104

104 Asian Pacific journal of Tropical Medicine (2015)104-111

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IF: 0.926
Asian Pacific Journal of Tropical Medicine

ELSEVIER journal homepage:www.elsevier.com/locate/apjtm

Document heading doi: 1O.1016/S1995-7645(14)60299-6

Total phenolic content, in vitro antioxidant activity and chemical

composition of plant extracts from semiarid Mexican region
Jorge E. Wong-paz\Juan C. Contreras Esquivel', Raw Rodrfguez-Herrera', Maria L. Carrillo-
r

1
Inungaray'', Lluvia I. Lopez', Guadalupe V. Nevarez-rMoorillon'', Cristobal N. Aguilar *
lLaboratory of Bioprocesses and Natural Products, Food Research Department, School of Chemistry, Universidad Autonoma de Coahuila, Saltillo,
25280, Mexico
2Laboratory of Biochemistry, Multidisciplinary Academic Unit-Huasteca, Universidad Autonoma de San Luis Potosi, 79060, Cd. Valles, S.L.P.,

Mexico
3Department ofMicrobiology, School of Chemistry, Universidad Autonoma de Chihuahua, 31125 Chihuahua, Mexico

AR TICLE INFO ABSTRACT

Article history: Objective: To determine the extraction suitable conditions of total phenolic content (fPC) by
Received 15 November 2014 heat-reflux system, antioxidant activities and HPLC characterization of the aqueous-ethanolic
Received in revised fonn 20 December 2014 extracts of Jatropha dioica (]. dioica) (Dragon's blood), Flourensia cemua (F. cemua) (far bush),
Accepted 15January 2015 Eucalyptus camaldulensis (E. camaldulensis) (Eucalyptus) and Tumera diffusa (T. diffusa)
Available online 20 February 2015 (Damiana). Methods: TPC was evaluated by the well-known colorimetric assay using Folin-
Ciocalteu reagent. The antioxidant activities were assayed by three methods based on scavenging
of DPPH, ABTS and by lipid oxidation inhibition. The chemical composition of the extracts
Keywords:
obtained was subject to HPLC analysis. Results: TPC in the plant extracts ranged from 2.3 to
Plant-extracts
Polyphenols 14.12 mg gallic acid equivalents/g for]. dioica and E. camaldulensis, respectively. The plant
Natural antioxidants extracts of F. cemua, E. camaldulensis and T. diffusa showed similar strong antioxidant activities
Mexican plants on scavenging of DPPH and lipid oxidation inhibition. In contrast, J. dioica extracts had lowest
Heat-reflux system potential antioxidant in three assays used. HPLC assay showed the presence of several phenolic
compounds in the extracts used. Conclusions: The results obtained suggest that F. cemua,
E. camaldulensis and T. diffusa are potential sources to obtain bioactive phenolic compounds
with high antioxidant properties which can be used in the factories as antioxidant agents or for
treatments in diseases.

and pharmaceutical manufacturers. For thousand years the

1. Introduction plant natural products have been used in the medicine,
cosmetics, nutrition and flavoring without or less harmful
Recently synthetic products are being restricted in the effects. Thus, plant extracts appear to be a feasible
industries, because of harmful effects observed such as alternative for this problem and the industries have put the
human toxicity and environmental pollutionltl and besides attention in the bioactive phytochemicals present in the
have been reported to be carcinogenicl2,3J. Additionally, plants.
the new demands by the consumers which pressure on the Generally, phytochemicals present in the plant extracts
industries particularly for safer products are presentedli.sl. are nontoxic, effective at low concentrations, low cost and
From this point of view, an increasing tendency towards friendly with the environment. Besides, recent studies have
the use of natural products instead of synthetic products shown that the ingestion of vegetables, fruit and herbs
has been observed in a high demand for food, cosmetics is associated with prevention of some bactericidal, anti-
viral, analgesic, anti-inflammatory and anti-carcinogenic
*Corresponding author: Cristobal N. Aguilar, School of Chemistry. Universidad disorders, due to their antioxidant activities!5-7J. In the
Aut6noma de Coahuila, Saltillo, Coahuila, Mexico.
food industry, phytochemicals are interesting due to these
E-mai l: cristobal.aguilar@uadec.edu.mx
Foundation projec t: It is supported by program Master in Foods Science and compounds retard oxidative degradations of lipids, improve
Technology in UAdeC.
 Jorge
Jorge E.
E. Wong-Paz et al.!Asian
Wong-Paz et al./Asian Pacific
Pacific Journal
Journal of
of Tropical
Tropical Medicine
Medicine (2015)104-111
(2015)104-111
105
105

the food quality and nutritional valuelsl and contributing to plants were transported to the Department of Food Research
prevent microbial deterioration[4,8-lOl. at Universidad Autonoma de Coahuila, in black plastic bags.
Plant extracts consist in a complex mixture of several Immediately the samples were dried for 24-48 h at 60 "C in
compounds as alcohols, esters, aldehydes, ketones, an oven (LABNET International, Inc.). The dry samples were
carbohydrates, terpenes, polyphenols, etc[llJ. In addition, ground in a mechanical mill and screened at 0.6-0.8 mm
crude extracts, purified fractions and pure compounds size particle. The fine and dried powder was stored in
have already been used in an antioxidant approachesuzl black plastic bags with sealed hermetically and at room
and several responses have been obtained. Generally, there temperature under darkness.
is not compound or extract that can be used as universal
Table
Table 11
antioxidant. However, nowadays is necessary search Source
Source and
and tissue
tissue of
of the
the plants
plants used.
used.
new sources and compound of specific antioxidants for Plant
Plant material
material Area
Area ofof Period
Period of
of Tissue
Tissue
determined objectives. Phenolic compounds are commonly recollection
recoHection recollection
recoHection
reported to have the most antioxidant activitylisl, J. dioica
]. dioica Parras
Parras dede la
la January, 2011
January,20ll Stem
Stem and
and root
root
Fuente,
Fuente, Coahuila
Coahuila
In this context, Mexico is an attractive country for its large F. cernua
F. cernua Saltillo,
Saltillo, Coahuila
Coahuila February, 2011 Stem
February,20ll Stem and
and leaf
leaf
endemic plant variety. In the semiarid regions of Mexico camaldulensis Saltillo,
E. camaldulensis
E. Saltillo, Coahuila
Coahuila May,
May, 2011
2011 Leaf
Leaf
several plants take part of a great source of antioxidative T.
T. diffusa
diffusa Saltillo
Saltillo Coahuila
Coahuila June,
June, 2011
2011 Stem
Stem and
and leaf
leaf
compounds, mainly because their ability to grown under
extreme climatic conditions[13,14J. This kind of plants have 2.2. Preparation ofthe plant extracts
developed in most cases a pool of chemical compounds
produced as secondary metabolites as environmental defense Phenolic extracts were obtained by heat-reflux extraction
mechanism. Chemical compounds of semiarid region plants system evaluating two independent factors. It was necessary
to select the best extraction time to assure the complete TPC
has been well studied to develop potential agents against
extraction. Then, the first factor evaluated was the extraction
certain pathogenic and phytopatogenic microorganisms
time of heat-reflux. Mixtures of water and ethanol were
present in the food and agricultural industries[4,10,13-16l. In
used because it is well known the phenolic compounds
addition, semiarid region endemic plants are commonly
are soluble in polar solvents as water and hydroalcoholic
used in the Mexican traditional medicine. Among the
solutions, in addition, ethanol is a safe solvent (FDA 2012).
plants most used are: Jatropha dioica (1. dioica) (Dragon's
However pure water is not the best solvent for phenols
blood), Flourensia cemua (F. cemua) (far bush), Eucalyptus
extraction[19,20J. Each dried powder sample (5 mg) (tissues
camaldulensis (E. camaldulensis) (Eucalyptus) and Tumera
ratio 1:1, w/w, where was necessary) were placed in an
dijfusa (T. dijfusa) (Damiana),
Erlenmeyer flask covered with aluminum foil to avoid light
J. dioica has been used as analgesic in toothache and T.
exposure and mixed with aqueous-ethanol (70%, v/v) (solid-
diffusa as aphrodisiaclrrl, On the other hand F. cemua is
liquid ratio 1:4, w/v, except in J. dioica, solid-liquid ratio
widely used to treat diarrhea, rheumatism, venereal disease,
1:15, w/v was used). The mixture of the material vegetal and
sores, bronchitis, chicken pox and common cold[15,18J.
solvent was heat-refluxed in a water bath at 60 'C. A kinetic
Nevertheless, actually there is a lack of knowledge about
study on the total phenolic content extracted was carried out
some plants that have not been extensively studied and
at different times: 0, 2, 4, 6, and 8 h of extraction.
neither theirs phytochemical compounds which could be
Once selected the best extraction time the second factor
used as antioxidants on control of certain diseases or in the
evaluated was the aqueous-ethanol concentration. Three
food industry. Therefore, the purpose of this work was to
aqueous-ethanol concentrations were tested: 0%, 35% and
evaluate the extraction conditions, antioxidant potential and
70% (v/v), where 0% is water without ethanol. Then, the heat-
chromatographic profiles (HPLC) of extracts from J. dioica,
reflux extraction was performed under the same conditions
F. cemua, T. dijfusa and E. camaldulensis.
as in the first experiment.
After extraction, each extract was filtered using a gauze for
eliminate the big particles. Briefly, extracts were centrifuged
2. Materials and methods at 3 500 rpm during 10 min and subsequent were filtered
through a filter paper (fine pore, 0.45 p. m) under vacuum.
2.1. Plant collection The filtered extracts were dehydrated in an oven at 60 "C
for 24 h and the extraction yields were obtained in dry base
Plant materials were proportioned by the company (%, w/w). The dried extracts were stored at 4 'C in a dark
Fitokimica Industrial de Mexico SA de CV (Table 1). The place before their use in the quantification of total phenolic
106
106 Jorge E.
Jorge E. Wong-Paz
Wong-Paz et
et al.!Asian
al./Asian Pacific
Pacific Journal
Journal of
of Tropical Medicine (2015)104-111
Tropical Medicine (2015)104-111

content. The experiments were performed in triplicates. In 2.4.1. 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical
all the next determinations the extracts were re-suspended scavenging activity
in water (1 mg/mL). First, the antioxidant activity in the extracts was evaluated
as the DPPHfree radical-scavenging activity. This assay was
2.3. Determination oftotal phenols content (TPC) following the method reported by Molyneux[23l: briefly, the
solution of free radical DPPH (2.9 mL, 60 t-t M, in methanol)
TPC was quantified according the methodology reported was added to the sample (0.1 ml.) and the controls (0.1 mL
by Makkariztl with some modificationslzzl, First, 800 t-t L of water) respectively. The reaction was 30 min standing in
of the sample were mixed with 800 t-t L of Folin-Ciocalteu a dark room at room temperature and after the absorbance
reagent (Sigma-Aldrich), shaken and left for 5 min. Then 800 was measured at 517 nm on a Cary-50-Bio Varian
t-t L of N~C03 (O.OlM) were added and shaken and left for
spectrophotometer. The free radical-scavenging activity of
5 min again. Finally, the solution was diluted with 5 mL of
the extracts was expressed as percentage inhibition of DPPH
distilled water and the absorbance was read at 790 nm. TPC
and was calculated according to the formula:
was expressed as gallic acid equivalents per gram of vegetal
material (mg GAElg).
b (Cabs-Sabs)
(Cabs-Sabs)
Percentage inhibition
Percentage of DPPH=
inhi ition of DPPH= x100
伊 100
2.4. Antioxidant activity C
Cabs
abs

where Cabs is the control absorbance and Sabs is the

Antioxidant activity was measured in the extracts that
sample absorbance.
presented the higher amount of total phenolic content in
each vegetal material for three methodologies (fable 3).
2.4.2. ABTS (2,2-azino-bis 3 ethyl benzothiazoline-6-
Table 22
Table sufonic acid) radical cation scavenging activity
Extraction yield
Extraction yield and
and total
total phenolic
phenolic content
content of
of the
the extracts
extracts obtained
obtained in
in
the kinetic
the kinetic of
of extraction.
extraction.
ABTS radical cation was prepared adding potassium
Plant material
Plant material Extraction time
Extraction time Extraction
Extraction Total
Total phenolic
phenolic content
content persulphate solution (2.45 mM) and an ABTS aqueous
(h)
(h) yield (%)
yield (%) (mg GAE/g )
(mgGAE/g) solution (7 mM) and stand in the dark at room temperature
J. dioica
]. dioica 22 9.89依0.15 2.1±0.30
9.89±0.15 2.1依0.30 for 12 h before use. After 12 h, the final solution was diluted
F. cernua
F. cernua 22 11.03依0.87 7.9依0.93
11.03±0.87 7.9±0.93
with ethanol to an absorbance of 0.70±0.02 at 734 nm.
E. camaldulensis
E. camaldulensis 22 14.85依0.58
14.85±0.58 12.8依0.96
12.8±0.96
T. diffusa
T. diffusa 22 9.64依3.11 2.5±0.52
9.64±3.11 2.5依0.52 Briefly, the extract (50 t-t L) or the control (50 t-t L of water)
Note: An
Note: All values
values are
are expressed
expressed as
as mean±SD
mean依SD of of three
three replicates.
replicates. was mixed with ABTS solution (1 mL) and immediately the
Results of 4,
Results of 4, 66 and
and 88 hh not
not shown
shown because
because no
no difference
difference were
were observed
observed time was taken and the absorbance was read after 1 min
at P<0.05.
at P<0.05.
using a Cary-50-Bio Varian spectrophotometer. The sample
Table 33
Table absorbance was compared with the control absorbance.
Effect solvent
Effect solvent concentration
concentration on
on the
the extraction
extraction yield
yield and
and total
total
The total antioxidant capacity was calculated as percent
polyphenolic content.
polyphenolic content.
Plant material
Plant material Aqueous-ethanol
Aqueous-ethanol Total phenolic
Total phenolic content
content inhibition of ABTS radical using the following equation:
concentration
concentration (mg GAE/g)
(mgGAE/g)
0% 1.36
l.36 bb (Cabs-Sabs)
J. dioica
]. dioica 0% b (Cabs-Sabs)
35%
35% 1.55
1.55"a Percentage in
Percentage inhibition of ABTS=
hi ition of ABTS= x100
伊 100
70%
70% 2.34a*
2.34"' C
Cabs
abs

F. cernua
F. cernua 0%
0% 2.09bb
2.09
35%
35% 10.24
1O.24aa*' where Cabs is the control absorbance and Sabs is the
70%
70% 7.95a
7.95" sample absorbance[24J.
E. camaldulensis
E. camaldulensis 0%
0% 9.56bb
9.56
35%
35% 14.12
14.12aa*'
70%
70% 12.88
12.88 aa
2.4.3. Lipid oxidation inhibition assay
T. diffusa
T. diffusa 0%
0% 2.59bb
2.59 The potential antioxidant of the extracts was obtained by
35%
35% 4.70aa*'
4.70 lipid oxidation inhibition assay with the finality of measure
70%
70% 2.54bb
2.54 the antioxidant activity in a medium close to lipids in a
Note: Values
Note: followed by
Values followed by different
different lower-case
lower-case letters
letters are
are significantly
significantly biological systemlel. First, the linoleic acid solution was
different at
different at P<0.05.
P<0.05.
** Extracts
Extracts selected
selected for
for the
the antioxidant
antioxidant activity
activity assays.
assays. prepared by placing 0.56 g oflinoleic acid and 1.5 g of Tween
20 in 8 mL of ethanol (96%, v/v). Then, the plant extract (50L)
was mixed with linoleic acid solution (100 t-t L) and acetate
 Jorge E.
Jorge E. Wong-Paz
Wong-Paz et
et al.!Asian
al./Asian Pacific
Pacific Journal
Journal of
of Tropical Medicine (2015)104-111
Tropical Medicine (2015)104-111
107
107

buffer (1.5 mL, 0.02 M, pH 4.0). Controls contained 50 f1- L of 3. Results

distilled water. The samples were homogenized in vortex and
incubated at 37 DC for 1 min. Once achieved 1 min, 750 f1- L 3.1. Extraction of total phenols content
of 50 M FeClz solution (0.099 4 g FeClz and 0.168 g EDTA
diluted to 1 L with distilled water) were added to induce the The kinetic extraction of polyphenols allowed to establish
lipid oxidation and incubated for 24 h at 37 DC. Two aliquots the time required for recovery the TPC maximum (Table
(250 f1- L) were withdrawn during this period, at 1 and 24 h. 2). In the four plants was noted that exist a significant
Each aliquot was processing in the moment as following: the difference in the TPC in the control time (0 h) compared
aliquot were added to NaOH solution (1 mL, 0.1 M, in ethanol to other extraction times (2, 4, 6 and 8 h). However, no
at 10%, v/v) to stop the oxidation process; after ethanol (2.5 mL, significant difference was found in the times 2, 4, 6 and 8 h
10%, v/v) was placed to dilute the sample. Then, the in four plants evaluated. Thus, with the purpose of select
absorbance of the samples was measured at 232 nm. Ethanol the suitable time for extraction of phenols, the productivity
(10%, v/v) was used as blank. Percent inhibition of linoleic in each vegetal material was determined (Figure 1). It was
acid oxidation was calculated with the following equation: observed in all cases that the productivity was higher at 2 h
of extraction and decreased at rest of the times evaluated.
(A-B)
(A-B) Using this analysis was possible select 2 h of extraction
Lipid oxidation inhibiton(%)=
Lipid oxidation inhibiton(%)= x伊100
100
A
A time as the suitable time for the TPC extraction in the four
plants and it was used in the evaluation of the second factor,
where A is the difference between the absorbance of aqueous-ethanol concentration.
the control sample (distilled water) after 24 hand 1 h of
77
incubation, and B is the difference between the absorbance aa 2h
2h

of each extract sample after 24 h and 1 h of incubation[25J. 6

6
4h
4h

55 6 hh
1ii1 6
2.5. High performance liquid chromatography analysis
aa
44
o 8h
8h
mg GAE. g-1/h

--'?
mg GAE.g-1.h

eo b
b
The characterization of the phenols compounds present oil
«: 3
0
in the selected extracts (Table 3) was carried out using blJ b
S
b
b b
2
2
high performance liquid chromatography (HPLC) analysis
c aa
aa
following the method reported by Ruiz et al[lsl. A Varian c b
1
b b b b
Pro-Star 330 with DAD detector and Photodiode detection b
00 -,
at 280 nm was used. Fractionation of the samples was J. dioica
}. dioica F. cernua
F. cernua E. camaldulensis
E. camaldulensis T.diffusa
T.difJusa
performed on an Optisil ODS column (5 f1- m, 4.6 mm x 250
mm) under following analytical conditions: using a mobile
phase consisting of 3% acetic acid and acetonitrile, during Figure 1. Productivity of the heat-reflux system.

25 min with flow rate of 1 mLlmin (sample previously

The second factor evaluated was the aqueous-ethanol
filtered through a 0.22 f1- m nylon membrane; with injection
concentration on the extraction of polyphenols from the four
volume of 10 f1- L). All the standard solutions of the different
plant used. In this experiment, the extraction of TPC from E.
phenolic compounds used were injected under the same
camaldulensis, F. cemua and T. diffw>a showed a quadratic
conditions.
effect (Table 3). When aqueous-ethanol concentration was
increased from 0% to 35% (v/v) more phenolic compounds
2.6. Statistics analysis
were extracted. But lower TPC yield was observed when
aqueous-ethanol concentration was increased until 70% (vI
The experiments were established under a blocks
v), On the other hand, in J. dioica extracts was observed
completely randomized design for which material vegetal
an increase in the TPC extracted which was proportional at
independently. The dates were transformed by log X and
the increase of aqueous-ethanol concentration (Table 3).
analyzed using the software SAS V 9.0. where the comparison
It is possible to mention that under the suitable conditions
test of means of multiple range Tukey (a = 0.5) was used. In
of extraction time and aqueous-ethanol concentration, E.
the graphic the dates observed are the true values.
camaldulensis and F. cemua were the species with higher
amount of TPC while T. dif/usa and J. dioica were the
108
108 Jorge E.
Jorge E. Wong-Paz
Wong-Paz et
et al.!Asian
al./Asian Pacific
Pacific Journal
Journal of
of Tropical Medicine (2015)104-111
Tropical Medicine (2015)104-111

species with lower TPC yield content (fable 3). 3.2.3. Lipid oxidation inhibition assay
In the light of the differences among the wide number of
3.2. Antioxidant capacity ofthe extracts test systems available for the antioxidant activity, the results
of a single-assay can give only a limited suggestion of the
Four plants of the semiarid Mexican regions were used to antioxidant properties of plant extracts[25,26J. In this study,
three assays were carried out for have a better interpretation
assess the antioxidant potential by three methods.
of the antioxidant activities of the obtained extracts.
Figure 2c shows the inhibition activity of the extracts
3.2.1. Free radical scavenging activity on DPPH
against linoleic acid peroxidation caused by FeC12 as an
Figure 2a shows the DPPH scavenging activity of the
oxidant initiator. In this assay the values obtained without
extracts obtained under the TPC suitable extraction antioxidants were taken for 100% lipid peroxidation. As do
conditions for the four plants. F. cemua, E. camaldulensis DPPH and ABTS assays, lower values (13.95%) were obtained
and T. dif.fusa extracts had similar capacity antioxidant with]. dioica extract at the concentration used. The rest
ranging between 76.03% to 91.96%. These results were of the extracts (F. cemua, E. camaldulensis and T. dif.fusa)
higher (70%) that obtained for J. dioica [(15.40±1.88)%]. To presented an average of 65% in the lipid oxidation inhibition.
our knowledge, there are not concise studies to compare the Anew E. camaldulensis extract maintained good antioxidant
J. dioica potential antioxidant. On the other hand, Salazar activity compared with the other extracts. These last findings
et al[13l evaluated the antioxidant activity using the DPPH for E. camaldulensis extract in the lipid oxidation inhibition
assay in F. cemua and T. dif.fusa reporting values ranged are close to results in the DPPHassay.
from 75.3% to 86.8% and from 27.9% to 88.1%,respectively for
100 a)

Lipid oxidation inhibition(%) ABTS+ radical inhibition(%) DPPH radical inhibition(%)

100 a)
individual tissues. It is according with our findings since we
80
80
used a mix of the tissues where antioxidant capacity of each 60
60
tissue was slight modified. 40
40
20
20
0
3.2.2. Free radical scavenging activity on ABTS o F.cermua E.camal~is
J.diokaKcermua
J.dioka E.camaldulensis Tsdlfussa
T.difussa
100
100 b)
b)
Antioxidant activity in the extracts was also investigated
80
80
using the well-known ABTS method. The results in ABTS
60
60
assays show that only E. camaldulensis extract maintained 40
40
a high antioxidant activity very similar in both DPPH and 20
20

ABTS assays with radical inhibition of 88% approximately o0

J.dioka F.cermua
].dioka F.cermua E.camaldulensis
E.camaldulensis T.difussa
T.difussa
under the condition used. While, F. cemua and T. dif.fusa 100
100 c)
c)

extracts were more effective in DPPH radical scavenging 80

80
60
60
than ABTS radical scavenging with a loss of 50% in the
40
40
antioxidant activity decreasing from 80% in DPPH assay to 20
20
30% in ABTS assay in both cases (Figure 2). In contrast, J. 0
o ~
J.dioka F.cermua
}.dioka F.cermua E.camaldulensis
E.camaldulensis T.difussa
T.difussa
dioica extract had a negligible increasing of 9% inhibition
approximately when the ABTS assay was evaluated. In
Figure 2. Antioxidant activity of the phenolic extracts selected (1 mg
general terms, E. camaldulensis extract showed more free
dried extractlmL).
radicals scavenging capacity as antioxidant activity.
a) DPPH Free radical scavenging activity; b) radical cation ABTS+
scavenging activity; c) linoleic acid oxidation inhibition.

Table 44
Table
Phenolic compounds
Phenolic compounds detected
detected by
by HPLC
HPLC assay
assay in
in the
the different
different extracts
extracts selected.
selected.
STD
STD PG
PG GA
GA RS
RS CHA
CHA MG
MG CUA
CVA CAT
CAT HA
HA EA
EA QE
QE CIA
CIA
].J. dioica
dioica - - - - - - - - - - -
F. cernua
F. cernua - - - +
+ - +
+ - - - +
+ -
Extracts E.camaldulensis
Extracts E.camaldulensis -- - - - - - + - - - -
+
T. diffusa
T. diffusa - - - - - - - - - +
+ -
RT
RT 6.5
6.5 7.46
7.46 9.51
9.51 11.05
11.05 12.99
12.99 15.09
15.09 15.65
15.65 17.76
17.76 18.83
18.83 20.11
20.11 21.05
21.05
Retention time
Retention time (RT),
(RT), standard
standard (STD),
(STD), pyrogaHol
pyrogallol (PG),
(PG), gaJIic
gallic acid
acid (GA),
(GA), resorcinol
resorcinol (RS),
(RS), chlorogenic
chlorogenic acid
acid (CHA),
(CHA), methyl
methyl gaHate
gallate (MG),
(MG), coumaric
coumaric
acid (CVA),
acid (CUA), catechin
catechin (CAT),
(CAT), 2-hydroxycinnamic
2-hydroxycinnamic acid
acid (HA),
(HA), eHagic
ellagic acid
acid (EA),
(EA), quercetin
quercetin (QE),
(QE), cinnamic
cinnamic acid
acid (CIA),
(CIA), presence
presence (+)
(+) and
and absence
absence
(-).
(-).
 Jorge
Jorge E.
E. Wong-Paz et al.!Asian
Wong-Paz et al./Asian Pacific
Pacific Journal
Journal of
of Tropical
Tropical Medicine
Medicine (2015)104-111
(2015)104-111
109
109

3.3. HPLC assay small size particle increase the contact surface in the vegetal
material and therefore, increases the extraction yields of
Using the HPLC analytical assay was possible detect and the target compoundstnl. Our findings are also according
confirm the presence of the some phenolic compounds in J. with previously reports by Castillo et a1[32], where these
dioica, F. cemua, T. diffusa and E. camaldulensis extracts. authors reported J. dioica had lower values of TPC than T.
Table 4 shows a summary of the analyzed samples. It was diffusa and F. cemua before and after fermentation, It is
noted that only J. dioica extract presented none phenolic well documented that phenol content in the plants varies
compound. Possibly because of this is a crude extract and depending the specie, the plant tissue and environmental
no purified then the phenolic compounds are present in factors as temperatures, water stress, light conditions as
low concentration. Nevertheless, recently Aguilera et al[27] well as phenological developmentlzcl. It explains the large
confirmed the presence of ellagic acid by HPLC in a differences obtained among the plants used.
methanolic hydrolyzed extract in some arid Mexican plants It is interesting to note that despite of that T. diffusa
including]. dioica, F. cemua and T. diffusa. However, these extract had lower TPC levels respect to F. cemua and
authors reported that the concentration was low (1.8-2.5 mgJ E. camaldulensis extracts, it showed a good potential
g). On the other side, the HPLC assay in the others three antioxidant. Several reports have showed that the phenolic
extracts, in special F. cemua, permitted to detect almost one compounds are not the unique phytochemicals to possess
compound (fable 4). Ruiz et al[18] studied the phytochemical antioxidant properties[331.
profile of the F. cemua extracts and fractions, reporting two On the other hand Amakura et al[34] evaluated the
important phenolic compounds namely 2-hidroxicinnamic antioxidant activity by DPPH assay of eucalyptus extract
and ellagic acids. In our screening these compound was not and, gallic and ellagic acids isolated from the eucalyptus
detected. However, three different polyphenolic compounds extract. They observed that the isolated compounds had
were clearly detected as chlorogenic and coumaric acids higher antioxidant activity than BHA and BHT compounds
and quercetin. Quercetin and catechin were identified in T. (synthetic antioxidants), isolated terpenes and phloroglucinol.
diffusa and E. camaldulensis extracts respectively. Finally, the authors concluded that the main antioxidant
activity in eucalyptus extract can be attributing to gallic and
ellagic acids present in the extracts. As expose above, E.
4. Discussion camaldulensis extract maintained a high antioxidant activity
very similar in both DPPH and ABTS assays. These findings
The TPC extracted from each plant was very different shows that one or several antioxidant compounds present
among plants despite of the extraction yields were similar in the E. camaldulensis extract have the capacity to act in
in all the samples. Similar reports have been published by two different mechanisms for the free radical scavenging
other authorslasl, These authors observed that no existed activity, through a single electron transfer reaction (ABTS
significant differences in the extraction yield evaluating assay)[6,35] and a hydrogen transfer reaction (DPPH assay)[23,36l.
times from 3 to 8 h with heat-reflux system using other In this sense, F. cemua and T. diffusa extracts had more
plants. Hence, they concluded that highest quantity of capacity for donate a hydrogen atom instead of transfer an
compounds extracted is achieved in a lesser time than electron. However, it is important to note that antioxidant
the times evaluated. Few studies had been developed compounds can respond in a different manner to different
concerning the polyphenolic content from F. cemua[15,29,30j. radical or oxidant compounds[371. It could be explained
Interestingly, in the present study was possible to obtain according the mechanism of oxidation proposed by Huang
higher TPC. These differences in the results can be et al[6] and Mishra et al[36] for DPPH assay and Choe and
attributed at solvent used in their studies as acetone and Ming[33] for lipid oxidation inhibition, both methods are
hexane which extracted certain compounds of low or no based on the oxidation process could be stopped when
polarity. In contrast, in our study, the solvent employed was another molecule have the capacity of transferring a
aqueous-ethanol which is a solvent with higher polarity hydrogen atom to the radical. However, the assay lipid
and it is known that the most phenolic compounds are oxidation inhibition is an antioxidant test stricter than DPPH
polar permitting easier the diffusivity in the ethanol and method due to chain reactions are involvedlsel, Hence, the
water mixture. Other factor could affect positively the yield antioxidant compounds should be more specific, resistant
extraction was the particle size. In our study was used a and higher stoichiometrically for stabilized the several kinds
small particle size between 0.6-0.8 mm. It is known that a free radical formed; besides exist the possibility that extracts
110
110 Jorge E.
Jorge E. Wong-Paz
Wong-Paz et
et al.!Asian
al./Asian Pacific
Pacific Journal
Journal of
of Tropical Medicine (2015)104-111
Tropical Medicine (2015)104-111

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