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A review on biological, nutraceutical and

clinical aspects of French maritime pine bark
extract
ARTICLE in JOURNAL OF ETHNOPHARMACOLOGY OCTOBER 2010
Impact Factor: 3 DOI: 10.1016/j.jep.2010.10.041 Source: PubMed
CITATIONS
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52
76
4 AUTHORS, INCLUDING:
Alya Maimoona
Ismat Naeem
Lahore College for Women University
13 PUBLICATIONS 170 CITATIONS
SEE PROFILE
SEE PROFILE
Zeb Saddiqe
SEE PROFILE
Available from: Alya Maimoona

Retrieved on: 19 March 2016
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Journal of Ethnopharmacology 133 (2011) 261277
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Review
A review on biological, nutraceutical and clinical aspects

of French maritime pine bark extract
Alya Maimoona a, , Ismat Naeem a , Zeb Saddiqe a , Khalid Jameel b
a
b
Department of Chemistry, Lahore College for Women University Lahore, Lahore, Pakistan
Combined military hospital, Kharian Cantt., Pakistan
a r t i c l e
i n f o
Article history:
Received 20 April 2010
Received in revised form 17 October 2010
Accepted 18 October 2010
Available online 31 October 2010
Keywords:
Pinus pinaster
Antioxidant
Nutritional supplement
Anti-inammatory
Proanthocyanidins
a b s t r a c t
Bark extract of Pinus pinaster has a long history of ethnomedicinal use and is available commercially
as herbal dietary supplement with proprietary name pycnogenol. It is used as a food supplement to
overcome many degenerative disorders. Rohdewald (2002) wrote the rst comprehensive review of
extract highlighting its antioxidative nature and its role in different diseases. Later, Watson (2003) and
Gulati (2005) in their reviews about cardiovascular health, described the extract as a best neutraceutical
agent in this regard. The objective of this paper is to review the current research on this extract in terms of
extraction methods, its pharmacological, toxicological and nutraceutical effects and clinical studies. Web
sites of Google Scholar, Pubmed and Medline were searched for articles written in English and published
in peer-reviewed journals from 2006 to 2009 and sixty-nine research articles were extracted. Of these,
two are about extraction advancement and analysis while the rest relate to its clinical, biological and
nutraceutical aspects.
2010 Elsevier Ireland Ltd. All rights reserved.
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Taxonomy and description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extraction and nger print analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pharmacokinetics of the extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biological effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Antioxidant activity of pine bark extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.1.
Free radical scavenging activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.2.
Synergism of PBE with synthetic antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.3.
Protecting biomolecules against oxidative damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.4.
Stimulation of androgen synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.5.
Protective effect against I/R-induced oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
262
262
262
263
263
263
263
263
263
263
263
Abbreviations: A, adrenaline; ACE, angiotensin converting enzyme; AD, Alzheimers disease; ADHD, attention decit hyperactivity disorder; ADRP, adipose
differentiation-related protein; AIDS, acquired immunodeciency syndrome; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AP, activator protein; ASA, acetylsalycylic acid; AST, aspartate aminotransferase; BHT, butylated hydroxytoluene; BUN, blood urea nitrogen; CAP, child attention problem; COX-1, cyclooxygenase-1; COX-2,
cyclooxygenase-2; CPRS, Conners Parents Rating Scale; CTARS, Conners Teacher Rating Scale; CVD, cardiovascular disease risk; CVI, chronic venous inefciency; DFO, desferrioxamine; DHEA, dehydroepiandrosteriod; DNP, dinitrophenyl; E, estrogen; EMC, encephalomyocarditis; EMCV, encephalomyocarditis virus; eNOS, endothelial nitric
oxide synthase; ESR, electron spin resonance; FBF, forearm blood ow; FLAP, ve-lox activating protein; fMLP, formyl-methionyl-leucyl-phenyalanine; FRAP, ferric reducing
antioxidant power; GE, grape extract; Gn-RHa, gonadotropin-releasing hormone agonist; GSH, glutathione; GSSH, oxidized glutathione; GST, glutathione-S-transferase; HPLC,
high performance liquid chromatography; ICVSTZ, intracerebro ventricular streptozotocin; IgE, immunoglobulin E; IOP, intraocular pressure; IR, ischemiareperfusion; LDH,
lactate dehydrogenase; LOX, lipooxygenase; LPS, lipopolysaccharide; MDA, malonaldehyde; MIC, minimum inhibitory concentration; MIDAS, migraine disability assessment; MMP, matrix metalloproteinases; MNCV, motor nerve conduction velocity; Mn-SOD, manganese superoxide dismutase; MPO, myeloperoxidase; NADPH, nicotin
amide adenine diphosphate hydrogenase; NAGA-N, acetyl beta-d-glucosaminidase; NA, noradranaline; NS, not signicant; NSAIDs, non-steroidal anti-inammatory drugs;
OA, osteoarthritis; OPC, oligomeric proanthocyanidins; PBE, pine bark extract; PC, protein carbonyl; PLA2, phospholipid A2; PMNL, polymorpho nuclear leukocyte; PMN,
polymorph nuclear neutrophils; PBE, pine bark extract; ROS, reactive oxygen species; RPMC, rat peritoneal mast cells; SNP, sodium nitroprusside; STZ, streptozotocin; TAS,
total antioxidant status; TBARS, thiobarbituric acid reactive substances; TLR4, toll like receptors 4-mediated signal; TXB2, thromboxane B2; VE, vitamin E; WBEA, whole
blood electrical aggregate; WHQ, womens health questionnaire; WOMAC, western Ontario McMaster; TXA2, thromboxane A2.
Corresponding author. Tel.: +92 42 992038019x245.
E-mail address: alya maimoona@yahoo.com (A. Maimoona).
0378-8741/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2010.10.041

262
A. Maimoona et al. / Journal of Ethnopharmacology 133 (2011) 261277
4.2.
Anti-inammatory activities of PBE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Stimulation of eNOS synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Antimicrobial activity of PBE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.
Antiviral activities of PBE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.
Clinical effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Cardiovascular disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.1.
Protective role against atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.2.
Endothelium-dependent vasodilatation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.3.
Platelet function and PBE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.4.
Hypertension and PBE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.5.
Chronic venous insufciency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.6.
Role of PBE in myocardial remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
PBE as anti-diabetic agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.1.
Effect of PBE on uptake of glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.2.
Effect of PBE on diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.3.
Effect of PBE on diabetic ulcers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.4.
Effect of PBE on camps and muscular pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.5.
CVD risk factors reduction in diabetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.6.
Lowering thromboxane level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
PBE and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.
Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.1.
Effect of PBE on glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.2.
Asthma and PBE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.3.
Osteoarthritis and PBE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.4.
PBE and skin care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.5.
PBE role in sexual disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.
Role of PBE in neurological disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
Cognition improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
PBE and ADHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.
Role of pine bark extract in migraine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.
Prevention of injuries caused by oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.
Non-clinical effects of PBE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1.
Nutritional effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.
Adverse effects/toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conict of interest disclosure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
For thousands of years, natural products have played a promising role throughout the globe in the treatment and prevention
of human diseases. To date, many natural products have been
extracted from barks of plants. A wide variety of products of diverse
chemical nature have their origin from bark, e.g. salicylic acid, the
acetylated form of which is aspirin, is obtained from Salix alba bark
(Tulp and Bohlin, 2004); avoring agent cinnamon from Cinnamomum zeylanicum (Jaradat, 2005) and alkaloids like Quinine from
Cinchona calisaya and Cinchona pubescens bark (Farnsworth and
Soejarto, 1985) and yohimbine from the bark of Corynanthe yohimbe
(Giampreti et al., 2008). The bark extracts which are a rich source
of phytochemicals with biological and physiological properties and
potential to be used as a medicine are of interest to humans.
Use of pine bark to reduce inammation can be traced back
to Hippocrates, the Father of Medicine (400 BC) (Packer et al.,
1999). Pine bark extract (PBE) from European coastal pine (Pinus
pinaster) was used by native Indians of Quebec. They introduced
French explorer Jacques Cartier and his crew, the pine-bark tea
during the winter of 1534 which proved wonderful in preventing
scurvy, a disease caused by deciency of vitamin C. Fascinated by
this information, Professor Jack Masquelier who was working on
bioavonoids suspected that bioavonoids might be used in the
treatment of scurvy. Later he determined that pine bark extract
was rich in bioavonoids including organic acids. These phytonutrients exhibit best free-radical scavenging activities. Such remedies
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are the human need because with better health cure facilities and
improved living conditions, the average life span of human beings
has increased adding a vast number of patients with degenerative
disorders caused by free radicals.
The present article encompasses all the research reports on
PBE for the period mentioned above. However, only controlled
human and animal trials, with better design and dose indication
are tabulated in Appendix A. Pilot studies with no control group
or untreated control group or methodological limitations are only
discussed.
1.1. Taxonomy and description
Pinus is the largest extant genus of the conifers in the family
Pinaceae (Farjon, 1984) with more than 100 species (Price et al.,
1998; Farjon, 2001). Some of them are cultivated world-wide (Le
Maitre, 1998). Kramer and Green (1990) placed the genus Pinus in
family Pinaceae under the class Pinatae in the subdivision Coniferophytina of Gymnosperms.
Pinus pinaster is a medium-sized pine up to 30 m tall with bright
reddish brown bark. Needles in the leaf spur are paired. Its cones
are oval, brown in colour and up to 2 cm long (Pullaiah, 2006).
2. Extraction and nger print analysis
Braga et al. (2008) revolutionized the extraction of antioxidants
from Pinus pinaster bark. Fractionated supercritical uid extraction

(FSFE) was done in two consecutive steps at different pressures and

temperatures using CO2 and CO2 + EtOH (10%) mixtures.
Quantication of avonoids was done by HPLC, and gas chromatography was used for characterization of oils. By applying
high pressure fractioned extraction methodology, two fractions
differing widely in wt.% and antioxidant activity were obtained.
Eighty four percent of the total extract with 2962% antioxidative activity was obtained in the rst step (CO2 ) whereas
second step extract (CO2 + EtOH) showed 6084% activity containing higher catechin + epicatechin (0.0510.346 g/mg d.b.) than the
ones obtained in the rst reaction (0.039 g/mg d.b.) inuencing
the antioxidant activity of the extract. Better extraction technique
made it possible to obtain extract fractions differing widely in their
antioxidant action.
Chen et al. (2009) introduced a ngerprint analysis for the bark
extract of Pinus pinaster (PBE dietary supplements) using an HPLC
method based on the USP monograph, which requires measurement of peak areas and identication of the four components of the
extract, i.e. caffeic acid, catechin, ferulic acid, and taxifolin. Additional qualitative information for the analysis of pycnogenol dietary
supplements (PDSs) can be obtained by this ngerprint analysis.
Standard PBE extract was helpful in assessing the efcacy of commercially available PDSs.
3. Pharmacokinetics of the extract

PBE when dried is a brown powder which remains stable
if kept in dark and dry place. Being rich in proanthocyanidins,
PBE is highly water soluble. It is quickly absorbed after oral
ingestion and its distribution in the body tissues is fast. In a
study by Grimm et al. (2006a,b), eleven volunteers, who were
kept on avonoid free diet for 24 h, were given a single dose of
300 mg of PBE. Their blood samples were taken after 14 h and
were analyzed using HPLC combined with ion pair reagents, UV
and electrochemical means. Plasma analysis, revealed maximum
concentrations (Cmax ) of catechin 107 ng/ml, caffeic acid 17 ng/ml,
ferulic acid 15 ng/ml, taxifolin 33 ng/ml and the metabolite M1
(delta-(3,4-dihydroxy-phenyl)-gamma-valerolactone) at 4 ng/ml.
Moreover ten unknown compounds were also identied and their
plasma levels and steady state were dened. Experiment was
then repeated by giving a dose of 200 mg to ve volunteers and
their plasma samples were taken after 4 h. The analysis of steady
state plasma samples revealed compounds as conjugates of sulfate
and/or glucuronic acid indicating phase II metabolism in the liver.
O
OH
OH
O
OH
H3CO
HO
OH
Caffeic Acid
Ferulic Acid
4. Biological effects
As PBE is a mixture of varying groups of chemicals, it is of no
surprise that it exhibits different modes of actions. Its major actions
include (I) antioxidant as radical scavenger, (II) antiinammatory
effect and (III) action through stimulation of eNOS synthesis.
Apart from the above mentioned actions, there are some studies
showing its antimicrobial and antiviral activities.
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4.1. Antioxidant activity of pine bark extract

4.1.1. Free radical scavenging activity
Jerez et al. (2007) compared the bark extracts of Pinus pinaster
and Pinus radiata for antiradical activities. Partial fractionation of
the crude ethanol extracts produced aqueous extracts of pFA and
rFA for Pinus pinaster and Pinus radiata respectively. The extracts
were found to contain the bulk of procyanidins (6587%) differing
in mean degree of polymerization (mDP) in the two sources. The
whole rFA exhibited best antiradical activity but for Pinus pinaster,
fractions with DP 722 showed best results.
4.1.2. Synergism of PBE with synthetic antioxidants
PBE enhances the activity of synthetic antioxidants showing
good synergism (Sivonova et al., 2006). PBE was found to increase
the antioxidant activity of synthetic antioxidants ascorbic acid and
trolox in model system of lipid peroxidation detected as conjugated dienes in liposomes and as protein carbonyls on oxidation
of proteins. Copper acetate and tert-butylhydroperoxide initiated
lipid peroxidation which was inhibited by bark extract and trolox
in a statistically signicant way depending on concentration and
time as compared to untreated unilamellar liposomes. An additive
preventive effect of PBE and trolox was observed in the oxidized
liposomes. On oxidation by two oxidative systems H2 O2 /FeSO4 and
HOCl, formation of carbonyl compounds in BSA and plasma proteins was strongly inhibited by bark extract in synergism with other
synthetic antioxidants ascorbic acid and trolox.
4.1.3. Protecting biomolecules against oxidative damage
PBE protects the biomolecules (proteins) against oxidative damage (Voss et al., 2006). Ansari et al. (2008) showed the protective
effect of PBE on induced oxidative damage in human neuroblastoma SH-SY5Y cell cultures.
4.1.4. Stimulation of androgen synthesis
PBE can stimulate the synthesis of physiologically active androgen DHT in the presence of nicotine which remained unaffected
by its toxic effects (Figuero et al., 2006). Interactive antioxidative
action of PBE with nicotine, coenzyme Q 10 (CoQ) and phytoestrogens was analyzed in a cell culture model. Human periosteal
broblasts and osteoblasts were incubated with 14C-testosterone
as substrate, in the presence or absence of CoQ (20 g/ml), bark
extract (150 g/ml), and phytoestrogens (10 and 40 g/ml), alone
and in combination with nicotine (250 g/ml). Medium was solvent
extracted and testosterone metabolites were separated by thinlayer chromatography and quantied using a radioisotope scanner
after incubation period. Pine extract was found to be active in
stimulating the synthesis of physiologically active androgen DHT,
individually and in combination with nicotine in both cell types.
4.1.5. Protective effect against I/R-induced oxidation
In vivo studies support the protective effect of bark extract
against I/R-induced oxidative renal damage (Sehirli and Sener,
2009). Destruction through oxygen free radicals is a well documented mechanism of action of ischemia induced injury. For this
purpose unilateral nephrectomy was done and Wistar rats were
subjected to 45 min ischemic insult by renal pedicle occlusion followed by 3 h of reperfusion. PBE (10 mg kg1 , i.p.) or saline was
administered at 15 min prior to ischemia and immediately before
the reperfusion. At the end of the study period (3 h), decapitation
was done. Trunk blood was collected for the analysis of different
parameters including creatinine, blood urea nitrogen (BUN), lactate dehydrogenase (LDH) TNF-alpha, IL-1beta, and IL-6 levels.
Tissue samples were taken for the determination of tissue malondialdehyde (MDA), glutathione (GSH) levels, Na+, K+-ATPase activity,
myeloperoxidase (MPO) activity and direct histologic examination

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of tissue. The results revealed that ischemia/reperfusion caused a

signicant decrease in tissue GSH level and Na+, K+-ATPase activity, while signicant increases in the renal MDA level and MPO
activity were noted. Similarly, saline-treated I/R group showed high
levels of serum creatinine and BUN, as well as LDH and IL-1, IL6, and TNF- level, as compared to saline-treated control group.
Conversely, PBE treated group showed reversion of all these biochemical indices, as well as histopathological alterations that were
induced by ischemia and reperfusion I/R.
Busserolles et al. (2006) studied the in vivo effects of procyanidin rich-extract on oxidative stress in rats in two conditions, i.e.
rats were given diet supplemented with grape extract (GE) and
pine extract (PE) either for a period of 8 weeks or given single
dose only to observe the postprandial effect. At the end, a signicant increase in total antioxidant capacity in plasma was observed
in the GE and PE treated group which was given supplemented
diet for 8 weeks as compared to control and postprandial groups.
However heart susceptibility to peroxidation and plasma TBARS
concentration did not differ signicantly between the groups. In the
postprandial study 2 h after ingestion, experimental group which
was given procyanidin-rich extracts (500 mg/kg of body weight)
showed higher concentration of ferric reducing antioxidant power
(FRAP) as compared with the control of this group and difference
was signicant in GE group.
4.2. Anti-inammatory activities of PBE

The mechanism of action of bioavonoids from Pinus maritime
in inhibiting the gene expression of the proinammatory cytokines
was studied by Cho et al. (2006). They selected RAW264.7 cells for
the study of cytokine interleukin-1 (IL-1) and Jurkat E6.1 cells
for interleukin-2 (IL-2). Strong scavenging activity was exhibited by
PBE against H2 O2 generated oxygen reactive species in RAW264.7.
ELISA, immunoblot analysis, and competitive RT-PCR indicated that
IL-1 and its mRNA levels are reduced dose dependently if LPSstimulated RAW 264.7 cells were pretreated with PBE. Moreover,
major factors involved in the expression of IL-1, i.e. nuclear factor
B (NF-B) and activator protein-1 (AP-1) are also blocked by PBE.
LPS-induced IB degradation is abolished by PBE. Inhibitory effect
of PBE on IL-2 gene expression in phorobol 12-myristate 13 acetate
plus ionomycin (PMA/Io)-stimulated human T-cell line Jurkat E6.1
was different. Both NF-AT and AP-1 chloramphenicol acetyltransferase (CAT) in transiently transfected Jurkat E6.1 were inhibited
but NF-k B CAT activity was not affected. PBE has destabilizing effect
on PMA/Io-induced IL-2 mRNA by posttranscriptional regulation. It
can be concluded that bioavonoids can serve as broad spectrum
therapeutic agents in treating many inammatory, autoimmune,
and cardiovascular diseases (Figs. 1 and 2).
The anti-inammatory response of PBE on the arachidonic
acid pathway in human polymorphonuclear leukocytes (PMNL)
was observed by Canali et al. (2009). PBE in dose of 150 mg/day
was given for ve days to healthy volunteers with age limit
of 3550. Blood was drawn before and after supplementation
Fig. 1. Radical scavenging action of PBE.
isolating PMNL which were primed with lipo-polysaccharide

(LPS) and stimulated with the receptor-mediated agonist formylmethionyl-leucyl-phenylalanine (fMLP) to activate the arachidonic
acid pathway and leukotrienes biosynthesis. Gene expression of 5lipoxygenase (5-LOX) and (COX-2) and activity of phospholipids
A2 (PLA2) was blocked by PBE supplementation associated with
a compensatory up-regulation of COX-1 gene expression. Interdependency between 5-LOX and 5-LOX activating protein (FLAP)
expression was suspended by PBE. The anti-inammatory response
of PBE is due to COX-2 and 5-LOX gene expression reducing
leukotriene biosynthesis in human PMNL.
A prospective study was conducted to evaluate the efcacy of
PBE as a potent antiallergic drug (Choi and Yan, 2009) by suppressing the immune-mediated allergic responses. PBE inhibited
liberation of histamine and other cytokines when they are stimulated through IgE mediated immune response. Both in vitro and
in vivo models were used in this study. In in vivo study of rats,
oral administration of extract resulted in signicant inhibition of
anti dinitrophenyl (DNP) IgE mediated passive skin anaphylaxis.
The in vitro study, conducted on rat peritoneal mast cells (RPMCs),
triggered by anti DNP IgE, revealed the following effects:
a. Dose dependent reduction of histamine release by rat peritoneal
(RPMC) mast cells.
b. Inhibition of protein expression and secretion of tumor necrosis
factor-alpha (TNF-Alpha) and interleukin 6 (IL 6).
c. Reduction of calcium uptake by RPMC.
d. Suppression of nuclear factor-kappa B activation.
Fig. 2. Inhibition of COX-2 expression.

The animal study looks promising but clinical trials in humans

are needed.
The anti-inammatory activity of PBE was assessed by observing
its effects on carrageenan-induced inammation in rat model (Ince
Yesil-Celiktas et al., 2009). The parameter for anti-inammatory
activity was paw edema in mice. Paw volume was measured
before and after different intervals of carrageen injection. PBE was
administered intraperitoneally that inhibited paw swelling dosedependently at 2, 3, 4, 5 and 6 h after carrageen injection. PBE
showed signicant anti-inammatory activity at doses of 75 and
100 mg/kg body weight between 2 and 4 h after administration
(P < 0.05), respectively.
The inhibitory effect of PBE on cyclooxygenases (COX-1 and
COX-2) activity in the serum samples of human volunteers after
intake of PBE was observed by Schafer et al. (2006). Blood samples
from ve healthy persons before and after 200 mg oral administration of PBE for 5 days were taken. COX-1 and COX-2 activities were
inhibited moderately. In the next step, a single dose of 300 mg PBE
was given to ten volunteers followed by collection of blood samples only after 30 min of ingestion. Statistically signicant increase
in the inhibition of both COX-1 (P < 0.02) and COX-2 (P < 0.002) was
observed. This inhibition supports the anti-inammatory activity
of PBE in human.
In another study, the presence of sufcient amount of active
principles to inhibit key mediators of inammation in human
plasma after oral ingestion of PBE was determined (Grimm et al.,
2006a,b). After ve days administration of 200 mg PBE/day, blood
samples from seven healthy volunteers were taken. Inhibition of
release of matrix metalloproteinase 9 from human monocytes and
NF-kB activation was statistically signicant.
Maroon et al. (2006) highlighted the deleterious side effects
of non steroidal drugs often prescribed to athletes facing musculoskeletal injuries and reviewed the natural supplements as
alternative for pain relief. Procyanidins from pine bark were highly
appreciated due to their anti-inammatory nature.
4.3. Stimulation of eNOS synthesis
The endothelium-dependent vasodilation facilitated by NO is an
important factor of vascular function. PBE activates the endothelial
nitric oxide synthase accompanied by NO release from endothelial
cells (Nishioka et al., 2007) and it can be reversed to cardiac remodeling induced by an inhibitor (L-NAME) of NO synthase (Zibadi et al.,
2007).
4.4. Antimicrobial activity of PBE
Owing to resistance of Helicobacter pylori strain to antibiotics,
alternative therapy for the treatment of this infection was tried in
the form of PBE (Rohdewald and Beil, 2008). This study was carried
on in liquid medium as well as in vitro model using sessile bacteria attached to AGS cells. Gastric cells, PBE and Helicobacter pylori
were co-incubated in vitro to determine the adherence. PBE was
effective with an MIC (50) of 12.5 g/mL in inhibiting the growth
of Helicobacter pylori in suspension. A reduction of 10% of the control value was observed by 125 g/mL PBE and adherence to gastric
cells was reduced to 70% with the same concentration after incubation of 3 h. PBE showed signicant, yet limited antibacterial activity.
For clinical purpose, in vivo study is needed.
Comparative antimicrobial effect of Pinus pinea bark extract and
PBE against Staphylococcus aureus in cooked red meat was analyzed
by Hames Kocabas et al. (2008). To raw meat, 1% concentration of
Pinus pinea extract and PBE were added along with Staphylococcus aureus inoculated at 103 cfu/concentration. Then the meat was
stored at 4 C for 9 days. Reduction in numbers of Staphylococcus
aureus was detected as 7.9 102 cfu/g after 6 days that reduced to
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7.1 102 cfu/g after 9 days; and 8.6 102 cfu/g and 9.8 102 cfu/g
for Pinus pinea and PBE respectively. For the control, values of
13.7 102 and 17.2 102 cfu/g were obtained after 6 and 9 days
of storage respectively indicating the antimicrobial effect of bark
extracts. From the study it can be concluded that these extracts
might be suggested as natural preservative in food industry.
4.5. Antiviral activities of PBE
Inhibition of NF-B-dependent gene expression revealed
diverse anti-inammatory activity of PBE. Keeping this in view,
effect of PBE on viral myocarditis was analyzed (Matsumori et al.,
2007). Intraperitoneal inoculation with 10 plaque-forming units
(pfu) of the encephalomyocarditis (EMCV) virus were given to
four-week-old inbred male DBA/2 mice. Oral administration of
PBE at a dose of 1 or 10 mg/kg/day for the histological study and
10 or 100 mg/kg for gene expression study was given from the
rst day of viral inoculation. PBE was found to exert inhibitory
effects on viral myocarditis by decreasing viral replication and
by suppressing expression of pro-inammatory cytokine. With
increasing dose of PBE (10 mg/kg), the myocardial inltration and
necrosis was 16.2 8.9% and 19.2 9.7%, respectively, compared
with controls 27.6 15.0% and 30.1 15.7%, respectively, on 7th
day of inoculation. Non-signicant trend towards less myocardial inltration in the PBE treated (1 mg/kg) group was observed.
In group treated with 1 mg/kg of PBE, myocardial virus concentrations were 8.4 0.3 103 pfu/mg on day 7 while control mice
exhibited 2.7 0.6 104 pfu/mg on the same day showing statistically signicant difference (P < 0.05). A 100 mg/kg PBE treatment
results in signicant suppression of gene expressions of tumor
necrosis factor, type-I procollagen, stem cell factor, and mast cell
tryptase.
Matsumori (2007) working on EMCV animal model, found the
inhibitory effect of PBE on viral replication by suppressing the
expression of pro-inammatory cytokines and mast cell-related
mediators and thereby improving the inammation and myocardial necrosis. Like Pimobendan and FTY720, PBE is a promising
agent against viral myocarditis.
It was reported that replication of human immunodeciency
virus type-1 (HIV-1) after entry in susceptible cells in vitro was
also inhibited by PBE in addition to binding to host cells. PBE
caused prominent alterations including elevated expression of
an intracellular antioxidant protein, manganese superoxide dismutase (Mn-SOD), and the inhibition of phosphorylation of the
ribosomal S6 protein. Interestingly, ectopic expression of Mn-SOD
inhibited HIV-1 replication as well suggesting its potential as a new
anti-HIV-1 agent (Feng et al., 2008).
5. Clinical effects
5.1. Cardiovascular disorders
Though PBE is found to have many different clinical effects but
so far the most well studied use is in cardiovascular disorders.
Many controlled human clinical trials in vascular diseases have
been operated.
5.1.1. Protective role against atherosclerosis
Atherosclerosis is a major vascular disorder responsible for most
of the cardiovascular ill health, e.g. MI (heart attacks) and CVA
(strokes). Radical scavenging effect of PBE strengthens antioxidant defense system of the body at cellular level. Combined with
endothelium dependent vasodilation through NO synthase leads to
better health and function of endothelium.

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Recently an important role has been assigned to adipose

differentiation-related protein (ADRP) in the pathogenesis of
atherosclerosis (Gu et al., 2008). It has been demonstrated that Tolllike receptor (TLR) 4-mediated signals, involved in atherosclerotic
plaque formation, enhance the expression of ADRP in macrophages.
LPS enhances ADRP expression in macrophages through TLR4mediated signals as macrophages from TLR4-decient mice fail
to respond to this stimulus. This LPS effect in ADRP expression
is at transcriptional level with synthesis of new protein resulting
in the enhancement of ADRP promoter activity, i.e. stimulation of
AP-1 binding to the Ets/AP-1 element. Moreover LPS also induces
the expression of interleukin (IL)-6, IL-1alpha, and interferon-beta
mRNAs, all of which in turn stimulate the ADRP expression. This is
strengthened by the fact that antibodies against these cytokines
or inhibitors of c-Jun NH(2)-terminal kinase and nuclear factor
(NF)-kappa B suppress the ADRP mRNA level. PBE, suppresses
the expression of ADRP and other cytokines. Additionally PBE
also suppresses the ADRP promoter activity and enhancer activity
of AP-1 and NF-kappa B, resulting in decreased ADRP expression. However it did not affect the LPS-induced DNA binding of
these factors.
Prevention of atherosclerosis will have a profound effect in controlling vascular events but this in vitro study is quite limited.
Further studies and controlled human trials are required to assess
the role of PBE in this very important disease.
5.1.2. Endothelium-dependent vasodilatation
The effect of PBE on endothelium-dependent vasodilations in
humans was carried out in a double-blind, randomized, placebocontrolled study (Nishioka et al., 2007). Responses of forearm
blood ow (FBF) to acetylcholine; an endothelium-dependent
and sodium nitroprusside (SNP); an endothelium-independent
vasodilators in 16 healthy young men in two groups before and after
2 weeks of daily oral PBE administration (180 mg/day) or placebo
were analyzed. Forearm or systemic hemodynamics remained
unaffected in both PBE and placebo. But an increase in FBF response
to Ach (acetylcholine) from 13.1 7.0 to 18.5 4.0 ml/min/100 ml
tissue (P < 0.05) in PBE group was found. SNP showed same vasodilation before and after treatment in both groups. PBE induced FBF
response to Ach was completely abolished by the administration
of NO synthase inhibitor; NG-monomethyl-l-arginine supporting
that PBE played its role in endothelium-dependent vasodilation by
increasing NO production. This study is limitated by small number
of subjects.
5.1.3. Platelet function and PBE
The in vitro ability of PBE in improving the efcacy of acetylsalicylic acid (ASA) was evaluated (Golanski et al., 2006). Blood from
thirty-eight volunteers was anticoagulated with hirudin and incubated with PBE at concentrations 10, 50, and 100 g /ml and/or
ASA at concentrations 25 and 100 g/ml for 20 min at room temperature. Whole-blood electrical aggregation (WBEA), and PRP
aggregation was employed for the investigation of platelet functions. Platelet agonists were collagen (1 g/ml) and ADP (5 g/ml).
Using ethanol-dissolved PBE, a synergistic effect of PBE and ASA in
inhibiting collagen-induced aggregation in PRP was observed.
These studies are encouraging regarding the improvement of
impaired responses to ASA therapy. ASA combined with PBE may
be favorable but in vivo trials are needed to support the idea.
5.1.4. Hypertension and PBE
Antihypertensive treatment may cause edema in essential
hypertension by increasing the capillary exchange surface and capillary inltration. In an 8-week, placebo-controlled, single-blinded
study, value of PBE in preventing antihypertensive treatmentinduced edema was evaluated (Belcaro et al., 2006a,b). Two
treatment groups of hypertension treated with calcium antagonist (nifedipine) or angiotensin-converting enzyme inhibitor for
at least 4 months were selected to see the effectiveness of
PBE in edema prevention with dose of 150 mg/day. Capillary ltration was calculated by strain-gauge plethysmography, which
measured the size enlargement or reduction of tissue at the
levels of the foot. PBE groups showed signicant decrease in
capillary ltration (2.44 mL/min/100 cm3 vs. 1.56 mL/min/100 cm3
of the tissue respectively; P < 0.05) as compared to nifedipine (2.48 mL/min/100 cm3 vs. 1.61 mL/min/100 cm3 of the tissue
respectively; P < 0.05).
Though this study shows the benecial effect of PBE in preventing antihypertensive drug (Nifedipine)-induced edema but it
is limited by poor design and short duration of the study.
5.1.5. Chronic venous insufciency
Cesarone et al. (2006a,b,c) highlighted the clinical efcacy of
oral PBE in patients with severe chronic venous insufciency (CVI).
A total of 21 patients were included in the treatment group and 18
equivalent patients were observed as control (no treatment during the observation period). All 21 patients in the treatment group
completed the 8-week study. Also the 18 controls completed the
follow-up period. There were no drop-outs. Ambulatory venous
pressure was 59.3 (SD 7.2; range 5068) on average with a relling
time shorter than 10 s (average 7.6; SD 3). Ambulatory venous pressure or relling time between the treatment and control patients
were not different. On average, disease duration was 5.7 years from
the rst signs/symptoms. A progressive decrease in skin ux, a
signicant decrease in capillary ltration, a signicant improvement in the symptomatic score, and a reduction in edema had been
observed in all PBE-treated subjects after 4 and 8 weeks. Controls
showed no visible effects.
Again study design does not meet the criteria of a good trial as
control group was not offered treatment.
Comparative effect of oral PBE and Daon (a combination of
diosmin and hesperidin) in patients with severe CVI was observed
in a prospective, randomized, and controlled study extended up to
8 weeks (Cesarone et al., 2006a,b,c). Oral PBE (capsules) 150 mg or
300 mg daily for 8 weeks or Daon, 1000 mg/day was given to 3
groups of 86 patients with severe CVI, venous hypertension, ankle
swelling and with a history of venous ulcerations. Microcirculatory results indicated a signicant level (p < .05) of improvement
achieved after 4 weeks of treatment in most patients of the PBE
group while only 6 subjects in the Daon group showed signicant
improvement. Overall effects of PBE after 8 weeks were signicantly superior in comparison to the Daon group.
This well-designed and properly cared out trial shows that PBE
may be a successful treatment for chronic venous insufciency.
However the dose of Daon used in this study is low to prove the
efcacy of Daon.
5.1.6. Role of PBE in myocardial remodeling
Reversing cardiac remodeling is the real target in cardiac therapy. In a study by Zibadi et al. (2007) cardiac remodeling was
induced in old C57BL/6N mice by blocking nitric oxide synthase
activity by NG -nitro-l-arginine methyl ester (l-NAME) administration in a model of heart failure. Dilated cardiomyopathy at
compensated stage was characterized by signicant increase of
pro-matrix metalloproteinase (MMP)-9 gene expression and activity resulting in the reduction of total and cross-linked collagen
content of the heart and a marked decrease in pro-collagen III1
gene expression was also observed. When l-NAME-exposed mice
was given PBE 30 mg/kg, a pronounced decrease in gene expression of pro-MMP-2, -9, and -13 and MMP-9 activity was seen
accompanied by signicant increase in cardiac tissue inhibitor of
metalloproteinase (TIMP)-4 expression. These results in reversing

of cardiac remodeling showed the positive role of PBE in improving

myocardial remodeling.
This study indicates that PBE supplement may be helpful in limiting the cardiac tissue damage in patients with heart failure.
5.2. PBE as anti-diabetic agent
5.2.1. Effect of PBE on uptake of glucose
Studies on diabetics after peroral ingestion of PBE revealed
its efcacy in delaying the uptake of glucose from a given meal.
PBE was found to be 190 times more potent than acorbase
(a prescription medicine used for the purpose) in preventing
the typical high-glucose peak in the blood stream after a meal
(Schafer and Hogger, 2007). PBE has no effect on insulin secretion, rather it inhibits synthesis of alpha-glucosidase (intestinal
enzyme involved in carbohydrate digestion). The inhibitory activity
of PBE, green tea extract and acarbose towards alpha-glucosidase
was analyzed. To explore the component responsible, different fractions of PBE having diverse masses from polyphenolic
monomers, dimers and higher oligomers were used. The higher
concentrations of procyanidins (avonoids) were found to be
responsible for these excellent results. This crucial study showed
that PBE is the most potent inhibitor of (IC50 about 5 g/ml)
alpha-glucosidase as compared to green tea extract (IC50 about
20 g/ml) and acarbose (IC50 about 1 mg/ml) proving vital role
of oligomeric procyanidins in glucose lowering effects of PBE in
diabetic patients.
5.2.2. Effect of PBE on diabetic retinopathy
Diabetic retinopathy is the result of weakened enzymatic
antioxidant defenses by uncontrolled diabetes mellitus. Kamuren
et al. (2006) established that a balanced oxidative condition can
be maintained by a low-carbohydrate, high-fat diet either alone
or supplemented with PBE. In the study normal and induced
diabetic rats were fed either a regular or low-carbohydrate diet
for 30 or 90 days. In addition, normal and diabetic rats on the
chronic (90-days) low-carbohydrate diet were treated with daily
intraperitoneal PBE doses (10 mg/kg) for 14 consecutive days.
Assays on retinal enzymes activities were done by fractionating
the retina. After 30 days, the glycemic parameters got reduced
by low-carbohydrate diet and aspartate aminotransferase activity
was normalized in diabetic animals, suggesting less organ damage. Cataract formation was inhibited in treated diabetic animals
whereas all diabetic control animals developed cataracts bilaterally. Increased activities of retinal glutathione peroxidase and
glutathione reductase enzymes with PBE supplemented diet, suggested that a low-carbohydrate diet plus PBE may be an effective
remedy and antihyperglycemic therapy against diabetic retinopathy and cataract formation.
The protective effects of PBE on the immediate onset of retinopathy were observed and visual improvement was perceived by 18
out of 24 patients treated with PBE (Steigerwalt et al., 2009).
5.2.3. Effect of PBE on diabetic ulcers
Diabetic microangiopathy is the cause of lower limb ulcers that
takes much time to heal. Belcaro et al. (2006a) analyzed the effect of
PBE on diabetic ulcers in a controlled trial. Subjects were classied
into 4 groups and medications were used as: (1) PBE systemic and
local application combined; (2) local PBE only; (3) oral PBE; and (4)
medications only (control group). Improvement of ulcerated areas
and symptom scores were found to be in the order accordingly, i.e.
best result with the combined oral and local treatment (P < 0.05).
Oral and local treatment separately was less effective, but still better in comparison to the control group. Healing percentage at six
weeks were 89%, 84%, 85% and 61% respectively. Single treatment
groups showed better microcirculation when given the combined
267
treatment. Healing percentages in treatment groups did not differ widely but difference was comparable to control group. The
study showed that combined treatment of PBE offers a speedy treatment of diabetic ulcers and is more effective than the prescribed
medication. However, the study conducted is not randomized and
blinded.
In another trial the clinical efcacy of oral PBE was analyzed
in diabetic patients in a 4-week study while controls completed
the follow-up period without any dropout. All the patients with
same microangiopathy were treated. Three groups were managed
one of which was without a history of diabetic ulcerations and
remaining two were affected with severe microangiopathy. Of the
affected groups, one was kept as control, i.e. no treatment given.
Subject group and one patient group received oral PBE (50 mg
capsules, 3 times daily for a total of 150 mg daily for 4 weeks).
Treatment groups complete duration of diabetes (from the rst
signs/symptoms) was on average 7.5 years (SD = 3). At the end,
clinical results indicated an improvement in the level of microangiopathy in all treated subjects as compared to control (Cesarone
et al., 2006a,b,c). The study is un-controlled, non-blinded and nonrandomized.
5.2.4. Effect of PBE on camps and muscular pain
The preventive action of PBE on cramps and muscular pain in
different groups of subjects and patients was assessed and efcacy
of PBE after withdrawal was also evaluated. The average number
of episodes was reduced from 4.8 (1.2) events per week to 1.3 (1.1)
at 4 weeks (p < 0.05) in normal subjects. In patients with venous
insufciency, the observed decrease in events was from 6.3 (1.1)
to 2.6 (0.4) per week (p < 0.05). In athletes the number of episodes
decreased from 8.6 (2) to 2.4 (0.5) (p < 0.05). The decrease was still
present at 5 weeks in the 3 groups, to levels signicantly lower than
inclusion values (p < 0.05). In the second part of the study, patients
with intermittent claudication and diabetic microangiopathy were
evaluated and treated (4 weeks). The groups treated with PBE and
the control, placebo groups were comparable. There was a signicant decrease in the number of cramps episodes (p < 0.05) and in the
score concerning muscular pain (p < 0.05) in claudicants and diabetics. No signicant effects were observed in the placebo groups
(Vinciquerra et al., 2006).
The effects of PBE at various doses on levels of pre and postprandial glucose, thiobarbituric acid reactive substances (TBARS),
N-acetyl-beta-d-glucosaminidase (NAGA) and on motor nerve conduction velocity (MNCV) in STZ-induced diabetes rats was analyzed
(Jankyova et al., 2009). PBE was found to be effective in lowering glucose level at all doses and specically at doses of 10 mg/kg
b.wt./day and 20 mg/kg b.wt./day. It signicantly decreased elevated levels of postprandial glycaemia but failed to induce a
signicant decrease in the same at a dose of 50 mg/kg b.wt./day.
Signicant improvement in impaired MNCV with PBE at doses
of 10 and 20 mg/kg b.wt./day was observed but levels of TBARs
and NAGA remain unaffected despite the treatment of PBE. The
effect of PBE on postprandial sugar levels and MNCV was not dosedependent.
5.2.5. CVD risk factors reduction in diabetics
Kilmas et al. (2009) studied the role of PBE in cardiac oxidative stress and the protein expression of reactive oxygen species
(ROS)-producing molecules [gp91(phox)-containing NADPH oxidase and NO-signalling proteins] by using streptozotocin induced
diabetic cardiomyopathy in rat. In the study hyperglycemia and left
ventricular catheterization in vivo were used to measure extent
of experimental diabetes mellitus and impaired cardiac function.
Reduction in fasting plasma glucose and normalization of basal
cardiac function was noted with PBE. Excessive oxidative stress
in streptozotocin (STZ) induced rats (suggested by 40% increase

268
of thiobarbituric acid reactive substances TBARS concentration)

was associated with increased expression of gp 91(phox) iNOS
and alpha-tubulin but unchanged expression of eNOS and its
alosteric regulators, as compared to control. PBE failed to affect
these expression abnormalities. The study concluded that PBE can
control diabetic cardiac dysfunction, without affecting the molecular maladaptations of ROS-producing enzymes and cytoskeletal
components.
In a randomized, double-blind, placebo-controlled trial with
parallel-group design, effect of PBE on blood pressure, fasting
plasma glucose, low-density lipoprotein LDL cholesterol, glycosylated hemoglobin (HbA1c), serum endothelin-1, and urinary
albumin was evaluated. 58.3% of subjects with PBE treatment
achieved BP control at the end of the 12 weeks with 50% reduction in individual pretrial dose of ACE-inhibitors (P < 0.05). There
was 3.9 pg/ml decrease observed in plasma endothelin-1 in PBEtreated group vs. 0.5 pg/ml increase in control group (P < 0.001).
Mean HbA1c shows decline by 0.8% in PBE-treated group (P < 0.05),
whereas it decreases by 0.1% in control group. Fasting plasma glucose level lowered to 23.7 mg/dL in PBE-treated group vs. 5.7 mg/dl
in control group (P < 0.0001). LDL also got improved signicantly
in PBE-treated group, declining by 12.7 mg/dl (P < 0.001). A pronounced decrease was observable in urinary albumin level at 8
weeks compared with the control group (P < 0.05) but not significant after 12 week supplementation. Improved diabetes control,
lowered CVD risk factors, and reduced antihypertensive medicine
use vs. controls were achieved with PBE supplementation (Zibadi
et al., 2008).
5.2.6. Lowering thromboxane level
Diabetes is associated with increased thromboxane level causing severe vascular complications. The antithrombotic effect of PBE
was studied by Nocun et al. (2008). The researchers assessed the
level of plasma thromboxane A2 (TXA2) and thromboxane B2
(TXB2) by enzyme-linked immunosorbent assay in Wister male
rats, induced to insulin-dependent diabetes. The effects of long
term PBE intake on these enzymes were also evaluated. PBE
(5 mg/kg b.wt.) and acetylsalicylic acid (10 mg/kg b.wt.) were found
to have signicant inhibitory effect when given separately and
effect was higher in the case of acetylsalicylic acid. Moreover no
improvement in effectiveness of ASA-mediated decrease in TXB2
generation by simultaneous administration of PBE and ASA was
observed.
5.3. PBE and cancer
In a study to explore the chemo preventive effect of PBE, ovarian
cell lines and polymorph nuclear neutrophils (PMN) were treated
with talc and with PBE followed by talc. Soft agar assay was used to
measure cell viability, reactive oxygen species (ROS) generation and
neoplastic transformation. Effect of talc was time-dependent in the
ovarian cells and dose-dependent in PMN setting the induction of
neoplastic transformation and increased cell proliferation and ROS
generation. PBE pretreatment showed inhibitory effect on all talcinduced activities proving its worth in cancer chemoprevention
(BuzZard and Lau, 2007).
The efcacy of PBE in alleviating the adverse effects of chemo
and radio therapies of cancer patients was explored (Belcaro et al.,
2008a,b). Two groups of cancer patients were treated with PBE
(150 mg) and placebo in a single-blinded design. All the investigated side effects were less frequent in 25 PBE treated patients
which had undergone radiotherapy as compared to 21 placebos,
though difference was limited in many categories. The incidence of
vein thrombosis in PBE and control group was 2.9% and 10% respectively. Similarly effects of chemotherapy on 34 patients treated
with PBE prior to chemotherapy were better as compared to 30
patients in control. All patients with rst session of chemotherapy

were included in the study. Better tolerance of chemotherapy and
less days of hospitalization of patients treated are suggestive of PBE
role in this condition. Study is limited by its design.
5.4. Miscellaneous
5.4.1. Effect of PBE on glaucoma
Glaucoma (intraocular hypertension) is an important eye disease which is a leading cause of non-cataract blindness. In this
condition IOP is raised. In a product evaluation study, the effects of
MirtogenolTM (Mirtoselect and PBE), a food supplement, on intraocular pressure (IOP) were evaluated using 38 asymptomatic subjects
with intraocular hypertension, Mirtogenol was found to be a preventive agent of glaucoma by lowering the IOP and improving the
ocular blood ow (Steigerwalt et al., 2008). Twenty subjects were
given Mirtogenol leaving 18 untreated taken as control. The mean
IOP decreased from a baseline of 25.2 mm Hg to 22.2 mm Hg and
22.0 mm Hg after two and three months of treatment respectively.
After three months with Mirtogenol, IOP was signicantly lowered
(P < 0.05) in comparison to control. No further change was observed
after six months. More studies are needed to evaluate the role of
PBE alone in this case.
5.4.2. Asthma and PBE
Watson et al. (2007), based on the literature review, presented
the role of PBE as a nutritional therapy for asthma risk factors
in desert environment of Southwest of USA where fungal spores,
Bermuda grass pollens and extreme xeric condition have increased
the severity of the disease. PBE was found to be effective in reducing
the symptoms in both old age and childhood asthma.
5.4.3. Osteoarthritis and PBE
Osteoarthritis is a degenerative joint disease caused by the damage to the cartilage covering the bones. It primarily involves the
weight bearing joints, e.g. knee and hips. The effect of PBE on knee
osteoarthritis (OA) was observed. In a randomized, double-blind,
placebo-controlled trial with parallel-group design, 37 osteoarthritis patients were undertaken. Patients were given either PBE or
placebo pills (50 mg, three times daily). Western Ontario and
McMaster Universities (WOMAC) Osteoarthritis Index was used to
evaluate monthly osteoarthritis clinical symptoms in addition to
the assessment of non-steroidal anti-inammatory drugs (NSAIDs)
or selective (COX-2) inhibitors usage. A signicant improvement in
WOMAC score was observed in test group lowering the frequency
of NSAIDs or COX-2 inhibitors usage compared to placebo group
with increased medication. The study indicated the efcacy of PBE
in osteoarthritis (Farid et al., 2007). This promising trial in a very
common disease effecting millions of people needs further studies
with large number of patients.
Cisar et al. (2008) studied the effect of PBE on knee osteoarthritis stages I and II in a double-blind, placebo-control trial treating
100 patients with 150 mg/day PBE for three months. After that
patients lled the WOMAC questionnaire every 2 weeks reporting
any change in the use of previously prescribed anti-inammatory
medication during the study period. Using a Visual Analogue
Scale for pain intensity, weekly pain symptoms were evaluated.
An improvement of WOMAC index (P < 0.05) was observed along
with signicant pain alleviation (P < 0.04) in comparison with
placebo group having no effect. Study is not randomized. Trial
pattern is similar to Farid et al. (2007) but with larger group
size.
In a double-blind, placebo-controlled study, the efcacy of
PBE in dose of 100 mg/day taken orally for 3 months in victim
of osteoarthritis (OA) was evaluated. WOMAC scores and their
walking performance (treadmill) were used for the evaluation

of OA symptoms and mobility respectively. All the parameters

including age, sex, WOMAC scores, walking distances and use of
anti-inammatory drugs were comparable in both treatment group
of 77 patients and placebo group of 79 patients. At the end of
the study, treatment group showed a decline of 56% (P < 0.05)
in WOMAC scores vs. 9.6% in the placebo group. Walking distance in the treatment group was improved from 68 m to 198 m
(P < 0.05), in treatment group vs. placebo from 65 m to 88 m (NS).
Use of drugs was reduced to 58% (P < 0.05) in treatment group
and 1% under placebo. Treatment group showed 63% gastrointestinal improvement as compared to 3% under the placebo. Foot
edema was reduced to 79% (P < 0.05) in the PBE treated group vs.
1% in controls. PBE was found to be a best alternative of the antiinammatory drugs (Belcaro et al., 2008a,b). The study appears to
be well-designed and executed.
All these studies in this common problem which effects large
number of people show encouraging results and PBE is shown to
reduce the symptoms of OA.
5.4.4. PBE and skin care
The ability of PBE to inhibit tyrosinase activity and melanin
biosynthesis was investigated using cultured B 16 melanoma cells
(B 16). Using electron resonance spectrometer, suppressive effect
of PBE against peroxynitrile (ONOO ), superoxide (O2 ), nitric oxide
(NO) and hydroxyl radical (OH)-scavenging activities were also
examined to know about anti-oxidative power of PBE. In vitro
assays, PBE suppressed these radicals and up-regulated the reduced
glutathione/oxidized glutathione ratio in B 16 cells. So PBE showed
anti-melanogenic activity (Kim et al., 2008).
The effect of antioxidant mixture of vitamin C, vitamin E, PBE,
and evening primrose oil given orally was studied for UVB-induced
wrinkle formation (Cho et al., 2007). Using mouse dorsal skin, possible molecular mechanism of photo protection against UVB through
inhibition of collagen-degrading activity or through enhancement
of procollagen synthesis was also investigated. For the purpose,
hairless SKH-1 female mice were selected and in two groups treated
with antioxidant mixture taken as test group and vehicle as control group accompanied by 10 weeks UVB irradiation three times
a week. The intensity of irradiation was gradually increased from
30 to 180 ml/cm2 . At the end of 10 weeks the evaluation of wrinkle formation was done through microtopographic and histological
assessment of the dorsal skins. Changes in the balance of collagen
synthesis and collagen degradation were investigated by Western
blot analysis and antioxidant mixture proved to be signicantly
effective in reducing chronic UVB-induced wrinkle formation by
inhibiting UVB-induced MMP activity along with the enhancement
of collagen synthesis.
Chang et al. (2009) studied the effects of a mixture of vitamin E, vitamin C, PBE and evening primrose oil (mixture LGNC-5)
on UV-induced pigmentation and wrinkle reductions of normal
healthy human volunteers. A dose of 4 capsules of the mixture
LGNC-5 (282.5 mg/capsule) was given to 54 subjects in the ABC
group or placebo in the group Ganada. Subjects in both groups
were irradiated 2.5 MED UV on the upper arms and whitening
effect was measured by colorimeter-based L value. Image analysis
using skin replica around the crow feet was done to determine the
levels of wrinkle reduction and the levels of vitamin E was determined before and after the treatment. A signicant reduction in
skin pigmentation and wrinkles (P = 0.011 and P = 0.000005 respectively) compared with placebo group was observed after 12-week
oral administration. Treated group showed signicantly increased
(P = 0.0001) level of serum vitamin E on completion of treatment as
compared to placebo group.
It can be concluded that LGNS-5 as a dietary supplement might
prevent the UV-induced skin pigmentation and wrinkles without
any side effects.
269
In two different articles, Berson (2008) and Bordan Allemann

and Baumann (2008) summarized the antioxidants from natural
sources which when applied topically can replenish the antioxidant
capacity of the skin disturbed by the environment. As PBE is shown
to have strong antioxidant effect, it can have signicant role in skin
care.
5.4.5. PBE role in sexual disorders

Kohama et al. (2007) designed a study to observe the efcacy
of PBE as a therapeutic alternative to gonadotropin-releasing hormone agonist (Gn-RHa) in the treatment of endometriosis. Study
include fty-eight women with a history of endometriosis and all
of them were followed at 4, 12, 24, and 48 weeks after starting
treatment for sign and symptoms of disease, including the changes
in CA-125 and estrogen levels (E2). Thirty-two patients included
in the study took 60 mg/day PBE orally for 48 weeks. Twenty-six
patients received Gn-RHa in the standard way. Gn-RHa reduced the
scores more efciently but this is not a permanent remedy because
recurrence of signs was observed after 24 weeks of the end of treatment. While PBE was found to act slowly and steadily reducing
the symptoms scores but had no inuence on menstrual cycles or
E2. CA-125 decreased in both groups. PBEs effects were more pronounced in patients with smaller endometriomas as compared to
larger endometriomas. Gn-RH lowered the CA-125 concentrations
far more effectively but this was not sustainable. So PBE can be an
effective alternative for endometriosis.
The effect of PBE on climacteric syndrome was investigated
(Yang et al., 2007). For the purpose, 200 peri-menopausal women
were selected and given 200 mg PBE daily in a double-blind,
placebo-controlled study. Following the womens Health Questionnaire (WHQ) climacteric symptoms were evaluated. A total of 155
women completed the study. PBE favorably altered the LDL/HDL
ratio increasing the antioxidative status and all climacteric symptoms were improved without any side effects suggesting PBE might
be an alternative treatment of this syndrome.
In a prospective, non-randomized clinical study, 50 infertile men
with subnormal testosterone levels had been treated with Prelox, a
combination of 3 g l-arginine aspartate and 120 mg PBE along with
120 mg testosterone propionate for 11 months. Blood lipids and
seminological parameters were determined before and after treatment. Endothelial nitric oxide synthase (eNOS) was analyzed in the
semen specimens. A Global Efcacy Questionnaire was followed for
erectile function rating. Signicant improvement in seminal motility and penetrating capacity was observed by enhancing the activity
of eNOS in sperms. 76% of the patients restored erectile function
and 40% achieved fertilization during treatment (Stanislavov et al.,
2008).
Suzuki et al. (2009) analyzed the effect of pycongenol on dysmenorrhea in a double-blind, placebo-controlled trial. For the
purpose, 116 women in age range of 1848 were selected. After
passing 2 menstrual cycles as control period, women received
either PBE supplement (60 mg/day) or a placebo in identical capsule form in the subsequent 2 menstrual cycles. After the end of
treatment period, one cycle was used for monitoring and assigning
of women to different groups pertaining to different degrees of pain
either low or dysmenorrhea. As a result of the study, it was found
that women with low menstrual pain history did not respond to
PBE treatment signicantly, however, women with dysmenorrhea
had a signicantly lower pain score and the need of analgesic medication was signicantly reduced during the treatment. Moreover,
the number of days for which analgesic medication was required,
were lowered signicantly and condition prevailed even after the
discontinuation of the treatment. PBE was found to be persistent
analgesic-sparing agent; however, there is doubt for the effects to
be clinically meaningful.

270
6. Role of PBE in neurological disorders

Degeneration of the nerve tissue results in neurological disorders which include loss of memory and attention, deciency of
learning ability, Parkinsons and Alzheimers disease. PBE has been
tried for various degenerative neurological disorders like:
6.1. Cognition improvement
Cognitive decline in Alzheimers disease (AD) progresses in
oxidative stress. The role of PBE and vitamin E (Vit E) in improving oxidative damage and cognitive decit in the hippocampus
and cerebral cortex of intracerebroventricular streptozotocin (ICVSTZ)-infused rats was studied by Ishrat et al. (2009). A pretreatment
of PBE (10 mg/kg), vitamin E (100 mg/kg) and vehicle (intraperitoneal; once daily for 3 weeks) was given to rats followed by
bilateral injection with ICV-STZ (3 mg/kg) while other group (sham
rats) receive the same volume of vehicle. Rats were tested for cognitive performance after 2 weeks and then killed for biochemical
assays. Signicant decline in cognitive performance and cholineacetyltransferase activity in the hippocampus was observed by
ICV-STZ induction, signicantly attenuated with PBE and Vit E.
From this study it can be concluded that in oxidative stress-related
neurodegenerative conditions such as AD, PBE and Vit E can prove
to be a promising treatment.
A range of cognitive and biochemical measures in healthy
elderly individuals affected by antioxidant PBE was analyzed. Ryan
et al. (2008) studied hundred and one elderly participants (6085
years) consuming a daily dose of 150 mg of PBE for a period of three
months are used in a double-blind, placebo-controlled, matchpaired design. Both the groups were matched by age, sex, body
mass index, micronutrient intake and intelligence. Participants
were assessed at intervals of 1, 2, and 3 months of treatment in
addition to baseline. PBE group showed signicant interactions for
memory-based cognitive variables and lipid peroxidation products
by displaying improved working memory and decreased concentrations of F2-isoprostans relative to the control group.
6.2. PBE and ADHD
Attention decit hyperactivity disorder (ADHD) is a neurodevelopmental disorder and is assumed to be the result of genetic and
non-genetic factors and one of the non-genetic factor is certainly
oxidative stress. Activities of the some enzymes like SOD, eNOS
are stimulated by PBE owing to its powerful antioxidant nature.
Dvorakova et al. (2006) focused on the level of GSH and GSSH glutathione and total antioxidant status (TAS) by PBE administration
in children suffering from ADHD in a randomized, double-blind,
placebo-controlled trial. A signicant decrease in GSSH accompanied by highly signicant increase in GSH levels improving the
GSH/GSSH ratio was observed in PBE group compared with placebo
group. PBE normalizes the TAS of ADHD children which is usually
less in comparison with reference values.
ADHD is associated with DNA damage. Chovanova et al.
(2006) analyzed the effects of PBE on the levels of oxidized
purines represented by 8-oxo-7,8-dihydroguanine (8-oxoG) and
total antioxidant status (TAS) in children suffering with this disease. For the purpose, DNA damage was compared in both ADHD
and control group (healthy group) and then ADHD children were
grouped into treated and placebo category. It was observed that
1-month treatment of PBE lowered 8-OxoG signicantly in comparison to beginning state and placebo group. TAS was also found
to be lower in ADHD children but after one month of treatment,
it was elevated statistically signicantly. PBE caused decrease in
DNA damage, an improvement in TAS followed by improvement of
attention in ADHD children.
Information regarding dose of the extract is lacking. Justication

of reciprocal relation between oxidative damage to DNA, TAS and
symptoms of ADHD and mechanism of PBE action needs further
research.
The effect of PBE on ADHD symptoms was evaluated in a randomized, placebo-controlled, double-blind study (Trebaticka et al.,
2006). PBE in a dose of 1 mg/kg/day was given to sixty-one children
for 4 weeks vs. placebo group. Standard questionnaires of Child
attention problem (CAP), teacher rating scale, Conners Teacher
Rating Scale (CTRS), the Conners Parent Rating Scale (CPRS) and
a modied Wechsler Intelligence Scale for children were used for
examination at different time periods, i.e. at the start of the trial,
on completion and after one month of completion of the trial. A
signicant reduction in hyperactivity accompanying improvement
in attention and visualmotoric coordination and concentration in
the children with ADHD was observed with no positive effects in
placebo group. But symptoms relapsed on termination of treatment. Nevertheless, these results should be further conrmed by
studies involving a large number of patients, especially girls for prolonged periods. Results suggest that PBE could be used as safe and
effective natural supplement to relief symptoms of ADHD in boys
and not in girls.
In another randomized, double-blind, Placebo controlled study,
PBE inuence on excretion of catecholamines and concentration
of noradrenalin was analyzed. As the concentration of adrenaline
(A) and noradrenalin (NA) were positively correlated with level of
oxidized glutathione in plasma, PBE treatment resulted in lowering A and NA level and increased GSH/GSSH ratio by decreasing
dopamine (D). Data supported the fact that over-activity of noradrenergic system is responsible for ADHD increasing release of A.
PBE normalized the enhanced excretion of catecholamine leading
to reduced oxidative stress and hyperactivity as well (Dvorakova
et al., 2007).
Though the basic mechanism of action is yet unknown, urinalysis exposed a minor excretion of catecholamines in the PBE group
than placebo group, signifying control of PBE on catecholamine
formation or metabolism.
The impact of PBE on levels of trace elements like Cu, Zn, Se,
Fe, ferritin and transferrin in plasma of ADHD children (aged 614
years) had been investigated in a randomized, double-blind and
placebo-controlled study (Viktorinova et al., 2009). Dose of PBE
was 1 mg/kg/day or placebo for 4 weeks. A group of 56 healthy
children was kept as control. ADHD children compared with control group showed lower Zn level (P < 0.05), higher Cu/Zn ratio
(P < 0.05) and higher Cu level. Se, Fe, ferritin and transferrin levels
were like those of healthy children. PBE administration signicantly decreased Cu levels (P < 0.01), Cu/Zn ratio (P < 0.05) and Fe
levels (P < 0.05). The levels of Zn, Se, ferretin and transferrin were
not affected. PBE inuenced antioxidant status by decreasing Cu,
Fe levels and Cu/Zn ratio, thereby improving clinical symptoms of
ADHD.
6.3. Role of pine bark extract in migraine
Migraine is a common medical problem which effect nearly
one fourth of the population. Twelve patients who had migraine
resistant to conventional therapies like -blockers, antidepressants, anticonvulsants, and 5-hydroxytryptamine receptor agonists
were included in the study. These were treated with antioxidant
formulation of 120 mg PBE, 60 mg vitamin C, and 30 IU vitamin
E in each capsule daily for a period of three months. For the
baseline measurement, enrolled patients were given migraine disability assessment (MIDAS) questionnaire to judge the severity
of their migraine before treatment. At the end of the treatment
period, a second MIDAS was carried out to study the response.
Compared to the baseline, there was signicant improvement in

MIDAS score (P < 0.05). Moreover, severity score and the number of headache days were also reduced (44 of baseline to 26.0
days) and mean headache severity lowered down from 7.5 of 10 to
5.5. So antioxidant therapy could be a useful remedy for migraine
(Chayasirisobhon, 2006).
7. Prevention of injuries caused by oxidative stress

Ansari et al. (2008) had reported their work on the human
neuroblastoma SH-SY5Y cells cultures exploring the protective
effect of PBE on acrolein-induced oxidative cell cytotoxicity. PBE
strengthened the intracellular antioxidant defense system including the glutathione levels. Treatment of SH-SY5Y with acrolein
is found to be co-related with increased NADPH oxidase activity, free radical production, protein oxidation/nitration (protein
carbonyl, 3-nitrotyrosine) and lipid peroxidation (4-hydroxy-2nonenal). This study showed that acrolein-induced cytotoxicity,
protein damage, lipid peroxidation, and cell death might be avoided
by pretreatment with PBE.
Mojzisova et al. (2009) observed the cardioprotective role of
quercetin, naringenin and PBE and synthetic antioxidant trolox
against daunorubicin-induced toxicity. MTT test and determination
of extracellular lactate dehydrogenase were used as parameters of cardiomycete protection. After 24 h of co-incubation
with daunorubicin, Quercetin (104 106 mol/L) and naringenin
(104 106 mol/L) were similar in their cytoprotective effect
showing signicantly increased cardiomyocyte survival in all
tested concentration (P < 0.001 and P < 0.01, respectively). PBE
was least effective of the three avonoids studied. However
all tested avonoids showed better protective effect as compared to trolox, i.e. protective action was in the order of
quercetin > naringenin > PBE > trolox. Daunorubicin induced leakage of lactate dehydrogenase was also prevented in accordance
with their cytoprotective activity.
The protective effect of PBE against CCL4 -induced oxidative
stress and hepatotoxicity in rats was analyzed. For the purpose,
4 groups of 6 rats each were constructed namely, vehicle control
group received the respective vehicles (distilled water and corn
oil) only; a CCL4 group received a 14-day repeated intraperitoneal
(i.p.) dose of distilled water and then a single dose of CCL4 at
1.25 ml/kg; CCL4 and PBE10 and CCL4 & PBE 20 groups received
a 14-day repeated i.p. dose of PBE 10 and 20 mg/kg respectively,
followed by single oral dose of CCL4 at 1.25 ml/kg. Measurement
of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, hepatic malondialdehyde (MDA) and
glutathione (GSH) concentrations, and catalase, superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities was
taken 24 h after the CCL4 treatment to assess the hepatotoxicity. The results conrmed that the single oral dose of CCl4
elevated levels of serum AST and ALT activities signicantly. Extensive liver injuries were observed histopathologically, characterized
by extensive hepatocellular degeneration/necrosis, fatty changes,
inammatory cell inltration, congestion, and sinusoidal dilatation. In addition, an increased MDA concentration and decreased
GSH, catalase SOD, and GST were observed in the hepatic tissues. But CCl4 -induced hepatotoxicity was prevented signicantly
by PBE treatment prior to the administration of CCL4 , including
the elevation of serum AST and ALT activities and histopathological hepatic lesions, in a dose-dependent manner. Pretreatment of
PBE efciently protected against CCl4 -induced oxidative damage
in rats indicated by the fact that MDA and GSH levels and catalase SOD and GST activities in hepatic tissues remain unaffected
after administration of CCL4 . PBE probably acted so by inhibiting
lipid peroxidation and increasing antioxidant activity (Yang et al.,
2008).
271
Toxicity of hexavalent chromium [Cr(VI)], correlated with overproduction of free radicals, is responsible for oxidative damage. It is
nephrotoxic in humans and animals. The efcacy of PBE in reducing
[Cr(VI)]-induced toxicity had been assessed (Parveen et al., 2009).
The Male Wistar rats were classied into 4 groups. First chosen
as control, the second group as control plus pre-treated with PBE
(10 mg/kg b.wt., in saline; intraperitoneally; once daily for 3 weeks)
as drug control and the third group saline pre-treated followed by
single injection of K2 Cr2 O7 (15 mg/kg, body wt.; in saline; intraperitoneally) as toxicant group. PBE pre-treated plus K2 Cr2 O7 injected
rats occupied the 4th group. Blood was drawn for estimating renal
injury markers in serum 48 h after K2 Cr2 O7 -treatment. Kidneys of
the dissected rats were assayed for biochemical and histopathological analysis. There were signicant increases in renal injury markers
in serum, including blood urea nitrogen (BUN), serum creatinine
(Scr), and alkaline phosphatase (ALP) in K2 Cr2 O7 -treated rats which
show signicant (P < 0.05) decrease with PBE pre-treatment. In kidney homogenate of K2 Cr2 O7 -treated rats, increased thiobarbituric
reactive substances (TBARS), malondialdehyde (MDA) and protein
carbonyl (PC) were signicantly (P < 0.05) decreased and oxidative
balance was improved by increasing glutathione (GSH) levels and
catalase activity by prophylactic pre-treatment with PBE suggesting that PBE was effective in preventing K2 Cr2 O7 -induced oxidative
mediated nephrotoxicity.
Radiotherapy for cancer treatment causes severe damage to
intestinal mucosa when applied to abdominal region. Deleterious
effects of x-radiation are due to production of radiolysis-induced
reactive oxygen species. PBE being a powerful antioxidant can be
a remedy of impairment (de Moraes Ramos et al., 2006). Keeping
this in view, irradiated rats had been given PBE orally prior to xradiation with 15 Gy. Dramatically better condition of the mucosal
layers was observed in PBE treated rats showing PBE could be a safe
guard against radiation.
8. Non-clinical effects of PBE
8.1. Nutritional effects
The antioxidant nature of PBE in increasing the nutritional value
and shelf life of dairy product yogurt was observed by Ruggeri et al.
(2008). PBE, when added to the yogurt, maintained the viability of
Lactobacillus delbrueckii, Lactobacillus bulgaricus and Streptococcus
thermophilus, pH, titratable acidity, macronutrients and folate content. Nutritional parameters of the yogurt were not modied during
storage. For the analysis of any possible degradation of PBE components by yogurt ora, estimation of total polyphenol contents
and some phenolic substances (catechin, epicatechins, chlorogenic
acid, and caffeic acid) was done at the beginning and at the end of
the study. Data indicated no change in PBE contents making it a
nourishing ingredient to enrich yogurt.
PBE can be used as oxidative stabilizer in food storage. Ahn et al.
(2006) tested the effect of different antioxidants on 3 days refrigerated cooked beef. ActiVin (Grape seed extract), PBE, and oleoresin
rosemary activity was evaluated by TBARS values, hexanal content
and sensory analysis to reduce warm-over avor (WOF). Compared
to control which was without any antioxidant, oxidative stabilizing
activity was in the order; ActiVin > PBE > Oleoresin rosemary.
9. Adverse effects/toxicology
PBE in the form of PBE has been declared GRAS (generally
regarded as safe) by an independent panel of toxicology experts
(Scientic and clinical monograph for PBE, 2010). This declaration is
based on the data obtained from 70 clinical trials including healthy
subjects and patients with particular disease history (n = 5723).

272
The frequency rate of adverse effects (AEs) is 2.4% and 0.19% in

patients and healthy subjects respectively. AEs include gastrointestinal discomfort, dizziness, headache and nausea. No alarming
side effects even at high dosages have been reported. So PBE at
dosage of 20100 mg per day for a long period extended for months,
and 100300 mg for shorter periods is considered nontoxic.
rmed by in vivo studies in humans because it would be a big revolt

in medicine to satisfy the needs of increasing number of diabetic
patients. Most of the in vivo studies use animal model. A few studies which are on human beings are limited either by their poor
design or less number of subjects included in the trial. However,
only in vivo reports including animal model and human volunteers
are summed up in Appendix A.
10. Conclusion
Conict of interest disclosure
From the review, biological, clinical, and nutraceutical signicance of the PBE is obvious. It has a wide spectrum of effects on
targets relevant for many degenerative diseases. Biologically, its
antioxidative nature can be used to prolong the shelf life of food
products. PBE has been found benecial in many diseases including most promising and potential role in cardiovascular health, but
still there is need to upraise the research in terms of better design,
number of subjects to make the results statistically signicant. To
get the status of a prescriptional drug or supplement, PBE needs
more studies using human model. There are many aspects of the
extract for which, only in vitro studies have been done so far and
it is important to go further e.g. PBE can control type II diabetes
(Schafer and Hogger, 2007) with IC50 of 5 g/mL. It must be con-
It is to state that neither authors nor their institution has a nancial or other relationship with any organization or people that may
inuence the authors work. Authors are not being paid by any organization or agency related with the product. There is no conict of
interest to be declared.
Acknowledgments
The authors would like to acknowledge Higher Education
Commission of Pakistan for funding provided under Indigenous
Fellowship Scheme for Ph.D.
Appendix A.
Author/year
Therapeutic goals
Design/duration
Dosage/preparation
Result
DB, PC and R.N = 16, 2 weeks
180 mg/day
Cesarone et al.,
2006a,b,c
Endothelium
dependent vasodilation
Chronic venous
inefciency
P, R, C n = 86
Zibadi et al., 2007
Myocardial remodeling
Old C57BL/6N mice
150 mg/day PBE (3

capsule of 50 mg) and
300 mg/day (6
capsules) 1000 mg/day
Daon
30 mg/kg
Vinciquerra et al., 2006
Muscular cramps and

pain
Kamuren et al., 2006
Diabetic retinopathy
Part 1: OL, n = 66 (22 normal

subjects with cramps intensity of 4
times/week, 21 patients with
venous disease and ramps
intensity 46 times/week and 23
athletes with frequent cramps
during exercise. Part II DB, PC
n = 47 mean age of 60 years, 25
with intermittment claudication,
22 patients with diabetic
microangiopathy
OL, normal and Diabetes induced
rats were subjects 3090 days
Increase in FBF response to

acetylcholine
Signicantly greater
improvement in CVI signs
and symptoms with PBE as
compared to baseline and
Daon
Pronounced decrease in
gene expression of
pro-MMP-2, -9 and -13 and
MMP-9
Frequency of cramps and
muscle pain score
decreased signicantly. No
effect in placebo treated
patients.
10 mg/kg for 14
consecutive days
Nocun et al., 2008
Lowering thromboxane
level
Wistar male rats induced to insulin

dependent diabetes
5 mg/kg b.wt. with ASA

in 10 mg/kg b.wt.
Jankyova et al., 2009
Lowering glucose level
STZ-induced diabetic rats were

used
10 mg/kg and 20 mg/kg

b.wt./day
Belcaro et al., 2008a,b
Alleviation of adverse
effects of chemo and
radio therapies
PC, SB, n = 4625 included in PBE

treated group and 21 in placebo
150 mg/kg b.wt.
In vivo effects of
procyanidin rich
extract on oxidative
stress.
Study was done in two conditions,

i.e. long time for a period of 8
weeks as food supplement and
given a dose singly for postprandial
analysis
Wistar rats were used for the
study. Time of study was 3 h
500 mg/kg of b.wt. in

single dose
Procyanidin rich extract is

effective against oxidative
stress
10 mg/kg
PBE reversed all the

oxidative indices
Cardiovascular health
Nishioka et al.
Antioxidant activity
Busserolles et al., 2006
Sehirli and Sener, 2009
Ischemia induced
injury
200 mg/day (50 mg

capsule 4 times or
100 mg capsule 2
time/day)
PBE proved to be an
effective remedy and
antihyperglycemic therapy
against diabetic
retinopathy and cataract
Signicant inhibitory effect
on production of
thromboxane.
Elevated level of
postprandial glycemia was
decreased signicantly
All the investigated side
effects were less frequent
in PBE group

273
Appendix A (Continued)
Author/year
Therapeutic goals
Design/duration
Dosage/preparation
Result
Inhibitory effect of PBE

on cyclooxygenases
In 1st step, PBE was administered

orally to 5 healthy persons for 5
days. In the 2nd step, 10 persons
were given a single dose.
200 mg for 5 days and

300 mg single dose and
blood was taken after
30 min of ingestion
Chovanova et al., 2006
Effect of PBE on key

mediators of
inammation
After treating 7 volunteers for 5

days, blood was drawn and
analyzed
200 mg/day
Ince Yesil-Celiktas
et al., 2009
Carrageenan induced
inammation rat
model
Paw edema was used as parameter

of anti inammatory activity
75 and 100 mg/kg b.wt.
Canali et al., 2009
Anti inammatory
response of PBE
supplementation on
the arachidonic acid
pathway in human
polymorphonucle ar
leukocytes (PMNL)
Healthy volunteers were given PBE

for 5 days and then
polymorphonucle ar leukocytes
(PMNL) were analyzed
150 mg/day
Statistically signicant
increase in the inhibition of
both COX-1 (P < 0.02) and
COX-2 (P < 0.002) was
observed
Matrix metalloproteinase 9
(MMP-9) released from
human monocytes and
NF-kB activation was
inhibited statistically
signicantly thus proving
that PBE exert anti
inammatory effects by
inhibiting proinammatory
gene expression.
PBE inhibited paw swelling
dose dependently at 2, 3, 4,
5 and 6 after carrageenan
injection showing
signicant anti
inammatory activity 2
and 4 h after
administration
PBE inhibits COX-2 and
5-LOX gene expression
reducing leukotrene
biosynthesis in human
PMNL
Viral myocarditis
Four-week-old inbred male DBA/2

mice were used
Oral administration of
PBE at a dose of 1 or
10 mg/kg per day and
10 or 100 mg/kg was
done for the
histological study, and
for gene expression
study respectively
from the rst day of
viral inoculation
The myocardial inltration

and necrosis was
signicantly smaller in PBE
treated (10 mg/kg) mice
hearts
Activity against
Helicobacter pylori
strain
This study was carried on in liquid

medium as well as in vitro model
using sessile bacteria attached to
AGS cells. Gastic cells
Antimicrobial activity was
analyzed in cooked red meat
MIC (50) of 12.5 g/mL
Signicant antibacterial
activity
1% conc. of Pinus pinea

bark extract and PBE
Reduction in numbers of
Staphylococcus aureus was
detected as 7.9 102 cfu/g
after 6 days that reduced to
7.1 102 cfu/g after 9 days;
and 8.6 102 cfu/g for
Pinus pinea and PBE
respectively. While after 6
and 9 days of storage,
values of 13.7 102 and
71.2 102 cfu/g were
obtained respectively for
the control indicating the
antimicrobial effect of bark
extracts.
Cognition
improvement in
Alzheimers disease
(AD)
ICV-STZ induced rats were

pretreated with PBE
PBE 10 mg/kg with

vitamin E
Effect of PBE on
cognition of healthy
elderly individuals
DB, PC and MP design 101 elderly

(6085 yrs) individuals were
treated with PBE for a period of 3
months
150 mg PBE
PBE attenuate cognitive

performance and
cholineacetyl transferase
activity lost by ICV-STZ
induction
PBE group showed
signicant interactions for
memory based cognitive
variables and lipid
peroxidation.
Anti-inammatory activity
Schafer et al., 2006
Antiviral activities of PBE

Matsumori et al., 2007
Antimicrobial
Rohdewald and Beil,
2008
Hames Kocabas et al.,

2008
Cognition improvement
Ishrat et al., 2009
Ryan et al., 2008
Antimicrobial effect of
pynogenol against
Staphylococcus aureus

274
Author/year
Therapeutic goals
Design/duration
Dosage/preparation
Result
Role of PBE in migraine control

Chayasirisobhon, 2006
Migraine
12 patients with long term history

of migraine, study period = 3
months
Antioxidant
formulation of 120 mg
pine bark extract,
69 mg vitamin C, and
30 IU vitamin E in each
capsule
Signicant improvement in
MIDAS with signicant
reductions in number of
the headache day and
severity
Osteoarthritis
Farid et al., 2007
Osteoarthritis
R, DB, PC Parallel group design
150 mg/day
Cisar et al., 2008
Osteoarthritis
150 mg/day
Belcaro et al., 2008a,b
Osteoarthritis
R, DB, PC trial n = 100 with primary

knee osteoarthritis and pain
including 32 men and 68 women
with mean age of 54 years.
Treatment period 3 months
R, DB, PC = 159 with primary
osteoarthritis including 78 men
and 78 women with mean age of
48 years. Period of treatment was 3
months.
A signicant improvement
in WOMAC score was
observed in test group. PBE
treatment resulted in
signicant decrease in use
of pain medicine compared
with placebo (P < 0.001)
An improvement of
WOMAC index between
treatments at 1.5, 2 and 3
months (P < 0.05)
compared with placebo.
Treatment group showed a
decline of 56 in WOMAC
scores. PBE is best
alternative of the anti
inammatory drugs
PBE and toxicology

Yang et al., 2008
Protective effect of PBE

against CCL4 induced
oxidative stress.
100 mg/day
Four groups of rats, one with

vehicle only, 2nd with CCL4 only,
and 3rd and 4th with 10 mg/kg and
20 mg/kg PBE respectively along
with CCL4 and all the four were
followed by single oral dose of
CCL4 at 1.23 mL/kg
R, DB, PC n = 61 (50 boys and 11
girls with age of 614 years 1
month treatment and 1 month
washout)
10 mg/kg and 20 mg/kg
PBE acts by inhibiting lipid

peroxidation and
increasing antioxidant
activity
1 mg/kg b.wt./day
PBE was found to be

effective in 2 of 4 ADHD
assessments. On child
attention problem rating
scale, teachers reported
signicant improvement in
hyperactivity and in
attention compared with
baseline and this returned
to baseline after one month
of washout. Evaluation of
the ADHD symptoms by
parents showed stability
compare with placebo and
baseline.
Pycnogenol inuenced
antioxidant status by
decreasing Cu, Fe, levels
and Cu/Zn ratio improving
clinical symptoms of ADHD
Trebaticka et al., 2006
Childhood ADHD
Viktorinova et al., 2009
ADHD control by
decreasing Cu, Fe levels
and Cu/Zn ratio
R, DB, PC n = 56 ADHD patients.

Period of study was 4 weeks
1 mg/kg/day
Photoprotection
against UVB
Hairless SKH-1 female mice was

used. Sub dorsal skin was used.
Study was done in two groups, i.e.
test group and control group 10
weeks
The antioxidant
mixture of vitamin C,
vitamin E, PBE and
evening primrose oil
was administered
orally
Kim et al., 2008
Anti-melanogenic
activity
Cultured B16 melanoma cells were

used
Chang et al., 2009
Photoprotection
against UV induced
pigmentation and
wrinkle formation
PC, mixture of vitamin E, C PBE and

evening primrose oil was given to
54 human volunteers orally
PBE and skin care

Cho et al., 2007
A dose of 4 capsules
(282.5 mg/Capsule)
Antioxidant mixture
proved to be signicantly
effective in reducing
chronic UVB induced
wrinkle formation
PBE showed
antimelanogenic activity
by suppressing free
radicals and upregulating
reduced
glutathione/oxidized
gluthathione ratio in B16
cells
A signicant reduction in
skin pigmentation and
wrinkles.

275
Author/year
Sexual disorders
Yang et al., 2009
Therapeutic goals
Design/duration
Dosage/preparation
Result
Climacteric syndrome
in perimenopause
R, DB, PC n = 200 perimenopausal

women in which menstrual cycles
had stopped for a period of 311
months and then start as normal
cycle. Treatment period 6 months
P, NR, clinical study. Prelox was
given for 11 months
200 mg/day
PBE favorably altered the

LDL/HDL ratio increasing
the antioxidative status
and all climacteric
symptoms were improved.
76% of the patients
restored erectile function
and 40% achieved
fertilization during
treatment.
Stanislavov et al., 2008
Male infertility
Suzuki et al., 2008
Dysmenorrhea
R, DB, PC n = 116 women with age

1848 years having symptoms of
menstrual pain. Treatment period
was 4 menstrual cycles
Pharmacokinetics of PBE
Grimm et al., 2006a,b
Pharmacokinetics
11 volunteers were selected for

this study. 6 were tested for single
dose and remaining 5 for steady
state. Period of study was 14 hours
and 5 days for single dose and
steady state analysis respectively
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A review on biological, nutraceutical and

Lahore College for Women University

Lahore College for Women University

13 PUBLICATIONS 170 CITATIONS

49 PUBLICATIONS 266 CITATIONS

Available from: Alya Maimoona

This article appeared in a journal published by Elsevier. The attached

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Contents lists available at ScienceDirect

A review on biological, nutraceutical and clinical aspects

Author's personal copy

A. Maimoona et al. / Journal of Ethnopharmacology 133 (2011) 261277

Author's personal copy

(FSFE) was done in two consecutive steps at different pressures and

3. Pharmacokinetics of the extract

4.1. Antioxidant activity of pine bark extract

Author's personal copy

A. Maimoona et al. / Journal of Ethnopharmacology 133 (2011) 261277

of tissue. The results revealed that ischemia/reperfusion caused a

4.2. Anti-inammatory activities of PBE

Fig. 1. Radical scavenging action of PBE.

isolating PMNL which were primed with lipo-polysaccharide

Fig. 2. Inhibition of COX-2 expression.

Author's personal copy

The animal study looks promising but clinical trials in humans

Author's personal copy

A. Maimoona et al. / Journal of Ethnopharmacology 133 (2011) 261277

Recently an important role has been assigned to adipose

Author's personal copy

of cardiac remodeling showed the positive role of PBE in improving

Author's personal copy

A. Maimoona et al. / Journal of Ethnopharmacology 133 (2011) 261277

of thiobarbituric acid reactive substances TBARS concentration)

patients in control. All patients with rst session of chemotherapy

Author's personal copy

of OA symptoms and mobility respectively. All the parameters

In two different articles, Berson (2008) and Bordan Allemann

5.4.5. PBE role in sexual disorders

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A. Maimoona et al. / Journal of Ethnopharmacology 133 (2011) 261277

6. Role of PBE in neurological disorders

Information regarding dose of the extract is lacking. Justication

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7. Prevention of injuries caused by oxidative stress

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A. Maimoona et al. / Journal of Ethnopharmacology 133 (2011) 261277

The frequency rate of adverse effects (AEs) is 2.4% and 0.19% in

rmed by in vivo studies in humans because it would be a big revolt

DB, PC and R.N = 16, 2 weeks

Zibadi et al., 2007

Old C57BL/6N mice

150 mg/day PBE (3

Vinciquerra et al., 2006

Muscular cramps and

Kamuren et al., 2006

Part 1: OL, n = 66 (22 normal

Increase in FBF response to

Nocun et al., 2008

Wistar male rats induced to insulin

5 mg/kg b.wt. with ASA

Jankyova et al., 2009