Inhibition of cyclooxygenase activity by standardized hydroalcoholic extracts of four Asteraceae species from the Argentine Puna

We determined the anti-inflammatory activity of standardized extracts of four medicinal plant species (Baccharis incarum, B. boliviensis, Chuquiraga atacamensis, Parastrephia lucida) that grow in the Argentine Puna (3800 m above sea level) and that are used to reduce oxidative stress and alleviate gout and arthritic pain. The extracts of plant aerial parts were standardized in terms of total phenolic compounds and flavone/flavanone content and free radical scavenging activity. All extracts showed high phenolic compound concentration (0.5-1.6 mg/mL), mainly flavones and flavonols (0.1-0.8 mg/mL). The extracts showed hydrogen donating ability (DPPH and ABTS) and reactive oxygen species scavenging activity (O2●, OH-, H2O2). The ability of the extracts to inhibit cyclooxygenase enzymes (COX-1 and COX-2) was determined by calculating percent inhibition of PGE2 production measured by enzyme immunoassay. All extracts inhibited both enzymes with IC50 values of 2.0 to 16.7 μg/mL. The anti-inflammatory activity of B. incarum and C. atacamensis extracts was higher than that of B. boliviensis and P. lucida. The IC50 values obtained for indomethacin were 0.11 and 0.78 μM for COX-1 and COX-2, respectively. The present results are consistent with the anecdotal use of these species in phytotherapic preparations.

The Asteraceae family is represented by more than 1,100 genera and 25,000 species distributed in different kinds of habitat, mainly in the mountainous tropical areas of South America. Baccharis incarum (Wedd.) Perkins, commonly known as "tola" or "lejía", Baccharis boliviensis (Wedd.) Cab. known as "tola", "tola limón" or "tolilla", Chuquiraga atacamensis Kuntze, known as "lengua de gallina" or "azafrán, "quebrolla" and Parastrephia lucida (Meyen) Cab., known as "romero", "tola" or "chachakoa", belong to the Asteraceae family and are used as ethnomedicinal plants, fodder plants and fuel wood. The leaves and stems of these plants maintained in ethanol are used as "rubbing" or topical preparation in order to treat rheumatism, fever and inflammation. Plant resin poultices are used in bruises and wounds and to consolidate luxations and fractures. The infusion or decoction of aerial parts of Baccharis are used also as a protective agent against live, prostate and stomach diseases, burns, skin wounds and ulcers, and as an antipyretic (1,2).
Some studies of the biological activity and phytochemistry of these plant species have been published (3)(4)(5)(6)(7)(8), but a survey of the literature revealed that there are no studies on the validation of the anti-inflammatory capacity of polar extracts from these plants.
Cyclooxygenase enzymes (COXs, prostaglandinendoperoxide synthases) catalyze two reactions, the first www.bjournal.com.br Braz J Med Biol Res 42 (9) 2009 being a cyclooxygenase function consisting of the addition of molecular oxygen to arachidonic acid (AA) to form prostaglandin G 2 (PGG 2 ). The second is the conversion of PGG 2 to PGH 2 by a peroxidase function. Hence, this COX enzyme performs the critical initial reaction in the AA metabolic cascade leading to the formation of pro-inflammatory prostaglandins, thromboxanes and prostacyclins. Prostaglandins regulate smooth muscle contractility, blood pressure and platelet aggregation and mediate pain and fever. Inhibition of cyclooxygenase activity is the mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) exert their analgesic, antipyretic, anti-inflammatory, and antithrombotic effects (9).
The constitutive form COX-1 is responsible for the maintenance of physiological prostanoid biosynthesis. In contrast, COX-2 is an inducible isoform linked to inflammatory cell types and tissues (10). Prolonged use of NSAIDs is also associated with severe side effects such as gastrointestinal hemorrhage due to COX-1 inhibition (9). The new COX-2 selective drugs do not seem to be free of risk either since several COX-2 inhibitors have been found to cause cardiovascular problems (11). Steroids have an obvious role in the treatment of inflammatory diseases but, due to their toxicity, they can only be used over short periods of time except in very serious cases for which the risks are acceptable. Consequently, there is a strong need for natural products with minimum side effects.
The aim of the present study was to evaluate the anti-inflammatory potential of standardized hydroalcoholic extracts of B. incarum, B. boliviensis, C. atacamensis, and P. lucida using a COX inhibition assay in order to validate their popular use as phytomedicines.
Total phenolic compound content was determined (12) and the results are reported as gallic acid equivalents. Flavone and flavonol content was measured by the method of Popova et al. (13) and quercetin was used as standard. Total flavanone and dihydroflavonol content was estimated using the colorimetric method for the DAB9 extract modified for propolis (13). Total flavanones and dihydroflavonols were calculated as naringenine from a calibration curve.
The ability of the extracts to inhibit the conversion of AA to PGH 2 by ovine COX-1 and human recombinant COX-2 was determined using a COX inhibitor screening assay kit (No. 560131; Cayman Chemical, USA). Cyclooxygenase catalyzes the first step in the biosynthesis of AA to PGH 2 . PGF 2α produced from PGH 2 by reduction with stannous chloride was measured by enzyme immunoassay (14). This assay is based on the competition between PGs and a PG-acetylcholinesterase conjugate (a PG tracer) for a limited amount of PG antiserum. The amount of PG tracer that is able to bind to the PG antiserum is inversely proportional to the concentration of PGs in the wells, since the concentration of the PG tracer is held constant while PG concentration varies. This antibody-PG complex binds to an anti-IgG antibody previously attached to the well. The plate is washed with a buffer solution and Ellman's reagent, which contains the substrate of acetylcholinesterase, is added to the well. The yellow product of this enzymatic reaction is determined spectrophotometrically in a Microplate Reader (BioRad, Japan) at 415 nm. The assay for obtaining 100% COX activity was performed with and without DMSO as solvent control. The inhibitory assays were performed in the presence of extracts at different concentrations (1 to 16 µg total phenolic compound/mL) or of a commercial anti-inflammatory drug such as indomethacin. Indomethacin (Sigma, Germany) was chosen because it is an inhibitor of both enzymes (COX-1 and COX-2). Pre-incubation time between enzyme and inhibitor was 10 min with 2-min incubation in the presence of AA at 37°C. Enzyme control was performed with COXs that had been inactivated by placing them in boiling water for 3 min. The detection limit was 29 pg PG/mL. The intra-and interassay coefficients of variations were 5 and 10%, respectively.
The anti-inflammatory effect of the test compounds was evaluated by calculating percent inhibition of PGE 2 production. The test compound concentration causing 50% inhibition of PGE 2 release (IC 50 ) was calculated from the concentration-inhibition response curve by regression analysis. The selectivity indices (SI) of the reference compounds and extracts (SI = IC 50 COX-2 / IC 50 COX-1) were calculated. The values reported are the means of three separate experiments.
In the present study, the ethanolic extracts of plant species that grow in the Argentine Atacama Puna were chosen to validate their traditional use in folk medicine as anti-inflammatory solutions.
A phytochemical study of the extracts was conducted in order to obtain a standardized phytotherapic preparation of each plant species (Table 1) The presence of terpenoids and flavonoids has been described in B. incarum, B. boliviensis, and C. atacamensis (3,(15)(16)(17)(18)(19). There are no bibliographical data about chemical studies on P. lucida. The standardized ethanolic extracts showed hydrogen donating ability and reactive oxygen scavenging activity (Table 1), inhibiting the production of reactive oxygen species. Since these plant species contain xanthine oxidase inhibitors, they reduce both uric acid and O 2 •production (IC 50 of 10 to 20 µg/mL) (8). Numerous pathological events such as inflammation processes, aging phenomena and degenerative dysfunction are associated with the generation of reactive oxygen species. Thus, the effectiveness of these plant extracts used in folk medicine to suppress inflammatory responses may be due to their capacity to reduce oxidative stress and to their inhibitory activity on COX enzymes. PGE 2 level increases markedly and provokes inflammation and pain in pathological events due to the activation of COX enzymes. Hence, the extracts were tested for their ability to inhibit COX-1 and COX-2. All extracts exhibited a significant anti-inflammatory activity in vitro by inhibiting PG production. The anti-inflammatory activities of all species were dose dependent in phenolic compound equivalents for COX-1 and COX-2 (Figure 1). At the same total phenolic compound concentration, the effects of the MI-1-3800 and MI-3-3800 extracts were higher than those of the MI-2-3800 and MI-4-3800 extracts. The greatest anti-inflammatory effect observed for MI-3-3800 could be due to a higher content of flavanones and dihydroflavonols.
The preliminary results presented in this paper provide some scientific basis for the traditional use of these plant species for managing inflammatory pains. There are no data in the literature on the anti-inflammatory compounds from the species included in this study, whereas data have been reported on the anti-inflammatory activity of terpenoids and flavonoids isolated from other Baccharis species (20). Further analysis, including additional purification of the extracts and chemical characterization of isolated compounds, should permit the identification of compounds possessing specific activities. These investigations, along with further studies in order to determine the effect of these extracts on these and other inflammatory parameters, are currently being conducted in our laboratory.