Modulatory activities of Agelanthus dodoneifolius (Loranthaceae) extracts on stimulated equine neutrophils and myeloperoxidase activity

Abstract

Abstract. Agelanthus dodoneifolius DC Danser (Loranthaceae)is used for the treatment of various diseases including asthma. The aqueous and hydroalcoholic extracts have been reported to have anti-inflammatory, spasmolytic and bronchorelaxant activities. The present study investigates the effects of the aqueous decoction and the diethyl ether, ethyl acetate and butanolic fractions of Agelanthus dodoneifolius DC Danser (Loranthaceae) on reactive oxygen species (ROS) production and myeloperoxidase (MPO) release by phorbol 12-myristate 13-acetate (PMA)-stimulated equine neutrophils and on purified equine MPO activity. ROS production and MPO release by the PMA-stimulated neutrophils were measured by the lucigenin-enhanced chemiluminescence and ELISA assays, respectively. Specific immunological extraction followed by enzymatic detection (SIEFED) was used to specifically measure the equine MPO activity. Identification and quantification of the individual and total phenolic and flavonoid compounds were performed using UPLC-MS/MS equipment and colorimetric methods involving Folin-Ciocalteu and AlCl 3 , respectively. All the tested extracts displayed dose-dependent inhibitory effects on the oxidant activities of neutrophils; a stronger effect was observed with the organic fractions than the aqueous decoction. These findings could be correlated with a high content of phenolic and flavonoid compounds. The results confirm the previously shown anti-inflammatory effect of Agelanthus dodoneifolius and its potential use for the treatment of neutrophil-dependent inflammatory diseases. IntroductionPolymorphonuclear neutrophils (PMNs) are the primary effector cells in host responses to injury and infection (1-4). The antimicrobial function of stimulated PMNs is based on their phagocytic capacity and their ability to release oxidant and proteolytic enzymes as well as reactive oxygen species (ROS) involved in the destruction of microorganisms. ROS play important roles in the pathogenesis of various serious diseases, such as neurodegenerative disorders, aging, cancer, cataracts, inflammation and cardiovascular diseases including atherosclerosis (5,6).Myeloperoxidase (MPO), a heme peroxidase enzyme stored in the azurophilic granules of neutrophils, plays a crucial role in the destruction of bacteria inside PMNs. The bacterial killing process mainly uses hydrogen peroxide (H 2 O 2 ) and the chloride anion (Cl -) to produce hypochlorous

Evaluation of Antiradical and Anti-Inflammatory Activities of Ethyl Acetate and Butanolic Subfractions ofAgelanthus dodoneifolius(DC.) Polhill & Wiens (Loranthaceae) Using Equine Myeloperoxidase and Both PMA-Activated Neutrophils and HL-60 Cells

“…When the half maximal inhibitory concentrations were compared, the inhibitory potency decreased in the following order: n -butanolic subfraction (IC 50 = 2.8 ± 1.2 μ g/mL) > gallic acid (IC 50 > 25 μ g/mL), data from previous work [ 13 ] ≥ ethyl acetate subfraction (IC 50 > 50 μ g/mL). Contrary to our study, Kroes et al [ 33 ] had previously reported an efficient inhibitory effect of gallic acid on MPO release by using another neutrophils activator: zymosan which was different in our model based on PMA stimulation.…”

“…Recently we have shown that butanolic and ethyl acetate fractions of Agelanthus dodoneifolius exhibited excellent anti-inflammatory and antioxidant properties that were attributed to their polyphenol components [ 13 ]. However, these interesting activities were not properly correlated to a specific type of polyphenols contained in each tested fraction.…”

“…ROS production by stimulated neutrophils was assessed by lucigenin-dependent chemiluminescence (CL), according to a method adapted from Benbarek et al [ 18 ] and described elsewhere [ 13 , 17 ]. Neutrophil suspensions (1 × 10 6 cells) were stimulated with PMA in the presence of increasing final concentrations of the extract in a 96-well microtiter plate (White Combiplate 8, Thermo Labsystems) and the CL response was monitored for 30 min at 37°C (Fluoroscan Ascent FL, Thermo Scientific, Belgium) and expressed as the integral value of the total CL emission.…”

“…MPO released by equine neutrophils stimulated with PMA was assessed by an original ELISA assay raised against equine MPO as previously reported [ 13 , 20 ] and distributed by BioPtis (Liège, Belgium). Briefly 1 × 10 6 cells in 1 mL PBS buffer were stimulated with PMA in the presence of the extract for 30 min at 37°C and further centrifuged 10 min at 600 ×g.…”

“…Among plants phytoconstituents, phenolics were the most abundant and those also reported to have scavenging properties based on various methods [ 11 , 12 ]. Our previous works have shown that crude fractions from Agelanthus dodoneifolius plant displayed interesting antioxidant activities that were related to their polyphenolic contents [ 13 ]. As those polyphenol components are very important at inducing antioxidant and anti-inflammatory effects, we were particularly interested to investigate which active component belonging to the subfraction is responsible of the antioxidant/antiradical activity.…”