Food Chemistry 142 (2014) 455460

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Total phenolics and antioxidant activity of Piper auritum

and Porophyllum ruderale
Lilia A. Conde-Hernndez, Jos . Guerrero-Beltrn
Departamento de Ingeniera Qumica, Alimentos y Ambiental, Universidad de las Amricas Puebla, Sta. Catarina Mrtir, Cholula, Puebla 72810, Mexico

a r t i c l e

i n f o

Article history:
Received 20 March 2013
Received in revised form 10 July 2013
Accepted 18 July 2013
Available online 25 July 2013
Keywords:
Antioxidant activity,
Total phenolic compounds
Mexican pepperleaf
Piper auritum
Papalo
Porophyllum ruderale

a b s t r a c t
Extracts from fresh and dried samples of Mexican pepperleaf (Piper auritum Kunth) and papalo
(Porophyllum ruderale) were obtained using a stirring or an ultrasound extraction system with ve types
of solvents (water, 50:50% v/v ethanol:water, 70:30% v/v ethanol:water, 85:15% v/v ethanol:1.5 N HCl,
and ethanol). Total phenolic compounds and antioxidant activity were evaluated with the phenol Folin
Ciocalteu reagent and the ABTS method, respectively. Total phenolic compounds (PC), trolox (T), and
ascorbic acid (AA), in the two herbs, were in the range of 6.7968.03 mg of galic acid (GA)/g dry solids
(d.s.), 4.8864.99 mg of T/g d.s., and 5.3149.84 mg AA/g d.s., respectively. Extracts from fresh papalo,
using ultrasound as the extraction system, had the highest amount of total phenolic compounds. The
fresh pepperleaf extract, obtained using ultrasound as the extraction method contained the highest
amount of antioxidant activity.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
The demand for natural additives, including antioxidants, has
grown worldwide in recent years. This requirement is coupled with
the development of the food industry and the introduction of new
technologies to meet the worldwide peoples requirements. Therefore, it would be worthwhile to nd new local sources of natural
antioxidants, and try to introduce these new alternatives to the
food industry.
The growing interest for replacing synthetic for natural antioxidants, obtained from parts of plants, has promoted the investigation for obtaining and identifying new antioxidants to be used in
foods. Oxidation reactions are not only important to the food
industry; antioxidants are also required to avoid deterioration of
products found in the cosmetics, pharmaceutical and plastic industries (Moure et al., 2001). It is also known that many plants used
for therapeutic purposes are excellent sources of phytochemicals
such as phenolic compounds. Many of these chemical compounds,
having antioxidant activity, are used in food products and a number of medical treatments (Li, Hao, Wang, Huang, & Li, 2009). Basically, antioxidants inhibit the spread of free radicals in biological
systems. To characterize such a property in parts of plants, and
other materials, the antioxidant activity should be determined.
The ABTS method has been widely used to measure the antioxidant
Corresponding author. Tel.: +52 222 229 2126; fax: +52 222 229 2727.
E-mail address: angel.guerrero@udlap.mx (J.. Guerrero-Beltrn).
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.07.078

activity of different substances (Re, Pellegrini, Proteggente, Annala,

Yang, & Rice-Evans, 1999).
Halliwell and Gutteridge (1990) have dened antioxidant compounds as substances that, when present at low concentrations,
may delay or prevent oxidative damages in cells due to the presence of oxidative chemical species. These oxidative species can undergo a redox reaction with phenolic compounds. Domnguez,
Nieto, Marin, Keck, Jeffery, & Cspedes (2005) have stated that
low concentrations of phenolic compounds might inhibit the polymerization chain initiated by free radicals and other subsequent
oxidizing reactions. According to Kuskoski, Asuero, Troncoso,
Mancini-Filho, & Fett (2005) the ABTS method, to determine the
ability of an antioxidant to scavenge free radicals, is considered a
very sensitive, practical, rapid, and stable method.
Piper auritum Kunth is a small shrub which grows in the tropic
area of Central America; it belongs to the Piperaceae family (Jaramillo & Manos, 2001). Depending on the culture and region where
it grows, P. auritum is commonly referred to as hoja santa, yerba
santa, anisillo, acuyo, or momo in Mexico and Central
America. In the United Stated of America, this herb is commonly
referred to as pepperleaf, eared pepper or root beer plant
(Gupta & Arias, 1985). The essential oil of P. auritum is composed
mainly of safrole (70%) and more than fourty other constituents
in smaller quantities; a number of those are mono and
sesquiterpenes. The essential oil can be used as a condiment in
the food industry and leaves as food for sh. It has been observed,
on the other hand, that extracts of P. auritum have reduced the

456

L.A. Conde-Hernndez, J.. Guerrero-Beltrn / Food Chemistry 142 (2014) 455460

growth of Colletotrichum gloeosporioides on Maradol papaya

(Baos-Guevara, Zavaleta-Meja, Colinas-Len, Luna-Romero, &
Gutirrez-Alonso, 2004).
Porophyllum ruderale (Jacq.) Cassini (Asteraceae) is a herb grown
in Mexico, Central and South America. It is known as papalo,
papaloquelite, tepelcasho, and tepegua (Martnez, 1979;
Rzedowski & Rzedowski, 2004). This species is an herb with a
strong and unique avor; the leaves and stems are commonly used
as salad dressings (Loayza et al., 1999). P. ruderale has been used in
traditional medicine for closing wounds and relief general pain
(Corra, 1984). The aerial parts of P. ruderale have been previously
analyzed by different research groups because of their volatile
compounds. Bohlmann, Jakupovic, Robinson, and King (1980) reported thiophene derivatives with unsaturated chains and a chemical compound derivative from thymol in petroleum ether extracts.
It is known that the essential oil can be used as a pesticide (Guillet,
Blanger, & Arnason, 1998).
The aim of this study was to evaluate the antioxidant properties
of P. auritum and P. ruderale extracts obtained by stirring or ultrasound procedures.
2. Materials and methods
2.1. Raw material
Fresh P. auritum (Mexican pepperleaf) and P. ruderale (ppalo)
stems with leaves, from the area growing of Atlixco, Puebla, were
purchased at the local market in Cholula, Puebla, Mexico. P. auritum and P. ruderale leaves were used in fresh and dried fashion.
2.2. Drying
Fresh leaves were dried in a Boekel Scientic 107905 oven (Festerville, PA, USA) at 40 C for 24 h. Dried samples were packed in
plastic bags, air evacuated, sealed, protected from light, and stored
at room temperature until being required for analysis. All tests
were performed in triplicate.
2.3. Moisture content
The moisture content of samples was measured in both fresh
and dried plants according to the 934.06 AOAC (2000) method.
2.4. Extracts
Extracts from P. auritum and P. ruderale were obtained according to Chizzola, Michitsch, and Franz (2008) method with some
modications. Water, 50:50% v/v ethanol:water, 70:30% v/v ethanol:water, 85:15% v/v ethanol:1.5 N HCl, and ethanol solutions
were used as the extracting agents. Two ways for extracting antioxidants were used. (a) Stirring method. One hundred or 300 mg
of dried (milled) or fresh (chopped) samples, respectively, were
placed in volumetric asks. Dried and fresh P. auritum samples
were placed in 10 mL volumetric asks. Dried and fresh P. ruderale
samples were placed in 25 mL volumetric asks. Flasks were added
with the corresponding solvent, mixed, and made up to the corresponding volume. Mixtures were poured into glass beakers, covered with aluminum foil and stirred for 2 h for the antioxidants
extraction. Mixtures were ltered through Whatman paper No. 1
under vacuum conditions and stored away from light until being
used for analysis. (b) Ultrasound method. Mixtures from the volumetric aks were transferred to dilution asks, capped, and treated
with ultrasound in a water bath for 30 min at room temperature
and 40 kHz of frequency. Three repetitions were carried out for
determining the antioxidants content and phenolics.

2.5. Total phenolic compounds

Total phenolic compounds were assessed according to the Gao,
Ohlander, Jeppsson, Bjrk, and Trajkovski (2000) method with
modications. Two milliliters of distilled water and 0.2 mL of FolinCiocalteau reagent (SigmaAldrich, St. Louis, MO, USA) were
mixed with 0.1 mL of the plant extract; the mixture was incubated
for 3 min at room temperature (25 C) and then 1 mL of 20% Na2CO3
was added. All mixtures were let stand for one hour at room temperature in a dark environment. Afterward, absorbance was measured at 765 nm in a 2800H UVVisible spectrophotometer
(UNICO, Shanghai, China). The total phenolic compounds content
was calculated as mg Gallic acid (GA)/g of dry solids (d.s.) for the
two types of plants. A standard curve was prepared with Gallic acid
(00.33 mg/mL) in order to evaluate the total phenolic compounds
from the plant extracts. The experimental standard curve was
Abs = 3.802 (Abs/mg GA/mL)C(mg GA/mL) + 0.024 Abs (R2 = 0.997).
2.6. Antioxidant activity
The antioxidant activity was evaluated by the ABTS method
using the 2,20 azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid)
diammonium salt (98%) reagent. The ABTS+ radical was obtained
according to Kuskoski et al. (2005) method with some
modications.
2.6.1. Radical formation
3.3 mg of potassium persulfate and 19.4 mg of the ABTS reagent
(SigmaAldrich, St. Louis, MO, USA) were weighed in a glass beaker, 5 mL of distilled water added and then perfectly mixed. This
solution was kept away from light and let stand for 16 h at room
temperature for the radical formation. Afterward, the ABTS+ solution was diluted with absolute ethanol (radical-ethanol solution)
until obtaining an initial absorbance of 0.700 0.020 (Ai) at
754 nm.
2.6.2. Antioxidant activity measurement
Eighty microliters of the plant extract was added to 3920 lL of
the ABTS+ radical-ethanol solution, placed in a quartz cell, perfectly homogenized, and let stand for 7 min. The nal absorbance
(Af) was measured at 754 nm in a 2800H UVvisible spectrophotometer (UNICO, Shanghai, China). Percentage of inhibition (I)
was calculated as I (%) = [AiAf]/Ai. The antioxidant activity was reported as mg of trolox equivalent to the antioxidant activity (T)/
g dry solid (d.s.) or mg of ascorbic acid equivalent to the antioxidant activity (AA)/g d.s.
2.6.3. Standard curve
Trolox (6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (97%)) (00.2 mg/mL) and ascorbic acid (00.14 mg/mL)
were used to prepare standard curves. Plant extracts were treated
similarly to the standard curve solutions. The standard curve for
trolox was I (%) = 387.13 (%/mg T/mL)C(mg T/mL) + 3.256%
(R2 = 0.996). The standard curve for ascorbic acid was I
(%) = 491.28 (%/mg AA/mL)C(mg AA/mL) + 0.358% (R2 = 0.996).
2.7. Gas chromatography mass analyzer
Volatile compounds from ethanol-extracts of dried samples
were identied using a 6850N gas chromatograph (Agilent Technologies, Sta. Clara, CA, USA) attached to a 5975 quadrupole mass
selective detector (Agilent Technologies, Sta. Clara, CA, USA). One
lL of each extract was injected. The separation of volatiles was performed using a HP5-MS column of 30 m in length and 0.25 mm in
diameter coated with a lm of 0.25 lm in thick. The analysis conditions were: 1.1 mL/min of helium as carrier gas, an injector

457

L.A. Conde-Hernndez, J.. Guerrero-Beltrn / Food Chemistry 142 (2014) 455460

temperature of 250 C, a 10:1 split ratio mode, an initial temperature of 60 C increasing at a rate of 4 C/min until reach 240 C. The
ionization energy was 70 eV. The scanning mass range was m/z 43350. The identication of compounds was based on comparing
their mass spectra with the database of the mass spectra NIST library and literature reports.
2.8. Statistical analysis
Data were analyzed using the Minitab 14 statistical program. An
analysis of variance (ANOVA) and Turkeys test for multiple comparisons were performed to determine signicant differences. Differences within results were tested at 5% (P < 0.05) to state
signicant differences. The Pearson correlation coefcients were
calculated in a bivariation correlation fashion (Montgomery,
2010). The standard deviation was calculated using the Excel
program.
3. Results and discussion
3.1. Moisture content of leaves
The moisture content of dried and fresh P. auritum was
8.07 0.51 and 83.2 1.31% w/w, respectively, and for P. ruderale
was 9.52 1.16 and 87.32 1.08% w/w, respectively.
3.2. Phenolic compounds
Table 1 shows the total phenolic compounds in extracts from
dried and fresh P. auritum and P. ruderale leaves. It is important
to take into account that solvents, such as water, ethanol and mixtures of them, used for making extracts from plants, could be safe
to be used for food purposes since solvents such as acetone and
methanol may leave toxic residues. The phenolic compounds content was between 6.79 and 68.04 mg GA/g d.s. for both types of
plants. These values are higher than those reported by Khknen
et al. (1999) for other herbal extracts. They reported values of total
phenols between 9.1 and 23.1 mg GA/g per d.s. of herb. Higher
quantities of phenolic compounds were observed as the amount
of ethanol was increased in mixtures. Moure et al. (2000) reported
that the total phenolic content in methanol and ethanol extracts of
hazelnut was higher than in acetone; the phenolic content was
three times higher in ethanol than in acetone.
For both types of plants, higher amounts of total phenolic compounds were observed in fresh plants when using the stirring or
the ultrasound system. In a general way, no signicant difference
(P > 0.05) was observed in the phenolic compounds content between the stirring and the ultrasound system for making the
extraction. Thus, the ethanolic extracts from powders or fresh
plant could be used in foods.

A higher content of total phenolic compounds was observed in

fresh material. These two types of plants could be used without
undergoing a drying process previously. The reason for advising
the use of fresh plants is explained because antioxidants might
be heat sensitive. Some compounds could be evaporated during
the drying process. However, the disadvantage of using fresh
plants is their high moisture content and their total weight.
The acidied ethanol extracts showed the highest phenolic
compounds content. Ethanol extracts, on the other hand, presented
the lowest amount of phenolic compounds. Phenolic compounds in
the acidied ethanol extracts were signicantly different (P < 0.05)
to the amount of phenolic compounds in other extracting agents.
Thus, the acidied extracting system improved the phenols extraction in the two types of plants due to the hydrolysis effect on
bound antioxidants. In a general way, the phenolic compounds
content obtained with both, the 50% and 70% of ethanol solutions
were not signicantly different (P > 0.05). Durling et al. (2007)
pointed out that the optimum conditions for extracting phenolic
compounds from sage (Salvia ofcinalis) were achieved using ethanolwater solutions in the range 5575%. They observed high
amounts of rosmarinic acid when using ethanolwater solutions
at the lower ratios; however, the carnosic-type lipophilic antioxidants were better extracted using high ethanolwater ratios.
Therefore, the ethanol concentration used for extracting phenolic
compounds is important when characterizing types of phenolic
compounds from plant materials.
Luthria and Mukhopadhanyay (2006) pointed out that total
phenolic compounds and chlorogenic acid were better extracted,
from Solanum melongena L., using a simple stirring system than
an ultrasound extraction system. Chizzola et al. (2008), on the
other hand, did not found more than 10% of difference when using
the two types of extracting systems for Thymus vulgaris using a 60%
ethanolwater solution; thus, they recommend using any of the
two systems for extracting the antioxidant active compounds. In
this study, extracts using 70% ethanolwater solution did not show
more than 10% of difference between both types of extracting systems (stirring and ultrasound).
3.3. Antioxidant activity
Tables 2 and 3, for trolox and ascorbic acid, respectively, depict
the antioxidant activity in fresh and dried P. auritum and P. ruderale
leaves. The antioxidant activity values ranged from 3.06 0.44 to
29.03 1.20, and from 14.82 2.88 to 64.99 3.97 mg trolox
equivalents/g d.s. for P. auritum and P. ruderale, respectively (Table 2). The acidethanol extracts (stirred and ultrasounded) for P.
auritum, at fresh and dry fashion, presented the lower amount of
antioxidant activity. The higher quantities of antioxidant activity
were observed in the 50% and 70% ethanolwater extracts (stirred
and ultrasounded) for both, P. auritum and P. ruderale in fresh and

Table 1
Total phenolic compounds from Piper auritum and Porophyllum ruderale leaves.
Material

Dry P. auritum
Fresh P. auritum
Dry P. ruderale
Fresh P. ruderale

Extraction

Stirring
Ultras.
Stirring
Ultras.
Stirring
Ultras.
Stirring
Ultras.

Water

14.11 0.41ax
14.25 0.66ax
27.55 2.85ax
32.19 2.73ay
20.26 0.83ax
25.62 2.17ay
42.90 2.29ax
37.78 5.34ax

Phenolic compounds content (mg GA/g d.s.)

E:W
(50:50%)

E:W
(70:30%)

E:HCl
(85:15%)

Ethanol

18.45 1.01cx
18.15 0.85cx
27.11 2.46ax
28.30 1.99abx
40.09 2.02bx
39.27 1.73cx
46.77 1.92ax
49.07 2.54by

18.67 1.56cx
17.76 2.04cx
27.83 3.73ax
30.39 1.88ay
42.13 2.67bx
39.66 1.90cy
46.74 9.22ax
49.96 4.86bx

22.69 0.71dx
23.38 0.74dx
39.81 5.96bx
31.32 4.52ay
51.29 3.04cx
52.04 3.88dx
67.22 7.14bx
68.04 2.55cx

8.72 1.69bx
6.79 1.41by
25.51 5.02ax
25.24 6.02bx
18.01 3.52ax
17.22 3.44bx
40.44 5.24ax
39.94 3.95ax

Different letters in a row indicates signicant differences (P < 0.05). x and y letters for the stirring and ultrasound procedures, for same type of sample, indicates signicant
differences (P < 0.05).

458

L.A. Conde-Hernndez, J.. Guerrero-Beltrn / Food Chemistry 142 (2014) 455460

Table 2
Antioxidant activity (as trolox equivalents) from Piper auritum and Porophyllum ruderale leaves.
Material

Dry P. auritum
Fresh P. auritum
Dry P. ruderale
Fresh P. ruderale

Extraction

Stirring
Ultras.
Stirring
Ultras.
Stirring
Ultras.
Stirring
Ultras.

Water

15.25 1.62ax
15.24 1.27ax
26.24 1.96ax
28.78 2.88ax
22.50 2.73ax
34.43 1.54ay
41.79 5.56ax
39.56 5.58ax

Antioxidant activity (mg Trolox/g d.s.)

E:W
(50:50%)

E:W
(70:30%)

E:HCl
(85:15%)

Ethanol

20.58 1.03cx
18.89 1.61cy
28.74 1.27ax
25.04 1.63a,cy
42.95 2.99cex
45.49 1.61cx
48.39 5.03ax
50.97 1.80cx

19.64 2.44cx
18.49 2.40cx
24.74 1.20abx
29.03 1.20ay
43.59 2.80cx
47.34 1.51cy
64.99 3.97cx
51.59 5.35cy

8.39 0.76dx
7.85 0.32bx
5.59 1.37dx
3.06 0.44dy
38.52 3.15dex
26.40 4.71dy
25.67 2.90bx
22.81 5.52bx

11.86 0.74bx
8.79 2.08by
15.35 2.69bx
22.67 5.38b,cy
14.82 2.88bx
14.95 1.93bx
26.20 5.31bx
28.60 3.18bx

Different letters in a row indicates signicant differences (P < 0.05). x and y letters for the stirring and ultrasound procedures, for same type of sample, indicates signicant
differences (P < 0.05).

Table 3
Antioxidant activity (as AA equivalents) from Piper auritum and Porophyllum ruderale leaves.
Material

Dry P. auritum
Fresh P. auritum
Dry P. ruderale
Fresh P. ruderale

Extraction

Stirring
Ultras.
Stirring
Ultras.
Stirring
Ultras.
Stirring
Ultras.

Water

12.66 1.28ax
12.65 0.99ax
21.85 1.55ax
23.77 2.27ax
19.36 2.15ax
28.76 1.21ay
36.81 4.38ax
35.05 4.40ax

Antioxidant activity (mg AA/g d.s.)

E:W
(50:50%)

E:W
(70:30%)

E:HCl
(85:15%)

Ethanol

16.86 0.81cx
15.53 1.27cy
23.82 1.00ax
20.90 1.28acy
35.47 2.36c,ex
37.48 1.27cx
42.01 3.96ax
44.04 1.42cx

16.12 1.92cx
15.21 1.89cx
20.67 0.95abx
24.04 0.95ax
35.98 2.21cx
38.93 1.19cy
55.09 3.13cx
44.53 4.22cy

7.26 0.60dx
6.83 0.25bx
5.57 1.08dx
3.58 0.35dy
31.99 3.97dex
22.44 3.71dy
24.11 2.28bx
21.85 4.35bx

9.99 0.58bx
7.57 1.64by
13.27 2.13bx
19.03 4.24bcy
13.31 2.27bx
13.41 1.52bx
24.53 4.18bx
26.42 2.51bx

Different letters in a row indicates signicant differences (P < 0.05). x and y letter for the stirring and ultrasound procedures, for same type of sample, indicates signicant
differences (P < 0.05).

dried fashion. Signicant differences (P < 0.05) were observed for

the antioxidant activity in the 50% ethanolwater extracts (stirred
and ultrasounded) from dried and fresh P. auritum. Signicant differences (P < 0.05) were also observed for the antioxidant activity
in the 70% ethanolwater extracts (stirred and ultrasounded) from
dried and fresh P. ruderale. Same tendency was observed for the
antioxidant activity reported as mg AA/g d.s. (Table 3). However,
the antioxidant activity content, reported as mg AA/g d.s. was lower, in dried and fresh plants, than the amount reported as mg
trolox/g d.s.
Guerrero-Beltran, Vergara-Balderas, and Hernandez-Reyes
(2010) reported values of antioxidant activity in Chenopodium
ambrosioides (epazote) extracts from 8.7 to 19.9 mg trolox/g d.s.
The antioxidant activity found in this work ranged from 3.061
to 64.991 mg trolox/g of dried P. auritum and P. ruderale. It is
important to point out that in this study, the absorbance was
recorded after 7 min of reaction. Some researchers have stated
that the ABTS reaction proceeds in about one minute. However,
Re et al. (1999) have pointed out that four minutes is an appropriate time for letting the ABTS to react. Sellappan, Akoh, and
Krewer (2002), on the other hand, have suggested measuring
absorbance after 7 min for pure compounds of plant extracts
or foods.
3.4. Total phenolic compounds and antioxidant activity correlation
It has been reported that the antioxidant activity of plants is
well correlated with phenolic compounds (Chizzola et al., 2008;
Djeridane et al., 2006) in some plants. The Pearson correlation coefcients (Cheung, Cheung, & Ooi, 2003) were calculated using the
antioxidant activity and total phenolic compounds found in
P. auritum and P. ruderale in this study using the ultrasound or

stirring extraction methods. However, no correlation was observed

between both the antioxidant activity and total phenolic compounds (Table 4).
The ultrasonic bath method was used by Saito et al. (2007).
They claimed that this extraction technique was a useful and rapid
procedure for evaluating antioxidants of green tea using low temperatures. According to Usaqun-Castro, Martnez-Rubio,
Aya-Baquero, and Gonzlez-Martnez (2006), no evidence or previous studies, that explain the effect of ultrasound on the antioxidant
activity of phenolic compounds, have been reported. Moure et al.
(2000) pointed out that the antioxidant activity of phenolic compounds may depend on factors such as growing conditions, quality
and origin (geographical location) of plants as well as the extraction and purication methods (type and polarity of solvents and
extraction conditions) used to determine the antioxidant activity.
It has been reported that polyphenols are located in the cytoplasm
of cells; therefore, the use of ultrasound may lead to a kind of tissue permeability by disrupting cell structures such as cell walls
and cell membranes, which are important for controlling mass
transfer (Usaqun-Castro et al. 2006) from the surrounding media.
Some researchers have revealed that, in contrast to conventional
methods, ultrasound may diffuse plant contents through the cell
walls causing cell disruption in a short period (Chemat, Lagha, Aitamar, Bartels, & Chemat, 2004; Li, Pordesimo, & Weiss, 2004). Results reported in this study indicate that the use of ultrasound
reduces the antioxidant activity of some extracts from P. ruderale
and P. auritum. It has been also reported that, depending on operating conditions, such as frequency and power, ultrasound not only
causes cell disruption but also molecular rearrangements that lead
to inhibition or acceleration of bio-reactions (McClements, 1995;
Sala, Burgos, Condn, Lopez, & Raso, 1995; Capelo, Maduro, &
Vilhena, 2005).

459

L.A. Conde-Hernndez, J.. Guerrero-Beltrn / Food Chemistry 142 (2014) 455460

Table 4
Pearson correlation coefcients for phenolic compounds and antioxidant activity relationship of Piper auritum and Porophyllum ruderale.
Material

Dry P. auritum
Fresh P. auritum
Dry P. ruderale
Fresh P. ruderale
*
**

Extraction

Stirring
Ultrasound
Stirring
Ultrasound
Stirring
Ultrasound
Stirring
Ultrasound

Water

0.303
0.388
0.088
0.678
0.356
0.592
0.813**
0.016

Correlation coefcients
E:W
(50:50%)

E:W
(70:30%)

E:HCl
(85:15%)

Ethanol

0.041
0.545
0.830
0.088
0.597
0.347
0.147
0.539

0.041
0.545
0.830
0.088
0.597**
0.347
0.147
0.539

0.741
0.429
0.069
0.573
0.288
0.355
0.695
0.176

0.957
0.581
0.886
0.936
0.380
0.120
0.962
0.095

P < 0.05.
P < 0.10.

Fig. 1. GCMS chromatograms of stirred ethanol-extract from dried Piper auritum.

Fig. 2. GCMS chromatograms of ultrasound ethanol-extract from dried Porophyllum ruderale.

3.5. Chemical compounds identication

Chemical compounds from P. auritum and P. ruderale were identied by the GCMS technique. Fig. 1 illustrates the chromatogram

for P. auritum extract obtained by stirring of dry powder for 2 h in

absolute ethanol. Three large peaks are observed in this gure.
Peaks 1, 2 and 3 corresponds to safrole, phytol, and 3,7,11,15-tetramethyl-2-hexadecen-1-ol, respectively. Gupta and Arias (1985)

460

L.A. Conde-Hernndez, J.. Guerrero-Beltrn / Food Chemistry 142 (2014) 455460

pointed out that safrole was the main compound found in essential
oil from P. auritum. Safrole is considered toxic and even carcinogenic. The Scientic Committee on Food of the European Commission 1979 (SCF, 1979) proposed a limit of 1 mg of safrole/kg of food
and beverages. Fig. 2, on the other hand, shows the chemical spectrum obtained for P. ruderale extract obtained by shaking dry powder during 2 h. Three main compounds are also observed. Peaks 1,
2 and 3 correspond to ethylcyclohexane, ciclogeraniolane, and triethylether glycerol, respectively. When making a comparison with
compounds reported by Loayza et al. (1999), no similarity is observed with compounds reported in this research. However, it is well
known that factors that may inuence the difference between compounds in biological materials may include cultivar type, country of
origin, and harvest time, among other environmental conditions.
4. Conclusions
The extract containing the higher amount of total phenols was
the acidied extract of fresh P. ruderale using an ultrasonic bath.
The higher amount of antioxidant activity was observed in the
70% ethanol extract of fresh P. ruderale obtained by stirring.
Although a number of antioxidants are present in P. ruderale and
P. auritum, phenolic compounds can make a signicant contribution to their antioxidant activity. The efciency of antioxidants
properties depends strongly on the conditions of oxidation; hence,
the ABTS method only gives an approximation of the possibilities
of how an extract acts as an antioxidant. P. ruderale extracts contained higher total phenolic content and antioxidant activity than
the P. auritum extracts.
Acknowledgements
This research was part of the project number 2007/62275-Z
supported by Consejo Nacional de Ciencia y Tecnologa (CONACyT)
in Mexico. Author Lilia A. Hernndez-Conde would like to thank to
CONACyT for the economic support provided for the completion of
her doctoral studies.
References
A.O.A.C. Ofcial Methods of Analysis, 2000. Association of Ofcial Analytical
Chemists. 16th ed. Gaithersburg, MD, USA.
Baos-Guevara, P. E., Zavaleta-Meja, E., Colinas-Len, M. A., Luna-Romero, I., &
Gutirrez-Alonso, J. G. (2004). Control biolgico de Colletotrichum
gloeosporioides en papaya maradol roja (Carica papaya L.) y siologa
postcosecha de frutos infectados. Revista Mexicana de Fitopatologa, 2, 198
205.
Bohlmann, F., Jakupovic, J., Robinson, H., & King, R. M. (1980). A dithienylacetylene
from Porophyllum ruderale. Phytochemistry, 19, 27592760.
Capelo, J. L., Maduro, C., & Vilhena, C. (2005). Discussion of parameters associated
with the ultrasonic solidliquid extraction for elemental analysis (total content)
by electrothermal atomic absorption spectrometry. An overview. Ultrasonic
Sonochemistry, 12, 225232.
Chemat, S., Lagha, A., Aitamar, H., Bartels, P. V., & Chemat, F. (2004). Comparison of
conventional and ultrasound-assisted extraction of carvone and limonene from
caraway seeds. Flavour and Fragrance Journal, 19, 188195.
Cheung, L. M., Cheung, P. C. K., & Ooi, V. E. C. (2003). Antioxidant activity and total
phenolics of edible mushroom extracts. Food Chemistry, 81, 249255.
Chizzola, R., Michitsch, H., & Franz, C. (2008). Antioxidative properties of Thymus
vulgaris leaves: Comparison of different extracts and essential oil chemotypes.
Journal of Agricultural and Food Chemistry, 56, 68976904.
Corra, M. P. (1984). Dicionario das plantas teis do Brasil e das exticas cultivadas
(vol. II. Brasil: IBDF-Ministerio da Agricultura (pp. 67). Brasil: IBDF-Ministerio
da Agricultura.
Djeridane, A., Yous, M., Nadjemi, B., Boutassouna, D., Stocker, P., & Vidal, N. (2006).
Antioxidant activity of some Algerian medicinal plants extracts containing
phenolic compounds. Food Chemistry, 97, 654660.

Domnguez, M., Nieto, A., Marin, J. C., Keck, A., Jeffery, E., & Cspedes, C. L. (2005).
Antioxidant activities of extracts from Barkleyanthus salicifolius (Asteraceae)
and Penstemon gentianoides (Scrophulariaceae). Journal of Agricultural and Food
Chemistry, 53, 58895895.
Durling, N. E., Catchpole, O. J., Grey, J. B., Webby, R. F., Mitchell, K. A., Foo, L. Y., et al.
(2007). Extraction of phenolics and essential oils from dried sage (Salvia
ofcinalis) using ethanolwater mixtures. Food Chemistry, 101, 14171424.
Gao, X., Ohlander, M., Jeppsson, N., Bjrk, L., & Trajkovski, V. (2000). Changes in
antioxidant effects and their relationship to phytonutrients in fruits of sea
buckthorn (Hippophae rhamnoides). Journal of Agricultural and Food Chemistry,
48, 14851490.
Guerrero-Beltran, J.A., Vergara-Balderas, F., & Hernandez-Reyes, C. (2010).
Antioxidant properties of epazote leaf powders. 2010 IFT Annual Meeting
and Food Expo. July 1720. Chicago, IL, USA.
Guillet, G., Blanger, A., & Arnason, J. T. (1998). Volatile monoterpenes in Porophyllum
gracile and P. ruderale (asteraceae): Identication, localization and insecticidal
synergism with a-terthienyl. Phytochemistry, 49, 423429.
Gupta, M. P., & Arias, T. D. (1985). Safrole, the main component of the essential oil
from Piper auritum of Panama. Journal of Naturals Products, 48, 330.
Halliwell, B., & Gutteridge, J. M. C. (1990). Role of free radicals and catalytic metal
ions in human disease: An overview. Methods in Enzymology, 186, 185.
Jaramillo, M. A., & Manos, P. S. (2001). Phylogeny and patterns of oral diversity in
the genus Piper (Piperaceae). American Journal of Botany, 88, 706716.
Khknen, M. P., Hopia, A. I., Vuorela, H. J., Rauha, J., Pihlaja, K., Kujala, T. S., et al.
(1999). Antioxidant activity of plant extracts containing phenolic compounds.
Journal of Agricultural and Food Chemistry, 47, 39543962.
Kuskoski, E. M., Asuero, A. G., Troncoso, A. M., Mancini-Filho, J., & Fett, R. (2005).
Aplicacin de diversos mtodos qumicos para determinar actividad antioxidante en
pulpa de frutos. Campinas, Brazil: Ciencia y Tecnologa Alimentaria, pp. 25, 726
732.
Li, H., Hao, Z., Wang, X., Huang, L., & Li, J. (2009). Antioxidant activities of extracts
and fractions from Lysimachia foenum-graecum Hance. Bioresource Technology,
100, 970974.
Li, H., Pordesimo, L., & Weiss, J. (2004). High intensity ultrasound-assisted
extraction of oil from soybeans. Food Research International, 37, 731738.
Loayza, I., Groot, W., Lorenzo, D., Dellacassa, E., Mondello, L., & Dugo, G. (1999).
Composition of the essential oil of Porophyllum ruderale (Jacq.) Cass. from
Bolivia. Flavour and Fragrance Journal, 14, 393398.
Luthria, S. L., & Mukhopadhanyay, S. (2006). Inuence of sample preparation an
assay of phenolic acids from eggplant. Journal of Agricultural and Food Chemistry,
54, 4147.
Martnez, M. (1979). Catlogo de nombres vulgares y cientcos de plantas mexicanas.
Mxico D.F.: Fondo de Cultura Econmica (pp. 56). Mxico D.F.: Fondo de
Cultura Econmica.
McClements, D. J. (1995). Advances in the application of ultrasound in food analysis
and processing. Trends in Food Science and Technology, 6, 293299.
Montgomery, D. C. (2010). Diseo y anlisis de experimentos. Mxico, D.F.: Limusa
Wiley.
Moure, A., Cruz, J. M., Franco, D., Domnguez, J. M., Sineiro, J., Domnguez, H., et al.
(2001). Natural antioxidants from residual sources. Food Chemistry, 72,
145171.
Moure, A., Franco, D., Sineiro, J., Domnguez, H., Nuez, M. J., & Lema, J. M. (2000).
Evaluation of extracts from Gevuina avellana hulls as antioxidants. Journal of
Agricultural and Food Chemistry, 48, 38903897.
Re, R., Pellegrini, N., Proteggente, A., Pannola, A., Yang, M., & Rice-Evans, C. (1999).
Antioxidant activity applying an improved ABTS radical cation decolorization
assay. Free Radical Biology Medicine, 26, 12311237.
Rzedowski, G. C., & Rzedowski, J. (2004). Manual de malezas de la regin de
Salvatierra, Guanajuato. Flora del Bajo y de regiones adyacentes. Mexico, D.F.:
Consejo Nacional de Ciencia y Tecnologa (CONACyT) y Comisin Nacional para
el Conocimiento y Uso de la Biodiversidad (pp. 7278). Mexico, D.F.: Consejo
Nacional de Ciencia y Tecnologa (CONACyT) y Comisin Nacional para el
Conocimiento y Uso de la Biodiversidad.
Saito, S. T., Gosmann, G., Saf, J., Presser, M., Richter, M. F., & Bergold, A. M. (2007).
Characterization of the constituents and antioxidant activity of Brazilian green
tea (Camellia sinensis var. Assamica IAC-259 Cultivar) extracts. Journal of
Agricultural and Food Chemistr, 55, 94099414.
Scientic Committee for Food, European Commission. 1979. Report of the Scientic
Committee for Food on Flavourings. Reports of the Scientic Committee for Food
(9th Series). Ofce for Ofcial Publications of the European Communities.
Luxemburg.
Sala, F. J., Burgos, J., Condn, S., Lopez, P., & Raso, J. (1995). Effect of heat and
ultrasound on microorganisms and enzymes. In G. W. Gould (Ed.), New methods
of food preservation (pp. 176204). Great Britain: Aspen Publishers.
Sellappan, S., Akoh, C. C., & Krewer, G. (2002). Phenolic compounds and antioxidant
activity of Georgia-Grown blueberries and blackberries. Journal of Agricultural
and Food Chemistry, 50, 24322438.
Usaqun-Castro, X., Martnez-Rubio, M., Aya-Baquero, H., & Gonzlez-Martnez, G.
(2006). Ultrasound assisted extraction of polyphenols from red-grape (Vitis
vinifera) residues. The International Union of Food Science and Technology
(IUFOST) World Congress. pp. 13151324.