El ácido p-cumárico (p-CA) es un metabolito ubicuo en plantas, con propiedades antioxidantes, anti-inflamatoria, y anticancerígenas. El presente estudio fue diseñado para evaluar los efectos preventivos de p-CA sobre la relajación dependiente de  endotelio, deteriorada por niveles altos de glucosa (HG) (D-glucosa 25 mM) en aorta torácica aislada de rata. Los anillos aórticos obtenidos  de ratas macho Sprague-Dawley se montaron en un baño de órganos y fueron pre-tratados durante 3 h con D-glucosa 5 mM, HG, y HG más  de p-CA (1, 10 y 100 μM). Después de este período de tiempo se evaluó la relajación dependiente de endotelio mediante la adición  acumulativa de acetilcolina (ACh) en anillos pre-contraídos con fenilefrina (PE) (0,1 μM). p-CA mostró un moderado efecto vasodilatador  dependiente de endotelio (Emax = 29,28  1,89%, N = 6; pD2 = 6,075  0,184, N = 6). Cuando los anillos aórticos se pre-incubaron durante  3 h con HG, la Emax para ACh se redujo drásticamente desde 87,69  2,59% (N = 6) a 40,54  1,78% (N = 6). El efecto negativo de HG se  revirtió parcialmente, de manera dependiente de concentración, en los anillos co-incubados con p-CA tal como lo muestra el valor de Emax  para cada concentración de p-CA: 1 μ M (48,57  1,82%), 10 μM (60,81  1,80%) y 100 μM (64,51  1,80%). La acción de p-CA se asoció  con un cambio significativo en la Emax. No se observó diferencia estadísticamente significativa en el pD2. Nuestros resultados muestran  claramente que p-CA protege la relajación dependiente de endotelio inducida por ACh, la cual es afectada por HG en anillos aislados de  aorta de rata

Araos, Patricio

Guzmán, Leda

Knox, Marcela

Vinet, Raúl

Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas

  
© 2014  
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 13 (3): 232 - 237 
ISSN 0717 7917 
www.blacpma.usach.cl 
 
 
Artículo Original | Original Article 
232 
 
p-Coumaric acid reduces high glucose-mediated impairment of 
endothelium-dependent relaxation in rat aorta 
 [Ácido p-cumárico reduce el deterioro mediado por alta glucosa de la relajación dependiente  
de endotelio en aorta de rata] 
 
Raúl VINET
1,2
, Patricio ARAOS
1
, Juan Carlos GENTINA
3
, Marcela KNOX
4
 & Leda GUZMÁN
4
 
 
1Laboratorio de Farmacología, Facultad de Farmacia, Universidad de Valparaíso, 2360102 Valparaíso, Chile. 
2Centro Regional de Estudios en Alimentos Saludables (CREAS), 2362696 Valparaíso, Chile. 
3Escuela de Ingeniería Bioquímica, Facultad de Ingeniería, Pontificia Universidad Católica de Valparaíso, 2362804 Valparaíso, Chile. 
4Departamento de Bioquímica, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, 2373223 Valparaíso, Chile. 
Contactos | Contacts: Raúl VINET - E-mail address: raul.vinet@uv.cl 
 
Abstract: p-Coumaric acid (p-CA) is an ubiquitous plant metabolite with antioxidant, anti-inflammatory, and anticancer properties. The 
present study was designed to evaluate the preventive effects of p-CA on endothelium-dependent impairment produced by high glucose 
(HG) (D-Glucose 25 mM) in isolated rat thoracic aorta. Aortic rings obtained from male Sprague-Dawley rats were mounted in an organ 
bath and pretreated for 3 h with D-Glucose 5 mM, HG and HG plus p-CA (1, 10 and 100 M). After this period of time endothelium-
dependent relaxation was tested by cumulative addition of acetylcholine (ACh) in pre-contracted rings with phenylephrine (PE) (0.1 μM). p-
CA elicited a moderate endothelium-dependent vasodilatory effect (Emax= 29.28  1.89%, N=6; pD2= 6.075  0.184, N=6). When aortic 
rings were pre-incubated for 3 h with HG, Emax for ACh decreased dramatically from 87.69  2.59% (N=6) to 40.54  1.78% (N=6). The 
negative effect of HG was partially reverted in rings co-incubated with p-CA in a concentration-dependent manner as shown for Emax 
values to each p-CA concentration: 1 M (48.57  1.82%), 10 M (60.81  1.80%) and 100 M (64.51  1.80%). The action of p-CA was 
associated with a significant change in Emax. No statistical difference in pD2 was observed. Our results clearly show that p-CA protect 
ACh-induced endothelial-dependent relaxation affected by HG in isolated rat aortic rings. 
 
Keywords: p-coumaric acid, rat aorta, endothelium, high glucose, acetylcholine  
 
Resumen: El ácido p-cumárico (p-CA) es un metabolito ubicuo en plantas, con propiedades antioxidantes, anti-inflamatoria, y 
anticancerígenas. El presente estudio fue diseñado para evaluar los efectos preventivos de p-CA sobre la relajación dependiente de 
endotelio, deteriorada por niveles altos de glucosa (HG) (D-glucosa 25 mM) en aorta torácica aislada de rata. Los anillos aórticos obtenidos 
de ratas macho Sprague-Dawley se montaron en un baño de órganos y fueron pre-tratados durante 3 h con D-glucosa 5 mM, HG, y HG más 
de p-CA (1, 10 y 100 μM). Después de este período de tiempo se evaluó la relajación dependiente de endotelio mediante la adición 
acumulativa de acetilcolina (ACh) en anillos pre-contraídos con fenilefrina (PE) (0,1 μM). p-CA mostró un moderado efecto vasodilatador 
dependiente de endotelio (Emax = 29,28  1,89%, N = 6; pD2 = 6,075  0,184, N = 6). Cuando los anillos aórticos se pre-incubaron durante 
3 h con HG, la Emax para ACh se redujo drásticamente desde 87,69  2,59% (N = 6) a 40,54  1,78% (N = 6). El efecto negativo de HG se 
revirtió parcialmente, de manera dependiente de concentración, en los anillos co-incubados con p-CA tal como lo muestra el valor de Emax 
para cada concentración de p-CA: 1 μ M (48,57  1,82%), 10 μM (60,81  1,80%) y 100 μM (64,51  1,80%). La acción de p-CA se asoció 
con un cambio significativo en la Emax. No se observó diferencia estadísticamente significativa en el pD2. Nuestros resultados muestran 
claramente que p-CA protege la relajación dependiente de endotelio inducida por ACh, la cual es afectada por HG en anillos aislados de 
aorta de rata. 
 
Palabras Clave: ácido p-cumárico, aorta de rata, endotelio, alta glucosa, acetilcolina. 
 
 
Recibido | Received: September 12, 2013 
Aceptado en versión corregida | Accepted in revised form: September 22, 2013 
Publicado en línea | Published online: May 30, 2014 
Declaración de intereses | Declaration of interests: This work was supported by Grant DI PIA 037.430/12 from DIPUCV, P. Universidad Católica de Valparaíso, Chile, Grant 
DIPUV-57/2011 from DIUV, Universidad de Valparaíso, Chile, and Grant CREAS CONICYT-REGIONAL, GORE Región de Valparaíso, R12C1001, Chile. 
Este artículo puede ser citado como / This article must be cited as: R Vinet, P Araos, JC Gentina, M Knox, L Guzman. 2014. p-Coumaric acid reduces high glucose-mediated 
impairment of endothelium-dependent relaxation in rat aorta. Bol Latinoam Caribe Plant Med Aromat 13(3): 232 – 237.  
Vinet et al. p-Coumaric acid protects endothelium-dependent relaxation  
 
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/233 
 
INTRODUCTION 
Since the early 50’s isolated thoracic aorta have been 
a fecund model to evaluate the bioactivity of many 
compounds, including phytochemicals (Vinet et al., 
2012). After the milestone discovery of the role that 
plays endothelium in the vasodilatation induced by 
acetylcholine (Furchgott and Zawadzki, 1980), 
isolated aorta -specially as rings- have been 
extensively used to assess the activity of natural 
compounds that cause endothelium - dependent 
vasorelaxation (Andriambeloson et al., 1998; Vinet et 
al., 2012).  
 A large number of studies have been 
performed to evaluate whether some phytochemicals 
prevent oxidation process associated with vascular 
dysfunction and progression of atherosclerosis 
(Howard and Kritchevsky, 1997; Traka and Mithen, 
2011) A positive, relationship between antioxidant 
activity and total phenolic content shows that 
phenolic compounds are the dominant antioxidant 
components in medicinal herbs (Cai et al., 2004). In 
relation to cardiovascular diseases, an increasing 
importance has been given to nutrition and 
specifically to plant products in the diet (Howard and 
Kritchevski, 1997; Traka and Mithen, 2011). 
 p-Coumaric acid (p-CA) (3-[4-
hydroxyphenyl]-2-propenoic acid), a phenolic acid, is 
a hydroxyl derivative of cinnamic acid. In plants, p-
CA is an intermediate product of the 
phenylpropanoid pathway, widely distributed in 
fruits, such as apples and pears, and in vegetables and 
plant products, such as beans, tomatoes, potatoes and 
tea. p-CA has been suggested to exhibit antioxidant, 
anti-inflammatory, and anticancer activities (Zang et 
al., 2000; Zhang et al., 2007; Lee et al., 2009; Kilic 
and Yesiloglu, 2013; Kong et al., 2013; Stojkovic et 
al., 2013; Yoon et al., 2013). In this work we 
evaluate the effect of p-CA as a protector against the 
impairment endothelium-dependent relaxation 
induced when aortic rings were pre-incubated with 
high glucose (HG, D-Glucose 25 mM). 
 
MATERIAL AND METHODS 
Animals 
Male Sprague-Dawley rats weighing 240-280 g were 
used. Rats were housed in a standard environmental 
condition. Food and water were freely available. 
Animals were used in accordance with the Guide for 
the Care and Use of Laboratory Animals (NRC, 
2011). 
 
Preparation of rat thoracic aorta rings and 
recording 
Rats were killed by decapitation and the thoracic 
aorta was carefully removed and mounted on a tissue 
chamber as previously described (Vinet et al., 1991). 
Briefly, aorta was carefully dissected, clean of 
connective tissue and divided into three rings 
segments of 5 mm. Caution was taken not to touch 
the endothelial surface to preserve the functional 
endothelium. In some experiments the endothelium 
was mechanically removed. The rings were 
suspended between two L-shaped stainless steel 
hooks and placed in a 30 mL organ chambers 
containing a modified Krebs-Henseleit buffer (KHB) 
with D-Glucose 5 mM, maintained at 37° C and 
oxygenated continuously with a 95% O2 - 5% CO2 
gas mixture. 
 Isometric tensions were measured using a 
Myobath II multi-channel isolated tissue bath system 
combined with Lab-Trax Data Acquisition System 
with a 4-channel amplifier and analyzed using the 
Data-Trax 2 software (World Precision Instruments, 
USA). Rings were allowed to equilibrate in the tissue 
bath for 60 min under an optimal resting tension of 
1.5 g. Mechanical stability of the system was 
accomplished by adding KCl 70 mM, three times. 
Integrity of endothelium was evaluated by testing the 
relaxation induced by ACh (1 μM) in pre-contracted 
rings with phenylephrine (PE) (0.1 μM). Aortic rings 
were repeatedly washed and allowed to re-equilibrate 
for additional 30 min.  
 The first ring was incubated in KHB 
(Control); the second ring was incubated in KHB 
with HG; and the third ring was incubated in KHB 
buffer with HG plus p-CA (1, 10, 100 μM). All rings 
were incubated for 3 h. After this period, rings were 
pre-contracted with phenylephrine (0.1 μM) and once 
a stable contraction was achieved, cumulative 
concentration-response curves to ACh were obtained. 
Maximum relaxation (Emax) and concentration 
producing the half-maximal effect (EC50) were 
determined from each concentration-response curve 
and expressed as pD2 (-log EC50). The relaxation 
from the pre-contracted level to the baseline was 
considered as 100% relaxation. 
 
Statistical analysis 
All statistics were run using GraphPad Prism version 
6. A one-way analysis of variance (ANOVA) with 
Vinet et al. p-Coumaric acid protects endothelium-dependent relaxation  
 
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/234 
 
Newman-Keuls Multiple Comparison Test or a paired 
t-test was used to analyze the data as appropriate. All 
data are expressed as means ± SEM. Differences 
were considered significant at P < 0.05. 
 
RESULTS 
Prior to investigate the effects of p-CA on rat aortic 
rings pre-incubated with HG, we evaluated the direct 
effect of p-CA on aortic rings with (+E) and without 
endothelium (–E) pre-contracted with PE (0.1 μM). 
As shown the concentration-response curve in Figure 
1A, p-CA displays a moderate endothelium-
dependent vasodilatory effect. Emax (%) was 29.28  
1.89 (N=6), and pD2 was 6.075  0.184 (N=6). 
 
 
0 600 1200 1800 2400
0
1
2
3
4
Time (s)
T
e
n
s
io
n
 (
g
)
+ E
- E
p-CA (-log M)
-8    -7    -6    -5    -4
¯        ¯        ¯        ¯        ¯
A
 
 
 
-10 -9 -8 -7 -6 -5
0
25
50
75
100
p-CA (log M)
R
e
la
x
a
ti
o
n
 (
%
)
B
-E
+E
 
 
Figure 1 
Relaxation induced by p-CA on rat aortic rings pre-contracted with PE (0.1 μM). (A) 
Representative record showing an endothelium-dependent relaxation induced by cumulative 
concentration of p-CA. p-CA produced moderated relaxation in aortic rings with endothelium 
(+E). No effect in aortic rings without endothelium (-E) was observed. (B) Concentration-
response curve for p-CA. Results (means + SEM) are expressed as percentage of inhibition of 
contraction induced by PE (0.1 μM). Emax (%) and pD2 were 29.28  1.89 (N = 6) and 6.075  
0.184 (N = 6), respectively. 
 
 
The next experiment was designed to evaluate the 
effect of p-CA as a protector of the impairment of 
endothelium-dependent relaxation induced by HG. 
As shows in Figure 2 and Table 1, when aortic rings 
Vinet et al. p-Coumaric acid protects endothelium-dependent relaxation  
 
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/235 
 
were pre-incubated for 3 h with HG, the maximum 
vasodilatory effect (Emax) of ACh decreased 
dramatically from 87.69  2.59% (N = 6) to 40.54  
1.78% (N = 6). 
 
-10 -9 -8 -7 -6 -5
0
25
50
75
100
ACh (log M)
R
e
la
x
a
ti
o
n
 (
%
)
Control
HG
HG + p-CA (1 µM)
HG + p-CA (10 µM)
HG + p-CA (100 µM)
 
Figure 2 
Relaxation induced by ACh on PE (0.1 M)-contracted rat aortic rings pre-incubated with 
HG and p-CA. Cumulative concentration-response curve for ACh (10
-10
 – 10-5 M) in aortic 
rings pre-incubated for 3 h in the presence of D-Glucose 5 mM (Control), D-Glucose 25 mM 
(HG), and HG plus p-CA (1, 10, 100 M). Results (means + SEM) are expressed as percentage of 
inhibition of contraction induced by PE (0.1 μM). Emax (%) and pD2 are shown in Table 1. 
 
However, the negative effect of HG was partially 
reverted in vessels co-incubated with p-CA: 1 M 
(48.57  1.82%), 10 M (60.81  1.80%) and 100 
M (64.51  1.80%). The protector effect of p-CA on 
HG endothelium-dependent relaxation induced by 
ACh was concentration-dependent. The action of p-
CA was associated with a significant change in 
Emax. No statistical difference in pD2 was observed.  
  
Table 1 
Effect of p-CA in the maximum response (Emax) and pD2 (-log EC50) to the ACh-induced 
endothelium-dependent relaxation in rat aortic rings pre-incubated with HG. 
 
Group Emax (%) pD2 
Control 
HG 
HG + p-CA (1 M) 
HG + p-CA (10 M) 
HG + p-CA (100 M) 
              87.69  2.59 
              40.54  1.78* 
48.57  1.82*# 
60.81  1.80*+ 
64.51  1.80*+ 
7.139  0.078 
7.097  0.114 
7.046  0.096 
7.097  0.076 
7.094  0.072 
*
 P < 0.0001 vs. Control; 
#
 P < 0.05 vs. HG; 
+
 P < 0.0001 vs. HG 
 
DISCUSSION 
Our results clearly show that p-CA protect ACh-
induced endothelial-dependent relaxation affected by 
high glucose (D-glucose 25 nM) in rat aortic rings. 
This effect is in accordance with experimental results 
in bovine aortic endothelial cells under oxidative 
stress induced by high glucose; in these endothelial 
cells p-CA acts as antioxidant preventing lipid 
peroxidation and cell death without affecting the 
production of reactive oxygen species (Lee et al., 
2009). The moderate capacity of p-CA to induce 
endothelium-dependent relaxation, observed in our 
study, may also be explained on the basis of their 
antioxidant activity, protecting nitric oxide (NO) 
from degradation, and thereby increasing their 
bioavailability. 
 Recently, radical scavenging and antioxidant 
capacity of p-CA have been clarified using different 
analytical methodologies (Kilic and Yesiloglu, 2013). 
Evidences from in vitro studies show that p-CA 
effectively scavenge OH in a dose-dependent 
manner, suggesting that p-CA antioxidant properties 
may involve the direct scavenging of ROS (Zang et 
al., 2000). According to an antioxidant mechanism p-
Vinet et al. p-Coumaric acid protects endothelium-dependent relaxation  
 
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/236 
 
CA also protects rat hearts against doxorubicin 
(DOX), an anticancer antibiotic whose therapeutic 
use is limited by its cardiotoxicity, which is 
associated to oxidative stress in the heart (Abdel-
Wahab et al., 2003). Additional evidence supporting 
the free radical scavenging activity of p-CA shows it 
prevents lysosomal dysfunction against cardiac 
damage induced by isoproterenol (Roy et al., 2013). 
 Interestingly, p-CA affects angiogenesis and 
glucose/lipid metabolisms. Thus, p-CA have been 
shown to inhibit the sprouting of endothelial cells in 
rat aortic rings, thereby inhibiting the tube formation 
and migration of endothelial cells; the mechanism of 
action of p-CA could be the downregulation of 
mRNA expression levels of key angiogenic factors, 
indicating that p-CA possesses potential anticancer 
properties due to the inhibition of angiogenesis in 
vivo (Kong et al., 2013). Furthermore, p-CA 
modulates glucose and lipid metabolism via AMPK 
activation in L6 skeletal muscle cells; on this basis 
the authors have proposed that p-CA may overcome 
metabolic disorders (Yoon et al., 2013). We are 
aware that p-CA effects indicated in this paragraph 
require an exposure time of several hours, so that 
does not explain our results that were obtained in a 
time frame of 3 h. 
 
ACKNOWLEDGEMENTS 
This work was supported by Grant DI PIA 
037.430/12 from DIPUCV, P. Universidad Católica 
de Valparaíso, Chile, Grant DIPUV-57/2011 from 
DIUV, Universidad de Valparaíso, Chile, and Grant 
CREAS CONICYT-REGIONAL, GORE Región de 
Valparaíso, R12C1001, Chile. 
 
REFERENCES 
Abdel-Wahab MH, El-Mahdy MA, Abd-Ellah MF, 
 Helal GK, Khalifa F, Hamada FM. 2003. 
 Influence of p-coumaric acid on doxorubicin-
 induced oxidative stress in rat's heart. 
 Pharmacol Res 48: 461 - 465. 
Andriambeloson E, Magnier C, Haan-Archipoff G, 
Lobstein A, Anton R, Beretz A, Stoclet JC, 
Andriantsitohaina R. 1998. Natural dietary 
polyphenolic compounds cause endothelium-
dependent vasorelaxation in rat thoracic 
aorta. J Nut 128: 2324 - 2333. 
Cai Y, Luo Q, Sun M, Corke H. 2004. Antioxidant 
activity and phenolic compounds of 112 
traditional Chinese medicinal plants 
associated with anticancer. Life Sciences 74: 
2157 - 2184. 
Furchgott RF, Zawadzki JV. 1980. The obligatory 
role of endothelial cells in the relaxation of 
arterial smooth muscle by acetylcholine. 
Nature 288: 373 - 376. 
Howard BV, Kritchevsky D. 1997. Phytochemicals 
and cardiovascular disease. A statement for 
healthcare professionals from the American 
Heart Association. Circulation 95: 2591 - 
2593. 
Kilic I, Yesiloglu Y. 2013. Spectroscopic studies on 
the antioxidant activity of p-coumaric acid. 
Spectrochimica acta. Part A, Mol Biomol 
Spectroscopy 115: 719 - 724. 
Kong CS, Jeong CH, Choi JS, Kim KJ, Jeong JW. 
2013. Antiangiogenic effects of p-coumaric 
acid in human endothelial cells. Phytother 
Res 27: 317 - 323. 
Lee SJ, Mun GI, An SM, Boo YC. 2009. Evidence 
for the association of peroxidases with the 
antioxidant effect of p-coumaric acid in 
endothelial cells exposed to high glucose plus 
arachidonic acid. BMB Reports 42: 561 - 
567. 
NRC. 2011. Guide for the Care and Use of 
Laboratory Animals, 8th edn, The National 
Academies Press.Washington DC, USA.  
Roy AJ, Stanely Mainzen Prince P. 2013. Preventive 
effects of p-coumaric acid on lysosomal 
dysfunction and myocardial infarct size in 
experimentally induced myocardial 
infarction. Eur J Pharmacol 699: 33 - 39. 
Stojković D, Petrović J, Soković M, Glamočlija J, 
Kukić-Marković J, Petrović S. 2013. In situ 
antioxidant and antimicrobial activities of 
naturally occurring caffeic acid, p-coumaric 
acid and rutin, using food systems. J Sci 
Food Agric 93: 3205 - 3208. 
Traka MH, Mithen RF. 2011. Plant science and 
human nutrition: challenges in assessing 
health-promoting properties of 
phytochemicals. Plant Cell 23: 2483 - 2497. 
Vinet R, Brieva C, Pinardi G, Penna M. 1991. 
Modulation of alpha-adrenergic-induced 
contractions by endothelium-derived relaxing 
factor in rat aorta. Gen Pharmacol 22: 137 - 
142. 
Vinet R, Knox M, Mascher D, Paredes-Carbajal C, 
Martínez JL. 2012. Isolated aorta model and 
its contribution to phytopharmacology. Bol 
Vinet et al. p-Coumaric acid protects endothelium-dependent relaxation  
 
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/237 
 
Latinoam Caribe Plant Med Aromat 11: 
35 - 45. 
Yoon SA, Kang SI, Shin HS, Kang SW, Kim JH, Ko 
HC, Kim SJ. 2013. p-Coumaric acid 
modulates glucose and lipid metabolism via 
AMP-activated protein kinase in L6 skeletal 
muscle cells. Biochem Biophys Res Comm 
432: 553 - 557. 
Zang LY, Cosma G, Gardner H, Shi X, Castranova 
V, Vallyathan V. 2000. Effect of antioxidant 
protection by p-coumaric acid on low-density 
lipoprotein cholesterol oxidation. Am J 
Physiol - Cell Physiol 279: 954 - 960. 
Zhang Y, Tie X, Bao B, Wu X, Zhang Y. 2007. 
Metabolism of flavone C-glucosides and p-
coumaric acid from antioxidant of bamboo 
leaves (AOB) in rats. Brit J Nut 97: 484 - 
494. 
 
 
 


Ácido p-cumárico reduce el deterioro mediado por alta glucosa de la relajación dependiente de endotelio en aorta de rata

http://www.revistas.usach.cl/ojs/index.php/blacpma/article/download/1509/1396/

Ácido p-cumárico reduce el deterioro mediado por alta glucosa de la relajación dependiente de endotelio en aorta de rata

Abstract

Similar works

Full text

Available Versions

Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas