Abstract
Context: Cyclolepis genistoides D. Don (Asteraceae) is an Argentinean endemic shrub, known by the vernacular name “palo azul” or “matorro negro”. It is widely used in folk medicine as a diuretic, an antirheumatic, and an antispasmodic agent.
Objective: The aim of this work was to describe the activity of two isolated compounds of C. genistoides, oleanolic acid (1) and deacylcynaropicrin (2), using the carrageenan-induced paw edema method in mice.
Materials and methods: Aerial parts of C. genistoides were dried, powdered, and extracted with petroleum ether, ethyl acetate, dichloromethane, and methanol for 48 h. The fractions obtained from the ethyl acetate extract yielded oleanolic acid, while deacylcynaropicrin was obtained according to Abdei-Mogib et al. Structures were elucidated by 1H-NMR and 13C-NMR. The products were administered intraperitoneally at doses of 40, 75, and 100 mg/kg.
Results and discussion: Compound 1 exhibited significant activity during the first 7 h of the inflammatory phase (at 1, 3, 5, and 7 h), exercising its inhibitory action on inflammation mediated by histamine, prostaglandins, serotonin, and kinins, while compound 2 showed a significant inhibition at 3 and 5 h contributing to this effect, acting in the intermediate phase.
Conclusions: According to the results of this work, the intraperitoneal administration of oleanolic acid and deacylcynaropicrin isolated from the aerial parts of C. genistoides produced a significant inhibition of carrageenan-induced inflammation at doses of 75 and 100 mg/kg. These results give support to the use of this plant as an anti-inflammatory in traditional medicine.
Introduction
Cyclolepis genistoides D. Don (Asteraceae) is an endemic shrub distributed in different Argentinean regions, known by the vernacular name “palo azul” or “matorro negro”. It is widely used in folk medicine as a diuretic, an antirheumatic, and an antispasmodic agent. Regarding its chemical composition, oleanolic acid and deacylcynaropicrin have been previously isolated from this species (CitationDe Heluani et al., 1997). In this work, we have investigated the anti-inflammatory properties of both compounds in the mouse paw edema model. Paw edema is characterized by an early phase caused by the release of histamine, 5-hydroxytryptamine, and bradykinin, followed by a sustained prostaglandin release (CitationDi Rosa & Willoughby, 1971). The aim of this work was to describe the activity of the isolated compounds of C. genistoides in the mouse paw edema test.
Material and methods
Plant material
C. genistoides was collected in the surroundings of Sierra de las Quijadas, San Luis province, Argentina, during April 2003. A voucher specimen (L. A. Del Vitto et al. # 8950) is preserved at the Herbarium of UNSL (National University of San Luis); it was identified by the collector.
Extraction and separation
Aerial parts (200 g) of C. genistoides were dried at 40°C, powdered, and sequentially extracted several times with petroleum ether, ethyl acetate, dichloromethane, and methanol for 48 h, and evaporated under vacuum using a rotatory evaporator (Büchi, Flawil, Switzerland). The plant material was filtered through Whatman No. 4 paper and evaporated to dryness under vacuum. The sample was then subjected to a pre-purification procedure by thin layer chromatography (TLC): silica gel (Merck 60G); mobile phase: petroleum ether and ethyl acetate (9.5:0.5); ultraviolet (UV) detection (254 nm); and was revealed with sulfuric acid/acetic acid/water (80:10:10) and heat.
The fractions obtained from the ethyl acetate extract were re-chromatographed on a Sephadex LH-20 column using methanol as an eluent agent, and yielded a white powder (85 mg, 1), while the other crystalline product was obtained according to CitationAbdei-Mogib et al. (1991) (25 mg, 2). The structures of 1 and 2 were elucidated by 1H-nuclear magnetic resonance (NMR) and 13C-NMR and by comparison with spectroscopic data in the literature.
Spectral data
Compound 1 1H-NMR (CDCl3, 300 MHz): δ 0.70, 0.71, 0.85, 0.88, 0.92, 0.93, 1.03 (s, 3H, -CH3 7′), 2.80 (dd, J = 14.0, 4.0 Hz, 1H, H-18), 3.14 (dd, J = 11.5, 4.5 Hz, 1H, H-3a), 5.20 (t, J = 3.5 Hz, 1H, H-12); 13C-NMR (CDCl3, 75 MHz): δ 180 (C-28), 124 (C-12), 139 (C-13), 76.5 (C-3), 54.5 (C-17), 52 (C-8), 40.2 (C-10), 46.5 (C-5), 42 (C-19), 41 (C-9), 40.5 (C-4), 39 (C-14), 38.8 (C-1), 38.5 (C-21), 32.4 (C-22), 31.5 (C-18), 30.7 (C-20), 27.7 (C-15), 27.5 (C-29), 27.1 (C-2), 25.9 (C-27), 24 (C-7), 23.6 (C-30), 23.4 (C-16), 22.9 (C-11), 18.3 (C-6), 17 (C-23), 16 (C-26), 15.5 (C-25), 15.3 (C-24).
Compound 2 1H-NMR (CDCl3, 300 MHz): δ 1.73 (ddd, J = 13.1, 11.0, 7.6 Hz, 1H, H-2b), 2.23 (ddd, J = 13.2, 7.3, 7.3 Hz, 1H, H-2a), 2.29 (dd, J = 14.0, 3.9 Hz, 1H, H-9a), 2.70 (dd, J = 14.0, 5.1 Hz, 1H, H-9b), 2.78 (m, 1H, H-7), 2.81 (m, 1H, H-5), 2.97 (ddd, J = 10.8, 8.3, 8.3 Hz, 1H, H-1), 3.96 (m, 1H, H-8), 4.15 (dd, J = 10.5, 9.1 Hz, 1H, H-6), 4.55 (m, 1H, H-3), 4.98 (s, 1H, H-14b), 5.13 (s, 1H, H-14a), 5.34 (dd, J = 1.7, 1.6 Hz, 1H, H-15b), 5.48 (dd, J = 1.7, 1.6 Hz, 1H, H-15a), 6.14 (dd, J = 3.1, 0.7 Hz, 1H, H-13b), 6.26 (dd, J = 3.4, 0.7 Hz, 1H, H-13a);13C-NMR (CDCl3, 75 MHz): δ 39.4 (t, C-2), 41.4 (t, C-9), 45.3 (d, C-1), 51.1 (d, C-7), 51.5 (d, C-5), 72.1 (d, C-8), 73.8 (d, C-3), 78.7 (d, C-6), 113.2 (t, C-15), 117.1 (t, C-14), 123.1 (t, C-13), 138.1 (s, C-11), 142.8 (s, C-10), 152, 6 (d, C-4), 169.7 (s, C-12).
Chemicals
All reagents were analytical grade; type IV carrageenan and phenylbutazone were purchased from Sigma Chemical Co. (USA).
Pharmacological studies
Animals
Rockland mice of both sexes, weighing 25–30 g, were used in this study. The animals were provided with food and water ad libitum. The mice were kept at a constant temperature of 22 ± 1°C and 60% humidity. All animals were acclimated for at least 1 day before the experiments. Experiments were carried out using three doses of product (40, 75, and 100 mg/kg), which correspond with the concentrations of infusions more frequently prepared in popular medicine.
The care and the handling of the rats were in accordance with the internationally accepted standard guidelines for animal use, and the protocol was approved by our institutional committee on the care of animals following the norms established by disposition no. 6344/96 of the National Administration of Medicines, Foods and Medical Technology (ANMAT, Argentina).
Anti-inflammatory test
Three groups of six animals each of both sexes were used to study carrageenan-induced mouse paw edema. The intraperitoneal administration of the reference drug was made 1 h before the subcutaneous injection of 0.05 mL of carrageenan (3.5% w/v in normal saline) (CitationSugishita et al., 1981) in the left hind paw of the mouse. The paw volume was measured by a plethysmometer (Ugo Basile; model 7150) before and at intervals of 1, 3, 5, and 7 h after carrageenan injection. The reference drug, phenylbutazone, was tested at 80 mg/kg i.p. Compounds 1 and 2 were tested at three different doses (40, 75, and 100 mg/kg). Edema volume was expressed as the difference found between the left hind paw and the control right paw, in each animal. The edema inhibition percentage was calculated for each group, in relation to the control group.
Statistical analysis
The numerical data are expressed as percentage (mean ± standard deviation). One-way analysis of variance (ANOVA) and Dunnett’s test were used to compare groups against control. A value of p < 0.05 was considered significant.
Results and discussion
Phytochemical studies of the aqueous extract of C. genistoides showed the presence of saponins, flavonoids, tannins, and anthraquinones. Instead, oleanolic acid (1) and the sesquiterpene lactone deacylcynaropicrin (2), which have been previously reported in this plant (CitationDe Heluani et. al., 1997), were isolated from the ethyl acetate extract. Their structures were elucidated by 1H-NMR and 13C-NMR and were coincident with literature data (CitationAgrawal & Jain, 1992; CitationDe Heluani et al., 1997). Both compounds were tested for anti-inflammatory properties in the mouse paw edema test and the results are shown in .
Table 1. Inhibition in paw edema in mice caused by intraperitoneal doses of compounds isolated from C. genistoides.
Compound 1 exhibited significant activity at 1, 3, 5, and 7 h, while compound 2 showed significant inhibition at 3 and 5 h. Previous studies using a carrageenan acute test suggested acute vascular responses, vasodilatation, and an increase of the vascular permeability due to the the sequential release of histamine, serotonin, bradikinin, and prostaglandins (CitationVane & Botting, 1987). Moreover, oleanolic acid has been detected in several other plant species with proven anti-inflammatory effects (CitationSingh et al., 1992; CitationAlvarez et al., 2000; CitationFan et al., 2004); the present work proved that such action is dose-dependent.
Histamine and serotonin are released mainly during the first 1.5 h after carrageenan injection (CitationDi Rosa et al., 1971; CitationVinegar et al., 1976, Citation1982). Kinins are released at 2.5 h and the last step of inflammation is continued at 5 h by prostaglandins. Other studies have suggested that the 1-arginine–nitric oxide pathway plays an important role in the inflammatory response induced by carrageenan. It has been reported that nitric oxide (NO) derived from constitutive nitric oxide synthase (cNOS) is involved in the development of inflammation in the early steps after the administration of carrageenan, and the NO produced by induced nitric synthase (iNOS) is responsible for the maintenance of the inflammatory response at later steps (CitationSalvemini et al., 1995). Inhibition of paw edema by the NOS inhibitors is associated with prostaglandin levels in the paw exudate. From the results obtained in this work, we can conclude that the intraperitoneal administration of both isolated compounds from C. genistoides showed a significant inhibition of the carrageenan-induced inflammation at doses of 75 and 100 mg/kg, while the dose of 40 mg/kg did not produce a significant anti-inflammatory effect.
Oleanolic acid exercises its inhibitory action on inflammation (mediated by histamine, prostaglandins, serotonin, and kinins) during the first 7 h of the inflammatory phase, while deacylcynaropicrin contributes to this effect, acting in the intermediate phase. Considering that compounds 1 and 2 were isolated from aerial parts of C. genistoides, these results give support to the use of this plant as an anti-inflammatory in traditional medicine.
Acknowledgements
The authors acknowledge SECyT-UNSL for financial support (Project 4-2-8702). The technical and botanical support of the Organic Chemistry Department and of Ing. Luis A. Del Vitto, University of San Luis, are grateful acknowledged.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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