19 Ren et al.20 reported thatorganically grown spinach and Chinese cabbage hadhigher antioxidant activities than the same vegetablesgrown conventionally. Despite increasing consumerdemand for organic produce, information aboutantioxidant activities of organically and conventionallygrown fruits and vegetables is limited.21

The objective of this study was to assess theinfluence of production method and environmentas well as growth stage on antioxidant capacity ofleafy vegetables. The study consisted of two separateexperiments. One experiment investigated the effect ofapplication rate of organic and conventional fertiliserson ORAC activity in pac choi (Brassica rapa L.Chinensis group) grown in high tunnels and openfield. A second experiment evaluated the effects ofgrowth stage at harvest and organic vs conventionalfertilisation on ORAC activity in lettuce (Lactucasativa L.), spinach (Spinacia oleracea L.) and pac choigrown in high tunnels and open field.

MATERIALS AND METHODSOrganically and conventionally managed hightunnel and open field plotsThe study was carried out in 2004 on a Kennebecsilt loam soil at the Kansas State UniversityHorticulture Research and Extension Center, Olathe.Six 9.8 m × 6.1 m high tunnels with 1.5 m sidewalls(Stuppy, North Kansas City, MO, USA) and sixadjacent 9.8 m × 6.1 m field plots were used forboth experiments. The tunnels were covered with asingle layer of 6 mil (0.153 mm) K-50 polyethylenefilm (Klerk’s Plastic Product Manufacturing, Inc.,Richburg, SC, USA). At establishment in 2002 thesix high tunnels were divided into three groups(blocks) and the two high tunnels in each blockwere randomly assigned for long-term organic orconventional management treatments. A similarset-up was used in the field plots. In 2003,organic plots were inspected and certified to be incompliance with USDA National Organic Programstandards.

Experiment 1Three pre-plant fertiliser rates based on nitrogen levelwere used, i.e. zero, medium and high. Conventionalfertiliser, NPK 13-13-13, was applied at 0, 78and 156 kg N ha−1 and organic fertiliser, Hu-More1-1-1 (composted cattle manure and alfalfa hay;Humalfa, Inc., Shattuck, OK, USA), was appliedat 0, 156 and 312 kg N ha−1. Twice as much totalnitrogen was applied for the organic treatment inorder to have roughly similar levels of nitrogenavailable to organic and conventional crops, assuming50% total nitrogen availability from the compost.22

Fertiliser treatments were applied to 1.8 m × 0.6 mplots separated by 0.6 m in a single-row bed ineach organically and conventionally managed hightunnel or open field plot. Eight plants of pac choicv. Mei Qing (Johnny’s Selected Seeds, Winslow,ME, USA) were grown in each experimental plotat a spacing of 0.2 m within rows. Transplants wereproduced in the greenhouse using 200-cell Speedlingflats (Speedling Incorporated, Sun City, FL, USA)with Jiffy mix (Jiffy Products of America Inc., Norwalk,OH, USA). Three-week-old transplants were plantedon 19 May and whole plants were harvested on 23June. Pyrethrin (PyGanic, McLaughlin Gormley KingCompany, Golden Valley, MN, USA) and permethrin(Pounce 3.0 EC, FMC Corporation, Philadelphia,PA, USA) insecticides were applied in organic andconventional plots respectively to control flea beetles(Phyllotreta crucifera) as required. At harvest, plants ineach plot were weighed and fresh weight per plant wasobtained. Insect damage was rated on a scale of 1 to5, where 1 = no damage and 5 = severe damage. Acomposite sample of six large inner leaves (from thewhorl immediately adjacent to the outermost leaves)taken from three heads (two leaves per head) per plotwas dried at 70 ◦C in a forced air oven for 72 h toconstant weight and the dry matter content (%) wasdetermined.23 Dry weight per plant was calculatedby multiplying fresh weight by dry matter content.Another pooled leaf sample from each plot preparedin a similar manner (mainly blades) was used forORAC analysis. Leaf samples were stored at −20 ◦Cprior to extraction.

Leaf sample extractionA 2 g portion of each leaf sample was extracted in15 mL of 75 mmol L−1 phosphate buffer (pH 7.4)using a mortar and pestle placed on ice, and thehomogenate was centrifuged at 20 000 × g and 4 ◦Cfor 30 min. The supernatant was transferred to vialsand stored at −20 ◦C for use as the hydrophilic extract.The pellet was resuspended in 10 mL of pure acetone,then, after shaking at room temperature for 20 min,the mixture was further centrifuged at 20 000 × g and4 ◦C for 20 min. The acetone supernatant was usedas the lipophilic extract. Prior to the ORAC assay,hydrophilic and lipophilic extracts were diluted 400-and 200-fold respectively with 75 mmol L−1 phosphatebuffer (pH 7.4).

J Sci Food Agric 87:2692–2699 (2007) 2693DOI: 10.1002/jsfa

X Zhao, T Iwamoto, EE Carey

Chemicals and apparatusFluorescein sodium and 6-hydroxy-2,5,7,8-tetra-methylchroman-2-carboxylic acid (Trolox) wereobtained from Sigma-Adrich (St Louis, MO,USA). 2,2′-Azobis(2-amidinopropane) dihydrochlo-ride (AAPH) was purchased from Wako ChemicalsUSA (Richmond, VA, USA). Polypropylene assayplates with 96 wells (Fisher Scientific, Hanover Park,IL, USA) were used for measurement. Fluorescencewas measured using a Varian Cary Eclipse fluores-cence spectrophotometer coupled with a microplatereader (Varian, Inc., Palo Alto, CA, USA).

ORAC assayThe ORAC assay was carried out following a modifiedmethod of Huang et al.24 This assay measures theability of antioxidants in test samples to protect thefluorescent probe (fluorescein) against the decline offluorescence induced by a peroxyl radical generator(AAPH). Briefly, 150 µL of 8.16 × 10−5 mmol L−1

fluorescein solution was pipetted into plate wells inparallel containing 25 µL of phosphate buffer blank(75 mmol L−1, pH 7.4) or 25 µL of Trolox standard(1, 2 or 4 µmol L−1 Trolox was used based on thesample concentration) or 25 µL of diluted samplesolution. After pre-incubation at 37 ◦C for 15 min theplate was immediately transferred to the plate reader.Then 25 µL of 153 mmol L−1 AAPH was added to thewells to initiate the reaction, bringing the total volumefor each well to 200 µL. Fluorescence was measuredevery minute for 35 min at an emission wavelengthof 515 ± 2.5 nm and an excitation wavelength of490 ± 2.5 nm. Blank, standard and sample wereanalysed in duplicate using a ‘forward-then-reverse’order to eliminate position effects.

The net area under the curve (AUC) of blanks,standards and samples was calculated as

AUC = 0.5 + f1/f0 + · · · fi/f0 + · · · f34/f0 + 0.5f35/f0

where f0 is the initial fluorescence measurement at0 min and fi is the fluorescence measurement at time i.

The ORAC value was calculated as

ORAC = ktc

[(AUCsample − AUCblank)/(AUCTrolox − AUCblank)]

where k is the dilution factor, t is the concentration ofTrolox (µmol L−1) and c is the ratio of unit conversion.The final results of ORAC value were calculated andexpressed as µmol Trolox equivalents g−1 fresh weight(FW).

Experiment 2High tunnel environment, organic fertilisation andgrowth stage of leafy greens were investigated inthis experiment. Replicated organically and conven-tionally managed high tunnel and open field plots(previously described) received pre-plant application

of 160 kg ha−1 Hu-More 1-1-1 (Humalfa, Inc.) and80 kg ha−1 NPK 13-13-13 respectively. High tunnelswere covered with 39% white shade cloth (Pak Unlim-ited, Norcross, GA, USA) throughout the experiment.On 29 July, pac choi cv. Mei Qing, spinach cv. Tyee,red leaf lettuce cv. Red Sails and green romaine let-tuce cv. Green Forest (Johnny’s Selected Seeds) weredirectly seeded on 1.2 m × 0.3 m beds in each hightunnel and open field plot. Plants were harvested attwo growth stages, i.e. baby size (suitable for salad mix)and mature head size. For the harvest at baby size, pacchoi from all plots and lettuce and spinach from hightunnel plots were harvested on 21 August, while let-tuce and spinach from open field plots were harvestedon 28 August. After harvest of baby greens, plots werethinned to allow for head growth of remaining plants.On August 24, shade cloth and polyethylene cover-ings on high tunnel plots were forcefully removed byhigh winds during a severe weather event. These plotsremained uncovered until harvest. Harvest of headswas conducted on 10 September for spinach from allplots and pac choi in high tunnel plots, while it was per-formed on 24 September for lettuce from all plots andpac choi in the open field. At each harvest, leaf sam-ples of each vegetable from all plots were collected forORAC measurement. Baby size leaf samples consistedof 12–20 randomly selected leaves, while mature headsamples comprised eight leaves from four randomlyselected plants. Sample extraction and ORAC assayfollowed the same procedure as in Experiment 1.

Statistical analysisIn Experiment 1, results were analysed as a split plotdesign with a partial nested treatment structure, whereenvironment (high tunnel vs open field) and fertilisersource (organic vs conventional) were whole plot andsubplot factors respectively and fertiliser rates werenested within the source of fertiliser. In Experiment 2,results were analysed as a split–split–split plot designwith environment (high tunnel vs open field) as wholeplot factor, fertilisation (organic vs conventional) assubplot factor, vegetable type as sub-subplot factorand harvest stage as sub-sub-subplot factor. Analy-sis of variance (ANOVA) was performed using theMIXED procedure in SAS Version 9.1 (SAS Insti-tute, Cary, NC, USA). Multiple comparisons wereconducted using Fisher’s least significant difference(LSD) test (α = 0.05).

RESULTS AND DISCUSSIONResults from the first experiment indicated markedinfluences of production environment and fertilisationon antioxidant capacity of pac choi (Table 1). Pacchoi grown in high tunnels showed significantlylower hydrophilic and total antioxidant capacitiesthan that grown in the open field (by 28.7 and27.3% respectively), while lipophilic antioxidantcapacities were not significantly affected by productionenvironment (Table 2). Plant phenolics are believed

2694 J Sci Food Agric 87:2692–2699 (2007)DOI: 10.1002/jsfa


to be major components contributing to antioxidantcapacity, and positive correlation between ORAC(especially hydrophilic ORAC) and total phenoliccontent has been demonstrated in a body ofliterature.14,15,25–28 Possibly, pac choi from the openfield plots contained larger amounts of phenolicscompared with that from the tunnels where lightintensity was reduced. Phenolic compounds, someof which act as UV protectants in plants, increasein response to high light.29,30 It was reported thatlettuce grown in a polycarbonate greenhouse had alower content of flavonoids than that grown in theopen field.31 On the other hand, plant fresh weightand dry weight decreased dramatically in open fieldplots, and there was a significantly higher rating ofinsect (flea beetle) damage (Tables 3 and 4). Themore pronounced insect damage on pac choi observedin the open field plots may also have contributedto the elevation of antioxidant activity, as phenolicaccumulation can also be induced by insect feeding.29

The validity of the experimental layout of high tunnelsand open field plots in adjacent parts of the field maybe questioned, but, considering that this was one ofthe potential randomisations of main plots and thatthe soil conditions in the whole field were uniform, thedesign was considered to be acceptable.

As shown by the significant interaction effect ofproduction environment and fertilisation, organic fer-tiliser significantly increased hydrophilic antioxidantactivity of pac choi in the open field but did not differfrom conventional treatment under the high tunnelenvironment (Tables 1 and 2). The main effect of fer-tilisation on lipophilic and total ORAC values wasindependent of production environment (Table 1);

Table 1. Analysis of variance of effects of production environment

(high tunnel vs open field), fertilisation (organic vs conventional) and

application rate on antioxidant capacity (ORAC values) of pac choi

grown in the late spring trial 2004 at Olathe, Kansas

EffectHydrophilic

ORACLipophilic

ORACTotal

ORAC

Environment (E) P = 0.0246 NS P = 0.0200Fertilisation (F) NS P = 0.0264 P = 0.0333E × F P = 0.0426 NS NSApplication rate

within F (R)NS NS NS

E × R NS NS NS

NS, non-significant.

however, the open field seemed to be more favourablefor ORAC enhancement by organic fertiliser relative tohigh tunnels, with an increase of 11.3% (Table 2). Thefinding of higher antioxidant capacity of pac choi underorganic production was in agreement with recent stud-ies indicating increased levels of phenolic compoundsin organically grown fruits and vegetables.20,32–35 Itis likely that, even though fertiliser rates were dou-bled under organic vs conventional treatment, a lowmineralisation rate of organic fertiliser and resultantnutrient limitation may have occurred during thisexperiment, considering the significant yield reduc-tion in the organic plots (Tables 3 and 5). Moreover,the higher ORAC value of organically grown pac choimight also have resulted from biotic stress, as insectdamage was more severe under organic fertilisation(Tables 3 and 5), possibly related to the differentialeffectiveness of organic and conventional insecticidesused to control flea beetles. These results were con-sistent with our previous finding of elevated totalphenolic levels in flea beetle-affected pac choi grownorganically.36

Table 3. Analysis of variance of effects of production environment

(high tunnel vs open field), fertilisation (organic vs conventional) and

application rate on plant fresh weight, dry weight and insect (flea

beetle) damage of pac choi grown in the late spring trial 2004 at

Olathe, Kansas

EffectPlant fresh

weightPlant dryweight

Insectdamage

Environment (E) P = 0.0029 P = 0.0091 P = 0.0290Fertilisation (F) P = 0.0025 P = 0.0029 P = 0.0050E × F NS NS NSApplication rate

within F (R)NS P = 0.0295 NS

E × R NS NS NS


Table 4. Plant fresh weight, dry weight and insect (flea beetle)

damage rating of pac choi grown under high tunnel and open field

environments in the late spring trial 2004 at Olathe, Kansas

Parameter High tunnel Open field

Fresh weight (g per plant) 301.2 ± 85.1a 122.7 ± 82.5bDry weight (g per plant) 16.4 ± 4.6a 8.7 ± 5.0bInsect damagea 3.2 ± 0.6b 3.7 ± 0.4a

Values are mean ± standard deviation. Means within a row followedby the same letter are not significantly different at P ≤ 0.05.a Scale 1–5, where 1 = no damage and 5 = severe damage.

Table 2. ORAC values (µmol Trolox equivalents g−1 FW) of pac choi grown organically and conventionally in high tunnels and open fields in the late

spring trial 2004 at Olathe, Kansas

High tunnel Open field

ORAC Organic Conventional Organic Conventional

Hydrophilic 30.1 ± 11.2c 30.4 ± 9.2c 45.0 ± 6.2a 40.0 ± 8.6bLipophilic 9.3 ± 3.8 7.9 ± 3.4 11.7 ± 4.0 10.3 ± 2.3Total 39.5 ± 12.3 38.4 ± 10.8 56.7 ± 8.3 50.3 ± 9.0

Values are mean ± standard deviation. Means within a row followed by the same letter are not significantly different at P ≤ 0.05.



Table 5. Plant fresh weight, dry weight and insect (flea beetle) damage rating of pac choi grown under three application ratesa of organic and

conventional fertilisers in the late spring trial 2004 at Olathe, Kansas

Organic Conventional

Parameter Low Medium High Low Medium High

Fresh weight (g per plant) 132.4 ± 79.8 187.9 ± 94.5 156.1 ± 67.0 218.1 ± 108.4 263.5 ± 76.8 313.6 ± 87.0Dry weight (g per plant) 7.9 ± 4.5c 11.3 ± 6.5bc 9.7 ± 3.9bc 12.0 ± 7.8b 15.2 ± 4.8ab 19.1 ± 3.5aInsect damageb 3.8 ± 0.4 3.8 ± 0.4 3.9 ± 0.2 3.1 ± 0.7 3.2 ± 0.4 3.1 ± 0.4

Values are mean ± standard deviation. Means within a row followed by the same letter are not significantly different at P ≤ 0.05.a Low, medium and high rates correspond respectively to 0, 156 and 312 kg N ha−1 for organic fertilisation and 0, 78 and 156 kg N ha−1 forconventional fertilisation.b Scale 1–5, where 1 = no damage and 5 = severe damage.

Plant fresh weight tended to increase as fertiliser rateincreased, but not at a significant level. Similar levelsof insect damage under different fertiliser rates werealso observed (Tables 3 and 5). However, dry weightof pac choi varied significantly with fertiliser rates,particularly under conventional production. Highrates of conventional fertiliser led to a significantlyhigher plant dry weight than did the low rate, whileorganically fertilised pac choi tended to have a higherplant dry weight at the medium rate (Tables 3 and 5).The influence of fertiliser rate on antioxidant capacityof pac choi was not significant in this experiment(Table 1). Nevertheless, an interesting tendency ofORAC values was observed in response to fertiliserrates. In general, hydrophilic and total ORAC valuesof pac choi tended to decrease at high fertiliserrates, particularly under conventional fertilisation,while lipophilic ORAC reached the highest levelsat the medium fertiliser rate (Fig 1). Antioxidantcapacity of leafy vegetables is likely to increase withreduced nitrogen application owing to higher levelsof phenolics. Phenolic compounds may accumulateowing to nitrogen deficiency.29,37 Elevation of totalphenolic content in apple leaves was reported tobe stimulated by low nitrogen supply.38 Similarly,flavones and soluble phenolic acids in barley leaveswere shown to increase with decreased applicationrates of animal manure.39

In the second experiment, vegetable type (speciesand cultivar) showed a highly significant effect onantioxidant capacity of the crops studied (Table 6).Spinach had the highest total ORAC value, followedby pac choi, red leaf lettuce and romaine lettuce(Table 7). Spinach has been shown previously to havea much higher antioxidant capacity than lettuce.27,40

Higher hydrophilic ORAC values of red leaf lettucethan of romaine lettuce were consistent with ourprevious study, where a red leaf lettuce was foundto possess significantly higher levels of phenoliccompounds in comparison with a green romainecultivar.41 Higher phenolic content in red leaf lettucethan in green types in general has also been reportedby other researchers.42,43 With respect to the lipophilicORAC, however, red leaf lettuce did not differfrom romaine lettuce (Table 7), which is consistentwith a previous report of similar concentrations ofcarotenoids and vitamin E in these lettuce types.44

05

101520253035404550556065

Low Medium High

OR

AC

(µm

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x eq

uiva

lent

s g−1

FW

)O

RA

C (

µmol

Tro

lox

equi

vale

nts

g−1 F

W)

OR

AC

(µm

ol T

rolo

x eq

uiva

lent

s g−1

FW

)Organic

Conventional

0

5

10

15

20

25

30

Low Medium High

Organic

Conventional

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101520253035404550556065

Low Medium High

Fertilizer rate

Organic

Conventional

A

B

C

Figure 1. (A) Hydrophilic, (B) lipophilic and (C) total ORAC values ofpac choi grown under three application rates of organic andconventional fertilisers in the late spring trial 2004 at Olathe, Kansas.Low, medium and high rates correspond respectively to 0, 156 and312 kg N ha−1 for organic fertilisation and 0, 78 and 156 kg N ha−1 forconventional fertilisation. Means are given with standard deviations.



Table 6. Analysis of variance of effects of production environment,

fertilisation, vegetable type and harvest date on ORAC values of leafy

greens in the late summer trial 2004 at Olathe, Kansas

EffectHydrophilic

ORACLipophilic

ORACTotal

ORAC

Environment (E) NS NS NSFertiliser (F) NS NS NSE × F NS NS NSVegetable type (V) P < 0.0001 P < 0.0001 P < 0.0001E × V P = 0.0001 P = 0.0011 P = 0.0006F × V NS NS NSE × F × V NS NS NSGrowth stage (G) P = 0.0122 P = 0.0033 P = 0.0101E × G NS NS NSF × G NS NS NSV × G NS NS NSE × F × G NS NS NSE × V × G P = 0.0225 NS P = 0.0192F × V × G NS NS NSE × F × V × G NS NS NS


In contrast to the first experiment, organic andconventional fertilisation resulted in similar levels ofantioxidant capacity of leafy vegetables in this trial(Tables 6 and 7), accompanied by comparable yieldsbetween organic and conventional treatments (datanot shown).

In the second study the removal of the polyethyleneand shade from the high tunnel environment immedi-ately following the harvest of crops at the baby stage(except for baby lettuce and spinach from the openfield, which were harvested 4 days after the removalof the covering) resulted in exposure of crops to simi-lar atmospheric conditions, including light, during thefinal weeks of growth. The influence of productionenvironment on antioxidant activity was reflected bythe significant interaction effects associated with veg-etable type and growth stage (Table 6). Comparedwith open fields, high tunnels significantly decreased

the lipophilic ORAC value in pac choi but not in theother vegetables (Table 7). High tunnel productionresulted in lower hydrophilic and total antioxidantcapacities in spinach at both baby size (by 54.8 and47.6% respectively) and mature head (by 62.1 and57.6% respectively) growth stages but did not causeany difference in the other vegetables at either growthstage (Table 7).

Maturation significantly enhanced the lipophilicORAC values, particularly in pac choi (Table 7).The influence of maturation on hydrophilic andtotal antioxidant capacities was dependent upon thevegetable type and production environment, sincesignificantly elevated values at the mature head stagewere only observed in pac choi from high tunnels andspinach from open fields (Table 7). Interestingly, pacchoi was the only crop to show markedly increasedantioxidant levels in mature heads in the high tunnelenvironment (following removal of the polyethylenecovering), indicating marked differences among cropsin responsiveness to stress conditions (in this case,presumably, increased light intensity). Relationsbetween antioxidant capacity and growth stage,vegetable type and production environment should befurther investigated in studies using more intensivesampling of crops over the course of the growthcycle. Growth stage control may have a particularimplication for leafy vegetables given that they areoften harvested at multiple stages for consumption.Cut-and-regrow is a common practice used in theproduction of baby salad greens, and it would be ofinterest to evaluate the antioxidant capacities of leavesfrom sequential harvests. Pandjaitan et al.45 reportedthat mid-size spinach leaves were significantly higherin total phenolics, total flavonoids and antioxidantcapacity on a dry weight basis than immature andfully mature leaves harvested from the same plant.They suggested that, in addition to the alterationof biosynthesis rate of phenolics, a nutrient dilutioneffect may come into play at a certain point during leaf

Table 7. Hydrophilic, lipophilic and total ORAC values (µmol Trolox equivalents g−1 FW) of different types of leafy vegetables at two growth stages

under high tunnel and open field environments in the late summer trial 2004 at Olathe, Kansas

High tunnel Open field

ORAC Vegetable Baby size Mature head Baby size Mature head

Hydrophilic Pac choi 36.6 ± 10.1b 75.8 ± 28.9a 49.5 ± 12.9ab 47.2 ± 9.7abSpinach 71.7 ± 36.0c 74.8 ± 33.9c 158.6 ± 54.2b 197.2 ± 74.0aRed leaf lettuce 9.2 ± 1.4a 13.6 ± 4.3a 8.9 ± 6.2a 11.9 ± 4.0aRomaine lettuce 4.4 ± 4.3a 4.7 ± 3.0a 3.2 ± 1.0a 4.6 ± 1.2a

Lipophilic Pac choi 4.2 ± 2.8b 10.1 ± 4.1a 11.5 ± 4.7a 13.3 ± 3.7aSpinach 8.3 ± 1.1a 10.3 ± 0.8a 7.4 ± 3.5a 8.8 ± 2.0aRed leaf lettuce 2.9 ± 2.2a 4.1 ± 2.5a 2.8 ± 2.2a 3.7 ± 1.7aRomaine lettuce 2.7 ± 1.2a 3.5 ± 1.3a 4.6 ± 1.2a 5.1 ± 2.4a

Total Pac choi 39.6 ± 10.8b 85.9 ± 29.9a 61.0 ± 17.2ab 60.5 ± 9.0abSpinach 87.4 ± 42.0c 87.4 ± 40.5c 166.9 ± 56.5b 206.0 ± 74.0aRed leaf lettuce 12.0 ± 2.0a 17.7 ± 2.7a 11.8 ± 4.9a 15.0 ± 5.4aRomaine lettuce 7.0 ± 5.5a 8.3 ± 4.0a 7.9 ± 1.6a 9.8 ± 1.7a

Values are mean ± standard deviation. Means within a row followed by the same letter are not significantly different at P ≤ 0.05.



growth, affecting the phenolic level and antioxidantcapacity in spinach.

The hydrophilic fraction accounted for a majorityof total ORAC values measured in both experiments(76% on average). A recent investigation of over 100different kinds of foods indicated that, in general, morethan 90% of total antioxidant capacity was attributableto hydrophilic ORAC.13 Our measurements of totalORAC values of lettuce matched the data fromthat study, although the lipophilic ORAC appearedto be higher, while the spinach ORAC valuesmeasured in this experiment were considerably higher.Compared with the study of Howard et al.14 in whichphycoerythrin was used as the fluorescent probe, ourORAC measurements for spinach were also muchhigher, indicating a need for further verification. Thelipophilic ORAC assay used in this study could befurther improved by using randomly methylated β-cyclodextrin as the solubility enhancer.46 Additionally,cellular measures of antioxidant activity may be usedin combination with the chemical ORAC assay tomore fully evaluate the complex antioxidant capacityof tested samples.25

CONCLUSIONSThe high tunnel environment exhibited the potentialfor reducing antioxidant capacities of pac choiand spinach. However, this effect might vary withgenotype, season and other conditions, requiringfurther exploration. Organic fertilisation may enhancethe antioxidant capacity of pac choi in contrast toconventional fertilisation, but this requires furtherinvestigation, since in the present study the fertilisereffect may have been confounded with nutrientavailability and insect attack, resulting in significantyield reduction under organic treatment. Totalantioxidant capacity of pac choi seemed to decrease asfertiliser rate increased, especially under conventionalfertilisation. The effect of nutrient supply levelon antioxidant activity of different leafy vegetablesdeserves further study. Hydrophilic antioxidants werethe major contributors to the total antioxidantcapacity. Among the vegetables tested in this study,spinach had the highest ORAC value, followed bypac choi, red leaf lettuce and romaine lettuce. Pacchoi and spinach had higher antioxidant capacitiesat mature head stage relative to baby size stage,but the maturation effect seemed to be related toproduction environment. Since many leafy vegetablesare consumed at various growth stages (leaf sizes),further study of antioxidant values of leafy vegetablesharvested at different growth stages is suggested.

ACKNOWLEDGEMENTSWe acknowledge the US Organic Farming ResearchFoundation for funding support. We are also gratefulto Dr John Tomich for assistance with laboratory

analysis and to Dr Rhonda Janke for input on themanuscript.

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Antioxidant capacity of leafy vegetables as affected by high tunnel environment, fertilisation and growth stage