Journal of the Science of Food and Agriculture J Sci Food Agric 79 :385–389 (1999)

Oat antioxidant extraction and measurement –towards a commercial process¹

Rita H Auerbach and David A Gray*Divis ion of Food Sciences , School of Biological Sciences , Univers ity of Nottingham,Sutton Bonington Campus , Loughborough,

LE12 5RD,UK

Abstract : Oat ýour has preserving properties that have been attributed to the antioxidant activity of

phenolic compounds, particularly hydroxycinnamic acid conjugates. This work compares extraction

methods based on methanol and propan-2-ol for the removal of oat antioxidants from milled oats.

The efficiency of the extraction method was determined by measuring the concentration of total phe-

nolic compounds and the antioxidant activity based on an oxidation reaction of b-carotene with lin-

oleic acid in an emulsion. A novel method was also used based on the quenching of light generated

from the chemiluminescence reaction between cytochrome c, tert- butylhydroperoxide (t-BHPO) and

luminol. The chemiluminescence method proved to be reliable, rapid and sensitive. Both propan-2-ol

and methanol extraction methods yielded oat extracts with antioxidant activity. The propan-2-ol

extract contained approximately 70% of the activity measured in the methanol extract. Despite the

lower yield, the industrial advantages of the latter method are discussed.

1999 Society of Chemical Industry(

Keywords: oat ; antioxidants ; phenolics ; chemiluminescence (CL); antioxidant activity ; hydroxycinnamicacid ; caþeic acid; ferulic acid

INTRODUCTION

Synthetic antioxidants such as butylatedhydroxy-toluene (BHT), butylatedhydroxyanisole (BHA) andpropyl gallate (PG) are used in some food productsto delay the onset of oxidative rancidity. As there isserious concern about the toxic and carcinogeniceþects of these substances,1 there is great interest insubstituting them with natural antioxidants.2

Oat is a grain with interesting properties. In addi-tion to its dietary übre content, oats are a source ofnatural antioxidants. The use of oat ýour to preventoils becoming rancid was patented in the 1930s.3The relatively high antioxidant properties of oatýour has been attributed to the phenolic compounds,predominantly ferulic and caþeic acid deriv-atives.4h11 Detailed qualitative analysis of these com-pounds revealed a range of long chain and(C26 C28)mono and diesters of caþeic and ferulic acids.6 Theirantioxidant capacity appears to be mediated throughtheir ability to react with free radicals and so breakthe chain propagation cycle associated with peroxi-dation reactions.

More recent work12h14 has highlighted the poten-tial of extracted oat antioxidants as a commerciallyviable source of natural antioxidants for the foodindustry. An essential aspect of this would be theselection of a cost-eþective process for extracting

antioxidants. Workers have used diþerent solventsand extraction procedures to obtain the polar lipidfraction, which is enriched with the phenolic com-pounds of interest.12,15 No researchers, however,have directly compared the activities of such extractswith a view to rationalising an industrial scaleprocess. Commercial considerations will also inýu-ence the methods used to measure the antioxidantactivity of the oat extracts. For example, continualmonitoring of the industrial process would berequired and so a method for measuring the anti-oxidant activity would be necessary. Some recentreports suggest that chemiluminescence is a conve-nient method for measuring antioxidant activ-ity.16h18

This report compares the yield and activity ofantioxidants extracted from oats by two previouslyreported extraction regimes. A new developmentapplied to both methods was the solid phase fraction-ation of crude extracts to yield the polar lipids. Theuse of chemiluminescence to measure the antioxidantactivity of polar lipid extracts from oats was alsoevaluated.EXPERIMENTAL

Materials

Oat (Avena sativa) groat (var Gerald, grown in trialplots by ADAS Rosemaund), was milled \400

¹ Bas ed on a paper pres ented at Ferulate ’98, IFR, Norwich,

8–11 July 1998.

* Corres pondence to : A Divis ion of Food Sciences ,David Gray,

School of Bioligical Sciences , Univers ity of Nottingham, Sutton

Bonington Campus , Loughborough, LE12 5RD, UK

Contract/grant s pons or : Home Grown Cereal Authority(Received 9 July 1998; revis ed vers ion received 3 September

1998; accepted 15 October 1998)

( 1999 Society of Chemical Industry. J Sci Food Agric 0022-5142/99/$17.50 385

RH Auerbach, DA Gray

mesh, UMIST, Manchester, UK). All solvents wereanalytical reagent grade and purchased from FisherScientiüc. All other chemicals were purchased fromSigma-Aldrich Chemical Co (unless otherwisestated). Tween 40 (polyoxyethylene sorbitanmono-palmitate), linoleic acid (L-1876, 99% pure), t-butylhydroperoxide (70% aq solution),

luminol (HPLC grade), cyto-Na2B4O7 · 10H2O,chrome c (horse heart, 99% pure), trans-b-carotene(Type I, synthetic, 95% pure), silicic acid (100mesh), tannic acid (code:T/0150/53; batch :975829347; MW 1701.22; purchasedC76H52O46from FisherScientiüc), oxygenated distilled water :pure oxygen was bubbled through triple distilledwater for 0.5h.

Methods

Lipid extractionMethanol extraction: adapted from Ref 14. Milled oatgroats (200g) were weighed into a ýask and methanol(1.4 litre) added. The suspension was left standingover 24h. On day 2 the suspension was ültered, andthe ültrate rotary evaporated, dissolved in 10mlmethanol and stored in the dark at 5¡C; the residuewas transferred back into the ýask and methanol (1.2litre) added. The suspension was then extracted foranother 24h. This procedure was repeated for 7 daysbut with diþerent volumes of methanol for theextraction (day 3: 1.2 litre, days 4–7: 800ml). On the8th day the methanolic extracts were combined,rotary evaporated and dissolved in 15ml methanol.The extract was further fractionated by columnchromatography.

Propan-2-ol extraction: adapted from Ref 15. Milledoat groats (100g) were weighed into a ýask andpropan-2-ol (1.2 litre) added. The ýask was thenplaced into a water bath at 70¡C and the suspensionwas stirred with a propeller stirrer at 300rpm for 2h.To obtain a clear solution the extract was centrifugedat 500] g. The supernatant was rotary evaporatedand dissolved in 7.5ml of methanol. Further frac-tionation by column chromatography was carriedout.

Fractionation of the crude lipid extractSilicic acid (50g of 100 mesh) was activated over-night at 105¡C. It was washed ürstly with three times100ml methanol, then with three times 100ml

petroleum ether. A petroleum ether slurry of thetreated silicic acid was packed into a column(25mm ] 300mm). The methanolic extract wasapplied to the top of the column. The ürst elutionwas carried out with 300ml petroleum ether toextract hydrophobic material and the second with300ml methanol to extract polar lipid material. Themethanol extract was concentrated on a rotary evapo-rated, dissolved in 15ml methanol and stored in thedark at [20¡C until further analysis.

Determination of total phenolic concentrationThe total phenolic content was measured by theAOAC Method (9.110)19 using tannic acid as stan-dard. Samples (0.4ml of extract) were mixed withFolin–Denis reagent and sodium carbonate (atambient temperature and at the prescribedconcentrations) and the absorbance at 760nm mea-sured after 30min.

Measurement of antioxidant activityb-Carotene bleaching method : adapted from Ref 20. b-Carotene (1mg) was dissolved in chloroform (10ml)and 10ml was added to a round-bottomed ýask thatcontained 20mg linoleic acid and 200mg Tween 40.Chloroform was removed by rotary evaporation at40¡C. Oxygenated distilled water (50ml) was thenslowly added to the ýask with vigorous stirring.

b-Carotene/linoleic acid solution (5ml) was addedto each polar lipid extract (0.2ml) in separate spec-trophotometer tubes. Measurements were made at470nm immediately after the addition of the emul-sion to the antioxidant solution. The tubes wereplaced in an agitating water bath at 50¡C. Absorb-ance measurements were made at 15min intervalsuntil the absorbance of the control read below 0.03.

Chemiluminescence method. Instrumentation – Theexperiments were carried out as depicted in Fig 1.The ‘Waters 2690’ pump has an automatic probesampler and an automatic injector with a sample loopof 50ll. Test samples were injected from this loopinto the chloroform–methanol mixture and pumpedto the chemiluminescence detector.

Calculation of antioxidant activity – A series of dilu-tions using methanol were made of each oat extract(1 : 4000, 1 : 3000 1 : 2000; 1 : 1500; 1 : 1000 and1 : 400) and t-butyl hydroperoxide added to a con-centration of 3lmol ml~1. The peak areas of thesedilutions were measured and compared to the area ofan equivalent solution minus oat extract which wasset to 100%. The antioxidant activity of each oatextract was calculated as an (inhibitionIC50

Figure 1. Flow diagram of the chemilumines cence method.

386 J Sci Food Agric 79 :385–389 (1999)

Oat antioxidant extraction and measurement

concentration) value, ie the concentration of the anti-oxidant that gives a peak area which is 50% of thecontrol. Unless otherwise stated, the values areIC50expressed in terms of the mass of oats equivalent tothis antioxidant concentration.

RESULTS AND DISCUSSION

Evaluation of chemiluminescence to measure oat

antioxidant activity

Propan-2-ol extracts from oats were used to evaluatechemiluminescence as a rapid, sensitive method formeasuring antioxidant activity. It was compared toanother antioxidant assay method based on the inhi-bition of b-carotene bleaching by antioxidants. Thismethod has been used to measure the antioxidantactivity of oats12 and is considered to be convenientand rapid compared to conventional acceleratedstorage trials.

For the b-carotene method, two solutions corre-sponding to 53 and 530mg oats were measured. Asthe results in Fig 2 show, an amount of extractequivalent to 53mg of oats has no eþect on the blea-ching of b-carotene within this system. A 10-foldhigher concentration of phenolics dramaticallyreduces the rate of b-carotene degradation.

Equivalent propan-2-ol oat extracts were used toevaluate antioxidant activity using chemilumine-scence. The higher the percentage quenching value,the more potent the antioxidant activity. Results (Fig3) show the eþect of oat extract concentration on thepercentage quenching ; as concentration increases, sodoes the percentage quenching of the chemilumine-scence reaction. Repeated measurements of an oatextract gave consistent results with negligible varia-tion. Besides its outstanding reproducibility, thismethod is at least two orders of magnitude more sen-sitive than the b-carotene method (compare results inFigs 2 and 3). There are other advantages of the che-miluminescence method. It is rapid and laboursaving, requiring only 15min of automated samplingper extract compared with 2h of continuous spectro-photometric measurement of several samples.Finally, chemiluminescence provides absolute IC50values which can be used to compare separatesamples and experiments. The b-carotene methodgives results that ýuctuate between experiments thusprecluding direct comparisons.

Figure 2. Carotene bleaching as s ay. Each value repres ents the

average of duplicate meas urements which gave a negligible

range.

Figure 3. Effect of oat polar lipid (derived from the is opropanol

extraction method) concentration on the chemilumines cence

reaction. and I repres ent the amount of chemilumines cenceI0

meas ured in the abs ence and pres ence of oat extract

res pectively. Each value repres ents the average of duplicate

meas urements , which gave a negligible range. valuesIC50

indicate the equivalent mas s of oats required to inhibit the

chemilumines cence reaction by 50%. They are calculated byins erting y\ 30.1 in the equation for the linear plots .

Comparison of methanol and propan-2-ol extraction

methods

The methanol extraction method yielded 156mg oftotal phenolics per kg of oats and the isopropanolmethod yielded 104mg kg~1 (Table 1). Both thepropan-2-ol and methanol methods used for extract-ing oat antioxidants in this study yielded total phe-nolic concentrations (104 and 156mg per kg of oats,respectively) which compare well with previouslyreported values (171mg kg~1 (Ref 6); 30mg kg~1(Ref 13) 230mg kg~1. (Ref 14)).

Although the methanol extraction method yieldedmore total phenolics compared to the propan-2-olmethod, the latter method is likely to be chosen bythe industrialist. The extraction time of 2-h and1.2 litres total isopropanol compared to 10 days and11 litres total methanol represents a signiücant costand time saving. A propan-2-ol based method for the

Table 1. Total phenolic concentration and antioxidant activity of

oat extracts a

Extraction method

Methanol Propan-2-ol

( SD) ( SD)

Total phenolic content 156 (^5.7) 104 (^13.4)(mg kgÉ1)

IC50oat equivalent 471 (^5.7) 635 (^58.5)

(lg)IC

50phenolic equivalentb 73 66

(ng)

a Each value repres ent the average of triplicate meas urements .

phenolic equivalent values indicate the mas s of phenolicb IC50

compounds which are pres ent in the oat extract giving 50%quench of the chemilumines cent reaction.

J Sci Food Agric 79 :385–389 (1999) 387

RH Auerbach, DA Gray

industrial extraction of oat oil has been patented.21Given the results from the present study, it would berelatively simple to add a column fractionation stepto this validated process and recover a commercialpolar lipid fraction enriched in active oat anti-oxidants.

Consideration of the values for the extractsIC50makes it possible to compare the antioxidant activitywithin these extracts to the amount of total phenol-ics. The phenolic equivalent values are veryIC50similar (Table 1). This strongly suggests that asimilar phenolic composition was recovered usingboth methods.

Since no other workers have measured oat anti-oxidant activity by chemiluminescence, it is hard tocompare the activity of our extracts with similar oatextracts which have been tested for antioxidant activ-ity. Xing and White14 added oat extract to soya beanoil heated to 60¡C and measured the peroxide valueover time. Parallel studies using a commercial anti-oxidant (tert-butyl hydroquinone, tBHQ) revealedthat, on an equivalent mass basis, the total oat phe-nolic extract displayed approximately 8% of thetBHQ activity. A similar comparison of the anti-oxidant activity in our extracts with the activity ofbutylated hydroxytoluene, BHT (another widelyused antioxidant in the food industry) using chemil-uminescence indicated that the total oat phenolicextract displayed 15% of the BHT activity. Danielsand Martin6 separated individual oat phenolic com-ponents and tested their antioxidant activity byadding them to oat oil and measuring the rate ofoxygen absorption. They reported activities, on anequivalent mass basis, which ranged from 30–60% ofthe activity of the commercially available antioxidantpropyl gallate. These workers used rigorous extrac-tion conditions where UV light was omitted. Thisprevented cis/trans isomerisation of the hydroxy-cinnamate thus enabling a detailed analysis of thestructures of phenolics present in the diethyl etherextract from oats. Although such rigorous conditionsmay not be compatible with an industrial process,further work could be done to test the eþect of suchmeasures on the activity of extracted oat anti-oxidants.

In this study, chemiluminescence has been provedto be a convenient, reliable, rapid and sensitivemethod for measuring oat antioxidant activity com-pared to the b-carotene bleaching method. Topredict the capacity of an antioxidant to slow downoxidative rancidity development in food materials, itis best to use a range of methods that cover bulk oiland aqueous emulsion phases.22 Accelerated storagetrials, involving temperatures signiücantly aboveambient, are routinely employed. Work has castdoubt on the validity of extrapolating results fromaccelerated storage trials to the prediction of shelflife under normal storage conditions.23 It was sug-gested that the mechanism by which antioxidantswork can be altered at elevated temperatures. Indeed

Burkow et al17 identiüed chemiluminescence as arapid method for analysing antioxidant potential at35¡C within 24h, thus avoiding the high tem-peratures of accelerated storage trials or the extendedtime course studies (days/weeks/months) of conven-tional room temperature storage trials.

CONCLUSION

Propan-2-ol extraction of oats (200g) at 70¡C for 2hfollowed by column fractionation, yields a polar lipidfraction with a concentration of total phenolic com-pounds comparable with values obtained fromsolvent-extracted oats previously quoted in the liter-ature. Antioxidant activity of this extract, as mea-sured by chemiluminescence, was at least comparablewith the activity of an oat polar lipid fraction14which was added to soya bean oil in an acceleratedstorage trial. Chemiluminescence oþers the potentialto screen oats rapidly for their antioxidant contentand to monitor any industrial process designed torecover the active antioxidants from oats. Signiü-cantly higher sensitivity (at least two orders ofmagnitude) was achieved using chemiluminescencecompared with using b-carotene bleaching tomeasure antioxidant activity. Consequently, largebatches of oats (kilograms) are not required forlaboratory-scale extraction of oat antioxidants ; gramor milligram batches of oats/oat ýour may be suffi-cient to extract and measure antioxidant activity.

ACKNOWLEDGEMENTS

The authors would like to thank Dr Rob Linforthand Shane Avison for their technical assistance andthe Home Grown Cereal Authority (HGCA) forsponsoring this work.

REFERENCES1 Branen A L, Toxicology and biochemistry of butylated

hydroxyanisole and butylated hydroxytoluene. J Am Oil

Chem Soc 52:59–63 (1975).2 Pratt D E, Natural antioxidants from plant material, in Phenol-

ic Compounds in Food and their Eþ ects on Health, Ed by M-THuang, C-T Ho and C Y Lee. American Chemical Society,New York, pp 55–71 (1992).

3 Musher S, Oil with grain material to stabilise against oxida-tion. United States Patent 2,049,017 (1936).

4 Collins F W, Oat phenolics : structure, occurrence and func-tion, in Oats Chemistry and Technology, Ed by Webster F H,American Association of Cereal Chemists, St Paul, MN, pp227–295 (1986).

5 Daniels D G and Martin H F, Isolation of a new antioxidantfrom oat. Nature 191:1302 (1961).

6 Daniels D G and Martin H F, Antioxidants in oats : monoesters of caþeic and ferulic acids. J Sci Food Agric 18:589–595 (1967).

7 Daniels D G and Martin H F, Antioxidants in oats : glycerylesters of caþeic and ferulic acids. J Sci Food Agric 19:710–712 (1968).

388 J Sci Food Agric 79 :385–389 (1999)

Oat antioxidant extraction and measurement

8 Daniels D G, King H G C and Martin H F, Antioxidants inoats : esters of phenolic acids. J Sci Food Agric 14:385–390(1963).

9 Durkee A B and Thivierge P A, Ferulic acid and other phenol-ics in oat seeds. J Food Sci 42:551–552 (1977).

10 Pokorny J, Natural antioxidant for food use. Trends Food Sci

Technol 2:223–227 (1991).11 White PJ, Novel natural antioxidants and polymerisation

inhibitors in oats, in Natural Protectants against Natural

Toxicants, Ed by Bidlack W R and Omaye S T. TechnomicPublishing Company, Lancaster, PA, USA, pp 35–49(1995).

12 Duve K J and White P J, Extraction and identiücation of anti-oxidants in oats, J Am Oil Chem Soc 68:365–370 (1991).

13 Tian L L and White P J, Antioxidant activity of oat in soybeanand cottonseed oils. J Am Oil Chem Soc 71:1079–1086(1994).

14 Xing Y and White P J, identiücation and function of anti-oxidants from oat groats and hulls. J Am Oil Chem Soc

74:303–307 (1997).15 Forssell P, Kervinen R, Alkio M and Poutanen K, Compari-

son of methods for separating polar lipids from oat oil. Fat

Sci Technol 9:355–358 (1992).

16 Ashida S, Okazaki S, Tsuzuki W and Suzuki T, Chemilumine-scence method for the evaluation of antioxidant activityusing lipid hydroperoxide-luminol. Anal Sci 7:93–96 (1991).

17 Burkow I C, Vikersveen L and Saarem K, Evaluation of anti-oxidants for cod liver oil by chemiluminescence and the ran-cimat method. J Am Oil Chem Soc 72:553–557 (1995).

18 Hirayama O, Takagi M, Hukumoto K and Katoh S, Evalu-ation of Antioxidant Activity by chemiluminescence. Anal

Biochem 247:237–241 (1997).19 AOAC, Official Methods of Analysis, Association of the Offi-

cial Analytical Chemists, Washington, DC (1984).20 Marco G J A, Rapid method for evaluation of antioxidants. J

Am Oil Chem Soc 45:594–598 (1968).21 Myllyma� ki O, Ma� lkki Y and Autio K, A process for fractionat-

ing crop into industrial raw material PCT Patent Appl WO89/01294 (1989).

22 Chen J H and Ho C T, Antioxidant activities of caþeic acidand its related hydroxycinnamic acid compounds. J Agric

Food Chem 45:2374–2378 (1997).23 Marinova E M and Yanishlieva N V, Eþect of temperature on

the antioxidative action of inhibitors in lipid antioxidation. J

Sci Food Agric 60:313–318 (1992).

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