La actividad antioxidante de exudados resinosos obtenidos desde las especies H. stenophylum y H. sinuatum, fue evaluada a través del ensayo ORAC  (Oxygen Radical Absorbance Capacity) en fase acuosa y en presencia de micelas de Triton X-100, usando como molécula prueba a pirogalol rojo (PGR) y  evaluando su reducción frente a la acción de radicales peróxidos obtenidos desde la termólisis de AAPH. Los resultados muestran que estos extractos  protegen al PGR de la acción de los radicales. Esta protección es reducida drásticamente en presencia de micelas de Tritón X-100. El mismo efecto fue  observado con el flavonoide mayoritario de estos extractos (3-O-metilgalangina). Estos resultados muestran la importancia de considerar el medio de reacción  de compuestos puros y/o extractos al momento de tomar en cuenta su uso como antioxidantes

Campos, Ana María

Chavez, Marjorie

Lissi, Eduardo

Modak, Brenda

Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas

  
© 2012 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 11 (6): 549 - 555 
 ISSN 0717 7917 
www.blacpma.usach.cl 
 
 
Artículo Original | Original Article 
549 
 
 
Antioxidant activity in heterogeneous and homogeneous system of the 
resinous exudates from Heliotropium stenophylum and H. sinuatum and of 
3-O-methylgalangin their main component 
 
 
 [Actividad antioxidante en sistema heterogéneo y homogéneo de los exudados resinosos de Heliotropium 
stenophyllum y H. sinuatum y de 3-O-methylgalangina su componente mayoritario] 
 
 
Ana María CAMPOS*, Eduardo LISSI, Marjorie CHAVEZ & Brenda MODAK 
 
 
Facultad de Química y Biología, Universidad de Santiago de Chile. 
Contactos | Contacts: Ana María CAMPOS - E-mail address: ana.campos@usach.cl 
 
Abstract  
The antioxidant activity of resinous extracts obtained from H. stenophylum and H. sinuatum species, was evaluated through ORAC index (Oxygen Radical 
Absorbance Capacity) in water phase and in presence of Triton X-100 micelles, using as test molecules to pyrogallol red (PGR) and evaluating  their 
reduction by the action of peroxyl radicals obtained from thermolysis of AAPH. The results show that these extracts protect to PGR of the action of the 
radicals. This protection is reduced drastically in the presence of Triton X-100 micelles. The same effect was observed with the main flavonoid of these 
extracts (3-O-methylgalangin). These results show the importance of the media of reaction of pure compounds and/or extracts at the time of to take into 
account their use as antioxidants. 
 
Keywords: antioxidant, flavonoid, ORAC-PGR, resinous exudate, micelles. 
 
Resumen 
La actividad antioxidante de exudados resinosos obtenidos desde las especies H. stenophylum y H. sinuatum, fue evaluada a través del ensayo ORAC 
(Oxygen Radical Absorbance Capacity) en fase acuosa y en presencia de micelas de Triton X-100, usando como molécula prueba a pirogalol rojo (PGR) y 
evaluando su reducción frente a la acción de radicales peróxidos obtenidos desde la termólisis de AAPH. Los resultados muestran que estos extractos 
protegen al PGR de la acción de los radicales. Esta protección es reducida drásticamente en presencia de micelas de Tritón X-100. El mismo efecto fue 
observado con el flavonoide mayoritario de estos extractos (3-O-metilgalangina). Estos resultados muestran la importancia de considerar el medio de reacción 
de compuestos puros y/o extractos al momento de tomar en cuenta su uso como antioxidantes.  
 
Palabras Clave: antioxidante, flavonoide, ORAC-PGR, exudados resinosos, micelas. 
 
 
 
 
 
  
Recibido | Received: August 31, 2012. 
Aceptado en versión corregida | Accepted in revised form: September 15, 2012 
Publicado en línea | Published online: November 30, 2012 
Declaración de intereses | Declaration of interests: This work was supported by FONDECYT Nº 1110295. . 
Este artículo puede ser citado como / This article must be cited as: Ana M. Campos, Eduardo Lissi, Marjorie Chavez, Brenda Modak. 2012. Antioxidant activity in 
heterogeneous and homogeneous system of the resinous exudates from Heliotropium stenophylum and H. sinuatum and of 3-O-methylgalangin their main component. Bol 
Latinoam Caribe Plant Med Aromat 11(6): 549 – 555.  
Campos et al. Effect of media on evaluation of antioxidant activity  
 
Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/550 
 
INTRODUCTION 
Currently, there is an increase in the interest by the use 
of antioxidant natural obtained from plants used in 
popular medicine (Ozsoy et al., 2008; Di Carlo et al., 
1999) and food products, such as soy, tea, coffee and 
red wine, among others (Avello and Suwalsky, 2006). 
The Heliotropium genus, Heliotropiaceae 
family, it is characterized by living in arid areas and be 
exposed to extreme environmental factors. For this, 
their species produce a resinous exudate that cover 
both leaves and stem and it is secreted by special 
glands called trichomes (Johnston, 1928). The resin is 
composed by terpenoids, lipids, waxed and flavonoids, 
that act as a protective barrier against aggression such 
as high temperature, radiation UV, water scarcity, 
herbivores and pathogens (Dell and Comb, 1978). 
Previous studies have shown that the components of 
this resin, have chemical and biological properties, 
such as: antimicrobial, antiviral and anti-fungal, 
increasing the resistance of the plants. (Martínez, 
2005; Modak et al., 2009a, Modak et al., 2009b, 
Modak et al., 2010). Of these properties, the 
antioxidant activity highlights (Modak et al., 2003; 
Modak et al., 2005; Modak et al., 2007; Modak et al., 
2009a; Modak et al., 2009b; Modak et al., 2010). 
In particular, H. stenophylum and H. sinuatum 
species produces a resinous exudate composed by 
flavonoids as main component accompanied of 3 H-
Spiro[1-benzofuran-2,1'-cyclohexane] derivatives in 
smaller amounts (Villarroel and Urzúa 1990; Torres et 
al., 1996). 
Previous studies of antioxidant activity of the 
resinous extracts of these species and of its flavonoids 
pure, using the discoloration of DPPH method in 
homogeneous phase, showed their ability to capture 
the radical free. However, the activity of these extracts 
and flavonoids resulted to be less to the vitamin E and 
Trolox activities (Lissi et al., 1999). Based on these 
results, arose the idea of study the effect of the 
environment in the antioxidant activity of flavonoid 
and of these mixtures. For this, the evaluation of 
antioxidant activity using the ORAC-PGR test 
(Romero et al., 2010) was carried out in homogeneous 
phase and in the presence of Triton X-100 micelles. 
The results are presented in this paper. 
 
MATERIALS AND METHODS 
Plant material 
Heliotropium stenophyllum Hook.& Arn. was 
collected during the flowering season in Los Vilos (IV 
region, Chile, 31º52`S,71º29`W). Heliotropuim 
sinuatum (Miers) was collected during the flowering 
season in the north of Vallenar (III region, Chile, 
29º57`S,71ºW).  Voucher specimens were deposited in 
the Herbarium of the Faculty of Biological Science of 
Catholic University of Chile. (ST2560 Hst; ST2563 
Hsin).  
 
Extraction and isolation of resinous exudate and 
flavonoid 3-O-methylgalangin. 
The resinous exudates were extracted by immersion of 
the fresh plant material in dichloromethane for 60 s at 
room temperature. The extracts were concentrated to a 
sticky residue. 
The extracts were purified by column 
chromatography (silica gel) using a hexane-
ethylacetate step gradient yielding 3-O-methylgalangin 
as major component (Torres et al.,1996). 
 
Antioxidant activity 
Evaluation of antioxidant capacity in phosphate 
buffer (pH 7.4, 75 mM) and in Triton X-100 (50 mM) 
solution 
To evaluate the consumption of PGR (5uM) caused by 
the addition of AAPH (10 mM), the absorbance was 
measured at 540 nm (UV-VIS spectrophotometer 
Shimadzu UV-160) as a function of time at 37° C. 
These measurements were realized in the presence of 
different concentrations of resinous exudates and pure 
compounds (3-O-methylgalangin and Trolox). All 
measurements were performed in triplicate and the 
results were expressed as mean ± standard deviation. 
 
Octanol/water partition 
The partition of 3-O-methylgalangin and resinous 
extracts in octanol/water was evaluated. Aqueous 
solutions (phosphate buffer 75 mM, pH 7.4) of each 
system were divided into two portions. A portion was 
added to decantation funnel and was treated with 
octanol, shaking by 20 minutes. The aqueous phase 
was separated and its antioxidant activity was 
evaluated and compared with the activity of the 
portion not treated with octanol.    
 
RESULTS AND DISCUSSION 
Figure 1 shows the kinetic profiles of the reaction of 
PGR and peroxyl radicals generated by AAPH 
thermolysis in absence and in presence of resinous 
extracts. The presence of the extracts produces a shift 
to the right of the curve, which is interpreted as a 
Campos et al. Effect of media on evaluation of antioxidant activity  
 
Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/551 
 
protection of the PGR against the action of the peroxyl 
radical. To compare the profiles obtained with both 
extracts, note that at equal concentration, the H. 
stenophyllum resin has greater antioxidant activity. 
These results coincide with those obtained using the 
radical DPPH (Lissi et al., 1999). 
 
 
 
0 500 1000 1500 2000 2500
0,0
0,2
0,4
0,6
0,8
1,0
A
/A
°
time /s
 
Figure 1 
Kinetic profiles of PGR consumption in presence of (▲) H. sinuatum 0.05 mg/mL; () H. stenophyllum 0.05 
mg/mL and (■) Control experiment (without extract) in homogeneous media.  
PGR 5 µM, AAPH 10 mM, 37° C. 
 
 
 
 
Figure 2 shows the bleaching of PGR elicited 
by peroxyl radicals in absence and in presence of 
Triton X-100 micelles. These data show that PGR 
consumption is nearly independent of the surfactant 
addition.  These results agree with those reported by 
Romero (2010), and indicate that, despite PGR 
incorporation to the micelles, its rate of consumption 
is not affected by the surfactant. 
In Figure 3, the kinetic profiles of 3-O - 
methylgalangin in absence and presence of micelles 
are given. From this figure it can be concluded that the 
protection provides by this flavonoid, decreases in 
micellar media. This reduced protection can be 
explained in terms of flavonoid incorporation into the 
micelles. This incorporation is favoured for its high 
hydrophobicity. This is demonstrated by the results 
obtained through the evaluation of antioxidant activity 
measured before and after treatment with octanol 
(Figure 4).  
 
Campos et al. Effect of media on evaluation of antioxidant activity  
 
Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/552 
 
0 500 1000 1500
0,0
0,2
0,4
0,6
0,8
1,0
A
/A
0
time /s
 
Figure 2 
Kinetic profiles of PGR consumption in presence (□) of Triton X-100 micelles (50 mM) and in absence (■) of 
micelles.  PGR 5 µM, AAPH 10 mM, 37° C. 
 
 
0 500 1000 1500 2000
0,00
0,25
0,50
0,75
1,00
A
/A
°
time /s
 
Figure 3 
Effect of 3-O-methylgalangin on the consumption of PGR induced by AAPH derived peroxyl radicals in the 
absence (▲) and presence of Triton X-100 micelles (●) to 1.5 mM and 1.8 mM respectively, and control 
experiment (■).  PGR 5 µM, AAPH 10 mM, a 37° C. 
 
 
 
Campos et al. Effect of media on evaluation of antioxidant activity  
 
Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/553 
 
0 500 1000 1500 2000 2500
0,0
0,2
0,4
0,6
0,8
1,0
A
/A
0
time /s
 
Figure 4 
Kinetic profiles of PGR consumption in presence of 3-O-methylgalangin (1.8 mM) before (●) and after (▲) 
of treatment with octanol. Control experiment (■). 
 
Figure 4 shows that the extraction with octanol 
drastically reduced the protection afforded to PGR, 
implying that 3-O-methylgalangin is retained almost 
completely in the organic phase. These results 
supports then those obtained by Romero (2010). This 
author have shown that the presence of micelles 
decreased significantly the protection of PGR afforded 
by lipophilic antioxidants (-carotene, octyl gallate), 
while no effect of micelles was observed for 
hydrophilic antioxidants such as Trolox, caffeic acid, 
gallic acid, and ascorbic acid. 
Table 1 shows the ORAC indexes obtained by 
both resins and its major flavonoid. These indexes 
were obtained in homogenous phase (ORAC-PGR) 
and in heterogeneous media. Also is included the ratio 
between both index (ORAC-PGR/ ORAC-PGRMIC). 
Values for reference antioxidants have been 
incorporated for comparison.  
 
Table 1 
Indexes ORAC obtained by the resinous extract and 3-O-methylgalangin in absence (ORAC-PGR) and 
presence of Triton X-100 micelles (ORAC-PGRMIC).* 
 ORAC-PGR ± s.d ORAC-PGRMIC  ± s.d 
ORAC-PGR/ ORAC-
PGRMIC     ± s.d 
3-O-
methylgalangin 
0.11 ± 0.03 0.05 ± 0.01 2.2 ± 0.9 
H. sinuatum 4.0 ± 0.1 0.37 ± 0.01 10.8 ± 0.1 
H. stenophyllum       11.1± 0.1 1.4 ± 0.1 7.9  ± 0.1 
Octyl gallate** 9.8 ± 0.3 1.6 ± 0.2 6.1 ± 0.9 
Methyl gallate**       12.0 ± 0.4 7.0 ± 0.6 1.7 ± 0.2 
Red wine**       36.0 ± 2.0 24 ± 3 1.5 ± 0.3 
*Values of the pure compounds are given relatives to Trolox efficiency. Values of the exudates are given in 
Trolox miliequivalents per gram of dry material. Red wine value is given in Trolox miliequivalents per litter. 
** Values obtained from Romero (2010). 
 
 
 
Campos et al. Effect of media on evaluation of antioxidant activity  
 
Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/554 
 
 
The results of the table 1 show that ORAC 
values of H.sinuatum are approximately one-third of 
those obtained with H. stenophyllum in both media. 
Furthermore, significant differences in the ORAC 
indexes are observed, in heterogeneous media for both 
extracts. In fact, the protection of PGR is reduced 91% 
and 88% with H. sinuatum and H. stenophyllum, 
respectively. This large reduction of protection implies 
that the active components of both extracts are located 
preferably in the interior of Triton micelles. 
Furthermore, a comparison of the ratio for 3-O-
methylgalangin (2.2) and those of the extracts allows 
concluding that this flavonoid marginally contributes 
to the antioxidant activity of the extracts. In fact, a 
simple calculation based on the efficiency of 3-O-
methylgalangin (Table 1) and its amount present in the 
exudates indicates that this compound contributes with 
ca. 1 % to the total antioxidant capacity of the 
exudates. 
Romero (2010) has proposed that the ratio 
ORAC-PGR/ORAC-PGRMIC represents the degree of 
hydrophobicity of the antioxidants in study. For 
example, the ratio ORAC-PGR/ORAC-PGRMIC of 
octyl gallate (6.1 ± 0.9) in relation to methyl gallate 
(1.7 ± 0.2) is greater than 3. This means that a greater 
degree of hydrophobicity, allows a greater addition to 
the micelle, which is reflected in a reduced protection 
of PGR consumption.  
If ORAC-PGR values of the extracts 
considered in the present study are compared with 
other mixtures, such as red wine, it can be concluded 
that the ratio of ORAC indexes between homogeneous 
and heterogeneous media are greater for the resinous 
extracts. This implies that the active compounds of red 
wine are less hydrophobic than the active components 
present in resinous extracts. 
3-O-methylgalangin, the major flavonoid in 
the resinous extracts, presents a low reactivity towards 
peroxyl radicals. This conclusion is similar to that 
reached regarding its reactivity towards DPPH radicals 
(Lissi et al., 1999) and can be explained in terms of 
the lack of HO groups in the B ring and the presence 
of only meta groups in the A ring. The ratio of ORAC 
values obtained for 3-O-methylgalangin (2.2 ± 0.9) is 
close to that reported for propyl gallate (1.9) by 
Romero (2010). This implies a rather high 
hydrophobicity compatible with the almost total 
extraction by octanol (Figure 4).  
 
 
 
CONCLUSION 
The extracts obtained from resinous exudates are able 
to trap peroxyl radicals. The antioxidants present in 
these extracts are of high hydrophobicity, as assessed 
by the ratio between ORAC-PGR values determined in 
buffer and in the presence of Triton X-100 micelles. 3-
O-methylgalangin, the most abundant flavonoid in the 
extracts presents low reactivity and only marginally 
contributes to the total antioxidant capacity of the 
exudates. The results obtained emphasize the 
importance that has, in microheterogenous systems, 
the distribution of the antioxidants between the 
different microenvironments. 
 
ACKNOWLEDGMENTS 
This work was supported by FONDECYT Nº 
1110295. 
 
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Actividad antioxidante en sistema heterogéneo y homogéneo de los exudados resinosos de Heliotropium  stenophyllum y H. sinuatum y de 3-O-methylgalangina su componente mayoritario

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Actividad antioxidante en sistema heterogéneo y homogéneo de los exudados resinosos de Heliotropium stenophyllum y H. sinuatum y de 3-O-methylgalangina su componente mayoritario

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Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas