Determination of Antioxidant Capacity of Papaya Fruit

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O R I G I N A L P A P E R

Determination of antioxidant capacity of papaya fruitand papaya-based food and drug integrators, using a biosensor

device and other analytical methods

Luigi Campanella

Tania Gatta

Emanuela Gregori

Mauro Tomassetti

Received: 28 May 2008 / Accepted: 28 July 2008/ Published online: 24 October 2008

Springer-Verlag 2008

Abstract Antioxidant and radical-scavenging properties

are alleged to form the basis of the therapeutic propertiesattributed to the papaya and papaya-based food and drug

integrators that have recently appeared on the market. In the

present research a biosensor-based method was used to

determine the antioxidant capacity of papaya fruit as is, that

of several food and drug integrators, and lastly of pure papain

alone. Results are compared using a reference spectrofluo-

rimetric method and two other spectrophotometric methods.

In addition pro-oxidant properties of the same samples were

also checked using two colorimetric methods.

Keywords Biosensor Superoxide dismutase enzyme

electrode Antioxidant, pro-oxidant papaya properties

Introduction

The weight-reducing properties of papain, a fundamental

constituent of papaya, as well as its anti-inflammatory

action, are now widely known [1]. The most recent studies

on papaya stem from the growing interest aroused by a

fermented papaya preparation (FPP) [2], the administration

of which is believed, among other things, to reduce the

symptoms of Parkinson’s disease. FPP is obtained from

the unripe fruit through a fermentation process and has

a wide range of different characteristics: it no longer

contains practically any papain or vitamins, but is rich in

oligosaccharides [2]. So far, no serious scientific study

has confirmed the above-mentioned effects. Nevertheless,antioxidant and immunostimulating properties believed to

underlie its therapeutic effects continue to be attributed to

FPP [3]. It actually seems probable that antioxidant and

radical-scavenging properties [4] underlie the therapeutic

properties claimed for these products. In order to verify

the antioxidant and thus ‘‘radical-scavenging’’ properties of

the principal papaya-based food and drug integrators on

the market exhaustively, in the present research the

following were used for the determination and comparison

of the antioxidant capacity: a biosensor method recently

developed in our laboratory [5], a spectrofluorimetric

reference method [6], and two spectrophotometric methods

[7, 8]. These analytical methods were applied both to the

fruit, with or without papaya skin, and to pure papain, as

well as to six papaya-based food or drug integrators and

one officinal pharmaceutical preparation.

Another aspect of the research in this field that we have

undertaken above all in recent times [9] is addressed to

ascertaining any possible inverse relationship between the

antioxidant and pro-oxidant capacity of plants and inte-

grators derived therefrom.

The majority of the pro-oxidant species are generated by

free radicals that, owing to their high reactivity, tend to

react with practically all organic molecules with which

they come into contact. This leads to the generation of

reactive oxygen metabolites (ROMs), comprising in prac-

tice many of the pro-oxidant species [10, 11]. The principal

pro-oxidants in animal and plant cells are represented by

the reactive oxygen species (ROSs), which may form under

conditions of high oxygen partial pressure. Two other

methods were consequently applied in the present research,

which allow an evaluation to be made of the pro-oxidant

characteristics of all the products tested in the form of both

L. Campanella T. Gatta E. Gregori M. Tomassetti (&)

Department of Chemistry,

University ‘‘La Sapienza’’,

00185 Rome, Italy

e-mail: [email protected]

1 3

Monatsh Chem (2009) 140:965–972

DOI 10.1007/s00706-008-0069-3



alcohol extracts and aqueous extracts obtained from them,

i.e., the lipoperoxide test and the d-ROMs test [12].

Results and discussion

The biosensor and the reference spectrofluorimetic meth-

ods used for antioxidant capacity measurements wereapplied to the aqueous extract of the samples both after

homogenization alone and after homogenization and cen-

trifuging; conversely, in the case of the spectrophotometric

methods, only the homogenized and then centrifuged

samples were analyzed. The tests were carried out imme-

diately after treatment of the sample in order to avoid any

likelihood of the determinations being affected by fer-

mentation or other modifications. The antioxidant capacity,

for equal weights of all the samples considered, obtained

using all the methods described in the ‘‘Materials and

methods’’ section, is compared in Figs. 1, 2, 3, and 4.

It is interesting to note that the value of total antioxidantcapacity for the same sample is always lower when it has

been subjected to both homogenization and centrifuging

than when it has only been centrifuged (Figs. 1, 2). This

may be accounted for by the fact that an appreciable

contribution to total antioxidant capacity can be made by

several relatively insoluble components of the sample.

Of course all the samples tested had the same weight so

as to allow a homogeneous comparison of their antioxidant

capacity. The results obtained using the biosensor method

are shown as histograms in Fig. 1, those obtained using the

spectrofluorimetric method in Fig. 2, and those obtained

using the spectrophotometric method ( N , N -diethyl- p-phe-

nylendiamine ?Fe3?) in Fig. 3; lastly, those obtained using

the OXY-adsorbent test are shown in Fig. 4.

Clearly, the comparison of the results obtained using

different methods cannot be based on absolute values, as

each method uses different units of measure, but rather on

the trends emerging from the histograms obtained. How-

ever, for the homogenized samples, the qualitative trends

displayed by the first two methods may be said to be vir-

tually identical, with only a very small inversion found in

Fig. 1 Histograms showing the antioxidant capacity determined by

SOD biosensor in the samples tested (for equal weight). White: values

for homogenized samples; black: values for homogenized and

centrifuged samples. a Papaya (pulp ? skin); b papaya (pulp);

c papain (enzyme); d fermented papaya {1}; e dry extract; f papaya

juice; g papaya (pharm. prep.); h fermented papaya {2}; i fermented

papaya {3}; l fermented papaya {4}


the ORAC method in the samples tested (for equal weight). White

values for homogenized samples; black values for homogenized and

centrifuged samples. a Papaya (pulp ? skin); b papaya (pulp);

c papain (enzyme); d fermented papaya {1}; e dry extract; f papaya

juice; g papaya (pharm. prep.); h fermented papaya {2}; i fermented

papaya {3}; l fermented papaya {4}


the spectrophotometric method (DMPD ? Fe3?

) in the samples

tested (for equal weight); all samples were homogenized and

centrifuged. a Papaya (pulp ? skin); b papaya (pulp); c papain

(enzyme); d fermented papaya {1}; e dry extract; f papaya juice;

g papaya (pharm. prep.); h fermented papaya {2}; i fermented papaya

{3}; l fermented papaya {4}


the OXY-adsorbent test spectrophotometric method in the samples

tested (for equal weight); all samples were homogenized andcentrifuged. a Papaya (pulp ? skin); b papaya (pulp); c papain

(enzyme); d fermented papaya {1}; e dry extract; f papaya juice; g

papaya (pharm. prep.); h fermented papaya {2}; i fermented papaya

{3}; l fermented papaya {4}

966 L. Campanella et al.

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the case of papain and powdered papaya. It is also observed

that the trends for the homogenized and centrifuged sam-

ples found in the first two methods are also in

comparatively good agreement, albeit with several small

additional inversions.

Also in the third method (the N , N -diethyl- p-phenylen-

diamine ?Fe3? spectrophotometric method), the trend is in

sufficient agreement with that of the first two methods,albeit with three relatively small inversions. The good

agreement between the ORAC method shown in Fig. 2

(selected as reference method) and each of the other two

methods in Figs. 1 and 3 is illustrated by the correlation

straight lines shown in Figs. 5 and 6, respectively.

It is also significant that, by using the equations of the

correlation straight lines found, it is also possible to express

all the values obtained using the three methods in the same

units of measure, for instance ORAC units, which are the

most commonly used.

Lastly, as several authors consider it important to know

the antioxidant capacity not only of the ‘‘whole’’ fruit, butalso of the dehydrated fruit sample, an experimental

determination was made of the percentage of water con-

tained in the papaya, with and without the skin, which was

found to be 82% by weight on average. It was thus possible

to compute also the antioxidant capacity respectively of the

anhydrous pulp and pulp skin for these two samples. The

values obtained using the three methods are shown as

histograms in Fig. 7.

It must be said with reference to these results that the

percentage of water lost in the process of drying papaya

fruit pulp was found to be slightly higher than for the whole

fruit, which probably accounts for the small inversion in

the results referring to the antioxidant capacity obtained

using anhydrous samples with or without skin (Fig. 7)

compared with the trends observed in Figs. 1 and 2.

With regard to the trend in the values obtained using the

OXY-adsorbent test, it must first of all be pointed out that,

in order to highlight the existence of a possible correlation

with the trends of the SOD and ORAC method, the data

from the OXY-adsorbent test were compared, in Fig. 4, as

the inverse of the experimental data effectively found (i.e.,

as 100 [1/(10-6 M)]. This is because the OXY-adsorbent

test determines the excess of equivalents of hypochlorous

acid and not directly the concentration of antioxidant

substances, and the former is inversely related to the latter.

Fig. 5 Correlation straight line for values obtained using the SOD

method and those obtained using the ORAC method ( y =

(38.5 ± 4.5) x ? (22.7 ± 1.8); R2= 0.8997)

Fig. 6 Correlation straight line for values obtained using theDMPD ? Fe3? method and those using the ORAC method

( y = (0.592 ± 0.088) x ? (24.607 ± 1.599); R2= 0.8509)


the first three methods for dried papaya samples of equal weight

Antioxidant capacity of papaya fruit and papaya-based food and drug integrators 967

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The observed trend in Fig. 4 displays some differ-

ences compared with the first three methods although,

except for the large inversion represented by the dry

extract, this trend is after all not substantially different

from those of the first three methods. What emerges is

certainly the widely varying antioxidant capacity dis-

played in this case by the fruit samples with or without

skin compared with the samples of all the food inte-grators and drug specialities.

Lastly, it is possible to compare the trends of data

referring to antioxidant capacity also with the trends of data

concerning pro-oxidant properties both for aqueous

extracts and for alcoholic extracts, shown respectively in

Figs. 8 and 9.

It would seem reasonable to assume that the pro-oxidant

properties would follow a trend that was roughly the

inverse of that of antioxidant capacity. However, as

observed also on a previous occasion [9], this trend is not

always respected. It must be pointed out, however, that in

papaya fruit (with or without skin), in which the highestantioxidant capacity is found, the concentration of pro-

oxidants, at least of those found in the aqueous extract,

proved to be the lowest (Fig. 8) of those found in all

samples tested, as reasonably expected.

The antioxidant capacity was determined in samples

of papaya fruit and in several papaya-based food and

drug integrators available on the market. The first three

analytical methods tested in the determination of anti-

oxidant capacity, although based on different functioning

principles, produced very similar results. This confirms

the accuracy of the trends obtained and thus the reli-

ability of the methods used. Furthermore, the accuracy,

precision, and robustness of the SOD biosensor method,

recently proposed by the present author and having

already emerged in previous studies [13–15], were again

confirmed by the present research. It is also significant

that the four applied methods should be complementary

as far as the information they produce is concerned: thesuperoxide dismutase (SOD) biosensor method, as well

as the ORAC method, determines the total antioxidant

capacity and represents a valid indicator for this feature,

but does not quantify the concentration of antioxidant

substances; lastly, the OXY-adsorbent test evaluates the

antioxidant power of the sample by measuring its

capacity to oppose the massive oxidative action of

hypochlorous acid. The two colorimetric methods, the

d-ROMs test and the ‘‘lipoperoxides’’ test, evaluate the

concentration of the prooxidant species contained

respectively in the aqueous fraction and in the lipidic

fraction of the samples; nevertheless, by these methods itshould therefore be possible to describe the pro-oxidant

properties of the different samples. However, while in

some cases the values referring to the pro-oxidant

properties are found to be quite convincing, as in the

case of whole papaya fruit or papaya pulp alone where a

very high antioxidant capacity actually corresponds to

relatively low pro-oxidant characteristics, generally

speaking, the same good inverse correlation is not found

in the case of the samples of papaya-based food inte-

grators and drug specialities. At the present state of our

research, in this and in other works [9], it has not been

possible to provide a definitive account of the foregoing

phenomena. However, it should also be borne in mind

that the measurement of pro-oxidant properties of the

test samples was performed exclusively using colori-

metric methods. Consequently, the reliability in the

values found cannot be as high as that assigned to the

anti-oxidant capacity values for which different methods

(biosensor, spectrofluorimetry, and spectrophotometry) all

gave the same results (that is, a good agreement among

their respective trends).

Fig. 8 Histograms showing hydroperoxide concentrations deter-

mined using the d-ROMs spectrophotometric method for samples of

equal weight; all samples were homogenized and centrifuged.

a Papaya (pulp ? skin); b papaya (pulp); c papain (enzyme);

d fermented papaya {1}; e dry extract; f papaya juice; g papaya

(pharm. prep.); h fermented papaya {2}; i fermented papaya {3};

l fermented papaya {4}

Fig. 9 Histograms showing lipoperoxide concentrations determined

using the ‘‘lipoperoxide test’’ spectrophotometric method for samples

of equal weight; all samples were homogenized and centrifuged.

a Papaya (pulp ? skin); b papaya (pulp); c papain (enzyme);

d fermented papaya {1}; e dry extract; f papaya juice; g papaya

(pharm. prep.); h fermented papaya {2}; i fermented papaya {3};

l fermented papaya {4}


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Conclusion

The most surprising result was, however, to observe that

the so-called ‘‘fermented papaya,’’ the one most publicized

from the therapeutic point of view, is the product that

displayed the lowest antioxidant capacity in three of the

four products tested, which certainly contain it. It may

therefore be postulated that the therapeutic propertiesapparently detected in fermented papaya are not to be

attributed solely to its antioxidant capacity, but also and

above all to some other specific active principle present in

the product. Moreover, as in previous research [16, 17]

comparing the antioxidant properties of plant products with

those of the corresponding papaya-based food integrators

or pharmaceutical specialities, also in this case it was found

that the antioxidant capacity of the latter was always

appreciably lower than in the whole fruit.

Materials and methods

Chemicals

3,7-Dihydro-1 H -purine-2,6-dione sodium salt (1), 2-[2-(bis

(carboxy methyl)amino)ethyl-(carboxymethyl)amino]ace-

tic acid (EDTA) (2), superoxide dismutase (SOD) 4,980 U/mg

(3), dialysis membrane (art. D-9777) (4), N , N -diethyl- p-

phenylendiamine (5), methanoic acid (6), ferric chloride

(7), sodium acetate anhydrous (8), phenol (9), and b-phy-

coerythrin (10) were supplied by Sigma (Milan, Italy).

Xanthine oxidase 0.39 U/mg (11) and cellulose acetate

(12) were supplied by Fluka AG, Buchs (Switzerland).

2,2’-Azobis(2-amidinopropan)dihydrochloride ( ABAP)

(13) was supplied by Waco Chemical (Richmond, VA).

Isopropylic acid (14), potassium dihydrogenphosphate

(15), and sodium hydrogenphosphate (16) were supplied by

Carlo Erba (Milan, Italy). Polyvinylacetate (17), acid 2-

carboxy-6-hydroxy-2,5,7,8-tetramethylchroman (Trolox)

(18) and cellulose triacetate (19) were supplied by Aldrich

(Germany). Kit Diacron for lipoperoxide assay (20), kit

Diacron OXY-adsorbent test (21), kit Diacron d-ROMs test

(22), and ROM-Diacron Standard (23) were supplied by

Diacron (Grosseto, Italy).

Apparatus

Ultra-Turrax homogenizer mod. T8 by Ika Labortechnik;Crison pH meter mod. GLP 22; spectrofluorimeter Perkin-

Elmer, mod. LS-5, equipped with a Perkin–Elmer recorder,

mod. 561; electrode mod. 4000-1 by Universal Sensor Inc.

New Orleans, LA, coupled with an Amel potentiostat mod.

551, connected to an Amel differential electrometer, mod.

631 and to an Amel analog recorder, mod. 868; spectro-

photometer UV-VIS Perkin Elmer mod. Lambda 5,

provided with printer; a 10 Yellowline Laboratory mill

from IKA Works Inc.

Samples

Papaya fruit purchased in local supermarkets, papaya-

based food, or drug integrators (marketed as tablets, cap-

sules, bags, or syrup solutions), purchased from specialized

shops or drugstores; pure papain (24) supplied by Sigma

Aldrich (Milan, Italy). The following were analyzed:

papaya fruit (pulp only or pulp plus skin), pure papain, a

dry papaya extract, papaya in tablet form, papaya juice, and

four different samples of powdered fermented papaya. All

the samples tested are listed in Table 1.

Treatment of samples

A sum of 1.0 g of each powdered sample was dissolved or

dispersed in 6 cm3 of phosphate buffer (pH = 7.5) and then

homogenized. In the case of papaya fruit samples, 1.0 g of

sample taken from half the entire fruit (pulp ? skin, after

eliminating all the internal seeds) as well as of 1.0 g of the

other half of the fruit without the skin were respectively

dispersed in 6 cm3 of phosphate buffer, then homogenized

Table 1 Analyzed samples

(n{1} ? n{4} different

fermented papaya samples)

Samples Itemized list Form

(a) Papaya Pulp ? skin Whole fruit

(b) Papaya Pulp Fruit (pulp only)

(c) Papain Enzyme Coarse powder

(d) Fermented papaya {1} Food integrator Powder in bag

(e) Dry extract Officinal pharmaceutical preparation Powder

(f) Papaya Food integrator Juice

(g) Papaya Pharmaceutical integrator Tablets

(h) Fermented papaya {2} Pharmaceutical integrator Powder in bag

(i) Fermented papaya {3} Pharmaceutical integrator Tablets

(l) Fermented papaya {4} Pharmaceutical integrator Powder in capsules


1 3



separately. All the samples were left to stand for 60 min at

ambient temperature. Each sample was then divided into

two parts; one of the two parts was analyzed as such. The

other part was instead centrifuged for 10 min at 4,000 rpm,

at ambient temperature, and the supernatant analyzed.

For the analyses carried out using the N , N -diethyl- p-

phenylen diamine (5) ? Fe3? method, the samples were

treated as described above, but diluted in 6 cm3 of acetatebuffer (pH = 5.25) before the analysis was performed.

Superoxide dismutase (SOD) biosensor method

The total antioxidant capacity was measured by SOD

biosensor operating as follows: the superoxide radical is

determined using a biosensor obtained by coupling a

transducer (an amperometric electrode for hydrogen per-

oxide) with the superoxide dismutase enzyme (3)

immobilized in kappa-carrageenan gel [5]. The gel con-

taining the enzyme is sandwiched between a cellulose

acetate (12) membrane and a dialysis membrane (4). Thewhole assembly is secured to the electrode with an O-ring.

A constant potential of ?650 mV with respect to an Ag/

AgCl/Cl- cathode is applied to the platinum anode. The

dialysis membrane is used to support the gel and to prevent

attack by the enzyme. The superoxide radical is produced

by the oxidation of 3,7-dihydro-1 H -purine-2,6-dione (1) in

aqueous solution to 7,9-dihydro-1 H -purine-2,6,8(3 H )-tri-

one in the presence of the xanthine oxidase (11) enzyme,

which is free in solution.

xanthine þ H2O2 þ O2 !xanthine oxidase

uric acid þ 2Hþ

þ O2 ð1Þ

The disproportion reaction of the superoxide radical,

catalyzed by the superoxide dismutase (3) immobilized

on the electrode, produces oxygen and hydrogen peroxide.

The hydrogen peroxide produced is oxidized at the anode,

generating an amperometric signal (in nA) that is

proportional to the concentration of the superoxide

radical present in solution.

O2 þ O

2 þ 2Hþ !superoxide dismutase

H2O2 þ O2 ð2Þ

The addition of a sample possessing antioxidant proper-

ties produces a decrease in the signal as, by reacting with

the superoxide radical, the concentration of these spe-

cies in solution is lowered. As a consequence, both the

released H2O2 and the intensity of the amperometric

current diminish. In practice, the electrochemical bio-

sensor was placed in a cell thermostated at 25C

containing 15.0 cm3 of phosphate buffer, 0.05 M, at pH

7.5, and allowed to stabilize under constant stirring. After

addition to the same solution of a fixed quantity of the

xanthine oxidase (11) enzyme (1.2 mg), three successive

additions of 0.2 cm3 of the solution of 3,7-dihydro-1 H -

purine-2,6-dione sodium salt (1) 0.01 M were made,

waiting for the signal to stabilize between successive

additions. The values of the recorded current variations

were thus plotted versus the 3,7-dihydro-1 H -purine-2,6-

dione (1) concentration and the slope value calculated.After washing the cell, all the same solutions were

renewed in the measuring cell, in which also the sample

under test for its antioxidant capacity was dissolved, or

dispersed. Lastly, a new straight line was recorded as

described above.

The value of the antioxidant capacity was expressed in

RAC units by the following algorithm:

ðRACÞ ‘‘Relative Antioxidant Capacity’’ ¼ 1 mc=mxð Þ

ð3Þ

mx = slope of the straight line obtained by means of suc-

cessive additions of 3,7-dihydro-1 H -purine-2,6-dione (1);mc = slope of the straight line obtained by means of suc-

cessive additions of 3,7-dihydro-1 H -purine-2,6-dione (1),

but in the presence of the sample possessing anti-oxidant

properties.

The superoxide dismutase (3) assembling and SOD

immobilization in kappa–carrageenan were described in

detail in previous papers [5, 13].

ORAC spectrofluorimetric method

The oxygen radical absorbance capacity (ORAC) spectro-

fluorimetric method is well known and extensively

described in the literature; it is usually chosen as a refer-

ence method as highly suitable and reliable, but also is too

expensive for routine analysis [7]. In the presence of free

radicals or oxidizing species, the protein b-phycoerythrin

(b-PE) (10) loses more than 90% of its fluorescence within

30 min. The addition of antioxidant species, which react

with the free radicals, inhibits the fluorescence of this

protein. This inhibition may be related to the sample’s

antioxidant capacity. In particular, 2,2-azobis(2-amidino-

propane) dihydrochloride (13) (ABAP) was used to

generate peroxide radicals (ROO.). To perform the mea-

surements, the wavelengths were set at k = 540 nm for

excitation and 565 nm for emission. Initially, 0.08 cm3 of

sample was placed in the cuvette together with 0.02 cm3

of phosphate buffer (0.075 M, pH 7), and 1.46 cm3 of

b-phycoerythrin (10) (0.183 M in phosphate buffer), pre-

pared and allowed to stand at 37C for 15 min before use.

The cuvette was placed in the spectrofluorimeter and the

initial fluorescence ( f 0) read off after 30 s. Then a further

0.02 cm3 of phosphate buffer was added to the solution in


1 3



the cuvette together with 0.02 cm3 of ABAP (3.2 10-4 M

in phosphate buffer). After stirring, the fluorescence was

read off after 0.5 s and then every 2 min, for a total time of

70 min. A similar procedure was also carried out using a 20

10-6 M solution of acid 2-carboxy-6-hydroxy-2,5,7,8-

tetramethylchroman (18) instead of sample.

The final results are expressed in ‘‘ORAC units’’

(micromoles of acid 2-carboxy-6-hydroxy-2,5,7,8-tetram-ethylchroman (18) equivalent per dm3 of sample):

ORAC Value ¼ 20k S sample S blank

ðS Trolox S blank Þ

ð4Þ

where k = dilution factor for the sample; S = integral of

the fluorescence curve of the sample, of the acid 2-car-

boxy-6-hydroxy-2,5,7,8-tetramethylchroman (18), or of the

‘‘blank.’’

Spectrophotometric method: N,N-diethyl-p-

phenylendiamine ?Fe3?

Following the method described in the literature [8]: in a

vessel containing 100 cm3 of acetate buffer (0.1 M at

pH = 5.25) 1.0 cm3 of a solution of N , N -diethyl- p-phe-

nylendiamine (5) 0.1 M and 0.2 cm3 of a solution of ferric

chloride (7) 0.05 M was added; this produced the purple

colored cation radical of N , N -diethyl- p-phenylendiamine

(5).

The final solution was placed in a quartz cuvette and the

absorbance at 514 nm read off. To this solution was then

added 0.15 cm3 of a suitably diluted sample or else a

solution of acid 2-carboxy-6-hydroxy-2,5,7,8-tetrame-

thylchroman (18) 1.0 mg cm-3; the absorbance at 514 nm

was then read off after 10 min during which the mixture

was maintained under constant stirring at a temperature of

25C. Only acetate buffer was placed in the reference

cuvette.

The results are reported as the percentage inhibition of

the signal ( I 514 %). The antioxidant capacity of the samples

is expressed in TEAC [antioxidant capacity in equivalent

acid 2-carboxy-6-hydroxy-2,5,7,8-tetramethylchroman

(18)] units using a calibration curve obtained using dif-

ferent amounts of acid 2-carboxy-6-hydroxy-2,5,7,8-

tetramethylchroman (18) and taking account of the fact that

absorbance inhibition at 514 nm is linear between (0.2 and

8.0) 10-6 g of acid 2-carboxy-6-hydroxy-2,5,7,8-tetrame-

thylchroman (18).

Oxy-adsorbent test method

The sample is subjected to massive oxidation through

HClO; the antioxidant substances contained in the sample

react with the acid and can be quantified by measuring the

excess of HClO.

The quantification of the non-reacted acid is carried

out by the spectrophotometric method (reading at k =

490 nm), after addition of suitable buffered chromogenous

agent, an aromatic alkyl diamine ( N , N -diethyl- p-phenyl-

endiamine (5)). The concentration of the colored complex

is directly proportional to the concentration of HClO and is

indirectly related to the antioxidant capacity. The results

are expressed as 10-6 M of non-reacted HClO.Practically, 1 g of homogenized sample is added to

4 cm3 of water in a Falcon tube. The suspension is

sequentially sonicated (15 min) and centrifuged (15 min,

4,000 rpm). The supernatant is filtered through a MILLEX

GV filter (0.45 9 10-6 m).

To perform the calibration curve for the quantitative

analysis, different solutions of ROMs DIACRON stabilized

Standard (23) (0.34 9 10-3 M as antioxidant) are used.

The concentrations of these standard solutions are,

respectively, 6.8, 3.4, 1.7, 0.85, and 0.43 9 10-6 M of

antioxidant. Measurements are performed simultaneously

both on the standards and the samples; 1 cm3 of oxidantsolution (hypochlorous acid), 0.01 cm3 of sample, or of

water for the blank, or of standard solution are placed in a

quartz cuvette (0.01 m path length). The samples are

incubated at room temperature for 10 min, then 0.01 cm3

of chromogenous reagent is added to each cuvette. After

gently mixing, the analysis is carried out by immediately

reading the absorbance at k = 490 nm. Sample absorbance

values are corrected for reagent blanks and concentrations

calculated using the standard calibration curve.

Colorimetric determination of peroxides

Peroxides are determined both after aqueous and alcoholic

extraction using two different colorimetric methods,

respectively called the ‘‘d-ROMs test’’ (22), the one used to

check the peroxides in aqueous phase, and the ‘‘lipoper-

oxide test’’ (20), the one used to check lipoperoxides in the

alcoholic phase.

Principle of method for aqueous extracts (d-ROMs test)

The d-ROMs test (22) is a spectrophotometric test based on

the capacity of transition metal ions to generate in vitro

alkoxyl and peroxyl radicals in the presence of hydroper-

oxides. A chromogenic reagent ( N , N ,-diethyl- p-phenylen-

diamine (5)) is then added to this solution. This chromogen

compound possesses the feature of being oxidized by

hydroperoxyl and alkoxyl radicals and transformed into a

pink- to red-colored cation. The concentration of the

colored complex is directly related to the hydroperoxide

levels of the sample.

A calibration curve is constructed to quantify the per-

oxide concentration. To this end, some standard solutions


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are prepared by dissolving different volumes of the

standard [ROM-DIACRON standard (23), title: 5.88 9

10-3 M as H2O2] in 1 cm3 of water. The calibration range

is (2.94–0.37) 10-3 M of H2O2.

Briefly, 1 g of homogenized sample is added to 4 cm3 of

water in a Falcon tube. The suspension has been previously

sonicated, centrifuged, and filtered as described in the

Oxy-adsorbent test method. At the same time, a workingmixture is prepared by mixing R1 reagent and R2 reagent

in the ratio of 1:100. The preparation of the cuvette is

carried out as follows: 1 cm3 of working mixture and

0.005 cm3 of sample, or of water for the blank reagent, or

of standard solution for the construction of the calibration,

are placed in a quartz cuvette (1.0-cm path length). The

samples are incubated at 37C for 75 min, and then the

analysis is carried out by immediately reading at

k = 490 nm. Absorbance values of the samples are cor-

rected for reagent blanks and concentrations calculated

using the standard calibration curve. The results are

expressed as 10-3 M of H2O2.

Principle of method for alcoholic extracts

(lipoperoxide test)

The method is based on the capacity of peroxides to cat-

alyze the oxidation of Fe2? to Fe3?. The Fe3? produced is

linked to the thiocyanate anion, yielding a red complex,

which is measured spectrophotometrically. The increase of

the absorbance is directly proportional to the concentration

of the peroxide present in the samples.

The sample (1 g in 4 cm3 of isopropyl alcohol) is pro-

cessed (sonicated, centrifuged, and filtered) as described

above, except that sonication is performed at 40C. Per-

oxide quantification is carried out by means of a calibration

curve. Several standard solutions, with concentrations of

(0.375, 0.750, 1.5 and 3.0) 10-3 Eq cm-3 of peroxides

[DIACRON’s standard (23)], are prepared by dissolving

different volumes of standard in 1 cm3 of isopropyl alcohol

(14).

The analyses are conducted as follows: 1.2 cm3 of R1

reagent (Fe2? salt) is added to 0.01 cm3 of each standard

solution, then 0.010 cm3 of R2 reagent (chromogenous

agent) is added and, after gentle mixing, allowed to stand

for 5 min. The spectrophotometric analysis is carried out at

k = 505 nm. The real samples undergo the same proce-

dure, but the working volumes used are different, i.e.,

0.4 cm3 of alcoholic extract, 0.6 cm3 of R1 reagent, and

0.01 cm3 of R2 reagent. Readings are corrected for the

blank reagent absorbance. The results are expressed as

10-3 Eq cm-3 of lipoperoxides.

Acknowledgments This work was financially supported by MIUR,

co-funded project Prin 2005.

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Determination of Antioxidant Capacity of Papaya Fruit

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