Antioxidant activity and inhibition of matrix metalloproteinases by metabolites of maritime pine bark extract (pycnogenol)

https://doi.org/10.1016/j.freeradbiomed.2003.12.017Get rights and content

Abstract

The procyanidin-rich maritime pine bark extract Pycnogenol has well-documented antioxidant and anti-inflammatory activity. After oral administration of Pycnogenol two major metabolites are formed in vivo, δ-(3,4-dihydroxyphenyl)-γ-valerolactone (M1) and δ-(3-methoxy-4-hydroxyphenyl)-γ-valerolactone (M2). We elucidated the effects of these metabolites on matrix metalloproteinases (MMPs) and determined their antioxidant activity to understand their contribution to the effects of maritime pine bark extract. We discovered strong inhibitory effects of M1 and M2 toward the activity of MMP-1, MMP-2, and MMP-9. On a microgram-per-milliliter basis both metabolites appeared more active than Pycnogenol. The metabolites were more effective than their metabolic precursor (+)-catechin in MMP inhibition. On a cellular level, we detected highly potent prevention of MMP-9 release by both metabolites, with concentrations of 0.5 μM resulting in about 50% inhibition of MMP-9 secretion. M1 was significantly more effective in superoxide scavenging than (+)-catechin, ascorbic acid, and trolox, while M2 displayed no scavenging activity. Both metabolites exhibited antioxidant activities in a redox-linked colorimetric assay, with M1 being significantly more potent than all other compounds tested. Thus, our data contribute to the comprehension of Pycnogenol effects and provide a rational basis for its use in prophylaxis and therapy of disorders related to imbalanced or excessive MMP activity.

Introduction

Pine bark extract has been used in traditional medicine in Europe and North America for inflammatory diseases and wound healing among other uses [1], [2]. Modern research with a standardized bark extract from the maritime pine, Pinus pinaster (Pycnogenol, Horphag Research Ltd., UK), indeed confirmed anti-inflammatory effects. In cell culture systems with endothelial cells or keratinocytes the maritime pine bark extract inhibited activation of the key molecule of inflammation, NF-κB, and expression of the adhesion molecules ICAM-1 and VCAM-1 [3], [4]. Furthermore it was demonstrated that the release of reactive oxygen species (ROS) was inhibited by pretreatment of cells with pine bark extract before challenge with tumor necrosis factor α (TNFα) [3]. Experiments with various rodent animal models supported the observation of anti-inflammatory effects of the herbal extract. Ingestion of the maritime pine bark extract resulted in a reduction of the inflammatory response in the rodent ear endema model [5]. After topical application it prevented erythema formation after UV radiation [6]. This erythema prevention after UV challenge was also observed in humans after oral ingestion of the extract [4]. Another anti-inflammatory action of the maritime pine bark extract was noticed in asthma patients who experienced a reduction of circulating leucotriene levels and an improvement of asthma symptom scores [7].

In inflammation, ROS play a special role as signaling molecules which contribute to cell injury and degenerative processes such as cartilage degradation in rheumatic diseases [8], [9]. In this context, matrix degrading enzymes, matrix metalloproteinases (MMPs), significantly contribute to the pathogenesis of various chronic inflammatory diseases. MMPs constitute a family of zinc-dependent proteolytic enzymes with so far 28 identified members [10]. Interestingly, MMPs can be activated by ROS [11], [12], [13], [14] so that both seem to contribute vitally to the inflammatory network. For the activation of MMPs by ROS various intracellular signaling pathways have been identified [15]. The maritime pine bark extract Pycnogenol has excellent radical scavenging properties [16], [17], [18] and enhances the production of antioxidative enzymes [19] which contributes to the anti-inflammatory effect of the extract.

An imbalance between MMPs and their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs), is generally assumed to result in proteolytic tissue damage or remodeling in pathophysiological conditions. In patients with arthritis, upregulation of MMP-1 (collagenase 1) or MMP-9 (gelatinase B) was determined [20], [21], [22]. MMP-1 also contributes to the photoaging of skin induced by UV exposure [14]. In asthma MMP-9 is upregulated and found to be involved in inflammation and remodeling processes [23], [24]. In pulmonary fibrosis MMP-2 (gelatinase A) plays an additional role [25]. Various MMPs contribute to cancer invasion and metastasis [26]. Based on these observations it is not surprising that the MMPs are regarded as a promising therapeutic target and the development of effective low-molecular-weight inhibitors is an important research focus of several pharmaceutical companies [27], [28].

The aim of our study was to determine the effects of Pycnogenol and its metabolites on the metalloproteinases that play a broad and important pathophysiological role, namely, MMP-1, MMP-2, and MMP-9. The extract is a mixture of procyanidins (proanthocyanidins), polyphenols, and phenolic acids that is either applied topically or administered orally [29]. The procyanidins are biopolymers comprising catechin or epicatechin monomer units in varying chain lengths. After oral intake ferulic acid and taxifolin were found in human urine [30]. These compounds are known components of the pine bark extract and ferulic acid was suggested as an excretion marker to determine bioavailability [31]. Most interestingly, two additional major metabolites (Fig. 1) were identified in human urine. These compounds, δ-(3,4-dihydroxyphenyl)-γ-valerolactone (metabolite 1, M1) and δ-(3-methoxy-4-hydroxyphenyl)-γ-valerolactone (metabolite 2, M2), are derived from their metabolic precursor catechin after various ring degradation and fission reactions [30]. We elucidated the effects of these metabolites on MMPs and determined their antioxidant activity to understand their contribution to the well documented anti-inflammatory action of maritime pine bark extract.

Section snippets

Enzymes

MMP-1 (proenzyme, human rheumatoid synovial fibroblast), MMP 2 (proenzyme, human rheumatoid synovial fibroblast), and MMP 9 (monomer, neutrophil, granulocyte) were all obtained from Calbiochem (Schwalbach am Taunus, Germany).

Inhibitors

A spray-dried extract from maritime pine barks (Pycnogenol) was generously provided by Horphag Research Ltd. (Geneva, Switzerland). Captopril was obtained from Fluka Chemie GmbH (Buchs, Switzerland). Tissue inhibitor of matrix metalloproteinase 1 (TIMP-1, human neutrophil

Inhibition of the enzymatic activity of MMP-1

Inhibition of the activity of MMP-1 was determined with two different substrates, collagen and gelatin (Fig. 2). With both substrates similar results were obtained. A comparison of the maritime pine bark extract Pycnogenol and its metabolites M1 and M2 revealed that all exhibit inhibitory activity. On a microgram-per-milliliter basis both metabolites were statistically significantly more potent than the whole extract while there was no significant difference between M1 and M2. Concentrations

Discussion

We determined effects of maritime pine bark extract (Pycnogenol) and its two major metabolites on pathophysiologically relevant matrix metalloproteinases. While there are several reports on clinical and molecular pharmacological effects of Pycnogenol [2], [29] little is known about the metabolites and their possible contribution to the acknowledged anti-inflammatory action of Pycnogenol.

The Pycnogenol metabolites M1 and M2 are not genuinely present in the pine bark extract, but are generated in

Acknowledgements

This work was supported by a research grant from Horphag Research.

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