Carnosic acid and carnosol potently inhibit human 5-lipoxygenase and suppress pro-inflammatory responses of stimulated human polymorphonuclear leukocytes

Abstract

Carnosic acid (CA) and carnosol (CS) are phenolic diterpenes present in several labiate herbs like Rosmarinus officinalis (Rosemary) and Salvia officinalis (Sage). Extracts of these plants exhibit anti-inflammatory properties, but the underlying mechanisms are largely undefined. Recently, we found that CA and CS activate the peroxisome proliferator-activated receptor gamma, implying an anti-inflammatory potential on the level of gene regulation. Here we address short-term effects of CA and CS on typical functions of human polymorphonuclear leukocytes (PMNL). We found that (I), CA and CS inhibit the formation of pro-inflammatory leukotrienes in intact PMNL (IC₅₀ = 15–20 μM [CA] and 7 μM [CS], respectively) as well as purified recombinant 5-lipoxygenase (EC number 1.13.11.34, IC₅₀ = 1 μM [CA] and 0.1 μM [CS], respectively), (II) both CA and CS potently antagonise intracellular Ca²⁺ mobilisation induced by a chemotactic stimulus, and (III) CA and CS attenuate formation of reactive oxygen species and the secretion of human leukocyte elastase (EC number 3.4.21.37). Together, our findings provide a pharmacological basis for the anti-inflammatory properties reported for CS- and CA-containing extracts.

Introduction

The o-diphenolic diterpene carnosic acid (CA) and its oxidation product carnosol (CS, Fig. 1) are major ingredients of extracts from Rosmarinus officinalis (Rosemary) and Salvia officinalis[1], [2], [3], [4] both of which possess anti-oxidant and anti-inflammatory properties [5], [6], [7], [8]. Dried herbs of Rosemary and Sage contain approximately 0.2–1% of CA and of CS, each [9], and in commercially available extracts CA and CS are strongly enriched (e.g. for Rosemary 10.3% CA and 4.4% CS was determined [10]). CA and CS have been shown to act as anti-oxidants, and anti-inflammatory, anti-proliferative, anti-tumorigenic and neuroprotective effects have been reported [2], [11], [12], [13], [14] (and references therein). Based on the abundant occurrence of CS and CA as herbal dietary ingredients, the development of natural remedies containing these diterpenes for the treatment of inflammatory diseases appears attractive. In support of this goal, it was shown that orally administered CA is present in the blood and in the brain in pharmacologically relevant concentrations [12].

Recently, we identified the peroxisome proliferator-activated receptor gamma (PPARγ) as a target selectively activated by CA and CS [14]. Activation of PPARγ causes long-term alteration of the expression of inflammatory gene products, implying the modulation of inflammatory cell functions in a comparably slow mode. In contrast, little is known about short-term effects of CS and CA on the molecular level or in pro-inflammatory cells (or animal models) that occur within minutes, distinct from gene expression-related mechanisms. CS has been shown to suppress the formation of pro-inflammatory leukotrienes (LTs) in rat leukocytes [15]. LTs are products of 5-lipoxygenase (5-LO, EC number 1.13.11.34) [16], [17]; however, a potential direct interference of CS with 5-LO was not investigated [15]. Furthermore, CS inhibits aggregation of rabbit platelets presumably by blocking collagen-induced Ca²⁺ signals and antagonizing the thromboxane receptor [18]. Despite these findings, it is presently unclear whether the anti-inflammatory properties of CS- and CA-containing extracts are primarily attributable to short-term actions, slow transcriptional events, or a combination of both.

Polymorphonuclear leukocytes (PMNL) are important early effectors of the innate immune response and play a crucial role in the development of an inflammatory phenotype [19]. They are highly responsive to chemotactic agonists such as the bacterial tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) [20], [21]. Activation of the G protein-coupled receptor for fMLP causes elevations of cytosolic [Ca²⁺] [22]. Ca²⁺ acts as second messenger and induces a number of distal signalling events, resulting in the elicitation of functional PMNL responses [23], [24] including the release of reactive oxygen species (ROS), predominantly generated by NADPH oxidase [25], [26]. Ca²⁺ also promotes the exocytosis of stored vesicles containing proteolytic enzymes like human leukocyte elastase (HLE, EC number 3.4.21.37) or cathepsin G [27]. Elevated ROS levels and enhanced protease release are typical early events in inflammatory reactions [28], [29], [30].

Here, we address the ability of CA and CS to directly interfere with typical short-term pro-inflammatory functions of human PMNL including LT formation, generation of ROS, and secretion of HLE. Moreover we show that the suppression of these PMNL functions correlates with reduced mobilisation of intracellular Ca²⁺, and we present 5-LO as a molecular target of CA and CS.