Original article
Inhibition of COX-1 and COX-2 activity by plasma of human volunteers after ingestion of French maritime pine bark extract (Pycnogenol)

https://doi.org/10.1016/j.biopha.2005.08.006Get rights and content

Abstract

There is evidence from several studies that supplementation with French maritime pine bark extract (Pycnogenol®) improves inflammatory symptoms in vivo. However, the molecular pharmacological basis for the observed effects has not been fully uncovered yet. Direct inhibitory effects of plant extracts or components upon cyclooxygenase (COX) activity have been repeatedly reported, but the question remained whether sufficiently high in vivo concentrations of bioactive compounds could be achieved in humans. The purpose of the present study was to determine a possible inhibition of the enzymatic activity of COX-1 and COX-2 by serum samples of human volunteers after intake of French maritime pine bark extract. This methodology considered that the serum samples would contain any bioavailable active principle. Therefore, we obtained blood samples before and after 5 days administration of 200 mg Pycnogenol to five healthy humans. The plasma moderately inhibited both COX-1 and COX-2 activities ex vivo. In a second approach, 10 volunteers received a single dose of 300 mg Pycnogenol. Only 30 min after ingestion of the pine bark extract the serum samples induced a statistically significant increase in the inhibition of both COX-1 (P < 0.02) and COX-2 (P < 0.002). This suggests a strikingly rapid bioavailability of bioeffective compounds after oral intake of the extract. Thus, we provide evidence that Pycnogenol exerts effects by inhibition of eicosanoid generating enzymes which is consistent with reported clinical anti-inflammatory and platelet inhibitory effects in vivo. The next challenge is to identify the active principle(s) that are rapidly bioavailable in human plasma.

Introduction

Pycnogenol is a standardized bark extract of the French maritime pine Pinus pinaster, Aiton, subspecies Atlantica des Villar (Pycnogenol®, Horphag Research Ltd., UK). The quality of this extract is specified in the United States Pharmacopeia (USP 28) [1]. Between 65% and 75% of Pycnogenol are procyanidins comprising of catechin and epicatechin subunits with varying chain lengths [2]. Other constituents are polyphenolic monomers, phenolic or cinnamic acids and their glycosides [2].

The effects of Pycnogenol have been explored in multiple in vitro and various animal and human in vivo studies [2], [3]. A considerable antioxidant activity was detected in simple in vitro or cell culture assays. Additionally to its radical scavenging activity an inhibition of NF-κB-dependent gene expression and decrease of the activity of various enzymes was observed for the Pycnogenol extract [3]. In vivo studies revealed distinct pharmacological actions [2]. These effects include anti-inflammatory and cardiovascular bioefficacy. Thrombus formation was prevented in passengers of long-haul flights after intake of Pycnogenol [4]. Supplementation of cardiovascular patients with maritime pine bark extract reduced platelet aggregation and improved microcirculation [5]. Anti-inflammatory effects of maritime pine bark extract were observed in experimental inflammatory bowel disease in animals [6]. Reduced urine [7] or serum [8] leukotriene concentrations were measured after Pycnogenol administration while asthma symptom scores and pulmonary function improved in asthma patients [7], [8]. Analgesic effects of Pycnogenol were observed in women with menstrual pain [9].

Thus, there is evidence from several in vivo studies that supplementation with French maritime pine bark extract improves inflammatory symptoms. It is tempting to explain the cellular or molecular mechanism of the observed in vivo effects with the results of effect studies on biological systems in vitro. There are two issues that warrant caution in interpretation and conclusions from in vitro effects. One issue is to consider whether the concentrations of the plant extract applied to cell cultures or enzymes in vitro would be realistic or achievable in vivo concentrations. In most cases this has to be denied due to the fact that plant extracts are typically not completely bioavailable and only certain fractions or components can be expected to be absorbed. French maritime pine bark extract, for example, comprises of high molecular weight components that cannot pass biological membranes in the gastrointestinal tract. The other issue that needs a second thought is that bioeffective compounds do not necessarily need to be present in the original extract, but might be formed in vivo due to intestinal bacterial and/or hepatic metabolism. After ingestion of Pycnogenol, for example, two metabolites derived from catechin were detected in human urine, δ-(3,4-dihydroxy-phenyl)-γ-valerolactone and δ-(3-methoxy-4-hydroxy-phenyl)-γ-valerolactone [10]. We recently demonstrated that these metabolites inherit an antioxidant activity as well as the potential to inhibit release and enzymatic activity of matrix metalloproteinase 9 in vitro [11]. Yet, it remained elusive whether sufficiently high in vivo concentrations of these metabolites could be attained.

A methodological approach that would consider both the absorption and possible metabolism of plant extract components would involve laboratory animals or human volunteers who donate blood samples before and after ingestion of the plant extract. The serum samples must contain any bioavailable active principle of the extract. These serum samples could be used in all kind of assays to uncover molecular pharmacological effects ex vivo in a realistic setting. The purpose of the present study was to determine a possible direct inhibition of the enzymatic activity of cyclooxygenase (COX) 1 and COX-2 ex vivo by serum samples after intake of regular doses of French maritime pine bark extract by human volunteers. We aimed to contribute to the explanation of the platelet aggregation inhibition, anti-inflammatory and analgesic effects of Pycnogenol observed in vivo.

Section snippets

Patients

Five and 10, respectively, healthy volunteers aged 18–30 years participated in this study. The study was approved by the ethical committee of the Comenius University, Bratislava, Slovak Republic, and all participants gave written informed consent. After 24 hours of a diet free of flavonoids (no vegetables, fruits and fruit juices or marmalades, tea, coffee, cocoa, wine and beer) blood samples were drawn to obtain basal values. Subsequently, five of the volunteers took tablets containing 200 mg

Results

After repeated intake of a daily dose of 200 mg French maritime pine bark extract incubation of five volunteers' plasma samples with COX-1 and COX-2, respectively, revealed direct inhibitory effect on cyclooxygenase activity (Fig. 1). An inhibition of COX-1 was obvious in three of five participants. The mean increase of cyclooxygenase inhibition after administration of Pycnogenol to volunteers was 13.8 ± 18.1% for COX-1 and 16.5 ± 35.3% for COX-2. The increase of cyclooxygenase inhibition was not

Discussion

The in vivo effects of plant constituents and extracts are studied with increasing interest [13], [14], but the elucidation of the molecular basis of biological or clinical effects remains a challenge. Bioavailability and metabolism factors must be considered for valid explanations of bioefficacy. Further complicating factors arise from the knowledge that plant extracts comprise of a complex mixture of various components and often enough it is not clear whether a single compound or a mixture of

Acknowledgments

This work was supported by an unrestricted research grant of Horphag Research.

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