Original Contributions
Procyanidins Extracted From Pinus maritima (Pycnogenol®): Scavengers of Free Radical Species and Modulators of Nitrogen Monoxide Metabolism in Activated Murine RAW 264.7 Macrophages

https://doi.org/10.1016/S0891-5849(97)00430-9Get rights and content

Abstract

Nitrogen monoxide (NO) has diverse physiological roles and also contributes to the immune defense against viruses, bacteria, and other parasites. However, excess production of NO is associated with various diseases such arthritis, diabetes, stroke, septic shock, autoimmune, chronic inflammatory diseases, and atheriosclerosis. Cells respond to activating or depressing stimuli by enhancing or inhibiting the expression of the enzymatic machinery that produce NO. Thus, maintenance of a tight regulation of NO production is important for human health. Phytochemicals have been traditionally utilized in ways to treat a family of pathologies that have in common the disregulation of NO production. Here we report the scavenging activity of Pycnogenol® (the polyphenols containing extract of the bark from Pinus maritima) against reactive oxygen and nitrogen species, and its effects on NO metabolism in the murine macrophages cell line RAW 264.7. Macrophages were activated by the bacterial wall components lipopolysaccharide (LPS) and interferon (IFN-γ), which induces the expression of large amounts of the enzyme nitric oxide synthase (iNOS). Preincubation of cells with physiological concentrations of Pycnogenol® significantly decreased NO generation. It was found that this effect was due to the combination of several different biological activities, i.e., its ROS and NO scavenging activity, inhibition of iNOS activity, and inhibition of iNOS-mRNA expression. These data begin to provide the basis for the conceptual understanding of the biological activity of Pycnogenol® and possibly other polyphenolic compounds as therapeutic agents in various human disorders.

Introduction

Nitrogen monoxide (NO) is produced in mammalian cells constitutively or can be induced by various cell activators through the oxidation of l-arginine by a family of enzymes known as nitric oxide synthase (NOS, l-arginine, NADPH oxidoreductase, NO forming EC1.1413.39).[1]

The expression of a distinct isoform of NOS (iNOS) may be induced in different cell types (macrophages, smooth muscle cells, epithelia) by various proinflammatory agents such as the endotoxin from bacterial wall lipopolysaccharide (LPS) or by interleukin-1β, tumor necrosis factor, and γ-interferon (IFN-γ). Other inducing agents include ultraviolet irradiation and ozone.2, 3

NO can be considered an immune modulator, owing to its complex activity during host cellular defense.[4]When macrophages are activated by the endotoxin from the bacterial wall components LPS or by IFN-γ, the inducible form of the enzyme NOS is significantly expressed and massive amounts of NO can be produced to exert a nonspecific immune response. NO has been reported to be effective against various foreign or infectious materials such as bacteria, parasites, helmints, and viruses, and also against tumor cells.[5]On the other hand, NO has a suppressive effect on T-lymphocyte proliferation leading to increased sensitivity to certain pathogens during chronic stages of the immune response.6, 7

During inflammation associated with different pathologies, such as arthritis or Crohn’s disease, NO production increases significantly and may become “self-destructive” as occurs in chronic inflammatory diseases. NO overproduction has been reported in autoimmune disease, transplanted organ rejection, and sepsis.4, 8Moreover, the free radical nature of NO and the high reactivity with O2•−, with subsequent generation of ONOO, renders NO a potent pro-oxidant molecule able to induce oxidative damage and potentially harmful toward cellular targets.9, 10NO and other reactive nitrogen species formed during the reaction of NO with superoxide or with oxygen have been reported to modify free and protein-bound amino acid residues, to inhibit enzymatic activities, to induce lipid peroxidation, and deplete cellular antioxidant levels. All these features may be associated with the development of different pathologies.11, 12, 13Thus, NO overproduction by iNOS can be considered as a biological process potentially leading to opposite outcomes, either physiological or pathological, depending on the ability of cells or tissues to control both the expression of iNOS activity and the nonspecific effects of NO.

Naturally occurring flavonoids, such as rutin and quercetin, have been reported to scavenge NO.[14]Complex mixtures of polyphenols such as Gingko biloba extract EGb761, which have been utilized in traditional medicine for the treatment of diseases now recognized to involve NO metabolism dysfunction, have also been recently reported to inhibit NO production in macrophages.[15]

Extracts from the bark of pine have also been used in the past in different parts of the world in traditional medicine and are believed to affect various pathologies ranging from vascular disease to arthritis. Pycnogenol® is a proprietary extract of the bark of the French maritime pine tree (Pinus maritima). The extract is prepared by a standardized procedure that includes an extraction of fresh pine bark with an aqueous solution of sodium chloride followed by extraction with ethylacetate. The resulting product is a mixture of at least 40 different molecules, mainly flavonoids as monomers (about 8% of the weight) such as catechin, epicatechin, and taxifolin and condensed polymers (85%), up to heptameric flavanols, designated as procyanidins. Pycnogenol® also contains phenolcarbonic acids and glycosylation products, i.e., sugar derivatives of phenolcarbonic acids and taxifolin, as minor constituents. All these molecules potentially have antioxidant properties[16]and may also act as modulators of metabolic enzymes.17, 18, 19and other cellular functions.[20]This mixture has been used in the past to correct symptoms of pathologies such as chronic inflammations or cardiovascular disease but also to overcome vitamin C deficiency. Taken together, these historical data suggest that this mixture may have an efficient antioxidant activity and some modulatory effect on both the immune system and on the vessels tone and functions. Data appearing recently in the literature reporting an effective biological activity of Pycnogenol®, as both antioxidant21, 22, 23, 24and as modulator of the immune response have been reviewed.24, 25, 26

In this investigation we report the effect of Pycnogenol® as a scavenger of reactive oxygen and nitrogen species and the effects of Pycnogenol® on NO production in the murine cell line of monocyte-macrophages RAW 264.7.

Section snippets

Materials and Methods

Pycnogenol® was a gift from Horphag Research Ltd (Guernsey, FRA). Interferon-γ (IFN-γ) and lipopolysaccharide (LPS) were purchased from Genzyme (Cambridge, MA). Fetal calf serum (FCS) was obtained from University of California, San Francisco cell culture facility. Oligonucleotide primers were obtained from Clontec (Palo Alto, CA). l-[U-14C] arginine was purchased from Du Pont (Wilmington, DE). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Labotec (Tokyo, Japan). l-Ascorbic acid

Antioxidant Activity of Pycnogenol®, and Its Major Components: Scavenging of NO

As a first step the antioxidant activity of Pycnogenol® was assessed. Flavonoids owing to the multiple phenolic groups in their molecular structure are potentially able to quench free radicals by forming more stable oxidized products. Some of the most important free radical in the biological environment, namely the reactive oxygen species O2•− and HO and the nitrogen reactive species NO, were investigated.

A reliable assessment of NO scavenging is determining its effect on the nitrite

Modulation of NO Metabolism

In this study it has been demonstrated that the complex mixture of flavonoids extracted from the bark of Pinus maritima significantly affected iNOS expression and activity in macrophages challenged by LPS and IFN-γ. The murine RAW 264.7 cell line, which is possibly the best characterized line for the study of NO metabolism and iNOS expression in immunocompetent cells, was used.[1]

In vitro, Pycnogenol® was found to have a remarkable modulatory effect on iNOS enzyme activity producing a slight

Acknowledgements

The authors acknowledge the contributions of Claude Saliou, for helpful support and suggestions throughout this work, and Yasuko Noda for ESR analysis of the oxygen free radical scavenging activity.

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    F. V. is on leave from the National Institute of Nutrition, Rome, Italy.

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    H. K. is on leave from the Institute of Medical Science, Center for Adult Diseases, Kurashiki, Japan.

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