ANTIMICROBIAL ACTIVITY OF PYCNOGENOL 647

Copyright © 2005 John Wiley & Sons, Ltd. Phytother. Res. 19, 647–648 (2005)

Copyright © 2005 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 19, 647–648 (2005)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1662

SHORT COMMUNICATIONAntimicrobial Activity of Pycnogenol®

Maria Angeles Calvo Torras1*, Carles Adelantado Faura1, F. Schönlau2 and P. Rohdewald2

1Veterinary Faculty, University Autonomous of Barcelona, Spain2Pharmaceutical Chemistry, University of Münster, Germany

Pycnogenol®, a standardized extract of Pinus pinaster bark, was tested for its antimicrobial activity against 23different pathogenic prokaryotic (gram-positive and gram-negative) and eukaryotic (yeast and fungi) micro-organisms. Pycnogenol® inhibited the growth of all the tested microorganisms in minimum concentrationsranging from 20 to 250 µµµµµg/mL. Thus, Pycnogenol® in concentrations as low as 0.025% could counteract thegrowth of all the strains investigated in our study. These results conform with clinical oral health care studiesdescribing the prevention of plaque formation and the clearance of candidiasis by Pycnogenol®. Copyright ©2005 John Wiley & Sons, Ltd.

Keywords: Pycnogenol; anti-bacterial; Candida; plaque.

Received 24 January 2005Accepted 27 January 2005

* Correspondence to: Dr M. A. Calvo Torras, Microbiology, VeterinaryFaculty, U.A.B. Campus Bellaterra 08193 Bellaterra, Barcelona, Spain.E-mail: Mariangels.calvo@uab.esContract/grant Sponsor: Horphag Research UK.

INTRODUCTION

Pycnogenol® is a phytochemical extracted from the barkof the pine tree species Pinus pinaster Ait., growingexclusively along the south-western coast of France.Pycnogenol® consists of standardized proportions ofmonomeric and oligomeric procyanidins and phenolicacids (derivatives of benzoic acid and cinnamic acid)(Rohdewald, 2002). Pycnogenol® is the registered trade-mark of Horphag Research Ltd UK, identificationand analysis are described in the US Pharmacopoeia(USP 28).

Pycnogenol® is used world-wide as a dietarysupplement as well as a food ingredient because ofits manifold health benefits related to antioxidant,protein-binding and endothelial nitric oxide synthesisactivities (Rohdewald, 2002). Chewing gum containingPycnogenol® was previously shown in a double-blindplacebo (plain chewing gum) controlled clinical studyto reduce gingival bleeding and plaque formation(Kimbrough et al., 2002). The latter effect made itplausible that the components of Pycnogenol® mightpossess bacteriostatic activities and this was the impetusfor the study presented here. A broad range of bothgram-positive and gram-negative bacteria as well yeastand fungi were tested to establish the minimum inhibi-tory dosages of Pycnogenol® using standard laboratorytechniques.

METHODS

Pycnogenol®, French maritime pine bark extract, wasprovided by Horphag Research UK Ltd. The standard-

ized extract corresponds to the monograph ‘MaritimePine Extract’ of the US Pharmacopoeia (USP, 28).

The tested strains were either available in house(Veterinary Faculty of Barcelona University = FVB) orobtained from the American Type Culture Collection(ATCC).

The susceptibility of the test organisms toPycnogenol® was determined by direct contact underliquid conditions. All experiments were carried out intriplicate.

The tested strains were harvested from cultures,washed and diluted in growth media to give 2 × 108 CFU/mL. Test tubes were prepared with culture medium(Mueller-Hinton agar) and inoculated with 1 mL of thetested microorganism. In the case of fungi, Shadomyagar with 2% malt extract was used. Serial dilutions ofPycnogenol® were prepared and 15 µL was added tothe test tubes. Samples of the culture medium weretaken at baseline and again after 5 min, 60 min, 2 h,1 week and 2 weeks, respectively. 100 µL of the samplewas added to Triptone Soy Agar in Petri dishes andsubmitted to further incubation for 24 h at 37 °C (yeastand bacteria) or 5 days at 28 °C (fungi). Antimicrobialactivity was evaluated by counting CFU per dishand comparison with the controls. The percentage ofsurvivors was presented with respect to the controlmixture without Pycnogenol®. Experiments in culturebroth allow the minimum quantity of Pycnogenol®

necessary to inhibit the growth of a microorganism tobe established. Furthermore, these experiments allowedthe possibility of a microbicidal activity of Pycnogenol®

to be disproved.

RESULTS AND DISCUSSION

As presented in Table 1 Pycnogenol® was foundto possess marked bacteriostatic activity against bothgram-positive and gram-negative strains. Moreover,Pycnogenol® was effective in inhibiting the growth ofeukaryotic microorganisms such as yeast (Candida) and

648 M. A. CALVO TORRAS ET AL.

Copyright © 2005 John Wiley & Sons, Ltd. Phytother. Res. 19, 647–648 (2005)

Table 1. Antimicrobial activity of Pycnogenol®

Microorganism Pycnogenol® MIC (µg/mL)

Salmonella sp.FVB 2345 100Staphylococcus aureusATCC 6538 20Enterococcus faecalisFVB 678 50Escherichia coliATCC 9337 20Klebsiella pneumoniaeFVB 543 20Proteus mirabilisATCC 32 20Pseudomonas aeruginosaATCC 10145 120Clostridium perfringensFVB 548 30Campylobacter sp.FVB 3567 250Streptococcus glucansFVB 688 50Bacillus cereusFVB 5970 30Candida albicansFVB1001 50Aspergillus oryzaeATCC 9102 90Penicillium funiculosumFVB 987 90Fusarium moniliformeFVB 934 80Streptococcus mutansFVB 345 30Lactobacillus acidophilusFVB 988 50Actinobacillus actinomycetemcomitansFVB 5467 230

between the MID values and the bacterial membranecomposition as reflected by gram-positive and gram-negative bacterial strains, respectively. Eukaryoticorganisms are growth-inhibited within the same MIDrange as for prokaryotes, suggesting a common mech-anism by which Pycnogenol® reversibly arrests thegrowth of all tested microorganisms.

Polyphenolic substances in general, originating fromvarious plant sources, have been suggested to inhibitthe growth of filamentous fungi, yeast and bacteria,as reviewed by Cowan (1999). These studies mostlyspeculate that the complexation of polyphenolicswith proteins via non-specific hydrogen bonding andhydrophobic interaction accounts for the antimicrobialactivity. As a result of the complexation microbialadhesins, enzymes, cell envelope transport proteinsetc. might be inactivated, and the cell cycle arrested.

These observations are in accordance with thepreviously described inhibition of plaque formationin humans by chewing sugar-free gum containingPycnogenol® (Kimbrough et al., 2002). Subjects wear-ing plastic stints to avoid tooth-brushing were advisedto chew either six pieces of sugar-free control chewinggum a day over a period of 2 weeks, or commerciallyavailable chewing gum with 5 mg Pycnogenol®. Whilethe control group showed significant development ofplaque, the group given Pycnogenol®-chewing gumsshowed no bacterial plaque.

Unpublished reports described immunosuppressedindividuals suffering from candidiasis of the throatchewing several Pycnogenol® tablets during the daywith a little water. After 1 month of treatment theinfection cleared (Dr Michael A. Ross, Victoria CA,personal communication).

In conclusion, Pycnogenol® may be an interestingingredient to control microbial growth. The mostresistant strain tested in our study, Campylobacter sp,required a Pycnogenol® concentration of 250 µg/mL foreffective growth inhibition. Thus, formulations bearing0.025% Pycnogenol® would yield antimicrobial activityagainst all the microorganisms presented in Table 1.

Acknowledgements

The author wishes to thank Horphag Research UK for support tocarry out this study.

moulds. As dilution of the Pycnogenol®-containingmedia re-initiated the proliferation of microorganisms,Pycnogenol® apparently does not display microbicidalactivity.

These experiments do not allow conclusions to bedrawn about the nature of the antimicrobial effectof Pycnogenol®. There appears to be no correlation

REFERENCES

Rohdewald P. 2002. A review of the French maritime pine barkextract (Pycnogenol®), a herbal medication with a diversepharmacology. Int J Clin Pharmacol Ther 40: 158–168.

USP 28 – NF 23. The United States Pharmacopoeia andNational Formulary 2005.

Cowan MM. 1999. Plant products as antimicrobial agents. ClinMicrobial Rev 12: 564–582.

Kimbrough C, Chun M, de la Roca G, Lau BHS. 2002.Pycnogenol® chewing gum minimizes gingival bleeding andplaque formation. Phytomedicine 9: 410–413.