DrugRes/2015-01-0941/24.3.2015/MPS
Original Article
Evaluation of Anti-inflammatory Activity of Seeds of Phalaris canariensis
Authors
D. Madrigales-Ahuatzi1, 2, R. M. Perez-Gutierrez2
Affiliations
1
Key words ▶ Phalaris canariensis ● ▶ cytokine ● ▶ antiinflammatory ●
Abstract
Departamento de Alimentos, Escuela Nacional de Ciencias Biologicas-IPN, Carpio S/N, Mexico D.F. Laboratorio de Investigación de Productos Naturales, Escuela Superior de Ingenieria Quimica e Industrias extractivas, Instituto Politecnico Nacional, Nacional S/N, Unidad Profesional Adolfo Lopez Mateos, Mexico D.F.
▼
Chloroform extract (ALC) from the seeds of Phalaris canariensis were assayed for antiinflammatory activity by carrageenan-induced oedema, cotton pellets-induced granuloma, histamineinduced inflammation, croton oil-induced oedema, activity of myeloperoxidase (MPO), adjuvantinduced arthritis, quantification of TNFα, IL-1β, PGE2 and LTB4 and nitric oxide (NO) assay. ALC exhibited significant anti-inflammatory activity in different chemically-induced edemas in a dose dependent manner. In the chronic model cotton pellets-induced granuloma showed decreased
Introduction received 17.01.2015 accepted 23.02.2015 Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1548764 Published online: 2015 Drug Res © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence R. M. Perez-Gutierrez Laboratorio de Investigación de Productos Naturales Escuela Superior de Ingenieria Quimica e Industrias extractivas, Instituto Politecnico Nacional, Av Instituto Politécnico Nacional S/N, Unidad Profesional Adolfo Lopez Mateos cp 07708 Mexico D.F. Tel.: + 52/55/57296 000 Ext.: 55142 [email protected]
▼
Chronic inflammation increases the risk of numerous pathological conditions, including cardiovascular and bowel diseases, cancer, diabetes, rheumatoid arthritis, and neurodegenerative disorders. Thus, limiting the frequency and extent of inflammation represents an effective strategy for the prevention and treatment of some diseases. However, the adverse effects of some nonsteroidal anti-inflammatory drugs has stimulated interest in identifying natural products for the prevention and treatment of inflammatory disorders [1]. The canary seed or alpiste, Phalaris canariensis L. is a member of a family of grasses (Graminaceae), and it is used in folk medicine in the form of tea as a coadjuvant in the treatment of hypertension, diabetes mellitus, anti-inflammatory and hypercholesterolernia [2]. There is one study related to the hypotensive effect of P. canariensis seed infusion in normotensive rats [3]; Canary seed peptides showed inhibition of dipeptidyl peptidase IV and angiotensin-converting enzyme activities [4]. In previous studies, effect of alpiste seeds was studied on diabetes rats induced streptozo-
formation of granuloma tissue. Also caused inhibition of ear inflammation edema and influx of polymorphonuclear cells, as evidence by a decrease in ear thickness and reduced myeloperoxidase activity and inhibit mediators of inflammation as TNFα, IL-1β, PGE2 and LTB4. When RAW 264.7 macrophages were treated with ALC together with LPS a significant inhibition of NO production was detected. These data provide evidence for antiinflamatory effect of P. canariensis by mechanisms that involve a reduced neutrophil influx and decreased production of inflammatory cytokines.
tocin, have shown potential antiobesity and hypoglycemic effect [5]. No studies found in the literature on the chemical composition of canary seed. Because the seeds are commonly used as anti-inflammatory the aim of the present study was to establish anti-inflammatory activity of the seeds of Phalaris canariensis attributed to this plant.
Materials and Methods
▼
Plant material
Seeds of P. canariensis were collected during march 2014 in Morelos State, Mexico. The seeds were identified by Biol. Edith Villafranca, A voucher specimen (No. 8054) has been deposited in the Herbarium of the National School of Biological Sciences, for further reference.
Preparation of plant extracts
A total of 1 000 g of the seeds of P. canariensis were dried and powdered in a mechanical grinder. The grinded material was extracted with 5 l of hexane (ALH), chloroform (ALC) and methanol (ALM) consecutively using a soxhlet assembly
Madrigales-Ahuatzi D, Perez-Gutierrez RM. Phalaris canariensis as Antiinflammatory … Drug Res
Downloaded by: Rutgers University. Copyrighted material.
2
DrugRes/2015-01-0941/24.3.2015/MPS
Original Article
Animals
Male Wistar rats weighing 150–200 g were acclimated for 1 week under 12 h light and 12 h dark cycle at room temperature of 22 ± 1 °C. Chow diet and water were provided ad libitum. Experiments reported in this study were carried following the guidelines stated in Principles of Laboratory Animal Care (National Institute of Health publication (NIH) 85-23, revised 1985 and the Mexican Official Normativity (NOM-062-Z001999). All animals procedures were performed in accordance with the recommendations for the care and use of laboratory animals (756/lab/ENCB).
Carrageenan-induced rat paw oedema
Acute inflammation was produced by the subplantar administration of 0.1 ml of 1 % carrageenan in normal saline in the right paw of the rats. The different groups were treated with ALH, ALC and ALM (50, 100, 200 mg/kg, p.o.), indomethacin (10 mg/kg, p.o.) and control vehicle were administered orally. The paw volume was measured at 0 and 3 h after carrageenan injection using plethysmometer (Plethysmometer 7 150, UGO Basile, Italy). The animals were pretreated with the extracts 1 h before the administration of carrageenan [7].
Cotton pellets-induced granuloma
After shaving, the rats were anaesthetized and 10 mg of sterile cotton pellets were inserted, one in each axilla. The extracts (50, 100, 200 mg/kg, p.o.) and indomethacin (10 mg/kg, p.o.) and control vehicle were administered orally for 7 consecutive days from the day of cotton pellet implantation. The animals were anaesthetized on the eighth day and cotton pellets were removed surgically and made free from extraneous tissues. The pellets were incubated at 37 °C for 24 h and dried at 60 °C to constant weight. Increment in the dry weight of the pellets was taken as measure of granuloma formation [8].
Induction of acute inflammation in rat hind paws by histamine
The anti-inflammatory activity of the extract was measured with phlogistic agents (viz. histamine, 5-HT) which act as mediator of inflammation. Acute inflammation in the hind paws was induced by the subcutaneous injection of 0.05 ml solution of histamine (1 %) into the right hind paws of the rats. The left hind paws without injection were used as control. The volumes (ml) of both hind paws of rat were measured using a plethysmometer at 1 h before induction and 0.5, 1, 2, 3, 4, 6 h after induction of the inflammation. Extracts (100, 50, 25, 12.5 mg/kg) or vehicle were intraperitoneally administrated 10 min prior to histamine injection [9].
Croton oil induced ear edema in mouse
The edema was induced by application of 20 µl of croton oil (200 µg) diluted in a solution of acetone/water (7:3) to the inner surface of the mouse’s left ear. The right ear received only the vehicle (20 µl). Immediately after injecting the phlogistic agent, in the groups of treated animals we applied 20 µl of the total extract (1.25, 2.5, 5.0, 7.5 mg) to the left ear. In the control group, 20 µl of the vehicle was applied to the left ear. After 6 h, the ani-
mals were killed, and the ears were sectioned in discs 6.0 mm in diameter and weighed (mg) in an analytical balance [10].
Activity of myeloperoxidase (MPO)
The MPO activity was evaluated in the supernatant of homogenates of the ear sections (controls and those treated with crude extract, 5.0 mg; or indomethacin, 1.0 mg), according to the technique described by Bradley el al., [11].
Adjuvant-induced developing arthritis in rat
Adjuvant arthritis was induced in rat by the subplantar injection of 0.02 ml freshly prepared suspension (5.0 mg/ml) of steam killed Mycobacterium tuberculosis (Difco, USA) prepared in liquid paraffin in the left hind foot pad [12]. The volume of the injected and uninjected paws were quantitated on day 23 after the adjuvant injection.
Quantification of TNFα, IL-1β, PGE2 and LTB4 in serum samples of arthritic rat
Samples of the serum obtained on day 14 from different groups of animals were prepared for the analysis of cytokine mediators [13]. TNFα, IL-1β, PGE2 and LTB4 were estimated using commercially available kits based on sandwich and competitive ELlSA technique (R&D Systems, MN, USA) according to the manufacturer’s instructions.
Nitric oxide (NO) assay
NO production was assayed by the measurement of nitrite, a stable NO oxidation product, from RAW 264.7 macrophage cells. RAW 264.7 cells were plated in 24-well culture plates at 1 × 105 cells/well and incubated for 24 h in a humidified 5 % CO2 incubator at 37 °C. Cells were pretreated with various concentrations of extract for 2 h and stimulated by 1 µg/ml of LPS (lipopolysaccharide); for 18 h. LPS-stimulated nitrite-production from RAW 254.7 cells was measured by the Griess reation [14]. Briefly, 100 µl of each supernatant was mixed with 100 µl of Griess reagent and the absorbance of the mixture was determined at 540 nm. 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) was used as positive control.
Statistical analysis
All data are expressed as mean ± SEM. The level of statistical significance was determined by analysis of variance followed by Duncan’s new multiple range test.
Results
▼
The hexane, chloroform and methanol extracts of P. canariensis seed were evaluated for anti-inflammatory activity in experimental animal models. The chloroform extract (ALC) exhibited significant (p
Original Article
Evaluation of Anti-inflammatory Activity of Seeds of Phalaris canariensis
Authors
D. Madrigales-Ahuatzi1, 2, R. M. Perez-Gutierrez2
Affiliations
1
Key words ▶ Phalaris canariensis ● ▶ cytokine ● ▶ antiinflammatory ●
Abstract
Departamento de Alimentos, Escuela Nacional de Ciencias Biologicas-IPN, Carpio S/N, Mexico D.F. Laboratorio de Investigación de Productos Naturales, Escuela Superior de Ingenieria Quimica e Industrias extractivas, Instituto Politecnico Nacional, Nacional S/N, Unidad Profesional Adolfo Lopez Mateos, Mexico D.F.
▼
Chloroform extract (ALC) from the seeds of Phalaris canariensis were assayed for antiinflammatory activity by carrageenan-induced oedema, cotton pellets-induced granuloma, histamineinduced inflammation, croton oil-induced oedema, activity of myeloperoxidase (MPO), adjuvantinduced arthritis, quantification of TNFα, IL-1β, PGE2 and LTB4 and nitric oxide (NO) assay. ALC exhibited significant anti-inflammatory activity in different chemically-induced edemas in a dose dependent manner. In the chronic model cotton pellets-induced granuloma showed decreased
Introduction received 17.01.2015 accepted 23.02.2015 Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1548764 Published online: 2015 Drug Res © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence R. M. Perez-Gutierrez Laboratorio de Investigación de Productos Naturales Escuela Superior de Ingenieria Quimica e Industrias extractivas, Instituto Politecnico Nacional, Av Instituto Politécnico Nacional S/N, Unidad Profesional Adolfo Lopez Mateos cp 07708 Mexico D.F. Tel.: + 52/55/57296 000 Ext.: 55142 [email protected]
▼
Chronic inflammation increases the risk of numerous pathological conditions, including cardiovascular and bowel diseases, cancer, diabetes, rheumatoid arthritis, and neurodegenerative disorders. Thus, limiting the frequency and extent of inflammation represents an effective strategy for the prevention and treatment of some diseases. However, the adverse effects of some nonsteroidal anti-inflammatory drugs has stimulated interest in identifying natural products for the prevention and treatment of inflammatory disorders [1]. The canary seed or alpiste, Phalaris canariensis L. is a member of a family of grasses (Graminaceae), and it is used in folk medicine in the form of tea as a coadjuvant in the treatment of hypertension, diabetes mellitus, anti-inflammatory and hypercholesterolernia [2]. There is one study related to the hypotensive effect of P. canariensis seed infusion in normotensive rats [3]; Canary seed peptides showed inhibition of dipeptidyl peptidase IV and angiotensin-converting enzyme activities [4]. In previous studies, effect of alpiste seeds was studied on diabetes rats induced streptozo-
formation of granuloma tissue. Also caused inhibition of ear inflammation edema and influx of polymorphonuclear cells, as evidence by a decrease in ear thickness and reduced myeloperoxidase activity and inhibit mediators of inflammation as TNFα, IL-1β, PGE2 and LTB4. When RAW 264.7 macrophages were treated with ALC together with LPS a significant inhibition of NO production was detected. These data provide evidence for antiinflamatory effect of P. canariensis by mechanisms that involve a reduced neutrophil influx and decreased production of inflammatory cytokines.
tocin, have shown potential antiobesity and hypoglycemic effect [5]. No studies found in the literature on the chemical composition of canary seed. Because the seeds are commonly used as anti-inflammatory the aim of the present study was to establish anti-inflammatory activity of the seeds of Phalaris canariensis attributed to this plant.
Materials and Methods
▼
Plant material
Seeds of P. canariensis were collected during march 2014 in Morelos State, Mexico. The seeds were identified by Biol. Edith Villafranca, A voucher specimen (No. 8054) has been deposited in the Herbarium of the National School of Biological Sciences, for further reference.
Preparation of plant extracts
A total of 1 000 g of the seeds of P. canariensis were dried and powdered in a mechanical grinder. The grinded material was extracted with 5 l of hexane (ALH), chloroform (ALC) and methanol (ALM) consecutively using a soxhlet assembly
Madrigales-Ahuatzi D, Perez-Gutierrez RM. Phalaris canariensis as Antiinflammatory … Drug Res
Downloaded by: Rutgers University. Copyrighted material.
2
DrugRes/2015-01-0941/24.3.2015/MPS
Original Article
Animals
Male Wistar rats weighing 150–200 g were acclimated for 1 week under 12 h light and 12 h dark cycle at room temperature of 22 ± 1 °C. Chow diet and water were provided ad libitum. Experiments reported in this study were carried following the guidelines stated in Principles of Laboratory Animal Care (National Institute of Health publication (NIH) 85-23, revised 1985 and the Mexican Official Normativity (NOM-062-Z001999). All animals procedures were performed in accordance with the recommendations for the care and use of laboratory animals (756/lab/ENCB).
Carrageenan-induced rat paw oedema
Acute inflammation was produced by the subplantar administration of 0.1 ml of 1 % carrageenan in normal saline in the right paw of the rats. The different groups were treated with ALH, ALC and ALM (50, 100, 200 mg/kg, p.o.), indomethacin (10 mg/kg, p.o.) and control vehicle were administered orally. The paw volume was measured at 0 and 3 h after carrageenan injection using plethysmometer (Plethysmometer 7 150, UGO Basile, Italy). The animals were pretreated with the extracts 1 h before the administration of carrageenan [7].
Cotton pellets-induced granuloma
After shaving, the rats were anaesthetized and 10 mg of sterile cotton pellets were inserted, one in each axilla. The extracts (50, 100, 200 mg/kg, p.o.) and indomethacin (10 mg/kg, p.o.) and control vehicle were administered orally for 7 consecutive days from the day of cotton pellet implantation. The animals were anaesthetized on the eighth day and cotton pellets were removed surgically and made free from extraneous tissues. The pellets were incubated at 37 °C for 24 h and dried at 60 °C to constant weight. Increment in the dry weight of the pellets was taken as measure of granuloma formation [8].
Induction of acute inflammation in rat hind paws by histamine
The anti-inflammatory activity of the extract was measured with phlogistic agents (viz. histamine, 5-HT) which act as mediator of inflammation. Acute inflammation in the hind paws was induced by the subcutaneous injection of 0.05 ml solution of histamine (1 %) into the right hind paws of the rats. The left hind paws without injection were used as control. The volumes (ml) of both hind paws of rat were measured using a plethysmometer at 1 h before induction and 0.5, 1, 2, 3, 4, 6 h after induction of the inflammation. Extracts (100, 50, 25, 12.5 mg/kg) or vehicle were intraperitoneally administrated 10 min prior to histamine injection [9].
Croton oil induced ear edema in mouse
The edema was induced by application of 20 µl of croton oil (200 µg) diluted in a solution of acetone/water (7:3) to the inner surface of the mouse’s left ear. The right ear received only the vehicle (20 µl). Immediately after injecting the phlogistic agent, in the groups of treated animals we applied 20 µl of the total extract (1.25, 2.5, 5.0, 7.5 mg) to the left ear. In the control group, 20 µl of the vehicle was applied to the left ear. After 6 h, the ani-
mals were killed, and the ears were sectioned in discs 6.0 mm in diameter and weighed (mg) in an analytical balance [10].
Activity of myeloperoxidase (MPO)
The MPO activity was evaluated in the supernatant of homogenates of the ear sections (controls and those treated with crude extract, 5.0 mg; or indomethacin, 1.0 mg), according to the technique described by Bradley el al., [11].
Adjuvant-induced developing arthritis in rat
Adjuvant arthritis was induced in rat by the subplantar injection of 0.02 ml freshly prepared suspension (5.0 mg/ml) of steam killed Mycobacterium tuberculosis (Difco, USA) prepared in liquid paraffin in the left hind foot pad [12]. The volume of the injected and uninjected paws were quantitated on day 23 after the adjuvant injection.
Quantification of TNFα, IL-1β, PGE2 and LTB4 in serum samples of arthritic rat
Samples of the serum obtained on day 14 from different groups of animals were prepared for the analysis of cytokine mediators [13]. TNFα, IL-1β, PGE2 and LTB4 were estimated using commercially available kits based on sandwich and competitive ELlSA technique (R&D Systems, MN, USA) according to the manufacturer’s instructions.
Nitric oxide (NO) assay
NO production was assayed by the measurement of nitrite, a stable NO oxidation product, from RAW 264.7 macrophage cells. RAW 264.7 cells were plated in 24-well culture plates at 1 × 105 cells/well and incubated for 24 h in a humidified 5 % CO2 incubator at 37 °C. Cells were pretreated with various concentrations of extract for 2 h and stimulated by 1 µg/ml of LPS (lipopolysaccharide); for 18 h. LPS-stimulated nitrite-production from RAW 254.7 cells was measured by the Griess reation [14]. Briefly, 100 µl of each supernatant was mixed with 100 µl of Griess reagent and the absorbance of the mixture was determined at 540 nm. 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) was used as positive control.
Statistical analysis
All data are expressed as mean ± SEM. The level of statistical significance was determined by analysis of variance followed by Duncan’s new multiple range test.
Results
▼
The hexane, chloroform and methanol extracts of P. canariensis seed were evaluated for anti-inflammatory activity in experimental animal models. The chloroform extract (ALC) exhibited significant (p
