Pycnogenol is a standardized bark extract of the French maritime pine Pinus pinaster
, Horphag Research Ltd., UK). It comprises of a concentrate of pine bark constituents such as polyphenolic monomers, procyanidins and phenolic or cinnamic acids and their glycosides [1
]. About 65–75 % of the Pycnogenol extract are procyanidins that consist of catechin and epicatechin subunits of varying chain lengths [1
]. The quality of this extract is specified in the United States Pharmacopeia (USP 28) [2
In human studies Pycnogenol revealed diverse anti-inflammatory actions [1
]. Double-blind, placebo-controlled studies in asthma patients showed reduced plasma [3
] or urine [4
] leukotriene concentrations after Pycnogenol supplementation, while asthma symptom scores and pulmonary function improved. Symptoms of osteoarthritis as pain and immobility of joints decreased in a double-blind, placebo-controlled study [5
]. Oral [6
] and topical [7
] application of Pycnogenol reduced inflammation and delayed skin-cancer formation following UV-radiation in controlled experiments in mice.
The anti-inflammatory mechanisms of maritime pine bark extract have been elucidated in a variety of in vitro
and cell culture studies [8
]. Additionally to its radical scavenging activity an inhibition of NF-κB-dependent gene expression and decrease of the activity of various pro-inflammatory mediators and adhesion molecules was observed after incubation of cells with the Pycnogenol extract [8
]. This experimental in vitro
design that pursues to uncover pharmacological effects by addition of plant extracts to cell cultures and subsequent measurement of cellular responses is widely employed. However, this methodology might inherit a couple of pitfalls.
Plant extracts often comprise of high molecular weight components that cannot be absorbed in the gastrointestinal tract and thus will never reach a target cell in vivo
. Furthermore, there are examples of metabolites that are not present in the original extract, but are formed in vivo
as a result of 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
]. Valerolactone derivatives were also found after ingestion of green tea [11
]. These newly formed metabolites may display significant efficacy and contribute to the observed in vivo
effects. We recently elucidated the cellular effects of δ-(3,4-dihydroxy-phenyl)-γ-valerolactone and δ-(3-methoxy-4-hydroxy-phenyl)-γ-valerolactone and uncovered an antioxidant activity as well as the potential to inhibit release and enzymatic activity of matrix metalloproteinase 9 (MMP-9) [12
Thus, pharmacokinetic issues of absorption and metabolism should be considered for valid identification of molecular pharmacological effects of plant extracts. A methodological approach that considers both the absorption and possible metabolism of plant extract components would involve laboratory animals or human volunteers who donate blood samples. These blood samples should contain all bioavailable active principles of the extract and allow an ex vivo
analysis in all kind of molecular pharmacological effects in cell culture assays (Figure ). There are only few examples of experimental settings described in literature that use this approach. Effects of nettle herb [13
] or willow bark extract [14
] on cytokine release and effect of Harpagophytum
extract on eicosanoid biosynthesis [15
] were elucidated in whole-blood assays of human volunteers after ingestion of the extract. Recently, a potent ex vivo
anti-HIV activity was detected in sera of volunteers after administration of Phyllanthus amarus
plant material [16
Schematic representation of the experimental procedure of the ex vivo experiments with human plasma incubated with monocytes.
The purpose of the present study was to determine molecular pharmacological effects of maritime pine bark extract ex vivo after intake of regular doses by human volunteers. Therefore, we obtained plasma samples before and after five days administration of Pycnogenol to seven healthy humans. These plasma samples were analyzed in two different experimental settings to evaluate the influence of bioavailable actives principles on cellular key components that contribute to inflammatory processes. We investigated a potential influence of the plasma samples on LPS-induced release of MMP-9 from human monocytes. Since MMP-9 induction and release might be initiated by NF-κB activation we also determined the effect of the plasma samples on LPS-induced NF-κB nuclear translocation.