Revista Mexicana

de Ingeniera Qumica
Academia Mexicana de Investigaci
on y Docencia en Ingeniera Qumica, A.C.

Revista Mexicana de Ingeniera Qumica

Volumen 11, N
umero 1, Abril 2012

Vol. 11,
No. 1 (2012) 1-10
CONTENIDO

ISSN 1665-2738

Volumen 8, nmero 3, 2009 / Volume 8, number 3, 2009

EFFECT OF SOLVENT-TEMPERATURE EXTRACTION CONDITIONS ON THE

INITIAL ANTIOXIDANT ACTIVITY AND TOTAL PHENOLIC CONTENT OF
MUITLE EXTRACTS AND THEIR DECAY UPON STORAGE AT DIFFERENT pH
1

213 Derivation and application of the Stefan-Maxwell equations

SOLVENTE-TEMPERATURA
EFECTO DE LAS CONDICIONES DE EXTRACCION
EN LA (Desarrollo
ACTIVIDAD
ANTIOXIDANTE
Y
CONTENIDO
DE FENOLES TOTALES EN
y aplicacin de las ecuaciones de Stefan-Maxwell)

EXTRACTOS
DE
MUITLE
Y
SU
P
ERDIDA
DURANTE
EL ALMACENAMIENTO A
Stephen Whitaker
DIFERENTES VALORES DE pH
E. Garca-Marquez1 , A. Roman-Guerrero1 , C. Perez-Alonso2 , F. Cruz-Sosa1 ,
Biotecnologa / BiotechnologyR. Jimenez-Alvarado3 and E.J. Vernon-Carter4
1

245 Modelado
de la biodegradacin
en biorreactores
lodos de hidrocarburos totales
del petrleo
Departamento
de Biotecnolog
a, Universidad
Autonoma de
Metropolitana-Iztapalapa.
San Rafael
Atlixco No. 186
Col. Vicentina, CP 09340 Mexico, D.F., Mexico
2
intemperizados
ensuelos
y sedimentos
Departamento
de Ingenier
a Qumica,
Facultad de Qumica, Universidad Autonoma del Estado de Mexico, Paseo
Colon esq. Paseo Tollocan s/n, Col. Residencial Colon, CP 50120, Toluca, Edo. Mex., Mexico.
(Biodegradation modeling of sludge bioreactors of total petroleum hydrocarbons weathering in soil
3
Universidad de la Canada, Jefatura de Carrera de Ingeniera en Agroindustrias, Carretera Teotitlan-San Antonio
and sediments)
Nanahuatip
an km 1.7 s/n, Paraje Titlacuatitla, CP 68540 Teotitlan de Flores Magon, Oaxaca, Mexico.
4
Departamento
de Ingeniera S.
deHuerta-Ochoa,
Procesos e Hidr
aulica,
Universidad Aut
noma Metropolitana-Iztapalapa.
San
S.A. Medina-Moreno,
C.A.
Lucho-Constantino,
L.oAguilera-Vzquez,
A. JimnezRafael Atlixco No. 186 Col. Vicentina, CP 09340 Mexico, D.F., Mexico.
Gonzlez y M. Gutirrez-Rojas

Received 9 yofadaptacin
Novemberde2011;
Accepted 15
of November
2011cidas
259 Crecimiento, sobrevivencia
Bifidobacterium
infantis
a condiciones
Abstract (Growth, survival and adaptation of Bifidobacterium infantis to acidic conditions)
The effect ofL.solvent
and extraction
on the initial
activity E.
(AA)
and total phenolicy content
(TPC) of Muitle
Mayorga-Reyes,
P.temperature
Bustamante-Camilo,
A.antioxidant
Gutirrez-Nava,
Barranco-Florido
A. Azaola(Justicia spicigera) extracts and their decay upon storage at different pH values was studied. Extraction with aqueous solvents,
Espinosa
at either 25 or 60 C, produced fresh extracts (pH 7.4) with higher initial TPC and AA. Extracts were adjusted to different pHs
265
Statistical
to optimization
of ethanol
Saccharomyces
cerevisiaebyina relatively
the
displayed different TPCapproach
and AA decay
rates upon storage.
TPCfermentation
had first orderby
decay
kinetics, characterized
quick
constant (k1 ) at relatively short storage times followed by a relatively slow constant (k2 ) at relatively large storage times. ABTS
presence
of Valfor
zeolite
and DPPH assays
estimated
different
AA NaA
in the extracts, attributed to compatibility differences between the bioactive species and
the free radicals.
(Optimizacin estadstica de la fermentacin etanlica de Saccharomyces cerevisiae en presencia de

Keywords: zeolita
Justicia
spicigera,
total
phenolic content, antioxidant activity, extraction conditions, decay kinetics.
Valfor
zeolite
NaA)
Resumen G. Inei-Shizukawa, H. A. Velasco-Bedrn, G. F. Gutirrez-Lpez and H. Hernndez-Snchez
Se estudio el efecto del solvente y la temperatura de extraccion en la actividad antioxidante inicial (AA) y el contenido de
fenoles totales (TPC) en extractos de Muitle (Justicia spicigera) y su perdida durante el almacenamiento a diferentes valores de
pH. La Ingeniera
extraccion de
conprocesos
solventes/ Process
acuosos,engineering
a 25 o 60 C, produjeron extractos frescos (pH 7.4) con mayor TPC y AA iniciales.
Los extractos
fueron ajustados
diferentes
valores Revisin
de pH mostrando
diferentes TPC
y tasas
de pempleados
erdida de AA
271 Localizacin
de unaa planta
industrial:
crtica y adecuacin
de los
criterios
en durante el
almacenamiento. TPC mostro una cinetica de perdida de primer orden caracterizada por una constante relativamente rapida (k1 )
esta decisin
a tiempos relativamente cortos seguida de una constante relativamente lenta (k2 ) a tiempos de almacenamiento largos. Pruebas
selection:
Critical AA
review
andextractos,
adequation
criteria aused
this decision)
de ABTS y (Plant
DPPHsite
estimaron
diferentes
en los
atribuido
las in
diferencias
de compatibilidad entre las especies
bioactivas y J.R.
los radicales
libres.
Medina, R.L. Romero y G.A. Prez

Palabras clave: Justicia spicigera, contenido de fenoles totales, actividad antioxidante, condiciones de extraccion,
cineticas de perdida.
Corresponding author. E-mail: [email protected]
Tel.: +52 55 5804 4934; fax: +52 55 5804 4900

Publicado por la Academia Mexicana de Investigacion y Docencia en Ingeniera Qumica A.C.

E. Garca-Marquez et al./ Revista Mexicana de Ingeniera Qumica Vol. 11, No. 1 (2012) 1-10

Introduction

Aqueous extracts of aerial parts of Muitle (Justicia

spicigera Schechtendal) have been used in traditional
medicine in Mexico and elsewhere as antipyretic,
blood depurative, anti-inflammatory, for treatment of
diarrhea and diabetes, uterine cancer, amenorrhea,
and have shown cytotoxic activities against human
leukemic cells (Wong, 1976; Mahabir and Gulliford,
1997; Marquez et al., 1999; Sepulveda Jimenez
et al., 2009). Likewise, ethanolic extracts have
demonstrated activity against Giardia duodenalis
(Ponce-Macotela et al., 2001). The main compounds
found in J. spicigera are kaempferitrin, O-sitosterol3--glycoside, allantoin and cryptoxanthin, and are
probably responsible for these activities (Euler and
Alam, 1982; Domnguez et al., 1990). However,
antioxidant activity studies of J. spicigera and other
species from this genus are scarce. SepulvedaJimenez et al. (2009) were the first in describing
the antioxidant activity from J. spicigera, where the
phenolic compounds and flavonoids contributed to this
activity, suggesting J. spicigera as potential source of
antioxidants for use against various free radical-related
disorders.
The interest in phenolic compounds has been
related primarily to their antioxidant activity; they
also show important biological activity in vivo and
may be beneficial in combating diseases related
to excessive oxygen radical formation, where the
antioxidant defense capacity of the human body
is exceeded (Morello et al., 2004; Mulero et al.,
2010). However, these natural antioxidants are fairly
unstable during harvesting, transportation, storage and
processing (Reyes-Mungua et al., 2009). On the
other hand, the initial phenolic content and antioxidant
activity of the extracts depends on the solvent and
extraction conditions used, mainly due to the complex
interrelationship between the solvents and phenolics
polarities and solubilities (Serrano-Maldonado et al.,
2011; Ares et al., 2010). Also it is important to
understand how storage time and pH of the phenolic
compounds affects their stability before proceeding to
use them in a commercial basis (Xu and Chang, 2007).
Thus, the aims of this work were: (a) to evaluate
the effect of the solvent and temperature extraction
conditions on the initial total phenolic content and
antioxidant activity of Muitle extracts; and (b) to
determine the decay of these two parameters in
extracts at different pH values stored for 20 days at
25 a C.
2

2.1

Materials and methods

Plant material and reagents

Leaves of Muitle (J. spicigera Schechtendal) from

adult plants were collected in the month of June from
Tepoztlan, Morelos, Mexico. Analytical reagents
and standards used for the determination of total
phenolic content (TPC), and antioxidant activity
(AA) were: Folin-Ciocalteau reagent (FCR) and
sodium azide (SA) both purchased from Hycel
de Mexico, S.A. de C.V., Mexico, D.F., Mexico;
2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid
(ABTS) 2,2-diphenyl-1- picrylhydrazyl (DPPH), 6hydroxy-2,5,7,8-tetramethylchroman- 2-carboxylic
acid (TROLOX), gallic acid standard (GAS),
potassium persulfate (PPS), glycerol (G, Sigma
Ultra, 99% GC) were obtained from Sigma-Aldrich
Quimica, S.A. de C.V., Toluca, State of Mexico,
Mexico; anhydrous sodium acetate (ASA), glacial
acetic acid (GAA), hydrochloric acid (HCl), methanol
(MeOH), and sodium hydroxide (NaOH) were
acquired from J.T. Baker, S.A. de C.V. (Xalostoc,
State of Mexico, Mexico); sodium carbonate (SC) was
provided by Qumica Meyer (Mexico, D.F., Mexico);
absolute ethanol (E100 ) was procured from Tecsiquim
S.A. de C.V. (Toluca, State of Mexico, Mexico); and
propylenglycol (PG) was gotten from Polioles, S.A.
de C.V. (Toluca, State of Mexico, Mexico). Deionized
water (W) was used in all the experiments.

2.2

Preparation of Muitle extracts

Muitle stems and leaves were dried for 72 h at

38 C in an oven (SW-17TA, Blue-M, New Columbia,
PA, USA). The dry vegetable material was finely
milled and sieved using a Mesh 35, and 2.5 g of
mill were extracted by maceration for 12 h with
100 g of different extraction solvents (two monocomponent and three binary systems) at 25 and
60 C, respectively. These temperatures were chosen
from preliminary experiments because high phenolic
compounds extraction and a relatively low degradation
caused by thermal effects were achieved. Codification
of extraction conditions was done in base to the
solvents abbreviation followed by the temperature
used, as follows: Water (W25, W60), absolute ethanol
(E100 25, E100 60), ethanol 500 g kg1 (E50 25, E50 60),
glycerol 50 g kg1 (G5 25, G5 60), and propylenglycol
50 g kg1 (PG5 25, PG5 60). The extracts were filtered
using Whatman 4 filter paper. Fresh Muitle extracts
presented an initial pH of 7.4, independently of the

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E. Garca-Marquez et al./ Revista Mexicana de Ingeniera Qumica Vol. 11, No. 1 (2012) 1-10

2.6

extraction conditions used.

DPPH radical scavenging activity

Fresh Muitle extracts were diluted with the

corresponding solvent system used in the extraction
to 500 g kg1 . The pH of the dilutions remained at
7.4. In order to obtain solutions at pH 3.5 and 5.5,
the requisite amount of 1.0N HCl was added. All the
extracts were stored for 20 days at 25 C at the three
pH values (3.5, 5.5, and 7.4), and their TPC and AA
were determined up to 20 days. These pH values were
chosen because they cover the pH range most used in
the beverage, food and pharmaceuticals industries.

Radical scavenging activity of the Muitle extracts

was evaluated using DPPH+ radicals as reported by
Yim et al. (2012). Stock solution of DPPH (0.075
mM) in methanol was prepared daily. 3.8 mL of
DPPH+ working solution was mixed with 0.02 mL of
Muitle extract. The mixture was shaken vigorously
for 1 min and left to stand at room temperature in
the dark for 30 min. Absorbance was measured
against a blank reagent at 517 nm. Radical scavenging
activity was determined by calculating the TEAC
concentration using the TROLOX calibration curve
mentioned above.

2.4

2.7

2.3

Characterization of Muitle extracts

Total
phenolic
determination

content

(TPC)

TPC was measured using the method Yim et al.

(2012), with slight modifications. In short, 0.02 mL
of Muitle extract was mixed with 0.1 mL of FCR (500
g kg1 ). After 3 min, 0.3 mL of SC (200 g kg1 )
were added to the mixture and adjusted to 2 mL with
deionized water. The mixture was allowed to stand
in dark environment for 90 min. Absorbance was
measured against the blank reagent at 725 nm using
a Spectronic GENESYS 2 UV-Vis Spectrophotometer
(Thermo Fisher Scientific, Inc., Waltham, MA, USA).
GAS was used for the calibration curve with a
concentration range of 80-1000 mg kg1 (R2 = 0.99)
and analyzed as above. Results were expressed as mg
of Gallic Acid Equivalent (GAE) per kg (mgGAE kg1 ).

2.5

ABTS radical scavenging activity

The capacity of the extracts to scavenge ABTS+ was

based on that reported by Gong et al. (2012), with
slight modifications. ABTS+ was prepared by mixing
an ABTS stock solution (7 mM) with 2.45 mM PPS.
This mixture was allowed to stand for 12-16 h at
room temperature in the dark. The ABTS+ working
solution was obtained by diluting the stock solution
in MeOH to an absorbance of 0.7 0.02 at 747 nm.
Then 0.02 mL of the appropriately diluted sample was
added to 3 mL of ABTS+ working solution and mixed
thoroughly. The reaction mixture was kept at room
temperature in the dark for 1 h, and the absorbance
was read at 747 nm during 20 days. A reagent control
was measured in the same way. The data were reported
as TROLOX equivalents (TEAC, mM). TEAC was
calculated from the calibration curve using a range of
0.5-10 mM of TROLOX.

Extracts TPC decay kinetics

The Muitle extracts obtained with different solvents

and adjusted to pH values of 3.5, 5.5, and 7.4 were
monitored for TPC during storage at 25 C, and the
decay in TPC determined using a first order kinetic
model with the following Eq. (1):
ln[T PC]t = ln[T PC]0 kt

(1)

where [TPC]t is the TPC concentration (mgGAE kg1 )

at time t (h), [TPC]0 is the initial TPC concentration in
Muitle extracts, and k is the decay rate constant (h1 ).
The variation in concentration with time provides
a highly detailed description of how fast a reaction
is occurring. In many circumstances, though, it is
desirable to have a simple, approximate measure of
the reaction rate, and the half-life (t1/2 ) provides such a
measure, and is given for a first order model (Walstra,
2002) as shown in Eq. (2).
t1/2 =

ln 2
k

(2)

t1/2 is the time it takes for one-half of the original

amount of material to react (assuming the compound
in question is a limiting reactant) expressed in days.
Intuitively, the faster the reaction proceeds the shorter
t1/2 will be.

2.8

Statistical analysis

Analyses were performed in triplicate for each sample

for all the tests, and data is presented as means
standard deviation (SD). Analysis of variance and
comparison of treatment means (LSD 5% level)
were performed using SPSS Statistics 17.0 (IBM
Corporation, NY, USA).

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E. Garca-Marquez et al./ Revista Mexicana de Ingeniera Qumica Vol. 11, No. 1 (2012) 1-10

3
3.1

Results and discussion

Effect of extraction solvent on initial
TPC

The solvent system used affected the fresh Muitle

extracts initial TPC as can be observed in Table 1. All
the fresh extracts had a pH of 7.4, independently of
the solvent used in the extraction with a solid content
ranged from 1.5-1.6% (w/w). Initial TPC was higher
than 700 mgGAE kg1 for most solvents, excepting
E100 . Given the poor TPC yield by E100 25, this extract
was not considered in the following assays. Nonsignificant highest initial TPC contents were displayed
by E50 25, E50 60, PG5 60 and G5 25, with W showing
significantly lower values than the former solvents,
but significantly higher than the E100 solvent. These
variations in the initial TPC can be attributed to the
difference in polarity of the solvent systems used.
The solvents with intermediate polarity, i.e., E50 [ =
52.5], PG5 [ = 77.6], and G5 [ = 78.3], showed
higher initial TPC than those with lower polarity (E100
[ = 25]) or higher polarity (W [ = 82]). It
has been reported that Ginkgo leaves (Ginkgo biloba
L.) and Henna leaves (Lawsonia inermis) extracts
showed a significant difference in phenolic compounds
content when extracted with solvents with different
polarities (Ding, 1999; Uma et al., 2010). According
to Li (1994), the solvents most used for extracting
phenolic compounds (flavonoids) include water and
aqueous methanol, ethanol, and acetone, without
considering the possible toxicity associated with the
residues of some of these solvents. While water is
rather safe to use and is a good solvent for some
flavonoid glycosides, its high polarity renders it a bad

solvent for other flavonoids. Mixtures of alcohols

and water are frequently used because they are more
efficient in extracting phenolic compounds than monocomponent solvent systems (Spigno and Faveri, 2007)
probably because a more polar medium is created
which facilitates the extraction of polyphenols. In this
work the solvents used for the extraction of Muitle
leaves was done based firstly on their safety and
secondly on their ability to provide high TPC yields.

3.2

Effect of extraction temperature on

initial TPC

The fresh Muitle extracts (pH 7.4) obtained with

solvents W, E50 and PG5 showed non-significant
differences in initial TPC when extraction temperature
was 25 or 60 C, but a significant effect was observed
on initial TPC when using E100 and G5 solvents (Table
1). While initial TPC decreased for G5 it increased
for E100 as extraction temperature was raised from
25 to 60 C. It has been reported that with certain
materials, an increase in temperature promotes solvent
extraction by enhancing both diffusion coefficients
and the solubility of polyphenol content (Al-Farsi
and Lee, 2008), and promotes the release of bound
polyphenols by breaking the cellular constituents of
plant cells (Wang et al., 2007). However, elevated
temperatures may not be suitable for all kinds of
phenolic compounds (Too et al., 2010), as they
may degrade or coagulate with proteins (Spigno and
Faveri, 2007). When the phenolic compounds are
thermally stable, it may be inferred that the rate of
extraction is higher than the rate of decomposition in
the temperature range studied (Liyana-Pathirana and
Shahidi, 2005).

Table 1. Initial TPC of Muitle extracts at different pH values

Muitle extract code

TPC

pH7.4

(mgGAE kg1 )

TPC

pH5.5

(mgGAE kg1 )

TPC

pH3.5

(mgGAE kg1 )

25
60

917.09 26.46 c,d

872.77 8.80 c

849.61 19.54 c
899.97 21.32 c

865.73 13.81 c
885.87 9.58c,d

E100

25
60

190.98 6.60 a
624.02 6.85 b

193.00 6.98 a
729.43 25.64 b

196.36 8.35 a
608.59 4.54 b

E50

25
60

1018.80 24.17 g
977.51 32.88 e,f,g

976.50 28.63 e,f

988.59 18.71 e,f

970.46 6.87 f
969.79 8.17 f

PG5

25
60

959.05 18.47 d,e,f

963.08 26.76 d,e,f,g

1018.13 26.29 f
849.61 25.60 c,d

914.06 15.98 d,e

883.85 14.23 c,d

G5

25
60

989.93 18.02 f,g

926.15 14.52 c,d,e

1006.72 16.76 e,f

952.33 20.12 d,e

936.22 16.87 e,f

968.45 13.81 f

Different lowercase letters in the same column represent data significantly different (P 0.05).

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E. Garca-Marquez et al./ Revista Mexicana de Ingeniera Qumica Vol. 11, No. 1 (2012) 1-10

W60
E10060

1.00

pH 7.4

E5060

pH 5.5

PG560

0.95

E5060

0.95

G560

W60
E10060

pH 3.5

E5060
PG560

0.95

G560

PG560
0.90

0.85

0.80

G560

0.90

0.85

C/C0

C/C0

0.90

C/C0

1.00

W60
E10060

1.00

0.85

0.80

0.80

0.75
0.70

0.75

0.75
0.65

a)

50

100

150

200

250

Time (h)

300

350

400

450

500

50

100

150

200

250

300

350

400

450

500

Time (h)

b)

50

100

150

200

250

300

350

400

450

500

Time (h)

c)

Fig. 1. Influence of solvent-time extraction conditions on the decay of total phenolic content of Muitle extracts
adjusted to different pH values and stored at 25 C for 20 days.

Fig1

Effect of pH on TPC during storage

The Muitle extracts were adjusted to different pH

values which caused slight variations in the initial
TPC content (Table 1). The extracts at different pH
values (3.5, 5.5 and 7.4) were stored at 25 C and
monitored for TPC for 20 days. In order to establish
a comparative basis of the relative TPC degradation of
the extracts, TPC values were normalized by plotting
the ratio of TPC at time = t and t = 0 versus
storage time. Fig. 1 shows the degradation profiles
of Muitle extracts obtained at 60 C with storage
time at different pH values. A similar behavior was
found for the extracts obtained at 25 C (data not
shown). The extraction conditions and pH affected
TPC degradation. TPC degradation from lowest to
highest was at pH 3.5: E100 60 < E50 60 < E50 25 < W25
< W60 < PG5 60 < PG5 25 < G5 25 < G5 60; at pH 5.5:
E50 25 < W60 < PG5 60 < W25 < G5 60 < E50 60 <
PG5 25 < E100 60; and at pH 7.4: E100 60 < E50 25 <
W60 < E50 60 < G5 60 < W25 < PG5 25 < PG5 60 <
G5 25, respectively.
The rate at which TPC-time experimental data
decreased were adjusted to several models (data not
shown), but the model that best fitted the experimental
data (R2 from 0.95 to 0.99) in all cases was a
first-order model (Eq. 1). The experimental data
fitting was made in two consecutive single processes
with different kinetic rates: (1) An initial stage
characterized by a steep gradient induced by the
high initial TPC loss, probably promoted by reactive
oxygen species (ROS), such as light and oxygen, that
react with the phenolic compounds in a relatively
short storage time (from t = 0 to t = t1 , with t1
varying between 31.3 to 80.4 h), and (2) a second
stage occurring at longer storage time (from t = t1 to t

= 480 h), where the TPC-time gradient is drastically

diminished, probably because most of the phenolic
compounds in the extracts have already reacted with
ROS, resulting in a slowing down in the rate of loss. A
representative graph depicting both the steep and the
relatively flat gradient stages for PG5 25 at pH 3.5 is
presented in Fig. 2. The rate constants corresponding
to the steep (k1 ) and relatively flat (k2 ) gradients of
TPC-time decay are given in Table 2. The variability
in the rate constants k1 and k2 for the Muitle extracts
obtained with different extraction conditions and pH
does not allow to establish a generalized behavior in
the degradation pattern of TPC during storage time,
other than the loss of TPC is considerable. Half-life
time (t1/2) was calculated using k1 values, because is
in the first gradient when a greater loss in TPC was
found.
0.12

0.10

Log (C0/C)

3.3

0.08

0.06

0.04

0.02

100

200

300

400

500

Time (h)

Fig. 2. Total phenolic content first order decay kinetics

for extract PG525 at pH 3.5: (), experimental data;
(....), fitted data for the steep gradient (k1 ); and (),
fitted data for the relatively flat gradient (k2 ).

Fig.2.

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E. Garca-Marquez et al./ Revista Mexicana de Ingeniera Qumica Vol. 11, No. 1 (2012) 1-10
Table 2. Rate constants corresponding to the steep (k1 ) and relatively flat (k2 )
gradients of TPC-time decay of Muitle extracts when fitting the experimental data to a
first-order kinetic model, and t1/2 values for the steep gradient.
pH

3.5 k1 (h1 )

t1/2 (days)

5.5 k1 (h1 )

t1/2 (days)

7.4 k1 (h1 )

t1/2 (days)

W25
W60
E100 60
E50 25
E50 60
PG5 25
PG5 60
G5 25
G5 60

7.23E-04
1.29E-03
2.71E-04
7.48E-04
3.78E-04
4.98E-04
8.77E-04
1.12E-03
1.04E-03

39.94
22.39
106.57
38.61
76.41
57.99
32.93
25.79
27.77

1.20E-03
2.16E-03
3.66E-03
4.77E-03
1.67E-03
4.29E-03
1.55E-04
7.38E-04
8.46E-04

24.07
13.37
7.89
6.05
17.29
6.73
186.33
39.13
34.14

5.48E-04
4.71E-04
4.49E-04
5.04E-04
7.08E-04
5.06E-04
9.44E-04
6.56E-04
6.98E-04

52.70
61.32
64.32
57.30
40.79
57.08
30.59
44.03
41.38

pH

3.5 k2 (h1 )

R2

5.5 k2 (h1 )

R2

7.4 k2 (h1 )

R2

W25
W60
E100 60
E50 25
E50 60
PG5 25
PG5 60
G5 25
G5 60

6.20E-05
2.10E-05
2.90E-05
7.20E-05
4.70E-05
8.90E-05
6.60E-05
6.50E-05
1.03E-04

0.96
0.98
0.96
0.97
0.97
0.99
0.96
0.94
0.98

4.88E-04
2.07E-04
4.77E-04
1.96E-04
5.57E-04
3.09E-04
1.55E-04
8.80E-05
1.01E-04

0.97
0.97
0.97
0.98
0.96
0.98
0.99
0.98
0.98

1.10E-04
1.02E-04
4.10E-05
7.40E-06
6.50E-05
7.04E-05
1.18E-04
1.72E-04
9.20E-05

0.99
0.99
0.97
0.96
0.98
0.95
0.97
0.98
0.94

Table 2 shows the t1/2 for the extracts at different

pH values. Since t1/2 is a function of the initial TPC
value, the TPC at t1/2 may be higher for a given
extract despite having a shorter t1/2 . This effect can be
clearly discerned from our experimental data. While
the highest half-life time (186.33 days) was displayed
by PG5 60 at pH 5.5, followed by E100 60 (106.57 days)
and E50 60 (76.41 days), the latter two at pH 3.5, their
TPC concentrations at t1/2 were of 424.81, 304.30,
and 484.90 mgGAE kg1 , respectively; whereas, the
lowest half-life times were exhibited by E50 25 (6.05
days), PG5 25 (6.73 days) and E100 60 (7.89 days), all
at pH 5.5, and their TPC concentrations at t1/2 were of
488.25, 509.07, and 364.72 mgGAE kg1 , respectively.

3.4

Effect of extraction solvent on initial

antioxidant activity

It has been reported that solvent clearly influences the

antioxidant capacity assay, but not all in the same way.
The two most used assays for determining antioxidant
activity of phenolic compounds are ABTS and DPPH.
In the case of ABTS, the more polar the solvent is,
the greater the ABTS value (Perez-Jimenez and SauraCalixto, 2006), while in the case of DPPH, the use of
6

solvents with high polarity, seem to give low values

for the extent of reduction (Molyneux, 2004).
Antioxidant activity was expressed as mM of
Trolox equivalents (TEAC) as a more meaningful
and descriptive expression than assays that express
antioxidant activity as the percentage decrease in
absorbance. Solvents used for polyphenol extraction
had significant effects on ABTS, where the blue
color due to the formation of ABTS free radicals
(ABTS+ ) is sensitive to the presence of antioxidants.
Discoloration following sample addition indicates that
ABTS radicals were quenched or reduced by the
antioxidants. The order from higher to lower initial
antioxidant activity determined by ABTS in the fresh
Muitle extracts at pH 7.4 was: W60 PG5 25, G5 60,
PG5 60, G5 25, E50 25 W25 > E50 60 > E100 60 (Table
3).
In the case of DPPH assay, which is a very fast
method to evaluate the AA, it is possible to determine
the antiradical power of an antioxidant by measuring
the decrease in absorbance of an alcoholic solution
of DPPH at 515 nm in the presence of a hydrogen
donating antioxidant. Resulting from a color change
from purple to yellow the absorbance decreased when
the DPPH was scavenged by an antioxidant through

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E. Garca-Marquez et al./ Revista Mexicana de Ingeniera Qumica Vol. 11, No. 1 (2012) 1-10
donation of hydrogen to form a stable DPPH molecule.
In the radical form this molecule had an absorbance
at 515 nm which disappeared after acceptance of
an electron or hydrogen radical from an antioxidant
compound to become a stable diamagnetic molecule
(Juntachote and Berghofer, 2005; Turkmen et al.,
2006). The scavenging activity in fresh Muitle extracts
from higher to lower was as follows: G5 60 W60
PG5 25, E50 25, E50 60 > PG5 60, G5 25,W25 > E100 60
(Table 3).
The differences in AA thrown by the ABTS
and DPPH assays may be due to the nature of the
solvent and the radicals, as the type of solvent and
polarity may affect the single electron transfer and the
hydrogen atom transfer, which are the main aspects
in the measurements of AA. Also the presence of
non-antioxidant compounds in extracts could affect the
results (Perez-Jimenez and Saura-Calixto, 2006).
In practical terms, our results indicate that
the Muitle extracts contain diverse phenolic
compounds whose extraction is directly related to
the compatibility of the compounds with the solvent
system, and the antioxidant activity is a result of the
reaction of the sum of the individual components of
the substrate with the free radical molecule used for
the assay, be it ABTS or DPPH. Thus, on the end,
AA will depend on the type of phenolic compounds
extracted by a given extraction system and on the
nature of the scavenging molecule (Molyneux, 2004).

3.5

Effect of extraction temperature on

initial antioxidant activity

The extraction temperature had a significant effect on

AA, determined by ABTS and DPPH, when extraction
was performed with W and E50 . With W, an increase
in extraction temperature produced an increase in AA,
but the opposite effect occurred with E50 . Extraction
temperature variation did not affect significantly AA
with the rest of the solvents, excepting G5 25, which
displayed an increase in AA (determined by DPPH)
as temperature increased (Table 3). These changes
could be due to the equilibrium principle, in which
higher temperatures could increase the extraction rate
and reach maximum phenolic compounds content
recovery and therefore higher antioxidant compounds
content. However, elevated temperatures may not
be suitable for all kinds of phenolic compounds.

Liyana-Pathirana and Shahidi (2005) reported that

only samples with higher proportions of thermally
stable polyphenols are more appropriate to extract
under elevated temperatures. Thus, from our results it
may be inferred that most of the phenolic compounds
extracted from J. spicigera are thermo-stable.

3.6

Effect of extract pH on antioxidant

activity during storage

Changing the pH of the fresh Muitle extracts from

7.4 to 3.5 and 5.5 affected differently the initial AA,
depending on the solvent system and on the assay
used to determine AA (Table 3). Because of the
lowest TPC yield displayed by E100 25, this extract
either was not considered in AA assays. With ABTS,
lowest initial AA occurred with E100 60 regardless of
the pH of the extract, whereas at pH 5.5 highest initial
AA corresponded to PG5 25, and at pH 3.5 to PG5 60,
respectively. With DPPH, also lowest AA happened
with E100 60 regardless of the pH of the extract, at pH
5.5 highest initial AA corresponded to G5 , and at pH
3.5 to G5 25 and E50 60, respectively. The results could
be attributed to the different conformations adopted
by the different phenolic compounds as result of the
different degree of dissociation of OH groups induced
by the changes in pH, affecting the number of active
ionized groups partaking in the reaction with the free
radical molecules of ABTS+ and DPPH+ , and causing
changes in AA (Akrem et al., 2007; Juntachote and
Berghofer, 2005).
Table 4 shows the loss in AA exhibited by the
Muitle extracts after 20 days of storage. With the
ABTS assay, the highest loss was displayed by E100 60
at all pHs tested. Non-significant lowest AA loss was
exhibited at pH 7.4 by W25 and G5 60, at pH 5.5 by
W25, G5 60, and E50 60, and at pH 3.5 by E50 25, E50 60,
PG5 25 and G5 60. With the DPPH assay the lowest AA
loss occurred for the ethanolic extracts (E100 and E50 ),
while the highest loss happened for W60, followed by
W25, all these results regardless of pH value. The
differences in the results obtained using the ABTS and
DPPH assays are most likely due to the compatibility
of the extracts with free radicals of different chemical
nature. Both methods are complementary and allow
for a more thorough understanding of the factors
affecting the stability and functionality of the Muitle
extracts.

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E. Garca-Marquez et al./ Revista Mexicana de Ingeniera Qumica Vol. 11, No. 1 (2012) 1-10
Table 3. Initial antioxidant activity in Muitle extracts determined by ABTS and DPPH assays.
Muitle
extract
code

ABTS pH7.4
(mM TEAC)

ABTS pH5.5
(mM TEAC)

ABTS pH3.5
(mM TEAC)

DPPH pH7.4
(mM TEAC)

DPPH pH5.5
(mM TEAC)

DPPH pH3.5
(mM TEAC)

25
60

6.290.12 c
6.740.10 d

5.800.13 c
6.340.16 e

5.760.28 b,c
6.280.17 c,d

4.450.06 b
5.850.18 c,d

5.720.05 d,e
5.480.05 c,d

4.820.13 c,d
4.240.04 b

E100

25
60

ND
4.330.04 a

ND
3.010.15 a

ND
2.770.19 a

ND
3.520.30 a

ND
2.900.07 a

ND
3.530.11 a

E50

25
60

6.420.06 c,d
5.950.13 b

4.830.10 b
6.100.11 c,d,e

5.320.64 b
6.190.15 b,c,d

5.630.15 c
5.520.08 c

3.230.09 b
5.880.07 e

5.030.08 d,e
5.460.10 g

PG5

25
60

6.610.18 c,d
6.540.12 c,d

7.350.19 f
6.340.18 d,e

5.740.16 b,c
7.130.13 e

5.740.29 c
5.560.22 c

6.350.13 f
5.430.25 c

5.200.16 e,f
4.340.03 b

G5

25
60

6.460.15 c,d
6.550.11 c,d

5.880.12 c,d
6.240.16 d,e

6.520.13 c,d
6.260.49 c,d

4.930.10 b
6.240.22 d

6.620.05 g
6.710.11 g

5.510.05 g
4.580.03 c

Different lowercase letters in the same column represent data significantly different (P 0.05); ND = not determined.

Table 4. Decay in antioxidant activity in Muitle extracts at different pH values after 20 days of storage.
Muitle
extract
code

ABTS pH7.4
(% )

ABTS pH5.5
(% )

ABTS pH3.5
(% )

DPPH pH7.4
(% )

DPPH pH5.5
(% )

DPPH pH3.5
(% )

25
60

42.751.90 a
50.441.79 c

42.882.06 a,b
59.992.66 c

57.224.18 b,c
60.651.55 c,d

68.312.56 e
93.160.35 f

66.971.60 e
80.240.34 f

69.602.89 e
82.690.51 f

E100

25
60

ND
87.531.20 e

ND
87.693.22 d

ND
93.483.17 e

ND
23.498.22 a

ND
16.683.14 a

ND
23.752.07 a

E50

25
60

54.511.40 d
49.731.86 c

44.411.55 b
41.813.22 a,b

51.986.03 a,b
50.221.86 a

33.879.61 c,d
21.455.10 a

21.353.68 b
16.490.91 a

28.022.29 b,c
22.141.56 a

PG5

25
60

46.422.45 b
48.921.90 b,c

58.502.92 c
61.341.76 c

51.372.03 a
64.643.42 d

32.293.64 b,c,d
39.315.76 d

22.341.85 b
31.212.96 c

28.343.02 c
33.422.12 d

G5

25
60

46.881.42 b
42.612.21 a

59.571.32 c
40.162.83 a

65.581.03 d
48.266.07 a

29.052.05 a,b,c
24.167.19 a,b

36.621.89 d
24.220.72 b

37.020.74 d
24.115.17 a,b

Different lowercase letters in the same column represent data significantly different (P 0.05); ND = not determined.

Conclusions
The solvent and temperature extraction conditions
significantly affected the initial total phenolic content
and the antioxidant activity of the Muitle extracts.
Adjusting the pH also had a significant effect
on the total phenolic contents and antioxidant
activity of the extracts with storage time. The
extracts obtained using solvents with higher polarity
displayed more effective radical-scavenging activity
than those obtained using less polar solvents.
These results indicate that the selective extraction
from botanicals sources, by appropriate solvents
and suitable methods, is important for obtaining
bioactive compounds with high phenolic contents and
8

antioxidant activities. Thus, this work establishes

the importance of employing adequate solvent
and temperature extraction conditions for obtaining
extracts from Justicia spicigera with high initial total
phenolic contents and antioxidant activities, and for
minimizing their degradation with storage time at
specific pHs, and contributes for a better performance
of these extracts in practical applications.

Acknowledgement
The authors wish to thank the Consejo Nacional de
Ciencia y Tecnologa (CONACyT) of Mexico for
partially financing this project through grant U-81157Z.

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E. Garca-Marquez et al./ Revista Mexicana de Ingeniera Qumica Vol. 11, No. 1 (2012) 1-10

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