Human Neutrophil Elastase Inhibitory Alkaloids from Chelidonium majus L.

Abstract

Abstract Human neutrophil elastase (HNE) represents a goodtherapeutic target for the treatment of inflammatory diseases aswell as invasion of microorganism. The methanol extract of aaerial part of Chelidonium majus L. showed high activity againstthe neutrophil elastase with an IC 50 value of 100µg/mL. Due toits potency, subsequent bioactivity-guided fractionation ofmethanol extract led to six alkaloids (1-6), which were identifiedas dihydrosanguinarine (1), (s)-stylopine (2), arnottianamide (3),(+)-chelidonine (4), spallidamine (5), and N-trans-feruloyltyramine (6). Among of them, three alkaloids (2, 5, and 6)inhibited HNE in a dose-dependent manner with IC 50 rangingbetween 11.6 and 51.0µM. Lineweaver-Burk and Dixon plots,and their secondary replots showed that alkaloids (2, 5, and 6)were mixed inhibitors of HNE. The analysis of K I and K IS valueproved that all inhibitors (2, 5, and 6) had reversible mixed typeI mechanism.Keywords Chelidonium majus L. · human neutrophil elastase ·inflammation · isoquinoline alkaloidIntroductionThe regulation of the enzymatic activity of human neutrophilelastase (HNE) has been the attractive field because neutrophilsare the first cells recruited to inflammatory sites and from theearliest line of defense against the invasion of microorganism(Brice et al., 2010). The human neutrophil elastase (EC 3. 4. 21.37) is the family of serine proteases that possess the ability tohydrolyze the extracellular matrix protein (Siedle et al., 2007). Itis present in azurophil granules in the neutrophil cytoplasm. HNEis a proteolytic enzyme involved in pathogenesis of emphysema,adult respiratory distress syndrome and rheumatoid arthritis(Crocetti et al., 2013). The catalytic site of HNE molecule iscomposed of the triad His41-Asp99-Ser173, of which the oxygenof serine attacks carbonyl group on the target substrate (Bode etal., 1989). The activity of HNE is controlled by inhibitor namedα1-antitrypsin produced in the liver. However, their HNE affinityis strongly decreased by oxidative stress and by proteases releasedfrom leukocytes that are recruited to inflammation sites (Brice etal., 2010). Thus, the imbalance between HNE and its inhibitors isimplicated in many inflammation diseases as like pulmonaryemphysema.Chelidonium majus L. is a perennial herbaceous plant of thefamily Papaveraceae, which is widely distributed around theworld. This plant has been traditionally used as an herbal medicinefor treatment of gastric ulcer, oral infection and liver disease(Lenfield et al., 1981). These effects are mainly due to the alkaloidspresent in milk sap. The major bioactive components of this plantare isoquinoline alkaloids such as chelidonine, chelerythrine,sanguianarine, bebeerine and coptisine (Barreto et al., 2003). Itincluded flavonoids and various acids such as ferulic, cumaric,caffeic and chelidonic acids (Barens J et al., 2007). The constituentsof C. majus L. have been found to exhibit antitumor, antiviral, andanti-inflammatory activities (Colombo and Bosisio, 1996). Additionally,the potent and selective acetylcholinesterase inhibition wasobserved on 8-hydroxydihydrochelerythrine in C. majus L (Cho etal., 2006).During a screening procedure on higher plant to find neutrophilelastase inhibitors, methanol extract of C. majus L. was shown toexhibit considerable inhibitory activity. In this study, we isolatedsix alkaloids targeting to HNE from the methanol extract of aerial