Online Series Monographs The Scientific Foundation for Herbal Medicinal Products

Myrtilli fructus Bilberry Fruit 2014

www.escop.com The Scientific Foundation for Herbal Medicinal Products

MYRTILLI FRUCTUS Bilberry Fruit

2014

ESCOP Monographs were first published in loose-leaf form progressively from 1996 to 1999 as Fascicules 1-6, each of 10 monographs © ESCOP 1996, 1997, 1999

Second Edition, completely revised and expanded © ESCOP 2003

Second Edition, Supplement 2009 © ESCOP 2009

ONLINE SERIES ISBN 978-1-901964-24-0

Myrtilli fructus - Bilberry Fruit

© ESCOP 2014

Published by the European Scientific Cooperative on Phytotherapy (ESCOP) Notaries House, Chapel Street, Exeter EX1 1EZ, United Kingdom www.escop.com

All rights reserved Except for the purposes of private study, research, criticism or review no part of this text may be reproduced, stored in a retrieval system or transmitted, in any form or by any means, without the written permission of the publisher.

Important Note: Medical knowledge is ever-changing. As new research and clinical experience broaden our knowledge, changes in treatment may be required. In their efforts to provide information on the efficacy and safety of herbal drugs and herbal preparations, presented as a substantial overview together with summaries of relevant data, the authors of the material herein have consulted comprehensive sources believed to be reliable. However, in view of the possibility of human error by the authors or publisher of the work herein, or changes in medical knowledge, neither the authors nor the publisher, nor any other party involved in the preparation of this work, warrants that the information contained herein is in every respect accurate or complete, and they are not responsible for any errors or omissions or for results obtained by the use of such information. Readers are advised to check the product information included in the package of each medicinal preparation they intend to use, to be certain that the information contained in this publication is accurate and that changes have not been made in the recommended dose or in the contraindications for administration.

Edited by Simon Mills and Roberta Hutchins Cover photograph © Szabi237 GFDL, CC-BY-SA-3.0 (Myrtilli fructus) Cover and text design by Martin Willoughby Typeset in Optima by Roberta Hutchins

Plant illustrated on the cover: Myrtilli fructus FOREWORD

It is a great pleasure for me to introduce the online era of ESCOP Monographs. Interest in herbal medicinal products continues to stimulate research on herbal substances and the body of knowledge in this field is steadily growing. ESCOP takes account of this by preparing new monographs and - as the only organisation in the field at the moment - particularly through regular revision of our published monographs. In order to provide readers and authorities with balanced compilations of scientific data as rapidly as possible, ESCOP Monographs will be published online from now on. This contemporary way of publishing adds further momentum to ESCOP’s endeavours in the harmonization of European standards for herbal medicinal products.

The Board of ESCOP wishes to express its sincere gratitude to the members of the Scientific Committee, external experts and supervising editors, and to Peter Bradley, the final editor of every monograph published up to March 2011. All have voluntarily contributed their time and scientific expertise to ensure the high standard of the monographs.

Liselotte Krenn Chair of the Board of ESCOP

PREFACE

Over the 15 years since ESCOP published its first monographs, initially as loose-leaf documents then as two hardback books, ESCOP Monographs have achieved a reputation for well-researched, comprehensive yet concise summaries of available scientific data pertaining to the efficacy and safety of herbal medicinal products. The Second Edition, published in 2003 with a Supplement in 2009, covered a total of 107 herbal substances.

The monograph texts are prepared in the demanding format of the Summary of Product Characteristics (SPC), a standard document required in every application to market a medicinal product for human use within the European Union and ultimately providing information for prescribers and users of individual products.

As a change in style, literature references are now denoted by the name of the first author and year of publication instead of reference numbers; consequently, citations at the end of a monograph are now in alphabetical order. This is intended to give the reader a little more information and perspective when reading the text.

Detailed work in studying the pertinent scientific literature and compiling draft monographs relies to a large extent on the knowledge, skills and dedication of individual project leaders within ESCOP Scientific Committee, as well as invited experts. After discussion and provisional acceptance by the Committee, draft monographs are appraised by an eminent Board of Supervising Editors and all comments are taken into account before final editing and approval. In this way a wide degree of consensus is achieved, but it is a time-consuming process.

To accelerate the publication of new and revised monographs ESCOP has therefore decided to publish them as an online series only, commencing in 2011. We trust that rapid online access will prove helpful and convenient to all users of ESCOP Monographs.

As always, ESCOP is indebted to the many contributors involved in the preparation of monographs, as well as to those who provide administrative assistance and hospitality to keep the enterprise running smoothly; our grateful thanks to them all. NOTES FOR THE READER

From 2011 new and revised ESCOP Monographs are published as an online series only. Earlier monographs are available in two books, ESCOP Monographs Second Edition (2003) and the Second Edition Supplement 2009, but are not available online for copyright reasons.

After purchase of a single monograph, the specific items to be downloaded are:

Front cover Title page Verso Foreword and Preface Notes for the Reader Abbreviations The monograph text Back cover

Information on the member organizations and people involved in ESCOP’s activities can be found on the website (www.escop.com):

Members of ESCOP Board of Supervising Editors ESCOP Scientific Committee Board of Directors of ESCOP ABBREVIATIONS used in ESCOP monographs

AA arachidonic acid ABTS 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) ACE angiotensin converting enzyme ADP adenosine diphosphate ALAT or ALT alanine aminotransferase (= SGPT or GPT) ALP alkaline phosphatase anti-IgE anti-immunoglobulin E ASA acetylsalicylic acid ASAT or AST aspartate aminotransferase (= SGOT or GOT) ATP adenosine triphosphate AUC area under the concentration-time curve BMI body mass index BPH benign prostatic hyperplasia b.w. body weight cAMP cyclic adenosine monophosphate CI confidence interval

Cmax maximum concentration of a substance in serum CNS central nervous system CoA coenzyme A COX cyclooxygenase CSF colony stimulating factor CVI chronic venous insufficiency CYP cytochrome P450 d day DER drug-to-extract ratio DHT dihydrotestosterone DNA deoxyribonucleic acid DPPH diphenylpicrylhydrazyl DSM Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association) ECG electrocardiogram

ED50 effective dose in 50% of cases EDTA ethylenediamine tetraacetate EEG electroencephalogram EMA European Medicines Agency ENT ear, nose and throat ER oestrogen receptor ERE oestrogen-responsive element FSH follicle-stimulating hormone GABA gamma-aminobutyric acid Gal galactose GFR glomerular filtration rate GGTP gamma-glutamyl transpeptidase GOT glutamate oxalacetate transaminase (= SGOT) GPT glutamate pyruvate transaminase (= SGPT) GSH glutathione (reduced) GSSG glutathione (oxidised) HAMA Hamilton Anxiety Scale 12-HETE 12-hydroxy-5,8,10,14-eicosatetraenoic acid HDL high density lipoprotein HIV human immunodeficiency virus HMPC Committee on Herbal Medicinal Products (of the EMA) HPLC high-performance liquid chromatography 5-HT 5-hydroxytryptamine (= serotonin)

IC50 concentration leading to 50% inhibition ICD-10 International Statistical Classification of Diseases and Related Health Problems, Tenth Revision ICH The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use ICSD International Classification of Sleep Disorders IFN interferon IL interleukin i.m. intramuscular iNOS inducible nitric oxide synthase INR International Normalized Ratio, a measure of blood coagulation (clotting) tendency i.p. intraperitoneal IPSS International Prostate Symptom Score i.v. intravenous kD kiloDalton KM Index Kuppermann Menopausal Index kPa kiloPascal LC-MS liquid chromatography-mass spectrometry

LD50 the dose lethal to 50% of animals tested LDH lactate dehydrogenase LDL low density lipoprotein LH luteinizing hormone 5-LOX 5-lipoxygenase LPS lipopolysaccharide

LTB 4 leukotriene B4 M molar (concentration) MAO monoamine oxidase MBC minimum bactericidal concentration MDA malondialdehyde MFC minimum fungicidal concentration MIC minimum inhibitory concentration Mr molecular MRS Menopause Rating Scale MRSA methicillin-resistant Staphylococcus aureus MTD maximum tolerated dose MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MW molecular weight NBT nitro blue tetrazolium NF-kB necrosis factor kappa-B NO nitric oxide NOS nitric oxide synthase n.s. not significant NSAID non-steroidal anti-inflammatory drug ovx ovariectomy or ovariectomized ORAC oxygen radical absorbance capacity PA pyrrolizidine alkaloid PAF platelet activating factor PCR polymerase chain reaction PEG polyethylene glycol PGE prostaglandin E PHA phythaemagglutinin p.o. per os POMS profile of mood states PVPP polyvinylpolypyrrolidone RANKL receptor activator of nuclear factor kappa-B ligand RNA ribonucleic acid RT-PCR reverse transcription polymerase chain reaction s.c. subcutaneous SCI spinal cord injury SERM selective oestrogen receptor modulator SGOT or GOT serum glutamate oxalacetate transaminase (= ASAT or AST) SGPT or GPT serum glutamate pyruvate transaminase (= ALAT or ALT) SHBG sex hormone binding globulin SOD superoxide dismutase SSRI selective serotonin reuptake inhibitor STAI state-trait anxiety inventory t1/2 elimination half-life TBARS thiobarbituric acid reactive substances TGF-b transforming growth factor-beta TNF tumour necrosis factor TPA 12-O-tetradecanoylphorbol-13-acetate URT upper respiratory tract URTI upper respiratory tract infection UTI urinary tract infection VAS visual analogue scale VLDL very low density lipoprotein MYRTILLI FRUCTUS 2014 Bilberry Fruit

DEFINITION

Bilberry fruit, dried consists of the dried ripe fruit of Vaccinium myrtillus L., containing a minimum of 1.0 percent of tannins, expressed as pyrogallol

(C6H6O3; Mr 126.1) calculated with reference to the dried drug.

Bilberry fruit, fresh consists of the fresh or frozen ripe fruit of Vaccinium myrtillus L., containing a minimum of 0.30 per cent of anthocyanins, expressed

as cyanidin-3-O-glucoside chloride (chrysanthemin, C21H21ClO11; Mr 484.8) calculated with reference to the dried drug.

Fresh bilberry fruit dry extract, refined and standardised, produced from Bilberry fruit, fresh using ethanol (96 per cent V/V) or methanol (minimum 60 per cent V/V). It contains 32.4 per cent to 39.6 per cent of anthocyanins, expressed

as cyanidin 3-O-glucoside chloride [chrysanthemin (C21H21ClO11); Mr 484.8) calculated with reference to the dried drug*.

The materials comply with the respective monographs of the European Pharmacopoeia [Bilberry fruit, dried; Bilberry fruit, fresh; Fresh bilberry fruit dry extract, refined and standardised].

CONSTITUENTS

Bilberry fruit, dried Tannins and tannin precursors: up to 4%, mainly catechol tannins, also catechin and epicatechin; Procyanidins: B-1, B-2, B-3 and B-4; Anthocyanins: 0.5% glycosides of mainly delphinidin, cyanidin and petunidin; Flavonoids: including hyperoside, isoquercitrin, quercetin, astragalin; Iridoids: asperuloside; Caffeic acid derivatives: chlorogenic and other caffeoylquinic acids; Other constituents: resveratrol, pectins, vitamin C, b-carotene [Friedrich 1973; Baj 1983; Jänicke 2003; Wichtl 2009].

Bilberry fruit, fresh Fresh bilberry fruit dry extract, refined and standardised Anthocyanins: mono- and di-saccharides of delphinidin, cyanidin, petunidin, peonidin and malvidin, highest amounts occurring in berry skins; Antho- cyanidins: delphinidin, cyanidin, petunidin, peonidin, malvidin; Catechins: (+)-catechin, (-)-epicatechin, epigallocatechin gallate; Flavonols: quercetin, myricetin; Phenolic acids: ferulic, p-coumaric, m-coumaric, caffeic, chlorogenic, protocatechuic, syringic and gallic; Procyanidins: dimers and trimers A-type derived from (-)-epicatechin; Stilbenes: trans-resveratrol; Other constituents: triterpenes, scopoletin, aliphatic organic alcohols [Jaakola 2002; Roy 2002; Du 2004; Viljanen 2004; Ehala 2005; Faria 2005; Määttä-Riihinen 2005; Burdulis 2007; Cassinese 2007; Milbury 2007; Bao 2008a; Bao 2008b; Latti 2008; Ogawa 2008; Burdulis 2009; Thomasset 2009; Akerström 2010; Karlsen 2010].

CLINICAL PARTICULARS

Therapeutic indications

Internal use Extracts of bilberry fruit enriched with anthocyanins: supportive treatment of problems related to varicose veins such as painful and heavy legs, peripheral vascular insufficiency and microcirculatory disorders of the eye [Ghiringhelli 1978; Passariello 1979; Grismondi 1980; Tori 1980; Nuti 1980; Scharrer 1981; Allegra 1982; Signorini 1983a; Signorini 1983b; Corsi 1985; Gatta 1988; Morazzoni 1996].

Dried bilberry fruit: supportive treatment of acute non-specific diarrhoea. In this indication the efficacy is plausible due to long-standing use [Schilcher 1997;

1*The HPLC-based assay defines anthocyanins (Syn. anthocyanosides) content of the Fresh bilberry fruit dry extract as 36%, which corresponds to the 25% specification determined by spectrophotometry based on anthocyanidins [Penman 2006; Cassinese 2007; Ulbricht 2009]. MYRTILLI FRUCTUS

Jänicke 2003; Wichtl 2009; Schilcher 2010]. Undesirable effects None reported. External use Topical treatment of mild inflammation of mucous membranes Overdose of the mouth and throat and for superficial wounds. In this No toxic effects reported. indication the efficacy is plausible due to long-standing use [Jänicke 2003; Wichtl 2009]. PHARMACOLOGICAL PROPERTIES Posology and method of administration Pharmacodynamic properties Internal use Dried bilberry fruit: For adults and children from 10 years, 20 In vitro experiments to 60 g daily, taken with water [Dorsch 2002; Jänicke 2003; Wichtl 2009; Schilcher 2010]. Antioxidant activity An anthocyanin-rich extract of bilberry fruit was reported to Decoction: 5-10 g of dried fruit boiled for 10 minutes in 150 be a potent scavenger of free radicals, behaving both as a mL water and drunk cold several times daily [Jänicke 2003; scavenger against superoxide anion [Salvayre 1981; Meunier Blaschek 2007; Wichtl 2009]. 1989; Martin- Aragón 1998; Martin- Aragón 1999] and as an inhibitor of lipid peroxidation induced by adenosine diphos- Cold macerate: 5-10 g dried fruit soaked in 150 ml water for phate (ADP)/Fe2+ and ascorbate in liver microsomes [Salvayre 2 hours and drunk several times daily [Jänicke 2003; Blaschek 1981; Martin- Aragón 1998; Martin- Aragón 1999]. 2007; Wichtl 2009]. The K+ loss induced by free radicals in human erythrocytes, as Fluid extract [1:1]: 3 to 6 ml daily [Mills 2005]. well as the cellular damage caused by oxidant compounds such as daunomycin and paraquat, was inhibited by an anthocyanin- Dry standardized extract containing 36% of anthocyanins: rich extract [Maridonneau 1982; Mavelli 1984]. 60 to 160 mg three times daily (equivalent to 65 to 173 mg anthocyanins daily) [Alfieri 1964; Bonacina 1973; Lietti An aqueous extract of bilberry fruit was shown to have a 1976; Robert 1977; Detre 1986; Virno 1986; Bertuglia 1995; potent protective action on human low density lipoprotein Sweetman 2008]. (LDL) during copper-mediated oxidation [Laplaud 1997] and a standardized extract containing 37% of anthocyanins prevented Topical use photo-induced oxidation of human LDL and fragmentation of A 10% decoction as a gargle or mouthwash [Blumenthal 2003; apoprotein [Rasetti 1996/7]. Sweetman 2008; Wichtl 2009]. The peroxy-radical scavenging capacity of berry extracts was Method of administration studied using the Oxygen Radical Absorbing Capacity (ORAC) For oral administration or local application. assay. The ORAC value (expressed using Trolox equivalents per g) for bilberry extract was 44 [Roy 2002]. Duration of use No restriction. The antioxidant activity of a bilberry extract (573 mg total phenolic If diarrhoea persists for more than 3-4 days medical advice compounds / g dry weight containing 94.8% anthocyanins) was should be sought. In microcirculatory disorders a use of at least investigated in a lactalbumin-liposome system at concentrations 4 to 8 weeks is recommended [Blumenthal 2003]. of 1.4, 4.2 and 8.4 µg / mL of liposome sample. The extracts showed an effective dose-dependent antioxidant activity towards Contra-indications protein and lipid oxidation. Anthocyanins were the major None known. contributors to the antioxidant effect by inhibiting both hexanal and protein carbonyl formation [Viljanen 2004]. Special warnings and special precautions for use None required. An ethanolic extract of fresh berries (258 mg/L total phenolics) had an antiradical activity (assessed by DPPH method) of 5.37 Interaction with other medicaments and other forms of (µM Trolox equivalents). The extract provided protection of a interaction liposomal membrane against peroxyl radicals by increasing the Due to the potential for bilberries to enhance the antiplatelet induction time of oxidation [Faria 2005]. effect of acetylsalicylic acid and other NSAIDs, bilberries should be used with caution by patients with haemorrhagic disorders To study structure-activity relationships and potential synergisms, and those taking warfarin or antiplatelet drugs [Morazzoni antioxidant activities of 15 bilberry phenolics were evaluated. 1990; Rasetti 1996; Laplaud 1997; Abebe 2002; Mills 2005]. The potency of activity towards the superoxide radical was in the order: delphinidin > petunidin > malvidin=cyanidin > Pregnancy and lactation (+)-catechin > peonidin > pelargonidin. The activity towards The dry extract is well tolerated in pregnancy. Bilberry has been the peroxynitrite radical (ONOO-) was: delphinidin > used safely in high doses in pregnant women with varicose cyanidin=petunidin > malvidin = (+)-catechin > peonidin > veins and haemorrhoids [Grismondi 1980; Teglio 1987; De pelargonidins [Rahman 2006]. Smet 1993]. In an assay using cytochrome C-enhanced 6-hydroxydopamine Scientific evidence for the safe use of bilberry during lactation oxidation reaction a 50% inhibition was observed with an is not available. aqueous bilberry extract (7 µM of total anthocyanin) [Yao 2007].

Effects on ability to drive and use machines Effects on vision None known. Pyridinium bisretinoid A2E, an autofluorescent pigment

2 MYRTILLI FRUCTUS

accumulating in human adult retinal pigment (ARPE) cells with in a dose-dependent manner. The effects were weaker than age and other retinal disorders, can mediate a detergent-like those produced by the bilberry extract (25% anthocyanins) at perturbation of cell membranes and light-induced damage to 30 µg/mL [Matsunaga 2010]. cells. Light-exposed A2E-laden ARPE cells exhibit a propensity for apoptosis via generation of epoxides which then interact with Effect on cardiovascular tissues DNA. Incubation of the cells with a bilberry extract (40mM) An anthocyanin-rich extract of bilberry fruit inhibited proteolytic reduced A2E-epoxidation, incidence of DNA damage and cell enzymes such as elastase [Jonadet 1983] and interacted with death [Sparrow 2003]. collagen metabolism by cross-linking collagen fibres, making them more resistant to collagenase action [Robert 1977] and A water-soluble fraction of a bilberry extract was found to reducing the biosynthesis of polymeric collagen [Boniface reduce A2E photooxidation. Separated single anthocyanins 1982]. (100 µM) suppressed the photooxidation of A2E. The ARPE cells that had been incubated with anthocyanins (100 µM) An anthocyanin-rich standardized extract (corresponding to exhibited an increased resistance to A2E-induced membrane 25% of anthocyanidins) had a slightly relaxing effect on various damage [Jang 2005]. isolated vascular smooth muscle preparations and reduced the response to contraction inducers such as serotonin and barium A bilberry extract (total anthocyanin content 28.1% cyanidin- [Bettini 1984a; Bettini 1984b; Bettini 1984c; Bettini 1985]. 3-glucoside equivalent; total phenol content 61.8% catechin equivalent) was found to upregulate the oxidative stress defence Application of a bilberry extract (12.1% total anthocyanins and enzymes haeme oxygenase-1 and glutathione S-transferase-pi 35.7% total phenolics) directly to pig coronary arterial rings in retinal pigment epithelial cells [Milbury 2007]. resulted in a dose- and endothelium-dependent vasorelaxation (% maximal relaxation at 5 mg total anthocyanins /L was 59 A bilberry extract (10 µg/mL) and anthocyanidins (cyanidin at ± 10) [Bell 2006]. 10 µM, delphinidin at 30 µM, malvidin at 10 µM) significantly inhibited 3-(4-morpholinyl) sydnonimine hydrochloride- A bilberry extract (standardized for 25% anthocyanidin) was induced retinal ganglion cell death and decreased intracellular evaluated for effects on ACE in cultured endothelial cells. Doses radical formation. Each inhibited the production of thiobarbituric of the extract (6.25, 12.5, 25, 50 and 100 µg/mL) resulted in a acid reactive substance (TBARS) in a concentration-dependent significant and dose-dependent inhibition of the ACE activity manner, the IC50 values being 1.0 µg/mL, 0.7, 1.9 and 8.3 µM (p<0.001). Reference anthocyanidins had no significant effect respectively versus vehicle (p<0.05) [Matsunaga 2009]. [Persson 2009].

A study examined the effects of anthocyanins on the regeneration Bilberry anthocyanins isolated from fresh ripe berries were of the photoreceptor rhodopsin using frog rod outer segment evaluated for their intracellular capacity to inhibit peroxy radical membranes. Cyanidin-3-glycosides stimulated the regeneration, formation on endothelial cells (EA.hy926). Anthocyanins were but the corresponding delphinidin glycosides showed no significantly active (p<0.001) at low concentrations (<0.5mg/L) significant effect [Matsumoto 2003]. with a saturable effect being observed >0.5 mg/L. Coronary flow (CF) of isolated rat hearts in experiments with 40-min In frog rod outer segment membranes cyanidin-3-glucoside ischaemia followed by reperfusion indicated a concentration- [C3G] was found to bind directly with rhodopsin depending dependent bi-phasic response: vasodilation at ≤ 1mg/L (p<0.05) on pH [Yanamala 2009]. Further work confirmed the C3G- and vasoconstriction at ≥10 mg/L up to 50 mg/L. Perfusion of enhanced regeneration [Tirupula 2009]. isolated rat hearts with low anthocyanin concentrations (0.01 – 1 mg/L) significantly attenuated the ischaemia-reperfusion Anti-angiogenic activity (I-R) injury as evidenced by decrease in the release rate of The antiangiogenic activity of a standardised bilberry extract lactate dehydrogenase (LDH), increase in postischaemic (25% anthocyanins) was evaluated using the chick chorio- coronary flow and decrease in the incidence and duration of allantoic membrane (CAM) assay. A 100 ng/50 µl concentration reperfusion arrhythmias (heart arrest, ventricular fibrillation, resulted in inhibition (Score -2) compared to control (Score +2) ventricular tachycardia, ventricular premature complexes). High [Ozgurtas 2008]. concentrations (5 - 50 mg/L) diminished cardioprotection and showed cardiotoxic activity [Ziberna 2010]. A bilberry extract (50 µg polyphenols/mL) showed a 96% inhibition of H2O2- induced vascular endothelial growth factor Antiaggregatory effects (VEGF) expression by human HaCaT keratinocytes, compared A standardized extract of bilberry fruit showed activity against to control (76%) (p<0.05) [Roy 2002]. aggregation induced by ADP, collagen and sodium arachidonate in rabbit platelet-rich plasma [Morazzoni 1990]. This was A bilberry extract (25% anthocyanins) at 0.3-30 µg/ml inhibited confirmed ex vivo on ADP- and collagen-induced aggregation both tube formation and migration of human umbilical vein of platelets obtained from the blood of 30 healthy volunteers endothelial cells (HUVECs) induced by vascular endothelial given 480 mg/day of the standardized extract orally for 30-60 growth factor-A (VEGF-A). In addition the bilberry extract days [Pulliero 1989]. inhibited VEGF-A-induced proliferation of HUVECs and VEGF- A-induced phosphorylations of extracellular signal-regulated An anthocyanin extract of bilberry fruit also inhibited in vitro kinase 1/2 and serine/threonine protein kinase family protein platelet aggregation induced by ADP or adrenaline in human kinase B, but not that of phospholipase C-gamma [Matsunaga plasma [Serranillos 1983]. 2007]. Wound healing effects In a second study the inhibitory effects induced by individual Pre-treatment (1 h) with a bilberry extract (25% anthocyanins) anthocyanidins (delphinidin, cyanidin and malvidin) at 3 and increased the viability of UV-A irradiated (20 J/cm2) keratinocytes 10µM significantly inhibited VEGF-induced tube formation (HaCaT cells) in a dose-dependent manner, with 100 mg/L (p<0.01) in a co-culture of HUVECs and fibroblasts. Each providing nearly 50% protection (p<0.05). Pre- and post- anthocyanidin (0.3 – 10µM) also scavenged the DPPH radical treatment (4 h) of the cells with the bilberry extract (25–100

3 MYRTILLI FRUCTUS

mg/L) attenuated the UVA-induced reactive oxygen species against Bacillus cereus, Salmonella enterica sv. Typhimurium (ROS) generation (p<0.05) [Svobodova 2008]. and sv. Infantis, Staphylococcus aureus, Clostridium perfringens and Helicobacter pylori. Activity is associated with phenolics, Further evaluation of the photoprotective effects of bilberry fruit mainly anthocyanins [Puupponen-Pimiä 2001; Puupponen- extract (25% w/w anthocyanins) on UVB-induced phototoxic Pimiä 2005; Nohynek 2006; Puupponen-Pimiä 2008]. stress (200 mJ/cm2) showed that pre-treatment (1 h) of HaCaT cells with the extract (5 – 50 mg/L) and post-treatment (4 h) Sugars and low molecular weight phenolic compounds of with 5 and 50 mg/L, reduced the extent of DNA breakage bilberry juice were found to inhibit Streptococcus pneumoniae (p<0.05), and laddering, together with caspase-3 and -9 activity binding to human bronchial cells (Calu-3). Adhesion inhibition (p<0.05). Pretreatment with the extract before UVB exposure was 52% with a fraction concentration of 9 mg/g [Huttunen also significantly (p<0.05) decreased reactive oxygen or 2011]. nitrogen species (RONS) generation and partially diminished IL-expression [Svobodova 2009]. Fractions of bilberry juice (mainly anthocyanins, proantho- cyanidins and flavonol glycosides) inhibited and reversed co- Treatment of fibroblasts with anthocyanins (cyanidin-3- aggregation of pairs of Streptococcus mutans with Fusobacterium glucoside (72%), delphinidin-3-glucoside (20%) and petunidin- nucleatum or Actinomyces naeslundii, species which are 3-glucoside (6%)) after 24 h showed a significant increase common in dental plaque [Riihinen 2011]. in migration at 100 µg/ml (p<0.05), while the migration of keratinocytes increased significantly at 50 and 100 µg/mL Polyphenolic fractions of bilberry juice containing mainly compared to control (p<0.05). Treatment with anthocyanins for anthocyanins and proanthocyanidins, between concentrations 48 h significantly stimulated the migration of both human dermal of 5-50 µg/mL, were found to significantly inhibit adhesion of fibroblasts and keratinocytes at 50 and 100 µg/mL (p<0.001). Neisseria meningitides to HEC-1B human epithelial cells when Treatment of cells with anthocyanins stimulated wound-induced compared to control (p<0.05) [Toivanen 2011]. VEGF production in fibroblasts and keratinocytes. However, anthocyanins inhibited ROS accumulation and VEGF production Antiproliferative and apoptotic activity in TNF-a-stimulated endothelial cells. Anthocyanin treatment A lyophilized ethanol extract of frozen bilberries (55.1 mg/g also reduced, in a dose-dependent manner, the adhesion of of total phenolics, 26.3 mg/g of total anthocyanins) showed a inflammatory monocytes to endothelial cells. Anthocyanins DPPH radical scavenging activity of 288 (µmol of Trolox/g). The also blocked both the translocation of NF-kB p65 into the extract decreased the number of viable human promyelocytic nucleus and the phosphorylation of the inhibitory factor kBa leukaemia (HL 60) cells by 84 and 88% at a concentration of [Nizamutdinova 2009]. 4 and 6 mg/mL respectively and the number of viable human colon carcinoma (HCT116) cells by 66% and 97% at 2 and Anti-inflammatory activity 4 mg/mL respectively. HL60 cell growth was inhibited when The effects of a standardised bilberry extract (36% anthocyanins) incubated with extract concentrations from 0.5 to 4 mg/mL for on genome-wide gene expression were investigated in an 24 and 48 h. In contrast HCT116 cell growth showed inhibition inflammatory cell model (RAW264). Cells were pretreated with at 1-4 mg/mL only after 48 h incubation. The bilberry extract or without 75 µg/mL bilberry extracts for 30 mins and then induced apoptotic cell bodies in HL60 cells and nucleosomal exposed to 40 ng/mL lipopolysaccharide (LPS) for another 6 DNA fragmentation in HL60 cells but not in HCT116 cells hrs. The induction of pro-inflammatory genes, including TNF, [Katsube 2003]. IL-1b, IL-6, TNC, PTGS2 and COX-2 were significantly reduced by bilberry (p<0.001 vs LPS only) [Chen 2008]. Pretreatment of cultured mouse JB6 cells with cyanidin-3- glucoside (C3G) inhibited UVB- and TPA-induced transactivation In cultured human monocytes (U937-3X kB-LUC cells) of NF-kB and AP-1 as well as expression of COX-2 and TNF-a. anthocyanins (100 mg/L) isolated from bilberries and black- These inhibitory effects appeared to be mediated through the currants efficiently suppressed LPS-induced activation of NF-kB inhibition of MAPK activity. C3G (10 - 40µM) significantly [Karlsen 2007]. inhibited, and at a concentration of 80 µM completely abolished, TPA-induced neoplastic transformation in JB6 cells (p≤ 0.05). In Human monocytic cells (ATCC CRL-1593.2) were co-incubated addition C3G (10 - 40µM) inhibited proliferation of a human with 13 polyphenols to evaluate their ability to modulate LPS- lung carcinoma cell line A549 [Ding 2006]. induced NF-kB activity. Quercetin inhibited the NF-kB activation in a dose-dependent manner as compared with control cells A methanolic extract from fresh bilberries (2:1) contained (77% and 95% at 25 and 50 µmol/L (respectively, both p<0.001). anthocyanins (12.8 mg/mL) and flavonols (0.29 mg/mL). For Resveratrol (50 µmol/L) and epicatechin (25 µmol/L) inhibited the determination of cell proliferation, HT-29 colon cancer the LPS-induced NF-kB activation by 79% (p=0.020) and 32% cells were exposed to 200 µL of culture medium containing (p<0.003) respectively. Petunidin (50 µmol/L) and delphinidin 0-60 mg/mL of berry extract or pure compounds (0-100 µg/ (50 µmol/L) increased LPS-induced NF-kB to respectively 245 mL) for 24 h. Bilberry extracts showed significant (p<0.001) and 146% of controls (p= 0.003 and p=0.007) [Karlsen 2010]. growth inhibition (30% cell reduction) at 10 mg/mL. The bilberry extract had no effect on DNA fragmentation after 48 Antimicrobial / gastroprotective activity h at concentrations up to 10 mg/mL. However, exposure of the A bilberry extract (0.25%) significantly (p<0.05) inhibited HT-29 cells to higher concentrations (20-60 mg/mL) resulted the growth of Helicobacter pylori (ATCC strain 49503) com- in pronounced fragmentation [Wu 2007]. pared to controls and increased susceptibility of H. pylori to clarithromycin (15 µg/mL). With a 1% concentration of the A bilberry extract was tested for growth inhibition (IC50) using extract, >90% inhibition was observed, with and without a human colorectal adenocarcinoma (HT29) cell line. The clarithromycin [Chatterjee 2004]. sulforhodamine B assay was applied to determine cell growth.

The IC50 for bilberry extract was found to be 32.2 µg/mL of Bilberry extract (1mg/mL) showed strong inhibition against cyanidin-3-glucoside equivalent [Jing 2008]. Escherichia coli (strains 50 and CM871) [Puupponen-Pimiä 2001]. Phenolic extracts have also been shown to be effective A standardised anthocyanin-rich bilberry extract (36%

4 MYRTILLI FRUCTUS

anthocyanins) reduced receptor tyrosine kinase (RTK) In another experiment the same anthocyanin-enriched bilberry domains of epidermal growth factor (EGFR, ErbB1) and extract (42% anthocyanins) was given orally to mice at 50, 100 ErbB2-4 receptors and the vascular endothelial growth and 200 mg/kg/day for 5 days. After the final treatment, potassium factor receptors (VEGFR-2 and VEGFR-3), with preferential bromate (KBrO3), an oxidizing agent and potent nephrotoxin, inhibition of VEGFR-2 and EGFR (≤ 3.4 µg/mL). Ligand- was given as a single intraperitoneal injection to mice (n=10) induced autophosphorylation of human vulva carcinoma at a dose of 200mg/kg. Serum blood urea nitrogen (BUN) and or porcine aortic endothelial cells was suppressed, with creatinine levels were significantly higher in the KBrO3-treated ErbB3 and VEGFR-3 being preferentially inhibited. The animals when compared with control (247.31 ± 3.22 mg/L vs extract completely abolished VEGFR-3 phosphorylation at 215.10 ± 2.82 mg/L; 22.26 ± 1.75 µg/mL vs 9.91 ± 0.86 µg/mL concentrations of ≥50 µg/mL [Teller 2009]. respectively). The 5-day oral intake of the extract significantly (p<0.001) reduced levels of BUN and serum creatinine to In human breast cancer cells (MCF7) a bilberry extract inhibited normal levels and decreased kidney MDA, NO and xanthine proliferation in a concentration-dependent manner, without oxidase levels. Improved ORAC levels in kidney tissue were inhibiting microtubule polymerization or mitosis. Significant also observed indicating a reduced degree of oxidative stress inhibition of proliferation (p<0.05) occurred with bilberry and kidney damage [Bao 2008b]. Administration of the extract + + extract concentrations >0.25 mg/mL, with an IC50 of 0.43 also elevated the Na /K -ATPase activity and mitochondrial mg/mL. At higher concentrations (0.5-1 mg/mL) inhibition potential which was reduced in the stressed group. Bilberry of polymerization (~30%) did occur with an increase in the extract had a protective effect on the liver by scavenging free fraction of cells at the G2/M phase, together with destruction radicals and attenuating mitochondrial dysfunction [Bao 2010]. of microtubules and formation of punctate tubulin aggregates in the cells [Nguyen 2010]. A standardized bilberry fruit extract (36% anthocyanins) was

evaluated in carbon tetrachloride (CCl4)-induced hepatic Various anthocyanins and anthocyanidins were exposed to fibrosis. Balb/C mice were treated i.p. with CCl4 dissolved in the human efflux transporters-multidrug resistance protein 1 olive oil (20% V/V; 2ml/kg) twice a week for 7 weeks. Control (MDR1) and breast cancer resistance protein (BCRP) using animals received the vehicle only. The bilberry extract in saline dye efflux, ATPase and vesicular transport assays. Malvidin, (1, 5 and 10 mg/kg to groups 4, 5 and 6 respectively) was then petunidin, malvidin-3-galactoside, cyanidin-3-galactoside, given daily for 15 days prior to sacrifice. Results showed that peonidin-3-glucoside and cyanidin-3-glucoside emerged as bilberry extract, in a dose-dependent manner, reduced hepatic potential BCRP substrates, while others (including cyanidin, hydroxyproline (an indicator of hepatic collagen) from 945 peonidin, pelargonidin, delphinidin) emerged as potential µg/g liver (CCl4 + saline) to 248 µg/g liver (CCl4 + BE 10 mg/ inhibitors of BCRP. Some exhibited bimodal activities, with kg; p<0.05) vs 198 µg/g liver (control). The extract (10 mg/kg) malvidin, malvidin-3-galactoside and petunidin serving as markedly attenuated oxidative stress, decreased TNF-a, TGF-b1 BCRP substrates at low concentrations and BCRP inhibitors at and a-smooth muscle actin (a-SMA) expression and eliminated higher concentrations. Aglycones exerted only mild inhibitory hepatic collagen deposits. The extract reduced liver fibrosis activity on MDR1 [Dreiseitel 2009]. by decreasing oxidative stress and inactivating hepatic stellate cells via down-regulation of the fibrogenic cytokines TNF-a Other effects and TGF-b1 [Domitrovic 2011]. Pre- and co-incubation of human colon adenocarcinoma cells (HT29) with a standardized bilberry extract (36% anthocyanins) Effects on vision / ophthalmic activity at concentrations ≥1µg/mL significantly suppressed (p≤ 0.001) An anthocyanin-rich extract of bilberry fruit, administered the strand-breaking effects of camptothecin and significantly intravenously to rabbits at 3.2 mg/kg, reduced the permeability diminished doxorubicin-mediated DNA strand breaks at of vessels of the ciliary body, which had been increased by concentrations ≥ 1µg/mL (p≤ 0.001) [Esselen 2011]. ocular puncture [Virno 1986].

Human HepG2 cells were incubated for 24 hrs with varying A mixture of anthocyanins administered intravenously to C3G concentrations (5–100 µmol/L) and assayed for cholesteryl rabbits at 160 mg/kg promoted dark adaptation after dazzling ester transfer protein (CETP) activity. Concentrations of 5 µmol [Alfieri 1964]. and above significantly suppressed CETP activity (p<0.001) compared to the untreated control [Qin 2009]. Senescence-accelerated OXYS rats with early senile cataract and macular degeneration were given control diets or In vivo experiments supplementation with 25% bilberry extract (20 mg/kg b.w. = 4.5 mg anthocyanidin) or vitamin E (40 mg/kg) for comparison. Antioxidant activity After 3 months 70% of control rats had cataracts and macular Oral pre-treatment of mice with 250 or 500 mg/kg of an degeneration. Supplementation with vitamin E showed no anthocyanin-rich extract of bilberry fruit inhibited liver per- significant effects. In contrast the extract completely prevented oxidation stimulated by FeCl2, ascorbic acid and ADP. The impairments in the lenses and retina. Both antioxidants decreased malondialdehyde content in the liver was significantly reduced lipid peroxides in the retina and serum [Fursova 2005]. (p<0.05) [Martin-Aragon 1999]. Administration of an ethanolic dry extract of bilberry to mice Oral administration of an anthocyanin-enriched bilberry extract by intravitreal injection (300 ng per eye) inhibited the formation (42% anthocyanins) to mice (n=10) at 50, 100 and 200 mg/ of neovascular tufts during oxygen-induced retinopathy, in kg/day for 5 days showed significant decreases (p<0.001) in comparison to saline [Matsunaga 2007]. plasma alanine aminotransferase (ALAT) levels at all doses, with the 200 mg/kg/day dose having an ALAT level of 17.23 ± 2.49 This extract was further evaluated for the effects on N-methyl- U/L compared to 107.68 ± 3.19 U/L 18 h in restraint stressed D-aspartic acid (NMDA)-induced retinal damage using mice and 18.08 ± 1.46 U/L in starved controls. Extracts also haematoxylin-eosin and terminal deoxynucleotidyl transfer- significantly (p<0.001) increased GSH and Vitamin C levels mediated dUTP nick-end labelling (TUNEL) staining. Intravitreal and significantly decreased MDA and nitric oxide (NO) levels injection of bilberry extract (100 µg per eye co-injected with in liver tissues at the highest dose [Bao 2008a]. NMDA at 5 nmol) significantly inhibited the morphological

5 retinal damage and increase in TUNEL-positive cells in the Treatment of rats with bilberry anthocyanins for 12 days before ganglion cell layer (both the GCL cell count and the IPL thickness) the induction of hypertension kept the blood-brain barrier [Matsunaga 2009]. permeability normal and limited the increase in vascular permeability in the skin and the aorta wall [Detre 1986]. Ocular inflammation (uveitis) was induced in mice by a foot- pad injection of lipopolysaccharide (100 mg/mouse). Oral When administered orally to rats at 100-200 mg/kg, or intra- administration of a bilberry extract (42% anthocyanins) at doses peritoneally or intramuscularly at 25-100 mg/kg, the extract of 50, 100 and 200 mg/kg/day for 5 days before challenge protected against capillary lesions induced by intradermal significantly reduced the ocular nitric oxide levels, especially injection of bradykinin [Lietti 1976]. at the highest dose (p<0.001). Also the malondialdehyde level was decreased, while increased levels were found in ORAC, The same extract was found to antagonise damage induced glutathione and vitamin C, as well as increased activities of by ischaemia-reperfusion in the hamster cheek-pouch micro- superoxide dismutase and glutathione peroxidase. Increased circulation model after oral administration at 100 mg/kg for 4 expressions of Cu/Zn superoxide dismutase, Mn superoxide weeks [Bertuglia 1995]. dismutase and glutathione peroxidase were also found [Yao 2010]. Effects of a bilberry extract on arteriolar vasomotion were assessed in cheek pouch microcirculation of anaesthetised ham- In a similar experiment an anthocyanin-rich bilberry extract sters and in skeletal muscle microvasculature of unanaesthetised (39% anthocyanins; 500 mg/kg b.w.) or PBS as control, were hamster skin fold window preparations. Intravenous injection administered orally to mice for 4 days. Three hours after the of the extract induced vasomotion in cheek pouch arterioles last administration animals received a single intraperitoneal and terminal arterioles with higher frequency in smaller injection of lipopolysaccharide (6 mg/kg b.w.) to induce uveitis vessels. In the skeletal muscle arteriolar networks the extract and retinal inflammation (EIU). Pretreatment with the bilberry increased vasomotion frequency and amplitude in all vessel extract had a protective effect on visual functioning during types. [Colantuoni 1991]. retinal inflammation. Anthocyanin treatment was found to preserve rhodopsin levels (p<0.05), suppress an increase of The effects of anthocyanins on skeletal muscle vascular the pro-inflammatory cytokine IL-6 (p<0.05), reduce oxidative permeabililty were evaluated in streptozotocin-induced diabetic stress (p<0.01), significantly suppress a-wave and b-wave rats. Animals (n=11) received bilberry extract (40 mg/kg) daily amplitudes (p<0.05 and p<0.01 respectively) and to suppress in drinking water or water only (n=10). Changes in vascular NF-kB (p<0.01) in the inflamed retina [Miyake 2012]. permeability were determined by measuring the capillary filtration of intravenously administered 99mtechnetium-labelled Effects on glucose and lipid metabolism (anti-diabetic activity) albumin (CFA), inducing venous compression in a hindquarter Bilberry extract (375 g anthocyanins/kg) included in the diet with a tourniquet and measuring the radioactivity externally (10 g anthocyanin/kg diet) of type 2 diabetic mice (male KK-Ay) on the limb before (time 0), during (after 6 weeks) and after for 5 weeks, significantly reduced blood glucose concentration removal of compression (after 12 weeks). Interstitial albumin and enhanced insulin sensitivity (p<0.05) via activation of retention (AR) was significantly higher in the diabetic rats than AMP-activated protein kinase (AMPK). Activation occurred the controls. In the groups receiving the extract AR decreased in white adipose tissue, skeletal muscle and the liver and was significantly (p=0.015) after 6 and 12 weeks [Cohen-Boulakia accompanied by upregulation of glucose transporter 4 in white 2000]. adipose tissue and skeletal muscle, and suppression of glucose production and lipid content in the liver. At the same time acetyl- Antiatherogenic (atheroprotective) activity CoA carboxylase was inactivated and PPARa, acyl-CoA oxidase An anthocyanin-rich extract of bilberry fruit, administered and carnitine palmitoyltransferase-1A were upregulated in the intraperitoneally at 100 mg/kg for 45 days to rabbits fed on a liver. After 5 weeks there was no difference in body weight gain cholesterol-rich diet, reduced the proliferation of intima and or adipose tissue weight between the test and control groups. calcium and lipid deposition on the aortic wall [Kadar 1979]. However, liver weight was significantly lower in the group receiving the bilberry extract (p<0.05). Compared with the Two bilberry extracts, one rich in anthocyanins extracted from control group total lipid, triglyceride and cholesterol contents bilberries (52% total anthocyanins) and one extracted from yeast- of the liver were significantly reduced in the test group, while fermented bilberries (almost devoid of typical anthocyanins but triglyceride and total cholesterol concentrations in serum were rich in complex polymers) were evaluated for their effects on also significantly lowered (p<0.05) [Takikawa 2010]. the development of atherosclerosis in apolipoprotein E-deficient mice. For 16 weeks mice received a diet supplemented with KK-Ay mice were fed control or control + 0.2% of C3G diet 0.02% of either extract. Both bilberry extracts caused a significant for 5 weeks. Dietary C3G significantly reduced blood glucose reduction in lipid deposits. The atherosclerotic lesions area was concentration and enhanced insulin sensitivity (p<0.05). C3G decreased in the anthocyanin-rich group by 15% (p<0.05) and upregulated the glucose transporter 4 and downregulated by 36% (p<0.0001) in the yeast-fermented group compared retinol binding protein 4 in the white adipose tissue, which to control. No significant differences in triacylglycerol and was accompanied by downregulation of the inflammatory total cholesterol levels in either plasma or liver were observed adipocytokines (monocyte chemoattractant protein-1 and between the groups [Mauray 2009]. TNF-a) (p<0.05 for both) [Sasaki 2007]. In a continuation of the experiment, apo E-deficient mice (n=40) Vasoprotective activity were fed ad libitum for two weeks either a control diet or the Anthocyanin-rich extracts of bilberry fruit exert modulating same diet supplemented with 0.02% of the anthocyanin-rich effects on capillary resistance and permeability, as was extract. After two weeks plasma lipid and antioxidant capacity demonstrated in various animal models [Lietti 1976; Robert were evaluated and the global genomic analysis carried out 1977; Detre 1986]. For example, an extract administered using pangenomic microarrays. Anthocyanin supplementation orally to rabbits at 200-400 mg/kg protected against capillary significantly (p<0.05) reduced total-cholesterol (mmol /L) permeability induced by topical application of chloroform from 7.0 (control) to 5.6, whereas plasmatic antioxidant status [Lietti 1976]. remained unchanged. Genomic analysis identified 1,261 genes

MYRTILLI FRUCTUS

in the aorta whose expression was modulated by the bilberry the activities of superoxide dismutase, catalase and glutathione extract [Mauray 2012]. peroxidase [Li 2008].

Further investigation of the anti-atherogenic effect of a bilberry Pretreatment of mice (male ddY) with a bilberry extract (25% extract (52% anthocyanins) on gene expression in the liver of anthocyanidins) at doses of 10, 30 and 100 mg/kg, 1 h before apo E-deficient mice was carried out. Mice received for 2 weeks HCl / ethanol administration, significantly inhibited gastric a standard diet supplemented with an anthocyanin-rich bilberry erosions in a dose-dependent manner (p<0.01 vs vehicle-treated extract (0.02%). After a 2-week supplementation plasma total mice) [Ogawa 2011]. cholesterol (mmol /L) and hepatic triglyceride levels (mg /g) were significantly reduced (p<0.05 and p<0.01 respectively), Neuroprotection / Memory while plasmatic antioxidant status remained unchanged. Rats given intraperitoneal injections of bilberry anthocyanins Transcriptional analysis showed that the expression of 2,289 (200 mg/kg/day) for 5 days had significantly more triiodo- genes was significantly altered, with over-expression of genes thyronine (T3) in their brains than rats given the solvent (26% involved in bile acid synthesis and cholesterol uptake into the alcohol). Bilberry-treated animals showed superior memory, liver, but down-regulation of expression of pro-inflammatory better vision and better control of sensory input [Saija 1990]. genes [Mauray 2010]. An aqueous suspension of lyophilised bilberries (equivalent to Wound healing effects 3.2 mg anthocyanin/ kg/day) was given orally to rats for 30 days Topical application of 5-10 mg of an anthocyanin-rich extract before first training (n=10 per group). Bilberries significantly of bilberry fruit accelerated the healing of experimental wounds enhanced short-term memory (p<0.05), but not long-term in rats [Curri 1976]. In another experimental model (healing memory in the inhibitory avoidance task, and induced an delayed by prednisone) the same extract applied topically at increase in the number of crossings in the first exposure to the concentrations of 0.5-2% for 3 days to skin wounds in rats open field. In the radial arm maze test, anthocyanin-treated rats promoted healing activity in comparison to prednisone-treated showed significant choice accuracy improvement (p<0.05) in controls [Cristoni 1987]. the early sessions, an effect which became attenuated in later sessions [Ramirez 2005]. Anti-inflammatory activity The vasoprotective effect could also be responsible for anti- The effects of long-term supplementation with bilberry extract inflammatory activity exhibited by anthocyanins (50-500 mg/ (2 g/kg of diet = 0.35 g anthocyanins/kg of food) and vitamin E kg given orally) against paw oedema induced by irritant agents (140 mg/kg of diet) were studied in young senescent-accelerated such as carrageenan, histamine or hyaluronidase [Bonacina OXYS and Wistar rats. The role of oxidative stress on brain bio- 1973; Lietti 1976]. molecules such as protein carbonyls, lipid peroxides, reduced glutathione, a-tocopherol and superoxide dismutase as well as Mice sensitized by an epicutaneous application of 2,4,6-trinitro- on learning ability was investigated. Bilberry extract and vitamin 1-chlorobenzene (TNCB; 100 µL of 1.1% v/v) to their shaved E improved cognitive deficits (tested by passive avoidance abdomen, were challenged with 10 µL/ ear of TNCB solution reaction retrieval) in OXYS rats. Supplementation with both on day 0. TNCB was reapplied to each ear 3 times per week vitamin E (p= 0.008) and bilberry extract (p=0.002) decreased until day 16. Daily oral administration (0.4 or 2 g/kg b.w.) of a lipid peroxides in rat brains. Bilberry extract significantly standardized bilberry extract (>25% anthocyanidins and >36% decreased MDA level only in OXYS rat’s brain (p=0.048), while anthocyanins) significantly decreased ear swelling (p<0.001 vs vitamin E significantly decreased MDA levels in both Wistar control) and suppressed bouts of scratching associated with and OXYS rats, when compared with their respective controls progressive and chronic allergic contact dermatitis (p<0.001 [Kolosova 2006]. vs control). The lower dose of bilberry was more effective [Yamaura 2011]. A bilberry extract was administered orally to mice (n=9; 100 mg/kg b.w.) for 7 days. Psychological stress was assessed by In a further experiment using the same methodology the whisker removal which is known to increase both protein standardized bilberry extract (400 mg/kg b.w.) was administered carbonyl formation and lipid peroxidation in the brain, heart, orally for 4 weeks to BALB/c mice with allergic contact kidney and liver. Treated mice showed less lipid peroxidation dermatitis induced by application of TNCB. Treatment with in all these tissues. The stress-induced shift of dopamine in the anthocyanin-containing extract (non-heated) significantly the brain was almost completely suppressed. [Rahman 2008]. attenuated the TNCB-induced increase in scratching behaviour compared to the vehicle (p<0.05). The anthocyanidin-rich (2-h Chemopreventive activity heated) extract had no effect. The non-heated extract (300 µg/ Bilberry extracts were assessed for their effects on multiple mL) resulted in significant inhibition of degranulation in RBL- biomarkers of azomethane-induced colon cancer in male rats 2H3 mast cells (p<0.05) [Yamaura 2012]. which were fed ad libitum for 14 weeks on a control diet or one supplemented with bilberry (containing 3.85 g monomeric Anti-ulcer/ gastroprotective activity anthocyanins / kg of diet). Total colonic aberrant crypt foci (ACF) A standardized extract (corresponding to 25% of anthocyanidins) were reduced (p<0.05) compared with the control. The number administered orally to rats at doses ranging from 25 to 200 of large ACF was also reduced (p<0.05) and colonic cellular mg/kg, showed antiulcer activity on ulcers induced by pyloric proliferation was decreased. Bilberry-fed rats had lower COX-2 ligation, non-steroidal-antiinflammatory drugs (NSAIDs) and mRNA gene expression. High levels of faecal anthocyanins and reserpine [Cristoni 1987]. increased faecal mass and moisture were found, as well as a significant reduction (p<0.05) in faecal bile acids [Lala 2006]. Oral pretreatment of rats with C3G (4 and 8 mg/kg body weight) resulted in a 36 and 80% inhibition respectively (P<0.05) ApcMin mice, a genetic model of human familial adenomatous of ethanol-induced gastric lesions in comparison to control polyposis, ingested C3G or a standardised bilberry extract (40% rats receiving only 80% ethanol. Pretreatment with C3G also anthocyanins) at 0.03, 0.1 or 0.3% in the diet for 12 weeks. inhibited elevation of lipid peroxide levels, and in gastric Ingestion of the highest doses of C3G and the bilberry extract tissues significantly (p<0.05) increased glutathione levels and showed a significant decrease in intestinal adenomas by 45%

7 MYRTILLI FRUCTUS

(p<0.001) or 30% (p<0.05) respectively compared to controls [Pulliero 1989]. Purified anthocyanins and in vivo metabolites of [Cooke 2006]. polyphenols were also found to have anti-thrombotic properties [Rechner 2005]. Multiple Intestinal Neoplasia/+ mice were fed modified high fat diets containing 10% freeze-dried bilberries (5.53 g total Clinical Studies anthocyanins, 0.104 g total flavonols /kg diet) or a similar berry- free diet as control for 10 weeks. The berry diet significantly Ophthalmic disorders inhibited the formation of intestinal adenomas in the distal small intestine, as indicated by a 15-30% reduction in tumour A systematic review of 30 relevant trials with bilberry antho- cell number (p<0.05) [Misikangas 2007]. cyanins on night vision, 12 of which were placebo-controlled, concluded that “negative outcome was associated with more The effect of C3G on xenograft growth of human lung carcinoma rigorous methodology but also with lower dose level and cell line A549 was evaluated. Nude mice were treated extracts from geographically distinct sources that may differ intraperitoneally with either PBS or C3G (9.5 mg/kg) three times in anthocyanoside composition. The hypothesis that bilberry per week beginning 2 days after s/c tumour cell implantation. anthocyanosides improve normal night vision is not supported C3G-treated mice showed a ~50% reduction in tumour size, by evidence from rigorous clinical studies. However when compared to control animals (p<0.02) and inhibited tumour all studies are taken together (controlled and un-controlled metastasis [Ding 2006]. trials with bilberry anthocyanins, controlled trials with related anthocyanins and evidence from animal studies), a sizeable Immunostimulation / myelo- and cyto-protective activity body of positive auxiliary evidence exists for the potential A single injection of 5-fluorouracil (5-FU) at 200 mg/kg into therapeutic role of bilberry” [Canter 2004; Lee 2005]. C57BL/6 mice induced severe peripheral erythrocytopenia, thrombocytopenia and leucopenia as well as hypocellularity of Daily treatment of 14 patients suffering from tapetoretinal the spleen and bone marrow. Oral administration of 500 mg/kg degeneration with 3 x 150 mg of a bilberry fruit extract resulted of an anthocyanin-rich bilberry extract for 10 days significantly in an improvement in light sensitivity of the retina starting from increased the number of red blood cells, neutrophils and the second day of treatment and remaining almost constant monocytes in peripheral blood to 1.2-fold, 9-fold and 6-fold during the 3-month treatment period [Zavarise 1968]. respectively, compared to 5-FU alone (p<0.05 - 0.001). The hypocellularity of the spleen and bone marrow caused by 5-FU Patients with glaucoma (n=8) were given a single dose (800 was also distinctly alleviated [Choi 2007]. mg) of a standardized bilberry extract (25% anthocyanins). Evaluation using electroretinography showed improvements Pharmacological studies in humans [Caselli 1985].

In a double-blind, placebo-controlled study involving 40 healthy The efficacy of a standardized extract (320 mg/day) was evaluated subjects the activity of an anthocyanin-rich extract, administered in 40 patients with refractory defects. Accurate ocular and orally at 240 mg daily for 3 months, was evaluated for pupillary electrofunctional examinations showed a significant increase movements through examination of the direct photomotor reflex. in the flash electroretinogram amplitude in medium (p=0.002) The study demonstrated a more efficient pupillary photomotor and high myopia (p=0.008), indicating an improvement in response after administration of the extract compared to placebo retinal sensitivity [Spadea 1991]. [Vannini 1986]. In another study, daily administration of 320 mg of the extract Young males with good vision, selected for a study using a for 3 months to 26 myopic patients resulted in improvement of double-blind, placebo-controlled, cross-over design, received electrophysiological functions [Contestabile 1991]. 160 mg of bilberry extract (25% anthocyanins) (n=7) or placebo (n=8) three times daily for 21 days, followed by a one-month Oral administration of a standardized extract of bilberry fruit washout period and the second three-week treatment period. at 480 mg/day for 180 days to 10 patients with type II diabetes After the three-month study no difference in night visual acuity mellitus and non-proliferative retinopathy resulted in improve- or night contrast sensitivity were found between placebo and ment of the diabetic retinopathy, with marked reduction or test subjects [Muth 2000]. disappearance of retinal haemorrhages [Orsucci 1983].

In a parallel-design, placebo-controlled clinical trial the effect of In a double-blind, placebo-controlled study, 40 patients a purified anthocyanin preparation (150 mg, equivalent to 100 g with diabetic or hypertensive retinopathy were treated with fresh bilberries, twice daily for 3 weeks) was studied in healthy 320 mg of a standardized extract of bilberry fruit or placebo adults (n=120). In the anthocyanin group, changes were observed daily for 30 days. An improvement in ophthalmoscopic and in (i) the NF-kB-controlled pro-inflammatory chemokine IL-8, angiographic patterns was observed in 77-90% of the verum (ii) RANTES (regulated upon activation, normal T cell expressed patients [Perossini 1987]. and secreted) and (iii) IFNa (an inducer of NF-kB activation) (45, 15 and 40% decreases from baseline, respectively) relative In another placebo-controlled study, involving 40 patients with to those in the placebo group (20, 0 and 15% decreases from diabetic retinopathy at a relatively early phase, a standardized baseline, respectively) (p<0.05). Similarly, changes in IL-4 and extract of bilberry fruit administered at 320 mg/day for 12 months IL-13, cytokines that mediate pro-inflammatory responses and promoted the regression of hard exudates, which is considered induce NF-kB activation, were found in the test group (60 and a reliable index of altered permeability [Repossi 1987]. 38% decreases from baseline respectively) compared with those in the placebo group (4 and 6% decreases) (p=0.056 and An open trial of bilberry extract (standardized to 25% p=0.089, respectively) [Karlsen 2007]. anthocyanins; 80-160 mg three times daily for 4 weeks) was conducted on 31 patients with retinal pathology from various Platelet aggregation was inhibited ex vivo in blood taken from causes; diabetic (n=20); retinitis pigmentosa (n=5); macular healthy volunteers after oral administration of bilberry extract degeneration (n=4) and haemorrhagic retinopathy from (480 mg/day; standardized to 36% anthocyanins) for 30-60 days anticoagulant therapy (n=2). Reduced vascular permeability

8 MYRTILLI FRUCTUS

and incidence of haemorrhage was reported in all patients, 54 cases of phlebopathies induced by stasis during pregnancy especially those with diabetic retinopathy [Scharrer 1981]. after oral administration of 320 mg of extract daily for 60-90 days [Grismondi 1980]. In a double-blind study, 14 diabetic and/or hypertensive outpatients with vascular retinopathy underwent therapy Cardiovascular health with bilberry anthocyanidins (160 mg twice daily) or placebo Unmedicated subjects with cardiovascular risk factors consumed (n=20) for 1 month. At the end of the month placebo patients berry (n = 35) or control products (n=36) for 8 weeks in a single- received the active for an additional month. Ophthalmoscopic blind, randomized, placebo-controlled intervention trial. Mean and fluoroangiographic findings obtained before and after daily intake from berry products was 837 mg polyphenols, of treatments showed improvements ranging from 77 to 90% in which 275 mg anthocyanins were from fresh bilberries (100 g). anthocyanin-treated patients [Bravetti 1989]. Berry consumption inhibited platelet function as measured with a platelet function analyser (using collagen and ADP as platelet Twenty-two subjects (14 males, 8 females; age range 23-59) with activator) [changes: 11% and -1.4% in the berry and control self-reported symptoms of dry eye were assessed at baseline by groups, respectively; p=0.018, analysis of covariance]. Serum the Ocular Surface Disease Index (OSDI), Tear Break-up Time HDL-cholesterol concentrations increased significantly more and Schirmer’s test. Patients were assigned to either bilberry (p=0.006, analysis of covariance) in the berry than in the control (80 mg orally, twice daily; no other details given) or placebo group (5.2% and 0.6%, respectively), but total cholesterol and for 30 days. The bilberry group showed a statistically significant triacylglycerol concentrations remained unchanged. Systolic improvement (p<0.01). The mean OSDI baseline for bilberry blood pressure decreased significantly (p=0.050, analysis of patients was 22.8 ±11.0, with post-treatment values of 13.7 ± covariance); the decrease mostly occurred in subjects with high 8.5 (p<0.01), while baseline for placebo was 27.9 ± 26.8 with baseline blood pressure (7.3 mm Hg in highest tertile; p=0.024, post treatment values of 19.6 ± 20.0 (p=n.s.) [Anderson 2011]. analysis of covariance). Polyphenol and vitamin C concentrations in plasma increased, whereas e.g. folate, tocopherols, sodium Peripheral vascular diseases and potassium were unaffected [Erlund 2008]. In 47 patients with lower limb varicose syndrome, oral treatment with bilberry fruit extract at 480 mg/day for 30 days led to Intake of anthocyanins (160 mg twice daily; cyanidin glycosides improvements in objective symptoms such as limb oedema 33% with delphinidin glycosides 58%) for 12 weeks by sub- and dyschromic skin phenomena, and subjective symptoms jects with elevated blood lipids (n=120, age 40-65 years) such as heaviness, paraesthesia and pain [Ghiringhelli 1977]. was compared with placebo in a double-blind, randomised, placebo-controlled trial. Anthocyanin consumption increased Improvements in vessel fragility and objective symptoms, after HDL-cholesterol concentrations (13.7% and 2.8% in the administration of bilberry fruit extract at 480 mg/day for 30-180 anthocyanin and placebo groups, respectively; p<0.001) and days, have been confirmed in trials performed on 22 diabetic decreased LDL-cholesterol concentrations (13.6% and -0.6% and dyslipidaemic patients [Passariello 1979], on 97 patients in the anthocyanin and placebo groups, respectively; p<0.001). with complaints induced by stasis of the lower extremities such Cellular cholesterol efflux to serum increased more in the as prevaricose syndrome, primary varicosis and post-phlebitic anthocyanin group than in the placebo group (20% and 0.2%, syndrome [Tori 1980], and on 42 patients with severe arterio- respectively; p<0.001). Anthocyanin intake decreased the mass sclerotic vascular disease of the lower limbs [Nuti 1980]. and activity of plasma cholesteryl ester transfer protein (CETP) by 10.4% and 6.3% respectively, in the anthocyanin group and In a double-blind, placebo-controlled study performed on 47 by -3.5% and 1.1% respectively in the placebo group (p<0.001). patients with peripheral vascular disorders of various origins, a In the anthocyanin group, the change in HDL cholesterol was standardized extract of bilberry fruit (480 mg/day for 30 days) negatively correlated with the change in CETP activity (rs=-0.33). reduced subjective symptoms such as heaviness, paraesthesia The change in LDL cholesterol was positively correlated with and pain, and improved oedema and mobility of finger joints the change in CETP mass activity (rs=0.354). The change in [Allegra 1982]. cellular cholesterol efflux to serum was positively correlated with

the change in HDL cholesterol activity (rs=0.485) [Qin 2009]. The efficacy of a standardized extract of bilberry fruit has been evaluated in two clinical studies, each in 15 patients with Anti-inflammatory activities venous insufficiency who were treated orally with 480 mg/day In a randomized controlled trial, subjects at elevated risk of for 2-4 months. Significant improvements in plethysmographic cardiovascular disease consumed either fresh bilberry juice (p<0.05) and duoregional rheographic (p<0.001) observations diluted to 1 litre with water (n=31) or 1 litre of water (n=31) were reported [Signorini 1983a, 1983b]. for 4 weeks. Participants in the test group consumed 330 mL bilberry juice /day. At the end of the intervention period plasma In 24 patients with chronic venous insufficiency, oral admin- concentrations of inflammatory mediators showed C-reactive istration of 480 mg of an anthocyanin-rich extract of bilberry protein (CRP), interleukin (IL)-6, IL-15 and monokine induced fruit daily for 60 days induced a decrease in the total time of by INF-g (MIG) were significantly decreased in the bilberry drainage after reactive hyperaemia evaluated by the strain gauge group (p=0.027, p=0.037, p=0.008 and p=0.047, respectively technique [Corsi 1985]. [Karlsen 2010].

The efficacy of the same extract (480 mg/day for 30 days) was In another randomized controlled trial, subjects with features further demonstrated in a single-blind, placebo-controlled of metabolic syndrome were given either a diet with added study carried out in 60 patients with different stages of venous bilberries equivalent to 400g fresh berries per day (n=15) or their insufficiency; this study showed significant activity of the extract normal diet alone (control group; n=12) for 8 weeks. Bilberry (p<0.01 to p<0.001) on subjective parameters, namely feeling supplementation decreased serum high-sensitivity C-reactive of pressure in the lower limbs and muscle cramps as well as protein, IL-6, IL-12 and LPS concentrations. An “inflammation oedema and leg and ankle girth [Gatta 1988]. score” based on changes of hsCRP, IL-6, IL-12 and LPS was significantly different between the groups (p=0.024) with the Significant improvements in subjective symptoms (p<0.01) and bilberry group showing decreased inflammation [Kolehmainen reductions in oedema and capillary fragility were observed in 2012].

9 MYRTILLI FRUCTUS

Other effects in a maximum anthocyanin plasma concentration of 0.26 µM. Colorectal cancer patients (n=25) scheduled to undergo resection Anthocyanins were detected in the liver (605 pmol/g), kidneys of primary tumour or liver metastases each received 1.4, 2.8 or (207 pmol/g), testes (149 pmol/g) and lungs (116 pmol/g), with 5.6 g daily of a standardised bilberry extract (36% anthocyanins; the malvidin glycosides being the predominant anthocyanins. containing 0.5, 1.0 or 2.0 g anthocyanins respectively) for 7 No anthocyanins were detected in the spleen, thymus, heart, days before surgery. Colorectal tumour sections, obtained before muscle, brain, white fat or eyes [Sakakibara 2009]. and after intake of the bilberry extract, were stained for Ki-67 (mouse anti-human monoclonal antibody) or cleaved caspase-3 ApcMin mice ingested cyanidin-3-glucoside (C3G) or a bilberry to determine proliferation of the tumour. In tumours from all extract (standardised to contain 40% anthocyanins) at 0.03, 0.1 patients receiving the bilberry extract the proliferation index or 0.3% in the diet (equivalent to 45, 150 or 450 mg/kg b.w./ (reflected by Ki-67 staining) showed a significant 7% reduction day) for 12 weeks. Anthocyanins were found in the plasma, the (p=0.003) compared with preintervention values. No significant intestinal mucosa and urine. At 0.3% in the diet, anthocyanin decrease in tumour tissue proliferation was found in patients levels were at the limit of detection in plasma, were 43 ng/g receiving 1.0 g and 2.0 g anthocyanins, while a significant tissue for C3G and 8.1 ng/g tissue for the bilberry extract in the 9% decrease (p=0.021) occurred in patients receiving 0.5 g intestinal mucosa and 7.2 and 12.3 µg/mL (C3G and extract anthocyanins [Thomasset 2009]. respectively) in the urine. Anthocyanin glucuronide and methyl metabolites were identified in the intestine and urine [Cooke Overweight and obese women (n=110) were recruited into a 2006]. study of randomized cross-over design to examine the effects of berries on variables of obesity-related diseases. Bilberries Fifteen minutes after oral supplementation of a mixture of 320 (100g daily, whole, frozen) were given for 33-35 days followed mg/kg b.w. C3G and 40 mg/kg b.w. cyanidin-3,5-diglucoside by a wash-out period of 30-39 days. Consumption of bilberries (Cy-dg), rats showed an increase to a maximum of 1563 µg (3490 resulted in a statistically significant (p=0.041) decrease in waist nmol) of C3G/L and 195 µg (320 nmol) of Cy-dg/L in plasma and circumference and a small decrease in weight (p=0.028). 0.067 µg (0.15 nmol) of C3G/g and a trace of Cy-dg together Significant decreases in vascular cell adhesion molecule with methylated metabolites in the liver [Miyazawa 1999]. (p=0.002) and TNF-a values (p=0.031) were also recorded [Lehtonen 2011]. Pharmacokinetics in humans

Pharmacokinetic properties A review of the data from human studies of cyanidins bio- availability shows that single doses of 188 – 3,570 mg Pharmacokinetics in animals total anthocyanins produced very low maximal plasma concentrations, of the range of 2.3 - 96 nM. The mean time Absorption, distribution, metabolism and elimination to reach these concentrations was around 1.5 h. Most studies After intravenous or intraperitoneal administration of an reported very low relative anthocyanin urinary excretions, anthocyanin-rich extract (equivalent to 25% of anthocyanidins) ranging from 0.018 to 0.37%. Maximal urinary excretion to rats at doses of 20-40 mg/kg or 25 mg/kg, respectively, was usually achieved in less than 4 h. Methylated derivatives, anthocyanins were rapidly distributed to the tissues. Elimination anthocyanidin glucuronide conjugates as well as anthocyanin occurred mainly in the urine (25-30% of the dose within 24 glycoside glucuronides have been identified in urine or plasma hours after administration) and to a lesser extent (15-20%) in [Galvano 2007]. bile [Lietti 1976]. All studies have shown that absorption is very low, with often After oral administration to rats of a single dose of 400 mg/kg, less than 0.15% of the ingested dose appearing in the urine a standardized extract of bilberry fruit was rapidly absorbed [McGhie 2003; McGhie 2007; Dreiseitel 2009]. from the gastrointestinal tract, reached a plasma concentration of Cmax of 2.47 µg/mL after 15 min, and rapidly declined within Colorectal cancer patients (n=25) received 1.4, 2.8 or 5.6 g 2 h [Morazzoni 1991]. daily of a standardised bilberry extract containing 36 % w/w anthocyanins for 7 days. Anthocyanins, as well as methyl- and A bilberry extract (containing 153.2 mg total anthocyanins/ glucuronide metabolites were identified in plasma, colorectal kg), with delphinidin galactoside as the main constituent (19.6 tissue and urine, but not the liver [Thomasset 2009]. mg/kg), was administered to rats orally (400 mg/kg b.w.) or intravenously (5 mg/kg b.w.). After oral administration most Thirty minutes after receiving a single oral dose of a mixture of 2.7 anthocyanins rapidly appeared in the plasma and reached their mg/kg b.w. C3G and 0.25 mg/kg b.w. cyanidin-3,5-diglucoside maximum level at 15 min, except galactosides of petunidin, (Cy-dg), human adults (n=12, 20-29 years, 40-70 kg) had an peonidin and malvidin that reached their maximum at 30 average of 24 nM of C3G and traces of Cy-dg in plasma. Neither min. The bioavailability of individual anthocyanins was in the cyanidin aglycone, nor conjugated or methylated metabolites range 0.6 to 1.8%, and 0.93% as the anthocyanin mixture. were found [Miyazawa 1999]. After intravenous administration anthocyanins recovered in urine and bile during the first 4 h were only 30.8 and 13.4% Healthy elderly women (n=4) having fasted overnight were respectively [Ichiyanagi 2006]. given a single oral dose of 720 mg anthocyanins (cyanidin-3- glucoside and cyanidin-3-sambubioside). Maximum plasma Oral administration of a bilberry extract to mice (dried, prepared concentrations of total anthocyanins varied from 55.3 to 168.3 from fresh berries; 100 mg/kg b.w.) resulted in a maximum plasma nM, with an average of 97.4 nM, and were reached within 71.3 anthocyanin concentration (1.18 µM) after 15 min. Urinary min (tmax). The elimination of plasma anthocyanins appeared excretion ceased by 24 h after a maximum between 0 and 6 to follow first-order kinetics. Half-life of cyanidin-3-glucoside h after administration. Total amount of anthocyanins excreted is 97.3 min and cyanidin-3-sambubioside is 168.9 min. No into the urine represented 1.88%. Malvidin-3-glucoside and anthocyanin glucuronides or sulphates were detected. The -3-galactoside were the principal anthocyanins in the plasma anthocyanins were excreted as glycosides in urine mostly during 60 mins after administration. Maintenance of the mice for 2 the first 4h (77.2 µg/h during the first 4 h and 13.4 during the weeks on a diet containing 0.5% of bilberry extracts resulted second 4 h) [Cao 2001].

10 MYRTILLI FRUCTUS

Adult males (n=3) received a dose of 721 mg cyanidin-3- Asymptomatic subjects with intraocular hypertension were given glycosides. The cumulative concentration of total anthocyanins either a combination of two phenolic extracts from bilberry (80 (parent and metabolites) detected in the serum (0-7 h) was 376.7 mg, standardised to 36% anthocyanins) and French maritime (nmol.h)/L, reaching a maximum concentration of 96.1 nM/L bark (40 mg, standardised to 70% procyanidins) (n=20) twice within 2.8 h. Parent anthocyanin glycosides represented only daily or were not treated (n=18). During 2 months of treatment, 32.0% of the total anthocyanins detected with 68% identified no side effects were observed [Steigerwalt 2008]. In a further as glucuronidated and methylated derivatives. Total urinary trial with this product, in patients (n=33) who received one excretion of anthocyanins over 24 h was 1072 µg, reaching a tablet daily, no side effects occurred [Steigerwalt 2010]. maximal excretion rate (203 µg/h) at 3.7 h [Kay 2005]. REFERENCES Preclinical safety data Abebe W. Herbal medication: potential for adverse interactions with Acute toxicity analgesic drugs. Review Article. J Clin Pharm Ther. 2002;27:391-401. Intraperitoneal and intravenous LD50 values of an anthocyanin- http://dx.doi.org/10.1046/j.1365-2710.2002.00444.x rich extract from bilberry fruit containing about 70% of antho- cyanins were determined as 4.11 g/kg and 0.84 g/kg in the Akerström A, Jaakola L, Bang U, Jäderlund A. Effects of latitude-related mouse, and 2.35 g/kg and 0.24 g/kg in the rat, respectively. No factors and geographical origin on anthocyanidin concentrations deaths were observed following oral doses of up to 25 g/kg in in fruits of Vaccinium myrtillus L. (Bilberries). J Agric Food Chem. the mouse and 20 g/kg in the rat [Pourrat 1967]. 2010;58:11939-45. http://dx.doi.org/10.1021/jf102407n

Acute oral LD50 of an anthocyanin-rich berry extract (containing Alfieri R, Sole P. Influence des anthocyanosides administrés par voie cyanidin, delphinidin, petunidin, malvidin) was greater than 5 parentérale sur l’adapto-électrorétinogramme du lapin. CR Soc Biol. 1964;158:2338-41. g/kg in rats. Acute dermal LD50 was greater than 2 g/kg [Bagchi 2006]. Allegra C, Pollari G, Criscuolo A, Bonifacio M. Antocianosidi e sistema Repeated dose and chronic toxicity studies microvasculotessutale. Minerva Angiol. 1982;7:39-44. Treatment of rats for 90 days with the same extract at a daily dose corresponding to approximately five times the human Anderson K, Anderson A, Connor C. Potential use of bilberry for clinical dose (i.e. 600 mg/day) did not produce any toxic effects dry eye relief. Optometry. 2011;82:380. http://dx.doi.org/10.1016/j. [Pourrat 1967]. optm.2011.04.081 Bagchi D, Roy S, Patel V, He G, Khanna S, Ojha N. Safety and whole- Mutagenicity and carcinogenicity body antioxidant potential of a novel anthocyanin-rich formulation of A bilberry extract (36% anthocyanins) did not show mutagenic edible berries. Mol Cell Biochem. 2006;281:197-209 http://dx.doi. activity in various tests of mutagenesis, with and without org/10.1007/s11010-006-1030-6 metabolic activation, and showed no teratogenic effect or influence on rat fertility [Eandi 1987]. Baj A, Bombardelli E, Gabetta B, Martinelli EM. Qualitative and quantitative evaluation of Vaccinium myrtillus anthocyanins by Cytotoxicity to HaCaT keratinocytes was determined using high-resolution gas chromatography and high-performance liquid a lactate dehydrogenase (LDH) leakage assay. Berry extracts chromatography. J Chromatogr. 1983;279:365-72. http://dx.doi. (50 µg/mL) were added to the cells and after 24 h media were org/10.1016/S0021-9673(01)93636-2 collected for assay. No significant cytotoxicity was noted with the bilberry extract (50 µg/mL) when compared to control Bao L, Yao X-S, Yau C-C, Tsi D, Chia C-S, Nagai H. Protective effects [Yasmin 2003]. of bilberry (Vaccinium myrtillus L.) extract on restraint stress-induced liver damage in mice. J Agric Food Chem. 2008a;56:7803-7807. http:// The effects of different concentrations of bilberry extract dx.doi.org/10.1021/jf800728m (containing 25% total anthocyanins) were studied using human corneal limbal epithelial cells (HCLEC). The extract (10-5 M) Bao L, Yao X-S, Tsi D, Yau C-C, Chia C-S, Nagai H. Protective effects promoted cell growth to 120% compared with the control of bilberry (Vaccinium myrtillus L.) extract on KBrO3-induced kidney group (p<0.05) after 24 h and 48 h incubation. Cytotoxicity damage in mice. J Agric Food Chem. 2008b;56:420-425. http://dx.doi. was not noted [Song 2010]. org/10.1021/jf072640s

Clinical safety data Bao L, Keiichi A, Tsang P, Xu J-K, Yao X-S, Liu H-W. Bilberry extract protect restraint stress-induced liver damage through attenuating The safety of the bilberry extract (36% anthocyanins; mostly mitochondrial dysfunction. Fitoterapia. 2010;81:1094-1101. http:// 160 mg twice daily for 1-2 months) was confirmed in a post- dx.doi.org/10.1016/j.fitote.2010.07.004 marketing surveillance study on 2295 subjects affected by: Bell DR, Gochenaur K. Direct vasoactive and vasoprotective properties lower limb venous insufficiency (24.23%), states of fragility of anthocyanin-rich extracts. J Appl Physiol. 2006;100:1164-1170. and altered permeability of the blood capillaries (21.51%), http://dx.doi.org/10.1152/japplphysiol.00626.2005 functional changes in retinal microcirculation (10.15%) and haemorrhoids (9.93%) [Eandi 1987]. Bertuglia S, Malandrino S, Colantuoni A. Effect of Vaccinium myrtillus anthocyanosides on ischemia-reperfusion injury in hamster cheek Daily intake of 100 - 335 mg of anthocyanins was considered pouch microcirculation. Pharmacol Res. 1995;31:183-187. http:// safe [Karlsen 2007]. Long-term oral administration in humans dx.doi.org/10.1016/1043-6618(95)80016-6 in doses equivalent to 180 mg/kg anthocyanins per day for six months produced no toxic effects [Eandi 1987]. Bettini V, Mayellaro F, Ton P, Zogno M. Interactions between Vaccinium myrtillus anthocyanosides and serotonin on splenic artery smooth In colorectal cancer patients, daily consumption of 5.6 g of a muscle. Fitoterapia 1984a;55:201-8. standardised bilberry extract (36% anthocyanins) for 7 days was well tolerated [Thomasset 2009]. Bettini V, Mayellaro F, Ton P, Zanella P. Effects of Vaccinium myrtillus

11 MYRTILLI FRUCTUS

anthocyanosides on vascular smooth muscle. Fitoterapia.1984b; to clarithromycin. Mol Cell Biochem. 2004;265:19-26. http://dx.doi. 55:265-72. org/10.1023/B:MCBI.0000044310.92444.ec

Bettini V, Mayellaro F, Patron E, Ton P, Terribile Wiel Marin V. Inhibition Chen J, Uto T, Tanigawa S, Kumamoto T, Fujii M, Hou D-X. Expression by Vaccinium myrtillus anthocyanosides of barium-induced contractions profiling of genes targeted by bilberry (Vaccinium myrtillus) in in segments of internal thoracic vein. Fitoterapia.1984c;55:323-7. macrophages DNA microarray. Nutr Cancer. 2008;60(S1):43-50. http:// dx.doi.org/10.1080/01635580802381279 Bettini V, Mayellaro F, Pilla I, Ton P, Terribile Wiel Marin V. Mechanical responses of isolated coronary arteries to barium in the presence of Choi EH, Yoon Y, Magnuson BA, Kim MK, Chun HS. Protective effect of Vaccinium myrtillus anthocyanosides. Fitoterapia.1985;56:3-10. anthocyanin-rich extract from bilberry (Vaccinium myrtillus L.) against myelotoxicity induced by 5-fluorouracil. BioFactors. 2007;29:55-65. Bilberry. In AltMedDex® System: Thomson Reuters Healthcare Inc. http://dx.doi.org/10.1002/biof.5520290106 Series 1974-2012. Cohen-Boulakia F, Valensi P, Boulahdour H. In Vivo Sequential Study Bilberry fruit, dried - Myrtilli fructus siccus. European Pharmacopoeia, of Skeletal Muscle Capillary Permeability in Diabetic Rats: Effects Council of Europe. of Anthocyanosides. Metabolism. 2000;49:880-885. http://dx.doi. org/10.1053/meta.2000.6754 Bilberry fruit, fresh - Myrtilli fructus recens. European Pharmacopoeia, Council of Europe. Colantuoni A,Bertuglia S, Magistretti MJ, Donata L. Effects of Vaccinium myrtillus anthocyanosides on arterial vasomotion. Arzneimittelforschung. Blaschek W, Ebel S, Hackenthal E, Holzgrabe U, Keller K, Reichling 1991;41:905-9. J, Schulz V. In Hagers Enzyklopädie der Arzneistoffe und Drogen, Band 16 Tri-Zyg. Stuttgart: Wissenschaftliche Verlagsgesellschaft mbH, Contestabile MT, Appolloni R, Suppressa F, D’Alba E, Pecorelli 2007:421-441. B. Trattamento prolungato con antocianosidi del mirtillo ad alto dosaggio: risposte elettrofisiologiche nel paziente miope. Boll Ocul. Blumenthal M, Brinckmann J, Wollschläger B. Bilberry. In: The American 1991;70:1157-69. Botanical Council Clinical Guide to Herbs. American Botanical Council, Austin,Texas, 2003:6-12. Cooke D, Schwarz M, Boocock DJ, Winterhalter P, Steward WP, Gesher AJ. Effect of cyanidin-3-glucoside and an anthocyanin mixture from Bonacina F, Galliani G, Pacchiano F. Attività degli antocianosidi nei bilberry on adenoma development in the Apc Min mouse model of processi flogistici acuti. Il Farmaco Ed Pr. 1973;28:428-34. intestinal carcinogenesis - relationship with tissue anthocyanin levels. Int J Cancer. 2006;119:2213-2220. http://dx.doi.org/10.1002/ijc.22090 Boniface R, Miskulin M, Robert L, Robert AM. Pharmacological properties of Myrtillus anthocyanosides: correlation with results of Corsi C, Pollastri M, Tesi C, Borgioli A, Boscarini A. Contributo allo treatment of diabetic microangiopathy. In: Farkas L, Gábor M, Kállay studio dell’attività degli antocianosidi sul microcircolo: valutazioni F, Wagner H, editors. Flavonoids and Bioflavonoids, Amsterdam: flussimetriche nell’insufficienza venosa cronica. Fitoterapia. Elsevier, 1982:293-301. 1985;56(Suppl 1):23-31.

Bravetti GO, Fraboni E, Maccolini E. Preventive medical treatment of Cristoni A, Magistretti MJ. Antiulcer and healing activity of Vaccinium senile cataract with Vitamin E and Vaccinium myrtillus anthocyanosides: myrtillus anthocyanosides. Il Farmaco Ed Prat. 1987;42:29-43. clinical evaluation. Annali di Ottalmologia e Clinica Oculistica. 1989;115;109-116. Curri BS, Lietti A, Bombardelli E. Modificazioni morfoistochimiche del tessuto di granulazione delle ferrite sperimentali indotte dall’apporto Burdulis D, Ivanauskas L, Dirse V, Kazlauskas S, Razukas A. Study of locale di antocianosidi. Giorn min Derm. 1976;111:509-515. diversity of anthocyanin composition in bilberry (Vaccinium myrtillus L.) fruits. Medicina (Kaunas) 2007, 43, 971-97. De Smet PAGM, Keller K, Hansel R. Chandler RF. Vaccinium myrtillus. In: Adverse Effects of Herbal Drugs, Vol. 2. Springer-Verlag, Berlin. Burdulis D, Sarkinas A, Jasutiene I, Stackeviciene E, Niklajevas L, Janulis 1993:307-314. V. Comparative study of anthocyanin composition, antimicrobial and antioxidant activity in bilberry (Vaccinium myrtillus L.) and blueberry Detre Z, Jellinek H, Miskulin M, Robert AM. Studies on vascular (Vaccinium corymbosum L.) fruits. Acta Poloniae Pharmaceutica-Drug permeability in hypertension: action of anthocyanosides. Clin Physiol Research. 2009;66:399-408. Biochem. 1986;4:143-9.

Cao G, Muccitelli H, Sanchez-Moreno C, Prior R. Anthocyanins are Ding M, Feng R, Wang S, Bowman L, Lu Y, Qian Y. Cyanidin-3-glucoside, absorbed in glycated forms in elderly women: a pharmacokinetic study. a natural product derived from blackberry, exhibits chemopreventive Am J Clin Nutr. 2001;73:920-6. and chemotherapeutic activity. J Biol Chem. 2006;281:17359-17368. http://dx.doi.org/10.1074/jbc.M600861200 Canter PH, Ernst E. Anthocyanosides of Vaccinium myrtillus (Bilberry) for night vision - a systematic review of placebo-controlled trials. Domitrovic R, Jakovac H. Effects of standardized bilberry fruit extract

Survey of Ophthalmology. 2004;49:38-50. http://dx.doi.org/10.1016/j. (MirtoselectR) on resolution of CCl4-induced liver fibrosis in mice. survophthal.2003.10.006 Food Chem Toxicol. 2011;49:848-54. http://dx.doi.org/10.1016/j. fct.2010.12.006 Caselli L. Clinical and electroretinopathic study on anthocyanosides. Arch Med Interna. 1985;37:29-35. Dorsch W, Loew D, Meyer-Buchtela E, Schilcher H. Kinderdosierung von Phytopharmaka, 3rd ed., Empfehlunger zur Anwendung und Cassinese C, De Combarieu E, Falzoni M, Fuzzati N, Pace R, Sardone Dosierung von Phytopharmaka, monographierte Arzneidrogen und ihre N. New liquid chromatography method with ultraviolet detection for Zubereitungen in der Pädiatrie. Bonn: Kooperation Phytopharmaka, analysis of anthocyanins and anthocyanidins in Vaccinium myrtillus fruit 2002. dry extracts and commercial preparations. J AOAC Int. 2007;90:911-919. Dreiseitel A, Oosterhuis B, Vukman KV, Schreir P, Oehme A, Locher S. Chatterjee A, Yasmin T, Bagchi D, Stohs SJ. Inhibition of Helicobacter Berry anthocyanains and anthocyanidins exhibit distinct affinities for the pylori in vitro by various berry extracts, with enhanced susceptibility efflux transporters BCRP and MDR1. Br J Pharmacol. 2009;158:1942-

12 MYRTILLI FRUCTUS

1950. http://dx.doi.org/10.1111/j.1476-5381.2009.00495.x fruit development. Plant Physiol. 2002;130:729-739. http://dx.doi. org/10.1104/pp.006957 Du Q, Jerz G, Winterhalter P. Isolation of two anthocyanin sambubiosides from bilberry (Vaccinium myrtillus) by high-speed counter-current Jänicke C, Grünwald J, Brendler T. Blaubeere-Vaccinium myrtillus. chromatography. J Chromatogr A. 2004;1045(1-2):59-63. http://dx.doi. In: Handbuch Phytotherapie. Stuttgart: Wissenschaftliche Verlags- org/10.1016/j.chroma.2004.06.017 gesellschaft, 2003:219-221.

Eandi M. Post-marketing investigations on TegensTM preparation Jang YP, Zhou J, Nakanishi K, Sparrow JR. Anthocyanins protect against with respect to side effects. In: Morazzoni P. Vaccinium myrtillus L. A2E photooxidation and membrane permeabilization in retinal pigment Fitoterapia 1996;67:3-29. epithelial cells. Photochem Photobiol. 2005;81:529-536. http://dx.doi. org/10.1562/2004-12-14-RA-402.1 Ehala S, Vaher M, Kaljurand M. Characterization of phenolic profiles of northern European berries by capillary electrophoresis and determination Jonadet M, Meunier MT, Bastide P. Anthocyanosides extraits de Vitis of their antioxidant activity. J Agric Food Chem. 2005;53:6484-6490. vinifera, de Vaccinium myrtillus et de Pinus maritimus. I. Activités http://dx.doi.org/10.1021/jf050397w inhibitrices vis-à-vis de l’élastase in vitro. II. Activités angioprotectrices comparées in vivo. J Pharm Belg. 1983;38:41-6. Erlund I, Koli R, Alfthan G, Marniemi J, Puukka P, Mustonen P. Favorable effects of berry consumption on platelet function, blood pressure and Jing P, Bomser JA, Schwarz SJ, He J, Magnuson BA, Giusti MM. Structure- HDL cholesterol. Am J Clin Nutr. 2008;87:323-331. function relationships of anthocyanins from various anthocyanin-rich extracts on the inhibition of colon cancer cell growth. J Agric Food Esselen A, Fritz J, Hutter, Teller N, Baechler S, Boettler U. Anthocyanin- Chem. 2008;56:9391-9398. http://dx.doi.org/10.1021/jf8005917 rich extracts suppress the DNA-damaging effects of topoisomerase poisons in human colon cancer cells. Mol Nutr Food Res. 2011;Suppl Kadar A, Robert L, Miskulin M, Tixier JM, Brechemier D, Robert AM. 1:S143-153. http://dx.doi.org/10.1002/mnfr.201000315 Influence of anthocyanoside treatment on the cholesterol-induced atherosclerosis in the rabbit. Arterial Wall 1979;5:187-206. Faria A, Oliviera J, Neves P, Gameiro P, Santos-Buelga C, De Freitas V. Antioxidant properties of prepared blueberry (Vaccinium myrtillus) Karlsen A, Retterstol L, Laake P, Paur I, Kjolsrud-Bohn S, Sandvik extracts. J Agric Food Chem. 2005;53:6896-6902. http://dx.doi. L. Anthocyanins inhibit nuclear factor-kB activation in monocytes org/10.1021/jf0511300 and reduce pro-inflammatory mediators in healthy adults. J Nutr. 2007;137:1951-1954. Fresh bilberry fruit dry extract, refined and standardised– Myrtilli fructus recentis extractum siccum rafinatum et normatum. European Karlsen A, Paur I, Bohn SK, Sakhi AK, Borge GI, Serafini M. Bilberry juice Pharmacopoeia, Council of Europe. modulates plasma concentration of NF-kappaB related inflammatory markers in subjects of increased risk of CVD. Eur J Nutr. 2010;49:345- Friedrich H, Schönert J. Untersuchungen über einige Inhaltsstoffe der 355. http://dx.doi.org/10.1007/s00394-010-0092-0 Blätter und Früchte von Vaccinium myrtillus. Planta Med. 1973;24:90- 100. http://dx.doi.org/10.1055/s-0028-1099474 Katsube N, Iwashita K, Tsushida T, Yamaki K, Kobori M. Induction of apoptosis in cancer cells by bilberry (Vaccinium myrtillus) and Fursova AZh, Gesarevich OG, Gonchar AM, Trofimova NA, Kolosova the anthocyanins. J Agric Food Chem. 2003:51:68-75. http://dx.doi. NG. Dietary supplementation with bilberry extract prevents macular org/10.1021/jf025781x degeneration and cataracts in senescence accelerated OXYS rats (Abstract only; Russian). Adv Gerontol. 2005;16:76-79. Kay C, Mazza G, Holub B. Anthocyanins exist in the circulation primarily as metabolites in adult men. J Nutr. 2005;135:2582-2588. Galvano F, La Fauci L, Vitaglione P, Fogliano V, Vanella L, Felgines C. Bioavailability, antioxidant and biological properties of the natural Kolehmainen M, Mykkänen, Kirjavainen P, Leppänen T, Moilanen free-radical scavengers cyanidin and related glycosides. Ann Ist Super E, Adriaens M, et al. Bilberries reduce low-grade inflammation in Sanita. 2007;43:382-393. individuals with features of metabolic syndrome. Mol Nutr Food Res. 2012;56:1501-1510. http://dx.doi.org.10.1002/mnfr.201200195 Gatta L. Gli antocianosidi del mirtillo nel trattamento della stasi venosa: studio clinico controllato su sesenta pazienti. Fitoterapia Kolosova NG, Shcheglova TV, Sergeeva SV, Loskutova LV. Long- 1988;59(Suppl 1):19-26. term antioxidant supplementation attenuates oxidative stress markers and cognitive deficits in senescent-accelerated OXYS rats. Ghiringhelli C, Gregoratti L, Marastoni F. Attività capillarotropa di Neurobiol Aging. 2006;27:1289-1297. http://dx.doi.org/10.1016/j. antocianosidi ad alto dosaggio nella stasi da flebopatia. Minerva neurobiolaging.2005.07.022 Cardioangiol. 1977;26:255-76. Lala G, Malik M, Zhao C, He J, Kwon Y, Giusti MM. Anthocyanin-rich Grismondi G. Treatment of pregnancy-induced phlebopathies. Estratto extracts inhibit multiple biomarkers of colon cancer in rats. Nutr Cancer. Minerva Ginecologica.1980;32:1-14. 2006;54:84-93. http://dx.doi.org/10.1207/s15327914nc5401_10

Huttunen S, Toivanen M, Arkko S, Ruponen M, Tikkanen-Kaukanen C. Laplaud PM, Lelubre A, Chapman MJ. Antioxidant action of Vaccinium Inhibition activity of wild berry juice fractions against Streptococcus myrtillus extract on human low density lipoproteins in vitro: initial pneumoniae binding to human bronchial cells. Phytother Res. observations. Fundam Clin Pharmacol. 1997;11:35-40. http://dx.doi. 2011;25:122-127. http://dx.doi.org/10.1002/ptr.3240 org/10.1111/j.1472-8206.1997.tb00166.x

Ichiyanagi T, Shida Y, Rahman MM, Hatano Y, Konishi T. Bioavailability Latti AK, Riihinen KR, Kainulainen PS. Analysis of anthocyanin variation and tissue distribution of anthocyanins in bilberry (Vaccinium myrtillus in wild populations of Bilberry (Vaccinium myrtillus L.) in Finland. J Agric L.) extract in rats. J Agric Food Chem. 2006;54:6578-6587. http://dx.doi. Food Chem. 2008;56:190-196. http://dx.doi.org/10.1021/jf072857m org/10.1021/jf0602370 Lee J, Lee HK, Kim CY, Hong YJ, Choe CM, You TW. Purified high-dose Jaakola L, Määttä K, Pirttilä AM, Törrönen, Kärenlampi S, Hohtola A. anthocyanoside oligomer administration improves nocturnal vision and Expression of genes involved in anthocyanin biosynthesis in relation clinical symptoms in myopia subjects. Br J Nutr. 2005;93:895-899. to anthocyanin, proanthocyanin and flavonol levels during bilberry http://dx.doi.org/10.1079/BJN20051438

13 MYRTILLI FRUCTUS

Lehtonen H-M, Suomela J-P, Tahvonen R, Yang B, Venojärvi M, Viikari inhibit cellular reactions of drugs producing oxy radicals. In: Cohen J. Different berries and berry fractions have various but slightly positive G, Greenwald RA, editors. Oxy Radicals: Their Scavenger Syst., Proc. effects on the associated variables of metabolic diseases on overweight Int. Conf. Superoxide - Superoxide Dismutase, 3rd. New York: Elsevier, and obese women. Eur J Clin Nutr. 2011;65:394-401. http://dx.doi. 1982(Pub 1983);2:326-9; through Chem. Abstr. 1984;100:150. org/10.1038/ejcn.2010.268 McGhie T, Ainge G, Barnett L, Cooney J, Jensen D. Anthocyanin Li CY, Xu HD, Zhao BT, Chang HI, Rhee HI. Gastroprotective effect glycosides from berry fruit are absorbed and excreted unmetabolized of cyanidin-3-glucoside on ethanol-induced gastric lesions. Alcohol. by both humans and rats. J Agric Food Chem. 2003;51:4539-4538. 2008;42:683-687. http://dx.doi.org/10.1016/j.alcohol.2008.08.009 http://dx.doi.org/10.1021/jf026206w

Lietti A, Cristoni A, Picci M. Studies on Vaccinium myrtillus McGhie T, Walton M. The bioavailablity and absorption of anthocyanins: anthocyanosides. I. Vasoprotective and antiinflammatory activity. towards a better understanding (Review). Mol Nutr Food Res. Arzneim-Forsch/Drug Res. 1976;26:829-32. 2007;51:702-713. http://dx.doi.org/10.1002/mnfr.200700092

Lietti A, Forni G. Studies on Vaccinium myrtillus anthocyanosides. II. Meunier MT, Duroux E, Bastide P. Activité antiradicalaire d’oligomères Aspects of anthocyanins pharmacokinetics in the rat. Arzneim-Forsch/ procyanidoliques et d’anthocyanosides vis-à-vis de l’anion superoxyde et Drug Res. 1976;26:832-5. vis-à-vis de la lipoperoxydation. Plantes Méd Phytothér. 1989;23:267-74.

Määttä-Riihinen KR, Kahkonen MP, Torronen AR, Heinonen IM. Milbury PE, Graf B, Curran-Celentano JM, Blumberg JB. Bilberry Catechins and procyanidins in berries of Vaccinium species and their (Vaccinium myrtillus) anthocyanins modulate heme oxygenase- and anitioxidant activity. J Agric Food Chem. 2005;53:8585-8491. http:// glutathione S-transferase-pi expression in ARPE-19 cells. Invest dx.doi.org/10.1021/jf050408l Ophthalmol Vis Sci. 2007;48:2343-2349. http://dx.doi.org/10.1167/ iovs.06-0452 Maridonneau I, Braquet P, Garay RP. Bioflavonoids protect human erythrocytes against the K+ -loss induced by free radicals. In: Farkas L, Mills S, Bone K. Bilberry Fruit. In: The Essential Guide to Herbal Safety. Gábor M, Kállay F, Wagner H, editors. Flavonoids and Bioflavonoids, Elsevier 2005:262-264 1981. Amsterdam: Elsevier, 1982:427-36. Misikangas M, Pajari A-M, Päivärinta E, Oikarinen S, Rajakangas J, Martín-Aragón S, Basabe B, Benedì JM, Villar AM. Antioxidant Martttinen M. Three Nordic berries inhibit intestinal tumorigenesis in action of Vaccinium myrtillus L. Phytother Res. 1998;12:S104-6. multiple intestinal neoplasia/+ mice by modulating b-catenin signalling http://dx.doi.org/10.1002/(SICI)1099-1573(1998)12:1+3.0.CO;2-O Miyake S, Takahashi N, Sasaki M, Kobayashi S, Tsubota K, Ozawa Y. Vision Martín-Aragón S, Basabe B, Bebedì JM, Villar AM. In vitro and in preservation during retinal inflammation by anthocyanin-rich bilberry vivo antioxidant properties of Vaccinium myrtillus. Pharmaceut Biol. extract: cellular and molecular mechanism. Lab Invest. 2012;92:102- 1999;37:109-13. http://dx.doi.org/10.1076/phbi.37.2.109.6091 109. http://dx.doi.org/10.1038/labinvest.2011.132

Matsumoto H, Nakamura Y, Tachibanaki S, Kawamura S, Hirayama Miyazawa T, Nakagawa K, Kudo M, Muraishi K, Someya K. Direct M. Stimulatory effect of cyanidin 3-glycosides on the regeneration intestinal absorption of red fruit anthocyanins, cyanidin-3-glucoside and of rhodopsin. J Agric Food Chem. 2003;51:3560-3563. http://dx.doi. cyanidin-3,5-diglucoside, into rats anfd humans. J Agric Food Chem. org/10.1021/jf034132y 1999;47:1083-1091. http://dx.doi.org/10.1021/jf9809582

Matsunaga N, Chikaraishi Y, Shimazawa M, Yokata S, Hara H. Vaccinium Morazzoni P, Magistretti MJ. Activity of Myrtocyan®, an anthocyanoside myrtillus (Bilberry) extracts reduce angiogenesis in vitro and in vivo. complex from Vaccinium myrtillus (VMA), on platelet aggregation and Evid Based Complement Alt Med. 2007;Oct 27 eCAM adhesiveness. Fitoterapia 1990;61:13-21.

Matsunaga N, Imai S, Inokuchi Y, Shimazawa M, Yokata S, Araki Y. Morazzoni P, Livio S, Scilingo A, Malandrino S. Vaccinium myrtillus Bilberry and its main constituents have neuroprotective effects against anthocyanosides pharmacokinetics in rats. Drug Res. 1991;41:128-131. retinal neuronal damage in vitro and in vivo. Mol Nutr Food Res. 2009;53:869-877. http://dx.doi.org/10.1002/mnfr.200800394 Morazzoni P, Bombardelli E. Vaccinium myrtillus L. Fitoterapia. 1996;67:3-29. Matsunaga N, Tsuruma K, Shimazawa M, Yokota S, Hara H. Inhibitory actions of bilberry anthocyanidins on angiogenesis. Phytother Res. Muth ER, Laurent JM, Jasper P. The effect of bilberry nutritional 2010;24:S42-S47. http://dx.doi.org/10.1002/ptr.2895 supplementation on night visual acuity and contrast sensitivity. Altern Med Rev. 2000;5:164-173. Mauray A, Milenkovic D, Besson C, Caccia N, Morand C, Michel F. Atheroprotective effects of bilberry extracts in Apo E-deficient mice. J Nguyen V, Tang J, Oroudjev E, Lee C, Marasigan C, Wilson L. Cytotoxic Agric Food Chem. 2009;57:11106-11111. http://dx.doi.org/10.1021/ effects of bilberry extract on MCF7-GFP-tubulin breast cancer cells. jf9035468 J Med Food. 2010;13:1-8. http://dx.doi.org/10.1089/jmf.2009.0053

Mauray A, Felgines C, Morand C, Mazur A, Scalbert A, Milenkovic D. Nizamutdinova IT, Kim YM, Chung JI, Shin SC, Seo HG, Lee JH. Nutrigenomic analysis of the protective effects of bilberry anthocyanin- Anthocyanins from black soybean seed coats stimulate wound rich extract in apo E-deficient mice. Genes Nutr. 2010;5:343-353. http:// healing in fibroblasts and keratinocytes and prevent inflammation in dx.doi.org/10.1007/s12263-010-0171-0 endothelial cells. Food ChemToxicol. 2009; 47:2806-2812. http:// dx.doi.org/10.1016/j.fct.2009.08.016 Mauray A, Felgines C, Morand C, Mazur A, Scalbert A, Milenkovic D. Bilberry anthocyanin-rich extract alters expression of genes related to Nohynek L, Alakomi H-L, Kahkonen M, Heinonen M, Helander IM, atherosclerosis development in aorta of apo E-deficient mice. Nutr Oksman-Caldetey K-M. Berry phenolics-antimicrobial properties and Metab Cardiovasc Dis. 2012;22:72-80. http://dx.doi.org/10.1016/j. mechanisms of action against severe human pathogens. Nutr Cancer. numecd.2010.04.011 2006;54:18-32. http://dx.doi.org/10.1207/s15327914nc5401_4

Mavelli I, Rossi L, Autuori F, Braquet P, Rotilio G. Anthocyanosides Nuti A, Curri SB, Vittori C, Lampertico M. Significance of anthocyanins

14 MYRTILLI FRUCTUS

in the treatment of peripheral vascular diseases. In: Reinis Z, Pokorný Rahman MM, Ichiyanagi T, Komiyama T, Sato S, Konishi T. Effects of J, Linhart J, Hild R, Schirger, editors. Adaptability of Vascular Wall. anthocyanins on psychological stress-induced oxidative stress and Prague: Springer-Verlag, 1980:572-3. neurotransmitter status. J Agric Food Chem. 2008;56:7545-7550. http:// dx.doi.org/10.1021/jf800930s Ogawa K, Sakakibara H, Iwata R, Ishii T, Sato T, Goda T. Anthocyanin composition and antioxidant activity of the crowberry (Empetrum Ramirez MR, Izquierdo I, Rasieira M, Zuanazzi JA, Barros D, Henriques nigrum) and other berries. J Agric Food Chem. 2008;56:4457-4462. AT. Effect of lyophilised Vaccinium berries on memory, anxiety and http://dx.doi.org/10.1021/jf800406v locomotion in adult rats. Pharmacol Res. 2005;52:457-462. http:// dx.doi.org/10.1016/j.phrs.2005.07.003 Ogawa K, Oyagi A, Tanaka J, Kobayashi S, Hara H. The protective effect and action mechanism of Vaccinium myrtillus L. on gastric ulcer in mice. Rasetti MF, Caruso D, Galli G, Bosisio E. Extracts of Gingko biloba L. Phytother Res. 2011; 25:1160-1165. http://dx.doi.org/10.1002/ptr.3413 leaves and Vaccinium myrtillus L. fruits prevent photo-induced oxidation of low density lipoprotein cholesterol. Phytomedicine 1996/1997;3:335- Orsucci PL, Rossi M, Sabbatini G, Menci S, Berni M. Trattamento della 8. http://dx.doi.org/10.1016/S0944-7113(97)80006-2 retinopatia diabetica con antocianosidi. Indagine preliminare. Clin Ocul. 1983;4:377-81. Rechner A, Kroner C. Anthocyanins and colonic metabolites of polyphenols inhibit platelet function. Thrombosis Res. 2005;116: 327- Ozgurtas T, Aydin I, Tapan S, Avci O, Erbil MK. Bilberry inhibits 334. http://dx.doi.org/10.1016/j.thromres.2005.01.002 angiogenesis in chick chorioallantoic membrane. BioFactors. 2008;33:161-164. http://dx.doi.org/10.1002/biof.5520330301 Repossi P, Malagola R, De Cadilhac C. Influenza degli antocianosidi sulle malattie vasali da alterata permeabilità. Ann Ottal Clin Ocul. Passariello N, Bisesti V, Sganbato S. Influenza degli antocianosidi sul 1987;113:357-61. microcircolo e sull’assetto lipidico in soggetti diabetici e dislipidemici. Gazz Med Ital. 1979;138:563-6. Riihinen K, Ryyanänen A, Toivanen M, Könönen E, Törrönen R, Tikkanen- Kaukanen C. Antiaggregation potential of berry fractions against pairs of Penman K, Halstead C, Matthias A, De Voss J, Stuthe J, Bone K. Streptococcus mutans with Fusobacterium nucleatum or Actinomyces Bilberry adulteration using the food dye amaranth. J Agric Food Chem. naeslundii. Phytother Res. 2011;25: 81-87. DOI : 10.1002/ptr.3228 2006;54:7378-7382. http://dx.doi.org/10.1021/jf061387d http://dx.doi.org/10.1002/ptr.3228

Perossini M, Guidi S, Chiellini S, Siravo D. Studio clinico sull’impiego Robert AM, Godeau G, Moati F, Miskulin M. Action of anthocyanosides degli antocianosidi del mirtillo (Tegens) nel trattamento delle of Vaccinium myrtillus on the permeability of the blood brain barrier. microangiopatie retiniche di tipo diabetico ed ipertensivo. Ann Ottal J Med. 1977;8:321-32. Clin Ocul 1987;113:1173-90. Roy S, Khanna s, Alessio HM, Vider J, Bagchi M, Sen CK. Anti-angiogenic Persson I, Persson K, Andersson R. Effect of Vaccinium myrtillus property of edible berries. Free Radic Res. 2002;36:1023-1031. http:// and its polyphenols on angiotensin-converting activity in human dx.doi.org/10.1080/1071576021000006662 endothelial cells. J Agric Food Chem. 2009;57:4626-4629. http:// dx.doi.org/10.1021/jf900128s Saija A, Princi P, D’Amico N, De Pasquale R, Costa G. Effect of Vaccinium myrtillus anthocyanins on triiodothyronine transport into brain in the Pourrat H, Bastide P, Dorier P, Pourrat A, Tronche P. Préparation et rat. Pharmacol Res. 1990;22:59-60. http://dx.doi.org/10.1016/S1043- activité thérapeutique de quelques glycosides d’anthocyanes. Chimie 6618(09)80029-7 Thérapeutique 1967;2:33-8. Sakakibara H, Ogawa T, Koyonagy A, Kobayashi S, Goda T, Kumazawa Pulliero G, Montin S, Bettini V, Martino R, Mogno C, Lo Castro G. Ex vivo S. Distribution and excretion of bilberry anthocyanins in mice. J Agric study of the inhibitory effects of Vaccinium myrtillus anthocyanosides Food Chem. 2009;57:7681-7686. http://dx.doi.org/10.1021/jf903424k on human platelet aggregation. Fitoterapia 1989;60:69-75. Salvayre R, Braquet P, Perruchot T, Douste-Blazy L. Comparison of the Puupponen-Pimiä R, Nohynek L, Meier C, Kahkonen M, Heinonen M, scavenger effect of bilberry anthocyanosides with various flavonoids. Hopia A. Antimicrobial properties of phenolic compounds from berries. In: Farkas L, Gábor M, Kállay F, Wagner H, editors. Flavonoids and J Appl Microbiol. 2001;90:494-507. http://dx.doi.org/10.1046/j.1365- Bioflavonoids, 1981. Amsterdam: Elsevier, 1982:437-42. 2672.2001.01271.x Sasaki R, Nishimura N, Hoshino H, Isa Y, Kadowaki M, Ichi T. Cyanidin- Puupponen-Pimiä R, Nohynek L, Hartmann-Schmidlin S, Kahkonen M, 3-glucoside ameliorates hyperglycemia and insulin sensitivity due to Heinonen M, Määttä-Riihinen KR. Berry phenolics selectively inhibit the downregulation of retinol binding protein 4 expression in diabetic mice. growth of intestinal pathogens. J Appl Microbiol. 2005;98:991-1000. Biochem Pharmacol. 2007;74:1619-1627. http://dx.doi.org/10.1016/j. http://dx.doi.org/10.1111/j.1365-2672.2005.02547.x bcp.2007.08.008

Puupponen-Pimiä R, Nohynek L, Ammann S, Oksman-Caldetey K-M, Scharrer A, Ober M. Anthocyanoside in der Behandlung von Buchert J. Enzyme-assisted processing increases antimicrobial and Retinopathien. Klin Mbl Augenheilk. 1981;178:386-89. http://dx.doi. antioxidant activity of bilberry. J Agric Food Chem. 2008;56:681-688. org/10.1055/s-2008-1057228 http://dx.doi.org/10.1021/jf072001h Schilcher H. Diarrhoea. In: Phytotherapy in Paediatrics - Handbook for Qin Y, Xia M, Ma J, Hao YT, Liu J, Mou HY. Anthocyanin supplementation Physicians and Pharmacists. Stuttgart: Medpharm Scientific, 1997:49-51. improves serum LDL- and HDL-cholesterol concentrations associated with the inhibition of cholesteryl ester transfer protein in dyslipidemic Schilcher H, Kammerer S, Wegener T. (Hrsg.) Leitfaden Phytotherapie subjects. Am J Clin Nutr. 2009;90: 485-492. http://dx.doi.org/10.3945/ 4.Auflage. München : Urban & Fischer, 2010 ajcn.2009.27814 Serranillos Fdez MG, Zaragoza F, Alvarez P. Efectos sobre la agregación Rahman MM, Ichiyanagi T, Komiyama T, Hatana Y, Konishi T. Superoxide plaquetaria “in vitro” de los antocianósidos del Vaccinium myrtillus L. radical- and peroxynitrite-scavenging activity; structure activity An Real Acad Farm. 1983;49:79-90. relationship and their synergism. Free Radic Res. 2006;40:993-1002. http://dx.doi.org/10.1080/10715760600815322 Signorini GP, Salmistrano G, Deotto GP, Maraglino GM. Ruolo delle

15 MYRTILLI FRUCTUS

moderne tecnologie strumentali angiologiche nella diagnostica juice polyphenolic fractions. Phytother Res. 2011;25 :828-832. http:// non invasiva delle agiolo-flebopatie periferiche. Parte I. Fitoterapia dx.doi.org/10.1002/ptr.3349 1983a;54(Suppl 3):3-18. Tori A, D’Errico F. Gli antocianosidi da Vaccinium myrtillus nella Signorini GP, Salmistrano G, Deotto GP, Maraglino GM. Ruolo delle cura delle flebopatie da stasi degli arti inferiori. Gazz Med Ital. 1980; moderne tecnologie strumentali angiologiche nella diagnostica 139:217-24. non invasiva delle agiolo-flebopatie periferiche. Parte II. Fitoterapia 1983b;54(Suppl 3):19-30. Ulbricht C, Basch E, Basch S, Bent S, Boon H, Burke D. An evidence- based systematic review of bilberry (Vaccinium myrtillus) by the Natural Song J, Li Y, Ge J, Duan Y, Cho-Wing Sze S, Tong Y. Protective effect of Standard Research Collaboration. J Dietary Suppl. 2009;6:162-200. bilberry (Vaccinium myrtillus L.) extracts on cultured human corneal http://dx.doi.org/10.1080/19390210902861858 limbal epithelial cells (HCLEC). Phytother Res. 2010;24:520-524. http:// dx.doi.org/10.1002/ptr.2974 Vannini L, Samuelly R, Coffano M, Tibaldi L. Studio del comportamento pupillare in seguito a somministrazione di antocianosidi. Boll Ocul. Spadea L, Giagnoli B, D’Amico M, Dragani T, Balestrazzi E. Antocianosidi 1986;65:569-77. del mirtillo ad alto dosaggio: risposte elettrofisiologiche nel soggetto normale, modesto miope e grande miope. Clin Ocul. 1991;3:122-5. Viljanen K, Kylli P, Kivikari R, Heinonen M. Inhibition of protein and lipid oxidation in liposomes by berry phenolics. J Agric Food Chem. Sparrow J, Vollmer-Snarr H, Zhou J, Jang Y, Jockush S, Itagaki Y. A2E- 2004;52:7419-7424. http://dx.doi.org/10.1021/jf049198n epoxides damage DNA in retinal pigment epithelial cells. J Biol Chem. 2003;278:18207-18213. http://dx.doi.org/10.1074/jbc.M300457200 Virno M, Pecori Giraldi J, Auriemma L. Antocianosidi di mirtillo e permeabilità dei vasi del corpo ciliare. Boll Ocul. 1986;65:789-95. Steigerwalt RD, Belcaro G, Morazzoni P, Bombardelli E, Burki C, Schönlau F. Effects of MirtogenolR on ocular blood flow and intraocular Wichtl M. In Teedrogen und Phytopharmaka. 5th ed. Wissenschaftliche hypertension in asymptomatic subjects. Mol Vis. 2008;14:1288-1292. Verlagsgesellschaft mbH Stuttgart 2009:460-462.

Steigerwalt RD, Belcaro G, Morazzoni P, Bombardelli E, Burki C, Wu QK, Koponen JM, Mykkänen, Törrönen AR. Berry phenolic extracts Schönlau F. Mirtogenol R potentiates Iatanoprost in lowering intraocular modulate the expression of p21WAF1 and Bax but not Bcl-2 in HT pressure and improves ocular blood flow in asymptomatic subjects. colon cancer cells. J Agric Food Chem. 2007;55:1156-1163. http:// Clin Opthalmol. 2010;4:471-476. dx.doi.org/10.1021/jf062320t

Svobodova A, Rambouskova J, Walerova D, Vostalova J. Bilberry Yamaura K, Shimada M, Ueno K. Anthocyanins from bilberry (Vaccinium extract reduces UVA-induced oxidative stress in HaCaT keratinocytes myrtillus L.) alleviate pruritis in a mouse model of chronic allergic : a pilot study. BioFactors 2008;33:249-266. http://dx.doi.org/10.1002/ contact dermatitis. Pharmacog Res. 2011;3:173-177. http://dx.doi. biof.5520330402 org/10.4103/0974-8490.85002

Svobodova A, Zdarilova A, Vostalova J. Lonicera caerulea and Vaccinium Yamaura K, Ishimatari M, Yamamoto M, Shimada M, Bi Y, Uena K. myrtillus fruit polyphenols protect HaCaT keratinocytes against Anthocyanins, but not anthocyanidins, from bilberry (Vaccinium UVB-induced phototoxic stress and DNA damade. J Dermatol Sci. myrtillus L.) alleviate pruritis via inhibition of mast cell degranulation. 2009;56:196-204. http://dx.doi.org/10.1016/j.jdermsci.2009.08.004 J Food Sci. 2012;77:H262-7. http://dx.doi.org/10.1111/j.1750- 3841.2012.02974.x. Sweetman S. ed. Martindale: The Complete Drug Reference; 1974 – 2008 Thomson Healthcare in AltMedDexR Yanamala N, Tirupula KC, Balem F, Klein-Seetharaman J. pH-dependent interaction of rhodopsin with cyanidin-3-glucoside. 1. Structural Takikawa M, Inoue S, Horio F, Tsuda T. Dietary anthocyanin-rich bilberry aspects. Photochem Photobiol. 2009;85:454-462. http://dx.doi. extract ameliortaes hyperglycemia and insulin sensitivity via activation org/10.1111/j.1751-1097.2008.00517.x of AMP-activated protein kinase in diabetic mice. J Nutr. 2010;140:527- 533. http://dx.doi.org/10.3945/jn.109.118216 Yao Y, Vieria A. Protective activities of Vaccinium antioxidants with potential relvance to mitochondrial dysfunction and neurotoxicity. Teglio L, Tronconi R, Mazzanti C, Guerresi E. Vaccinium myrtillus NeuroToxicol. 2007; 28:93-100. http://dx.doi.org/10.1016/j. anthocyanosides in the treatment of venous insufficiency of the neuro.2006.07.015 lower limbs and acute piles in pregnancy. Quad Clin Ostet Ginecol. 1987;42:221-231. Yao N, Lan F, He R-R, Kurihara H. Protective effects of bilberry (Vaccinium myrtillus L.) extract against endotoxin-induced uveitis. J Agric Food Teller N, Thiele W, Marcylo TH, Gescher AJ, Boettler U, Sleeman Chem. 2010;58:4731-4736. http://dx.doi.org/10.1021/jf904572a J. Suppression of the kinase activity of receptor tyrosine kinases by anthocyanin-rich mixtures extracted from bilberries and grapes. J Agric Yasmin T, Sen CK, Hazra S, Bagchi M, Bachi D, Stohs SJ. Antioxidant Food Chem. 2009;57:3094-3101. http://dx.doi.org/10.1021/jf803 capacity and safety of various anthocyanin berry extract formulations. 094a Res Commun Pharmacol Toxicol. 2003;8:IV-25-IV-35.

Thomasset S, Berry DP, Cai H, West K, Marczylo TH, Marsden D. Pilot Viljanen K, Kylli P, Kivikari R, Heinonen M. Inhibition of protein and study of oral anthocyanins for colorectal cancer chemoprevention. lipid oxidation in liposomes by berry phenolics. J Agric Food Chem. Cancer Prev Res. 2009;2:625-633. http://dx.doi.org/10.1158/1940- 2004; 52:7419-7424. http://dx.doi.org/10.1021/jf049198n 6207.CAPR-08-0201 Zavarise G. Sull’effetto del trattamento prolungato con antocianosidi Tirupula KC, Balem F, Yanamala N, Klein-Seetharaman J. pH-dependent sul senso luminoso. Ann Ottal Clin Ocul. 1968;94:209-14. interaction of rhodopsin with cyanidin-3-glucoside. 2. Functional aspects. Photochem Photobiol. 2009;85:463-470. http://dx.doi. Ziberna L, lunder M, Moze S, Vanzo A, Tramer F, Passamonti S. Acute org/10.1111/j.1751-1097.2008.00533.x cardioprotective and cardiotoxic effects of bilberry anthocyanins in ischaemia-reperfusion injury: beyond concentration-dependent Toivanen M, Huttunen S, Lapinjoki S, Tikkanen-Kaukanen C. Inhibition antioxidant activity. Cardiovasc Toxicol. 2010;10:283-294. http://dx.doi. of adhesion of Neisseria meningitidis to human epithelial cells by berry org/10.1007/s12012-010-9091-x

16 MOST RECENT VERSIONS

Title Common name Publication

ABSINTHII HERBA Wormwood Second Edition, 2003 AGNI CASTI FRUCTUS Agnus Castus Second Edition, 2003 AGRIMONIAE HERBA Agrimony Supplement 2009 ALCHEMILLAE HERBA Lady's Mantle Online Series, 2013 ALLII SATIVI BULBUS Garlic Second Edition, 2003 ALOE BARBADENSIS Barbados Aloes Supplement 2009 ALOE CAPENSIS Cape Aloes Second Edition, 2003 ALTHAEAE RADIX Marshmallow Root Second Edition, 2003 ANGELICAE RADIX Angelica Root Supplement 2009 ANISI FRUCTUS Aniseed Second Edition, 2003 ARNICAE FLOS Arnica Flower Second Edition, 2003 BALLOTAE NIGRAE HERBA Black Horehound Supplement 2009 BETULAE FOLIUM Birch Leaf Second Edition, 2003 BOLDI FOLIUM Boldo Leaf Second Edition, 2003 CALENDULAE FLOS Calendula Flower Second Edition, 2003 CAPSICI FRUCTUS Capsicum Supplement 2009 CARVI FRUCTUS Caraway Fruit Second Edition, 2003 CARYOPHYLLI AETHEROLEUM Clove Oil Online Series, 2014 CENTAURII HERBA Centaury Second Edition, 2003 CENTELLAE ASIATICAE HERBA Centella Supplement 2009 CHELIDONII HERBA Greater Celandine Second Edition, 2003 CIMICIFUGAE RHIZOMA Black Cohosh Online Series, 2011 CINNAMOMI CORTEX Cinnamon Second Edition, 2003 CRATAEGI FOLIUM CUM FLORE Hawthorn Leaf and Flower Second Edition, 2003 CRATAEGI FRUCTUS Hawthorn Berries Supplement 2009 CUCURBITAE SEMEN Pumpkin Seed Supplement 2009 CURCUMAE LONGAE RHIZOMA Turmeric Second Edition, 2003 CURCUMAE XANTHORRHIZAE RHIZOMA Javanese Turmeric Supplement 2009 CYNARAE FOLIUM Artichoke Leaf Supplement 2009 ECHINACEAE ANGUSTIFOLIAE RADIX Narrow-leaved Coneflower Root Supplement 2009 ECHINACEAE PALLIDAE RADIX Pale Coneflower Root Supplement 2009 ECHINACEAE PURPUREAE HERBA Purple Coneflower Herb Supplement 2009 ECHINACEAE PURPUREAE RADIX Purple Coneflower Root Supplement 2009 ELEUTHEROCOCCI RADIX Eleutherococcus Supplement 2009 EUCALYPTI AETHEROLEUM Eucalyptus Oil Second Edition, 2003 FILIPENDULAE ULMARIAE HERBA Meadowsweet Second Edition, 2003 FOENICULI FRUCTUS Fennel Second Edition, 2003 FRANGULAE CORTEX Frangula Bark Second Edition, 2003 FUMARIAE HERBA Fumitory Supplement 2009 GENTIANAE RADIX Gentian Root Online Series, 2014 GINKGO FOLIUM Ginkgo Leaf Second Edition, 2003 GINSENG RADIX Ginseng Second Edition, 2003 GRAMINIS RHIZOMA Couch Grass Rhizome Supplement 2009 GRINDELIAE HERBA Grindelia Supplement 2009 HAMAMELIDIS AQUA Hamamelis Water Online Series, 2012 HAMAMELIDIS CORTEX Hamamelis Bark Online Series, 2012 HAMAMELIDIS FOLIUM Hamamelis Leaf Online Series, 2012 HARPAGOPHYTI RADIX Devil’s Claw Root Supplement 2009 HEDERAE HELICIS FOLIUM Ivy Leaf Second Edition, 2003 HIPPOCASTANI SEMEN Horse-chestnut Seed Second Edition, 2003 HYDRASTIS RHIZOMA Goldenseal rhizome Online Series, 2013 HYPERICI HERBA St. John’s Wort Second Edition, 2003 JUNIPERI PSEUDO-FRUCTUS Juniper Second Edition, 2003 LAVANDULAE FLOS/AETHEROLEUM Lavender Flower/Oil Supplement 2009 LICHEN ISLANDICUS Iceland Moss Second Edition, 2003 LINI SEMEN Linseed Second Edition, 2003 LIQUIRITIAE RADIX Liquorice Root Second Edition, 2003 LUPULI FLOS Hop Strobile Second Edition, 2003 MALVAE FLOS Mallow Flower Supplement 2009 MARRUBII HERBA White horehound Online Series, 2013 MATRICARIAE FLOS Matricaria Flower Second Edition, 2003 MELALEUCAE AETHEROLEUM Tea Tree Oil Supplement 2009 MELILOTI HERBA Melilot Second Edition, 2003 MELISSAE FOLIUM Melissa Leaf Online Series, 2013 MENTHAE PIPERITAE AETHEROLEUM Peppermint Oil Second Edition, 2003 MENTHAE PIPERITAE FOLIUM Peppermint Leaf Second Edition, 2003 MENYANTHIDIS TRIFOLIATAE FOLIUM Bogbean Leaf Online Series, 2013 MILLEFOLII HERBA Yarrow Supplement 2009 MYRRHA Myrrh Online Series, 2014 MYRTILLI FRUCTUS Bilberry Fruit Online Series, 2014 OLIBANUM INDICUM Indian Frankincense Supplement 2009 ONONIDIS RADIX Restharrow Root Second Edition, 2003 ORTHOSIPHONIS FOLIUM Java Tea Online Series, 2014 PASSIFLORAE HERBA Passion Flower Second Edition, 2003 PAULLINIAE SEMEN Guarana Seed Supplement 2009 PIPERIS METHYSTICI RHIZOMA Kava-Kava Second Edition, 2003 PLANTAGINIS LANCEOLATAE FOLIUM/HERBA Ribwort Plantain Leaf/Herb Online Series, 2013 PLANTAGINIS OVATAE SEMEN Ispaghula Seed Second Edition, 2003 PLANTAGINIS OVATAE TESTA Ispaghula Husk Second Edition, 2003 POLYGALAE RADIX Senega Root Second Edition, 2003 PRIMULAE RADIX Primula Root Second Edition, 2003 PRUNI AFRICANAE CORTEX Pygeum Bark Supplement 2009 PSYLLII SEMEN Psyllium Seed Second Edition, 2003 RATANHIAE RADIX Rhatany Root Supplement 2009 RHAMNI PURSHIANI CORTEX Cascara Second Edition, 2003 RHEI RADIX Rhubarb Second Edition, 2003 RIBIS NIGRI FOLIUM Blackcurrant Leaf Second Edition, 2003 ROSAE PSEUDO-FRUCTUS Dog Rose Hip Supplement 2009 ROSMARINI FOLIUM Rosemary Leaf Second Edition, 2003 RUSCI RHIZOMA Butcher’s Broom Second Edition, 2003 SALICIS CORTEX Willow Bark Second Edition, 2003 SAMBUCI FLOS Elder flower Online Series, 2013 SALVIAE OFFICINALIS FOLIUM Sage Leaf Second Edition, 2003 SALVIA TRILOBAE FOLIUM Sage Leaf, Three-lobed Online Series, 2014 SENNAE FOLIUM Senna Leaf Second Edition, 2003 SENNAE FRUCTUS ACUTIFOLIAE Alexandrian Senna Pods Second Edition, 2003 SENNAE FRUCTUS ANGUSTIFOLIAE Tinnevelly Senna Pods Second Edition, 2003 SERENOAE REPENTIS FRUCTUS (SABAL FRUCTUS) Saw Palmetto Fruit Second Edition, 2003 SERPYLLI HERBA Wild Thyme Online Series, 2014 SOLIDAGINIS VIRGAUREAE HERBA European Golden Rod Second Edition, 2003 SILYBI MARIANI FRUCTUS Milk Thistle Fruit Supplement 2009 SYMPHYTI RADIX Comfrey Root Online Series, 2012 TANACETI PARTHENII HERBA Feverfew Online Series, 2014 TARAXACI FOLIUM Dandelion Leaf Second Edition, 2003 TARAXACI RADIX Dandelion Root Second Edition, 2003 THYMI HERBA Thyme Second Edition, 2003 TORMENTILLAE RHIZOMA Tormentil Online Series, 2013 TRIGONELLAE FOENUGRAECI SEMEN Fenugreek Second Edition, 2003 URTICAE FOLIUM/HERBA Nettle Leaf/Herb Second Edition, 2003 URTICAE RADIX Nettle Root Second Edition, 2003 UVAE URSI FOLIUM Bearberry Leaf Online Series, 2012 VACCINII MACROCARPI FRUCTUS Cranberry Supplement 2009 VALERIANAE RADIX Valerian Root Supplement 2009 VIOLAE HERBA CUM FLORE Wild Pansy Supplement 2009 VITIS VINIFERAE FOLIUM Red Vine Leaf Supplement 2009 ZINGIBERIS RHIZOMA Ginger Supplement 2009 Online Series Monographs The Scientific Foundation for Herbal Medicinal Products

The second edition of ESCOP Monographs, published as a hardback book in 2003 with a Supplement in 2009, has been widely acclaimed for its authoritative information on the therapeutic uses of herbal medicines. Monographs covering a total of 107 herbal substances include extensive summaries of pharmacological, clinical and toxicological data, and copious references to scientific literature form an important part of each text.

Although publication in the form of books was convenient in the past, ESCOP recognizes that online publication now offers a number of advantages, not least in facilitating rapid publication of individual monographs as soon as all stages of preparation have been completed. Commencing from 2011, therefore, new and revised monographs will be published online only.

The European legislative framework for herbal medicines has advanced considerably over the past decade. Directive 2004/24/EC introduced a simplified registration procedure for traditional herbal medicinal products in EU member states and imposed a 2011 deadline for the registration of certain products on the market. The Committee on Herbal Medicinal Products (HMPC), established in 2004 as part of the European Medicines Agency, has made substantial progress in the preparation of Community Herbal Monographs and associated documentation to provide a more harmonized approach to the scientific assessment of herbal medicinal products throughout the European Community

Whether the evaluation of a herbal medicine is based on evidence of clinical efficacy (well- established use) or on experience and historical use of that product (traditional use) those involved at all levels of the regulatory process need access to detailed, reliable and structured summaries of the available efficacy and safety data. ESCOP monographs meet that requirement and offer an invaluable source of scientific information on herbal medicines to regulators, manufacturers, academics, researchers, health professionals and numerous others.

www.escop.com ISBN 978-1-901964-.24-0

19