Antioxidant Activity and Chemical Composition of Colombian Propolis - Natural Antioxidants and Biocides from Wild Medicinal Plants - page 103

scavenging properties of antioxidant com-
pounds. In a methanol solution, DPPH acquires
a purple colour that changes to yellow when
an antioxidant substance is present.
2. Trolox Equivalent Antioxidant Capacity
Assay (TEAC), a technique similar to DPPH
assay. In the presence of sodium or potas-
sium persulfate, an ABTS (2,2´-azinobis
(3-ethylbenzthiazoline-6-sulfonic acid)) sta-
ble radical cation is generated. This oxidant
species (ABTS •− ) is blue, but in the presence
of an antioxidant compound turns to the
ABTS 2− colourless form.
3. Ferric ion reducing antioxidant power
assay (FRAP), a colorimetric assay carried
out in acidic conditions to evaluate the total
antioxidant capacity of compounds. This
technique uses a [Fe(III)-(TPTZ) 2 ] 3+ com-
plex, (TPTZ = 2,4,6-tri(2-pyridil)-s-triazine),
that, in the presence of an antioxidant
agent, is reduced to its ferrous form [Fe(II)-
(TPTZ) 2 ] 2+ , yielding a blue colour.
4. Oxygen radical absorbance capacity
(ORAC), an antioxidant scavenging activity
assay against peroxyl radicals formed by heat-
ing AAPH (2,2´-azobis(2-amidino-propane)
dihydrochloride), a free-radical generator com-
pound. In the method, radicals formed attack
fluorescein (a fluorescent probe). Therefore, if
a high fluorescence value remains, it is assumed
that the antioxidant is acting.

number of biological activities, such as anti-
cancer, anti-inflammatory, immunomodula-
tory, analgesic and antioxidant, among
others (Gómez et al ., 2006). Propolis are
rich in these kind of substances; however,
the phenolic concentration from one sam-
ple of propolis to another can vary consid-
erably depending on the origin. Therefore,
the presence of some biological activities
can be present or absent.
The reasons mentioned above support
the determination of total phenolic content
(TPC) as a quality-control parameter for
propolis. At present, this determination
is done by using the Folin-Ciocalteau color-
imetric method. This methodology is based
on the formation of a coloured substance
that absorbs at 760 nm, which is produced
by the oxidation of the phenolic com-
pounds with phosphoric molibdotungstate
(Singleton and Rossi, 1965). Some countries,
such as Brazil and Argentina, have estab-
lished official quality control standards for
raw propolis. They have a minimum limit of
50 mg AE/g of Ethanol Extract of Propolis
(EEP) for phenolics (Ministerio, 1999).
Furthermore, many studies have reported a
direct correlation between TPC and antioxi-
dant and antimicrobial properties of propo-
lis (Kumazawa et al ., 2004; Choi et al ., 2006;
Moreira et al ., 2008).

Furthermore, spectrophotometric assays
are the reference methods for determination
of total phenolic content and total flavonoid
content, because they are fast and cheap.
Popova et al . (2004) validated some pro-
cedures for the quantification of three of the
main bioactive groups of metabolites identi-
fied in propolis (phenols, flavones and fla-
vonols, flavanones and dihydroflavonols).
The authors assert that the joint quantifica-
tion of compounds with similar structures
correlates much better with biological activ-
ities and provide better information than
quantification of individual compounds.

7.4.2 Total flavonoid content (TFC)

Flavonoids are the main group of phenolic
compounds. Because of the many biological
and therapeutic activities attributed to them,
these compounds have been the object of
many scientific studies. Although other phe-
nolic compounds are also involved, high fla-
vonoid content is thought to account for
much of the activities in propolis.
An aluminium chloride (AlCl 3 ) colori-
metric assay is a method commonly used to
evaluate total flavonoid content (TFC) in
propolis samples (Woisky and Salatino,
1998; Ahn et al ., 2004, 2007; Kumazawa
et al. , 2004; Marquele et al ., 2005; Choi et al .,
2006; Gómez et al ., 2006; Soleo de Funari
et al ., 2007; Moreira et al ., 2008). In this

7.4.1 Total phenolic content

Phenolic compounds or polyphenols are
widely recognized for showing a great