Food Science and Food Biotechnology in Developing Countries

FF-27

Fruits of capuln (Prunus serotina) as a potential source of bioactive compounds

Villa-De la Torre, F.(1)*, Luna-Vzquez, F.(1), Ibarra-Alvarado, C.(1), Rivero-Cruz, F.(2), Rojas-Molina,
J.(3), Rojas-Molina, A. (1)

(1)Facultad de Qumica, Universidad Autnoma de Quertaro, Centro Universitario, s/n. Col. Las
campanas, Quertaro, 76010 Qro. Mxico. Tel. (442) 1921307, Fax (442) 1921304, e-mail:
qfbfabiolavilla@gmail.com
(2) Facultad de Qumica, Universidad Nacional Autnoma de Mxico. Ciudad Universitaria, s/n. Mxico
D.F., 04510, Mxico
(3) Facultad de Ciencias Naturales, Universidad Autnoma de Quertaro, campus Juriquilla. Avenida
de las Ciencias s/n. Col. Juriquilla., Quertaro 76230 Qro. Mxico.

Abstract

The fruits and leaves of Prunus serotina are used in traditional medicine for the treatment of diarrhea,
cough and hypertension. In the present study we found that the extracts and the essential oil obtained
from the fruits of capuln caused a concentration-dependent relaxation of aorta and trachea. The
dichloromethane extract and the essential oil elicited the highest vasodilatory response, suggesting the
presence of non polar compounds related to the vascular relaxation.

Keywords: Prunus serotina, rat aorta, guinea pig trachea, smooth muscle, vasorelaxant

Introduction
The World Health Organization (WHO) estimates that 80% of the world population uses plants to treat
diverse diseases. Mexico, with its floristic diversity, ethnic variety, long history of human civilization,
and centuries old record of the use of plants provides a rich and valuable reservoir of new bioactive
substances with nourishing and medicinal properties. A recent documental and ethnomedical survey of
medicinal plants grown in Mxico and widely used by the Mexican population revealed that the bark,
leaves, inflorescences and fruits of Prunus serotina, commonly known in Mexico as capuln, are
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highly valued to treat cardiac, pulmonary and intestinal ailments . Particularly, the fruits are used, alone
1
or prepared in syrups and liquors, for the treatment of cough and diarrhea .
This species has been the subject of few chemical studies. Kaempferol, quercetin and isorhamnetin
6,7,8
glycosides, ursolic acid derivatives and prunasin have been detected in the leaves . Cyanogenic
glycosides such as amygdalin and prunasin have been isolated from the seeds and anthocyanidins
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have been detected in the fruit skin . The only previous study reported in the bibliography, concerning
the pharmacological effects of this plant indicated that the methanolic extract prepared from the bark
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exhibits anti-proliferative activity in cancer cells of human colon .
In our continuing efforts to study plants used in Mexico for the treatment of gastrointestinal and
cardiovascular ailments, P. serotina was selected as a potential source of bioactive compounds.
Firstly, the ability of the aqueous extract prepared from the leaves and branches to relax the isolated
rat aorta was investigated. In that study, we found that the extract elicited a significant endothelium
independent vasorelaxation. Furthermore, bioactivity-guided fractionation of the methanol-
dichloromethane 1:1 extract of the leaves resulted in the isolation of hyperin and prunasin, which
induced a concentration-dependent relaxation of vascular smooth muscle. Both compounds were
approximately ten fold less potent than quercetin, which was employed as a positive control. However,
hyperin induced a maximum vasodilatory effect that was higher than that of quercetin.
Considering the traditional uses of the fruits of P. serotina, which suggested the presence of smooth
muscle relaxant agents, in the present study, we investigated the effect of the essential oil and the
aqueous and organic (ethanolic and dichloromethane) extracts obtained from the fruits of P. serotina
on rat aortic and guinea pig tracheal smooth muscle. The essential oil was analyzed by gas
chromatography coupled to mass spectrometry. In addition, considering that many plants contain a

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variety of polyphenols, and current evidence strongly suggest that these compounds contribute
significantly to the treatment and prevention of cardiovascular diseases due to their antioxidant and
vasorelaxing properties, in the present work the total phenolic and flavonoid contents of the fruits were
determined.

Materials and Methods

Plant material. Capulin fruits were collected in the locality of Huejotzingo in the State of Puebla,
Mexico. Samples were authenticated by Dr. Robert Bye from the Institute of Biology, National
Autonomous University of Mexico.

Extraction. Seeds were removed from the fruits and the pulp was stored at -70 C for later use. The
aqueous extract was prepared by decoction, while the ethanolic and dichloromethane extracts were
prepared by maceration at room temperature. The essential oil was obtained by hydrodistillation.

Experimental design. The extracts and essential oil were tested on aortic and tracheal smooth
muscle at the following non-cumulative concentrations: 1, 3.16, 10, 31.6, 100, 316 and 1000 g/ml.

Pharmacological evaluation. The relaxant effects of the extracts and the essential oil were evaluated
in rat aortic segments (Fisher male rats, 250 g) and guinea pig tracheal rings (male guinea pigs, 350-
500 g). Segments of both tissues were mounted in organ chambers filled with Krebs-Henseleit solution
at 37 C, gassed with 95% O2 and 5% CO2. Changes induced by the extracts on the isometric tension
2,3
were measured by Grass FT03 force-displacement transducers attached to a Grass 7D polygraph .

Pharmacological data analysis. The effects of the extracts and the essential oil were calculated as a
percentage of the initial contraction achieved with phenylephrine in the case of aorta, and carbachol in
the case of trachea. Results of the experiments were expressed as the mean S.D. from n = 4 to 6
experiments. Concentration-response curves for the extracts were plotted and fitted to the sigmoidal
concentration-response equation using the data analysis and graphics program Prism 4.0.

Total phenolic and flavonoid contents. 2 g of lyophilized pulp of the fruits of capulin were extracted
with 200 mL of distilled water at 100 C for 2 min. The extract was then centrifuged at 10000g for 5
min, and the resulting supernatant was used for determination of total phenolic and flavonoid contents.
The total phenolic content of the extract was determined according to the Folin-Ciocalteu colorimetric
method. Results are expressed as milligrams of gallic acid equivalents per gram of extract (GAE). The
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total flavonoid concentration was measured using a colorimetric assay developed by Zhishen et al. ,
diluted samples and a standard solution of epicatechin were analized. Absorbances were determined
against a prepared blank at 510 nm. The total flavonoid contents are expressed as milligrams of
epicatechin equivalents per gram of the extract (ECE). All data are reported as mean S.D. of
triplicate analyses.

Gas chromatography. This study was performed employing a gas chromatograph connected to a
mass spectrometer (GC-MS-TOF) LECO Pegasus 4D. A 180 m internal diameter and 10 m length
capillary column was used, employing helium as gas carrier.

Results and discussion

Pharmacological evaluation. All the tested extracts and the essential oil prepared from the fruits of
capuln caused a concentration-dependent relaxation of rat aorta. Figure 1A shows the concentration
response curves for the extracts and the essential oil. It is important to note that in the case of the
ethanolic and dichloromethane extracts, a clear maximum response was not obtained since it was not
possible to test higher extracts concentrations due to solubility problems. At a concentration of 1000
g/ml, which was the highest tested concentration, the dichloromethane extract displayed the highest
maximum vasorelaxant response of more than 60%, followed by the essential oil (E max = 55.8 5.8%)
and the ethanolic extract, whereas the aqueous extract elicited the lowest vasorelaxant effect (E max =
19.79 0.52%). Concerning the results obtained in the guinea pig trachea, the present study

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demonstrates that the extracts and essential oil obtained from the fruits of P. serotina promote tracheal
smooth muscle relaxation. In this model, the essential oil and the dichloromethane extract exhibited the
highest smooth muscle relaxant effect (Figure 1B).
The results derived from the pharmacological evaluation clearly indicated that the fruits of capuln
contain aortic and tracheal smooth muscle relaxant compounds. The tracheal relaxing effect displayed
by the extracts and the essential oil supports the ethnomedical use of the fruits of P. serotina for the
treatment of cough. Additionally, our findings showed that these fruits have alternative potential health
benefits in the therapy of cardiovascular ailments, such as atherosclerotic ischaemic heart disease,
coronary artery disease, and hypertension.

A B

Fig.1. Concentration-response curves for the A) vasodilatory and B) tracheal smooth muscle relaxant
effect elicited by the essential oil and the extracts obtained from the fruits of P. serotina.

Total phenolic and flavonoid contents. Since phenolic compounds are among the most abundant
plant secondary metabolites that have diverse biological activities, including antioxidant and
vasorelaxant effects, the total phenolic and flavonoid contents of the fruits were determined. The total
phenolic and flavonoid contents were 8.18 0.018 GAE and 6.33 0.005 ECE, respectively. These
results indicated that the fruits of capulin possess a significant content of these secondary
metabolites. Previous studies have demonstrated that phenolics can cause endothelium-dependent
4,9
vasodilation, via the release of endothelium-derived vasorelaxing factors . Therefore, this type of
polar compounds could be responsible for the vasodilatory effect elicited by the aqueous and ethanolic
extracts of the fruits. However, it is evident that other kind of non-polar secondary metabolites could
also be responsible for the vasodilatory properties of the essential oil and the dichloromethane extract.

Gas chromatography. Gas chromatography analysis showed that the essential oil obtained from the
fruits of P. serotina contains 39 compounds. The major constituents were 2-chlorociclohexanol
(32.66%), hexanedial (13.21%), butylated hydroxytoluene (10.44%), 2-butenal- 2-etenil (4.94 %) and
1,2- benzene dicarboxylic acid mono-2-ethylhexyl ester (4.69%). It is very likely that the most abundant

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detected compound, which is chlorinated, is not a natural product synthesized by the plant and rather
might be captured by the fruits from the air as a result of environmental pollution.

Conclusions
The results derived from this study provide a scientific basis for the traditional use of the fruits of P.
serotina for the treatment of cough. Our findings also showed that these fruits have alternative potential
health benefits in the therapy of cardiovascular diseases. Further investigations are currently in
progress in order to identify the metabolites that are responsible for the pharmacological activities and
to characterize their mechanisms of action.

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