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In vitro Antioxidant and Antibacterial Activities of Extracts from Annatto (

Bixa orellana L.) Leaves and Seeds

Article · July 2012

DOI: 10.1111/j.1745-4565.2012.00393.x

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Journal of Food Safety ISSN 1745-4565

IN VITRO ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF

EXTRACTS FROM ANNATTO (BIXA ORELLANA L.) LEAVES
AND SEEDS
MANUEL VIUDA-MARTOS2,3, GELMY L. CIRO-GÓMEZ1, YOLANDA RUIZ-NAVAJAS2,
JOSÉ E. ZAPATA-MONTOYA1, ESTHER SENDRA2, JOSÉ A. PÉREZ-ÁLVAREZ2 and JUANA FERNÁNDEZ-LÓPEZ2
1
Programa de Ofidismo/Escorpionismo, Universidad de Antioquia AA 1226, Sede de Investigación Universitaria (SIU) Carrera 53 # 61-30, Lab. 631
Torre 2, Medellín, Colombia
2
IPOA Research Group (UMH-1 and REVIV-Generalitat Valenciana), AgroFood Technology Department, Escuela Politécnica Superior de Orihuela,
Miguel Hernández University, Crta. Beniel km. 3,2, E-03312 Orihuela, Alicante, Spain

3
Corresponding author. TEL: +34-966749737;
FAX: +34-966749677; EMAIL:
mviuda@umh.es
Accepted for Publication July 30, 2012
doi: 10.1111/j.1745-4565.2012.00393.x
ABSTRACT
The aim of this work was to determine (1) the total phenolic compound (TPC),
total flavonoid compound (TFC) and bixin and norbixin content of polar extracts
of leaves (ALE) and seed (ASE) from annatto (Bixa orellana L.); (2) the antioxi-
dant activity, the ALE and ASE by means of different antioxidant tests, and (3) the
effectiveness of ALE and ASE on the growth inhibition of several bacterial strains.
Five different test systems were used to determine the antioxidant activity, while
the microdilution method was used to test for antimicrobial activity. ALE pre-
sented higher (P < 0.05) TPC and TFC than ASE. As regards antioxidant activity,
at all the concentrations tested and with all the methods, except the Rancimat test,
the ALE samples showed higher (P < 0.05) antioxidant activity than ASE samples.
As regards antibacterial activity, ASE was a stronger inhibitor (P < 0.05) of bacte-
rial growth than ALE. Both ALE and ASE could be used as alternative natural pre-
servatives in food matrices due to mainly their broad antioxidant activity and
lesser extent of their antibacterial activity.

PRACTICAL APPLICATIONS
The B. orellana seed and leaf extracts could be suitable for application in on the
food industry because It is an important source phenolic, flavonoids and caro-
tenoids compounds, the antioxidant properties of which could be very appreciated
in a big number of food processing to avoid its oxidation not only during process-
ing but also during storage period. However, their antimicrobial action is limited.
Other important reason for their suitability is their natural origin, which consum-
ers find comforting.

INTRODUCTION easily oxidizable materials, such as fats (Viuda-Martos et al.

Food spoilage is basically caused by enzymes and microor-
ganisms, which induce undesirable off-flavors and toxicity,
and affect the shelf life of many foods. To prevent this, the
food industry has traditionally used synthetic additives
(added or already present naturally in the foods), which
diminish microbial growth or inhibit microorganisms and
prevent or delay, to a significant extent, the oxidation of
2011a).
The addition of chemical preservatives has long been an
effective method to control microbial contamination and
the development of oxidative reactions, although in recent
years, popular demand has shown a marked aversion to
such synthetic chemical preservatives (Cushnie and Lamb
2005). This has resulted in a growing demand for natural
products, principally, plant extracts, which are, in the

Journal of Food Safety 32 (2012) 399–406 © 2012 Wiley Periodicals, Inc. 399
ANTIOXIDANT ACTIVITY OF ANNATTO EXTRACTS
consumers mind, safer, functional and provide nutritional
and health benefits. This demand has increased the impor-
tance of searching for alternative sources of natural preser-
vatives rich in phenolic compounds (Farag et al. 2003).
Bixa orellana L. (achiote, orlean, roucou or annatto),
from the Bixaceae family, is a native plant of Central and
South America, which accumulates several carotenoid
derivatives (bixin and norbixin), terpenoids, tocotrienols
and flavonoids in the seeds and leaves (Rodrigues et al.
2007). The medicinal uses of annatto seeds include the
treatment of diabetes, skin infections, burns, fever, measles,
gonorrhea, diarrhea and asthma (Rojas et al. 2006). Also,
some studies have demonstrated the antimicrobial and anti-
oxidant properties of annatto seed extracts. For example,
Galindo-Cuspinera et al. (2003) found that commercial
annatto extracts have antimicrobial activity against
several Gram-positive bacteria, especially Bacillus cereus,
Clostridium perfringes and Staphylococcus aureus. As regards
the antioxidant activity of annatto seeds extracts, Haila et al.
(1996) reported that bixin strongly inhibited the autoxida-
tion of rapeseed oil triglycerides. In the same way, Kiokias
and Gordon (2003) found that the norbixin inhibited the
oxidative deterioration of bulk olive oil and oil-in-water
emulsions. Likewise, Chisté et al. (2011) reported that
extracts of annatto seed have a high antioxidant capacity,
although bixin presented the lowest IC50 values.
On the other hand, the annatto leaves have also been used
in traditional medicine. Their pharmacological properties
are directly related to the chemical composition of the
leaves. Some of the chemical compounds that have been
isolated from the leaves include flavonoids, heterosides,
sulfated derivates, diterpenes, gallic acid, pyrogallol and
essential oil (Coelho et al. 2003). Another important prop-
erty of leaves is their antimicrobial activity (Fleischer et al.
2003; Navarro-Garcia et al. 2003). Ethnolic extracts of
annatto leaves are reportedly active anaist a range of Gram-
positive bacteria and Gram-negative bacteria and fungi
(Fleischer et al. 2003), while Navarro-Garcia et al. (2003)
studied and identified antimicrobial activity in leaf and
stem extracts of B. orellana against the fungi Trychophyton
mentagrophytes and Trychophyton rubrum. In addition to
their antimicrobial properties, the polyphenol content of
annatto leaf extracts has shown other biological properties
including antioxidant activity (Shilpi et al. 2006; Enciso
et al. 2010).
However, few reports jointly study the antimicrobial and
antioxidant activities of polar extracts of leaves and seeds
from annatto, so that the aim of this work was to determine
(1) the total phenolic compound (TPC), total flavonoid
compound (TFC) and bixin and norbixin content of polar
extracts of leaves (ALE) and seed (ASE) from annatto
(B. orellana L.); (2) the antioxidant activity, the ALE and
ASE by means of five different antioxidant tests (DPPH,
400
M. VIUDA-MARTOS ET AL.
FRAP, TBARS, FIC and Rancimat); and (3) the effectiveness
of ALE and ASE on the growth inhibition of several bacte-
rial strains (indicators of pathogenic bacteria, spoilage bac-
teria and toxin-producing bacteria) to ascertain if the polar
extracts of leaves and seeds from annatto could be used as
natural food ingredients.
MATERIALS AND METHODS
Polar Extract from Leaves and Seeds
of Annatto
Annatto leaves and seeds were collected in the municipality
of San Luis (Antioquia, Colombia). The seeds and leaves
were identified as B. orellana L. (red variety) by the Her-
barium of Antioquia University (Colombia). The leaves and
seeds were then dried in a conventional oven at 37 ⫾ 0.2C
for 48 h. The dry materials (500 g of seed or leaves) were
subjected to an extraction process with 95% ethanol
(1,000 mL) for 48 h in a single process. Ethanol extracts
were used in the present study because the extracts were
destined for use as in foods; whereas other extractant
agents, such as methanol or methanol : ethyl acetate, are not
accepted for such uses. The ethanolic extract was evapo-
rated to dryness using a rotary evaporator R-205 (Büchi,
Flawil, Switzerland) under reduced pressure (<100 mbar) at
40C. The polar extracts were filtered and properly stored in
sealed amber bottles for transport at a temperature of
4 ⫾ 0.2C. The polar extracts were then sent to the Miguel
Hernandez University laboratories (Orihuela, Spain) for
their analysis.
Total Phenol Content
TPC was determined using Folin–Ciocalteu’s reagent
(Singleton and Rossi 1965). A volume of 0.3 mL of the
methanolic solutions of polar extracts from ALE and ASE
(10 g/L) was introduced into test tubes followed by 2.5 mL
of Folin–Ciocalteu’s reagent (diluted 10 times with water)
and 2 mL of sodium carbonate (7.5% w/v). The tubes were
vortexed, covered with parafilm and incubated at 50C for
5 min. Absorption at 760 nm was measured with a HP 8451
spectrophotometer (Hewlett Packard, Cambridge, UK) and
compared with a gallic acid calibration curve. The results
being expressed in mg gallic acid equivalents (GAE)/L
sample as mean of three replicates.
Total Flavonoid Content
To assess TFC, a method based on that described by Blasa
et al. (2005) with some modifications was used. One millili-
ter of polar extracts from ALE and ASE (10 g/L) was mixed
with 0.3 mL NaNO2 (5%), and 0.3 mL AlCl3 (10%) was
Journal of Food Safety 32 (2012) 399–406 © 2012 Wiley Periodicals, Inc.
M. VIUDA-MARTOS ET AL.
added after 5 min. The polar extracts samples were mixed
and after 6 min, were neutralized with 2 mL NaOH solution
(1 M). For all the samples, the absorbance was read at
510 nm, and the quantification was carried out using a cali-
bration curve.
Different concentrations of rutin (8.5–170 mg/mL) were
used for calibration, giving a linearity of 0.997 (R2). The
results being expressed in mg rutin equivalents (RE)/100 g
of sample as mean of three replicates.
Bixin and Norbixin Content
The exact concentrations of bixin and norbixin in polar
extracts from annatto seeds were determined following the
indications of Bareth et al. (2002). For norbixin, 0.5 g of
ASE or ALE was place in a 25-mL volumetric flask, which
was filled with 2.5% KOH (w/w). For bixin, 1.3 g of ASE or
ALE was placed in a similar volumetric flask, which was
filled with CHCl3. The maximum extinctions were mea-
sured with an HP 8451 spectrophotometer (Hewlett-
Packard) at 482 nm for norbixin and 498 nm for bixin.
The exact concentrations were calculated with the follow-
ing equation:
β = ( Aλ × 10, 000) E
where: b is the concentration of the measured solution in
mg/L; Al is the measured absorbance for bixin at the
maximum of l = 498 nm in CHCl3 and for norbixin at
the maximum of l = 482 nm in 2.5% KOH (w/w); E is the
extinction value of annatto bixin and norbixin (2,870);
10,000 is the factor to convert (%) in (mg/L).
Antioxidant Activity
Determination of Antioxidant Activity Using
2,2⬘-Diphenyl-1-Picrylhydrazyl Radical Scavenging
Method. The antioxidant activity of polar extracts from
ALE and ASE was measured in terms of hydrogen-donating
or radical-scavenging ability, using the stable radical 2,2′-
diphenyl-1-picrylhydrazyl (DPPH) (Brand-Williams et al.
1995). For this, methanolic stock solutions of ALE and ASE
(50, 20, 10 and 5 g/L) were used. Each assay was carried out
in triplicate.
Ferric-Reducing Antioxidant Power. The ferric-
reducing antioxidant power (FRAP) of the polar extracts
was determined by using the potassium ferricyanide–ferric
chloride method (Oyaizu 1986). For this, methanolic stock
solutions of ALE and ASE of four different concentrations
(50, 20, 10 and 5 g/L) were used. The FRAP of a sample is
estimated in terms of Trolox equivalent antioxidant capacity
(TEAC) in mmol/L Trolox. Each assay was carried out in
triplicate.
Journal of Food Safety 32 (2012) 399–406 © 2012 Wiley Periodicals, Inc.
ANTIOXIDANT ACTIVITY OF ANNATTO EXTRACTS
Thiobarbituric Acid Reactive Species Test. The
method described by Daker et al. (2008) was modified to
determine the thiobarbituric acid reactive substance
(TBARS), a secondary product of lipid peroxidation. For
this, 0.2 mL of methanolic solutions of ALE and ASE (50,
20, 10 and 5 g/L) was added to mixture containing 1 mL
fowl egg yolk emulsified with 0.1 M phosphate buffer
(pH 7.4), to reach a final concentration of 25 g/L and
100 mL of 1 mM Fe2+. The mixture was incubated at 37C for
1 h, after which it was treated with 0.5 mL of freshly pre-
pared 15% trichloroacetic acid and 1 mL of 1% thiobarbitu-
ric acid. The reaction tubes were kept in a boiling water
bath for 10 min. Upon cooling with ice, the tubes were cen-
trifuged at 3,500¥ g for 10 min. The formation of TBARS
was measured by removing 100 mL of supernatant and mea-
suring the absorbance at 532 nm. BHT was used as stan-
dard. Each assay was carried out in triplicate.
Ferrous Ion-Chelating Ability Assay. Ferrous ion
(Fe2+) chelating activity was measured by inhibiting the for-
mation of the Fe2+–ferrozine complex after treating the test
material with Fe2+, following the method of Carter (1971).
For this, methanolic stock solutions of ALE and ASE (50, 20,
10 and 5 g/L) were used. Each assay was carried out in
triplicate.
Rancimat Assay. A Rancimat 743 (Methrohm, Switzer-
land) was used to determine the antioxidant lipid activity of
polar extracts from annatto leaves and seeds. A methanolic
solution (100 mL) of different concentrations ALE and ASE
(50, 20, 10 and 5 g/L) was added to previously melted lard
(2.5 g), giving a final concentration of 0.2%, 0.08%, 0.04%
and 0.02% of the polar extracts in the reacting system. The
lard with and without added antioxidant was heated at
120C, and an air flow of 20 L/h was constantly blown into
the mixture. The antioxidant activity index (AAI) was cal-
culated from the measured induction times, according to
the following formula described by Forster et al. (2001).
AAI = (induction period of lard with antioxidant/
Induction period of pure lard)
Microbial Strains
Polar extracts of annatto leaves and seeds were individually
tested against Listeria innocua, CECT 910; Aeromonas hydro-
phila, CECT 5734; B. cereus, ATCC 11778; and Pseudomonas
aeruginosa, ATCC 9027, species supplied by the Spanish
Type Culture Collection (CECT) of the University of Valen-
cia and American Type Culture Collection (ATCC). These
bacteria were chosen because they are commonly associated
with refrigerated foods and act as indicators of pathogenic
bacteria (L. innocua), spoilage bacteria (A. hydrophila and
P. aeruginosa) and toxin-producing bacteria (B. cereus).
401
ANTIOXIDANT ACTIVITY OF ANNATTO EXTRACTS
Antibacterial Activity Assay
Antibacterial activity was determined based on the colori-
metric broth microdilution method proposed by Abate et al.
(1998), with some modification. Bacterial strains A. hydro-
phila and P. aeruginosa were cultured for 24 h at 26C,
B. cereus was cultured for 24 h at 30C and L. innocua was
cultured for 24 h at 37C on Mueller Hinton broth (Merck,
Darmstadt, Germany) and adjusted to a final density of
106 cfu/mL before being used as inoculum. ALE and ASE
were dissolved in dimethyl sulfoxide (DMSO) to reach a
final concentration of 4,096 mg/mL. Serial twofold dilutions
were made in a concentration ranging from 4 to
4,096 mg/mL in sterile test tubes containing Mueller–Hinton
broth. The 96-well microplates (Becton Dickinson Labware,
San Agustin de Guadalix, Madrid, Spain) were prepared by
dispensing 95 mL of Mueller–Hinton broth and 5 mL of the
bacterial inoculums into each well. A 100 mL aliquot from
each ALE and ASE was added to the first wells. Then 100 mL
of serial dilutions were transferred into ten consecutive
wells. The last row containing 195 mL of Mueller–Hinton
broth without the compound, and 5 mL of the inoculums
was used as negative control. The final volume in each well
was 200 mL. Nisin (4–4,096 mg/mL) was prepared in
Mueller–Hinton broth and used as positive control. The
contents of each well were mixed on a plate shaker at
300 rpm for 30 s prior to incubation for 5 h at 26, 30 or
37C, depending on the bacterial inoculums. After incuba-
tion, 25 mL of 3-{4.5-dimethylthiazol-2-yl}-2,5-diphenyl
tetrazolium bromide (MTT) (Alfa Aesar, Germany), dis-
solved in DMSO (0.8 mg/mL) was added to each of the
wells and incubated for 1 h, to allow the viable microorgan-
isms to metabolize the yellow MTT dye into formazan
(purple crystals). The minimum inhibitory concentration
(MIC) value was considered as the concentration of the first
well that did not undergo color change (from yellow to
purple) and was confirmed by plating 5 mL samples from
clear wells on Mueller–Hinton agar medium. The procedure
was repeated three times for each microorganism.
Statistical Analysis
Conventional statistical methods were used to calculate
means and standard deviations of three simultaneous assays
carried out with the different methods. Normal distribution
was confirmed by running the normality test. Statistical
TPC (mg TFC (mg BC (mg
Extracts GAE/g ext) RE/g ext) bixin/g ext)
ALE 1.81 ⫾ 0.03 65.53 ⫾ 0.72 0.00 ⫾ 0.00
ASE 0.73 ⫾ 0.01 22.18 ⫾ 0.49 15.35 ⫾ 0.72
GAE, gallic acid equivalent; RE, rutin equivalent.
402
M. VIUDA-MARTOS ET AL.
analysis (ANOVA) was applied to the data to determine dif-
ferences (P < 0.05). To discover whether there were signifi-
cant differences between the levels of the main factor,
contrasts (Tukey’s test) between means were made (Afifi
and Azen 1979). For the antioxidant activity, ANOVAs with
two factors (polar extracts and concentration) were applied
for each parameter. The statistical analyses were made using
Statgraphics 5.1 for Windows (Statistical Graphics Corp.,
Rockville, MD).
RESULTS AND DISCUSSION
Total Phenol and TFC
TPC values of annatto extracts are presented in Table 1. The
TPC of annatto extracts, expressed as gallic acid equivalent,
ranged from 1.81 mg/g for ALE to 0.74 mg/g for ASE. Card-
arelli et al. (2008) reported a TPC content in an annatto
extract that ranged between 0.30 and 1.84 mg of gallic acid
equivalent per gram of dry seeds (depending on the solvent
used for extraction), while Chisté et al. (2011) mentioned
TPC values of 1.7 mg of gallic acid equivalent per gram of
wet seeds. The concentration and type of phenolic sub-
stances present in the extracts depended on several factors;
season and environmental factors, such as soil type and
climate, genetic factors and processing methods such as type
of solvent, etc.
As regards the flavonoid content (Table 1), ALE had a
higher (P < 0.05) content than ASE with values of 65.53 and
22.18 mg rutin equivalent/g extracts, respectively. Studies in
ASE flavonoids have revealed the presence of luteolin and
apigenin (Pino and Correa 2003) and hypolaetin, along
with a caffeoyl acid derivative (Chisté et al. 2011); while in
ALE the presence of flavonoid bisulfates was noted by Har-
borne (1975).
The phenolic and flavonoid contents can be used as pow-
erful indicators of the antioxidant capacity, which can be
used as a preliminary screen for any product when intended
as a natural source of antioxidants in functional foods
(Viuda-Martos et al. 2011b)
Bixin and Norbixin Content
The bixin and norbixin content of ASE and ALE are pre-
sented in Table 1. Neither of the carotenoids was found in
ALE; while in ASE, the concentration of bixin and norbixin
TABLE 1. TOTAL PHENOL (TPC), TOTAL
NBC (mg
FLAVONOID (TFC), BIXIN CONTENT (BC) AND
norbixin/g ext)
NORBIXIN CONTENT (NBC) CONTENTS OF
0.00 ⫾ 0.00 POLAR EXTRACTS FROM LEAVES AND SEED
4.92 ⫾ 0.22 OF ANNATTO
Journal of Food Safety 32 (2012) 399–406 © 2012 Wiley Periodicals, Inc.
M. VIUDA-MARTOS ET AL. ANTIOXIDANT ACTIVITY OF ANNATTO EXTRACTS

TABLE 2. ANTIOXIDANT ACTIVITY OF POLAR EXTRACTS FROM LEAVES AND SEEDS OF ANNATTO AT DIFFERENT CONCENTRATIONS (A = 5 G/L,
B = 10 G/L, C = 20 G/L, D = 50 G/L) MEASURED BY DPPH, TBARS, FRAP, FIC AND RANCIMAT METHODS

Method Sample A B C D *IC50

DPPH (% Inhibition) ALE 87.21 ⫾ 0.33 aA
90.75 ⫾ 0.66bA
92.55 ⫾ 0.33cA
94.20 ⫾ 0.64dA
3.2 ¥ 10-5
ASE 29.88 ⫾ 1.11aB 36.00 ⫾ 0.66bB 63.92 ⫾ 1.55cB 93.01 ⫾ 1.22dA 15.40
BHT 93.28 ⫾ 0.33aC 95.31 ⫾ 0.44bC 96.09 ⫾ 0.12cC 97.16 ⫾ 0.11dB 4.13 ¥ 10-11
TBARS (% Inhibition) ALE 48.54 ⫾ 1.51aA 60.67 ⫾ 0.51A 70.81 ⫾ 0.73cA 80.18 ⫾ 0.82dA 5.55
ASE 42.86 ⫾ 1.30aB 64.97 ⫾ 0.34bB 70.81 ⫾ 0.17cA 75.88 ⫾ 0.21dB 6.42
BHT 82.44 ⫾ 0.11aC 84.78 ⫾ 0.09bC 87.81 ⫾ 0.21cB 90.01 ⫾ 0.15dC 4.6 ¥ 10-4
FRAP (TEAC) ALE 8.71 ⫾ 0.65aA 18.51 ⫾ 1.52bA 31.20 ⫾ 0.76cA 73.74 ⫾ 1.20dA –
ASE 2.73 ⫾ 0.20aB 6.51 ⫾ 0.11bB 12.07 ⫾ 1.45cB 25.41 ⫾ 3.52dB –
BHT 74.23 ⫾ 0.00aC 92.45 ⫾ 0.01bC 101.34 ⫾ 0.10cC 125.44 ⫾ 0.00dC –
FIC (% Chelating effect) ALE 12.38 ⫾ 0.21aA 18.68 ⫾ 1.78bA 37.36 ⫾ 1.57cA 51.67 ⫾ 0.84dA 43.72
ASE 7.49 ⫾ 0.21aB 8.23 ⫾ 0.42bB 9.41 ⫾ 0.84cB 10.53 ⫾ 0.31dB 1.82 ¥ 1014
BHT 36.81 ⫾ 0.23aC 40.53 ⫾ 0.09bC 44.78 ⫾ 0.31cC 48.76 ⫾ 0.21dC 50.83
Rancimat (AAI) ALE 1.01 ⫾ 0.01aA 1.04 ⫾ 0.03abA 1.06 ⫾ 0.01bA 1.10 ⫾ 0.02cA –
ASE 1.12 ⫾ 0.00aB 1.14 ⫾ 0.02aB 1.21 ⫾ 0.02bB 1.34 ⫾ 0.07cB –
BHT 1.28 ⫾ 0.24aC 1.71 ⫾ 0.11bC 2.09 ⫾ 0.31cC 2.48 ⫾ 0.11dC –

* IC50: Concentration (g/L) for a 50% inhibition or chelating effect.

For a same method, values followed by the same small letter within the same line are not significantly different (P > 0.05) according to Tukey’s mul-
tiple range test.
For a same method, values followed by the same capital letter within the same column are not significantly different (P > 0.05) according to Tukey’s
multiple range test.
DPPH, 2,2′-diphenyl-1-picrylhydrazyl; TBARS, thiobarbituric acid reactive substance; FRAP, ferric-reducing antioxidant power; TEAC, Trolox equivalent
antioxidant capacity (mg Trolox/g extracts); AAI, antioxidant activity index.

was 15.35 and 4.92 mg/g extract, respectively. The results
obtained for bixin were similar to those reported by Chisté
et al. (2011) with values of 14 mg/g seed extracts. However,
Cardarelli et al. (2008) mentioned that the bixin content of
dry annatto seeds ranged from 0.45 to 4.92 mg/g dry seeds.
Bixin, which is a carotenoid with two carboxylic acid
groups, one of which is esterified, is the major pigment
present in annatto extract. Norbixin, which is derived from
bixin by hydrolysis of the ester group, is also sold as a food
pigment; and this molecule is water-soluble, whereas bixin
is oil-soluble (Kiokias and Gordon 2003)
Antioxidant Activity
There are many different methods for determining antioxi-
dant activity, which depends on different generators of free
radicals acting through different mechanisms (Huang et al.
2005). To assess the antioxidant activity of these extracts,
five different methods were used in this study because it is
generally accepted that a mix of methods should be used for
assessing antioxidant activities in vitro so that all aspects of
antioxidant efficacy are covered (Aruoma 2003). A single
method would only provide basic information about anti-
oxidant properties, but a combination of methods describes
the antioxidant properties of the sample in more detail (Číž
et al. 2010).
In the DPPH test, the ability of extracts to act as the
donor of hydrogen atoms or electrons in the transformation
of DPPH into its reduced form, DPPH-H, was measured
spectrophotometrically (Table 2). Both polar extracts were
able to reduce the stable, purple color of the radical DPPH
to yellow. ALE was a stronger inhibitor (P < 0.05) that ASE
at all the concentrations studied except the highest concen-
tration, where no statistically significant differences were
found (P > 0.05) between extracts. The positive control,
BHT, had a higher antioxidant effect (P < 0.05) at all con-
centrations than ALE and ASE. The inhibition of DPPH-H
reached 50% with an IC50 as follows: 3.2 ¥ 10-5 g/L for
annatto leaves and 15.40 g/L for annatto seeds. Although the
assay is usually classified as a single electron transfer (SET)
reaction, this radical indicator can be neutralized either by
direct reduction via electron transfer or by radical quench-
ing via H-atom transfer (Rojano et al. 2008). The DPPH
scavenging data suggest that the extract is capable of scav-
enging free radicals, thus preventing the initiation and
propagation of free-radical-mediated chain reactions
(Viuda-Martos et al. 2011b).
As regards the TBARS method (Table 2), a concentration-
dependent lipid peroxidation inhibitory effect was found in
both samples. The EC50 values ranged from 0.00046 to
6.42 g/L, and the lipid peroxidation inhibitory power was in
the order BHT > ALE > ASE. The TBARS method is prefer-
able for obtaining useful data in an environment that is
similar to the real-life situation and allows the testing of
both lipophilic and hydrophilic substances (Kulisic et al.
2004).

Journal of Food Safety 32 (2012) 399–406 © 2012 Wiley Periodicals, Inc. 403
ANTIOXIDANT ACTIVITY OF ANNATTO EXTRACTS
According to the FRAP values (Table 2), a concentration-
dependent reducing capacity was found for all concentra-
tions of ALE and ASE. The leaf extracts showed a higher
(P < 0.05) ferric-reducing capacity than the seeds extracts.
The positive control, BHT, had the highest antioxidant
effect (P < 0.05) at all concentrations. The reducing capacity
of a compound may serve as a significant indicator of its
potential antioxidant activity (Meir et al. 1995).
Analysis of the metal ion-chelating properties showed
that the annatto extracts studied were capable of chelating
iron (II) in a concentration-dependent manner (Table 2). At
all the concentrations assayed (5, 10, 20 and 50 g/L), ALE
showed higher values (P < 0.05) for chelating iron (II) than
ASE; although BHT, at all concentrations, was the best
chelator of iron (II) (P < 0.05) except in the case of the
maximum ALE concentration (P < 0.05). Transition metals
such as ion can stimulate the lipid peroxidation of generat-
ing hydroxyl radicals through fenton reactions and acceler-
ate lipid peroxidation into peroxyl and alkoxyl radicals,
thereby driving the chain reaction of lipid peroxidation
(Zhao et al. 2008). Chelating agents may inhibit radical gen-
eration by stabilizing transition metals, consequently reduc-
ing free radical damage.
Table 2 gives the Rancimat test values in terms of the AAI
of lard obtained with the annatto extracts added. The Ran-
cimat test is a very easy and inexpensive method, which
requires small sample volumes and achieves reproducible
results. It is commonly used in the food industry and gov-
ernmental analytical laboratories. This method is based on
measuring the changes of electrical conductivity of water
caused by the formation of short-chain compounds when
fats and oils are oxidized under high temperatures and
accelerated aeration. The higher the induction times of lard
compared with the control, the better the antioxidant activ-
ity of that compound (Schwarz et al. 2000). The antioxidant
activity index, as determined by the Rancimat method,
decreased in the order BHT > ASE > ALE. According to this
method, both ALE and ASE showed antioxidant activity
(AAI = 1.01–1.34), but far below the activity of synthetic
antioxidants (BHT, AAI = 2.48). Unlike other antioxidant
methods tested, ASE showed a higher degree of antioxidant
activity than ALE, perhaps because the compounds present
in this extract (including bixin and norbixin) have a high
affinity for lipophilic compounds like fat.
Few studies have examined the antioxidant activity of
annatto extracts, although, Shilpi et al. (2006) found that
B. orellana leaf extract showed a concentration-dependent
manner DPPH-free radical scavenging activity. Similarly,
Enciso et al. (2010) showed that the hydroalcoholic extract
from leaves of B. orellana contain a high concentration of
flavonoids, which reflected the high correlation with the
antioxidant capacity tested. Cardarelli et al. (2008) reported
that the highest free radical scavenging capacity of annatto
404
M. VIUDA-MARTOS ET AL.
extracts was observed in the extract obtained with the most
polar solvents, which also showed the highest phenolic
content, with TEAC values ranging from 2.04 to 9.55 mM of
Trolox equivalent per gram of dry seeds. Chisté et al. (2011)
showed that the ethanol : ethyl acetate and ethyl acetate
extracts of annatto seeds, which presented the highest levels
of hypolaetin and bixin, respectively, were the extracts with
the highest antioxidant capacity; although bixin presented
the lowest IC50 values.
As mentioned above, both ALE and ASE contained a
moderate concentration of carotenoids and phenolic com-
pounds. Carotenoids and phenolic compounds belong to
the group of bioactive compounds to which the ability to
act as antioxidant compounds has been attributed. The
antioxidant activity is essentially due to the presence of phe-
nolic compounds and flavonoids, although their mecha-
nism of action is not fully understood. Several explanations
have been provided; for example, Mathew and Abraham
(2006) suggested that activity is linked to the sequestration
of free radicals, hydrogen donation, metallic ion chelation
or even to a role as substrate for superoxide or hydroxyl
radicals.
Although the antioxidant properties of carotenoids have
been widely studied, our knowledge regarding how the con-
centration, structure and ratios of specific carotenoids affect
their activity is limited (Rubio-Díaz et al. 2010). The princi-
pal carotenoid of annatto seeds, bixin (methyl (9-cis)-
hydrogen-6,6′-diapo-Y,Y-carotenedioate) is known to be a
very efficient quencher of singlet oxygen (1O2) and the
triplet state of sensitizers (Rios et al. 2007). The possible
mechanisms that might be considered when carotenoids are
exposed to free radicals, such as peroxyl radicals and other
oxidizing agents, are electron transfer due to the presence of
many conjugated double bonds (11 in the bixin structure)
or hydrogen abstraction from the carotenoid molecule,
functioning, in this way as chain-breaking antioxidant
(Yeum et al. 2009).
Antibacterial Activity
The broth microdilution method was used to determine the
antimicrobial activity of annatto leaf and seed extracts and
nisin against Gram-positive and Gram-negative bacteria.
The MIC values of ALE were found to be 512 mg/mL for
P. aeruginosa and 4,096 mg/mL for B. cereus. This leaf
extract did not inhibit the growth of L. innocua and A. hy-
drophila in the range of concentrations tested. In a compari-
son with nisin, which is the only bacteriocin widely
accepted as a natural food preserver, the MIC values were
found to be 1,024, 256, 512 and 256 mg/mL for P. aerugi-
nosa, B. cereus, L. innocua and A. hydrophila, respectively.
As regards ASE, the MIC values were 128 and
1,024 mg/mL for P. aeruginosa and B. cereus, respectively. As
Journal of Food Safety 32 (2012) 399–406 © 2012 Wiley Periodicals, Inc.
M. VIUDA-MARTOS ET AL.
in the case of ALE, ASE did not inhibit the growth of L. in-
nocua and A. hydrophila in the range of concentrations
tested. Both annatto extracts were found to have higher
antimicrobial activity for P. aeruginosa than nisin.
The antibacterial activity of annatto extracts has been
widely demonstrated, but few authors have determined the
MIC values. In this respect, Ongsakul et al. (2009) men-
tioned that annatto ethanolic leaf extracts show antibacte-
rial activity against S. aureus with MIC values of 62.5 mg/
mL. Galindo-Cuspinera et al. (2003) found that commercial
annatto extracts have antimicrobial activity against several
Gram-positive bacteria, especially B. cereus, C. perfringes
and S. aureus, with MICs values of 0.08, 0.31 and 0.16%
(vol/vol), respectively. Fleischer et al. (2003) reported that
the ethanolic extracts of the leaves and seeds of B. orellana
showed activity against all the Gram-positive and Gram-
negative bacteria and the yeast-like fungus Candida albi-
cans. The activity appears to be more pronounced in the leaf
extract. In contrast, the results obtained in this study
showed that ASE was a stronger inhibitor of bacterial
growth than ALE.
The phytochemicals, phenolics acids, flavonoids and
carotenoids, are reported to have antibacterial activity
(Mahanom et al. 1990). Some of the simplest bioactive phy-
tochemicals consist of a phenolic ring, which is toxic to
microorganisms. The mechanisms thought to be respon-
sible for phenolic toxicity toward microorganisms include
enzyme inhibition by the oxidized compounds, possibly
through reaction with sulfhydryl groups or through more
nonspecific interactions with the proteins (Cowan 1999).
CONCLUSIONS
Annatto seed extracts and annatto leaf extracts may be
interesting alternatives for use as natural preservatives to
replace synthetic preservatives in food matrices due to their
broad antioxidant activity and to a lesser extent its antimi-
crobial action. This study is the first step toward determin-
ing the potential application of annatto extracts as food
ingredients, and it will be necessary to carry out further
studies in order to identify and quantify all the bioactive
compounds therein and determine their toxicity.
ACKNOWLEDGMENTS
We are grateful to the “Caja Murcia” for a research scholar-
ship to the author (MVM) and the project CYTED-
IBEROFUN codec: 110AC0386.
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