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Revista Mexicana de Ciencias Farmacuticas

ISSN: 1870-0195
rmcf@afmac.org.mx
Asociacin Farmacutica Mexicana, A.C.
Mxico

Mrquez-Flores, Yazmn K.; Montellano-Rosales, Hortensia; Campos Aldrete, Ma. Elena; Melndez-
Camargo, Ma. Estela
Anti-inflammatory activity of aqueous and methanolic extracts of Oenothera rosea L' Hr. ex Ait in the
rat
Revista Mexicana de Ciencias Farmacuticas, vol. 40, nm. 3, julio-septiembre, 2009, pp. 11-16
Asociacin Farmacutica Mexicana, A.C.
Distrito Federal, Mxico

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Anti-inflammatory activity of aqueous and methanolic


extracts of Oenothera rosea L Hr. ex Ait in the rat
Actividad antiinflamatoria de los extractos acuoso y metanlico
de Oenothera rosea L Hr. ex Ait en la rata

Yazmn K. Mrquez-Floresa, Hortensia Montellano-Rosalesb,


Ma. Elena Campos Aldretec, Ma. Estela Melndez-Camargoa

Departamentos de Farmaciaa, Morfologab y Qumica Orgnicac,


Escuela Nacional de Ciencias Biolgicas del Instituto Politcnico Nacional

Abstract
Oenothera rosea L Hr. ex Ait. (Onagraceae) is commonly known as hierba del golpe. It is used in Mexican Folk Medicine to
treat inflammatory, renal and bacterial diseases. The aim of this study was to evaluate the anti-inflammatory activity of aque-
ous and methanolic extracts of this plant. Aqueous extract at a dose of 500 mg/kg body weight (b.wt.) and methanolic extract
(100 mg/kg b.wt.) of Oenothera rosea were evaluated for anti-inflammatory activity using the cotton pellet-induced granu-
loma formation model in rat and histological techniques. Both extracts produced a significant decrease of the inflammatory
process in relation with the control groups (p<0.05). The anti-inflammatory effect of methanolic extract was similar to the ef-
fect of indomethacin. This data was supported by the histological results. No extracts produced gastrointestinal damage. The
LD50 of aqueous and methanol extracts, was higher than 40 and 8 g/kg, respectively. These results suggest that both extracts
of Oenothera rosea induced anti-inflammatory activity and it was considered not toxic.

Resumen
Oenothera rosea L Hr. ex Ait. (Onagraceae) es comnmente conocida como hierba del golpe y utilizada en la medicina Tra-
dicional Mexicana para el tratamiento de padecimientos inflamatorios, renales y bacterianos. El objetivo de este estudio fue
evaluar la actividad antiinflamatoria de los extractos acuoso y metanlico de esta planta. Los extractos acuoso (500 mg/kg de
peso corporal (p.c.)) y metanlico (100 mg/kg p.c.) de Oenothera rosea fueron evaluados mediante el modelo del granuloma
en ratas y tcnicas histolgicas. Ambos extractos indujeron una disminucin significativa del proceso inflamatorio en relacin
con los grupos testigo (p<0.05). El efecto antiinflamatorio del extracto metanlico fue similar al efecto de indometacina. Este
dato fue corroborado por los resultados histolgicos. Ningn extracto produjo dao gastrointestinal. La DL50 de los extractos
acuoso y metanlico fue mayor de 40 y 8 g/kg, respectivamente. Estos resultados sugieren que ambos extractos de Oenothe-
ra rosea produjeron actividad antiinflamatoria y fueron considerados no txicos.

Keywords: Oenothera rosea, Onagraceae, anti-inflammatory, Palabras clave: Oenothera rosea, Onagraceae, antiinflamato-
cotton pellet granuloma, indomethacin rio, modelo del granuloma, indometacina

Correspondencia: Telfono: 5729-6300 ext.: 62341 y 52380


Autor responsable: Ma. Estela Melndez Camargo Fax: 5729-6300 ext.: 62341
Departamento de Farmacia, Escuela Nacional de Ciencias Bio- e-mail: mcamargo@ipn.mx, emelendezc@hotmail.com
lgicas del Instituto Politcnico Nacional.
Prolongacin de Carpio y Plan de Ayala, Col. Santo Toms, Fecha de recepcin: 20 de agosto de 2009
11340 Mxico, D.F. Fecha de recepcin de modificaciones: 5 de octubre de 2009
Fecha de aceptacin: 6 de octubre de 2009

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Volumen 40 Nmero 3 Julio - Septiembre 2009

Introduction ing a brown syrup extract weighing 18.5g (yield: 11.56%). A


preliminary test for metabolites qualitative detection of both
The use of plants with therapeutic effects is a common practice extracts were made7.
among the Mexican population. There are a great number of
species with anti-inflammatory effects, among them is Oenothera Animals
rosea LHr. ex Ait (syn. Hartmannia rosea G. Don, Oenothera Adult female Wistar rats and male NIH mice were used for this
purpurea Lam., Oenothera rubra Cav., Oenthera virgata Ruz study. They were housed and maintained in the animal house
y Pavn, Hartmania virgata Spach). It is commonly known at room temperature (22-24C) and 50-55% relative humidity,
as hierba del golpe, and the whole plant possesses medicinal with day/night cycles of 12 x 12h. They were fed with standard
properties useful in the treatment of skin diseases1, renal and rodent diet and water ad libitum. Care and handling of the ani-
inflammatory diseases2,3,4, hepatic pain3, liver and skin prob- mals were in agreement with internationally accepted procedures
lems5, as well as anti-diarrheic effect 2,5. In Folk Medicine the and approved by our institutional committee following the rec-
whole plant is often used as an infusion for internal inflamma- ommendation included in the Mexican Technical Specifications
tion or as a cataplasm in topic swellings. for the Production, Care, and Use of Laboratory Animals8.

In spite of the traditional use of this plant, only a few detailed Anti-inflammatory activity
chemical and pharmacological studies that could support the Cotton pellet-induced granuloma formation in rats
anti-inflammatory activity have been reported. Meckes6 made a A sterilized cotton pellet granuloma weighing 50 5 mg was
preliminary screening of the effect produced by methanolic ex- introduced subcutaneously, under sterile conditions into axilla,
tract of Oenothera rosea on carrageenan-induced rat paw edema, in an anaesthetized rat9, 10, 11. The test groups of animals were
finding a higher anti-inflammatory effect of this extract during treated orally with 250 or 500 mg/kg of the aqueous extract and
the 7 hours after the treatment, that is why these authors pro- with 100 mg/kg of the methanolic extract, daily for 7 days. Ani-
posed this plant as a good candidate to be submitted for further mals, in the control and reference groups, received 5% sodium
investigation. bicarbonate solution, peanut oil and indomethacin (5 mg/kg in
5% sodium bicarbonate solution).
Due to its popular use in several diseases, in this study, the an-
ti-inflammatory activity of aqueous and methanolic extracts of The animals were sacrificed on the eighth day. The pellets sur-
Oenothera rosea was evaluated. Besides the phytochemical and rounded by granuloma tissue were dissected out carefully and
histological analysis and the acute toxicity were determined. recorded for wet weight, then, the pellets were dried at 60C
to a constant weight. The difference between the final weight
of the pellet after drying and its initial weight was taken as the
Materials and methods final granuloma weight.

Plant material Mean weight, of the granuloma tissue formed in each group,
Oenothera rosea LHr. ex Ait, hierba del golpe (Onagraceae) was obtained and the results were expressed in wet, dry and fi-
was collected in August 2004 (Mexico City) and botanically au- nal granuloma weight.
thenticated by Biologist Laura Doval Ugalde. The specimen was
compared with a voucher specimen deposited in the herbarium Histological analysis
of the National School of Biological Sciences, IPN (M. J. Daz After dissection of granuloma tissue, a sample of tissue in touch
No. 164). The whole plant was shade-dried and powdered. with the granuloma, was fixed in 10% formaldehyde, during 48
hours, and then included in paraffin.
Preparation of the aqueous and methanolic extracts
During the experiments, the aqueous extract was prepared as Later cuts of a 6 m thickness of were carried out, which were
an infusion every day (250 mg and 500 mg/mL water), just as mounted in a microscope slide and dyed with Hematoxilin-
the Mexican population has reported its use. Eosin (topographic technique), toluidine blue for metacromasia,
which allowed the demonstrate the presence of mast cells and
The methanolic extract was prepared with 200g of the dried with Mallory identify collagen fibers12, 13.
powdered material, which was extracted by reflux with 2.0 L
of methanol (analytical grade) for 9 hours. The dissolvent was Gastroduodenal damage study
replaced every three hours. The abdomen of the rats from the cotton pellet-induced gran-
uloma formation were opened. The stomach and duodenum
The extract was filtered and evaporated in a carefully regu- were removed and opened then the tissue was examined for
lated water bath that was maintained at 45C until yield- macroscopic lesions. The results were expressed as presence

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or absence of gastroduodenal irritation (redness and/or pres-
ence of ulcers).

Acute toxicity test


Acute toxicity study was performed in 48 male NIH mice weigh-
ing 28 5g, which were divided into 12 groups. Four animals
were used for complete evaluation in each dosage level. Aque-
ous extract of Oenothera rosea at doses from 5 to 40g/kg were
administrated to the first five groups, methanolic extract at doses
from 1 to 8g/kg were administrated to the other five groups.
Water and peanut oil, respectively, were administrated to both
remaining groups (control groups). The animals had access to
food and water ad libitum. During the first 6 hours, any motor
and respiratory activity change, as well as any different sign from
control group were observed and recorded, after the adminis-
tration of the aqueous and methanolic extracts. For the next 7
Figure 1. Anti-inflammatory effect of Oenothera rosea L Hr. ex
days the number of dead animals was also recorded.
Ait extracts on final granuloma weight. Data represent mean
S.E.M. *Significantly different from control p<0.05.
Statistical analysis
The data are presented as mean S.E.M and the statistical sig-
nificance between groups was analyzed by one-way analysis of
variance (ANOVA) followed by Bonferronis test. The differ- The methanolic extract showed a high anti-inflammatory effect
ences between groups were regarded as significant at p<0.05. in final granuloma weight, being this result very similar to the
one obtained with indomethacin (Figure 1).
Drug and chemicals The anti-inflammatory effect of some species has been attrib-
Indomethacin was purchased from Sigma Chemical Co (St Lou- uted to the presence of several secondary metabolites like the
is, MO). Sodium bicarbonate was Baker and peanut oil (PO), A. flavonoids, which have the aptitude to modify eicosanoid bio-
R. (Mexico). All other reagents were analytical grade. synthesis (antiprostanoid and anti-inflammatory responses) as
well as prevent the plaquet aggregation17, 18. Due to the fact that
this class of metabolites was present in Oenothera rosea extracts,
Results and discussion it is suggested that the same ones should be responsible for the
anti-inflammatory activity of this plant.
The preliminary test showed the presence of alkaloids, reduc-
ing sugars, tannins, coumarins, flavonoids and cardiotonic gly- Other metabolites classes with anti-inflammatory properties
cosides. are the alkaloids and coumarins, which also were present in
Oenothera rosea extracts. Alkaloids as the 5-Hydroxymethyl-1-
In relation to the pharmacological activity, cotton pellet-induced (1,2,3,9-tetrahydro-pyrrolo [2,1-b] quinazolin-1-yl)-heptan-1-
granuloma formation was induced in rat model. This model has one isolated from Sida cordifolia Linn.19, the eleagnine obtained
been widely used to assess the transdutative, exudative and a from the Chrysophyllum albidum seeds20 and some coumarins as
proliferative phase of inflammation, due to the formation of the umbelliprenin, distributed in the plants of Apiaceae family21
granuloma tissue. This is a typical feature of subacute or chronic have been described as anti-inflammatory compounds.
inflammatory reaction. The fluid absorbed by the pellet greatly
influences the wet weight of the granuloma, whereas the dry Another possible mechanism of the anti-inflammatory effect
weight correlates well with the amount of granulomatous tis- of this plant is related to the presence of essential fatty acids in
sue formed14, 15, 16. some species of the Onagraceae family, like Oenothera biennis,
of which, the seeds are used to obtain the oil known as evening
As shown in Figure 1, aqueous extract at doses of 250 and 500 primrose oil, used like anti-inflammatory, due to its high indexes
mg/kg decreased the wet, dry and final granuloma weight. of linolenic acid (71.5 %) and -linolenic acid (7 - 14 %)22,23. This
However, only at dose of 500 mg/kg the difference between test characteristic has caused that the evening primrose oil has being
group and control group was statistically significant (p<0.05) used mainly in circulatory, genital and dermatological disorders
in all cases. and rheumatism24.

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Volumen 40 Nmero 3 Julio - Septiembre 2009

Knorr and Hamburger25, reported that the evening primrose


oil contains lipophilic triterpenoids ethers as 3-O-trans-cafeol,
which present anti-inflammatory properties.

The nonsteroidal antiinflamatory drugs (NSAID), like indo-


methacin, act on the active site of two cyclooxygenase isoforms:
cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), in-
hibiting prostaglandin biosynthesis. The gastrointestinal damage
induced by these drugs is not selective, they inhibit the COX-2
isoform present in damaged or inflamed tissues and the COX-1,
the protective isoform. When COX-1 is inhibited, gastrointes-
tinal toxicity could present 26, 27, 28.
A
The results demonstrated that the groups treated with the aque-
ous and methanolic extracts, as well as the control groups, did
not produce gastroduodenal irritation. Possibly, this specie has
an anti-inflammatory mechanism COX-2 selective29.

The macroscopic analysis shown that the stomach and duodenum


of the groups treated with 5% sodium bicarbonate solution and
peanut oil presented a normal epithelium; in comparison with
the group treated with indomethacin, in which the presence
of irritation was observed along the duodenum and in some of
the cases gastrointestinal sores were present. In all the groups
treated with the aqueous and methanolic extracts of Oenothera
B rosea, there were no signs of irritation or ulceration in the gas-
troduodenal tract.

Also, group treated with indomethacin, a very thin capsule was


observed and in other occasions it was strongly adhered to the
granuloma, unlike the groups treated with the aqueous and
methanolic extracts in which the thickness of the capsule was
bigger and an increase in the exudation of liquid was observed.
In the groups treated with 5% sodium bicarbonate solution and
peanut oil, the thickest capsules were observed.

In the group treated with the aqueous extract (500 mg/kg),


methanolic extract (100 mg/kg) and indomethacin (5 mg/kg)
C a decrease in the number of conjunctive tissue dispersed leu-
kocytes was observed in comparison to the inside and adhered
blood vessels ones. This change in the leukocytary infiltration
is a defense mechanism, due to the fact that the leukocytes
consume the aggressor agents, they destroy bacteria and other
microorganisms, however their leucocytary products can also

Figure 2. Tissue in contact with granuloma of a rat treated with


indomethacin (5 mg/kg) (A), aqueous extract of Oenothera rosea
(500 mg/kg) (B), 5% sodium bicarbonate (C), methanolic extract of
Oenothera rosea (100 mg/kg) (D). Cells presence: a) neutrophil, b) blood
vessel, c) lymphocyte, d) mast cells with degranulation activity, e) mast
D cell without degranulation activity. Hematoxiline-Eosine technique 40X
(A), 100X (B). Toluidine blue technique 40X (C), 100X (D).

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induce tissue damage and increase the inflammation period10. In 7. Domnguez X.A. 1978. Mtodos de Investigacin Fitoqumi-
the groups treated with 5% sodium bicarbonate and aqueous ex- ca. 2 ed. Limusa, Mxico, pp. 39-44.
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phase, were observed, possibly these treatments are favoring the Pesca y Alimentacin. Norma Oficial Mexicana NOM-062
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tor and vasoactive intestinal peptide from this kind of cells that cuidado y uso de los animales de laboratorio.
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with indomethacin, the anti-inflammatory reference drug and D., Cardona C. P., Villareal M. M. E., Melndez C. M. E.
methanolic extract, decrease the degranulation activity, which 2003. Estudio del efecto antiinflamatorio de Sedum praeal-
allows to suppose that this ones are producing a cellular level tum en la rata. Revista Mexicana de Ciencias Farmacuticas,
recovery, diminishing the inflammatory process. 34(1):26-31.
10. Mrquez F. Y. K., Alvear R. S., Montellano R. H., Meln-
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