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Hyperici herba St. John's Wort 2018

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

HYPERICI HERBA St. John's Wort

2018

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 978-1-901964-61-5

Hyperici herba - St. John's Wort

© ESCOP 2018

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 Roberta Hutchins and Simon Mills Cover and text design by Martin Willoughby Typeset in Optima by Roberta Hutchins

Plant illustrated on the cover: perforatum FOREWORD

It is a great pleasure for me, on behalf of my colleagues in ESCOP, 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.

Dr. Tankred Wegener 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 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 the production of ESCOP monographs and other 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 ABTS 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) ACE angiotensin converting 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

CCl4 carbon tetrachloride

Cmax maximum concentration of a substance in serum CNS central nervous system CoA coenzyme A COX CSF colony stimulating factor CVI chronic venous insufficiency CYP d day DER drug-to-extract ratio DHT dihydrotestosterone DMSO dimethyl sulfoxide 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 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 (= )

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 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 radical absorbance capacity PA pyrrolizidine PAF platelet activating factor PCR polymerase chain reaction PEG polyethylene glycol PGE E Pgp P-glycoprotein PHA phythaemagglutinin p.o. per os POMS profile of mood states PVPP polyvinylpolypyrrolidone RANKL receptor activator of nuclear factor kappa-B RNA ribonucleic acid RT-PCR reverse transcription polymerase chain reaction s.c. subcutaneous SCI spinal cord injury SERM selective oestrogen 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 SSRI selective serotonin inhibitor STAI state-trait anxiety inventory t1/2 elimination half-life TBARS thiobarbituric acid reactive substances TC total cholesterol 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 TABLE OF CONTENTS: PHARMACOLOGICAL PROPERTIES Opioid withdrawal 24 Antiaggressive effects 24 Pharmacodynamic properties 4 Sexual dysfunction 24 Anti-inflammatory effects 25 experiements 4 Wound healing 26 Effects on enzyme activity 4 Bone formation 26 Effects on receptor binding and regulation 4 Gastrointestinal motility 26 Adrenergic receptors 4 Gastroprotective effects 26 Cholinergic receptors 4 Metabolic effects 27 Corticosteroid receptors 4 Reperfusion injury 27 (DA) receptors and DA transporters 4 Nephroprotection 28 GABA receptors 5 Antiviral effects 28 NMDA and glutamate receptors 5 Immunomodulatory effects 28 Oestrogen receptors 5 Antiproliferative effects 28 Opioid receptors 5 Antiangiogenic effects 28 Serotonin receptors 6 Antioxidant effects 29 Other receptors 6 Photosensitising and photodynamic effects 29 Effects on receptor regulation 6 re-uptake inhibition 6 Pharmacological studies in humans 29 Membrane interaction 7 Receptor binding 29 Effects on ion channels 7 29 Cholinergic effects 8 Cognitive function 30 Smooth muscle function 8 Effect on stress hormones 30 Immunological effects 8 Autonomic nervous function 30 Autoimmune inflammatory demyelination 9 Skin protection 30 Neuroprotection 9 Anti-inflammatory effects 10 Clinical studies Tissue repairing effects 10 Clinical studies: depressive disorders 30 Metabolic effects 10 Other clinical studies 40 Antiviral effects 11 Attention-deficit hyperactivity disorder (ADHD) 40 Antimicrobial effects 11 Somatoform disorder 40 Photosensitising and photodynamic effects 11 Social phobia 40 Antiproliferative effects 12 Obsessive-compulsive disorder 40 Antiangiogenic effects 13 Autism 40 Apoptosis 13 Menopause 40 Antioxidant effects 13 Premenstrual symptoms (PMS) 41 Penetration of blood-brain barrier 14 Irritable bowel syndrome (IBS) 41 Effects on nitric oxide synthase 14 Burning mouth syndrome (BMS) 41 Antiviral effects 41 In vivo experiments 14 Antibacterial effects 41 Biochemical findings 14 Wound healing 41 Neurotransmitter receptor density in the brain 15 Animal models of 15 Pharmacokinetic properties 41 Forced swimming test 15 Escape deficit test (learned helplessness) 17 in vitro 41 Tail suspension test (TST) 17 Interaction 41 Hot plate 17 Acute administration 42 Open field test (Locomotor activity) 17 Sub-chronic or chronic administration 42 Elevated plus maze 18 Pharmacokinetics in animals 43 Other pharmacological effects: Interactions 44 Stress response 18 Pharmacokinetics in humans 45 Post-stress grooming 18 Interactions 46 Influence on body temperature 18 Influence on sleeping time 19 Preclinical safety data 47 Effects on EEG 19 Acute toxicity 47 Effects on and hormones 19 Repeated dose and chronic toxicity studies 47 Cognitive effects 20 Mutagenicity and carcinogenicity 51 Anxiety 21 Genotoxicity 51 Neuroprotection 21 Carcinogenicity 51 Neurogenesis 21 Reproductive toxicity 51 Alzheimer’s disease 21 In vitro experiments 51 Parkinson’s disease 22 In vivo experiments 51 Movement disorders 22 Effects on offspring 51 Effects on seizures 22 52 Migraine 22 Phototoxicity 52 Antinociceptive effects 23 23 Clinical safety data 53 withdrawal 23 Pregnancy and lactation 53 withdrawal 24 Phototoxicity and photosensitization 53 HYPERICI HERBA 2018 St. John's Wort

DEFINITION

St. John’s wort consists of the whole or cut, dried flowering tops of L., harvested during flowering time. It contains not less than 0.08

per cent of total hypericins, expressed as (C30H16O8; Mr 504.4) and calculated with reference to the dried drug.

The material complies with the monograph of the European Pharmacopoeia 2013 [St. John’s wort].

CONSTITUENTS

The characteristic constituents are naphthodianthrones and . Naphthodianthrones (0.05-0.3%), consisting mainly of hypericin and pseudo- hypericin, accumulate primarily in the and buds [Nahrstedt 1997]. Other naphthodianthrones are the biosynthetic precursors protohypericin and protopseudohypericin (which are transformed into hypericin and pseudohypericin respectively on exposure to light), and a small amount of cyclopseudohypericin [Häberlein 1992; Krämer 1992; Schütt 1994].

The principal phloroglucinols are (2-4.5%) and (0.2- 1.8%) [Bergonzi 2001; Greeson 2001; Maisenbacher 1992]. Both compounds have limited stability [Bilia 2001; Fuzzati 2001; Orth 1999a; Orth 1999b; Trifunovic 1998] and their oxidated derivatives are also present [Bergonzi 2001].

Quercetin glycosides (2-4%), including , , isoquercitrin and are the main [Hölzl 1987; Nahrstedt 1997; Tekel'ová 2000]; a small amount of free [Tekel'ová 2000]. Biflavonoids such as I3,II8- biapigenin (0.1-0.5%) and I3’,II8-biapigenin (, 0.01-0.05%) occur exclusively in the flowers [Berghöfer 1987; Nahrstedt 1997; Tekel'ová 2000].

Other constituents include phenylpropanoids, such as and other caffeoylquinic and p-coumaroylquinic esters [Jürgenliemk 2002; Nahrstedt 1997]; polymeric and oligomeric proanthocyanidins [Brantner 1994; Nahrstedt 1997] including A2, B1, B2, B3, B5, B7 and C1 [Melzer 1991; Ploss 2001], together with and epicatechin monomers [Nahrstedt 1997; Ploss 2001]; trace amounts of xanthones such as 1,3,6,7-tetrahydroxyxanthone (0.0004% in the leaves and stems) [Nahrstedt 1997; Sparenberg 1993]; and (0.1-0.25%), containing mainly higher n-alkanes and monoterpenes [Nahrstedt 1997].

CLINICAL PARTICULARS

Therapeutic indications

Preparations based on hydroalcoholic extracts (50-68% ethanol, 80% ethanol or 80% methanol)

Preparations based on dry extract, extraction solvent ethanol (50-68% V/V):

Episodes of mild depressive disorders [Häring 1996; Harrer 1999; Hoffmann 1979; Hübner 1994; Kalb 2001; Kugler 1990; Laakmann 1998; Lenoir 1999; Philipp 1999; Quandt 1993; Schlich 1987; Sommer 1994; Witte 1995; Woelk 2000a].

Preparations based on dry extract, extraction solvent methanol (80% V/V), and dry extract, extraction solvent ethanol (80% V/V or 50% V/V):

Mild to moderate depressive episodes in accordance with ICD-10 categories F32.0, F32.1, F33.0 and F33.1 (see the boxes below) [Anghelescu 2006; Bjerkenstedt 2005; Brenner 2000; Fava 2005; Gastpar 2005; Gastpar 2006; Halama 1991; Hänsgen 1994; Häring 1996; Harrer 1994; Harrer 1991; Holsboer- Trachsler 1999; Hübner 2000; Hübner 1994; Kaehler 1999; Kasper 2006; Kasper

1 HYPERICI HERBA

2007a; Kasper 2007b; Kasper 2008; Lecrubier 2002; Lehrl 1993; Posology and method of administration Mannel 2010; Meier 1997; Montgomery 2000; Sarris 2012; Schmidt 1989; Schrader 2000; Schrader 1998; Shelton 2001; Dosage Singer 2011; Sommer 1994; Szegedi 2005; Uebelhack 2004; Vorbach 1997; Vorbach 1994; Wheatley 1997; Woelk 1994]. Preparations based on hydroalcoholic extracts (50-68% ethanol, 80% ethanol or 80% methanol) Other Preparations Adults and children from 12 years: Comminuted and powdered herbal substance as well as the 450-1050 mg daily of hydroalcoholic dry extracts with DERs of following preparations: 2.5-5:1, 4-7:1 or 5-7:1 [Anghelescu 2006; Bjerkenstedt 2005; Brenner 2000; Fava 2005; Gastpar 2005; Gastpar 2006; Halama • Dry extract (DER 4-7:1), extraction solvent ethanol 38% (m/m) 1991; Hänsgen 1994; Häring 1996; Harrer 1994; Harrer 1999; • Liquid extract (DER 1:13), extraction solvent maize oil or Holsboer-Trachsler 1999; Hübner 2000; Hübner 1994; Kaehler other suitable vegetable oil 1999; Kalb 2001; Kasper 2006; Kasper 2008; Laakmann 1998; • Tincture (DER 1:10), extraction solvent ethanol 45-50% (V/V) Lecrubier 2002; Lehrl 1993; Mannel 2010; Martinez 1994; Meier • Tincture (DER 1:5), extraction solvent ethanol 50% (V/V) 1997; Philipp 1999; Rapaport 2011; Rychlik 2001; Schrader • Liquid extract (DER 1:2), extraction solvent ethanol 50% (V/V) 2000; Schrader 1998; Shelton 2001; Sommer 1994; Szegedi • Liquid extract (DER 1:5-7), extraction solvent ethanol 50% 2005; Uebelhack 2004; Vorbach 1994; Wheatley 1997; Woelk (V/V) 2000a; Woelk 1994]. • Expressed juice from the fresh : 1200 – 1800 mg daily of hydroalcoholic dry extracts with Relief of temporary mental exhaustion [Behnke 2002; Engesser DERs of 2.5-5:1, 4-7:1 or 5-7:1 [Anghelescu 2006; Davidson 1996; Harrer 1991; Hoffmann 1979; Kugler 1990; Lenoir 1999; 2002; Kasper 2006; Rychlik 2001; Shelton 2001; Szegedi 2005; Quandt 1993; Rapaport 2011; Schlich 1987; Schmidt 1989; Vorbach 1997]. Warnecke 1986; Werth 1989]. Children from 6 to 12 years under medical supervision only. Liquid extract (DER 1:4-20), extraction solvent vegetable oil; tincture (DER 1:10), extraction solvent ethanol 45-50% (V/V); Other Preparations and tincture (DER 1:5), extraction solvent ethanol 50% (V/V): Symptomatic treatment of minor inflammations of the skin (such Comminuted and powdered herbal substance as well as the as sunburn) and as an aid in healing of minor wounds [Gerlach following preparations: 2008; Samadi 2010; WHO 2002; Wichtl 2004]. • Dry extract (DER 4-7:1), extraction solvent ethanol 38% (m/m) • Liquid extract (DER 1:13), extraction solvent maize oil or Comminuted herbal substance: other suitable vegetable oil Symptomatic relief of mild gastrointestinal discomfort: • Tincture (DER 1:10), extraction solvent ethanol 45-50% (V/V) [Grünwald 2004; Hänsel 1993; Wichtl 2004]. • Tincture (DER 1:5), extraction solvent ethanol 50% (V/V)

Classification of severity of depression based on ICD-10 criteria [WHO 2010]

Mild depressive episode Mild first manifestation or mild recurrence of at least two target [F32.0/F33.0] signs and at least two associated symptoms

Moderate depressive episode: Two or three target signs and at least three or four associated [F32.1/F33.1] symptoms (first manifestation or recurrent episode)

Severe depressive episode: All three target signs and at least four associated symptoms, some particularly [F32.2/F33.2/F32.3/F33.3] pronounced (first manifestation or recurrent, without or with psychotic symptoms)

Diagnostic features of depressive disorders according to ICD-10 Chapter V Primary Care Version [WHO 2010]

Target signs Associated symptoms Low or sad mood Disturbed sleep Loss of interest or pleasure Feelings of guilt and unworthiness or loss of energy Reduced self-esteem and self-confidence Poor concentration Disturbed appetite Decreased libido Suicidal thoughts or acts Agitation or slowing of movement or speech Weight loss

Symptoms of anxiety or nervousness are also frequently present

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• Liquid extract (DER 1:2), extraction solvent ethanol 50% (V/V) Caution is indicated at the beginning and end of treatment • Liquid extract (DER 1:5-7), extraction solvent ethanol 50% with concomitant , as well as when making dosage (V/V) changes of St. John’s wort products. • Expressed juice from the fresh herb: Treatment with St. John’s wort extract should be assessed with Single oral dose: respect to interactions with co- at least 5 days before 60 – 200 mg extract, liquid preparation 1-20 mL, comminuted elective surgery, and again upon discharge from the hospital. herbal substance () 1.5-2 g, or powdered herbal substance 300-500 mg. Interaction with other medication and other forms of interaction Daily oral dose: Oral administration of preparations with a daily dose of more 180-360 mg extract, liquid preparation 6-30 mL, comminuted than 1000 mg drug equivalent or more than 1 mg hyperforin: herbal substance (tea) 3-6 g, or powdered herbal substance Administration of St. John´s wort extracts and some of its 900-1000 mg. components can lead to an induction of several of the [Harrer 1991; Hoffmann 1979; Kugler 1990; Quandt 1993; cytochrome P450 enzyme family (CYP3A, CYP2B6, CYP2C9 Schlich 1987; Schmidt 1989; Warnecke 1986; Werth 1989; and CYP2C19), and the P-glycoprotein drug efflux transporter. Engesser 1996]. For this reason, interactions can occur with drugs including amitryptiline, fexofenadine, , , Undiluted preparations for cutaneous administration: liquid , finasteride, . The concomitant use of extract (DER 1:4-20), extraction solvent vegetable oil; tincture cyclosporine, for systemic use, amprenavir, (DER 1:10), extraction solvent ethanol 45-50% (V/V) or tincture and other protease inhibitors, and (DER 1:5), extraction solvent ethanol 50% (V/V) [Gerlach 2008; (such as or phenprocoumon) is contraindicated. Women Samadi 2010; WHO 2002; Wichtl 2004]. using oral hormonal contraceptives should be informed about possible mid-cycle bleeding as a result of interactions, and Comminuted herbal substance for tea preparation: single dose 2 g counselled on the use of additional contraceptive measures due and daily dose 4 g [Grünwald 2004; Hänsel 1993; Wichtl 2004] to a possible reduction in contraceptive efficacy [Johne 1999, 2002; Sugimoto 2001; Gurley 2002; Dresser 2003; Eich-Hochli Elderly: dose as for adults. 2003; Mai 2004; Müller 2004; Murphy 2005; Lundahl 2009].

Method of administration Several cases of serotonergic effects following concomitant For oral administration and topical application. use of St. John’s wort preparations with certain have been reported [Gordon 1998; Lantz 1999; Prost 2000; Duration of administration Dannawi 2002; Mannel 2004; Bonetto 2007]. The attribution of these cases to St. John’s wort remains unclear. • Mild to moderate depressive episodes: no restriction. If symptoms persist for more than 6 weeks or worsen, medical Patients taking other medicines on prescription should consult advice should be sought. a physician or pharmacist before taking St. John’s wort.

• Relief of temporary mental exhaustion: if symptoms persist for Pregnancy and lactation more than 1 week or worsen, medical advice should be sought. Studies generally do not show an impact of St. John’s wort on pregnancy and lactation, although some animal studies showed • Symptomatic treatment of minor inflammations of the skin equivocal results [Borges 2005; Dugoua 2006]. Several of these (such as sunburn), as an aid in healing of minor wounds studies offer exposure data in humans [Demling 2004; Klier and symptomatic relief of mild gastrointestinal discomfort: if 2006; Lee 2003; Moretti 2009]. symptoms persist for more than 2 weeks or worsen, medical In accordance with general medical practice, the product advice should be sought. should not be used during pregnancy and lactation without medical advice. Contra-indications Known hypersensitivity to the active substance. Effects on ability to drive and use machines Clinical studies indicate no negative influence on general per- Oral administration of preparations with a daily dose of more formance or the ability to drive [Friede 1998; Herberg 1994; than 1000 mg drug equivalent or more than 1 mg hyperforin: Schmidt 1991; Schmidt 1993]. Concomitant use with certain immunosuppressants (, tacrolimus or for systemic use), certain anti-retroviral Undesirable effects HIV-drugs from the group of non-nucleoside reverse transcriptase At therapeutic dose levels (up to the equivalent of 6 g of inhibitors (e.g. ), protease inhibitors (e.g. indinavir, drug), occasional mild gastrointestinal disturbances, , amprenavir), certain cytostatics (e.g. , irinotecan), and restlessness, fatigue, , insomnia or allergic reactions oral anti-coagulants of the coumarin type (e.g. warfarin). See have been reported. also section on Interactions. Onset of mania and hypomania were reported in several Special warnings and special precautions for use patients with latent bipolar disorders [Fahmi 2002; Moses 2000; Very rarely, and especially in fair-skinned persons, unwanted Nierenberg 1999]. reactions of the skin (sunburn-like redness) may occur following ingestion of St. John’s wort preparations and subsequent Phototoxicity exposure to sun light. Intense UV-exposure should be avoided Very rarely, and especially in fair-skinned people, reactions of during treatment. the skin (sunburn-like redness) may occur after ingestion of St. John’s wort preparations and subsequent exposure to sun light. Oral administration of preparations with a daily dose of more Intense UV-exposure should be avoided during treatment [He than 1000 mg drug equivalent or more than 1 mg hyperforin: 2004; Schempp 2003b].

3 HYPERICI HERBA

Overdose of the opioid receptor family, the GABA system, modulation A 16-year old girl experienced seizures and after of oestrogen, N-methyl-D-aspartate (NMDA) and glutamate ingestion of up to 4.5 g of an unspecified St. John’s wort extract receptor binding and the corticosteroidal system in the per day in the preceding 2 weeks and an additional 15 g before CNS. Consequently, antidepressants increase the levels of admission to the hospital [Karalapillai 2007]. On day 6 EEG neurotransmitters, e.g. by reuptake inhibition or by inhibiting was normalised and the patient discharged. the degradation. NMDA receptor-mediated processes or binding to opioid receptors have also been considered to be involved in the mechanism of action of conventional antidepressants. PHARMACOLOGICAL PROPERTIES Adrenergic receptors Pharmacodynamic properties Methanolic extracts, hypericin and (at 100 µg/mL) showed either no or only weak inhibition of the adrenergic

In vitro experiments a1, a2 and b1-receptors [Müller 1996; Raffa 1998; Rolli 1995].

Effects on enzyme activity Hypericin potently inhibited binding to rat b1 and b2 receptors, A potent irreversible inhibition of monoamine oxidase type A whereas pseudohypericin had no effect on either receptor. Rutin and B (MAO-A and MAO-B) was found for hypericin with IC50 and showed significant binding inhibition at human -4 -4 values of 6.8 × 10 M and 4.2 × 10 M respectively [Suzuki a2C adrenergic receptors (52.81% and 59.60% respectively) 1984]. As other studies did not confirm the MAO inhibitory [Butterweck 2002b]. effects of hypericin [Cott 1997; Demisch 1989; Sparenberg 1993; Thiede 1994], the MAO inhibition was explained as Cholinergic receptors being due to a purity of only 80% of the tested compound with Hypericin at 1 µM displayed weak affinity (12%) to rat cortex a possible 20% content, for which MAO inhibition nicotinic acetylcholine (Ach) receptors, and moderate affinity has been confirmed. to muscarinic Ach receptors (49%) [Raffa 1998].

An extract (not further specified) as well as flavone, xanthone At human muscarinic Ach receptors M1-M5, hypericin (10 µM) and kaempferol (IC50 of 1 to 3 µM) led to >90% inhibition of was a weak inhibitor of M2 receptor binding. Inhibition of MAO-A [Demisch 1989]. binding to human muscarinic receptor subtypes was observed

for miquelianin (M2: 53.75%; M5: 55.20%) and rutin (M5: Hyperforin (35 µmol/L) showed a 30.3% inhibition of MAO-A. A 53.82%). Affinity of rutin to M5 receptors was Ki = 35.13 ± 13 significant inhibition of MAO-A activity (66 – 96.2%), and to a nM (reference: pirenzepine, Ki = 20.59 ± 2.4 nM) [Butterweck lesser extent MAO-B activity (2.2 – 29.9%), was demonstrated for 2002b]. quercetin, quercitrin, and kaempferol at concentrations of 39 – 65 µmol/L [Sparenberg 1993]. Corticosteroid receptors Corticotropin-releasing hormone (CRH) is a family of neuro-

Inhibition of MAO-A in rat brain homogenates was only shown humoral factors which act on receptors including CRH1. The at non-physiological concentrations of 1-10 mM of a methanolic corticosteroidal system seems to be particularly important in (80% V/V) extract or a flavonoid-rich fraction thereof. Hypericin the pathogenesis of depression by changes in the regulation of did not inhibit MAO-A. Additional ex vivo experiments did the hypothalamic-pituitary-adrenal axis. Elevated levels of CRH not show a relevant inhibition of MAO-A or MAO-B after i.p. have been found in the cerebrospinal fluid of depressed patients administration of 100 mg/kg b.w. of 1,3-dihydroxyxanthone, [Arborelius 1999], and drugs are considered to quercitrin or the extract [Bladt 1994]. normalise CRH hypersecretion [Stout 2002].

An undefined extract of fresh flowers and buds (containing 0.1% Bisanthraquinone glycosides isolated from an ethanolic (50%) 125 hypericin), at a concentration of 5 µg/mL, inhibited MAO-A by extract inhibited binding of [ I]-sauvagine to CRH1-receptors 93% and MAO-B by 49% [Cott 1997]. with IC50 values of 1 to 4 µM. The IC50 values of hyperforin and hypericin were 10 and 6 µM [Wirz 2000].

A weak inhibitory effect (IC50 = 120 and 370 µg/mL for MAO-A and MAO-B respectively) was obtained with a methanolic (80% A methanolic extract (0.2% hypericin and 5% hyperforin) 125 V/V) extract containing 0.3% hypericin and 1.5% hyperforin weakly inhibited [ I]-astressin binding to the human CRH1 [Müller 1997]. A hyperforin-enriched (38%) CO2 extract (>500 receptor with an IC50 of 50 µg/mL whereas hypericin (IC50 µg/mL) demonstrated no inhibition [Müller 1998]. approximately 300 nM) and pseudohypericin (IC50 of 2 µM) were more active. Hyperforin did not show any inhibition up Hyperforin at doses of up to 50 µg/mL did not inhibit MAO-A to 10 µM [Simmen 2001]. or MAO-B activities [Chatterjee 1998a]. The effect of hypericin, pseudohypericin and hyperforin on On fractionation of an extract, an inhibition of catechu-O- corticotropin-releasing factor (CRF)-stimulated cAMP formation methyl-transferase (COMT) was found for the hydrophilic was studied in recombinant Chinese hamster ovary cells. Only fractions containing mainly flavonoids, as well as hypericins pseudohypericin selectively antagonised CRF (KB 0.76 µM) and xanthones. Hypericin did not inhibit COMT from pork [Simmen 2003]. at concentrations up to 1 mM (500 mg/L) [Thiede 1994]. Dopamine (DA) receptors and DA transporters Several studies have shown an inhibition of dopamine-b- Hypericin and pseudohypericin (10 µM) markedly inhibited hydroxylase by ethanolic extracts, hypericin and pseudohypericin binding to rat D3- and D4- receptors (approximately 70-80% [Kleber 1999]. inhibition), with pseudohypericin being more potent on D4- receptors. Inhibition by hyperforin (10 µM) was 57% for the

Effects on receptor binding and regulation binding to D1- and 63% to D5-receptors. Amentoflavone (10 µM) Depression has been attributed to reduced availability of showed a complete inhibition of binding to the D3-receptor and serotonin (5-HT), or dopamine, disturbances a 70% inhibition at the DA transporter [Butterweck 2002b].

4 HYPERICI HERBA

Weak interaction with the rat DA transporter system was shown No inhibition of binding to rat NMDA receptors was observed for a methanolic extract (IC50 24.5 µg/mL) and a CO2 extract for the constituents hypericin, hyperforin, pseudohypericin, (IC50 of 12.9 µg/mL) [Gobbi 2001]. amentoflavone, quercitrin, isoquercitrin, hyperoside and miquelianin [Butterweck 2002b]. GABA receptors

An ethanolic extract inhibited binding to human GABAA- In contrast, a hydromethanolic (80%) extract inhibited receptors with an IC50 value of 1 to 3 µg/mL [Simmen 1999; radioligand binding (CGP 39653) to NMDA receptors with an Simmen 2001]. A hydromethanolic extract (4.5% hyperforin, IC50 of 7.0 ± 2.7 µg/mL [Wonnemann 1997]. 0.5% hypericin and 0.4% biapigenin) showed an inhibition of binding to GABAA and GABAB receptors with IC50 values of Hyperforin (at 10 µM) inhibited NMDA-induced influx 5.5 and 8.0 µg/mL respectively [Gobbi 2001; Gobbi 1999]. in rat cortical neurons demonstrating binding and antagonistic activity at NMDA receptors. In hippocampal preparations from A hydromethanolic (80%) extract inhibited radioligand binding male Wistar rats, hyperforin inhibited NMDA-receptor-mediated of muscimol to the GABAA-receptor with an IC50 of 1.6 ± 0.5 release of from phospholipid membrane [Kumar 2006]. µg/mL [Wonnemann 1997]. Oestrogen receptors A extract, obtained by extraction with methanol followed Binding of oestradiol to the oestrogen receptor (ERa) was by acetone, inhibited binding of flumazenil to weakly inhibited (IC50 200 µg/mL) by an ethanolic (50%) receptors in rat brain with an IC50 of 6.8 µg/mL. Leaf extracts up extract. Oestrogen binding to ERa receptor was inhibited by to 200 µg/mL showed 25% inhibition of flumazenil binding. Of biapigenin (IC50 1 µM) [Simmen 1999]. Other flavonoids did the single constituents tested, amentoflavone had an IC50 of 14.9 not significantly inhibit binding to ER-a (IC50 > 10 µM) [Simmen nM, whereas hypericin, flavones and glycosylated flavonoids did 1999; Simmen 2001]. not inhibit binding [Baureithel 1997]. Amentoflavone binding activity at the benzodiazepine receptor with an IC50 of 6 nM Opioid receptors was comparable to the affinity of [Nielsen 1988]. An affinity of 1 µM hypericin with more than 40% inhibition was shown for non-selective muscarinic cholinergic receptors Various undefined extracts were investigated for their effect and for non-selective s-opioid receptors [Raffa 1998]. on neurotransmitter re-uptake. Dopamine re-uptake was studied in synaptosomes from male Wistar rats, all other Binding of [3H]- to human recombinant µ- and k-opioid uptake mechanisms (5-HT, norepinephrine (NE), GABA and receptors was inhibited by an ethanolic (50%) extract with IC50- L-glutamates) in those from female NMRI mice. Adhyperforin, values of 25 – 40 µg/mL and 90 – 200 µg/mL respectively. The like hyperforin, showed a strong inhibition in all systems. extract was approximately 10 times less active than a methanolic Hyperforin-free preparations were able to inhibit the uptake extract with approximately 0.2% hypericin and 5% hyperforin. in a weak to moderate manner. Moreover, a weak to moderate Corresponding activity was observed for the hexane fraction inhibition was found for a fraction containing oligomeric of the ethanolic extract containing phloroglucinols, and the procyanidins [Wonnemann 2001]. pure compounds hyperforin and adhyperforin. Hypericin and pseudohypericin inhibited binding to opioid receptors at 1-4 µM. Amentoflavone (10 µM) inhibited binding to rat GABA receptors No activity was observed with procyanidins, rutin, hyperoside, by 98%. Quercitrin, isoquercitrin, hyperoside and miquelianin isoquercitrin, quercitrin, quercetin and I3,II8-biapigenin at exhibited only a weak effect. The affinity of amentoflavone concentrations of 10 µmol/mL [Simmen 1999; Simmen 2001; to the rat benzodiazepine receptor was Ki = 6.17 ± 1.2 nM Simmen 1998]. (reference: diazepam, Ki = 10.6 ± 2.2 nM) [Butterweck 2002b]. In a screening of an ethanolic extract, binding of radiolabelled

Examination of the binding sites of amentoflavone suggested ligands was strongly inhibited at the opioid receptors µ (IC50 a complex mechanism of interaction at the GABAA receptor. 4 µg/mL), k (IC50 3 µg/mL) and d (IC50 5 µg/mL) [Simmen 1999]. Amentoflavone appeared to act as an antagonist at the classical benzodiazepine site, whereas the functional effects A hydromethanolic extract (0.5% hypericin, 4.5% hyperforin at GABAA receptors were most likely mediated by mechanisms and 0.4% biapigenin) and a CO2 extract (0.1% hypericin, 24% distinct from the benzodiazepine and lorecazole binding sites hyperforin and trace amounts of biapigenin) only weakly inhib- [Hansen 2005]. ited ligand binding at rat brain cortex s-receptors. Hypericin

showed an inhibition IC50 of 2.8-7.3 nM. The effect was light- In a screening of an ethanolic extract, binding of radiolabelled dependent, with increasing IC50-values under exclusion of light. ligands was strongly inhibited at the GABAA receptor (IC50 Hyperforin did not inhibit ligand binding at rat brain cortex 1 µg/mL) [Simmen 1999]. s-receptors [Gobbi 2001].

A hydromethanolic extract demonstrated reduced expression In contrast, a methanolic extract with approximately 0.2% of serotonin receptors in a neuroblastoma cell line model hypericin and 5% hyperforin inhibited binding to opioid receptor [Müller 1993]. sub-types µ, k and d at low micromolar ranges. Corresponding activity was also observed for hyperforin and adhyperforin NMDA and glutamate receptors [Simmen 2001]. Binding of the selective ligand Ro 63-6599 to the human metabotropic glutamate receptor 2 (mGlu2) was not inhibited At a concentration of 10 µM neither hypericin nor pseudo- by an ethanolic (50%) extract or a methanolic/acetonic extract hypericin inhibited binding to the human opioid d receptor. A containing 0.2% hypericin and 5% hyperforin [Simmen 2001]. slight inhibition was found for hypericin at the µ-receptor, and for pseudohypericin at the k-receptor. No binding at opioid This was supported by a study finding that an unspecified receptors was determined for quercitrin, isoquercitrin, rutin, extract did not inhibit binding to NMDA receptors. However, hyperoside and miquelianin. Binding to opioid receptor d hypericin did bind to NMDA receptors with moderate affinity was inhibited by amentoflavone (75%, Ki of 36.5 ± 14 nM) (Ki approximately 1 µg/mL) [Cott 1997]. [Butterweck 2002b].

5 HYPERICI HERBA

Serotonin receptors of 65 ± 6% (p<0.01), an effect fully reversed by co-incubation After fractionation of an ethanolic (50%) extract the most distinct with 100 µM a-tocopherol. The extract led to a down-regulation inhibition of binding was found at the 5-HT6 (IC50 1 µg/mL) and of 56 ± 9% compared to untreated controls (p<0.001). On 5-HT7 (IC50 4 µg/mL) receptors for the hexane fraction containing co-incubation with a-tocopherol, receptor down-regulation hyperforin-related compounds, whereas the CH2Cl2 (pigments) was significantly inhibited (88.6±5.3% of control, p<0.001) and methanolic fractions (flavonoids and hypericins) were less following exposure to the extract [De Marchis 2006]. active. Hypericin inhibited binding with an IC50 of 6 µM at 5-HT6, and an IC50 > 10 µM at 5 HT7. Hyperforin inhibited binding to Pre-incubation of rat C6 glioblastoma cells with hyperforin 5-HT6 (IC50 of 2 µM) and 5-HT7 (IC50 of 3 µM). Flavonoids and and hyperoside for 3 days led to a significant reduction in I3,II8-biapigenin did not exhibit any effect on ligand binding b2-AR sensitivity, comparable to , and a loss of to the serotonin receptors 5-HT6 and 5-HT7 at concentrations cAMP generation after b2-stimulation with terbutaline (p<0.05) up to 10 µM [Simmen 1999; Simmen 2001] [Prenner 2007].

A methanolic (80%) extract inhibited binding to 5-HT1A-receptors Similar results were obtained with a methanolic (80%) extract, with an IC50 of approximately 50 µg/mL [Rolli 1995]. which caused a dose-dependent reduction in the expression of serotonin (5-HT) receptors in a rat neuroblastoma cell line (PC- A methanolic (80%) extract had a slight effect on binding to 12) after short-term exposure over 2 to 6 hours [Müller 1993]. mouse brain 5-HT2 receptors at a concentration of 100 µg/mL [Müller 1996]. In a further study, the same extract affected cellular mRNA levels

of the glucocorticoid receptors GRa and its inhibitory counterpart An unspecified extract from fresh flowers and buds (containing GRb at clinically relevant concentrations of hyperforin (125 ng/ approximately 0.1% of hypericin) showed 54% inhibition of mL) via a primary and transient up-regulation of GRa mRNA binding (Ki = 25 µg/mL) to the 5-HT1 receptor [Cott 1997]. levels (at a concentration of 2.5 µg/mL) and a down-regulation of GRb mRNA levels (at a concentration of 10 µg/mL) after A hydromethanolic extract (0.5% hypericin, 4.5% hyperforin 16 h of treatment [Enning 2011]. and 0.4% biapigenin) and a CO2 extract (0.1% hypericin, 24% hyperforin and trace amounts of biapigenin) did not inhibit Neurotransmitter re-uptake inhibition ligand binding at recombinant rat 5-HT6 and 5-HT7 receptors Inhibition of neurotransmitter re-uptake from the synaptic cleft [Gobbi 2001]. has long been assumed to be the principal biochemical mech- anism underlying the therapeutic effects of antidepressants such

Amentoflavone (10 µmol/L) inhibited binding to the 5-HT1D as antidepressants, selective 5-HT re-uptake inhibitors receptor (85%). Inhibition at the rat 5-HT2C receptor was weaker (SSRIs), norepinephrine (NE) re-uptake inhibitors (NRIs) and (46%) [Butterweck 2002b]. 5-HT–NE reuptake inhibitors (SNRIs).

Other receptors A methanolic (80%) extract inhibited serotonin re-uptake by

A hydromethanolic extract (0.5% hypericin, 4.5% hyperforin rat synaptosomes with an IC50 of 6.2 µg/mL [Perovic 1995]. and 0.4% biapigenin) and a CO2 extract (0.1% hypericin, 24% hyperforin and trace amounts of biapigenin) did not inhibit ligand In mice synaptosomal preparations a CO2 extract (38.8% binding at neuropeptide NPY1 and NPY2 receptors from rat brain hyperforin) demonstrated pronounced reuptake inhibition of cortex and rat brain hippocampus respectively [Gobbi 2001]. 5-HT (IC50 0.26 µg/mL), NE (IC50 0.25 µg/mL), DA (IC50 0.056 µg/mL), GABA (IC50 0.12 µg/mL) and glutamate (IC50 2.83 µg/mL) Either no or weak inhibition of binding to histamine H1 receptors and for hyperforin (IC50 0.11 µg/mL). IC50 values of a methanolic was found for an ethanolic (50%) extract, hypericin at 1 µM, extract (1.5% hyperforin) were about 10 times higher than those hyperforin at 10 µM, and the flavonoids amentoflavone, of the CO2 extract [Chatterjee 1998a]. quercitrin, isoquercitrin, hyperoside and miquelianin [Butterweck 2002b]. A hydromethanolic extract (hyperforin content < 5%) inhibited the accumulation of [3H]-5-HT in rat brain cortical synaptosomes

Hypericin at 1 µM had no significant activity (<10%) at the rat with an IC50 of 7.9 µg/mL, compared to 1.8 µg/mL for hyperforin. adenosine A1 and A2-receptors, the rat angiotensin AT1 receptor, The extract at 3-10 µg/mL and hyperforin at 0.3-1 µg/mL induced the cloned human B2 receptor, the unspecific mouse marked tritium release from synaptosomes preloaded with 3 glucocorticoid receptor and the rat vasopressin V1 receptor. [ H]-5-HT [Gobbi 1999]. Weak affinity was found at the rat CCKA-receptor and the cloned human endothelin ETA-receptor [Raffa 1998]. A hydromethanolic extract (0.3% hypericin, 4.9% hyperforin) dose-dependently inhibited 5-HT and NE uptake in astrocytes

No inhibition of binding to nicotinic, , vasopressin with IC50 values of 25 µg/mL and 10 µg/mL respectively [Neary receptors V1-V3 and oxytocin receptors was found for hypericin, 1999]. Another hydromethanolic extract inhibited synaptosomal pseudohypericin, amentoflavone, quercitrin, isoquercitrin, uptake of 5-HT, DA and NE with IC50 values of 2.43, 0.85 and hyperoside and miquelianin [Butterweck 2000]. 4.47 µg/mL respectively [Müller 1997].

Effects on receptor regulation Hyperforin was tested on voltage- and ligand-gated ionic Exposure of confluent rat astrocytoma C6 cell cultures to an conductances to measure neuronal responses to stimulation ethanolic (50%) extract over 8 days resulted in a dose-dependent of NMDA or AMPA (a-amino-3-hydroxy-5-methylisoxazole- b-adrenoreceptor (AR) down-regulation equal to desipramine 4-propionic acid) receptors. At concentrations between 3 and [Kientsch 2001]. 100 µM, antagonistic effects on the NMDA receptor and on responses mediated by AMPA or GABA were seen [Chatterjee A dry extract (DER: 4-7:1; ethanol 57.9% V/V) demonstrated a 1999]. Hyperforin non-competitively inhibited the synaptosomal reduction in b-AR numbers in cultured cells comparable with uptake of [3H]-glutamate and [3H]-GABA [Wonnemann 2000]. desipramine (DMI) and (FLU). Seven-day incubation of cell cultures with DMI resulted in a reduction of b-AR numbers An ethanolic (50% m/m) extract led to a dose-dependent

6 HYPERICI HERBA

inhibition of NE- and 5-HT-uptake into rat brain slices. The caused an immediate decrease in fluorescence and the addition extract was more selective for the uptake of NE than for that of of 1 µM carbonyl-cyanide p-trifluromethoxyphenylhydrazone 5-HT. The maximal extent of uptake inhibition by the extract (FCCP) abolished this effect. H+-ATPase dependent proton pump- was comparable to that of the positive controls , ing was inhibited by hyperforin in a dose dependent manner (IC50 desimipramine and , but lower for NE transport = 1.9×10-7 M). Hyperforin acted similarly to the protonophore than the controls [Kientsch 2001]. FCCP, abolishing the ATP induced fluorescence quenching (IC50 = 4.3×10-7 M). Hyperforin and FCCP had similar potencies for A study assessed the effects on neurotransmitter reuptake of two inhibiting rat brain synaptosomal uptake of [3H]-monoamines different extracts (extracts 1 and 2: 3.34% and 0.56% hyperforin, as well as vesicular monoamine uptake. The efflux of [3H]-5-HT 0.59% and 0.05% adhyperforin, respectively), methanolic from synaptic vesicles was sensitive to both drugs, thus 50% of and n-heptane fractions, as well as purified hyperforin and preloaded [3H]-5-HT was released in the presence of 2.1×10-7 adhyperforin. Adhyperforin was shown to have a slightly more M FCCP and 4×10-7 M hyperforin [Roz 2003]. potent inhibitory effect than hyperforin on the uptake of DA

(IC50 1.8 and 6 ng/mL), 5-HT (IC50 15 and 65 ng/mL) and NE The efficacy of different commercial extracts was investigated (IC50 7.9 and 18 ng/mL) [Jensen 2001]. by measuring their activity to inhibit 5-HT uptake. The products with different concentrations of pharmaceutically relevant

A methanolic (80%) extract inhibited uptake of 5-HT and NE ingredients also differed in their IC50 values between 3.07 and in cultures of rat cortical astrocytes. The extract also inhibited 17.9 µg/mL. The activity of the products was correlated to their 5-HT uptake in neuronal cultures from serotonergic-rich raphe amount of hyperforin (p<0.05) [Schulte-Löbbert 2004]. nuclei. The extract was 25 times more potent in inhibiting 5 HT uptake in neurons than in astrocytes. The extract also inhibited The immediate effects of an ethanolic extract (50% m/m) with NE re-uptake in SK-N-SH cells, a human neuroblastoma cell a low content of hyperforin on the specific DA uptake were line enriched in NE transporters. Hyperforin was about 10 to 20 studied in rat striatal brain slices and compared with the effects times more potent than the extract in inhibiting neurotransmitter on NE and 5-HT uptake in rat cortical brain slices. Specific uptake in astrocytes and neuronal cells; this is consistent with DA uptake was inhibited in a dose-dependent manner. The the concentration of 5% hyperforin in the extract. The extract NE uptake inhibition was around 30-times higher than that stimulated a persistent activation of extracellular signal regulated for 5-HT, and seven times higher than that of the DA uptake protein kinase (ERK) [Neary 2001]. [Ruedeberg 2010].

The effects of a methanolic (80%) extract (10 µg/mL) and pure Adhyperforin inhibited uptake of [3H]-5-HT, [3H]-NE, and[3H]- hyperforin (300 nM) on human blood platelet 5-HT uptake and DA (IC50 4.1, 2.64, and 0.89 µg/mL, respectively) in synapto- efflux were compared to those of (AMI), somes of the frontal cortex, and displayed robust binding affinities

(VEN), and . In contrast to AMI and VEN, the extract and to the 5-HT, NE but not DA transporters (Ki values 18.75 and hyperforin did not inhibit 5-HT uptake in platelet-rich plasma. In 4.03 µg/mL respectively) [Tian 2014]. contrast to controls and AMI, a loss of accumulated [14C]-5-HT after a prolonged incubation period was observed, suggesting Membrane interaction that both hyperforin and the extract might affect the storage The effects of hyperforin on the fluidity of crude brain mem- of 5 HT in platelet vesicles. In the presence of a comparably branes from young guinea pigs was investigated using three high concentration of reserpine (2.5 µM), the degree of 5-HT different fluorescent probes. Diphenylhexatriene (DPH) depletion in storage vesicles was lower than in the presence and trimethylammonium-diphenylhexatriene (TMA-DPH) of 1 μM hyperforin [Uebelhack 2001]. anisotropy measurements were inversely correlated with membrane flexibility (annular and bulk fluidity). Incubation of The effect of hyperforin on 5 HT re-uptake was examined in brain membranes with 10 µM hyperforin salt resulted Sprague-Dawley rat cortex synaptosomal preparations. Total re in increased DPH (p<0.05) and decreased TMA-DPH (p<0.01) uptake of radiolabelled choline was significantly inhibited by anisotropy, respectively, indicating that hyperforin modifies 10 µM hyperforin (-32%, p<0.05). The effect was exclusively specific membrane structures in different ways. It decreases the based on the high affinity system, but not the non-sodium- flexibility of fatty acids in the membrane hydrocarbon core, but dependent low affinity re-uptake (total inhibition 68%, IC50 of fluidises the hydrophilic region of membrane phospholipids. hyperforin 8.5 µM) [Buchholzer 2002]. Lower concentrations (0.3 µM) significantly (p<0.05) decreased the annular fluidity of lipids close to membrane proteins. A hyperforin-rich heptane extract inhibited the sodium- However, bulk fluidity was unchanged by this low hyperforin dependent uptake of 5-HT, DA and NE into rat brain synapto- concentration [Eckert 2001]. somes. Hyperforin inhibited the uptake of all three monoamines non-competitively, which was in contrast to the competitive An ethanolic (60%) extract (containing 5% hyperforin) and inhibition exhibited by FLU, GBR12909 and DMI. Hyperforin hyperforin sodium salt were given at single oral doses of 300 had no inhibitory effect on the binding of [3H], mg/kg or 15 mg/kg, respectively, to female NMRI mice. Three [3H]-GBR12935 and [3H]-nisoxetine to membrane presynaptic hours later in the isolated brains the properties of murine brain transporters for 5-HT, DA and NE respectively. Hyperforin also membrane fluidity were determined. Oral administrations non-competitively inhibited the uptake of these monoamines to resulted in hyperforin brain levels of 29.5 ± 18.7 pmol/g and rat brain synaptic vesicles but did not affect the direct binding 53.7 ± 18.7 pmol/g. Treatment of mice with hyperforin lead to of [3H]-dihydrotetrabenazine, a selective vesicular monoamine decreased annular- (p<0.05) and bulk fluidity (p<0.001) and transporter ligand, to rat forebrain membranes [Roz 2002]. increased (p<0.01) acyl-chain flexibility of brain membranes [Eckert 2004]. The mechanism of inhibition of synaptic vesicle transport of monoamines by hyperforin was investigated. Since the pH Effects on ion channels gradient across the synaptic vesicle membrane, induced by In rat cerebellar Purkinje neurons, hyperforin inhibited ion + vacuolar type H -ATPase, is the major driving force for vesicular currents at the NMDA and AMPA receptors (IC50 3.2 µM and monoamines uptake and storage, proton transport was measured. 4.6 µM respectively) in a dose- and time-dependent manner Adding Mg-ATP to the medium containing and synaptic vesicles [Chatterjee 1999].

7 HYPERICI HERBA

Spasmolytic properties of hyperforin were studied in the hamster Neutrophils from patients with Behçet’s disease (BD) were vas deferens smooth muscle cell line DDT1-MF2. Hyperforin divided into three subgroups and incubated with either an (0.3-10 μg/mL) caused a concentration-dependent increase of unspecified extract (containing 3.6% hyperforin), voltage- 2+ intracellular calcium concentration ([Ca ]i) and extracellular gated calcium channel (VGCC) blockers (+, acidification rate (ECAR). Both effects were independent of V+D) or a non-specific TRPM2 channel blocker (2-aminoethyl extracellular Ca2+. Since the ECAR stimulation was inhibited by diphenylborinate, 2-APB). They were subsequently stimulated the intracellular Ca2+ chelator BAPTA, as well as by inhibitors with fMLP as a Ca2+ agonist and oxidative stress generator. + 2+ 2+ 2+ of plasmalemmal and mitochondrial Na /Ca exchangers, The high cytosolic-free Ca [Ca ]i, caspase-3, and caspase-9 but not by inhibitors of the Na+/H+ antiporter, the intracellular values found in the patient groups decreased markedly after Ca2+ increase seems to be essential for this hyperforin effect treatment with the extract and low GSH and GSH-Px values 2+ [Koch 2001]. increased. The [Ca ]i also decreased in the groups with V+D and 2-APB incubations. The extract induced protective effects Isolated patch-clamped rat central and peripheral neurons ex- on oxidative stress by modulating Ca2+ influx in BD patients. posed to rapid changes in the compartment of external medium [Naziroglu 2014a; Naziroglu 2014b]. were used to investigate the modulation of various voltage- and ligand-gated channels by hyperforin and other constituents Cholinergic effects of Hypericum perforatum. At nanomolar concentrations, The effect of hyperforin on choline (Ch) uptake was studied hyperforin induced inhibition of various ion channels. On P-type in brain synaptosomes from Sprague-Dawley rat cortices. Ca2+ channels hyperforin acted via interaction with calmodulin Hyperforin inhibited high-affinity choline uptake with an or through calmodulin-activated pathways involving at least IC50 of 8.5 μM, whereas low-affinity uptake was not affected one second messenger [Krishtal 2001]. [Buchholzer 2002].

In rat PC12 cells, hyperforin (10 µM) increased intracellular The spontaneous release of Ach in the neuromuscular junction concentrations of Na+ and Ca2+. This increase was dependent of Charles River male mice was unaffected by a hydroalcoholic on the activation of a non-selective cation channel, TRPC6, extract at doses from 0.2 to 1.0 mg/mL indicating that neither a transient receptor potential channel. Blockade of TRPC6 presynaptic nor postsynaptic functions were modified by the channels inhibited the hyperforin effect in PC12 cells [Leuner extract. Both the frequency and amplitude of the miniature 2007]. This was confirmed by experiments in cortical neurons end-plate currents (mepcs) were unmodified by the extract. The from embryonic murine brain [Gibon 2010]. mepcs decay time, i.e. the apparent cholinergic channel life time, was significantly increased after treatment. The amplitude of the evoked end-plate currents was constantly and in a dose- Hyperforin inhibited various ion channels in the patch-clamp dependent manner increased by the extract [Re 2003]. model in peripheral and central neuronal cells isolated from 2+ Wistar rats. In P-type Ca channels it was confirmed that Smooth muscle function hyperforin acts via an interaction with calmodulin or through Contractions of rat bladder strips were induced by electrical calmodulin-activated pathways involving at least one second field stimulation (EFS) or by exogenous a,b-methylene ATP. An messenger. An extract (5% hyperforin) inhibited almost all unspecified hydromethanolic extract was significantly (p<0.001) tested ligand-gated ion channels. Quercitrin was detected as a more active in inhibiting the EFS-induced contractions than the potent inhibitor of ATP-induced conductance; biapigenin and a,b-methylene ATP-induced contractions. The inhibitory effect hyperoside inhibited ATP- and AMPA-induced conductances, of the extract on EFS-induced contractions was unaffected by whereas hypericin was inactive in all cases [Krishtal 2001; methysergide, , phentolamine and Leuner 2007; Treiber 2005; Wang 2010]. (antagonists blocking the action of 5-HT, DA, and NE on their own receptors), the L-type calcium channel antagonist The effect of an unspecified extract and hypericin on cell verapamil, (blocking the vanilloid receptor), or viability, human chorionic gonadotrophin (hCG) production, CB1 receptor antagonist SR141716A. However, Ca2+ uptake, and Ca2+ transport proteins expression in JEG-3 the opioid receptor antagonist naloxone significantly (p<0.001) cells was analysed. Toxicity was seen at high concentrations reduced the inhibitory effect of the extract on EFS-induced (≥150 μg/mL), but with no effect on hCG production with the contractions [Capasso 2004]. extract (25 μg/mL) or hypericin (7.5 and 75 ng/mL). Cells treated with the extract or hypericin exhibited increased intracellular An unspecified hydromethanolic extract (1-300 µM) exhibited Ca2+ concentrations after 24 h but not 10 min of incubation. a concentration-dependent decrease in the amplitude of A significant (p<0.01) decrease in translationally controlled contractions in isolated rat and human vas deferens induced tumour protein Ca2+ handling protein was only caused by by both EFS and a,b-methylene ATP [Capasso 2005]. the extract. Hypericin increased the protein expression of the transient receptor potential vanilloid 6 Ca2+ channel and 28- Immunological effects kDa calcium-binding protein and decreased that of plasma A hydromethanolic extract caused pronounced suppression membrane Ca2+-ATPase 1/4 [da Conceicao 2010]. of interleukin-6 (IL-6) release in human blood samples after stimulation with phytohaemagglutinin [Thiele 1993]. Hypericin Hyperforin induced TRPC6-independent H+ currents in HEK-293 at the micromolar range inhibited phorbol myristate acetate- cells, cortical microglia, chromaffin cells and lipid bilayers. The and TNF-a-induced activation of NF-kB, whereas hyperforin latter demonstrated that hyperforin acts as a protonophore. This and an ethanolic extract (60%, w/w; 0.15% hypericin and 5% activity caused cytosolic acidification, which strongly depended hyperforin) were inactive [Bork 1999]. The myeloperoxidase- on the holding potential, and fueled the plasma membrane catalysed dimerisation of enkephalins was inhibited by a sodium-proton exchanger. Thereby the free intracellular sodium methanolic extract [Denke 1999]. concentration increased and the neurotransmitter uptake by Na+ co-transport was inhibited. Additionally, hyperforin depleted Mononuclear cells from healthy donors were incubated and reduced loading of large dense core vesicles in chromaffin with a hydromethanolic extract or with SSRIs and tested for cells, which requires a pH gradient in order to accumulate natural killer cell activity. The extract was tested at very low monoamines [Sell 2014]. concentrations (0.5-20 ng/mL), paroxetine (PAR) at 120 ng/mL

8 HYPERICI HERBA

and norfluoxetine at 100 ng/mL. In contrast to the SSRIs, the concentration, the flavonoids reduced the formation of amyloid- extract failed to enhance natural killer cell activity at these induced ROS in microglia. No influence of the extract on the concentrations [Helgason 2000]. capacity of microglia to phagocytose sub-toxic concentrations

of fibrillar Ab1-40 was observed [Kraus 2007]. Seven unspecified commercial extracts were studied for their effect on (SP)-induced synthesis of IL-6 in human The neuroprotective effect of a methanolic extract was investi- astrocytoma cells. A potent and dose-dependent inhibition of gated in astrocytes from male Swiss albino mice. Treatment with SP-induced IL-6 synthesis by various extracts was demonstrated the extract for 7 days (300 mg/kg/day p.o.) following a challenge [Fiebich 2001]. on day 1 with 4 i.p. injections of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) (20 mg/kg in 2 h intervals) resulted Autoimmune inflammatory demyelination in reduced MPTP-induced striatal area astrocyte activation and Hyperforin inhibited IFN-g production, with down-regulation a signfiicant inhibition of MAO-B activity (p<0.05) compared of a Th1 gene expression marker and upregulation of a Th2 to MPTP alone [Mohanasundari 2007]. marker, on IL-2/PHA-activated T cells. In parallel, a strong down-regulation of the chemokine receptor CXCR3 expression Quercetin, kaempferol and biapigenin significantly (p<0.05) on activated T cells was shown. In vivo, hyperforin (150 mg/kg reduced chemically induced neuronal death in hippocampal in food pellets) significantly (p<0.001) attenuated the severity neurons from Wistar rat embryos. Neuroprotection was of symptoms of experimental allergic encephalomyelitis, a correlated with prevention of delayed calcium deregulation Th1-mediated autoimmune disease of the CNS [Cabrelle 2008]. and with the maintenance of mitochondrial transmembrane electric potential in brain mitochondria. The compounds Neuroprotection reduced mitochondrial lipid peroxidation (p<0.05 to p<0.01) The effect of hyperforin was studied on the processing of the and loss of mitochondrial transmembrane electric potential amyloid precursor protein (APP) in PC12 cells, stably transfected caused by oxidative stress. Biapigenin significantly (p<0.05) with human wild type APP. A transiently increased release of affected mitochondrial function and decreased the capacity of secretory APP fragments was observed upon hyperforin treatment mitochondria to accumulate calcium [Silva 2008a]. (50 nM - 10 µM). These results suggest that hyperforin is an activator of secretory processing of APP [Froestl 2003]. Hypericin significantly interacted with peptides in b-sheet conformation by hindering the formation of fibrils and thereby The neuroprotective role of an ethanolic (80%) extract and interfering with early stages of the polymerisation process hexane, ethyl acetate, and butanol fractions in amyloid-b peptide responsible for neurodegenerative disorders [Bramanti 2010]. (Ab)-induced cell death in rat cultured hippocampal neurons was investigated. Induced lipid peroxidation was significantly An unspecified extract (31.25 ng/mL-5 µg/mL) significantly (p<0.05) inhibited by fractions containing flavonol glycosides, (p<0.05) decreased the survival of human SH-SY5Y neuro- flavonol and biflavone aglycones, and by a fraction containing blastoma cells at the highest dosage, whereas lower mainly chlorogenic acid-type phenolics (21%, 77% and 98% concentrations exerted no significant effects. The treatment of respectively). Lipid peroxidation evaluated after incubation cells with 5 µg/mL led to a significant (p<0.05) increase of ATP with 25 µM Ab, was significantly (p<0.05) inhibited by the levels, whereas treatment with a concentration of 500 ng/mL extract. The extract, and fractions containing flavonol glycosides, had no significant effect. The extract did not show any cytotoxic flavonol and biflavone aglycones, reduced Ab-induced cell activity [Schmidt 2010]. death by 65%, 58% and 59% respectively. Ab induced a decrease in cell volume, chromatin condensation and nuclear Quercetin and kaempferol stimulated depression-related fragmentation, effects not evident in the presence of the extract signalling pathways involving brain-derived neurotrophic and fractions containing hypericins (hypericin = 11.0 µM), or factor (BDNF), phosphorylation of cAMP response element fractions containing flavonoids (quercetin = 21.1 µM). Dendritic binding protein (CREB), postsynaptic density proteins PSD95, lesions were observed following insult with Ab, but prevented and reduced amyloid-b peptide (Ab) in neurons isolated from after exposure to the peptide plus the fractions [Silva 2004]. double transgenic AD mice (TgAPPswe/PS1e9). In addition, enhanced BDNF expression and reduction of Ab oligomers

An unspecified extract protected PC12 cells from H2O2-induced were confirmed in the hippocampus [Hou 2010]. damage in a dose-dependent manner within 24-hour treatment. Intra- and extra-cellular ROS levels decreased significantly to Hyperoside (2.5, 5, 10 or 20 µM) significantly inhibited cyto-

71.9% and 50.0% of the control at 20 µg/mL, respectively. toxicity and apoptosis in A-b25-35-induced primary cultured The extract blocked DNA fragmentation and prevented H2O2- cortical neurons from embryonic Sprague-Dawley rat foetuses by induced apoptosis of the cells at concentrations of 10 to 100 reversing A-b25-35-induced mitochondrial dysfunction, including µg/mL [Lu 2004]. mitochondrial membrane potential decrease, ROS production (p<0.01), and mitochondrial release of cytochrome c (p<0.01). An ethanolic extract (50% V/V; 0.37% hypericins, 10.1% The compound activated the PI3K/Akt signalling pathway, total flavonoids and 0.7% hyperforin) was evaluated for its resulting in inhibition of the interaction between Bad and antioxidative properties in glutamate-induced cell death of BclXL and inhibited the mitochondria-dependent downstream hippocampal HT22 cells. The maximum protective effect was caspase-mediated apoptotic pathway (p<0.05 to p<0.01). observed at 0.1%. The effects were related to an attenuation It protected Ab-induced primary cultured cortical neurons of calcium fluxes and cellular energy statuses [Breyer 2007]. via PI3K/ Akt/ Bad/ BclXL-regulated mitochondrial apoptotic pathway [Zeng 2011]. In a microglial cell line pre-treated with an ethanolic (50%) extract, cell death evoked by treatment with Ab25-35 and Ab1-40 An ethanolic extract (60% V/V; 0.3% hypericin and 0.61% was attenuated significantly (p<0.01 to p<0.001) in a dose- hyperforin) administered to male Wistar rats for 21 days at a dependent manner. Investigation of the single compounds dose of 300 mg/kg per day demonstrated increased mRNA levels revealed that (+) catechin and (-)-epicatechin increased cell for mdr1 by 275% (p<0.001), for oatp1c1 by 41% (p<0.05), viability slightly, whereas the quercetin, rutin, hyperoside and a reduced mRNA level (by 68.5%, p<0.05)) for mrp1 in and quercitrin showed no effect on cell viability. At the same hippocampal tissue [Mrozikiewicz 2014].

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Anti-inflammatory effects The inhibitory effect of the 4 component system on LPS-induced

Hypericin (1, 5, 10 µg/mL) significantly (p<0.05) and dose- prostaglandin E2 and NO production in RAW 264.7 mouse dependently inhibited IL-12 production in LPS-activated macrophages was compared to ethanolic extracts and extract macrophages from female DBA/2 mice (IC50 = 1.45 μg/mL) fractions. Pseudohypericin was found to be the most important and potently inhibited the activation of an IL-12 gene promoter for anti-inflammatory activity. Most of the extracts reduced LPS-

[Kang 2001]. induced PEG2 and NO production [Huang 2012].

A methanolic (80%) extract and hyperforin (0.03, 0.1, 0.3, Hyperoside exerted an anti-inflammatory effect through 1, 3 and 10 µM) were investigated for inhibition of 5-lipoxy- inhibition of iNOS expression and suppressed production genase (5-LO) and (COX). Hyperforin directly of TNF-a, IL-6 and NO in LPS-stimulated mouse peritoneal inhibited 5-LO (IC50 ~ 90 nM) in freshly isolated human macrophages. Furthermore, it inhibited NFkB activation by polymorphonuclear leukocytes (PMNL). In thrombin- or inhibiting LPS-induced degradation of the inhibitory subunit ionophore-stimulated human platelets, hyperforin suppressed IkB-a, a key step for NFkB-induced transcription of certain

COX-1 activity with IC50 values of 0.3 and 3 µM respectively, proinflammatory genes including iNOS [Kim 2011]. being about 3- to 18-fold more potent than . Hyperforin did not interfere with COX-2 product formation and did not An unspecified extract was studied at doses of 10, 25 and 50 significantly inhibit 12-LO in platelets or 15-LO in leukocytes. µg/mL on adipocyte differentiation and for anti-inflammatory Inhibition of 5-LO was slightly more prominent for equimolar effects in 3T3-L1 pre-adipocytes. The extract promoted adipocyte amounts of the extract (calculated for hyperforin; IC50 22 µg/mL) differentiation, while immunoblots indicated that the extract [Albert 2002]. increased the expression of peroxisome proliferator activated receptor gamma (PPARg), a nuclear receptor regulating A methanolic (80%) extract exhibited antiinflammatory adipocyte differentiation, and adiponectin, an anti-inflammatory activity on PMNL myeloperoxidase (MPO) activity. The extract adipokine. The anti-inflammatory activity of the extract was reduced the peroxidative and chlorinating activity of MPO in demonstrated by its inhibition of the activation of NF-kB. a concentration-dependent manner by 18.5% (at 20 µg/mL) to Stimulation of mature 3T3 L1 adipocytes by TNF-a decreased 44.1% (at 40 µg/mL) [Pabuccuoglu 2003]. the expression of the NF-kB inhibitor IkBa, and increased its phosphorylation. Treatment with the extract decreased the TNF-a Hyperoside exerted an anti-inflammatory action through induced perturbation in IkBa expression and phosphorylation, suppressed production of TNF-a, IL-6, and NO in LPS-stimulated indicating that the extract mediated the inhibition of NF-kB mouse peritoneal macrophages. The maximal inhibition rates activation. In addition, the extract decreased the TNF-a induced of TNF-a, IL-6, and NO production by 5 μM hyperoside were increase in the mRNA levels of pro-inflammatory adipokines, 32.31 ± 2.8%, 41.31 ± 3.1%, and 30.31 ± 4.1% respectively. IL-6, and monocyte chemoattractant protein-1 [Hatano 2014]. In addition, hyperoside inhibited NF-kB activation and IkB-a degradation [Kim 2011]. Tissue repairing effects The tissue repairing effects of an alcoholic (50%) extract were Various extracts (acetonic, 96% ethanolic and 50% ethanolic) evaluated in comparison with dexpanthenol on cultured chicken and isolated constituents (flavonoids and dianthrones) showed embryonic fibroblasts. The extract exhibited a tissue repairing a comparable and dose-dependent inhibition (>80% inhibition activity mainly due to an increase in the stimulation of fibroblast at 0.15%) of 5-LO. The strongest effect was seen for hyperoside collagen production and the activation of fibroblast cells in -6 (IC50 3.89 x 10 M), whereas quercetin, quercitrin, isoquercitrin polygonal shape [Öztürk 2007]. -5 and rutin were less active (IC50 of 3.7 – 9.0 x 10 M). The weakest effects were seen for hypericin and pseudohypericin (IC50 of Metabolic effects 1.5 and 6.3 x 10-4 M) [Oblozinsky 2006]. In the INS-1E b-cell line an unspecified hydroalcoholic extract and hyperforin (at 1-3 µM) prevented cytokine (IFN-g, 150 In isolated PMNL hyperforin inhibited the generation of reactive U/mL + IL-1b 10 U/mL + TNF-a 50 U/mL) induced impairment oxygen species (ROS) as well as the release of leukocyte elastase of -stimulated insulin secretion, and protected cells with an IC50 of about 3 µM and blocked receptor-mediated against apoptosis in a dose-dependent manner. iNOS expression 2+ Ca mobilisation (IC50 0.4 and 4 µM) in PMNL and monocytic (p<0.05) as well as cytokine-induced activations of STAT-1 and cells [Feisst 2004]. NF-kB were down-regulated by the extract and hyperforin (range 0.5-5 µM) [Menegazzi 2008].

The inhibition of (PGE2) production by various extracts and some constituents was investigated in RAW 264.7 Ethanolic extracts, one from leaves and one from flowers, mouse macrophage cells. The anti-inflammatory activity of all inhibited differentiation of 3T3-L1 adipocytes. Both extracts extracts was light-independent. The extracts significantly reduced substantially inhibited insulin sensitive glucose uptake in mature

LPS-induced PGE2 production by 73 to 82% (each p<0.0001) 3T3-L1 adipocytes. Treatment with the flower extract resulted [Hammer 2007]. An extract fraction used in the previous study, in a time- and dose-dependent inhibition of insulin stimulated and a combination of four synergistic constituents at levels glucose uptake in mature adipocytes [Amini 2009]. identified from that fraction called the 4 component-system (0.07 µM amentoflavone, 0.08 µM quercetin, 0.2 µM chlorogenic acid An unspecified flower extract at a dose of 50 µg/mL significantly and 0.03 µM pseudohypericin), were studied to identify key (p<0.02) reduced insulin-sensitive glucose uptake in human gene targets involved in the anti-inflammatory activity. The adipocytes compared to control. The extract inhibited insulin reduction in PGE2 in LPS-stimulated macrophages observed receptor substrate-1 (IRS-1) tyrosine phosphorylation in with both treatments may be accounted for by genes involved both murine and human fat cells. Hypericin (0.02–0.3 μM) in Janus kinase, as well as a signal transducer and activator and hyperforin (0.17–2.8 μM) did not modulate adipocyte of transcription (JAK–STAT) and eicosanoid pathways. COX-2 development and insulin activity in mature adipocytes [Richard appeared to be a major target of both the 4-component-system 2012]. and the fraction, and upstream pathways leading to COX-2 expression were also affected by both treatments [Hammer A hydroalcoholic extract (4.1% hyperforin) at doses of 100 2010]. and 200 µg/mL and hyperforin at 1 and 2 µM were applied to

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isolated rat and human islets exposed to cytokines (rat or human assays, ten of the oils inhibited Trypanosoma brucei rhodesiense

IFN-g 1000 µg/mL + IL-1b 100 U/mL + TNF-a 1000 U/mL) over (IC50 15.9-64.5 µg/mL), while only one oil exerted antimicrobial 20 h. In both rat and human islets, the extract and hyperforin activity towards Staphylococcus aureus (IC50 88.7 µg/mL) counteracted cytokine-induced functional impairment and [Orhan 2013]. down-regulated mRNA expression of pro-inflammatory target genes, such as iNOS, CXCL9, CXCL10 and COX-2. Cytokine- Hypericin (0-2.5 μM) resulted in a dose- and light-dose induced NO production from cultured islets was significantly dependent (1 – 8 J/cm2) cytotoxicity (cellular destruction and (p<0.01 to p<0.001) reduced in the presence of the extract and induction of early apoptosis as a prominent mode of cell death) hyperforin. The increase in apoptosis and necrosis following in CNE-2 nasopharyngeal carcinoma cells [Xu 2010]. 48-h exposure to cytokines was fully prevented by the extract, but only partially by hyperforin. Ultrastructural morphometric Various extracts (MeOH; petroleum ether; chloroform, ethyl analysis in human islets exposed to cytokines for 20 h showed acetate) showed growth inhibition of Gram-positive bacteria. that the extract or hyperforin avoided early beta-cell damage The ethyl acetate extract was the most active (MIC 12.5 - 50 µg/mL). [Novelli 2013]. Incubation of selected microorganisms with flavonoids, hypericins or hyperforins resulted in a growth inhibition by hypericin (MIC Antiviral effects 12.5 - >1000 µg/mL), hyperforin (MIC 25 – 100 µg/mL) and its Hypericin showed antiretroviral activity against equine infect- dicyclohexilammonium salt (MIC 25 – 100 µg/mL). Flavonoids ious anaemia virus (EIAV). Treatment with 10 µg/mL hypericin appeared to be inactive [Avato 2004]. reduced the virus production completely [Kraus 1990]. Photosensitising and photodynamic effects In primary human lymphocytes hypericin exhibited anti- The methanolic fraction (PMF) of an unspecified extract was HIV-1 activity via an inhibition of HIV-1 reverse transcriptase examined for activity against urinary bladder carcinoma using [Schinazi 1990]. the T24 (high grade metastatic cancer) and RT4 (primary low grade papillary transitional cell carcinoma) human bladder Hypericin and protohypericin inhibited virus replication of cancer cells. PMF was excited using 630 nm laser light. PMF human cytomegalovirus strain AD-169 in human foreskin (Hs- exhibited significant photocytotoxicity in both cell lines at a 2 68) and lung fibroblast cells (MRC-5) with IC50 values of 1.8 concentration of 60 µg/mL, with 4-8 J/cm light dose, resulting in and 5.7 µM respectively [Barnard 1992]. apoptosis from 80% to 86%. At 20 µg/mL PMF was not active in either cell line. Photofrin®, a clinically approved photosensitiser, Hypericin (500 ng/mL) exhibited strong antiviral activity against exhibited less photocytotocixity at 4 µg/mL than PMF in both vesicular stomatitis virus, Herpes simplex virus types 1 and 2, tested cell lines [Stavropoulos 2006]. parainfluenza virus and vaccinia virus (up to 3.5 log10 reduction of viral production) [Andersen 1991]. In human epidermoid carcinoma cells (A431) the time- and dose- dependence of hypericin-photodynamic treatment (PDT)-based Hypericin (0.12 - 0.25 µg/mL) inactivated murine cytomegalo- induction of cytotoxicity and apoptotic cell death was studied. virus, sindbis virus, and human immunodeficiency virus type Induction of apoptosis by hypericin-PDT was dose-dependent 1 (HIV-I), especially on exposure to fluorescent light [Hudson with necrotic cell death at higher doses. Time-course analysis 1991]. of an almost homogenous apoptotic population of cells (at 1.44 J/cm2) showed a rapid increase in nuclear fragmentation Hypericin was active against duck hepatitis B virus (DHBV). and activation of caspases reaching a maximum 5 hours after After 1 h incubation with hypericin at 15 µg/mL, cells stably- irradiation. Using specific caspase substrates, significant transfected with DHBV stopped virus replication for several activation of caspase-2, -3, -6 and -9 was found. Mitochondrial days, though virus-like particles continued to be released into involvement during hypericin-PDT-induced apoptosis was the culture medium. Hypericin was only slightly virucidal against proven by a rapid reduction of the mitochondrial membrane DHBV, and culture medium from treated cultures did not block potential; the level of intracellular adenosine-5'-triphosphate initiation of infection when added to DHBV susceptible cultures (ATP) remained at control level for up to 6 hr post irradiation prior to a challenge with infectious DHBV [Moraleda 1993]. suggesting upregulation of glycolysis as a compensating mechanism for energy supply [Berlanda 2006]. An ethanolic extract at doses of 0.32 to 40 µg/mL was tested for anti-hepatitis B virus (HBV) activity in HepG2 2.2.15 cells. The modulation of hypericin-PDT by pre-treatment with n-3 and The extract effectively suppressed the secretion of HBsAg and n-6 fatty acids was studied in HT-29 and HeLa tumour cells. A HBeAg in a dose-dependent manner, as well as the extracellular stimulation of cytotoxic effects by docosahexaenoic acid (n-3) HBV DNA. In addition, the extract selectively inhibited the and arachidonic acid (n-6) was observed in both cell lines. activity of HBV promoter Fp [Pang 2010]. Treatment with either fatty acids or photodynamic therapy alone induced apoptosis in a dose- and time-dependent manner. The Antimicrobial effects effect was even more striking with the combination applied as Various extracts were evaluated for their antibacterial activity pre-treatment with fatty acids prior to PDT. The combination in- against Bacillus subtilis, P. aeruginosa, Sarcina lutea and E. duced changes in membrane lipid composition leading to alteration coli at concentrations of 1, 10, 25 and 50 μg/μL. The n-hexane in cell membrane fluidity. Increased toxicity from the combined and dichloromethane extracts were inactive against all tested treatment was also confirmed by the presence of oxidative stress organisms. The n-butanol and ethyl acetate extracts showed as demonstrated by ROS production, RNS accumulation and activity against all the tested bacterial strains at concentrations increased presence of lipoperoxides [Kello 2010]. of 25 and 50 μg/μL while the methanolic extract was only active against B. subtilis at concentrations 25 and 50 μg/μL A hydroalcoholic extract was assessed for its anti-proliferative [Naeem 2010]. activity in A375 human melanoma cells after irradiation with 2 UVA (1.8 J/cm ) and demonstrated phototoxicity at IC50 78 Twenty-one samples of traditionally-prepared and commercial µg/mL. The extract caused a dose-related inhibition of NO St. John's Wort macerates were screened for their production in murine monocytic macrophage cell line RAW antimicrobial and antiprotozoal activity. In the antiprotozoal 264.7 with an IC50 of 342 µg/mL [Menichini 2013].

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Antiproliferative effects suggesting a role for this pathway [Vantieghem 2002]. Hypericin and pseudohypericin showed a strong inhibitory effect on protein kinase C (IC50 of 1.7 and 15 µg/mL respectively) which The effect of an unspecified extract on antiproliferative activity resulted in an anti-proliferative effect in BALB 3T3/H-ras cells of anticancer drugs and the expression of MDR1 mRNA were with IC50 values of 6.3 and 28 µg/mL respectively; associated examined using HeLa and its MDR1-overexpressing subline. with an antiretroviral effect [Takahashi 1989]. The effects on MDR1-mediated transport were evaluated using [3H]-digoxin and MDR1 overexpressing LLC-GA5-COL150 cells. Rather than stabilising topoisomerase II (topo II) in covalent The effects of hypericin were compared with those of the extract. complexes with DNA (cleavage complexes), hypericin inhibited The extract (60 µg/mL containing 0.1 µM hypericin), but not the enzyme prior to DNA cleavage. The assays indicated that hypericin, reversed the cytotoxicity of PXL and of daunorubicin hypericin is a potent antagonist of cleavage complex stabilisation slightly, down-regulated MDR1 mRNA, and inhibited MDR1- by etoposide and amsacrine. This antagonism appears to be mediated transport [Wada 2002]. due to the ability of hypericin to intercalate or distort DNA structure, thereby precluding topo II binding and/or DNA Hyperforin inhibited the growth of leukaemia K562 and U937 cleavage. Supporting its non-DNA damaging, catalytic inhibition cells, brain glioblastoma cells LN229 and normal human of topoisomerase II, hypericin was shown to be equitoxic to astrocytes with GI50 values between 14.9 and 19.9 µM. both wild-type and amsacrine-resistant HL-60 leukaemia cell Hyperforin and hypericin acted synergistically on leukaemic lines. Hypericin was incapable of stimulating DNA damage- (K562, U937) cell growth. Cell death occurred by apoptosis responsive gene promoters that are activated by etoposide. As after 24 h treatment with hyperforin. A dose-dependent loss of with the topo II assay, antagonism of DNA damage stimulated membrane phospholipid asymmetry associated with apoptosis by 30 μM etoposide was evident in leukaemia cells pre-treated was induced in all cell lines [Hostanska 2003b]. with 5 μM hypericin [Peebles 2001]. Different extracts (A: ethanol-water macerate of fresh flowering Photoactivated hypericin and (PXL) similarly increased tops, B: macerate of flowering tops dried in the dark, C: the activity of caspase-8 and caspase-3. Drug-induced apoptosis aqueous-ethanolic percolation of the light-dried whole aerial was completely blocked by inhibitors of caspase-8 and parts), containing various amounts of hyperforin (A: 3.25%; caspase-3. The involvement of death receptors was analysed B: 2.21%; C: 0.21% w/w) and flavonoids (A: and B: 5.3%; C: using neutralising monoclonal antibodies against Fas (SM1/23), 10% w/w), but similar amounts of naphthodianthrones (0.3%), FasL (NOK-2) and TNF-R1 (MAB225), and a polyclonal rabbit inhibited growth of K562 and U937 cell lines in the WST-1 anti-human TNF-related apoptosis-inducing ligand (TRAIL) assay. The GI50 for the extracts was 248.3-621.3 µg/mL in K562 antiserum. TRAIL antibody blocked TRAIL-induced and photo- and 378.2 911.7 µg/mL in U937 cells. The values of the three activated hypericin-induced, but not PXL-induced apoptosis of main constituents were 1.6 3.9 µM for naphthodianthrones, 1.0- Jurkat cells. In contrast, PXL-induced, but not hypericin-induced 40.7 µM for phloroglucinols and 30.5 68.5 µM for flavonoids apoptosis was blocked by the SM1/23 and NOK-2 antibodies. [Hostanska 2003a]. Anti-TNF-R1 antibody had no effect. These findings suggest that hypericin photo-induced apoptosis of Jurkat cells is mediated The influence of a methanolic extract and hypericin on the in part by the TRAIL/TRAIL-receptor system and subsequent human erythroleukaemic cell line K562 was tested. After 1 h activation of upstream caspases [Schempp 2001]. exposure to increasing concentrations (as hypericin content: 0.5 to 5 µg/mL) of both agents in the dark, leukaemic cells Hyperforin inhibited growth of various human and rat tumour were grown for 24 h and 48 h. Hypericin showed only a cell lines with IC50 values between 3 – 15 µM. Treatment weak inhibitory effect on cell growth and no effect in inducing with hyperforin resulted in a dose-dependent generation of apoptosis. In contrast, the extract showed a significant (p<0.05) apoptotic oligonucleosomes, typical DNA-laddering and concentration-dependent inhibition of cell growth, and induced apoptosis-specific morphological changes. In MT-450 mammary apoptosis [Roscetti 2004]. carcinoma cells hyperforin increased the activity of caspase-9 and caspase-3, and hyperforin-mediated apoptosis was A lipophilic petroleum ether extract induced apoptosis with blocked by the caspase inhibitor zVAD.fmk. Hyperforin, but LC50 values of 5 and 7 µg/mL in T24 and NBT-II bladder cancer not paclitaxel, induced a rapid loss of the mitochondrial trans- cells respectively. The most active fraction from this extract membranal potential Δ ψ m, and subsequent morphological displayed 60% cell growth inhibition at a concentration of 0.95 changes such as homogenisation and vacuolisation of µg/mL. Hyperforin concentrations in the extract and its most mitochondria. Hyperforin-mediated mitochondrial permeability active fraction were 0.94 µg/mL, and 0.17 µg/mL respectively; transition was not prevented by zVAD.fmk. The compound was considerably lower than the active cytotoxic concentration of capable of releasing cytochrome c from isolated mitochondria. pure hyperforin which appeared to be in the range of 1.8 µg/mL - These findings suggest that hyperforin activates a mitochondria- 5.0 µg/mL [Skalkos 2005]. mediated apoptosis pathway [Schempp 2002a]. Hypericin inhibited protein kinase C in H-ras transfected mouse Photoactivated hypericin did not cause Bcl-2 degradation but BALB 3T3 cells and induced apoptosis. This mechanism seemed induced Bcl-2 phosphorylation in a dose- and time-dependent to be linked to the photoactivated state, whereas in the dark no manner in HeLa cells. Bcl-2 phosphorylation was induced relevant cytotoxicity was observed [Kubin 2005; Lavie 1995; by sublethal PDT doses; increasing the photodynamic stress Takahashi 1989]. immediately led to apoptosis, during which Bcl-2 was neither phosphorylated nor degraded. The co-localisation of hypericin Hyperforin promoted apoptosis in B-CLL cells, as shown by with a-tubulin and the aberrant mitotic spindles observed time- and dose-dependent stimulation of phosphatidylserine following sublethal PDT doses suggested that photodamage externalisation and DNA fragmentation, by disruption of the to the microtubule network provokes the G2/M phase arrest. mitochondrial transmembrane potential, caspase-3 activation Bcl-2 phosphorylation is selectively suppressed by the and cleavage of the caspase substrate PARP-1 and induced down- cyclin-dependent protein kinase (CDK)-inhibitor roscovitine, regulation of the two antiapoptotic proteins Bcl-2 and Mcl-1. completely blocked by the protein synthesis inhibitor Hyperforin also down-regulated two proteins overexpressed by cycloheximide and enhanced by the over-expression of CDK1, patients' B-CLL cells, the cell cycle inhibitor p27kip1 through

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caspase-dependent cleavage into a p23 form, and the inducible non-phosphorylation by AKT1. In primary CLL cells, hyperforin NO synthase resulting in a reduction of NO production, known acted by stimulating the expression of the pro-apoptotic Bcl- to be antiapoptotic in B-CLL cells. Preventing effects of the 2 family member Noxa. Other hyperforin targets included caspase inhibitor z-VAD-fmk indicated that hyperforin-promoted matrix metalloproteinase-2 in AML cells and VEGF and matrix apoptosis of B-CLL cells was mostly caspase dependent. B metalloproteinase-9 in CLL cells. Hyperforin also lowered the lymphocytes from healthy donors appeared less sensitive to activity of Pgp which is involved in the resistance of leukaemia hyperforin-induced apoptosis than B-CLL cells [Quiney 2006]. cells to chemotherapeutic agents [Billard 2013].

Hyperforin-mediated induction of the mitochondrial pathway of In this study hypericin was absorbed by melanoma cells and caspase-dependent apoptosis was associated with up-regulation partially co-localised in the endoplasmic reticulum, mito- of Noxa, a BH3-only protein of the Bcl-2 family. Hyperforin was chondria, lysosomes and melanosomes, but not the nucleus. shown to induce an interaction between Noxa and a pro-survial Light activation of hypericin induced a rapid, extensive protein Mcl-1, with the proapoptotic protein Bak becoming modification of the tubular mitochondrial network into a activated after being displaced from its complex with Mcl-1 beaded appearance, loss of structural details of the endoplasmic [Zaher 2011]. These steps are known to cause mitochondrial reticulum and concomitant loss of hypericin co-localisation. The membrane permeabilisation, accompanied by release of opposite was found for lysosomal-related organelles, suggesting apoptogenic factors, and subsequent caspase cascade leading that melanoma cells might use these intracellular organelles for to apoptosis [Merhi 2011]. hypericin-PDT resistance. However, hypericin-PDT was effective in killing both unpigmented (A375 and 501mel) and pigmented Hypericin induced an enhanced degradation of hypoxia- (UCT Mel-1) melanoma cells by specific mechanisms involving inducible factor 1a (HIF-1a) in two human tumour cell lines the externalisation of phosphatidylserines, cell shrinkage and (U87-MG glioblastoma cells and RCC-C2VHL-/- renal cell loss of cell membrane integrity. The treatment resulted in carcinoma) and promoted ubiquitin ligation of HIF-1a mediated extrinsic (A375) and intrinsic (UCT Mel-1) caspase-dependent by lysosomal cathepsin-B enzymes through a hypericin- apoptotic cell death, as well as a caspase-independent apoptotic mediated reduction in intracellular pH [Barliya 2011]. mode that did not involve apoptosis-inducing factor (501 mel) [Kleemann 2014]. The effect of hyperforin in the up-regulation of Noxa, a mito- chondrial protein with pro-apoptotic activity, was investigated in Antiangiogenic effects cells from patients with chronic lymphocytic leukaemia CLL and Hyperforin blocked angiogenesis of human dermal microvascular the chronic B-cell leukaemia cell line MEC-1. Hyperforin caused endothelial cells (HDMEC) and reduced proliferation of HDMEC an increase in Noxa expression in a time- and concentration- in a dose-dependent manner, without displaying toxic effects dependent manner. Noxa silencing by siRNA partially reduced or inducing apoptosis [Schempp 2005]. hyperforin-elicited apoptosis [Zaher 2011]. In bovine aortic endothelial cells hyperforin treatment resulted

The effects of hyperforin on acute myeloid leukaemia (AML) in strong inhibitory effects on angiogenesis (IC50 7 ± 3 µM). cell dysfunction were investigated in cell lines defining distinct Capillary tube formation on Matrigel was abrogated completely AML subfamilies and primary AML cells. Hyperforin inhibited by 2.5 µM hyperforin. Hyperforin also exhibited a clear inhibitory the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) effect on the invasive capabilities of endothelial cells at 10 µM. by inducing apoptosis in a time- and concentration-dependent Zymographic assays showed that hyperforin treatment produced manner. It also induced apoptosis in primary AML blasts, a complete inhibition of urokinase and a remarkable inhibition whereas normal blood cells were not affected. The apoptotic of matrix metalloproteinase 2 [Martinez-Poveda 2005]. process in U937 cells was accompanied by down-regulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, Hyperforin inhibited neutrophil and monocyte chemotaxis and mitochondrial membrane depolarisation, activation of angiogenesis induced by angiogenic chemokines (CXCL8 or procaspases and cleavage of the caspase substrate PARP 1. CCL2) [Lorusso 2009]. The caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly Apoptosis (p<0.05) attenuated apoptosis. Hyperforin-mediated apoptosis Pre-treatment with an unspecified extract significantly (p<0.05) was associated with dephosphorylation of active Akt1 (at decreased apoptosis in human SK-N-MC neuroblastoma cells Ser(473)) and Akt1 substrate Bad (at Ser(136)) which activates as shown in different assays. In addition, pre-treatment with the

Bad pro-apoptotic function. Hyperforin suppressed the kinase extract inhibited H2O2-induced increase in caspase-3 activity activity of Akt1, and combined treatment with the allosteric [Jang 2002]. Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells [Merhi 2011]. Antioxidant effects Two extracts, standardised to either 0.3 – 0.5% hypericin or A dried aqueous extract showed protective effects against 3% hyperforin (not further specified), diluted between 1:1 to oxidative DNA damage in HT29 colon cells (1 and 5 µg/mL). 1:20, were evaluated in a /xanthine oxidase assay in The extract and its main phenolic compounds quercetin and both cell-free and human vascular tissue. A pro-oxidant effect rutin (10 µM) resulted in a DNA protection of 40% (p<0.05). The was seen at the highest concentration. Lower concentrations alkylating damage repair was increased significantly (p<0.05) revealed dose-dependent antioxidant properties of the extracts by 1 µg/mL extract (50%) and chlorogenic acid (80%) by base with the strongest suppression at the most diluted concentration excision repair [Ramos 2013]. [Hunt 2001].

Anti-leukaemic effects of hyperforin were shown by induction Strong antioxidant effects were observed for hyperforin in the of apoptosis in chronic lymphoid leukaemia (CLL) and acute oxidative burst of polymorphonuclear cells after stimulation myeloid leukaemia (AML) cells known for their resistance to with N-formyl-methionyl-leucyl-phenylalanine (IC50 1.8 µM). chemotherapy. In AML cells, hyperforin inhibited the kinase No antioxidant activity was found after stimulation with activity of the serine/ threonine protein kinase B/AKT1, leading opsonised zymosan or in the H2O2/ peroxidase to activation of the pro-apoptotic Bcl-2 family protein Bad via system [Heilmann 2003].

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Pre-incubation with an ethanolic extract for 24 h at 1-100 µg/ TRPM2 channels in dorsal root ganglion (DRG) neurons from mL significantly (p<0.05) prevented the marked reduction in male Wistar rats using whole-cell patch-clamp, Fura-2 and 2+ cell survival observed following 8 h exposure of PC12 cells to antioxidant experiments. TRPM2 current densities and [Ca ]i in H2O2 (300 µM). Different concentrations of the standardised the neurons were reduced by the extract at doses of 10, 20, 30 extract (0.1-100 µg/mL) also attenuated the increase in caspase-3 and 40 µg/mL (2 and 24 h after administration). Cytosolic Ca2+ activity and suppressed the H2O2–induced reactive oxygen mobilization was reduced by the extract and VGCC blockers species generation [Benedi 2004]. (verapamil+diltiazem, V+D). Glutathione peroxidase activity and GSH values in the DRG were high for the extract, and lipid The free radical-scavenging properties of an ethanolic extract peroxidation was low [Naziroglu 2014a]. were studied using DPPH (EC50 = 49 μg dwb/mL), 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) (EC50 = 50 μg dry Penetration of blood-brain barrier weight biomass (dwb)/mL) and lipid peroxidation induced by The penetration of [3H]-amentoflavone through the blood-brain ascorbate/ (EC50 = 28 μg dwb/mL). The extract was able to barrier (BBB) was studied in primary cell cultures of porcine brain scavenge NO and HOCl [Silva 2008b]. capillary endothelial cells (PBCEC). The concentration-depend- ent uptake (37 2000 nM) was neither saturable nor temperature- The antioxidant activity of an unspecified flavonoid-rich extract sensitive, indicating passive diffusion as the major uptake was investigated to demonstrate protection against hydrogen mechanism as confirmed by transport experiments through peroxide-induced apoptosis in PC12 cells. After exposure of PBCEC monolayers (> 2% of applied dose was transported

PC12 cells to H2O2, a significant (p<0.001) decrease in cell after 30 min). Co-administration of an extract increased viability and increased levels of lactate dehydrogenase (LDH) amentoflavone transport significantly (amentoflavone only: -6 release were observed. Pre-treatment of PC12 cells with the permeability coefficient Papp 4.59 x 10 cm/s; amentoflavone -6 extract elevated cell viability, decreased levels of LDH release, + extract: Papp 6.74 x 10 cm/s) indicating that the extract decreased occurrence of apoptotic cells and inhibited DNA enhanced amentoflavone transport. In experiments with the laddering in PC12 cells [Zou 2010]. Pgp over-expressing cell line P388-MDR, amentoflavone uptake was significantly enhanced by addition of the Pgp inhibitor Pronounced antioxidant activity was observed for fractions of verapamil [Gutmann 2002]. an extract in the DPPH assay (lowest IC50 0.52 μg/mL), for NO scavenging (6.11 μg/mL), superoxide scavenging (1.86 μg/mL), PBCEC were used as a model of the BBB and epithelial cells lipid peroxidation (0.0079 μg/mL) and ferric reducing ability of of the plexus chorioidei as a model of the blood-CSF barrier plasma (highest reduction capacity 104 mg Fe2+ equivalents/g). (BCB). Results indicated no active transport of miquelianin in The activity of the fractions was attributed to flavonoids and one direction. Although moderate, the permeability coefficients phenolic acids, while phloroglucinols and naphthodianthrones (BBB: Pc = 1.34 ± 0.05×10-6 cm/s; BCB: Pc = 2.0 ± 0.33×10-6 showed no significant activity [Orcic 2011]. cm/s) indicated the ability of miquelianin to cross both barriers to reach the CNS [Juergenliemk 2003]. The antioxidant activities of ethanolic, acetonic and petroleum ether extracts were determined in the DPPH assay. The IC50 The effects of a methanolic (80%) extract and selected constit- values were <7.8, 105.9, 5.99 and 12.77 μg/mL for the ethanolic uents on the BBB transporter Pgp and on protein kinase C (PKC) extract, the acetonic extract, and the two reference antioxidants, were assessed in cultured PBCEC and freshly isolated pig brain ascorbic acid and butylated hydroxytoluene (BHT), respectively. capillaries from. Pgp function was studied in PBCEC using a The petroleum ether extract did not demonstrate antioxidant calcein-AM uptake assay and in isolated pig brain capillaries activity [Mašković 2011]. using the fluorescent ciclosporin (CyA) derivative NBD-CSA and confocal microscopy. The extract (0.1 to 5 µg/mL) and the

An H2DCFDA-assay was performed in porcine HaCaT keratino- constituents hyperforin, hypericin and quercetin (0.1 to 10 µM) cytes irradiated with solar simulated radiation. Hyperforin decreased Pgp transport activity in a dose- and time-dependent

(IC50 0.7 µM) was much more effective than Trolox (IC50 12 µg/ manner. The extract and hyperforin directly inhibited Pgp activity, mL) and N acetylcysteine (IC50 847 µg/mL), without showing whereas hypericin and quercetin modulated transporter function phototoxicity. The radical protection factor of a cream (containing through a mechanism involving protein kinase C. Quercetin

1.5% (m/m) hypericin-free CO2 extract with 44.3% hyperforin) decreased Pgp transport activity at high concentrations, but was 200 x 1014 radicals/mg, indicating a high radical scavenging increased transporter function at low concentrations [Ott 2010]. activity. The cream ex vivo on porcine ear skin significantly (p<0.05) reduced radical formation after infrared irradiation Effects on nitric oxide synthase [Meinke 2012]. Quercetin and hyperoside showed concentration-dependent inhibitory activity on nitric oxide synthase (NOS) in blood and

An ethanolic (70%) extract was investigated for phototoxicity cerebral homogenate of rat. The IC50 values of quercetin in rat in A375 human melanoma cells. peroxidation cerebral homogenate and blood were 63.06 and 57.54 µM, and DPPH tests were used to assess antioxidant activity, and those of hyperoside 56.23 and 158.49 µM, respectively while the MTT assay allowed evaluation of anti-proliferative [Luo 2004]. activity of A375 cells after irradiation with a UVA dose of 1.8 J/cm2. Inhibition of NO production of macrophages was In vivo experiments also investigated. The extract showed greater antioxidant activity in the beta-carotene bleaching test than in the DPPH assay Biochemical findings

(IC50 = 0.89 µg/mL); significant phototoxicity in A375 cells at After acute and repeated oral treatment of mice with 10 mg/kg 78 µg/mL concentration resulted in cell destruction of 50%. of an extract, the levels of serotonin and the serotonin metabolite The extract caused significant (p<0.05) dose-related inhibition 5-hydroxyindolacetic acid (5-HIAA) were increased in the of NO production in murine monocytic macrophage cell line hypothalamus and hippocampus indicating increased turnover;

RAW 264.7 with an IC50 value of 342 µg/mL [Menichini 2013]. 5-HIAA levels were also increased in other regions of the brain [Yu 2000]. An unspecified ethanolic extract was assessed for its protective role on Ca2+ entry and oxidative stress through modulation of The effects of acute oral administration (24 h and 1 h before

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testing) of a methanolic extract (62.5-500 mg/kg; 0.3% of control values respectively, and increased locomotor activity hypericin, 6% flavonoids) or an extract enriched in flavonoids in non-habituated rats. DOPAC and HVA levels were markedly (62.5-500 mg/kg; 0.3% hypericin, 50% flavonoids) on the reduced. 5-HT concentrations were elevated moderately, while levels of , 5-HT, 5-HIAA, NE and DA in the cortex, the NE levels were not affected by any treatment. Whole-tissue diencephalon and brainstem of rats were evaluated. Results analysis revealed that the extract increased, whereas the MAO were compared with the effects of fluoxetine (10-80 mg/kg). A/B inhibitor decreased, DA and 5-HT turnover The two extracts and fluoxetine increased 5-HT levels in the [Yoshitake 2004]. cortex. The flavonoid-enriched extract increased 5-HT and 5-HIAA levels in the diencephalon, and 5-HT and NE levels Neurotransmitter receptor density in the brain in the brainstem. Both extracts increased NE and DA levels Sprague-Dawley rats were treated daily for 26 weeks with an in the diencephalon [Calapai 1999]. Comparable results for 80% methanolic extract (2700 mg/kg p.o.). In preparations of the flavonoid-enriched extract were obtained in a subsequent the brain without cerebellum and brain stem, the number of study using similar techniques; it did not modify tryptophan both 5-HT1A and 5-HT2A receptors were significantly (p<0.01) content, but significantly enhanced 5-HT and 5-HIAA levels increased as compared to controls, whereas the affinity of both in the cortex, diencephalon and brainstem at 125-500 mg/kg, serotonergic receptors remained unaltered [Teufel-Mayer 1997]. and increased NE and DA in the diencephalon and NE in the brainstem at 250-500 mg/kg [Calapai 2001]. Following sub-acute oral treatment of rats with 240 mg/kg of an 80% methanolic extract for 14 days, b-adrenoreceptor Rats were treated orally with 300 mg/kg of a hydromethanolic down-regulation was observed in the frontal cortex, a common extract three times (23.5 h, 5 h and 1 h before forced swimming characteristic finding for many antidepressant drugs. The test). The extract significantly reduced immobility time (p<0.01). simultaneous up-regulation of 5-HT2 receptors observed was No significant changes in monoamine levels were detected in in contrast to the 5-HT2 down-regulation observed with many cortical or hippocampal brain regions 1 hour and 24 hours classic antidepressants [Müller 1997]. after the last dose [Calapai 2001]. Using the push-pull superfusion technique, neurotransmitter The effects of imipramine (IMI; 15 mg/kg), an 80% methanolic concentrations in the locus coeruleus of rats were studied extract (500 mg/kg) and hypericin (0.2 mg/kg) on the levels of after i.p. injection of hyperforin at 10 mg/kg. Extracellular 5-HT, NE, DA and their metabolites in the hypothalamus and concentrations of DA, NE, 5-HT and glutamate increased, hippocampus were investigated after short-term (2 weeks) whereas 5-HIAA, GABA, , aspartate, serine and arginine and long-term (8 weeks) daily oral administration to rats. All remained unchanged [Kaehler 1999]. three treatments significantly increased 5-HT levels in the hypothalamus (p<0.05) after 8 weeks, but not after 2 weeks; The short- (2 weeks) and long-term (8 weeks) effects of an 80% levels in the hippocampus were unchanged. 5-HT turnover methanolic extract (500 mg/kg p.o.), a CO2 extract (15 mg/kg (the ratio of 5-HIAA to 5-HT) was significantly reduced in both p.o.), hypericin (0.2 mg/kg p.o.), hyperoside (0.6 mg/kg p.o.) and brain regions (both p<0.05) after 8 weeks of treatment with hyperforin-trimethoxybenzoate (8 mg/kg p.o.) on b-adrenoceptor the extract but not with hypericin; IMI reduced 5-HT turnover (b-AR) binding were investigated by quantitative radioligand only in the hippocampus. Consistent changes in catecholamine receptor-binding-studies in the frontal cortex of CD rats, and levels were only detected in hypothalamic tissues after long- compared to IMI (15 mg/kg p.o.) and FLU (10 mg/kg p.o.). [125I]- term treatment. Comparable to IMI, the extract and hypericin cyanopindolol binding to b-AR was decreased after short as well significantly decreased 3,4-dihydroxyphenylacetic acid and as long-term treatment with IMI (36%, p<0.01; 40%; p<0.01). homovanillic acid levels in the hypothalamus (p<0.01). The Short-term treatment with FLU decreased the number of b-AR data showed that long-term, but not short-term, administration (17%; p<0.05) while it elicited an increase (14%; p<0.01) in of St. John’s wort and its active constituent hypericin modified b-AR-binding in long-term treatment. This effect was comparable levels of neurotransmitters in brain regions involved in the to that of the CO2 extract which decreased b-AR-binding (13%; pathophysiology of depression [Butterweck 2002a]. p<0.05) after two weeks and slightly increased the number of b-AR's after 8 weeks (9%). Short-term treatment with the The effects of acute and repeated administration of an undefined methanolic extract decreased b-AR-binding (14%); no effects extract on extracellular levels of 5-HT, NE, DA, and their were observed after 8 weeks. Treatment with hypericin led to major acidic metabolites, in the brain of anaesthetised male a significant down-regulation (13%; p<0.05) of b-AR's in the Sprague Dawley rats were studied using in vivo microdialysis frontal cortex after 8-weeks, but not after 2 weeks; hyperforin- in the prefrontal cortex (PFC), ventral hippocampus and trimethoxybenzoate and hyperoside were ineffective in both striatum. The extract inhibited the reuptake of DA, 5-HT and cases [Simbrey 2004]. NE after i.p. administration of 60 mg/kg, whereas a single oral dose of 300 mg/kg only showed an effect on DA in the Animal models of depression PFC. In rats treated for 14 days with a daily dose of 300 mg/ Various test models of depression allow the detection of anti- kg p.o., the last dose of the extract increased the extracellular depressant activity. Behavioural experiments have indicated concentrations of all monoamines in the order DA >> 5-HT that St. John’s wort extracts are centrally active, with a variety > NE, whereas the levels of metabolites were reduced in the of extracts showing antidepressant-like properties in different order 3,4-dihydroxyphenylacetic acid << homovanillic acid animal models of depression. Antidepressant-like effects < 5-hydroxyindoleacetic acid. The most pronounced effect of comparable in magnitude with those induced by classical hypericum in both the acute and subchronic regimens was seen antidepressants have been obtained in the rat forced swimming on the re-uptake and metabolism of DA [Kehr 2002]. test (FST) and in related models, such as the mouse FST, the mouse tail suspension test (TST) and the rat learned helplessness The effects of a hydroalcoholic extract on extracellular 5-HT, test. The extracts also induced an antidepressant-like profile with NE and DA levels and the acidic metabolites (3,4-dihydrox- respect to in vivo monitored field potentials in freely moving rats. yphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-HIAA) were examined by in vivo microdialysis in the prefrontal Forced swimming test cortex of awake rats. Single doses of the extract (60 mg/kg i.p. or An 80% methanolic extract (0.24-0.32% total hypericin), at 300 mg/kg p.o.) increased DA concentrations to 165 and 140% an oral dose of 125-1000 mg/kg b.w., significantly reduced

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(p<0.05 to p<0.001) the duration of immobility in the FST in In the 24h preceding a FST, male CD-COBS rats were admin- male Sprague Dawley rats. The effects on immobility time were istered three i.p. injections of either one of two methanolic eliminated by dopamine D2 antagonists [Winterhoff 1995]. extracts (containing hyperforin at 4.5% (A) or 0.5% (B)), the dicyclohexylammonium (DCHA) salt of hyperforin or a vehicle. A 50% ethanolic extract (250 mg/kg i.p.) was tested in mice Immobilisation time was significantly decreased by extract A at in the FST, with DMI (30 mg/kg i.p.) and (TRI) doses of 3.12 and 6.25 mg/kg (p<0.05) and hyperforin DCHA (20 mg/kg i.p.) serving as controls. All treatments significantly at doses of 0.19 and 0.38 mg/kg (p<0.01), but not by extract decreased swimming time of male albino mice (p<0.05) [Öztürk B [Cervo 2002]. 1997; Öztürk 1996]. Three extracts were tested in the FST in male CD rats. Extract Fractionation of an 80% methanolic extract yielded two A (3.2% hyperforin, 0.15% hypericin) was prepared with fractions with high activity in the FST with male CD rats, one 60% ethanol. Extract B (0.15% hypericin) was prepared from contained mainly flavonoids and the other naphthodianthrones. extract A by removal of hyperforin. Extract C (approximately Hypericin was inactive in the FST at a dose of 0.8 mg/kg. 12% flavonoids) was prepared from extract B by removing

However, procyanidins (mainly B2) present in the naphthodianthrones. Immobility time was significantly reduced naphthodianthrones fraction markedly increased the solubility by oral administration of all three extracts (500 mg/kg) and IMI and bioavailability of hypericin; in combination with procyanidin (30 mg/kg). Only extract C significantly reduced immobility

B2 even 0.009 mg/kg p.o. hypericin was significantly active. time when the dose was reduced to 250 mg/kg. However, after Comparable results were obtained with pseudohypericin. In 14 days of pre-treatment, similar effects were observed for all addition to hypericins, a flavonoid fraction from a methanolic three extracts [Butterweck 2003a]. extract and the flavonoids hyperoside, isoquercitrin and miquelianin showed significant activity in the FST after acute The effects of hyperoside at single i.p. doses of 7, 35 and 70 mg/kg and sub-acute administration [Butterweck 2000; Butterweck was investigated in the FST in male BIC57 mice. With the increasing 1998; Butterweck 1997]. doses, immobility time increased by 10.1%, 25.8% and 38.6% respectively, as compared to controls [Bach-Rojecky 2004].

The effect of oral administration of a CO2 extract (38.8% hyper- forin) was compared to an ethanolic extract (4.5% hyperforin) An ethanolic (60%; V/V) extract, rutin and isorhamnetin were in the FST in rats [Chatterjee 1998a] and mice [Bhattacharya compared to IMI in the FST after oral administration for 9 days. 1998]. Both extracts were active, with the effect of 5, 15 and The extract and the two flavonoids were found to significantly

30 mg/kg of the CO2 extract being comparable to that of 50, decrease the duration of immobility, with isorhamnetin (3 and 150 or 300 mg/kg of the ethanolic extract. 10 mg/kg) exhibiting the strongest effect, more pronounced than after IMI administration. The administration of 200 mg/kg or more Immobility time in the rat FST was significantly reduced by of the extract was comparable to 30 mg/kg IMI [Paulke 2008]. acute i.p. administration of 80% methanolic (p<0.05) and 50% ethanolic p<0.01) extracts. Results for these extracts (5-40 mg/kg) The effects of an ethanolic (A: 50%; (m/m), 0.08% hyperforin) were comparable to i.p. administration of IMI (3-30 mg/kg) and and a methanolic (B: 80%; V/V, 4.5% hyperforin) extract were FLU (3-30 mg/kg) [De Vry 1999]. evaluated and compared to FLU (1 mg/kg p.o.) in male Wistar rats in the FST and an open field test over 28 days. The rats Daily administration for 3 days of a 50% ethanolic extract, at 100 were given an oral daily dose of either one of four different and 200 mg/kg p.o. to CF rats and Wistar mice, demonstrated doses of extracts A (1.78, 3.57, 7.14, and 14.28 mg/kg) or B comparable effects to acute administration of IMI at 15 mg/kg (3.21, 6.43, 12.86, and 27.71 mg/kg), FLU or vehicle. The tests i.p. in various animal models of depression [Kumar 1999]. were carried out before treatment commenced and on days 7, 14, 21 and 28 of treatment. After 21 and 28 days of treatment, An extract (0.3% hypericin, 3.8% hyperforin) was administered both extracts significantly (p<0.05) increased the latency to the intragastrically to male msP rats at 3 doses of 250 mg/kg. The first immobility and decreased the total immobility time in a anti-immobility effect of the extract in the FST was completely dose-dependent manner. FLU significantly (p<0.05) increased suppressed by i.p. pre-treatment with the s-receptor antagonist the latency to first immobility and total immobility time. FLU . Intracerebroventricular pre-treatment with exhibited antidepressant activity after 14 days, whereas with 5,7-dihydroxytryptamine, which produced a marked depletion both extracts 21 days were required [Lozano-Hernandez 2010]. of brain 5-HT, also reduced the anti-immobility effect [Panocka 2000]. Adult male CF1 mice were treated with hyperoside (10, 20 or 40 mg/kg i.p.), IMI (20 mg/kg i.p.), or vehicle 30 min before the After acute oral administration to rats at 250-500 mg/kg, an FST. Compared to vehicle, a significant (p<0.001) antidepressant- unspecified extract enriched in flavonoids (0.3% hypericin, like effect was observed for hyperoside at doses of 10 and 20 4.5% hyperforin and 50% flavonoids) significantly and dose- mg/kg, comparable to IMI. In male Wistar rats, hyperoside dependently decreased immobility time (p<0.001 compared to (1.8 mg/kg/day p.o.) and IMI both showed a significantly (both saline). Concomitant treatment with the extract (500 mg/kg) and p<0.01) decreased immobility time. The antidepressant-like sulpiride (a DA receptor antagonist), metergoline (a serotonin effect in rats was prevented by the D2 antagonist sulpiride receptor antagonist) or 6-hydroxydopamine (which destroys (50 mg/kg i.p.) [Haas 2011; Schulte Haas 2011]. NE-containing neurons) significantly increased the period of immobility by 22-57%, the largest effect was observed with An unspecified extract at an oral dose of 25 mg/kg was compared metergoline (57%; p<0.001) [Calapai 2001]. to FLU (10 mg/kg i.p.) in male albino rats. Both treatments showed a significant (p<0.05) antidepressant effect in all behavioural Several ethanolic (60%) and methanolic (80%) extracts demon- studies and also significantly increased brain neurotransmitter strated a significant (p<0.01) increase in activity in male Sprague- levels, especially that of 5-HT. The effects of the extract were Dawley rats in the FST, with the exception of one methanolic comparable to FLU [Kulkarni 2012]. extract containing a reduced level of rutin. Addition of rutin to this extract produced a pharmacological effect comparable to The effects of a standardised extract (not further specified) the other extracts [Nöldner 2002]. at doses of 30-90 mg/kg i.p. were compared with standard

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antidepressants in the FST when given 1 h before the test in representing inhibitors of serotonin or NE synthesis respectively, male rats. The extract caused a dose-dependent reduction in prevented the effect of rutin in the TST [Machado 2008]. immobility time with the maximal effect of 53% at 90 mg/kg (p<0.001). This effect was reversed at higher doses (100 mg/kg) Hot plate showing a U-shaped dose response curve. FLU and IMI (30 No activity of hyperoside (10, 20 and 40 mg/kg i.p.) was found -70 mg/kg i.p.) produced a similar reduction in the immobility on the hot-plate in male Swiss mice [Schulte Haas 2011]. time in rats, whereas VEN exhibited only a 34% reduction [Bukhari 2013]. Open field test (Locomotor activity) Oral application for 3 days of a methanolic extract (80%, Adhyperforin at an oral dose of 16 mg/kg significantly (p<0.05) V/V; 500 mg/kg/day) or of hypericin (1.5 mg/kg/day) had no decreased the immobility time of male Swiss mice [Tian 2014]. impact on the spontaneous motility of male and female CD rats [Winterhoff 1995]. Escape deficit test (learned helplessness)

Male Charles Foster rats received either a CO2 extract (no The effect of an ethanolic (50%) extract on locomotor activity hypericin, no , 38.8% hyperforin) or an ethanolic in the rota-rod test was assessed. Male albino mice received extract (0.3% hypericin, 4.5% hyperforin) before the inescapable i.p. either the extract (250 mg/kg), DMI (30 mg/kg) or TRI (20 electrical shock test. Control animals, trained to the inescapable mg/kg). The extract demonstrated a significant decrease in the shock situation, exhibited marked increases in escape failures walking time of mice on the rotating rod (p<0.05). While DMI under escapable conditions (72.5%), as compared to animals not and TRI caused a more significant decrease (p<0.005) [Öztürk previously trained to helplessness. Dose-dependent decreases 1997; Öztürk 1996]. of escape failures were observed for both extracts [Bhattacharya 1998; Chatterjee 1999]. Oral doses of pseudohypericin (166 µg/kg) and hypericin (9 -28 µg/kg) were administered to male CD rats concomitantly Sprague-Dawley rats received 250, 500, 1000 or 1500 mg/kg with a fraction of flavonoids. The number of line crossings in of a hydroethanolic extract (0.3% hypericin) p.o., 60 minutes comparison to the control group were not increased, suggesting before an inescapable stress test. A significant dose-dependent that reported positive results in the FST (see above) were not reduction in escape deficit 24 h later was found for the 1000 mg related to stimulation of locomotor activity [Butterweck 1998]. (p<0.05) and 1500 mg (p<0.01) doses as compared to control. A tail-flick test and hot plate test confirmed that the result was not Hyperoside, miquelianin and isoquercitrin at an oral dose of caused by a non-specific analgesic effect. Co-administration of 0.6 mg/kg did not change locomotor activity in male CD rats the DA D1 receptor antagonist SCH 23390 or the 5-HT1A- and [Butterweck 2000]. b-AR antagonist pindolol significantly reduced the efficacy of the extract. After pre-treatment of rats with daily doses of 500, In contrast, a lyophilised aqueous infusion administered i.p. 1000 or 2000 mg/kg p.o. for 15 days or IMI (5 mg/kg, i.p., twice to male Charles River mice at doses of 25, 50, and 100 mg/kg daily), the extract at 1000 mg showed significantly (p<0.05) significantly diminished the number of crossings and rearings greater activity (increased escapes) than control, and the 2000 (p<0.01), whereas immobility times were raised (p<0.05) mg dose had a significant effect comparable with IMI (p<0.01) [Coleta 2001]. [Gambarana 1999]. A methanolic extract (80%, V/V; 500 mg/kg) or hypericin Tail suspension test (TST) (0.1 mg/kg p.o.), administered alone or in combination with

A methanolic (80%, V/V) extract at an oral dose of 500 mg/kg procyanidin B2, did not induce changes in locomotor activity caused a significant decrease in immobilisation time of NMRI of male CD rats [Butterweck 2001a]. mice. The effect was completely abolished by D,L butyro- lactone (an inhibitor of neuron function), and by An unspecified methanolic extract was tested at doses from 2.5 a-methyltyrosine (an inhibitor of DA and adrenaline synthesis) to 200 mg i.p. in adult male CD1 mice. The extract increased [Butterweck 1997]. motor activity at 10 mg/kg i.p. and decreased it at the highest dose [Diana 2007]. Three different extracts were studied at an oral dose of 500 mg/kg in female NMRI mice: (A) an ethanolic (60%) extract The effects of an ethanolic (50%) extract were tested on -

(3.2% hyperforin, 0.15% of hypericin); (B) a CO2 extract (no induced locomotor activity in male Swiss-Webster mice. Caffeine hyperforin, 0.14% hypericin) and (C) a sub-fraction of extract (4 - 16 mg/kg i.p.) produced significant (p<0.0001) increases in (B) (no hyperforin or hypericin, approximately 12% flavonoids). locomotor activity. The extract (6 - 24 mg/kg i.p.) significantly IMI (30 mg/kg p.o.) was used as control. Extracts B and C reduced (p=0.016) blocked the caffeine-induced locomotor hyperactivity. immobility time significantly (p<0.05) and were comparable to Pre-treatment with L arginine (1 g/kg) reversed the inhibitory IMI, whereas extract A was less active. Hyperforin produced a effect of the extract (6 mg/kg i.p.) without any significant effect U-shaped dose-response curve: below 4 and above 8 mg/kg when administered alone [Uzbay 2007]. hyperforin remained inactive [Butterweck 2003a]. The effects of an ethanolic (50%) extract on behavioural changes The effects of hyperoside at single oral doses of 7, 35 and 70 were assessed in streptozotocin (STZ)-diabetic Wistar rats. In mg/kg were investigated in the TST in adult male CF1 mice. horizontal and vertical spontaneous locomotor activities, a With the increasing doses, the antidepressant effect increased diabetes mellitus-induced decrease was observed which was by 12.7%, 16.5% and 24.5% compared to controls [Bach- not restored by insulin (2 IU/kg/day). Treatment with the extract Rojecky 2004]. (125 and 250 mg/kg) for seven days resulted in a significant (p<0.05 and p<0.01) improvement [Can 2011b]. The administration of rutin (0.3 - 3 mg/kg, p.o.) reduced the immobility time of male Swiss mice in the TST without Adult male CF1 mice treated with single doses of hyperoside a change in locomotor activity. Pre-treatment of mice with at 20 and 40 mg/kg i.p. showed reduced (p<0.001) exploratory p-chlorophenylalanine methyl ester (100 mg/kg, i.p., for 4 behaviour in the open field test compared to controls [Schulte consecutive days) or alpha-methyl-p-tyrosine (100 mg/kg, i.p.), Haas 2011].

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The effects of a standardised extract (details unknown) were investigated in Wistar rats: group 1 was exposed to HRS, group compared with antidepressants in the open field test at doses 2 was the untreated control, groups 3-5 received the extract (25, of 1-30 mg/kg i.p. in rats. The extract caused a dose-dependent 50 and 100 mg/kg/day p.o.), and group 6 received ranitidine (50 reduction in locomotor counts with a maximal effect of 80% at mg/kg s.c.) as the positive control. Macroscopic analysis of the 30 mg/kg (p<0.001) compared to vehicle. FLU (30-70 mg/kg stomach lesions showed that treatment with the extract at each i.p.), dothiepin (30 mg/kg i.p.) and VEN (50 mg/kg i.p.) produced dose significantly reduced lesions compared to stressed controls similar reductions in the number of locomoter counts (89%, by 65, 95 and 75% respectively (p=0.001). Treatment with p<0.001; 70%, p<0.001 and 62%, p<0.001, respectively) ranitidine also improved ulcers significantly. Histopathologic [Bukhari 2013]. analyses indicated that 50 mg/kg/day of the extract produced the most significant effect [Cayci 2009]. Elevated plus maze

A CO2 extract (no hypericin, 38.8% hyperforin) and an ethanolic In a restraint stress model, male Albino mice were immobilised extract (0.3% hypericin, 4.5% hyperforin) were tested in Wistar for a period of 6 h resulting in severe anxiety like behaviour, mice in the elevated plus maze (EPM). Both extracts at the antinociception and impaired locomotor activity as compared highest dose (300 mg/kg) significantly (p<0.05) reduced the to unstressed animals (p<0.05). An unspecified extract was time spent in the open arm [Bhattacharya 1998]. administered at doses of 50 and 100 mg/kg i.p. for five days 30 min before acute immobilised stress. Various behavioural Lyophilised aqueous extracts at i.p. doses of 25, 50 and 100 mg/kg parameters for anxiety, locomotor activity and nociceptive were tested in the elevated plus maze in male Charles River mice. threshold were assessed, followed by biochemical assessments The extract significantly reduced (p<0.01) the total number of (malondialdehyde (MDA) level, GSH, catalase activity (CAT) entries and the number of closed entries [Coleta 2001]. and nitrite). Biochemical analyses revealed an increase in MDA and nitrites concentration, and depletion of reduced GSH and A diabetes mellitus-induced decrease in exploratory CAT, as compared to unstressed animal brain. Treatment with behaviour, indicating increased anxiety in STZ-diabetic Wistar the extract significantly (all p<0.05) attenuated restraint stress- rats, was not restored by daily insulin (2 IU/kg/day i.p.), but induced behavioural changes (improved locomotor activity, was significantly (p<0.001) improved by treatment with an reduced tail flick latency and effect) and oxidative ethanolic (50%) extract (125 and 250 mg/kg, i.p.) for seven damage as compared to control [Kumar 2010]. days [Can 2011a]. An ethanolic (80%) extract was studied at doses of 125 to Other pharmacological effects 750 mg/kg p.o. in a chronic restraint stress model. Stressed rats showed decreased time in open field activity compared Stress response to unstressed rats; an effect reversable by the extract and FLU A 14-day mild, unpredictable and inescapable foot shock (10 mg/kg p.o.). Chronic restraint stress significantly decreased stress (FSS) test induced depression, suppressed male sexual thymus and spleen indices (p<0.05). The extract produced a behaviour, cognitive dysfunction, marked gastric ulceration and significant and dose-dependent increase in both thymus and a significant increase in adrenal gland weight with concomitant spleen indices compared to the stressed controls (p<0.05), as decrease in spleen weight in albino rats. All these FSS-induced did FLU. Both treatments significantly reduced stress-induced perturbations were attenuated dose-dependently by the extract increases in plasma ACTH and levels (p<0.05 to at 100 and 200 mg/kg p.o. [Kumar 2001b]. p<0.01) as well as TNF-a levels (p<0.05) [Grundmann 2010].

Administration of a dried St. John´s wort preparation (350 mg/ Post-stress grooming kg/day p.o.) for 21 days significantly enhanced recall of passive Post swim grooming behaviour of Wistar mice was signific- avoidance behaviour, but had no effect on the acquisition of antly (p<0.01) increased by IMI (10 mg/kg i.p.), and dose- conditioned avoidance responses in chronically (2 h daily for dependently and significantly (p<0.05 to <0.001) increased 21 days) stressed rats. Impaired recall of passive avoidance by oral administration of an ethanolic extract (0.3% hypericin, behaviour following chronic administration of corticosterone 4.5% hyperforin; 50-300 mg/kg/day), whereas a CO2 extract (no (5 mg/kg/day for 21 days) was also reversed [Trofimiuk 2006]. hypericin, 38.8% hyperforin; 5-30 mg/kg/day) had a significant effect only at the highest dose (p<0.05) [Bhattacharya 1998]. Male laca mice were sleep deprived for 72 hours using the grid suspended over water method. An ethanolic extract (60%; Influence on body temperature 0.3% hypericin, 4.1% hyperforin, 3.3% rutin, 1.8% hyperoside, The decrease in body temperature after i.p. application of 1.0% isoquercitrin, 1% quercetin, 0.3% bioapigenin) or IMI reserpine to female NMRI mice was counteracted by oral were administered for five days, starting two days before sleep administration of a hydroalcoholic extract (0.015% hypericin) deprivation. Sleep deprivation significantly altered body weight [Winterhoff 1993]. and locomotor activity, and produced anxiety-like behaviour and oxidative damage (depleted reduced GSH and CAT activity; Oral treatment of male Charles Foster rats with either a CO2 (no increased lipid peroxidation and nitrite activity) as compared hypericin, 38.8% hyperforin) or an ethanolic (0.3% hypericin, to naive animals placed on sawdust (p<0.05). Treatment with 4.5% hyperforin) extract did not induce changes in body the extract (200 and 400 mg/kg p.o.) or with IMI (10 mg/kg temperature, except for a slight increase at the highest dose of i.p.) significantly improved body weight, locomotor activity, 300 mg/kg/day. Reserpine-induced hypothermia was completely antianxiety and antioxidant effects as compared to the sleep antagonised by 15 mg/kg/day of the CO2 extract and 300 mg/ deprived control (p<0.05). Co-administration of the extract kg/day of the ethanolic extract. The activity of both extracts in (200 mg/kg p.o.) with IMI (10 mg/kg i.p.) further improved reserpine-treated animals was similar to IMI (10 mg/kg i.p.) body weight, locomotor activity and the antianxiety effect, as [Bhattacharya 1998]. well as reducing oxidative damage in sleep-deprived animals as compared to their effect per se (p<0.05) [Kumar 2007]. A methanolic (80%; V/V) extract was administered orally to female NMRI mice (500 mg/kg). One hour after administration The effect of a methanolic (80%) extract on gastric mucosal basal body temperature was significantly elevated (p<0.005) damage induced by hypothermic restraint stress (HRS) was [Winterhoff 1995].

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Exposure of male BL6/C57J mice to open field stress significantly antagonist WAY-100635. The effects were less pronounced after increased body temperature by 1.8 ± 0.13°C (p<0.05). Oral sub-chronic treatment with the extract (20 and 100 mg/kg/day administration of an ethanolic extract (80%; V/V; 0.3% for two weeks). Acute treatment with the extract and hyperforin hypericin, 4% hyperforin and 9.4% flavonoids) significantly significantly decreased plasma prolactin (PRL), whereas sub- reduced the body temperature increase at doses of 250 and chronic treatment with the extract at 100 mg/kg/day significantly 500 mg/kg (p<0.01). Hypericin (0.1 mg/kg p.o.) administered (p<0.01) increased PRL [Franklin 2000; Franklin 2001]. 60 min prior to testing significantly reduced the increase (p<0.001), whereas hyperforin (1 10 mg/kg p.o.) had no effect. Oral treatment of male Sprague-Dawley rats for 2 weeks with Hyperoside, isoquercitrin and quercitrin (all at 0.6 mg/kg p.o.) a methanolic (80%, V/V) extract (in a diet containing 3 g/kg) and rutin (1 mg/kg p.o.) only partially blocked open field-induced resulted in significantly reduced corticosterone (p<0.002) hyperthermia (p<0.05). Miquelianin (1.2 mg/kg p.o., p<0.05) and PRL (p<0.04) responses to the post-synaptic 5-HT2A and amentoflavone (0.1 mg/kg p.o., p<0.001) also significantly receptor agonist 2,5-dimethoxy-4-iodophenyl-2-aminopropane decreased the stress-induced hyperthermia. Diazepam (5 mg/ [Franklin 2003]. kg p.o; p<0.001) and the 5-HT1A receptor agonist buspirone (10 mg/kg p.o.; p<0.01) significantly reduced the temperature Brain concentrations of corticosterone and cortisol (COR) were increase, whereas IMI (10 mg/kg p.o.) and fluoxetine (10 mg/kg significantly reduced in rats receiving an oral dose of 3 g/kg of p.o.) had no effect [Grundmann 2006]. a methanolic (80%, V/V) extract for 2 weeks, whereas serum concentrations remained unaffected [Franklin 2004]. Influence on sleeping time Oral pre-treatment of female NMRI mice with 500 mg/kg of an Using in situ hybridisation histochemistry, the effects of short- ethanolic extract (containing 0.015% hypericin), 2 h prior to term (2 weeks) and long-term (8 weeks) oral administration of injection of , significantly (p<0.05) reduced sleeping a CO2 extract (containing about 23.7% hyperforin and 6.2% time [Winterhoff 1993]. adhyperforin) and hyperforin-trimethoxybenzoate were studied in male CD rats. FLU and haloperidol were used as positive and Oral administration of a methanolic extract (80%, V/V; 50 to negative controls respectively, to assess the expression of genes 1500 mg/kg) demonstrated dose-dependent activity in the same involved in the regulation of the hypothalamic-pituitary-adrenal model, with fully expressed activity at 500 mg/kg (p<0.001). (HPA). FLU (10 mg/kg p.o.) daily for 8 weeks, but not 2 weeks, Dose reduction to 200 mg/kg led to a less pronounced reduction significantly (p<0.05) decreased levels of CRH mRNA by 22% in sleeping time (p<0.05), while the effect with 50 mg was not in the paraventricular nucleus of the hypothalamus and tyrosine significant. IMI (20 mg/kg) led to a prolongation of sleeping hydroxylase (TH) mRNA by 23% in the locus coeruleus. FLU time, whereas (20 mg/kg), a dopamine re-uptake increased (p<0.05) levels of mineralocorticoid (MR) (17%), inhibitor, had the same effect as the extract. Hypericin, at glucocorticoid (GR) (18%) and 5-HT1A receptor (21%) mRNAs doses equivalent to that found in the tested extract, exhibited in the hippocampus at 8, but not 2, weeks. Comparable to less reduction in sleep duration when compared to the extract haloperidol (1 mg/kg p.o.), neither the hyperforin-rich CO2 [Winterhoff 1995]. extract (27 mg/kg p.o.) nor hyperforin-trimethoxybenzoate (8 mg/kg p.o.) altered mRNA levels in brain structures relevant Effects on EEG for HPA axis control at either time point [Butterweck 2003c]. Power changes in the frontal cortex and hippocampus of male Fisher-344 rats were detected after oral administration of an An ethanolic extract (DER 3.6:1; containing 0.13% hypericin ethanolic (50%, m/m) extract at doses of 0.5 and 1 mg. Frequencies and pseudohypericin, 3.03% hyperforin) was given orally at in these brain areas changed predominantly in the direction of 20 to 240 mg/kg to male Sprague-Dawley rats over 8 weeks. effects known from antidepressant drugs [Dimpfel 1997]. Chronic IMI treatment (5 mg/kg/day i.p. for 8 weeks) significantly (p<0.05 both) down-regulated circulating plasma levels of Continuous EEG was recorded in male rats after oral application adrenocorticotropic hormone (ACTH) and corticosterone of a CO2 extract (30.1% hyperforin, practically no naphtho- compared to controls. The extract had no effect on plasma ACTH dianthrones and flavonoids) and a methanolic extract (80%, V/V; or corticosterone, even at the highest doses tested [Frost 2003]. 4.7% hyperforin). The dosage was adjusted for the application of identical amounts of hyperforin. Both extracts produced Hypericin (0.2 mg/kg), hyperoside (0.6 mg/kg), isoquercitrin nearly identical patterns of electrical power changes during the (0.6 mg/kg), miquelianin (0.6 mg/kg) and IMI (15 mg/kg), were first two hours. The changes mainly consisted of reproducible administered daily to male CD rats by gavage for two weeks. power increases within the a1 band of the striatum, similar to The treatments significantly down-regulated circulating plasma the reaction following the application of SSRIs. The methanolic levels of ACTH (p<0.05) and corticosterone (p<0.05 to p<0.01) extract developed a late action (increased d-activity) not seen by 40 - 70%. However, none of the compounds had an effect on with the CO2 extract, which matched data obtained with plasma ACTH and corticosterone levels after chronic treatment NMDA-antagonists like MK 801 or [Dimpfel 1998]. over 8 weeks [Butterweck 2004].

Effects on neurotransmitters and hormones In a study on the expression of genes involved in the regulation Three weeks of oral treatment with 125 mg/kg/day of a of the hypothalamic-pituitary-adrenal axis, short (2 weeks) and methanolic (80%, V/V) extract significantly reduced basal long term (8 weeks) oral administration to male CD rats of a prolactin (p<0.05) and corticosterone levels (p<0.01) in male methanolic extract (80%, V/V; 500 mg/kg), hypericin 0.2 mg/ CD rats [Winterhoff 1995]. kg) and IMI (15 mg/kg) resulted in decreased levels of mRNA for CRH (p<0.01) by 16 - 22% in the paraventricular nucleus,

The effects of a methanolic extract (80%, V/V; 200 mg/kg i.p.), and mRNA for 5-HT1A-receptor (p<0.05 to p<0.01) by 11 - 17% hypericin (0.4 mg/kg i.p.) and hyperforin (9.3 mg/kg i.p.) on 5-HT in the hippocampus. Only IMI decreased tyrosine hydroxylase and DA-mediated neuroendocrine responses were measured mRNA levels in the locus coeruleus by 23% (p<0.01) after 8 in male Sprague-Dawley rats. Plasma corticosterone was weeks [Butterweck 2001b]. significantly (p<0.05 to p<0.001) and equivalently increased in all three treatment groups. The effects were blocked by the 5-HT2 An ethanolic extract (0.3% hypericin, 4.5% hyperforin), but receptor antagonist ketanserin, but not by the 5-HT1A receptor not a CO2 extract (no hypericin, 38.8% hyperforin), dose-

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dependently and significantly (p<0.001) potentiated L-DOPA- with sodium nitrite. The extract at both doses and piracetam induced changes in behaviour of Wistar mice. Treatment with facilitated the acquisition and retention of AA and attenuated apomorphine (0.5 mg/kg i.p.) led to stereotypical behavioural the scopolamine and sodium nitrite-induced impaired retention changes which were increased by IMI (10 mg/kg i.p.) (p<0.01). of AA [Kumar 2000]. The ethanolic extract, and to a lesser extent the highest dose of the CO2 extract, significantly (p<0.001 and p<0.05 respectively) The effect of an ethanolic (80%, V/V) extract was tested on mem- increased the apomorphine stereotypy score [Bhattacharya 1998]. ory retrieval 24 h after training on a one-trial passive avoidance task in male Swiss albino mice. Acute i.p. administration at doses The effects of a methanolic extract (80%, V/V; 4.67% hyperforin; of 4.0, 8.0, 12.0, and 25.0 mg/kg before retrieval testing increased

31.25, 62.5 and 125 mg/kg/day) and a CO2 extract (30.14% (p<0.01) the step-down latency during the test session. The hyperforin; 2.42, 4.84 and 9.68 mg/kg/day) were investigated same doses of the extract failed to reverse scopolamine-induced in male CD rats as single oral doses acutely and over 14 days. amnesia of a two-trial passive avoidance task. Pre-treatment

IMI (20 mg/kg/day p.o.) and vehicle served as controls. Both of the animals with serotonergic 5-HT1A receptor antagonist extracts increased DA and 5-HT levels, as measured by in vivo (-)-pindolol (0.3, 1.0, and 3.0 mg/kg s.c.), serotonergic 5-HT2A microdialysis. The dose-response curve followed an inverse receptor blocker spiperone (0.01, 0.03, and 0.1 mg/kg i.p.), U-shape. Repeated administration caused a rapid tolerance of a-AR antagonist phentolamine (1, 5, and 10 mg/kg i.p.), b-AR DA but not of 5-HT neurons. Similar changes were observed antagonist propranolol (5, 7.5, and 10 mg/kg i.p.), dopaminergic after acute and repeated administrations of IMI. The potency D1 receptor antagonist SCH 23390 (0.01, 0.05, and 0.1 mg/kg of hyperforin surpassed that of imipramine in the acute release i.p.), and dopaminergic D2 receptor antagonist sulpiride of both DA and 5-HT by the nucleus accumbens. The effect (5, 7.5, and 10 mg/kg i.p.) revealed the involvement of adrenergic of hyperforin correlated with its inhibiting potency on high- and serotonergic 5-HT1A receptors in the facilitatory effect of affinity DA and 5-HT uptake. The inhibition of neuronal GABA the extract on retrieval memory [Khalifa 2001]. transport occurred at higher concentrations of hyperforin [Rommelspacher 2001]. The effects of a methanolic (80%, V/V) extract and hyperforin sodium salt were evaluated in rat and mouse avoidance tests. In The effect of hyperforin on striatal ACh release and associated a conditioned rat avoidance response, 7 days oral administration choline levels was studied using microdialysis in adult Sprague- of the extract (50 mg/kg/day) and hyperforin (1.25 mg/kg/day) Dawley rats. Local infusion of hyperforin (10 μM and 100 μM) improved learning ability from the second day until day 7. via the dialysis probe caused changes in brain ACh and choline The memory of the learned responses acquired during the 7 levels. Systemic administration of hyperforin (1-10 mg/kg i.p.) consecutive days of administration and training was largely led to therapeutic plasma levels of hyperforin and caused a retained after 9 days without further treatment or training. A single significant elevation of striatal ACh release. The higher hyperforin dose of the extract at 25 mg/kg did not reveal any significant dose (10 mg/kg) revealed a reduction of locomotor activity in effects in the passive avoidance response test in mice, whereas CD1 mice [Buchholzer 2002]. a single oral dose (1.25 mg/kg) of hyperforin almost completely reversed scopolamine-induced amnesia [Klusa 2001]. Male rats were treated orally with two different doses (30 and 100 mg/kg) of an unspecified extract for 3 or 15 days. A 3-day The Morris water maze (MWM) was used to test the effects of treatment was not able to modify PRL serum levels, whereas a St. John's wort tablets containing dried crude herb standardised 15-day treatment with the extract at the higher dose significantly to 0.3% hypericin, administered orally for 9 weeks at doses (p<0.01) inhibited PRL production [Di Carlo 2005]. equivalent to 4.3 and 13 μg/kg hypericin to male Wistar rats. In the acquisition phase (days 1 to 4) latencies were significantly The effects of chronic treatment with intragastric injections of a (p<0.001) reduced due to training. The learning effect was greater methanolic (80% V/V) extract on neurochemical markers of 5-HT, (p<0.05) in the high dose group than in the low dose group or DA and opioid systems in mesolimbic regions of fawn-hooded control. In the probe trial on day 5, the high dose group crossed male FH rats were investigated by quantitative autoradiography. the correct annulus in the target quadrant significantly (p<0.05) After 10 days' treatment, the extract increased [3H]- more often than the other groups. After completion of the binding to 5-HT transporters in multiple mesolimbic regions. experiment, the concentrations of monoamines and metabolites In contrast, the extract resulted in a region-specific alteration were determined in selected brain regions (prefrontal cortex, of [3H]-mazindol binding to DA transporters, such as increased hippocampus and hypothalamus) demonstrating significant binding of [3H]-mazindol in the olfactory tubercle and decreased (p<0.05 to p<0.001) differences in their contents between the binding in the ventral tegmental area. In addition, the extract treatment groups and control [Widy-Tyszkiewicz 2002]. resulted in differential modulation of the binding properties of

5-HT1A-, 5-HT2A- and µ-opioid receptors in a region-specific Hippocampal acetylcholine levels were increased (p<0.01 to manner [Chen 2003]. p<0.001) by 50-100% in adult Sprague-Dawley rats after i.p. administration of hyperforin at doses of 1 and 10 mg/kg. The Cognitive effects effect was almost completely suppressed by local perfusion with The effect of a 50% ethanolic extract was investigated in various calcium-free buffer or with tetrodotoxin (1 µM) [Kiewert 2004]. experimental models of learning and memory, viz. transfer latency (TL) on elevated plus-maze, passive avoidance (PA), The potential for prevention of working memory impairments active avoidance (AA), and scopolamine- and sodium nitrite- was tested in male Wistar rats orally administered 350 mg/kg of induced amnesia (SIA & NIA) in albino rats. The extract was an extract (solvent not specified; 0.05-0.3% hypericin, 2-4.5% administered orally (100 and 200 mg/kg) for three days, while hyperforin, 2-4% flavonoids and 8-12% procyanidins) for 21 piracetam (500 mg/kg i.p.), a nootropic agent, was admin- days. Spatial working memory impairments caused by chronic istered acutely. The extract and piracetam significantly (p<0.01) restraint stress or medication with exogenous corticosterone shortened TL on days 2 and 9, and antagonised the amnesic in the Barnes maze and MWM were prevented (p<0.001). The effects of scopolamine and sodium nitrite on TL. The extract treatment significantly improved hippocampus-dependent had no significant effect on the retention of PA. The higher dose spatial working memory in comparison with control (p<0.01) produced a significant (p<0.05) reversal of scopolamine-induced and alleviated other negative effects of stress on cognitive PA retention deficit but no significant reversal was observed functions [Trofimiuk 2008; Trofimiuk 2005].

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Middle-aged male Wistar rats (18 months) displayed a decline was administered orally at doses of 150 and 300 mg/kg acutely, in the acquisition of spatial working memory in the MWM. subchronically (7 days) and chronically (21 days) to adult male Chronic oral administration of the same extract (350 mg/kg for Swiss albino mice. PAR at 5 mg/kg was used as a positive control. 21 days) significantly (p<0.001) improved processing of spatial Anxiolytic-like and antipanic-like effects were assessed with the information and increased the levels of phosphorylated cAMP mouse defence test battery. On day 21 both treatments showed response element binding protein in the hippocampus (p<0.01). reduced flight reactions to the presence of the predator. After Locomotor impairments caused by ageing as tested in the OFT 21 days PAR increased the number of approaches/withdrawals remained unaffected [Trofimiuk 2010]. and reduced the number of upright postures, while the extract only reduced the number of upright postures [Beijamini 2003]. An ethanolic (50%) extract significantly improved glucose metabolism in STZ-induced diabetic Wistar rats when given at A hydroalcoholic extract (not further specified) was tested for i.p. doses of 125 and 250 mg/kg over one week and significantly the treatment of co-morbid mood disturbances and anxiety in improved learning parameters of animals in a shuttle-box test diabetic rats. Type 2 diabetes mellitus was induced by a single (p<0.01 to p<0.001) compared to controls [Can 2011a]. i.p. injection of STZ (65 mg/kg), 15 min after an i.p. injection of (120 mg/kg). The extract was administered orally The effect of an aqueous dry extract (3% (m/m) hyperforin and (100 and 200 mg/kg) to diabetic animals for 14 days. Anxiolytic > 20% (m/m) flavonoids) on diabetes-induced learning and activity was evaluated using the open-field exploration test memory impairment was assessed in streptozotocin-induced (OFT) and elevated plus maze (EPM) test. Diabetic rats showed diabetic rats. After oral administration of 6, 12 and 25 mg/kg a significant increase in anxiety in OFT (p<0.05) and EPM for 30 days, passive avoidance learning and memory were (p<0.05) compared to non-diabetic control rats. Treatment with significantly (p<0.05 to p<0.001) improved by the two highest the extract significantly (p<0.001) improved anxiety symptoms doses [Hasanein 2011]. in the OFT and (p<0.001) reduced elevated blood glucose levels in diabetic rats [Husain 2011a]. The potential for alleviation of stress- and corticosterone-related memory impairments by restoring levels of the synaptic plasticity Neuroprotection proteins, neuromoduline (GAP-43) and synaptophysin (SYP), in In male Sprague-Dawley rats hyperforin (6 µM) injected in the hippocampus and prefrontal cortex was tested in male Wistar rats hippocampus bilaterally (i) decreased Ab deposit formation after which displayed a decline in the acquisition of spatial working injection of amyloid fibrils in the hippocampus, (ii) decreased memory in the Barnes maze. Chronic oral administration of a the neuropathological changes and behavioural impairments dried St. John’s wort preparation (350 mg/kg for 21 days) potently in a model of amyloidosis, and (iii) prevented Ab-induced improved processing of spatial information in stressed and neurotoxicity in hippocampal neurons both from amyloid fibrils corticosterone-treated rats (p<0.001). Treatment significantly and Ab oligomers, avoiding the increase in ROS associated with increased levels of GAP-43 and SYP in hippocampus and amyloid toxicity. The effects were explained by the capacity prefrontal cortex (p<0.05) and prevented the deleterious effects of hyperforin to disaggregate amyloid deposits in a dose and of both chronic restraint stress and prolonged corticosterone time-dependent manner and to decrease Ab aggregation and administration on working memory. It significantly (p<0.01) amyloid formation [Dinamarca 2006]. improved hippocampus-dependent spatial working memory in comparison with control and alleviated other negative effects The effect of an unspecified extract (0.34% hypericin, 4.1% of stress on cognitive functions [Trofimiuk 2011]. hyperforin, 5% flavonoids, 10% ) given at an oral dose of 30 mg/kg was evaluated in an experimental model of spinal The effect of an ethanolic extract (80%; 250 and 500 mg/kg p.o.) cord injury in adult CD1 mice. The degree of (a) spinal cord and FLU (10 mg/kg p.o.) on genes involved in the pathogenesis inflammation and tissue injury (histological score), (b) nitro- of depression were studied using a chronic restraint stress tyrosine, (c) poly(adenosine diphosphate-ribose), (d) neutrophils model in rats. Hypothalamic and hippocampal tissues were infiltration, and (e) the activation of signal transducer and analysed using the Affymetrix gene chip Rat Genome 230 activator transcription 3 were markedly reduced in spinal cord 2.0 Array. Genes involved in the pathways of inflammatory tissue [Genovese 2006b]. processes (Mapk8), oxidative stress (Gpx3, Gstm3, Sod3) and Alzheimer's disease (Sncb, Apbb1ip) were altered by both FLU Wistar rats (18 months old) displayed a decline in the acquisition and the extract [Jungke 2011]. of spatial working memory (p<0.001) in the MWM. Admin- istration of a dried St. John´s wort preparation (0.3 % hypericins, Anxiety 6% hyperforin, 2-4% flavonoids and 8-12% procyanidins), at an A methanolic (80%, V/V) extract was given to male Wistar rats oral dose of 350 mg/kg for 21 days, significantly improved the at doses of 62.5-500 mg/kg p.o., either acutely or as chronic processing of spatial information (p<0.001). The preparation also treatment for 14 days. The animals were tested in the elevated increased pCREB in the hippocampus (p<0.01). Aging caused T-maze (ETM), the light/dark transition, and the cat odour significant locomotor impairments in the OFT (p<0.001) but no test. The results showed that acute treatment (125 mg/kg) changes in the MWM test; factors unaffected by the treatment impaired ETM inhibitory avoidance without altering escape [Trofimiuk 2010]. performance. Chronic treatment (62.5-250 mg/kg) enhanced avoidance latencies only in animals pre-exposed to the open Neurogenesis arms of the maze, with pre-exposure shortening escape latency. A methanolic extract was administered at an i.p. dose of 30 In contrast to IMI (15 mg/kg p.o.), chronic treatment with the mg/kg/day for 3 weeks to adult CD1 mice with corticosterone- extract did not impair escape from the open arms of the maze. induced stress. Treatment with the extract reversed the decrease Similarly to IMI, the extract increased the number of transitions in hippocampal progenitor cell proliferation induced by between the two compartments in the light/dark transition corticosterone. Stressed mice also exhibited a reduced dendritic model. Treatment regimens with the extract and IMI did not spine density that was significantly (p<0.05) ameliorated by the alter behavioural responses of rats to a cloth impregnated with extract [Crupi 2011]. cat odour [Flausino 2002]. Alzheimer’s disease A methanolic extract (80%, V/V; 0.3% hypericin, 3.3% hyperforin) Quercetin and kaempferol stimulated depression-related

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signalling pathways involving brain-derived neurotrophic factor repressed the epileptic manifestations in 40% of animals, (BDNF), phosphorylation of cAMP response element binding whereas lipid-soluble constituents in the ether fraction potenti- protein CREB and postsynaptic density proteins PSD95, and ated the epileptic activity [Ivetic 2002]. reduced Ab peptide in neurons isolated from double transgenic AD mice (TgAPPswe/PS1e9). Administration of the flavonols The anticonvulsant activity of undefined aqueous and ethanolic enhanced BDNF expression and reduced Ab oligomers in extracts was studied in mice in the pentylenetetrazole (PTZ) hippocampus of the mice; this correlated with cognitive and the maximal electroshock seizures (MES) tests. In the PTZ improvement [Hou 2010]. test, the extracts (0.1-1 g/kg i.p.) delayed the onset of tonic convulsions and protected mice against mortality. In the MES A methanolic extract (80%, V/V; 5% hyperforin) was given orally test, neither extract demonstrated anti-seizure activity. L-NAME (1250 mg/kg/day) to 30-day-old male C57BL/6J-APP/PS1(+/-) (1-10 mg/kg i.p.) reduced the anticonvulsant activity of the mice for 60 or 120 days. When compared to the untreated extracts [Hosseinzadeh 2005]. controls, mice receiving the extract showed significant (p<0.05 to p<0.01) reductions in plaque load for both Ab1-40 and Ab1-42, Water, n-butanol and ether fractions of an ethanolic (70%) and significant (p<0.001 to 0.0001) increases in cerebrovascular extract were tested on epileptogenesis in Chinchilla rabbits of Pgp expression [Brenn 2014]. both sexes in the kindling model of epilepsy. Epileptic focus was induced by stimulation of the hippocampus. Animals treated Parkinson’s disease with the ether fraction required significantly weaker minimum A study evaluated the protective effect of chronic administration of current strengths for development of epileptogenic focus, and two standardised extracts, (A) rich in hyperforin (6%) and (B) poor displayed longer after-discharge (AD) times, while the number in hyperforin (0.2%), and quercetin, against neurodegeneration of electro-stimulations necessary for full kindling was less. In induced by chronic administration of rotenone in rats.The contrast, animals treated with water and n-butanol fractions animals received pre-treatment with extract A (4 mg/kg i.p.), required increased electro-stimulations for the development extract B (4 mg/kg i.p.) or quercetin liposomes (25 and 100 of epileptic discharge, and displayed shortened AD durations mg/kg i.p.) 60 min before rotenone injection (2.5 mg/kg) for versus controls [Ivetic 2011]. 45 days. Pre-treatment with both extracts efficiently blocked deleterious toxic effects of rotenone, revealing normalisation A methanolic (80%) extract was given at doses of 25 to 200 mg/ of catalepsy and amelioration of neurochemical parameters. kg i.p. to male adult mice. After 20 min, seizures were induced Extract A also reduced neuronal damage by diminishing the by 10 mg/kg i.p. picrotoxin. Latency to seizure increased with substantia nigra dopaminergic cell death caused by rotenone the extract at 50 mg/kg (p<0.01). The highest dose (200 mg/kg) and inhibited the apoptotic cascade by decreasing Bax levels. significantly decreased duration of seizure (p<0.01) and death Extract A was more active than extract B [Gomez del Rio 2013]. latency (p<0.01) [Etemad 2011].

Movement disorders The effect of a standardised extract (not futher specified) The effects in male Wistar rats of an unspecified aqueous extract on PTZ-induced convulsions was compared with standard (300 mg/kg/day p.o.) were evaluated in models of abnormal antidepressants (FLU 30 and 50 mg/kg i.p.; dothiepin 30 movements induced by (25 mg/kg/day i.m.) or and 50 mg/kg i.p.; VEN 30 mg/kg i.p.). Male mice were reserpine (0.5 mg/kg, s.c. every 2 days for 6 days). The number injected with either saline, the extract (50-500 mg i.p.) or the of vacuous chewing movements (VCMs) and locomotor activity antidepressants. Sixty minutes later, animals received 60 mg/ were measured. In experiment 1, rats received a single dose kg i.p. pentylenetetrazole. In a dose-dependent manner, the of fluphenazine enanthate and/or the extract for 7 days. In extract significantly (p<0.05 to p<0.001) reduced latency to experiment 2, rats received reserpine and/or the extract for convulsion, with the maximum reduction of about 43% at 100 16 days beginning 10 days before the first administration and 200 mg/kg. The extract also dose-dependently increased of reserpine. The extract failed to protect against orofacial mouse mortality, with 100% mortality occurring at 400 mg/ movements induced by fluphenazine or reserpine [Reis 2013]. kg. FLU, dothiepin and VEN at 30 mg/kg produced maximum reductions in latency of 33, 44 and 37.6% respectively. VEN An ethanolic (70%) extract was tested in a rat model of Parkin- caused 80% mortality at 30 mg/kg, while at the same dose son’s Disease. Unilateral intrastriatal lesions were induced FLU and dothiepin caused 20 and 40% mortality respectively by 6-hydroxydopamine (6-OHDA) in adult male Wistar rats. [Bukhari 2013]. Treatment with the extract (200 mg/kg/day i.p.) was started 1 week pre-surgery and continued for 1 week post-surgery. The Migraine extract attenuated apomorphine-induced rotational behaviour, The pain-relieving property of an unspecified extract was decreased the latency to initiate and the total time in the narrow measured in male Swiss albino mice. The NO donors beam task, lowered striatal level of malondialdehyde, enhanced nitroglycerin (GTN; 10 mg/kg i.p.) and sodium nitroprusside striatal catalase activity and reduced GSH content, normalised (SNP; 0.5, 1 and 2 mg/kg i.p.) induced allodynia (cold plate striatal expression of glial fibrillary acidic protein and tumour test) and hyperalgesia (hot plate test). A single oral dose of necrosis factor alpha with no significant effect on mitogen- the extract (5 mg/kg p.o.) produced a prolonged relief from activated protein kinase, lowered nigral DNA fragmentation, and pain hypersensitivity. Pre-treatment with the extract delayed prevented damage of nigral dopaminergic neurons with a higher the onset of hyperalgesia and reduced the duration of the striatal tyrosine hydroxylase immunoreactivity [Kiasalari 2015]. painful symptomatology. No behavioural side effects or altered locomotor activity were observed [Galeotti 2013a]. Effects on seizures The effects of different fractions of an ethanolic (80%) extract (A: An unspecified extract (5 mg/kg p.o.) reduced meningeal water, B: ether, C: n-butanol; at 100 mg/mL i.m.) on the kindling nociception in mice induced by GTN and SNP, which epileptic discharges in Chinchilla rabbits were analysed using produced a delayed meningeal inflammation, as shown by electrodes implanted in cortical structures and hippocampus. the upregulation of IL-1b and iNOS, and a prolonged cold The effect correlated with the polarity of the fractions. The most allodynia and heat hyperalgesia with a time-course consistent polar constituents found in the water fraction exerted the highest with NO-induced migraine attacks. A single oral administration antiepileptic activity in 100% of animals. The butanol fraction of the extract counteracted the nociceptive behaviour and the

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over-expression of IL-1b and iNOS. The increased expression extract at low doses (30 60 mg/kg p.o.) reversed mechanical and phosphorylation of pro-inflammatory factors was prevented hyperalgesia up to 180 min after administration. Hyperforin and by the extract and hypericin (10 µg/kg p.o.) [Galeotti 2013b]. hypericin were responsible for the antihyperalgesic properties whereas flavonoids were ineffective. Treatment with the extract, Antinociceptive effects hyperforin and hypericin did not result in any behavioural side The i.p. administration of 30-100 mg/kg of an unspecified extract effects or signs of altered locomotor activity [Galeotti 2010b]. produced significant analgesic effects in acetic acid-induced writhing (up to 88% inhibition, p<0.001) and formalin licking The antinociceptive effect of an unspecified extract on hyper- tests (up to 83% inhibition, p<0.01) in NMRI mice. The effect sensitivity induced by GTN and SNP was assessed by cold was twice as potent as (100 mg/kg i.p.) in the acetic and hot plate tests in male Swiss albino rats. A single oral acid-induced writhing test [Bukhari 2004]. administration of low doses of the extract or hypericin reversed the induced prolonged allodynia and hyperalgesia, whereas Oral administration of an unspecified extract (25 mg/kg) hyperforin and flavonoids were ineffective. The increased displayed antinociceptive activity in the tail electric stimulation CREB and STAT1 phosphorylation, the activation of NF-kB in (p<0.05) and hot plate (p<0.01) tests. Acetic acid-induced periaqueductal grey matter and thalamus, and the increased writhing was significantly (p<0.05) reduced by oral FLU (50 expression and phosphorylation of PKCg/e isoforms after GTN mg/kg) or (72 mg/kg), but not by the extract. The and SNP administration were prevented by the extract and nociceptive response caused by i.p. injection of hypericin [Galeotti 2012]. (1.6 µg/paw) was unaffected by the extract, but reduced by FLU (p<0.05). Injection of the extract (50, 125 or 250 mg/kg Alcohol withdrawal s.c.) to pylorus-ligated rats decreased gastric acid secretion, In cAA rats, a genetic model of alcoholism, acute i.p. admin- but increased indomethacin-induced gastric mucosal lesions istration of IMI (3-30 mg/kg), FLU (1-10 mg/kg) or an ethanolic dose-dependently [Abdel-Salam 2005]. extract (50% m/m; 10-40 mg/kg) dose-dependently reduced alcohol intake in a 12-h limited access two-bottle [ethanol An unspecified hydroethanolic extract was studied in acetic 10% (V/V) versus water] choice procedure; with minimal acid-induced abdominal constriction in Balb-C mice. The effective doses of 30, 5 and 20 mg/kg respectively. The anti- extract (10-20 mg/kg i.p.), opium (10-30 mg/kg i.p.), morphine alcohol effects of FLU and the extract appeared to be specific, (0.75-3.0 mg/kg i.p.) and acetylsalicylic acid (50-100 mg/kg as reductions in alcohol intake co-incided with reductions in i.p.) showed dose-dependent antinociceptive effects. In animals alcohol preference [De Vry 1999]. treated with naloxone (0.5 mg/kg s.c.), the antinociceptive effect of the extract was significantly (p<0.05 to p<0.01) reduced Upon administration of single oral doses of 100, 200, 400, similar to that of opium, while the effect of acetylsalicylic acid 600 and 800 mg/kg of a methanolic extract (80%, V/V; 0.22% remained unchanged [Subhan 2007]. hypericin, 4.05% hyperforin), alcohol intake was dose- dependently reduced in alcohol-preferring rats. Repeated A study investigated the antinociceptive properties of an application of 400 mg/kg for 15 days did not induce tolerance unspecified extract in male ICR mice. Oral pre-treatment with of this effect [Rezvani 1999]. the extract (100 1000 mg/kg) significantly (p<0.05) attenuated licking/biting times, in both the first and second phases following Acute intragastric administration of 125 - 250 mg/kg of an formalin injection in a dose-dependent manner. Naloxone ethanolic extract (50%; 0.3% hypericin) reduced alcohol intake (5 mg/kg s.c.) did not attenuate the effect. Formalin injection of alcohol-preferring male msP rats by 30 - 40% [Perfumi resulted in an increase in nitrites/nitrates (NOx) in mouse 1999]. spinal cord which was significantly (p<0.05) attenuated by the extract. Pre-treatment with the extract significantly (p<0.01) The application of an ethanolic extract (50%; 0.3% hypericin potentiated the antinociceptive effect of morphine (0.3 mg/kg and 3.8% hyperforin) by 3 intragastric administrations of 250 s.c.), although concentrations of morphine in plasma and brain mg/kg to male msP rats produced positive results in the model were not significantly changed [Uchida 2008]. of the alcohol-preferring rat. The inhibition observed was neither modified by the s receptor antagonist rimcazole nor An unspecified dried extract induced a prolonged antinoci- by the neurotoxin 5,7-dihydroxytryptamine [Panocka 2000]. ceptive effect for 120 minutes after intracerebroventricular (i.c.v.) injection to male Swiss albino mice in acute thermal Administration of a methanolic extract (0.3% hypericin, 3.8% and chemical pain. Thermal antinociception was prevented by hyperforin; E1) or a CO2 extract (24.33% hyperforin, very naloxone and by the protein kinase C (PKC) activator PMA, low hypericin content; E2) to alcohol preferring male msP whereas the chemical antinociception was prevented only rats dose-dependently reduced ethanol intake, with E2 being by PMA. A chloroform (CHL) and a methanol (MET) fraction approximately 8-times more potent than E1. Food and water increased pain threshold with a time course comparable to intakes were not affected [Perfumi 2001]. the extract. The CHL antinociception was antagonised by naloxone, the MET antinociception by PMA. Hyperforin and In the same model the effect on ethanol intake of msP rats hypericin showed an antinociceptive efficacy comparable to was investigated 1h after intragastric administration of a CO2 CHL and MET respectively. The flavonoids remained without extract. Naloxone (NAL:1, 3, or 5 mg/kg) or naltrexone (NTX; effect. The presence of hypericin was fundamental to inducing 0.5, 1, or 3 mg/kg) were given by i.p. injection 10 min before thermal and chemical antinociception through the inhibition the extract. The extract alone reduced ethanol intake at 31 or of the PKC activity, whereas hyperforin selectively produced a 125 mg/kg, but only at 7 mg/kg when combined with NAL. thermal opioid antinociception [Galeotti 2010a]. These doses neither modified food or water intake, nor reduced 0.2% saccharin intake. NAL alone significantly reduced ethanol Neuropathic pain and food intake at 3 or 5 mg/kg, but not at 1 mg/kg. When

An unspecified dried chloroform extract was investigated for its NAL was combined with each dose of the CO2 extract, the effect on neuropathic pain due to chronic constriction injury attenuation of ethanol intake was more pronounced than after or the repeated administration of oxaliplatin (3.4 mg/kg i.p.) in the administration of the extract alone. NTX alone reduced male Sprague Dawley albino rats. Acute administration of the ethanol intake at 1 and 3 mg/kg, but not at 0.5 mg/kg. When

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NTX 0.5 mg/kg was combined with the CO2 extract at 7 mg/kg, antagonist WAY 100635 (1 mg/kg i.p.). Brain 5-HT metabolism ethanol intake was markedly reduced [Perfumi 2003]. was evaluated in the cortex 30 days after nicotine withdrawal through evaluation of 5-HT, 5-HIAA and the 5-HIAA/5-HT In C57BL/6J alcohol-preferring mice oral administration of a ratio. The total abstinence score was significantly (p<0.01) crude methanolic extract was compared with i.p. administration reduced in mice given the extract (500 mg/kg) after nicotine of a hyperforin-rich CO2 extract (45% hyperforin). The dose of withdrawal compared to the nicotine control group. WAY the hyperforin-rich extract required to significantly (p<0.05) 100635 significantly (p<0.01) inhibited the reduction of total reduce 10% ethanol intake was 5 mg/kg, and was 125-fold less abstinence score induced by the extract. A reduction of 5-HT than that required for the methanolic extract (625 mg/kg), and content was observed after nicotine withdrawal. A significant comparable to the dose of FLU (10 mg/kg i.p.). None of the increase of cortical 5-HT content was found in animals treated treatments significantly affected water intake [Wright 2003]. with the extract, with a concomitant significant increase of

5-HT1A receptors [Mannucci 2007]. Intragastric administration of a CO2 extract (24.33% hyper- forin, 0.08% hypericin) significantly reduced ethanol self- Opioid withdrawal administration in alcohol preferring male msP rats at doses of 31 In morphine-dependent adult male Wistar rats signs of or 125 mg/kg but not 7 mg/kg, when given 1h prior to ethanol withdrawal were induced by naloxone (3 mg/kg i.p.) given access. It did not modify saccharin self-administration. The 4 h after the last injection of morphine on day 7 of morphine extract at 31 and 125 mg/kg abolished the increased ethanol treatment. The manifestations of withdrawal were significantly intake following ethanol deprivation (p<0.05 and p<0.01 (p<0.001) inhibited by co-administration of the morphine (dose respectively) [Perfumi 2005b]. increased daily from 2.5 to 50 mg/kg) with an unspecified aqueous extract (p.o. 0.4 mL/200 g, 0.8 mL/200 g and 1.2 In a similar study, repeated intragastric administration of a mL/200 g per gavage) or clonidine (0.2 mg/kg i.p.). Clonidine

CO2 extract, alone or combined with NTX (0.5 or 3 mg/kg was more effective than the extract at 0.4 mL/200 g. There was i.p.) daily for 12 days, markedly reduced ethanol intake at 125 no significant difference in the mean frequency of withdrawal mg/kg (p<0.01), but not at 7 mg/kg. The same dose of the CO2 signs between clonidine and the extract at 0.8 mL/200 g. extract slightly reduced the simultaneous intake of food, but The extract at a dose of 1.2 mL/200 g exhibited significantly only on days 3 and 11 of treatment. Rats promptly recovered (p<0.05) greater attenuation of morphine withdrawal signs than baseline ethanol intake when treatment did not precede access clonidine [Feily 2009]. to ethanol (on day 8) or after the end of treatment (days 13 and 14). Repeated i.p. treatment with NTX reduced ethanol intake Sprague-Dawley rats were treated orally with an opium extract at 3 mg/kg (p<0.001), but not at 0.5 mg/kg. The extract (7 mg/ (increasing from 80–650 mg/kg) along with an ethanolic St. kg) combined with NTX (0.5 mg/kg) synergistically evoked a John’s wort extract (70%; 20 mg/kg), either twice daily for significant (p<0.001) reduction of ethanol intake. The effect on 8 days (chronic use), or acutely as a single dose 1 h before ethanol intake of the combined treatment remained stable over induction of opium withdrawal with naloxone. Chronic use the 12 days of treatment [Perfumi 2005a]. of the extract significantly (p<0.05) reduced withdrawal signs including stereotype jumps and wet dog shake number when In ethanol-dependent adult male Wistar rats an ethanolic extract compared to saline control, and decreased rectal temperature (50%; 25-200 mg/kg i.p.) given just before ethanol withdrawal at 30 min (p<0.05), 60 min (p<0.01) and 120 min (p<0.05). produced a dose-dependent and significant (p<0.05) inhibition Acute use of the extract reduced withdrawal-induced diarrhoea of locomotor hyperactivity 2 and 6 hours after withdrawal, (p<0.05 vs. saline) [Khan 2014]. and significantly (p<0.05) reduced the number of stereotyped behaviours. The extract produced significant inhibitory effects on Antiaggressive effects tremor (50 and 100 mg/kg; p<0.05) and audiogenic seizures (100 After oral administration of hydroethanolic preparations with mg/kg, p<0.05) during the withdrawal period [Coskun 2006]. defined amounts of hypericin (equivalent to 2-12 mg/kg), aggressive behaviour was significantly reduced in mice after Nicotine withdrawal 3 weeks of daily treatment with extract equivalent to 6 or 12 Male Swiss mice received nicotine (2 mg/kg, s.c. four injections mg/kg of hypericin [Okpanyi 1987]. daily) for 14 days. An extract was administered at various doses (125-500 mg/kg p.o.) immediately after the last nicotine The antiaggressive activity of hyperforin was investigated in injection. In a further experiment a dose of 500 mg/kg of extract several rodent models using adult male Charles Foster rats was administered with nicotine at various points during the and male Wistar mice. Animals were screened for aggressive study period. The reduction of locomotor activity induced by behaviour, divided into three groups and treated once daily, nicotine withdrawal was abolished by the extract, which also for seven consecutive days, with A: lorazepam (2.5 mg/kg i.p.), significantly (p<0.05 to p<0.01) and dose-dependently reduced B: hyperforin (10 mg/kg i.p.) or C: vehicle. Hyperforin treatment the total nicotine abstinence score when injected after nicotine significantly (p<0.001) reduced various aggressive parameters withdrawal [Catania 2003]. in isolation induced aggression, resident-intruder aggression and foot shock-induced aggression. In the water competition Male Swiss mice were divided into several treatment groups test, hyperforin treatment significantly (p<0.001) reduced the (n=6 each): Group 1: saline i.p. injection, 4 times daily for 14 duration of water consumption and frequency of water spout days; Group 2: nicotine (2 mg/kg i.p.) 4 times daily for 14 days to possession [Kumar 2009]. induce nicotine dependence; Groups 3-5: nicotine protocol plus administration (125 to 500 mg/kg p.o.) of a flavonoid-enriched Sexual dysfunction ethanolic extract (60%; 0.3% hypericin, 4.5% hyperforin, 50% A hyperforin-rich CO2 extract was examined in an experimental flavonoids) immediately after the last nicotine injection for 30 model for the expulsion phase of ejaculation in anesthetised days after nicotine withdrawal; Group 6: saline protocol plus Sprague Dawley rats. The ejaculation model involved inducing the extract at 500 mg/kg after the last saline injection for 30 days rhythmic bulbospongiosus (BS) muscle contractions in male (no nicotine). In a further group, animals treated with nicotine rats under urethane anaesthesia (1.2 g/kg s.c.) by transiently (14 days), alone or with the extract, were co-administered raising the internal urethral pressure with saline infusion. with a single s.c. dose of saline or the selective 5-HT receptor Injection of the 5-HT1A agonist 8-hydroxy-2-(di-N-propylamino)

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tetralin (8-OH-DPAT) (0.4 mg/kg s.c.) intensified the BS muscle markedly reduced irinotecan-induced diarrhoea (p=0.002) contractions induced by the increases in urethral pressure. The and intestinal lesions (p<0.05) and significantly suppressed extract (5 to 80 mg/kg i.p.) reduced the effects of 8-OH-DPAT the intestinal epithelial apoptosis induced by irinotecan over on ejaculation with maximum effect at a dose of 10 mg/kg days 5-11 (p<0.05 to p<0.001). The extract further significantly i.p. The reduction in the amplitude of bursts with the extract inhibited the expression of TNF-a mRNA in the intestine over remained unchanged after a midthoracic spinal transsection, days 5-11 (p<0.01 and p<0.001) [Hu 2006]. suggesting that the action of hyperforin is either at the spinal ejaculation generator or directly on the neurons innervating In the carrageenan-induced rat paw oedema test, ethanolic the BS muscles [Thomas 2007]. extracts from various Hypericum species were each admin- istered to adult male Wistar rats at doses of 25, 50, 100 and Anti-inflammatory effects 200 mg/kg p.o. and compared to indomethacin at 1, 2, 4 and Anti-inflammatory effects of St. John’s wort extracts were studied 8 mg/kg p.o. or vehicle. All tested doses of the Hypericum in carrageenan-induced paw oedema test [Savikin 2007; Zdunic perforatum extracts demonstrated significantly (p<0.001) greater 2009], the indomethacin-induced gastric mucosa damage test anti-inflammatory activity than the vehicle-treated controls

[Zdunic 2009] in rats and the croton-oil induced ear oedema with mean ED50 between 47.6 mg/kg and 50.9 mg/kg. The ED50 test in mice [Sosa 2010] after topical [Sosa 2010], oral [Savikin of indomethacin was 2.48 mg/kg. Anti-inflammatory activity 2007; Zdunic 2009] and i.p. [Feisst 2009] administration. was not found to be correlated with the hypericin content of the extracts [Savikin 2007]. A standardised ethanolic (50%) extract was examined for its putative anti-inflammatory and analgesic activity at the daily An unspecified extract improved the inflammatory and immune doses of 100 and 200 mg/kg p.o. The experimental paradigms response of colonic mucosa in male Wistar albino rats with used were carrageenan-induced paw oedema and cotton pellet- inflammatory bowel disease (IBD) induced by intracolonic induced granuloma for anti-inflammatory activity; the tail flick, administration of 25 mg 2,4,6-trinitrobenzene sulfonic acid. hot plate, and acetic acid-induced writhing methods were Treatment with the extract (50, 150 and 300 mg/kg/day i.p.) used to assess analgesic activity. Indomethacin (20 mg/kg i.p.) resulted in significant (p<0.05 to p<0.001) improvement in the was used as an anti-inflammatory active control, while penta- macroscopic scoring of colonic damage, blood levels of catalase, zocine (10 mg/kg i.p.) and aspirin (25 mg/kg i.p.) were used as GSH, tissue levels of GSH reductase (GR), MPO activity and analgesic active controls. The extract showed anti-inflammatory GSH-Px [Dost 2008]. and analgesic activity at both dose levels, in all the paradigms used. In addition, the extract potentiated the anti-inflammatory Hyperforin (4 mg/kg i.p.) significantly reduced the exudate activity of indomethacin and analgesic activities of volume (63%, p<0.001), the number of migrating cells (50%, and aspirin [Kumar 2001a]. p<0.001) and the leukotriene B4 (LTB4) formation in pleural exudates (50%, p<0.001) of carrageenan-treated rats [Feisst A hydroethanolic extract reduced croton oil-induced ear 2009]. oedema in rodents by 50% (p<0.05). From fractionation it was concluded that the anti-inflammatory principle is concentrated Of the three different oil extracts tested, an extract prepared in the lipophilic fractions [Brondz 1982]. by maceration with 96% ethanol followed by extraction with sunflower oil, given at an oral dose of 1.25 mL/kg, exhibited the Administration of an unspecified extract to Sprague-Dawley highest anti-inflammatory (95.24 ± 11.66%) and gastroprotective rats at 50, 150 or 300 mg/kg s.c. dose-dependently produced activity (gastric damage score of 0.21 ± 0.12) in carrageenan- a significant (p<0.001) inhibition of carrageenan-induced induced rat paw oedema and indomethacin-induced rat gastric oedema, by 53.7, 61.3 and 75.3% respectively, compared to mucosa damage. Its major components quercetin and I3,II8- 90% with FLU at 50 mg/kg s.c. and 60.7% with etodolac (ETO) biapigenin (at 8 mg/kg p.o.) showed a significantly (p<0.05 and at 72 mg/kg s.c. [Abdel-Salam 2005]. p<0.01 respectively) greater anti-inflammatory activity than the sunflower oil control and comparable with indomethacin A methanolic extract (0.34% hypericin, 4.1% hyperforin, 5% (8 mg/kg p.o.; p<0.001), as well as significant gastroprotective flavonoids, 10% tannins) ameliorated acute pancreatitis induced activity (p<0.05), compared to indomethacin [Zdunic 2009]. by caerulein in male CD mice. The degree of (a) pancreatic inflammation and tissue injury (histological score), (p<0.05), (b) Topical anti-inflammatory effects of a hydroalcoholic extract, expression of ICAM-1, (c) the concentration of nitrotyrosine and a lipophilic extract and an ethylacetate fraction, as well as poly(ADP-ribose), and (d) MPO activity was markedly reduced isolated constituents, were investigated in croton oil-induced after administration of the extract (30 mg/kg p.o.). Moreover, ear oedema in mice. The hydroalcoholic extract (100 - 1000 the treatment significantly (p<0.01) reduced the mortality rate µg/cm2), the lipophilic extract (100 - 1000 µg/cm2), and 5 days after caerulein administration [Genovese 2006a]. the ethylacetate fraction (30 - 1000 µg/cm2) were applied topically to mouse ears. The hydroalcoholic extract was less The same extract was given at 30 mg/kg p.o. prior to the potent than the lipophilic extract and ethylacetate fraction 2 2 2 carrageenan in male CD mice with carrageenan-induced (ID50 > 1000 µg/cm , 220 µg/cm and 267 µg/cm respectively). pleurisy. All parameters of inflammation were attenuated by the Amentoflavone, hypericin, hyperforin dicyclohexylammonium 2 extract. The carrageenan-induced upregulation of the expression salt and adhyperforin showed ID50 values of 0.16 µmol/cm , of ICAM-1, and the increases in nitrotyrosine and poly (ADP- 0.25 µmol/cm2, 0.25 µmol/cm2 and 0.30 µmol/cm2 respectively. 2 ribose), were all significantly (p<0.01) reduced by the extract. Indomethacin resulted in an ID50 of 0.26 µmol/cm [Sosa 2010]. The extract also inhibited inflammation-induced activation of NF-kB and STAT-3 in the lung [Menegazzi 2006]. In carrageenan-induced rat paw oedema in male Wistar Han rats administration of hyperforin (4 mg/kg i.p.) significantly reduced

The effect of a methanolic (80%) extract on various pro- the exudate volume (64%; p<0.001) and suppressed LTB4 inflammatory cytokines (IL-1b, IL-2, IL-6, IFN-g, TNF-a and on formation (50%; p<0.01) in pleural exudates. The effects were intestinal epithelium apoptosis, was investigated in rats treated similar to i.p. administration of 10 mg/kg of the 5-LO inhibitor with irinotecan (60 mg/kg i.v.) for 4 days in combination with (77%, p<0.001 and 65%, p<0.001, respectively) the extract or vehicle (400 mg/kg p.o.) for 8 days. The extract [Feisst 2009].

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Various extracts inhibited several members of the immunoglobulin pouch was scratched with a sterile needle on days 1 and 2. The superfamily of cell adhesion molecules, specifically intercellular extract was administered orally (300 mg/kg) and topically (10% cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion extract gel) on days 12-17. Both of the extract treatment groups molecule-1 (VCAM-1) [De Paola 2005; Genovese 2006a; demonstrated a significant (both p<0.01) relief in oral mucositis Menegazzi 2006] as well as P-selectin in mice and rats [De compared to the control and base gel group [Tanideh 2014]. Paola 2005]. The efficacy of an unspecified extract for prevention of post- A methanolic extract (0.34% hypericin, 4.1% hyperforin, 5% operative left uterine horn adhesion formation was studied in flavonoids, 10% tannins) was given orally for eight days at a female Wistar rats. Group 1 (control): closure of abdominal dose of 2 mg/kg/day to male Sprague Dawley rats following incision. Group 2: closure after i.p. administration of Ringer's ligature-induced periodontitis. The extract exerted potent anti- lactate (RL) solution. Group 3: closure after i.p. administration inflammatory effects, significantly (p<0.05 to p<0.01) reducing of olive oil (vehicle). Group 4: closure after i.p. administration all measured parameters of inflammation [Paterniti 2010]. of the extract. Fourteen days later, re laparatomy was performed and surgical adhesion scores, inflammation and fibrosis scores An olive oil extract (A), an ethanolic (96%) extract (B) and were determined. Groups did not differ in inflammation and fractions of the ethanolic extract (n-hexane, chloroform and fibrosis scores [Hizli 2014]. ethyl acetate) were assessed for their anti-inflammatory activity in acetic acid-induced increased capillary permeability. A dose- Bone formation dependent anti-inflammatory activity was found for the ethanol The effect of an aqueous extract on bone formation in the extract, the ethyl acetate subfraction and fractions containing expanded premaxillary suture model was studied in male Wistar hyperoside, isoquercitrin, rutin, (-)-epicatechin and hypericin rats. Four groups of rats were investigated: control (A); only [Süntar 2010]. expansion (B); the extract given (21 mg/day p.o.) only during the expansion and retention period (a total of 17 days) (C); and An unspecified extract (150, 300 and 450 mg/kg p.o.) given for the extract given during the nursery phase before expansion 26 days improved irritable bowel syndrome (IBS) induced by (40 days), and during the expansion and retention periods (a 5-day restraint stress in male Wistar rats. FLU and loperamide total of 57 days) (D). After the 5-day expansion period was (LOP) were used as positive controls. Gastric emptying, small completed, the rats in the B, C, and D groups underwent 12 days bowel and colon transit, as well as the levels of TNF-a, MPO, of mechanical retention, following which they were killed, and lipid peroxidation, and antioxidant power in colon homogenates their premaxilla dissected and fixed. Histological examination were determined. A significant reduction in small bowel (p<0.05) was performed to determine the number of osteoclasts and and colonic (p<0.001) transit (450 mg/kg), TNF-a (p<0.001), capillaries, as well as the number of osteoblasts, inflammatory MPO (p<0.001), and lipid peroxidation (p<0.05 to p<0.001) cell infiltration, and the amount of new bone formation. The and an increase (p<0.001 each) in antioxidant power in all number of osteoclasts and capillaries, and the inflammatory extract-treated groups were seen as compared with the control cell infiltration, as well as new bone formation, were higher group [Mozaffari 2011]. in the extract groups C and D than in the other groups. Among these two groups, all parameters were higher (p<0.01) in group Administration of hyperforin (4 mg/kg i.p.) to rats reduced exu- D than C [Halicioglu 2015]. date volume and leukocyte numbers in carrageenan-induced pleurisy associated with reduced PGE2 levels, and inhibited Gastrointestinal motility carrageenan-induced mouse paw oedema formation with an The effect on gastric motility of a standardised extract (details

ED50 (1 mg/kg) superior to that of indomethacin (ED50 = 5 mg/kg) unknown) at doses of 100, 300 and 600 mg/kg/day p.o. was [Koeberle 2011]. investigated in male Wistar rats. The extract delayed gastric emptying (p<0.05). The effect of the extract on electrical field An unspecified ethanolic extract (30-300 mg/kg i.p.) dose- stimulation (EFS)-induced contractions was unaffected by drugs dependently reduced -induced lethality in male inhibiting intrinsic inhibitory nerves or by tachykinin antagonists, Swiss mice (p<0.05). The paracetamol-induced increase in but reduced by the 5-HT antagonist methysergide. The inhibitory plasma AST and ALT concentrations, in hepatic MPO activity and effect of the extract on ACh-induced contractions was reduced by IL-1b, TNF-a and IFN-g concentrations, as well as reduced GSH the sarcoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic concentrations and decreased capacity to reduce 2,2’-azinobis- acid (p<0.001), but not by the L-type Ca2+ channel blocker (3-ethylbenzothiazoline-6-sulfonate radical cation) were or by methysergide. Among the chemical constituents inhibited (p<0.01) by the extract at 300 mg/kg [Hohmann of the extract, hyperforin (p<0.05 to p<0.001) and, to a lesser 2015]. extent, the flavonoids kaempferol (p<0.01) and quercitrin (p<0.05), inhibited ACh-induced contractions [Capasso 2008]. Wound healing An olive oil extract (A), an ethanolic (96%) extract (B), and Gastroprotective effects fractions of the ethanolic extract (n-hexane, chloroform and Injection of an unspecified extract (50, 125 or 250 mg/kg s.c.) to ethyl acetate), showed wound healing activity in excision and pylorus-ligated rats decreased gastric acid secretion (p<0.05), but incision wound models in male Sprague-Dawley rats. Extract dose-dependently increased (p<0.05) indomethacin-induced A had a significant wound healing effect on excision (5.1- gastric mucosal lesions [Abdel-Salam 2005]. 82.6% inhibition, p<0.001) and circular incision (20.2-100.0% inhibition, p<0.001) wounds. The ethyl acetate fraction was the Three oil extracts, prepared according to traditional prescriptions, most active, inhibiting excision wounds by 17.9% to 100.0% were studied in the indomethacin-induced rat gastric mucosa and incision wounds by 9.4% to 100.0% [Süntar 2010]. damage test. All extracts possessed gastroprotective activity. The extract prepared by maceration with 96% ethanol, followed A study investigated the therapeutic efficacy of the topical and by extraction with sunflower oil, had the highest content of systemic administration of an unspecified extract on chemo- quercetin and I3,II8-biapigenin (129 ± 9 µg/mL and 52 ± 4 µg/mL therapy induced-mucositis. Oral mucositis was induced in 72 respectively). Quercetin and I3,II8-biapigenin given separately male golden hamsters by administration of 5-fluorouracil (5- at a dose of 8 mg/kg p.o. exhibited significant (both p<0.05) FU; 60 mg/kg i.p.) on days 0, 5 and 10 of the study. The cheek gastroprotective activity [Zdunic 2009].

26 HYPERICI HERBA

Gastric mucosal damage induced by hypothermic restraint A hydroalcoholic extract (50%; 3% hyperforin, 0.3% hypericin) stress in Wistar rats was improved by an unspecified extract given at doses of 100 and 200 mg/kg orally over 15 days led to (25, 50 or 100 mg/kg/day p.o.) given over 3 days. The extract lowered total cholesterol and low-density cholesterol in normal significantly reduced lesions compared to by 65, 95 Charles Foster rats. In high-fat-fed rats, the extract significantly and 75% respectively (p=0.001). Treatment with ranitidine (5 (p<0.001) inhibited weight gain. In fructose-fed rats, the extract mg/kg s.c.) also reduced ulcers significantly (p=0.001) compared normalised the -induced dyslipidaemia (p<0.01) and with the placebo groups [Cayci 2009]. improved insulin sensitivity (p<0.001) [Husain 2011b].

Metabolic effects A 95% ethanolic extract was given orally to diet-induced obese The hypocholesterolaemic effect of a flavonoid-rich extract (DIO) C57BL/6J mice at doses of 50 and 200 mg/kg. The extract (FEHP) was studied in male Wistar rats receiving either a significantly improved glucose and lipid metabolism, lowered cholesterol-rich (2%) or a standard diet for 16 weeks combined glucose AUC (p<0.01 and p<0.001) and fasting insulin levels with a daily oral dose of FEHP (0, 25, 75 and 150 mg/kg). (both p<0.01), improved insulin receptor sensitivity (HOMA-IR) The cholesterol-rich diet induced hypercholesterolemia with (p<0.01 and p<0.001) and decreased serum triglyceride levels elevated serum levels of total cholesterol (TC) and triglycerides (p<0.05 and p<0.01) as compared to control [Tian 2015]. (TG), and lowered LDL. Daily medium (75 mg/kg) and high doses (150 mg/kg) of FEHP decreased (all p<0.05) the serum Reperfusion injury levels of TC, TG and LDL, and increased (p<0.05) serum levels The effect of an unspecified extract on splanchnic artery of HDL. Serum and liver contents of MDA were decreased occlusion (SAO) shock-mediated injury was studied in male (p<0.05) after FEHP compared with cholesterol-rich diet Sprague-Dawley rats. SAO shock was induced by clamping the alone. FEHP increased (p<0.05) the activity of SOD in serum superior mesenteric artery and the celiac trunk for 45 min. After and liver, whereas the activity of CAT was elevated only in the 1 h of reperfusion, SAO-shocked rats developed a significant liver [Zhou 2005]. (p<0.01) fall in mean arterial blood pressure. Treatment of rats with an unspecified extract (applied at 25 mg/kg i.v. over A study evaluated the effect of an unspecified extract (0.1% 15 min before reperfusion) significantly (p<0.01) reduced hypericin, 3.8% hyperforin) in an experimental model of binge the fall in mean arterial blood pressure and the migration of eating (BE). BE for highly palatable food (HPF) was evoked polymorphonuclear cells caused by SAO-shock. The extract also in female Sprague-Dawley rats by three 8-day cycles of food attenuated the ileum injury (histology) as well as the increase restriction/re-feeding and acute stress on the test day (day 25). in the tissue levels of MPO (p<0.01) and MDA caused by SAO Stress was induced by preventing access to HPF for 15 min, shock in the ileum. The extract markedly reduced the intensity while rats were able to see and smell it. Only rats exposed to and degree of P-selectin (p<0.01) and ICAM 1 (p<0.01) in tissue both food restrictions and stress exhibited BE. The oral doses of section from SAO-shocked rats. The extract also significantly 250 and 500 mg/kg but not 125 mg/kg of the extract significantly (p<0.01) improved survival [De Paola 2005]. (p<0.05 and p<0.01 respectively) reduced the BE episode. The same doses did not affect HPF intake in the absence of BE An ethanolic (70%) extract was studied for its effects on hepatic [Micioni Di Bonaventura 2012]. ischaemia-reperfusion (I/R) injury in rats which were subjected to 45 min of hepatic ischaemia followed by 60 min of reperfusion. Ovariectomised (OVX) rats were treated orally for 6 weeks with The extract (50 mg/kg i.p. 15 min prior to hepatic ischemia) a 70% ethanolic extract of whole (A) or a 70% ethanolic significantly (all p<0.05) decreased the ALT, AST, LDH and extract of flowers and leaves (B), b-oestradiol-3-benzoate (50 MDA levels, and markedly increased (p<0.05) activities of CAT µg/kg/day) or vehicle. OVX increased body weight gain and and GPx in tissue homogenates compared to I/R-induced rats adiposity, showed higher food efficacy ratio and increased without treatment [Bayramoglu 2013]. serum cholesterol as well as insulin resistance while reducing uterus weight and uterine epithelial proliferation rate. Extracts The effects of and an unspecified extract against A and B showed an oestrogen-like effect on body weight gain, I/R-induced intestinal damage were investigated. Hamsters adipose tissue weight and food efficacy ratio in OVX rats. The were divided into 4 groups after midline abdominal laparatomy: extracts prevented hypercholesterolaemia induced by OVX more control, I/R group, montelukast and I/R (MIR) group, and effectively (p<0.05) than b-oestradiol-3-benzoate [You 2014]. extract and I/R (HPIR) group. After 60 min of ischaemia through obstruction of the superior mesenteric artery, 24 h of reperfusion An ethanolic (50%) extract significantly improved glucose was maintained. Ten minutes prior to the reperfusion period, metabolism in STZ-induced diabetic Wistar rats (p<0.01, the MIR group received montelukast (7 mg/kg i.p.) and the p<0.001; 125 and 250 mg/kg i.p. for seven days) [Can 2011a]. HPIR group received the extract (7 mg/kg i.p.). Montelukast and the extract significantly reduced MDA levels and increased In STZ-diabetic Charles Foster rats, an ethanolic (50%) extract GSH levels compared to the I/R group (p<0.008). A significant given orally at 100 and 200 mg/kg significantly (p<0.001) difference was also found between the I/R group and MIR and reduced diabetes-induced elevated blood glucose levels HPIR groups in terms of MPO levels (p<0.008). The MIR and [Husain 2011a]. HPIR groups showed increased cardiotrophin-1 levels compared to the control and I/R groups (p<0.008 for all) and significantly Male STZ-induced diabetic albino rats received an ethyl acetate lower histological scores compared to the I/R group (p=0.03 extract at 50, 100 and 200 mg/kg p.o. for 15 days. At the end of and p=0.007 respectively) [Ocak 2014]. the treatment the extract showed a dose-dependent decrease in fasting blood glucose (FBG). Thirty minutes after administration, Albino rats were subjected to 45 min of hepatic ischaemia FBG was reduced significantly (p<0.01) when compared with followed by a 60 min reperfusion period. An ethanolic extract controls. The extract caused a significant reduction in (p<0.05 (70%; 50 mg/kg) was injected as a single i.p. dose, 15 min prior to p<0.01) plasma glucose level, serum total cholesterol, to hepatic ischaemia. Biochemical investigations of serum and triglycerides and glucose-6-phosphatase levels. Tissue glycogen liver tissue resulted in significantly increased ALT, AST, LDH content, HDL and glucose-6-phosphate dehydrogenase were and MDA levels whereas CAT and GPx were significantly significantly (p<0.01) increased compared with diabetic controls decreased in I/R-induced control compared to normal control [Arokiyaraj 2011]. (p<0.05). Treatment with the extract significantly decreased

27 HYPERICI HERBA

the ALT, AST, LDH and MDA levels, and markedly increased ethanol vehicle treated mice. Suppressor of cytokine signalling activities of CAT and GPx compared to I/R-induced rats (all 3 (SOCS3) transcription was increased by the extract both in p<0.05) [Bayramoglu 2014]. A549 cells and BALB/c mice, which could have interrupted the anti-viral immune response and thus led to the inefficient The effect of an ethanolic (96%) extract on I/R injury of the liver viral clearance and increased lung inflammation. The extract was investigated in male Wistar-albino rats divided into five resulted in a minor reduction of viral titre without affecting groups: Sham group, I/R group (who underwent partial liver I/R body weight when mice were inoculated with a lower dose 5.0 protocol), carboxymethyl cellulose (CMC) group administered (~10 EID50) and the extract was applied in the later phase 0.5 % carboxymethyl cellulose for a week before I/R, and the of infection. Mice challenged intranasally with the high dose extract group (HP) receiving 0.5 % carboxymethyl cellulose of influenza virus suffered from a higher mortality rate when with the extract (400 mg/kg) for a week before I/R. Blood levels dosed with the extract [Huang 2013]. of ALT, TNF-a, IL-6 and MDA were significantly (p<0.05) lower in the extract group compared with the I/R and CMC groups Immunomodulatory effects (p<0.05). There was no difference between the liver injury The immumodulatory potential of an ethanolic (95%) extract scores of I/R and CMC groups [Aydin 2014]. was investigated in male BALB/c-mice randomly allocated to two groups and immunised with sheep red blood cells (SRBCs) Nephroprotection and complete Freund's adjuvant. Mice treated with the extract Male Wistar rats were treated with an unspecified extract (110 mg/kg/day i.p.) for the duration of the 2 week study (400 mg/kg/day p.o.) for 10 consecutive days, and were injected showed a significant (p<0.001) increase in the level of anti- with cisplatin (5 mg/kg i.v.) one day after the final extract admin- SRBC antibody and a significant decrease (p<0.01) in the level istration. Cisplatin treatment increased (p<0.01) the serum of cellular immunity (a lower percentage of foot pad thickness) creatinine level to 1.5 ± 0.2 mg/dL (mean±SE) from 0.3 ± 0.1 mg/ compared to control [Froushani 2015]. dL (control) on day 5 after injection. This increase was improved significantly (p<0.01) to 0.9 ± 0.1 mg/dL by pre-treatment with Antiproliferative effects the extract. Cisplatin-induced histological abnormality of the Treatment with hypericin (10 mg/kg i.p.) reduced the growth kidney was blocked by the pre-treatment. Extract administration rate of metastases in breast adenocarcinoma (DA3) in female resulted in increases in renal metallothionein (MT) and hepatic BALB/c mice and squamous cell carcinoma (SQ2) in male multidrug resistance protein 2 (Mrp2) to 164.8 ± 13.0% and BALB/c mice after resection of primary tumours at stages with 220.8 ± 39.3% of control levels respectively. GSH levels in presence of lung micro-metastases. Long-term survival in the kidney and liver were not changed. Total and free cisplatin DA3 tumour-excised groups increased (p<0.001) from 15.6% concentration in serum was not influenced by the extract in controls to 34.5% after treatment with hypericin (5 mg/kg [Shibayama 2007]. on day 1, 2.5 mg/kg on day 2 and 1.25 mg/kg on days 3-5 i.p.) prior to surgery. In mice bearing SQ2 tumour metastases, Antiviral effects therapy with hypericin increased animal survival from 17.7% A dose-dependent anti-viral effect of hypericin and pseudo- in tumour resected controls to 46.1%. Median survival time hypericin against retrovirus infection with Friend leukaemia virus was prolonged (p<0.001) from 52.5 days to 97 days (resected was confirmed in BALB/cJ, BALB/cBy and C57BL/6J mice after group) and to 190 days in the combination group (surgery + a single i.v. dose of up to 150 µg. Both compounds inactivated hypericin). The hypericin content in lungs bearing metastases infectivity and reverse transcriptase activity of intact viruses. was approximately 2-fold higher than in the lungs of healthy Hypericin was more effective than pseudohypericin [Lavie animals, demonstrating the tendency of hypericin to accumulate 1989]. In a further experiment the efficacy of both compounds in primary and metastatic tumours [Blank 2004]. against radiation leukaemia virus was shown [Meruelo 1988]. Hyperforin (daily injections of 0.2 µM over 14 days) inhibited The efficacy of an unspecified extract (50 to 200 mg/kg b.w. the growth of autologous MT-450 breast carcinoma in immuno- p.o. twice daily for 5 days) against influenza A virus (IAV) was competent female Wistar Furth rats to a similar extent as pacli- investigated in BALB/c mice. Protection rate, mean survival taxel, without any signs of acute toxicity [Schempp 2002a]. days, lung index and viral titre, as well as the secretion of IL-6, IL-10, TNF-a and IFN-g in lung tissue and serum were measured Antiangiogenic effects on days 3 and 5 post-infection. The extract dose-dependently After s.c. injection with MT-450 mammary carcinoma cells, reduced the lung index and viral titre (p<0.05 to p<0.01) of Wistar rats were treated with peritumoural injections of infected mice, decreased mortality, and prolonged the mean hyperforin or solvent. Hyperforin (1 – 5 µg/mL) significantly survival time (p<0.01). It decreased the concentration of IL-6 (p<0.05) inhibited tumour growth, induced apoptosis (p<0.05) at the highest dose (p<0.05) and TNF-a (p<0.01) in lung tissue of tumour cells and reduced (p<0.05) tumour vascularisation and serum on day 5 post-infection. The lung and serum levels of [Schempp 2005]. IL-l0 (p<0.01) and IFN-g (p<0.01) on days 3 and 5 post-infection were enhanced in a dose-dependent manner [Xiuying 2012]. In Wistar albino rats, extensive angiogenesis induced in the cornea and iris by intraocular administration of basic fibroblast The immune-regulatory effect of an ethanolic (95%) extract growth factor (FGF-2) was effectively inhibited by treatment was evaluated in A549 lung epithelial cells and BALB/c mice with hypericin (2 mg/kg i.p.) [Lavie 2005]. exposed to influenza A/PR/8/34 H1N1 virus. In A549 cells, the extract (30 µg/mL) significantly (p<0.05) inhibited influenza An unspecified extract (15 mg/kg/day) or hypericin (15, 45 or virus induced monocyte chemotactic protein (MCP)-1 and 135 µg/kg/day) were given orally to C57BL/6 neonatal mice IFN-g induced protein 10 kD (IP-10) (p<0.05), but significantly to investigate retinal neo-vascularisation. Treatment with (p<0.05) increased IL-6. In mice inoculated intranasally with a the extract or hypericin significantly inhibited the degree of 7.9 high dose of of influenza A/PR/8/34 H1N1 (10 EID50), daily retinal neovascularisation (p<0.05 to p<0.001), but did not oral treatment with the extract at 110 mg/kg increased lung viral affect the area of retinal vasoobliteration. Treatment with the titre, bronchoalveolar lavage (BAL), pro-inflammatory cytokine extract or hypericin reduced (both p<0.05) phosphorylation of and chemokine levels, and the infiltration of pro-inflammatory extracellular signal-regulated MAP kinases (ERK) in the retina cells in the lung 5 days post-inoculation, as compared to [Higuchi 2008].

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Antioxidant effects over 14 days. Hypericin acetate dose-dependently induced A dry ethanolic extract administered orally to rats at 250 mg/ equal or more severe phototoxic responses compared with kg/day significantly reduced immobilisation stress-induced Me-ALA [Boiy 2008]. lipid peroxidation (measured ex vivo in liver homogenate; 42% protection after 30 days, p<0.001) [Tripathi 1999]. Pharmacological studies in humans The effect of a methanolic extract (80%, V/V; 3 × 300 mg/day The effects of an unspecified extract (0.34% of hypericin, 4.1% p.o. for 14 days) on plasma concentrations of testosterone, di- of hyperforin, 5% flavonoids, 10% tannins) on the development hydrotestosterone (DHT), sulfate, sex of zymosan-induced multiple organ dysfunction syndrome hormone-binding globulin and the combined concentrations of were investigated in male CD1 mice. Treatment with the extract androsterone sulfate (AoS) and epiandrosterone sulfate (epiAoS) (30 mg/kg i.p.), 1 and 6 h after zymosan (500 mg/kg i.p.), was determined in 12 healthy subjects (6 males, 6 females). The attenuated the zymosan-induced peritoneal exudation and extract significantly decreased the combined concentrations of migration of polymorphonuclear cells, pulmonary, intestinal AoS and epiAoS in all subjects (p=0.02), and in males (p=0.04). and pancreatic injury, and renal dysfunction as well as the The testosterone to DHT ratio was increased without significance increase in MPO in the lung and intestine. Immunohistochemical in both men and women [Donovan 2005]. analysis showed that the increase of iNOS, nitrotyrosine, and poly(ADP-ribose) in lung and intestine tissues from zymosan- A double-blind, randomised, crossover study examined injected mice was markedly reduced in mice treated with the whether a methanolic (80%) extract could reverse low-dose extract [Di Paola 2007]. ketamine-induced changes in auditory evoked potentials (AEP) in healthy subjects. Sixteen healthy subjects were given either Male albino Wistar rats received pre-treatment with an 2 × 750 mg of the extract or placebo for 1 week. On day 7, a unspecified extract (4 mg/kg) or quercetin liposomes (25 and test-battery including AEP, the oculodynamic test (ODT) and 100 mg/kg) 60 min before injection with rotenone (2 mg/kg). a cognitive test were performed before and after an infusion All treatments were given i.p. for 45 days. Rotenone increased with 4 mg of S-ketamine over a period of 1 hour. S-ketamine activities of SOD, GSH GPx and levels of MDA, and decreased led to a significant (p=0.004) decrease in the N100-P200 peak the content of reduced GSH (p<0.05). Rotenone significantly to peak (ptp) amplitude after placebo treatment, whereas ptp (p<0.05) induced the gene expression of CuZnSOD, MnSOD, was significantly increased by S-ketamine infusion in subjects CAT and GPx in brain. In contrast, the extract exerted an treated with the extract. Cognitive testing and the ODT revealed antioxidant effect which was related to a decrease of MnSOD no significant effect of ketamine-infusion and, therefore, no activity and mRNA levels of some antioxidant enzymes. interaction between the treatments [Murck 2006]. Treatment with quercetin liposomes resulted in a preservation of the activities of antioxidant enzymes and a decrease (p<0.05) Receptor binding in the mRNA levels of these enzymes [Sanchez-Reus 2007]. Patients with major depressive disorder (n=13) were treated twice daily for 6 weeks with either an unspecified extract In a chronic restraint stress model, inflammatory markers, (600 mg) or the MAO-A inhibitor (300 mg). hormonal levels and enzymatic markers of antioxidant capacity Assessment with [11C]-harmine positron emission tomography such as SOD, GPx and CAT were determined in the hippocampus found MAO-occupancy of less than 5% for the extract, but and hypothalamus of male Sprague-Dawley rats. Oral treatment 74% for moclobemide [Sacher 2011]. with an ethanolic (80%, V/V) extract at doses of 125-750 mg/ kg resulted in normalisation of the activities of antioxidant Smoking cessation enzymes [Grundmann 2010]. In an open, uncontrolled pilot study, 28 smokers of 10 or more cigarettes per day for at least one year were randomised to receive The protective properties of an unspecified extract on oxidative a methanolic (80%) extract at 300 or 600 mg daily for one week stress, apoptosis, and Ca2+ entry through transient receptor before and continuously for 3 months after a target quit date. potential melastatin 2 (TRPM2) and transient receptor potential In addition, all participants received motivational/behavioural vanilloid 1 (TRPV1) channels in spinal cord injury (SCI)-induced support from a trained pharmacist. The point prevalence and dorsal root ganglion (DRG) neurons of rats were investigated. continuous abstinence rates were both 18% at 3 months and Rats received 30 mg/kg p.o. of the extract for three successive 0% at 12 months [Barnes 2006]. days after SCI induction. The SCI-induced TRPM2 and TRPV1 currents and cytosolic free Ca2+ concentration were reduced by A methanolic (80%) extract was assessed for its feasibility of use the extract. The SCI-induced decrease in GPx and cell viability and efficacy in reducing withdrawal symptoms after smoking were ameliorated by the extract, as well as the SCI-induced cessation. A one-arm, uncontrolled study utilised a two-stage increase in apoptosis, caspase 3, caspase 9, cytosolic ROS group sequential design with a 1-week run-in period between production, and mitochondrial membrane depolarisation values the start of the extract treatment and the designated quit date in DRG of SCI [Ozdemir 2015]. in smokers (smoking ≥ 10 cigarettes/day). The extract (450 mg) was taken orally twice daily along with cessation counselling Photosensitising and photodynamic effects messages. Smoking status was determined through self-report The time course and skin histopathology of the phototoxic and bioverification using carbon monoxide (CO) testing. Among response after a single topical application of hypericin (0.1- evaluable subjects, the 12-week quit rate was 37.5% (9/24). The 1%) and hypericin acetate (0.015-1.5%), with subsequent extract was generally well tolerated [Lawvere 2006]. irradiation (10 J/cm2, 20 mW/cm2) were investigated. Methyl- aminolaevulinic acid (Me-ALA) was used as positive control. In a double-blind study, smokers stopping smoking were Application of hypericin on mouse ears resulted in limited randomised to oral treatment with 900 mg methanolic (80%) phototoxicity, probably due to confined penetration into extract or placebo daily. All participants received weekly the epidermal layers. Extended penetration achieved by behavioural support. The primary endpoints were biochemically administration of hypericin acetate evoked a more severe and confirmed prolonged abstinence and mean weight gain in prolonged response after irradiation, characterised by intense abstinent smokers 4 weeks after quitting. Six of 71 (8.5%) erythema and ear swelling. Skin damage induced by 0.15% participants treated with the extract and 9/72 (12.5%) on placebo hypericin acetate completely recovered without scar formation achieved prolonged abstinence at 4 weeks. At 6 months, 4.2%

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of the extract group and 8.3% placebo participants were still on 4 different days, on the COR, ACTH, GH and PRL secretions abstinent [Parsons 2009]. were examined in 12 healthy male volunteers. A significant (p=0.044) stimulatory effect of the extract on ACTH secretion A standardised hydroalcoholic extract (0.3% hypericin) was was observed, whereas COR, GH and PRL secretions were not given orally to 118 participants at doses of 900 or 1800 mg significantly influenced [Schüle 2004]. per day in a randomised, blinded, placebo-controlled, three- arm, dose-ranging together with behavioural In a double-blind, randomised, placebo-controlled, crossover interventions over 12 weeks. The dropout rate was high (43%). study, 16 healthy subjects (11 men and 5 women) received By intention-to-treat analysis, no significant differences were either an ethanolic extract (60%; 300 mg three times daily) observed in abstinence rates at 12 and 24 weeks between the or placebo for 7 days. IMI treatment (50 mg three times daily) extract and placebo. The extract neither attenuated withdrawal in 7 subjects served as a positive control. The extract had no symptoms in abstinent subjects nor reduced abstinence rates effect on plasma concentrations of NE and its main metabolite, [Sood 2010]. dihydroxyphenylglycol, whereas plasma DOPAC (the main metabolite of DA) concentrations were significantly (p=0.04) The neurocognitive effects of an ethanolic (50% m/m) extract increased (1661 ± 924 pg/mL) compared to placebo (1110 ± were compared with nicotine replacement therapy (NRT) and 322 pg/mL) [Schroeder 2004]. the combination of NRT plus extract during smoking abstinence in 20 smokers aged between 18 and 60 years over a period of The effects of two doses of a methanolic (80%) extract on 10 weeks during smoking cessation. A spatial working memory evening salivary cortisol and NE-mediated were (SWM) task was completed at baseline, 4 weeks prior to quitting, studied. Twenty healthy male volunteers received either a low as well as at the completion of the study, following the 10 weeks (2 x 300 mg/day) or high (2 × 900 mg/day) dose of the extract of treatment. Brain activity was recorded during the completion for 7 days. The extract significantly (p<0.01) increased salivary of the SWM task using steady-state probe topography. Reaction COR in the low dose group but had no discernible effect in time and accuracy on the SWM task were not significantly the high dose group. Salivary melatonin was not increased in different between treatment groups at retest. Differences in either group [Franklin 2006]. the steady state visually evoked potentials (SSVEP) treatment profiles were noted: a stronger SSVEP amplitude increase in Autonomic nervous function posterior-parietal regions for the extract and NRT groups, and In a randomised, double-blind, placebo-controlled study in greater fronto-central SSVEP phase advance in the extract group 12 healthy male subjects, the effects of an unspecified extract [Camfield 2013]. (standardised to 900 µg hypericin) on the autonomic responses of blood vessels and sweat glands were compared with those of Cognitive function AMI and placebo. Subjects received oral doses of 255 to 285 Thirteen healthy volunteers received a methanolic extract mg of the extract, 25 mg of AMI and placebo 3 times daily for (80%, V/V; 900 or 1800 mg/day), AMI (25 mg/day, positive 14 days each, with at least 14 days washout. The extract affected control) or placebo in a double-blind, placebo-controlled study. neither the vasoconstrictory response (VR) of cutaneous blood Cognitive and psychomotor tests, and subjective measures of flow nor the skin conductance response (SR). Whereas AMI sedation, were evaluated before and 1, 2 and 4 hours after led to a reduction of SR and a prolonged dilation phase of VR drug administration. AMI impaired performance on a battery of (p<0.05) [Siepmann 2004]. psychological tests, which included critical flicker fusion, choice reaction time, digit symbol substitution test, profile of mood The effects on heart rate variability of the same extract and AMI states and the line analogue rating scale, while the extract did (at the same dosages) were compared to placebo in 12 healthy not influence performance in these tests [Timoshanko 2001]. male volunteers in a randomised, double-blind, crossover study. The extract did not affect heart rate variability, whereas AMI In a randomised, double-blind, crossover study healthy male significantly (p<0.001) decreased it [Siepmann 2002]. volunteers received oral doses of either 255-285 mg of an un- specified extract (900 μg hypericin), 25 mg AMI or placebo, Skin protection three times daily for periods of 14 days each with at least 14 A cream containing 1.5% w/w of a hyperforin-rich (44.3%) CO2 days wash out. Psychometric tests and quantitative EEG (qEEG) extract was tested on 20 volunteers in a randomised, double- were performed before start of medication and on the last blind, vehicle-controlled study. It significantly (p<0.01) reduced treatment day. Neither the extract nor AMI had an influence on UVB-induced erythema as compared to vehicle. Occlusive cognitive performance such as choice reaction, psychomotor co- application of cream on non-irradiated test sites did not cause ordination, short-term memory and responsiveness to distractive any skin irritation [Meinke 2012]. stimuli. AMI, but not the extract, decreased self rated activity (p<0.05). Both drugs caused significant qEEG changes. The Topical treatment with a 1.5% hyperforin-rich cream increased extract increased theta power density (p<0.05). AMI increased the radical protection of the skin during VIS/NIR irradiation in 11 theta as well as fast a power density (p<0.05) [Siepmann 2002]. volunteers. After a single application, VIS/NIR-induced radical formation was completely inhibited by both verum and placebo Effect on stress hormones (p<0.05). After application for 4 weeks, radical formation was The effects of daily oral administration of a methanolic extract significantly reduced by 45% (p<0.05) by placebo and 78% (80%; 300 or 600 mg) or placebo on COR, GH and PRL secretion (p<0.01) by verum. Skin lipids in both verum and placebo were examined in twelve healthy subjects. No PRL stimulation groups increased directly after a single application but only was observed. A small but significant (p<0.05) elevation in GH significantly (p<0.05 and p<0.01) for ceramide [AP], ceramide AUC values occurred after 300 mg of the extract. After 600 mg [NP1] and squalene. After long-term application, concentration of the extract, a distinct COR stimulation was observed (p<0.05 of cholesterol and ceramides increased without significance to p<0.01), occurring from 30 up to 90 min after administration [Arndt 2013; Haag 2013; Haag 2014]. [Schüle 2001]. Clinical studies: depressive disorders In a similar study, the effects of oral administration of a The efficacy and safety of standardised hydroalcoholic St. John’s methanolic (80%) extract, at doses of 600, 900 and 1200 mg Wort extracts have been assessed in 54 controlled, double-blind

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studies involving 8,327 patients [Anghelescu 2006; Behnke Clinical studies and post-marketing surveillance studies in 2002; Bjerkenstedt 2005; Brenner 2000; Engesser 1996; Fava mild to moderate depression performed with hydromethanolic 2005; Gastpar 2005; Gastpar 2006; Halama 1991; Hänsgen extracts (80% methanol) 1996; Häring 1996; Harrer 1994; Harrer 1991; Harrer 1999; 2002; Hoffmann 1979; Hübner 1994; Kalb 2001; Kasper The DER of the extract used in the following studies was 3-6:1 2006; Kasper 2007a; Kasper 2008; Kugler 1990; Laakmann 1998; Lecrubier 2002; Lehrl 1993; Lenoir 1999; Mannel 2010; In a randomised, placebo-controlled study involving 50 patients Martinez 1994; Montgomery 2000; Moreno 2006; Philipp suffering from psychovegetative depressive symptoms (ICD-09: 1999; Quandt 1993; Randløv 2006; Rapaport 2011; Sarris neurotic depression or depressive mood of short duration), 2012; Schlich 1987; Schmidt 1989; Schrader 2000; Schrader 3 × 300 mg daily, in some cases reduced to 2 × 300 mg after 1998; Shelton 2001; Singer 2011; Sommer 1994; Szegedi 2005; week 2, of an extract led to a substantial improvement after 4 Uebelhack 2004; van Gurp 2002; Vorbach 1997; Vorbach 1994; weeks. A reduction of at least half from their initial HAMD total Warnecke 1986; Werth 1989; Wheatley 1997; Witte 1995; score or a score below 10 after 4 weeks was achieved by 50% Woelk 2000a] and 15 open studies, drug monitoring studies of patients under verum (p<0.01), but by none in the placebo and numerous case reports [Albrecht 1994; Brattström 2009; group. Other parameters, such as the von Zerssen Complaint Demling 2004; Findling 2003; Grube 1999; Holsboer-Trachsler Score and CGI score, showed similar differences between verum 1999; Hübner 2000; Meier 1997; Melzer 2010; Mueller 1998; and placebo [Halama 1991]. Rychlik 2001; Schakau 1996; SDSGrube 1996; Sepehrmanesh 1999; Simeon 2005] have involved a further 15,359 patients. In a multicentre, placebo-controlled study, 50 patients (mean age 49 years) with depressive symptoms (ICD-09: 300.4 The major indication in most of the studies was mild or mild neurotic depression; or 309.0 brief depressive reaction) were to moderate depressive disorders. Three studies were designed treated daily for 4 weeks with either 3 × 300 mg of an extract for treatment of severe depressive disorders [Davidson 2002; or placebo. HAMD scores improved from 23.7 to 17.4 in the Shelton 2001; Vorbach 1997]. A significant improvement in verum group and from 21.6 to 16.8 in the placebo group, the main symptoms (mood, loss of interest and activity) and other difference was not significant. In an evaluation of cognitive symptoms of the depressive syndrome (sleep, concentration, performance using the short test for general basic capacities somatic complaints) has been demonstrated in many of of information processing, patients on verum showed slightly these trials. The activity has been studied in comparison to better improvement than those on placebo (p<0.1) [Lehrl 1993]. placebo and various antidepressants (AMI, IMI, , FLU, (SERT), citalopram (CIT)). Nine meta-analyses In a multicentre, uncontrolled drug surveillance study, 1060 [Kim 1999; Linde 2005a; Linde 2009; Linde 1996; Liu 2013; patients (mean age 51 years) with mild to moderate depressive Liu 2014; Raak 2012; Rahimi 2009; Whiskey 2001] and 18 symptoms were assessed over a 4 week period of treatment systematic reviews [Clement 2006; Gaster 2000; Hammerness with 3 × 300 mg daily of an extract. By the end of the study, 2003; Hazell 2009; Henderson 2002; Izzo 2001; Izzo 2009; the average HAMD score had decreased from 18.4 to 5.4 and Knüppel 2004; Linde 1998; Linde 2005b; Linde 2005c; Linde the D-S score from 21.1 to 7.3. According to CGI evaluation, 2009; Linde 2011; Posadzki 2013; Raak 2012; Whiskey 2001; 66% of patients had improved and 27% were symptom-free Whitten 2006] of clinical trials with different selection criteria [Albrecht 1994]. have confirmed the efficacy of various St. John’s wort extracts in mild to moderate depression, but not in severe depression. In a multicentre, randomised, double-blind study, 105 patients (mean age 45 years) with depressive symptoms (ICD-09: 300.4 The studies summarised below are grouped according to the or 309.0) were allocated to 3 × 300 mg of an extract, or placebo preparations tested. Comparability is assumed for hydroalcoholic daily for 4 weeks. By the end of the study initial HAMD scores dry extracts within the range 60% ethanol to 80% methanol on (15.8 points in both groups) had dropped to 7.2 in the verum the basis of their similar active constituent profiles [Gaedcke group and 11.3 in the placebo group (p<0.01). Responder 2000]. An overview of the controlled studies is given in Table 1. rates, defined by a total endpoint score of less than 10 or a

ABBREVIATIONS used in the summaries of clinical studies

BDI Beck Depression Inventory [Beck 1972] D-S Depression scale (von Zerssen) [von Zerssen 1973] CGI Clinical Global Impressions scale [Guy 1967] DSM-III-R Diagnostic and Statistical Manual of Mental Disorders, 3rd ed., revised [Ocak 2014] DSM-IV Diagnostic and Statistical Manual of Mental Disorders, 4th ed. [Martinez 1994] GAF Global Assessment of Functioning [Hall 1995] HAMA Hamilton Anxiety Scale [Hamilton 1959] HAMD Hamilton Depression Scale [Williams 1988] ICD-09 International Classification of Diseases, Ninth Revision: Mental Disorders [WHO 1975] ICD-10 International Statistical Classification of Diseases and Related Health Problems, Tenth Revision [Sell 2014; WHO 2010]. ITT Intention-to-treat MADRS Montgomery-Asberg Scale for Depression [Williams 2008] MDD Major depression disorder MRS Menopause Rating Scale [Greene 2002] MOS Medical Outcome Study (older form of SF-36) Functioning scores PHQ-9 Patient Health Questionnaire [Kroenke 2001] SF-36 The Short Form (36) Health Survey [Jenkinson 1996]

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TABLE 1: OVERVIEW OF CONTROLLED CLINICAL STUDIES

First author Number Type of preparation Daily dosage Reference Duration Mean Severity of Superi- Year of Extraction solvent therapy (days) initial depression ority ? patients HAMD 2) Halama 1991 50 Dry extract, 80% methanol 3 × 300 mg 1) Placebo 28 18.3 Mild to moderate Yes Lehrl 1993 50 Dry extract, 80% methanol 3 × 300 mg 1) Placebo 28 22.7 Mild to moderate No Sommer 1994 105 Dry extract, 80% methanol 3 × 300 mg1) Placebo 28 15.8 Mild Yes

Hübner 1994 39 Dry extract, 80% methanol 3 × 300 mg Placebo 28 12.5 Mild Yes Hänsgen 1996 102 Dry extract, 80% methanol 3 × 300 mg Placebo 42 20.7 Mild to moderate Yes Häring 1996 28 Dry extract, 80% methanol 3 × 300 mg Placebo Not Not stated Mild stated Montgomery 248 Dry extract, 80% methanol 3 × 300 mg Placebo 84 No 2000 Shelton 2001 200 Dry extract, 80% methanol 3-4 × 300 mg Placebo 56 > 20.0 Severe No Kasper 2006 332 Dry extract, 80% methanol 1 × 600 mg Placebo 42 22.8 Mild to moderate Yes 2 × 600 mg 22.6 Yes

Kasper 2007a 161 Dry extract, 80% methanol 1 × 600 mg Placebo 120 22.5-23.3 Mild to moderate No 2 × 600 mg Relapse Kasper 2008 425 Dry extract, 80% methanol 3 × 300 mg Placebo 546 23.8 Mild to moderate Yes Kasper 2007b Dry extract, 80% methanol 600 – 1200 mg Placebo 217 ≤ 20 Mild Yes Mannel 2010 206 Dry extract, 80% methanol 2 × 300 mg Placebo 56 12.4 Mild Yes Harrer 1994 102 Dry extract, 80% methanol 3 × 300 mg Maprotiline 28 20.5 Mild to moderate No Vorbach 1994 135 Dry extract, 80% methanol 3 × 300 mg Imipramine 42 20.2 Mild to moderate No Wheatley 1997 165 Dry extract, 80% methanol 3 × 300 mg Amitriptyline 42 20.6 Mild to moderate No Vorbach 1997 209 Dry extract, 80% methanol 3 × 600 mg Imipramine 42 25.3 Severe No Brenner 2000 30 Dry extract, 80% methanol 2-3 × 300 mg Sertraline 49 21.3 Mild to moderate No HDTSG 2002 340 Dry extract, 80% methanol 3-5 × 300 mg Sertraline 8 23.1 Moderate to severe No Placebo No Sarris 2012 [Continuation 124 26 No HDTSG 2002] No Lecrubier 2002 375 Dry extract, 80% methanol 3 × 300 mg Placebo 42 24.2 Mild to moderate Yes Bjerkenstedt 163 Dry extract, 80% methanol 3 × 300 mg Fluoxetine 28 24.9 Mild to moderate No 2005 Placebo No Fava 2005 135 Dry extract, 80% methanol 3 × 300 mg Fluoxetine; 84 19.6 Mild to moderate Yes Placebo Yes Szegedi 2005 251 Dry extract, 80% methanol 3 × 300 mg Paroxetine 42 25.5 Moderate to severe Yes 3 × 600 mg Anghelescu 133 Dry extract, 80% methanol 3 × 300 mg Paroxetine 145 25.3 Moderate to severe No 2006 3 × 600 mg Gastpar 2006 388 Dry extract, 80% ethanol 900 mg Citalopram 42 21.9 Mild to moderate No Placebo Yes Singer 2011 [extra analysis of 154 900 mg Citalopram 180 No Gastpar 2006] Placebo No Uebelhack 2004 140 Dry extract, 80% ethanol 900 mg Placebo 42 22.8 Mild to moderate Yes Laakmann 1998 147 Dry extract, 60% ethanol 3 × 300 mg Placebo 42 20.8 Mild to moderate [WS 5572; 5% hyperforin] Yes [WS 5573; 0.5% hyperforin] No Philipp 1999 263 Dry extract, 60% ethanol 3 × 350 mg Imipramine 56 22.7 Moderate No Placebo Yes Harrer 1999 149 Dry extract, 60% ethanol 2 × 400 mg Fluoxetine 42 16.9 Mild Lenoir 1999 348 Fresh extract 30, 60, 180 mg - 42 16.4-16.9 Mild Yes 60% ethanol Kalb 2001 72 Dry extract, 60% ethanol 3 × 300 mg Placebo 42 19.9 Mild to moderate Yes

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First author Number Type of preparation Daily dosage Reference Duration Mean Severity of Superi- Year of Extraction solvent therapy (days) initial depression ority ? patients HAMD 2) Witte 1995 97 Dry extract, 50% ethanol 2 × 100-120 mg Placebo 42 24.6 Moderate Yes Schrader 1998 162 Dry extract, 50% ethanol 2 × 250 mg Placebo 42 19.5 Mild to moderate Yes Schrader 2000 240 Dry extract, 50% ethanol 2 × 250 mg Fluoxetine 42 19.6 Mild to moderate No Woelk 2000a 324 Dry extract, 50% ethanol 2 × 250 mg Imipramine 42 22.4 Mild to moderate No Gastpar 2005 161 Dry extract, 50% ethanol 615 mg Sertraline 168 22.0 Mild to moderate Yes Behnke 2002 70 Unspecified 2 × 150 mg Fluoxetine 42 20.0 Mild to moderate No Moreno 2006 72 unspecified 900 mg Fluoxetine 8 - Mild to moderate No Placebo No Randløv 2006 150 unspecified 270 mg Placebo 6 13.9/14.1 Mild to moderate No Rapaport 2011 unspecified 810 mg Citalopram 84 13.6 Mild No (minor depression) van Gurp 2002 87 unspecified 900 to 1800 mg Sertraline 84 16 Mild to moderate No Warnecke 1986 60 Tincture, 50% ethanol 3 × 1.5 ml Diazepam 94 --- Mild Hoffmann 1979 60 Tincture, 50% ethanol 3 × 1.5 ml Placebo 42 --- Mild Schlich 1987 49 Tincture, 49% ethanol 3 × 1 ml Placebo 28 23.5 Mild Schmidt 1989 40 Tincture, 49% ethanol 3 × 1.5 ml Placebo 28 29.4 Mild to moderate Harrer 1991 120 Tincture, 49% ethanol 3 × 1.5 ml Placebo 42 21.3 Mild to moderate Quandt 1993 88 Tincture, 49% ethanol 3 × 1.5 ml Placebo 28 17.6 Mild Werth 1989 30 Tincture, 49% ethanol 3 × 1.5 ml Imipramine 23 --- Mild Kugler 1990 80 Tincture, 49% ethanol 3 × 1.5 ml Bromazepam 28 --- Mild Lenoir 1999 348 Dry ethanolic extract from 3 × 60 mg Different 42 16.5 Mild fresh shoot tips dose levels of same extract Engesser 1996 19 Tea preparation Not stated Milfoil tea 14 --- Mild

1) Daily dosage was up to 50% lower due to up to 50% of excipients in the amount stated [Schulz 1998]. 2) In the St. John’s wort group drop of at least 50% from baseline values, were 67% and 28% 3 × 25 mg daily of IMI. The patients (n = 135; mean age 53 respectively [Sommer 1994]. years) had diagnoses in accordance with DSM-III-R criteria of depression with a single episode (296.2), several episodes In a placebo-controlled, randomised, double-blind study, 40 (296.3), depressive neurosis (300.4) or adjustment disorder with patients (mean age 51 years) suffering from mild depression with depressed mood (309.0). Initial HAMD scores declined from somatic symptoms (ICD-09: 300.4; 309.0) were treated with 20.2 to 8.8 in the extract group and from 19.4 to 10.7 points 3 × 300 mg of an extract or placebo daily for 4 weeks. Initial in the IMI group. Group comparisons showed no significant mean HAMD scores of 12.5 dropped to 5.5 in the verum group differences. Analysis of von Zerssen depression scale (D-S) compared to 10.8 in the placebo group (p<0.05). Patients in and CGI patterns in the two groups also revealed comparable the placebo group had an improved score of 9 after 2 weeks, results. Subgroups with initial HAMD scores of > 21 performed but this subsequently increased. Responder rates, defined by an significantly better (p<0.05) with the extract (n = 26) than IMI endpoint score of < 10 or a drop of at least 50% from baseline (n = 25) with regard to HAMD score and CGI [Vorbach 1994]. value, were 70% and 47% respectively [Hübner 1994]. In a multicentre, uncontrolled drug surveillance study, 3250 In a 4-week randomised, double-blind, comparative study, the patients (mean age 51 years), of whom 49% had mild, 46% efficacy and tolerability of 3 × 300 mg daily of an extract was moderate and 3% severe depressive symptoms, were treated compared with 3 × 75 mg daily of maprotiline in 102 patients for 4 weeks with 3 × 300 mg daily of an extract. D-S scores (mean age 46 years) with moderate depression (ICD-10: F dropped from 23.2 to 11.8 over the 4 week period. At the end 32.1). Initial 17-item HAMD scores decreased from 20.5 to of treatment, 82% of patients were assessed as improved or 12.2 in the St. John’s wort group and from 21.5 to 10.5 in the symptom-free by the physicians and 79% by self assessment. maprotiline group. Responder rates, defined by a total endpoint The therapeutic effect was slightly better in patients less than score of less than 10 or a drop of at least 50% from baseline 50 years old. Cases of mild or moderate depression responded values, were 61% and 67% respectively, with no significant to treatment to the same extent, while severe cases improved differences between groups [Harrer 1994]. less [Woelk 1994].

In a 6-week multicentre, randomised, double-blind, compar- In an open, randomised, single-blind study involving patients ative study, 3 × 300 mg daily of an extract was compared to (mean age 46 years) suffering from seasonal affective disorder

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(major depression with a seasonal pattern in accordance with endpoint HAMD score of less than 10 or a drop of at least 50% DSM-III-R), daily treatment for 4 weeks with 3 × 300 mg of an from baseline). HAMD scores dropped to 10 in the extract group extract was combined with phototherapy: either bright light and to 6 in the amitriptyline group after 6 weeks, a difference (3000 lux; n = 10) or dim light (< 300 lux, n = 10) for 2 hours per significantly in favour of amitriptyline (p<0.05). Analysis of the day. Average HAMD scores at baseline decreased significantly Montgomery-Asberg rating scale for depression (MADRS) also in both groups (p<0.001) [Martinez 1994]. favoured amitriptyline (p<0.05) [Wheatley 1997].

In 102 outpatients (mean age 52 years) with depression of In a 6-week multicentre, randomised, double-blind comparative mild or moderate severity (DSM-III-R: 296.2, 296.3), daily treat trial, 209 patients (mean age 49 years) were allocated to 3 × 600 ment with 3 × 300 mg of an extract for 4 weeks was compared mg daily of an extract or 3 × 50 mg daily of IMI. The primary to placebo. Average HAMD scores at baseline were 21 in the target parameter, mean 17-item HAMD scores, dropped from verum group and 20.4 in the placebo group. After 4 weeks the 25.3 to 14.4 points in the extract group and from 26.1 to 13.4 average score was significantly lower (p<0.001) in the verum points in the IMI group, significantly in favour of IMI (p=0.021). group (8.9) than in the placebo group (14.4). Similar results Equivalence of the two treatments was shown with respect to were obtained on the von Zerssen depression scale (D-S) response rates, defined as the percentage of patients showing (p<0.001). Responder rates were 70% under verum and 24% a reduction of at least 50% in HAMD total score over the study under placebo (p<0.001). The randomised, double-blind period period: 35.3% in the extract group and 41.2% in the IMI group was followed by a further 2-week period when all patients were [Vorbach 1997]. openly treated with the extract; HAMD scores decreased further, to 6 in the former verum group and more substantially to 8.7 In a hospital-based, double-blind, randomised study, 30 out- in the former placebo group [Hänsgen 1996]. patients (18 to 65 years) with an initial 17-item HAMD score ≥ 17 and a DSM-IV diagnosis of major depressive disorder (single In a prospective, placebo-controlled, double-blind pilot study, or recurrent episodes), dysthymic disorder, adjustment disorder 28 outpatients undergoing chemotherapy for solid tumours with depressed mood, or depressive disorder not otherwise were randomly assigned to 3 × 300 mg of an extract or placebo specified, were given daily doses of either 2 × 300 mg of an daily for 2-3 chemotherapy treatment cycles. Although the extract or 50 mg SERT. After one week, the extract dose was average HAMD score in the verum group showed a slight trend increased to 3 × 300 mg and SERT to 75 mg daily for a further towards improvement, results from the two treatment groups 6 weeks. Mean HAMD and CGI scores significantly improved were comparable with respect to a quality of life analysis, the during both treatments (p<0.01), and no significant differences D-S scale and the CGI scale [Häring 1996]. were observed between the treatments. Comparable clinical response (≥ 50% reduction in HAMD scores) was obtained in In a 5-week drug monitoring study, the efficacy of an extract (3 47% of patients receiving the extract and 40% receiving SERT × 135-225 mg providing 900 µg of total hypericin daily) was [Brenner 2000]. assessed in 114 patients (mean age 48 years) with mild depressive symptoms. The treatment was evaluated by the D-S scale, the Adult outpatients (n = 200; mean age 42 years) suffering from distribution of characteristic psychic and somatic symptoms recurrent depression (64%) or a single episode of depression and a global rating by the physician. From an initial score of (35%) or melancholia (40%) with a DSM-IV diagnosis of major 21 the D-S score dropped to 15 after 2 weeks and to 2 after 5 depression and an initial HAMD total score (17-item scale) of at weeks. The physicians rated 39% of patients as improved and least 20 participated in a randomised, double-blind, placebo- 35% as symptom-free after the 5 weeks [Grube 1996]. controlled study. The duration of the current major depressive disorder episode was more than 2 years on average and onset of In a 12-week uncontrolled drug-monitoring study, the efficacy the initial major depressive disorder was more than 10 years ago. of an extract (3 × 135-225 mg, providing 900 µg of total hyper- After a 1-week single-blind run-in with placebo, patients were icin daily) was assessed in 111 women (mean age 52 years) treated for 8 weeks with either an extract, 3 × 300 mg daily for 4 with menopausal symptoms. Treatment was evaluated by the weeks, increased thereafter to 4 × 300 mg daily in the absence menopause rating scale (MRS), a questionnaire for assessing of adequate response, or placebo. The primary outcome measure sexuality and the CGI scale. Menopausal complaints diminished (rate of change in HAMD score over the treatment period) was or disappeared completely in 79% of the women as rated by significantly decreased by both treatments (p<0.001), with no the physicians (CGI rating better or symptom-free). Sexual differences between treatments (p=0.16). Similar results were well-being was favourably affected in approximately 60%. obtained for the secondary outcome measures, HAMA and The average MRS total score decreased from an initial 63.4 CGI. 26.5% of patients in the verum group and 18.6% in the (corresponding to a marked intensity of symptoms) to 23.5 placebo group were responders. A significantly higher (p=0.02) (slight intensity) after 12 weeks [Grube 1999]. remission rate (defined as an endpoint HAMD score of 7 or less and CGI-I score of 1 or 2) was determined for the extract In an uncontrolled drug-surveillance study, 647 patients with (14.3%) compared to placebo (4.9%) [Shelton 2001]. mild to moderate depression were treated daily for 6 weeks with 3 × 300 mg of an extract. Improvement was shown in In a multicentre, randomised, placebo-controlled, double-blind, 75% of the patients. The D-S score dropped from 19.8-21.2 at three-armed study, 340 patients (mean age 42 years) with major baseline to 8.1-9.3 after 6 weeks. Patients older than 65 years depression of moderate severity (according to Clinical Interviews improved at a slightly slower rate but the severity of depression for Axis I DSM-IV disorders, initial 17-item HAMD score ≥ did not appear to affect the outcome [Holsboer-Trachsler 1999]. 20 and a Global Assessment of Functioning (GAF) score ≤ 60) were assigned to one of three treatments daily for 8 weeks: 3 In a 6-week multicentre, randomised, double-blind, comparative × 300 mg of an extract (standardised to 0.12-0.28% hypericin study, 165 mildly to moderately depressed patients (mean age and 3.1% of hyperforin), 50 mg of SERT (divided into 3 doses) 40 years) diagnosed in accordance with DSM-IV were treated or placebo. Responders at week 8 could continue blinded with 3 × 300 mg daily of an extract or 3 × 25 mg daily of treatment for another 18 weeks. Daily dosage was increased amitriptyline. Initial 17-item HAMD scores were between 17 after week 3 to 1500 mg of the extract or to 100 mg of SERT if and 24 (mean 20.7). No significant difference between groups warranted; the mean highest daily doses prescribed during the was observed with respect to the response rate (defined as an 8-week period were 1299 mg of the extract, 75 mg of SERT or

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placebo equivalent. With respect to the change from baseline The DER of the extract used in the following studies was 3-7:1 HAMD score and full response rates, defined as an endpoint HAMD score of 8 or less and a CGI-I score of 1 or 2, neither In a double-blind, randomised, placebo-controlled study, 375 the extract nor SERT were significantly different to placebo. out-patients (aged 18 to 25 years) after a one-week placebo run- Mean reductions in HAMD scores were 8.7 for the extract, in period received 3 × 300 mg daily of an extract (standardised 10.5 for SERT and 9.2 for placebo. Full response rates were to 3-6% hyperforin and 0.12-0.28% hypericin) or placebo for 23.9% for the extract, 24.8% for SERT and 31.9% for placebo 6 weeks. Patients had a current major depressive episode of at [HDTSG 2002]. least 2 weeks duration according to DSM-IV (mild or moderate depression, single or recurrent episode), and a total score on the Continuation data from the above study was used to analyse the 17-item HAMD score between 18 and 25 and a score on item 124 participants (responders) that continued treatment with an 1 (“depressed mood”) of 2 or higher at screening and baseline. extract (900 – 1500 mg), SERT (50 – 100 mg) or placebo from The HAMD score changed from 21.9 to 12.0 in the verum week 8 until week 26. At week 26, the primary outcome HAMD group and from 21.9 to 13.8 in the placebo group (p=0.03). scores were: extract (6.6 ± 4.5), SERT (7.1 ± 5.4) and placebo (5.7 Significantly (p=0.03) more patients had a treatment response ± 5.4), with a significant effect for time (p=0.036). Differences or remission in the verum group (52.7%) compared to placebo between all treatments were not significant (p=0.61). The effects (42.3%). There was a trend towards greater reduction in the on BDI, CGI-severity, CGI-improvement, and on intention-to- MADRS score for the verum group (p=0.06). The depression treat analyses were also non-significant [Sarris 2012]. sub-score of SCL-58 was not significantly different between treatments [Lecrubier 2002]. In a double-blind, randomised, placebo-controlled study, 163 out-patients (mean age 50.3 years) with major depressive disorder In a double-blind, randomised, active-controlled, double-dummy, (MDD) received, after a 3 to 7 day placebo run-in period, one non-inferiority study, 251 out-patients (mean age 47.3 years) with of the following treatments for 4 weeks: 3 × 300 mg daily of an MDD received one of the following treatments over 6 weeks: extract, 20 mg daily fluoxetine or placebo. Patients had a current 3 × 300 mg daily of an extract (standardised to 3-6% hyperforin major depressive episode with mild to moderate intensity and a and 0.12-0.28% hypericin) or 20 mg daily PAR. Patients had single minimal total score of 21 on the 21-item HAMD score, as well or recurrent moderate or severe episodes of MDD according as a history of at least two episodes of non-psychotic MDD. to DSM-IV persisting between two weeks and one year. At During the four weeks treatment, a reduction in HAMD total screening and baseline all participants had to have a total score score was observed from 24.9 to 15.0 in the extract group, from ≥ 22 points on the 17-item HAMD scale and ≥ 2 points for the 23.8 to 14.9 in the fluoxetine group and from 25.2 to 9.7 in the item “depressive mood”. HAMD decreased from 25.5 to 11.1 placebo group. There was no significant difference for HAMD in the extract group and from 25.5 to 14.1 in the PAR group, and treatment response between the treatments. However, with demonstrating that the extract was as effective as PAR. At the regard to remission, the extract (24%; p=0.02) and fluoxetine end of the treatment, 71% of patients in the extract group and (28%; p=0.005) were significantly superior to placebo (7%). 60% in the PAR group had responded to treatment (p=0.08), Almost identical results for the three groups were attained with and 50% and 35% showed remission (p=0.02) respectively. the MADRS and CGI [Bjerkenstedt 2005]. Patients in the St. John’s wort group showed a significantly greater decrease of the MADRS (p=0.01) and BDI scores (p=0.01). Item In a double-blind, randomised, placebo-controlled study, 1 of the CGI score was signficiantly (p=0.02) improved in ≥ 2 135 out-patients (mean age 37.3 years) with MDD received categories in the extract group [Szegedi 2005]. one of the following treatments for 12 weeks after a 1 week placebo run-in period: 3 × 300 mg daily of an extract, 20 mg In a multicentre, randomised, double-blind, active controlled daily fluoxetine (FLU) or placebo. Patients had a current MDD study, 133 patients received after a 3-7 days run-in phase, 2 episode according to DSM-IV of at least 2 weeks duration and × 300 mg daily of an extract or 20 mg daily PAR for 6 weeks. a total 17-item HAMD score ≥ 16. In comparison to baseline, Patients suffered from single or recurrent episodes of moderate treatment with the extract was associated with a significantly or severe intensity MDD, without psychotic features, according (p<0.05) greater decrease in HAMD scores compared with to DSM-IV with a duration of at least two weeks and not more fluoxetine at all post-baseline visits, except visit 5 (week 8). than 1 year. A total score of at least 22 points on the 17-item There was no significantly greater reduction in HAMD score in HAMD scale and an item score of at least 2 for ‘depressive mood’ the extract and FLU groups when compared with placebo and were required for inclusion. Patients with a HAMD total score non-significantly higher rates of remission (HAMD < 8) in the decrease of ≥ 50% during the 6 weeks of acute treatment were extract group (38%) compared to FLU (30%) or placebo (21%). asked to continue the treatment for another 16 weeks to prevent No significant differences were observed for CGI-S, CGI-I and relapse. Patients who received the extract in the acute phase BDI score between the treatments [Fava 2005]. were randomised to maintenance doses of either 900 (n = 33) or 1800 mg of the extract daily (n = 38). Patients who received In an 8-week double-blind, placebo-controlled, randomised trial, PAR in the acute phase were randomised to maintenance doses 600 mg daily of an extract or placebo were given to patients with of either 20 (n = 28) or 40 mg daily (n = 34) of PAR. Between vegetative features of atypical depression, e.g. hyperphagia or baseline of the acute phase and end of continuation treatment hypersomnia. One-hundred patients with a mild- and another the total HAMD score decreased from 25.3 ± 2.5 (mean ± SD) 100 patients with a moderate severity major depressive episode to 4.3 ± 6.2 points for the extract and from 25.3 ± 2.6 to 5.2 ± according to ICD 10, who met a score of 2 in at least one of the 5.5 points for PAR (p=0.49). Remission (total HAMD score < items 22–26 of the 28-item HAMD scale, and had an episode 8) occurred in 81.6% of the patients in the extract group and duration of at least 3 months, were randomized for treatment. in 71.4% of the PAR group (p=0.29). Three patients relapsed The absolute reduction of the 17-item HAMD was significantly (HAM-D increase > 5 points during continuation treatment) in (p<0.05) greater for the extract than placebo. No significant benefit the extract group and two patients in the PAR group. None of was observed for the sum score of the atypical vegetative items of the secondary parameters showed any significant difference the 28-item HAMD; however, the sum score of the hypersomnia between the treatments [Anghelescu 2006]. items showed a significant superiority for the extract. The HAMA, PHQ-9, and CGI-I scales demonstrated superiority of the extract In a double-blind, randomised, placebo-controlled, multicentre (p<0.01) [Mannel 2010]. study an extract at two doses (600 mg/day; 1200 mg/day) or

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placebo were given for 6 weeks to 332 patients with mild to determined individually by the responsible physician. Relief moderate, single or recurrent, major depressive episodes as of symptoms, tolerability and satisfaction with the therapy was defined by the DSM-IV. Average total HAMD scores decreased assessed by physicians and parents by questionnaires. Physicians in all three groups between baseline and day 42 (p<0.001 each). rating of effectiveness as ‘good’ or ‘excellent’ was 72% after 2 Both verum groups showed a significantly (p<0.001) greater weeks, 97% after 4 weeks and 100% after 6 weeks. The ratings decrease than placebo, from 22.8 to 11.2 (600 mg/day) and by parents were very similar [Hübner 2001]. from 22.6 to 11.8 (1200 mg/day) versus 23.6 to 17.6 (placebo). CGI, MADRS, SF-36 and global self-rating scores improved Clinical studies and post-marketing surveillance studies in significantly (p<0.001) for both verum groups compared to mild to moderate depression performed with hydroethanolic placebo [Kasper 2006]. extracts (ethanol 80%)

In a double-blind, randomised, placebo-controlled study, The DER of the extract used in the following studies was 3-6:1 relapse of depressive symptoms following 6-weeks single blind treatment with an extract was studied in 426 patients In a post-marketing surveillance study in 4188 patients suffering (18–65 years old) suffering from a recurrent episode of major from psychovegetative disorders, depressive states, anxiety depression (ICD-10 F33.0 or F33.1, and DSM-IV 296.3), and and/or nervous unrest, the effect of an extract (900 mg per day with a history of 2 or 3 previous episodes according to ICD- p.o.) was studied over 12 weeks. The HAMD score decreased 10 and DSM-IV criteria. After a 1 week single-blind placebo from an average of 15.8 to 9.5 after 4 weeks and finally to run-in period, the 6 weeks single blind acute treatmen took 4.6. The percentage of responders (decrease in HAMD-score place. Patients classified as responders to acute treatment were ≥ 50%) was about 78%. In the subgroup of patients with a randomised (2:1) to continue in a double-blinded manner with HAMD-baseline score of ≥ 17 the percentage of responders either 3 × 300 mg extract or placebo for 26 weeks. Patients was about 77%; of this group, two-thirds of the patients older who did not relapse during continuation treatment were entered than 65 years and two-thirds of those with an illness duration into a 52-week double-blind maintenance treatment phase. > 1 year experienced an improvement. The efficacy of the extract Patients under placebo continued on the same regimen, those was rated "very good" or "good" by 81.1% of the doctors and treated with the extract were re-randomised at a ratio of 1:1 76.6% of the patients. The tolerability was rated "very good" to maintenance treatment with either 3 × 300 mg/day extract or "good" by 95.6% of the doctors and 92.4% of the patients or placebo. Relapse was defined as a 17-item HAMD score [Demling 2004]. ≥ 16, or clinical diagnosis of a depressive episode according to ICD-10 criteria, or premature treatment termination related In a double-blind, randomised, placebo-controlled, prospective to lack of efficacy. In the intention-to-treat (ITT) analysis, 51 study, an extract at a dose of 900 mg daily was compared to of 282 (18.1%) patients on verum and 37 of 144 (25.7%) on placebo. Out-patients (n=140) suffering from moderate MDD placebo relapsed (p=0.035). The mean time until relapse was according to ICD-10 (F32.1 or F33.1) and DSM-IV with a total 177 days (verum) and 163 days (placebo), p=0.034. Compared 17-item HAMD score of 20 to 24 were included. Following to baseline, HAMD scores were decreased after the 6 week acute a single-blind placebo run-in period of 7 days, the patients treatment phase from 23.8 to 8.6 in the group randomised to were randomised to the extract or placebo for 6 weeks. While receive verum continuation treatment and from 23.6 to 8.6 in the total HAMD score decreased in both groups compared to the group randomised to receive placebo. In the continuation baseline, from 22.8±1.1 to 11.8±4.4 in the verum group and treatment period, verum signficantly (p=0.04) reduced the from 22.6±1.2 to 19.2±3.8 in the placebo group, there was a HAMD score by a further 1.3, compared to 0.1 with placebo. significantly greater reduction in the verum group (p<0.001). CGI improved significantly (p=0.01) during verum continuation Comparable differences in favour of the extract were revealed treatment, whereas BDI was comparable [Kasper 2008]. by the D-S and CGI scales and a global efficacy assessment [Uebelhack 2004]. The DER of the extract used in the following studies was 4-7:1 Patients (n=388) suffering from a first episode or recurrent In an uncontrolled, multicentre, drug monitoring study involv- MDD defined by ICD-10 (F32.1 or F33.1) and DSM-IV (major ing 1606 patients (mean age 52 years) suffering from typical depressive (296.2 x) and recurrent major depression (296.3 x) depressive symptoms such as low mood, disturbed sleep, loss with a total 17-item HAMD score of 20-24 received 900 mg of interest and energy, more than 90% were treated 2-3 times extract or 20 mg CIT or placebo daily for 6 weeks. From baseline daily with 300 mg of an extract (standardised to 900 µg of total values of 21.9 ± 1.2 points (extract), 21.8 ± 1.2 points (CIT) hypericin). After a mean treatment period of 5 weeks, the intensity and 22.0 ± 1.2 points (placebo), the total HAMD scores were of dominating symptoms was markedly reduced. The efficacy reduced to 10.3 ± 6.4 (extract), 10.3 ± 6.4 (CIT) and 13.0 ± was rated as very good or good by 81% of the physicians and 6.9 (placebo). A therapeutic equivalence of the extract to CIT 76% of patients. No age-dependent correlation of efficacy was and superiority over placebo (p<0.0001) were demonstrated. reported [Sepehrmanesh 1999]. At the end of treatment, 54.2% (extract, p<0.003 vs. placebo), 55.9% (CIT, p<0.001 vs. placebo) and 39.2% (placebo) of the In an uncontrolled, multicentre, drug monitoring study, 313 patients were assessed as therapy responders (HAMD ≤ 10). patients received an extract at a dose of 3 × 300 mg daily for No significant difference in the responder rate was observed 12 months. Patients were suffering from mild to moderate MDD between both active treatments. Regarding changes in D-S and (DSM-IV 296.2 and 296.3), dysthymic disturbances (300.4) or CGI scales, the extract was not significantly different from CIT, but adaptation disturbances (309.0) and had a baseline 17-item was significantly (p<0.001) superior to placebo [Gastpar 2006]. HAMD score between 12 and 22, as well as a CGI score of 4 to 6. At the end of treatment, the HAMD score decreased from In the above study, 188 patients were classified as responders 17.4 to 7.1 (p<0.001). Responder rate (HAMD score < 10 and to treatment, as defined by a final HAMD score of ≤ 10 at the HAMD reduction of at least 50%) was 68.7% [Hübner 2000]. end of the trial. Duration of response and occurrence of relapse/ recurrence was measured in 154 patients by re-analysing data In an uncontrolled study an extract was given for 4 to 6 weeks from the clinical trial. In total, 30 (19.5%) of the responders were to paediatric patients (n = 101; 1 to 12 years) with symptoms diagnosed with a relapse. The number of patients exhibiting of depression and psychovegetative disturbances. Doses were relapse was the highest in the CIT group (14 of 54), whereas

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patients who were treated with the extract showed the lowest suffering from a first episode of mild or moderate depression relapse rate (8/54); patients from the placebo group showed a (ICD-10: F32.0 or F32.1). During treatment, average HAMD relapse rate of 8/46. No difference in the severity of relapse was scores decreased from 16.6 to 7.9 in the extract group and observed. The duration of response was longest for the extract from 17.2 to 8.1 in the FLU group. The efficacy of the two group (1817 days), intermediate for the CIT group (1755 days) medications was found to be equivalent in both mild and and shortest for the placebo group (802 days) [Singer 2011]. moderate depressive episodes. Responder rates (total HAMD score < 10 or a reduction of at least 50% from initial score) A controlled observational study with a follow-up duration of were 71% in the extract group and 72% in the fluoxetine group six months involved 514 patients with moderate depression [Harrer 1999]. (intention-to-treat group). Of the included patients, 362 (70.4%) received an extract and 152 (29.6%) were treated with an In a further uncontrolled drug monitoring study, 607 patients SSRI, in most cases CIT. Outcome measures were efficacy and (mean age 50.5 years) with depressive symptoms were treated tolerability of treatment under day-to-day conditions. Efficacy once or twice daily for 6 weeks with 425 mg of an extract. was comparable between the groups, assessed by physicians The initial mean 17-item HAMD score of 19.2 decreased to as “good” to “very good” in 86.1% of extract group patients 7.4 points by the end of the treatment. Similar results were and 87.9% of SSRI group patients. The extract was significantly obtained for the D-S scale score decreasing from 22.2 to 8.9 (p<0.001) better tolerated than the SSRIs, with tolerability judged points. Core symptoms such as low mood, lack of energy and as “very good” to “good” in 97.0% of extract patients compared concentration improved by 50% [Mueller 1998]. to 82.1% of SSRI patients [Kresimon 2012]. The DER of the extract used in the following studies was 2.5-5:1. Clinical studies and post-marketing surveillance studies in mild to moderate depression performed with hydroethanolic In a multicentre, randomised, double-blind, placebo-controlled extracts (ethanol 60%) study, 147 patients (mean age 49 years) suffering from mild or moderate depression in accordance with DSM-IV criteria, and The DER of the extract used in the following studies was 5-7:1 with initial total 17-item HAMD scores ≥ 17, were treated daily for 6 weeks with placebo or 3 × 300 mg of one of two extracts In an uncontrolled drug surveillance study, 2404 patients (mean (5% hyperforin or 0.5% hyperforin). The average HAMD score at age 50 years) with depressive symptoms of varying severity baseline was 20.8. At the end of the study, the group receiving the were treated with an extract at an average dose of 120-180 extract with 5% hyperforin had the largest reduction in HAMD mg twice daily for an average treatment period of 5 weeks. score from baseline (10.3 ± 4.6), followed by the group with the Typical symptoms of depression decreased in both frequency lower content of hyperforin (8.5 ± 6.1) and the placebo group and intensity, the response rates were 73% for depressed mood, (7.9 ± 5.2). The proportions of responders were 49% in the 5% 81% for loss of interest, 66% for reduced self-esteem and self- hyperforin group, 39% in the 0.5% hyperforin group and 33% confidence, 69% for lack of concentration and 68% for anxiety. in the placebo group. HAMD score reduction was significantly Overall, 90% of patients responded to treatment and more superior to placebo only in the 5% hyperforin group (p=0.004). than 50% noticed an improvement in symptoms after 2 weeks Results were similar for other outcome measures (CGI and D-S of therapy. Using the CGI scale, efficacy was rated as good or depression scales) [Laakmann 1998]. very good in 77% of patients (about 80% for patients under 54 years, decreasing to 73% with older patients). A correlation In a multicentre, randomised, double-blind, placebo-controlled was evident between severity of condition and response rates study, 72 patients with mild to moderate MDD (in accordance (82% for mild, 79% for moderate, 64% for marked severity) with DSM-IV) were given either a daily dose of 3 × 300 mg [Schakau 1996]. of an extract (5% hyperforin) or placebo for 6 weeks. Group differences in favour of the verum were significant after 4 weeks In a multicentre, randomised, double-blind, three-armed study, (p=0.011) and 6 weeks (p<0.001). Average 17-item HAMD 263 patients (mean age 47 years) with moderate depression scores decreased from 19.7 to 8.9 after verum and from 20.1 to (ICD-10: F32.1 and F33.1), and a mean initial total 17-item 14.4 after placebo. Analysis of responders showed a reduction HAMD score of 22.6 points, were allocated daily treatment of at least 50% from HAMD baseline scores in 62.2% of the for 8 weeks with either 3 × 350 mg of an extract (0.2-0.3% verum group and 42.9% of the placebo group (p=0.10). The total hypericin, 2-3% hyperforin), 100 mg of IMI (50 mg + 25 mg difference was larger when ‘60%-responders’ were considered + 25 mg) or placebo. The extract reduced HAMD scores more (51.4% vs. 17.1%) [Kalb 2001]. effectively than placebo (mean decrease after 6 weeks of 13.4 versus 10.3). Mean decreases from baseline at 8 weeks were In a multicentre, observational study, the effectiveness of an similar for the extract and IMI (15.4 versus 14.2). More patients extract was examined in 2166 patients (average age 50 years) receiving the extract had ≥ 50% improvement in HAMD suffering from mild to moderate depression. Most of the patients scores than patients receiving placebo (p=0.027); whereas were suffering from a first episode, with one third suffering from the proportions did not differ between the extract and IMI recurrent depression. Initially, 1385 patients took 600 mg and groups (p=0.14). Comparable results were found for HAMA 781 patients 1200 mg of the extract daily. Moderate depression and CGI scores, and were most pronounced for the Zung self- at baseline (based on CGI) was reduced to less than "mild" rating depression score. Quality of life, as measured by the after an average observation time of seven weeks in 83.7% of SF-36 standardised mental component scale, was improved patients at 600 mg and 88.6% at 1200 mg. The improvement to a greater extent by both active treatments than by placebo in symptoms as measured by 17-item HAMD scores was of (40.5% by the extract, 30% by IMI). Using the SF-36 physical clinical relevance [Rychlik 2001]. component scale, compared with placebo quality of life was markedly improved only by the extract (74.5% as compared The DER of the extract used in the following studies was 3.5-6:1 to 23% by IMI) [Philipp 1999]. In an observational study, 1778 patients with depressive The effects of 2 × 400 mg of an extract or 2 × 10 mg of FLU symptoms received an extract (425 – 1700 mg) for 12 weeks. daily were compared in a 6 week randomised, double-blind, At inclusion, the mean daily dose was 822.5 mg and at the comparative trial in 149 elderly outpatients (mean age 69 years) last visit 754.4 mg. At the last visit, the ICD-10 sum score had

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decreased by 63.1% and the proportion of patients described in the IMI group. None of the differences between treatment as ‘normal to mildly ill’ (GCI-S) had increased from 21.6% to groups reached significance, except for a difference in HAMD 72.4%. Regarding the GCI-I, 77% of the patients had improved anxiety-somatisation subscale scores in favour of the extract ‘very much’ or ‘much’, consistent with their self-assessment (p=0.03). Responder rates, defined by a drop of at least 50% (76%). Lower age and shorter duration of the disorder were of the baseline HAMD values, were 43% in the extract group associated with significantly better outcomes [Melzer 2010]. and 40% in the IMI group [Woelk 2000b].

Clinical studies and post-marketing surveillance studies in An observational study in 11,296 patients, diagnosed in most mild to moderate depression performed with hydroethanolic cases with mild to moderate depression, was performed using extracts (50% ethanol) an extract. After eight weeks of treatment symptom scores (modified HAMD) were improved by 60%. Efficacy was rated The DER of the extract used in the following studies was 5-8:1 as “good” to “very good”by the physicians in 80.7% of cases. Mild adverse effects were reported in only 0.2% of cases, and In a randomised, double-blind, multicentre trial, 97 patients did not show a systematic pattern [Volz 2000]. (mean age 43 years) with a moderate depressive episode (ICD- 10, F32.1) received 2 × 100-120 mg of an extract or placebo A 12-week observational study assessed the effects on quality of daily for 6 weeks. Initial HAMD scores dropped from 24.6 to life and safety of a once daily dosing scheme (dose not specified) 7.9 points in the verum group and from 22.6 to 10.3 in the of an extract in 4337 patients with mild to moderate depression. placebo group (p=0.019). Responder rates (total score at the Quality of life was assessed using the SF-12 symptom score, endpoint < 10 or decrease of at least 50% from initial score) and showed a significant improvement, reaching average values were 79% and 56% respectively [Witte 1995]. of the normal population. Only four patients (0.15%) reported mild adverse events [Rudolf 2004]. The DER of the extract used in the following studies was 4-7:1 A controlled, double-blind, multicentre study aimed to In an open, uncontrolled, multicentre drug surveillance study, demonstrate the non-inferiority of an extract (612 mg once daily; 170 patients (mean age 49 years) with masked, mild, moderately n = 123) compared to SERT (50 mg; n = 118) in patients with a severe or severe depression received 2 × 250 mg of an extract diagnosis of moderate depressive disorder (according to ICD-10 (0.2% total hypericin) daily for an average period of 66 days. criteria). The initial study duration was 12 weeks, followed by HAMD scores were evaluated in 84 patients. Treatment response an additional 12 weeks of treatment for 81 participants in the was satisfactory in patients with masked, mild and moderate extract group and 80 participants in the SERT group. As measured depression, but not in those with severe depression. Typical by the primary efficacy variable, the 17-item HAMD total score depressive symptoms decreased by 40% in the first group but at the end of the first 12 weeks of treatment, the extract was only by 12.5% in the subgroup with severe depression. In the found not to be inferior to SERT (p<0.0001) [Gastpar 2005]. group of patients whose HAMD scores were evaluated, the mean initial score of 36.3 dropped to 27.2 by the end of treatment. In The long-term safety and effects of an extract were evaluated a further subgroup analysis, severe cases did not significantly in 440 out-patients suffering from mild to moderate depression benefit from the treatment (p=0.46). Global efficacy, assessed according to ICD-10 in an open multicentre study. Patients in 94 of the patients, was rated as good or very good in 78%, were treated for up to 1 year with 500 mg of the extract per slight in 3% and insufficient in 19% [Meier 1997]. day. Mean HAMD scores decreased from 20.58 at baseline to 12.07 at week 26 and to 11.18 at week 52. Mean CGI scores In a randomised, double-blind, multicentre trial with 162 patients decreased from 3.99 to 2.20 at week 26 and 2.19 at week 52 (mean age 43 years) suffering from mild to moderate depression [Brattström 2009]. (ICD-10: F32.0, F32.1) the treatment was either 2 × 250 mg of an extract (0.2% total hypericin) or placebo daily for 6 weeks. Clinical studies performed with tinctures (49-50% ethanol) Over the course of the study 21-item HAMD scores dropped from 20.1 to 10.5 in the verum group and from 18.8 to 17.9 In a randomised, double-blind, placebo-controlled study, 60 in the placebo group (p<0.001). Responder rates (defined as depressive patients (mean age 49 years) were treated daily for reduction in HAMD ≥ 50% and/or actual HAMD score of ≤ 10) 6 weeks with 3 × 1.5 mL of a tincture (50% ethanol, daily dose were 56% and 15% respectively [Schrader 1998]. equivalent to 0.9 mg of total hypericins) or placebo. Using a self-developed, graded scale with 52 symptoms, a considerable In another randomised, double-blind, multicentre study, involv- reduction of 61.4% in the total score for the verum group ing 240 patients (mean age 47 years) with mild to moderate compared to only 15.8% for the placebo group. Responder depression (ICD-10: F32.0, F32.1) and 21-item HAMD scores rates, defined by the investigators’ assessment of good or very of 16 to 24 (mean initial score 19.6), the treatment was either good improvement, were 63% in the verum group and 10% 2 × 250 mg of an extract (0.2% total hypericin) or 1 × 20 mg of in the placebo group [Hoffmann 1979]. FLU daily for 6 weeks. After 6 weeks, HAMD scores dropped significantly from 19.65 to 11.5 in the extract group compared In another randomised, double-blind, placebo-controlled to 19.5 to 12.2 in the FLU group (p<0.09). The extract was study, 49 patients (mean age 42.3 years) with mild to moderate superior to fluoxetine with respect to responder rates (defined depressive symptoms were treated with 3 × 1 mL of a tincture as reduction in HAMD ≥ 50% and/or actual HAMD score of ≤ (49% ethanol, 0.4:1) or placebo daily for 4 weeks. In the placebo 10) (p=0.005; 60% for the extract and 40% for fluoxetine) and group the mean number of symptoms increased from 24 to CGI item 1 score (p<0.03) [Schrader 2000]. 29.6 after 4 weeks, whereas patients treated with the tincture experienced an average reduction from 22.9 to 16.4 symptoms In a randomised, double-blind, multicentre study, 324 out- (p<0.05) [Schlich 1987]. patients (mean age 46 years) with mild to moderate depression (ICD-10: F32.0, F32.1, F33.0, F33.1) were treated daily for 6 In a two-centre, double-blind, placebo-controlled study, 40 weeks with either 2 × 250 mg of an extract (0.2% total hypericins) patients (mean age 47 years) with depressive symptoms received or 2 × 75 mg of IMI. The 17-item HAMD scores decreased daily either 3 × 1.5 mL of a tincture (49% ethanol, 0.4:1) or from 22.4 to 12.0 in the extract group and from 22.1 to 12.75 placebo. After 4 weeks the initial HAMD total scores had

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decreased from 29.25 to 9.75 in the verum group (n = 16) and years old) and compared to milfoil tea (Achillea millefolium) as from 29.5 to 19.5 in the placebo group (n = 12). Responder a control. Each treatment period lasted 14 days, separated by rates (a reduction of at least 50% from baseline HAMD scores a wash-out period of 3 days. Thirteen patients reported better or an endpoint score of < 10) were 62.5% in the verum group results with St. John’s wort tea, while five patients had better and 33.3% in the placebo group [Schmidt 1989]. results with milfoil tea and one patient showed no difference. The pilot study indicated a trend towards a better mood in In a multicentre, double-blind, placebo-controlled study, 120 patients treated with St. John’s wort tea (p=0.06) [Engesser patients (mean age 48.5 years) suffering from mild depressive 1996]. symptoms (ICD-09: 304.4 and 309.9; initial HAMD scores of 16-20) were treated with 3 × 1.5 mL of a tincture (49% ethanol; In a randomised, controlled, double-blind study, 70 patients 0.25 mg of total hypericins per mL) or placebo daily for 6 (mean age 49.7 years) with mild to moderate depression received weeks. Results from 116 patients showed a marked reduction twice daily for 6 weeks either an unspecified extract (150 mg) or of 57.9% in HAMD total scores in the verum group compared FLU (20 mg). Both treatments significantly (p<0.001) decreased to only 18.1% in the placebo group. Comparable results were the total HAMD score (extract: 50%; FLU: 58%) and the D-S obtained using the HAMA and D-S scale. Responder rates scale (42% and 52% respectively). The extract achieved 83% (total HAMD score < 10 at endpoint or a drop of at least 50% (HAMD) and 78% (D-S) of the effects of fluoxetine, with the from baseline scores) were 65.9% after verum and 25% after differences not significant (p=0.23). Assessments by physicians placebo [Harrer 1991]. and patients indicated considerable improvement with no differences between the treatments [Behnke 2002]. In another randomised, double-blind, placebo-controlled, multicentre study, 88 patients (mean age 43.3 years) suffering In a double-blind, randomised trial, treatment for 12 weeks with from mild to moderate depressive symptoms (ICD-09: 300.4; an unspecified extract (900 to 1800 mg/day) was compared with HAMD score at least 16) were treated with either 3 × 1.5 mL of SERT (50 to 100 mg/day) in 87 men and women with major a tincture (49% ethanol; 0.25 mg of total hypericins per mL) or depression and an initial HAMD score of ≥ 16. No differences placebo daily for 4 weeks. HAMD scores decreased from 17.8 in the change to mean HAMD and BDI scores (using intention- to 5.2 in the verum group (p<0.001) and from 17.3 to 15.5 in to-treat analysis), with and without adjustment for baseline the placebo group. Responder rates (defined as reduction in demographic characteristics, were observed between the two HAMD ≥ 50% and/or actual HAMD score of ≤ 10) were 70.7% groups at 12 weeks. Significantly (p<0.05) more side effects after verum and only 7.1% after placebo [Quandt 1993]. were reported in the SERT group than in the extract group at 2 and 4 weeks' follow up [van Gurp 2002]. In other studies, the same preparation at the same daily dosage was compared to 50 mg of IMI daily in 30 patients with A pilot study examined the efficacy, safety, tolerability, and depressive states after surgery [Werth 1989], and to 6 mg of of an unspecified extract in the treatment bromazepam daily in 80 patients suffering from psychogenic of 33 outpatient youths (6 to 16 years; mean (SD) age 10.5 depressive symptoms [Kugler 1990]. Comparable efficacy results (2.9)) diagnosed with major depressive disorder (DSM-IV). for the tincture and the reference medication were reported in In this 8-week, prospective, open-label study, patients were both studies. initially prescribed 150 mg of the extract three times daily. At the end of week 4, in the 22 patients who did not meet the a In an open, comparative study, patients (mean age 52.9 years) priori response criteria, the dose was increased to 300 mg three with climacteric complaints were treated daily with either times daily. Twenty-five of the patients met the response criteria 3 × 1.5 mL of a tincture (50% ethanol; daily dose equivalent (Children’s Depression Rating Scale – Revised (CDRS-R) score to 0.9 mg of total hypericins; n = 40) or 3 × 2 mg of diazepam ≤ 28 and a CGI-S score ≤ 2) after 8 weeks of treatment. Overall, (n = 20). In the investigator’s judgement, 77.5% of patients the extract was well tolerated [Findling 2003]. treated with the tincture and 50% of patients treated with diazepam were fully remitted after 3 months. The score for the An uncontrolled study evaluated the efficacy of treatment depression component of the HAMA scale dropped from 2.80 for 8 weeks with an unspecified extract (0.3% hypericin, 3% to 1.73 after 1 month and to 0.79 after 3 months in the tincture hyperforin; 3 × 300 mg daily) in 26 adolescents with MDD (12–17 group, and from 2.95 to 1.86 after 3 months in the diazepam years, mean age 14.8 years). Of the 11 patients completing group. Similar results were obtained from D-S score evaluation the study, 9 (82%) showed significant clinical improvement [Warnecke 1986]. based on CGI change scores. Of the 15 patients (58%) who did not complete the study, 8 were non-compliant and 7 were Clinical studies performed with other preparations discontinued because of persisting or worsening depression [Simeon 2005]. In a randomised, double-blind, multicentre, three-armed study involving 348 outpatients suffering from mild to moderate An unspecified extract was compared with FLU in a randomised, depression, diagnosed in accordance with ICD-10, three double-blind trial in 72 patients with mild to moderate preparations containing a dry extract (DER 4-5:1; standardised depression. Patients were treated for 8 weeks with either a to 0.17 mg, 0.33 mg or 1 mg of total hypericins per day) were standardised extract (not futher specified; 900 mg/day), FLU assessed for 6 weeks. The highest daily dose corresponded to (20 mg/day) or placebo. Intention-to-treat analysis showed 3 × 60 mg of the extract. Initial average HAMD scores of 16- no differences in changes to the mean scores of HAMD-21, 17 dropped to 8-9 after 6 weeks in all three groups. Response Montgomery-Asberg Rating Scale and CGI between the three rates (defined as reduction in HAMD ≥ 50% and/ or actual groups. Patients receiving the extract had the lowest remission HAMD score of ≤ 10) were 62%, 65% and 68% respectively. rates (12%, p=0.016) compared to FLU (34.6%) and placebo No significant differences between the three groups were (45%) [Moreno 2005]. detected [Lenoir 1999]. In a three-arm, randomised clinical trial, the effects of treatment In a randomised, double-blind, crossover study, the anti- for 12 weeks with an unspecified extract (810 mg/day) were depressant effect of a tea taken twice daily (at least 0.28 mg of compared with CIT (20 mg/day) and placebo in 81 patients total hypericins per day) was assessed in 19 patients (at least 60 with minor depression or milder forms of MDD (with at least

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of two to four symptoms of major depression according to treated patients, 45.4% were classified as responders compared DSM-IV). Additional criteria included HAMD scores between with 20.9% for placebo (p=0.0006). Tolerability of the extract 10 and 17, a GAF score of less than 70, and either an MOS treatment was equivalent to placebo [Müller 2004]. Social Functioning score of 75% or less or an MOS Emotional Role Functioning score of 67% or less. Due to a high placebo Social phobia response on all outcome measures, neither the extract nor CIT Forty patients with generalised social were differed from placebo [Rapaport 2011]. randomised to 12 weeks of treatment with a flexible dose (600- 1800 mg) of a methanolic (80%) extract or placebo. Subjects Clinical studies - other conditions with co-morbid depression (HAMD>16) were excluded. No significant difference (p=0.27) in mean change on the Liebowitz Attention-deficit hyperactivity disorder (ADHD) Social Anxiety Scale was observed between the extract (11.4) An unspecified standardised extract (0.3% hypericin) was admin- and placebo (13.2) [Kobak 2005b]. istered in a randomised, double-blind, placebo-controlled trial to children aged 6 to 17 years who met DSM IV criteria for Obsessive-compulsive disorder ADHD. After a placebo run-in phase of 1 week, participants In a randomised, double-blind, placebo-controlled, parallel were randomly assigned to receive either placebo or 300 group study sixty subjects with obsessive-compulsive disorder mg of the extract 3 times daily for 8 weeks. No significant (OCD) were randomised to 12 weeks of treatment with a difference was found in the change in ADHD Rating Scale-IV methanolic (80%) extract in a flexible-dose schedule (600-1800 scores from baseline to week 8 between the treatment and mg/day p.o.) or placebo. The mean change in the Yale-Brown placebo groups: inattentiveness improved by 2.6 points (95% Obsessive-Compulsive Scale (Y-BOCS) with the extract (3.43) confidence interval [CI], -4.6 to -0.6 points) with the extract vs. as well as Y-BOCS subscales were not significantly different 3.2 points (95% CI, -5.7 to -0.8 points) with placebo (p=0.68) from the mean change with placebo (3.60). The percentage of and hyperactivity improved by 1.8 points (95% CI, -3.7 to patients rated as 'much' or 'very much' improved at endpoint 0.1 points) with the extract vs. 2.0 points (95% CI, -4.1 to 0.1 was not significantly different between the extract treatment points) with placebo (p=0.89). There was also no significant (17.9%) and placebo (16.7%) (p=0.905) [Kobak 2005a]. difference in the percentage of participants who met criteria for improvement (score ≤ 2) on the CGI Improvement Scale. Autism No difference between groups was found for adverse effects An unspecified extract was assessed in an open trial in three [Weber 2008]. male patients with autistic disorder. Parent and mentor ratings on the Aberrant Behaviour Checklist, irritability, stereotypy An unspecified extract was given at a daily oral dose of 30 and inappropriate speech factors improved slightly during mg for 4 weeks in an open trial to three 14-16-year-old male treatment. Ratings such as the Psychiatric Rating Scale Autism, psychiatric outpatients with ADHD. After this treatment, patients Anger and Speech Deviance factors, Global Assessment Scale received a placebo for a further 4 weeks. Assessed by the Conner and CGI efficacy did not improve significantly [Niederhofer Scale and by the Continuous Performance Test, at baseline and 2009]. at the end of each treatment, patients' mean scores improved for Conners' hyperactivity, inattention and immaturity factors Menopause [Niederhofer 2010]. Women aged from 40 to 65 years (n=47) experiencing hot flushes (≥ 3 per day, Heart and /Progestin Replacement Study Somatoform disorder scale) were randomly assigned to receive an ethanolic extract The efficacy of a methanolic (80%) extract was studied in a (300 mg 3 times daily) or placebo. After 12 weeks of treatment, multicentre, double-blind, placebo-controlled trial in 151 a non-significant difference favouring the extract group was out-patients suffering from somatisation disorder (ICD-10: observed in the daily hot flush frequency (extract -2.3 ± 3.6; F45.0), undifferentiated somatoform disorder (F45.1), or placebo -1.0 ± 2.2; p=0.11) and the hot flush score ( 3.8 ± 8.3 somatoform autonomic dysfunctions (F45.3). Patients received and -1.8 ± 6.5 respectively; p=0.10). After 3 months of treatment, the extract (600 mg/day p.o.) or placebo for 6 weeks. The extract the women in the extract group reported significantly better demonstrated significantly (p=0.001) greater efficacy in the quality of life (p=0.01) and significantly less sleep problems primary outcome criterion Hamilton Anxiety Scale, subfactor (p=0.05) compared to placebo [Al-Akoum 2009]. somatic anxiety (HAMA-SOM) [decrease from 15.39 to 6.64 in the extract group and from 15.55 to 11.97 in the placebo group]. Women (n=100, mean age 50.4 years) with climacteric com- The extract was also significantly more effective as measured plaints according to the Blatt-Kupperman Index were treated with by the CGI (p=0.006), HAMA-total score (p=0.0001), subscore 20 drops 3 times per day of an extract (containing 0.2 mg/ml psychic anxiety (p=0.001), HAMD (p=0.001), Self-Report hypericin; not further specified) or placebo for 8 weeks. Although Symptom Inventory 90 items – revised (SCL-90-R; p=0.001), and both groups responded to the interventions, the differences SCL-90-R, subscore somatic anxiety (SCL-90-R-Som; p=0.001). between the groups in frequency, duration, and severity of hot Tolerability of the extract was excellent [Volz 2002]. flushes were significant (p<0.05). The duration of hot flushes was significantly decreased in the extract group compared with In a prospective, randomised, double-blind, placebo-controlled, placebo in week 8 (p<0.001), but not in week 4 (p=0.085). The parallel group study, outpatients with somatisation disorder decrease in frequency of hot flushes was significantly greater (ICD-10 F45.0), undifferentiated somatoform disorder (F45.1) in the extract group compared to placebo in weeks 4 and 8 and somatoform autonomic dysfunction (F45.3), received either (p=0.05 and p<0.001 respectively). The decrease in the severity 300 mg of a methanolic (80%) extract twice daily or placebo of flushes was more evident in the extract group in weeks 4 and for 6 weeks. Somatoform Disorders Screening Instrument-7 8 (p=0.004 and p<0.001 respectively) [Abdali 2010]. days (SOMS-7), somatic subscore of the HAMA, SCL-90-R, subscores "improvement" and "efficacy" of the CGI, and the St. John’s wort was found to be significantly superior to placebo global judgment of efficacy by the patient were measured. In (standard mean difference = -1.08; 95% confidence interval: 1.38 the intention-to-treat population, for each of these measures to -0.77) in a meta-analysis comparing the efficacy and adverse as well as for the combined test, significant superiority of events for extracts of St. John's wort, combination products and treatment over placebo was demonstrated (p<0.0001). Of the placebo in menopausal women [Liu 2013].

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Premenstrual symptoms (PMS) of patients with adequate relief was also significantly higher in Women with (n=19) took a methanolic the placebo group (p=0.02) [Saito 2010]. (80%) extract for two complete menstrual cycles (300 mg extract per day standardised to 900 µg hypericin). The Hospital Anxiety Burning mouth syndrome (BMS) and Depression (HAD) scale and a modified Social Adjustment A double-blind, randomised, placebo-controlled, single-centre Scale were measured at baseline and after one and two menstrual study evaluated patients with BMS (n=39) receiving oral doses cycles. There were significant (p<0.05) reductions in all outcome (300 mg, 3 times daily) of an unspecified extract (hypericin measures. The degree of improvement in overall premenstrual 0.31%, hyperforin 3.0%) or placebo for 12 weeks. Burning pain, syndrome scores between baseline and the end of the trial measured using a visual analogue scale (VAS), was only slightly was 51%, with over two-thirds of participants demonstrating more improved in the extract treated group [Sardella 2008]. at least a 50% decrease in symptom severity [Stevinson 2000]. Antiviral effects In a randomised, double-blind, placebo-controlled, crossover In a phase I dose escalation study, the antiviral activity of study 36 women (18-45 years) with regular menstrual cycles hypericin was assessed in 19 patients with chronic HCV infection. (25-35 days), who were prospectively diagnosed with mild For 8 weeks, 12 patients received 0.05 mg/kg and 7 patients PMS, underwent a two-cycle placebo run-in phase. They then 0.1 mg/kg hypericin orally once a day. At the end of the 8 week received either 900 mg/day of a methanolic extract (80%; period, plasma HCV RNA level did not change more than 1.0 standardised to 0.18% hypericin; 3.38% hyperforin) or placebo log10 units. No anti-HCV activity was noted [Jacobson 2001]. for two menstrual cycles. After a placebo wash-out month the treatments were crossed over for two further cycles. The extract Antibacterial effects was superior to placebo in improving physical and behavioural In a randomised, placebo-controlled, double-blind study, symptoms of PMS (p<0.05) but not superior for mood- and pain- 21 patients with mild-to-moderate atopic dermatitis (mean related symptoms. Plasma hormone (FSH, LH, E2, , SCORAD score 44.5) were treated twice daily for 4 weeks with PRL and testosterone) and cytokine (IL-1b, IL-6, IL-8, IFN-g and either a cream containing an unspecified extract (1.5% hyper- TNF-a) levels, as well as weekly reports of anxiety, depression, forin) or placebo (vehicle). The intensity of the eczematous aggression and impulsivity, did not differ significantly between lesions was significantly (p<0.05) improved with the verum treatments (p>0.05) [Canning 2010]. compared to the vehicle at all clinical visits (days 7, 14, and 28). Skin colonisation with Staphylococcus aureus was reduced Patients with PMS (n=170) received an unspecified extract (two by verum and placebo treatments, with a trend towards better 680 μg “hypericin tablets” per day) or placebo for 8 weeks. antibacterial activity for the verum (p=0.064) [Schempp 2003a]. Treatment with the extract resulted in significantly lower PMS scores compared with baseline (p<0.001) and control (p<0.001). Wound healing The biggest improvements in score occurred for crying (71%) In a randomised, double-blind clinical trial (n=144), ointments and depression (52%). The drop-out rate due to adverse events containing either an oily extract or placebo were applied was higher in the extract group (p=0.02), although there were topically to surgical wounds 3 times daily for 16 days, starting no significant differences in adverse events. Symptom ratings from 24 h after caesarean section. An additional control group were reduced by 40% after extract treatment [Ghazanfarpour remained without any post-operative intervention. Compared 2011]. to both placebo and control groups, there were significant improvements observed in the extract group for wound healing Irritable bowel syndrome (IBS) on the 10th day (p<0.005) and scar formation on the 40th day An unspecified extract was given for 8 weeks at an oral dose postpartum (p<0.0001). Significantly lower pain (p<0.001) of 900 mg/day to 30 female patients with IBS. Patients and 20 and pruritus (p<0.001) were reported by the treatment group healthy controls underwent serial autonomic nervous system compared with the placebo and control groups on day 40 function tests before and after 8 weeks of treatment. Five-minute- [Samadi 2010]. short-time heart rate variability spectral analysis was assessed at rest and during three different types of stress test (somatic, Pharmacokinetic properties psychological and visceral stimuli). HAMD and HAMA scores were used to measure depression and anxiety; and patients Pharmacokinetics in vitro completed a gastrointestinal (GI) symptom diary once daily Variations in the matrices of different extracts affected their during the 2 weeks before therapy and for 2 weeks up to the last absorption and efflux as shown for three extracts submitted scheduled therapy session. Compared to the untreated control to a human intestinal Caco-2 cell transport experiment. group, the IBS-group had higher initial scores in the HAMA and Transepithelial transport across the Caco-2 cell monolayer HAMD (both p<0.01); with these scores significantly decreasing was different for rutin, hyperoside and isoquercitrin founds in post-treatment (p<0.05). Compared to the resting state, the IBS extracts when compared to the respective pure compounds. group exhibited significant increases in the low frequency band/ The results clearly revealed that the product matrices affected high frequency band ratio (L/H) during each period of stress permeability of the key components [Gao 2010]. testing (p<0.01). Treatment with the extract for 8 weeks led to a significantly decreased (p<0.05) L/H response to the various The metabolism of hyperforin was characterised using human stressors when compared to baseline. GI symptoms of IBS were liver microsomes and CYPs, CYP-selective chemical inhibitors also significantly (p<0.05) relieved by the treatment [Wan 2010]. and correlation with CYP-recombinant heterologously expressed CYP enzymes. Among 57 detected metabolites six In a randomised, double-blind, placebo-controlled trial, 70 mono¬hydroxylated compounds (M1-M6) were identified, patients with IBS received either an unspecified extract at an while others were higher hydroxylated. A combined approach oral dose of 900 mg or placebo for 12 weeks. Of the patients, of cDNA-expressed recombinant specific marker activities 29% had constipation dominated IBS (C-IBS), 37% diarrhoea indicated a central role of the CYP2C and CYP3A families in dominated IBS (D-IBS), and 31% had mixed IBS. Both groups the metabolism of hyperforin [Hokkanen 2011]. reported decreases in overall bowel symptom score from baseline, though the placebo arm had significantly lower scores Interactions at 12 weeks (p=0.03) compared with the extract. The percentage After administration of St. John's wort extracts and some of its

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components, two types of qualitatively different pharmacokinetic probe substrate, inhibited CYP3A4 activity. Hypericin had interactions have been observed: no effect on any of the enzymes tested [Komoroski 2005; Komoroski 2004]. • After acute administration: inhibition of metabolic enzymes and drug transporters [Budzinski 2000; Chaudhary 2006; Human hepatocytes were exposed to hyperforin (0.1, 0.5 or Dostalek 2011; Dostalek 2005; Foster 2004; Hokkanen 2011; 1.5 µM) or rifampin (10 µM) for 48 hours. The medium was Komoroski 2005; Komoroski 2004; Lee 2006; Obach 2000; then replaced by one containing (100 µmol/L). Ott 2010; Patel 2004; Schwarz 2011; Tuna 2010; Volak 2010; Docetaxel metabolism was characterised by HPLC/MS after Wada 2002; Wang 2004a; Weber 2004]. 1 hour: metabolism induced by rifampin was 6.8- to 32-fold higher than that in control cultures. Hyperforin induction was • After subchronic or chronic administration: induction of dose-dependent and, at maximum, was 2.6- to 7-fold greater several enzymes of the cytochrome P450 (CYP) enzyme than that in controls. CYP3A4 and CYP2C8 were involved in family (CYP1A1, CYP1A2, CYP3A, CYP2B6, CYP2C8 and metabolite formation [Komoroski 2005]. CYP2C9, C2C19 and CY2E1) [Chen 2004; Gutmann 2006; Hellum 2009; Komoroski 2005; Komoroski 2004; Krusekopf Seven flavonoids in St. John's wort and were screened 2005; Sahi 2009], UDP-glucuronyltransferases (UGT1A1, for their inhibition of recombinant human CYP1B1 and CYP1A1. UGT1A6) [Volak 2010], and several drug efflux transporters While , apigenin, kaempferol, quercetin, amento- from the ATP-binding cassette transporters superfamily flavone, quercitrin and rutin were slightly more selective for

(ABC transporters, such as P-glycoprotein (MDR1/ABCB1) CYP1B1 ethoxyresorufin-O-deethylase (EROD) inhibition (Kis or MRP2 (ABCC2), MRP3 (ABCC3)) [Gutmann 2006; Ott 0.06 - 5.96 µM) compared to CYP1A1 (Kis 0.20 - 1.6 µM), the 2010; Weber 2004], shown in vitro and in animal trials. differences in Kis for the P450s were not significantly different. One mechanism of the interactions is the activation of the Rutin did not inhibit CYP1A1 at concentrations up to 10 µM. In (PXR) by hyperforin [Moore 2000; kinetic analyses apigenin and amentoflavone were competitive Watkins 2003; Wentworth 2000]. The extent of PXR inhibitors of CYP1B1, while quercetin showed mixed type mediated gene induction was proportional to the extent of inhibition. Five flavonoids were studied further for their ability to hyperforin in the extracts [Gödtel-Armbrust 2007; Mueller inhibit TCDD-induced EROD activity in 22Rv1 human prostate 2009; Mueller 2006]. cancer cells. 22Rv1 cells expressed constitutive and TCDD-

inducible CYP1A1 and CYP1B1 mRNA. The IC50 values were Acute administration similar to those measured for the recombinant CYP1A1 except

Unspecified extracts in methanolic (1%) solution were for amentoflavone. Quercetin (IC50: 4.1 µM), kaempferol (3.8 examined (using microsomal enzyme preparations) for their µM), myricetin (3.0 µM) and apigenin (3.1 µM) caused significant inhibition towards CYP1A2, CYP2C9, CYP2C19, CYP2D6 and (p<0.05) inhibition of EROD activity whereas amentoflavone CYP3A4. CYP2D6 was the most sensitive. In the assay of single did not cause inhibition [Chaudhary 2006]. components, CYP1A2 was inhibited by I3,II8-biapigenin (IC50 3.7 µM) and quercetin (IC50 7.5 µM), CYP2C9 by hyperforin Further investigations confirmed this activity of hyperforin, (IC50 4.4 µM), I3,II8-biapigenin (IC50 4.0 µM) and hypericin (IC50 demonstrating the inhibition of recombinant CYP3A4 (IC50 3.4 µM), CYP2C19 by hyperforin (IC50 31 µM), I3,II8-biapigenin 0.63 μM) [Lee 2006] and of CYP3A4 (IC50 4.4 - 9.6 µM) and (IC50 28 µM) and hypericin (IC50 37 µM), CYP2D6 by hyperforin CYP2D6 (IC50 7.3 µM) [Hokkanen 2011]. (IC50 1.6 µM), I3,II8-biapigenin (IC50 5.7 µM), quercetin (IC50 24 µM) and hypericin (IC50 8.5 µM) and CYP3A4 by hyperforin A study of the inhibition of CYP1A1-mediated 2-hydroxylation (IC50 2.3 µM), I3,II8-biapigenin (IC50 0.082 µM), quercetin (IC50 of oestradiol by a methanolic (80%, V/V) extract and single 22 µM) and hypericin (IC50 8.7 µM) [Obach 2000]. components found IC50 values of 19.5 µg/mL for the extract, 9.8 µM (hypericin), 6.8 µM (pseudohypericin), 3.2 µM (quercetin), Two ethanolic extracts (A: 50% m/m; B: 60% V/V) and a 2.2 µM (kaempferol) and 2.2 µM (myricetin). No effects were methanolic extract (C: 80% m/m), as well as hypericin, pseudo- seen for hyperforin and rutin [Schwarz 2011]. hypericin, hyperforin, rutin and quercetin, were investigated for their inhibitory effects on carcinogen activation by CYP1A1 using The effects of hyperforin and hypericin on Pgp activity were purified enzyme preparations. The extracts potently inhibited compared to CyA in the MDR1 over-expressing cell line NIH-

CYP1A1 activity (IC50 values: 24.0 ± 3.9 µg/mL (A), 8.0 ± 1.0 3T3-G 185. Both compounds inhibited Pgp activity with an µg/mL (B), 1.3 ± 0.2 µg/mL (C)). All constituents, except rutin, IC50 of about 30 µM, which was about 15-fold higher than for possessed strong inhibitory potencies with IC50 values of 0.5 ± ciclosporin (2 µM) [Wang 2004a]. 0.1 µM (hypericin), 8.0 ± 0.1 µM (pseudohypericin), 1.2 ± 0.2 µM (hyperforin) and 1.5 ± 0.2 µM (quercetin) [Schwarz 2003]. A methanolic (80%, m/m) extract and some of its components were investigated for their effect on Pgp activity in the calcein-AM The inhibition of human cytochrome P450 enzymes by 18 St. assay using porcine brain capillary endothelial cells (PBCEC) as John’s wort products containing a wide variety of hyperforin, a model of the blood-brain barrier as well as in a human MDR1 hypericin and pseudohypericin levels was assessed. The aqueous over-expressing lymphocytic leukaemia-derived cell line VLB extracts markedly inhibited cytochrome P450-mediated (CYP 100. The extract inhibited Pgp activity above a concentration of 2C9*1, 2C9*2, 2C19, 2D6, 3A4, 3A5 and 3A7) metabolism and 100 µg/mL (p<0.05) in both cell lines. Hyperforin (1 - 10 µM), inhibited P-glycoprotein (Pgp) activity [Foster 2004]. hypericin (0.03 - 30 µM), quercetin (0.01 - 3 µM), hyperoside (0.3 and 3 µM), rutin (10 µM) and biapigenin (10 and 30 µM) In MDR1 transfected MDCK cells, hypericin and quercetin at showed significant (p<0.05 to p<0.001) inhibitory effects on concentrations of 5 to 200 µM showed a significant (p<0.05) Pgp activity in PBCEC. Amentoflavone activated P-glycoprotein and dose-dependent inhibition of Pgp-mediated efflux of [3H]- at 3 and 30 µM (p<0.05), quercetin and biapigenin activated and inhibited CYP3A4 activity (hypericin IC50 0.87 µM, Pgp at the lowest concentration and inhibited it at the higher quercetin IC50 0.53 µM, kaempferol IC50 0.26 µM) [Patel 2004]. concentrations (p<0.05). In VLB 100 cells, only 10 µM hyperforin inhibited Pgp (p<0.01) [Weber 2004]. In primary cultures of human hepatocytes, hyperforin at high concentrations of 5 and 10 µM, 1 hour before and along with A methanolic extract (80%, V/V; 0.1 – 5 µg/mL), hyperforin

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(0.1 – 10 µM), hypericin (1 – 50 µM) and quercetin (1 – 50 at 8 and 80 µg/mL (p<0.05), while a strong inhibition was µM) decreased Pgp transport activity in a dose- and time- observed at 800 µg/mL (p<0.05). CYP2E1 activity was increased dependent manner in PBCEC cells and freshly isolated porcine by 65% (p<0.05) at 8 µg/mL and completely inhibited at brain capillaries. The extract and hyperforin directly inhibited 800 µg/mL [Hellum 2009]. Pgp activity, whereas hypericin and quercetin modulated transporter function through a mechanism involving protein Pharmacokinetics in animals kinase C. Quercetin decreased Pgp transport activity at high After intravenous injection of 350 µg hypericin (about 17.5 concentrations (> 10 µM), but increased transporter function mg/kg), peak plasma concentrations in BALB/c mice were 27.8 at low concentrations (≤ 10 µM) [Ott 2010]. µg/mL at 10 minutes after administration, which decreased to 0.010 µg/mL after 240 h. The AUC was 285 µg×h/mL, the Hypericin bound and inhibited the major glutathione-S-trans- volume of distribution 12.6 mL and the elimination half-life ferase isoforms GST-a (Ki 0.16 ± 0.02 µM to 1.91 ± 0.21 µM) 38.5 h [Liebes 1991]. and GST p (0.55 ± 0.07 µM to 2.46 ± 0.43 µM) [Tuna 2010]. After oral administration of [14C]-labelled hypericin and pseudo- Many UDP-glucuronosyltransferases (UGTs) require hypericin to 8 female mice, radioactivity was measured in the phosphorylation by protein kinase C (PKC) for glucuronidation GI tract and in liver, kidney, muscle, blood and brain. At 1.5 activity. Hypericin, a non-selective PKC inhibitor, inhibited and 6 h after administration, 42% and 19% of the hypericin paracetamol glucuronidation by human LS180 cells (IC50 = dose and 69% and 44% of the pseudohypericin dose were 7.1 ± 0.6 μM). In UGT1A6-infected Sf9 insect cells, hypericin detected in the . A proportion of 31% and showed a three times more potent inhibition of serotonin 11% radioactivity from hypericin was found in muscle after 1.5 glucuronidation in treated whole cells versus cell lysates (IC50 and 6 hours. In blood and brain minor quantities were found for = 0.59 ± 0.05 μM) [Volak 2010]. both compounds, in the brain 0.7% and 0.6% of the hypericin dose and 0.4% and 0.9% of the pseudohypericin dose at 1.5 Sub-chronic or chronic administration and 6 hours respectively [Stock 1991]. LS180 cells were exposed for 72 hours to a methanolic (80%) extract at concentrations of 3 to 300 µg/mL or hyperforin (0.03 After an i.v. administered dose of [14C]-hypericin and [14C]- to 3 µM) for 3 days. Pgp expression was strongly and dose- pseudohypericin in mice, 80% of hypericin and 60% of pseudo- dependently induced by the extract (400% increase at 300 µg/mL) hypericin was absorbed. Tissue distribution was followed for 2, and by hyperforin (700% at 3 µM). In Caco-2 cell monolayers, 4, 6, 8, 24, 72 and 168 hours. The highest tissue concentrations the extract and hyperforin caused a moderate inhibition of Pgp- were observed in the lung with over 3.5 µg/g at 4 h, gradually mediated transport at the highest concentration [Perloff 2001]. declining to less than 2% of the maximum after 7 days. The concentrations in the tissues were: lung > spleen > liver > blood LS180 cells were cultured for 48 hours in the presence and > kidney > heart > gut > tumour > stomach > skin > muscle > absence of three doses of an extract (not further specified) con- brain [Chung 1994]. taining 5.61% hyperforin. The extract increased the expression of CYP1A2 in a dose-dependent manner [Karyekar 2002]. The tissue distribution of hypericin (2, 5 or 20 mg/kg i.p.) in DBA/2 mice bearing subcutaneously implanted P388 lymphoma In primary cultures of human hepatocytes, after 48 hours cells resulted in a very high uptake in liver and spleen. Clearance incubation with 10 µM hyperforin or rifampin, CYP2B6 from plasma followed a two-phase exponential decay: a first expression was induced approximately 4-fold and 15-fold phase of rapid clearance (half-life 6.9 hours) was followed by respectively [Goodwin 2001]. Treatment for 96 h resulted in a slower phase (half-life 37.9 hours) [Chen 2000]. significant (p<0.05) increases in mRNA and protein expression, as well as activity of CYP3A4 and CYP2C9, but had no effect After oral administration of an ethanolic extract containing on CYP1A2 or CYP2D6 [Komoroski 2005; Komoroski 2004]. 5% hyperforin to rats at 300 mg/kg, maximum plasma levels of 370 ng/mL hyperforin were reached after 3 hours. Estimated HepG2 cells were treated with 0.1% (V/V) of an ethanolic (50% half-life and clearance values were 6 hours and 70 mL/min/kg m/m) extract or 0.1 µM hyperforin for 24 hours and RNA was [Biber 1998]. subjected to gene array analysis. The extract and hyperforin affected the expression of genes that mediate metabolism (e.g. In Sprague-Dawley rats, i.p. administration of 10 mg/kg CYP3A4, CYP1A1, CYP1A2) and transport exogenous and hyperforin led to plasma levels in the low micromolar range endogenous compounds (e.g. MRP2), but also of genes involved (3 - 5 µM). No hyperforin was detected in the cerebrospinal in energy metabolism, intracellular calcium regulation, cell fluid. At 1 mg/kg the compound was detected in the plasma proliferation and apoptosis [Krusekopf 2005]. [Buchholzer 2002].

An extract and hyperforin but not hypericin increased the In a study of the metabolic profile of hyperforin in liver microsomes expression of Pgp in LS180 cells. Incubation with the extract from male and female Sprague-Dawley rats, with or without (20 to 75 µg/mL) for 24 hours led to a dose-dependent increase induction by or , four major phase of MDR1 mRNA expression that exceeded the positive control I metabolites (19-hydroxyhyperforin, 24 hydroxyhyperforin, rifampin (10 µM). Removal of the extract resulted in restoration 29-hydroxyhyperforin and 34-hydroxyhyperforin) were of basal Pgp levels within 48 hours [Tian 2005]. identified. Results suggested hydroxylation as a major bio- transformation of hyperforin in rat liver, mainly by inducible An ethanolic extract (50% m/m, low hyperforin content) did CYP3A and/or CYP2B [Cui 2004]. not affect MDR1 or CYP3A4 expression in LS180 cells. MDR1 expression was significantly (p<0.01) up-regulated by hypericin In male CD rats, procyanidin B2 increased the oral bioavailability and hyperforin (each 10 µM), and CYP3A4 expression by of hypericin by approximately 58% and hyperoside by 34%. hypericin, quercitrin and hyperforin [Gutmann 2006]. The compounds had a different influence on the plasma kinetics of hypericin; median maximal plasma levels of hypericin were

An ethanolic (60%) extract incubated for 72 hours increased the detected after 360 min (Cmax 8.6 ng/mL) for B2, and after 150 metabolic activity of CYP2C19 in primary human hepatocytes min (Cmax 8.8 ng/mL) for hyperoside [Butterweck 2003b].

43 HYPERICI HERBA

Unspecified extracts (containing either 4.5 or 0.5% hyperforin) more elevated in females (93 ± 7 ng/mL) than in males (32 ± and hyperforin dicyclohexylammonium (DCHA) were given to 3 ng/mL) [Radu 2009]. male CD-COBS rats. The 4.5% extract, administered as three i.p. injections in 24 h (3.12-6.25 mg/kg), resulted in dose-related An experiment investigating fractions of extracts for their plasma concentrations of hyperforin which were of similar antidepressant activity showed that the amount of hypericin magnitude to those found following the administration of hyper- needed for significant antidepressant activities in the FST was forin DCHA that contained hyperforin at a level equivalent very high (0.23 mg/kg p.o.), whereas a hypericin-rich fraction to the 4.5% extract (0.14 and 0.28 mg/kg). Hyperforin was was active at doses of 0.028 - 0.16 mg/kg. The fraction contained undetectable in rat brain [Cervo 2002]. procyanidin B2 which increased solubility of hypericin in water, which for hypericin alone was 0.5 µg/mL compared to more

The determination of plasma levels of hypericin in rats in than 60 µg/mL with increasing amounts of procyanidin B2. the presence and absence of procyanidin B2 or hyperoside Higher solubility of hypericin led to increased bioavailability showed increased oral bioavailability of hypericin by ca. 58 % and to increased in vivo activity as shown by co-administration procyanidin B2 and 34% (hyperoside). Median maximal plasma of hypericin with procyanidin B2, thereby increasing the AUC levels of hypericin were detected after 360 min (Cmax: 8.6 ng/ to 160% [Nahrstedt 2010]. mL) for procyanidin B2, and after 150 min (Cmax: 8.8 ng/mL) for hyperoside [Butterweck 2003b]. Hypericin was administered as an i.v. bolus of 2 mg/kg (n = 3) or 5 mg/kg (n = 1) to male Rhesus monkeys. Plasma and In mice treated with 15 mg/kg hyperforin, either as the sodium cerebrospinal fluid (CSF; ventricular or lumbar) were sampled salt or as a 5% extract, average brain tissue concentrations of prior to administration and at frequent intervals for up to 50 h hyperforin were found to be higher for the sodium salt group after administration of the drug. Mean peak plasma concentration (28.8±10.1 ng/g) than the extract group (15.8±10.9 ng/g) of hypericin following the 2 mg/kg dose was 142 ± 45 µM. [Keller 2003]. Elimination of hypericin from plasma was biexponential, with

an average distribution half-life of t½,a of 2.8 ± 0.3 h and an After administration of 300 mg/kg p.o. of an ethanolic extract average terminal half-life of t½,b of 26 ± 14 h. The 2 mg/kg dose (4.5% hyperforin) to male CD1 mice, hyperforin rapidly was sufficient to maintain plasma concentrations above 10 µM reached the systemic circulation, with a Cmax of approximately for up to 12 h. No hypericin was detected in the CSF of any 1 µM within 60 min. Blood levels peaked faster (30 minutes) animal (LOD 0.1 µM) [Fox 2001]. after the application of hyperforin DCHA salt at 18.1 mg/kg. Hyperforin was quantifiable up to 4 - 6 hours post administration. Interactions

Interestingly, the Cmax of hyperforin was reduced by approxim- The effects of a co-administered methanolic (80%) extract on the ately 50% after repeated dosing (4 days), correlating with an pharmacokinetics of CPT-11 were evaluated in rats pre-treated induction of CYP3A enzymes by hyperforin [Cantoni 2003]. with the extract (400 mg/kg p.o. for 8 days, 6 mL/kg) or vehicle. CPT-11 was given at a dose of 60 mg/kg per day by i.v. injection Wistar rats treated with an unspecified extract (1000 mg/kg/ via tail vein for 4 consecutive days. Co-administration with the day) for 1, 3 or 7 days were administered a single oral dose extract significantly (p<0.05) altered the pharmacokinetics of of at 10 mg/kg after their final extract dose. Oral both CPT-11 and its metabolite SN-38, and reduced the AUC clearance of midazolam in the extract treated rats increased of the SN-38-glucuronide by 31.2% [Hu 2005]. time dependently, and was significant (p<0.01) after 7 days of treatment with the extract. The midazolam-1-hydroxylation Swiss Webster mice received either A) an unspecified ethanolic activity in liver microsomes obtained from the extract treated extract for 2 weeks (oral dose not clearly defined); B) the same rats was significantly (p<0.01) higher than in controls. dose of the extract for 1 week; C) a single dose of the extract; Linear correlation was observed between oral clearance and D) no extract; E) hypericin (10 µg); or F) control. All groups midazolam-1-hydroxylation activity in the liver microsomes, received a single dose of procainamide (100 mg/kg p.o.), 24 suggesting that CYP3A induction in the liver mainly decreased h after the last extract dose in groups A and B, and 1 h later the midazolam concentration in plasma. Immunoblotting in group C. The mean procainamide concentration 1 h after revealed that CYP3A was induced within 3 days of extract administration was highest in group C (11.59 µg/mL) followed treatment [Qi 2005]. by group A (9.92 µg/mL), whereas group B (7.44 µg/mL) and the control group D (7.36 µg/mL) showed comparable values. After single oral doses of an ethanolic extract (60%; 1600 mg/kg) The concentration in group C was significantly higher than and isoquercitrin (100 mg/kg) to male Sprague Dawley rats, in the control group D (p=0.03). N-Acetyl procainamide maximal CNS levels for quercetin (340 ng/g) and isorhamnetin/ concentrations and estimated half-life of procainamide were tamarixetin (50 ng/g) were found at 4 h. With repeated daily comparable between the groups. With hypericin, a significant doses over 8 days the maximal cumulation for quercetin (367 (p<0.05) increase in bioavailability (53%) of procainamide ng/g) occurred after 5 days, whereas isorhamnetin/tamarixetin was observed compared to control group F [Dasgupta 2007]. (640 ng/g) did not reach its maximal cumulation level within the 8-day test period [Paulke 2008]. Oral administration of either 150 or 300 mg/day of an unspecified extract for 15 days significantly reduced plasma levels of After administration of an unspecified extract, brain concen- indinavir. A perfusion study demonstrated that both the small trations of amentoflavone were below the limit of quantification. intestine and the liver contributed significantly (both p<0.05) Levels were consistently detected only after i.p. amentoflavone to the reduction of indinavir bioavailability. The effect was flow (10 mg/kg), with a mean brain-to-plasma ratio of about 0.02. rate-dependent. The small intestine was the major site for the These concentrations were possibly related to the compound's pre-systemic metabolism of indinavir, with or without extract contribution from residual blood, but in any case are too low to pre-treatment [Ho 2009]. support any interaction with central mechanisms so far tested [Colovic 2008]. An unspecified extract was given at an oral dose of 400 mg/kg/day to male Wistar rats. Daily administration for 10 days increased In mice treated for a month with an unspecified extract (1500 respectively the amounts of MRP2, glutathione S-transferase-P mg/kg p.o.), hypericin plasma levels were significantly (p<0.05) (GST-P) and CYP1A2 in the liver to 304%, 252% and 357%

44 HYPERICI HERBA

of control levels, the amounts of P-gp and MRP1 were not hypericin, observed about 4 hours after administration, were changed. MRP2 did not appear to increase in the kidney. The 0.028, 0.061 and 0.159 mg/L respectively [Brockmöller 1997]. increase in the levels of each protein was maximal at 10 days after extract treatment and lasted for at least 30 consecutive Oral administration to 13 healthy volunteers (at intervals of at days [Shibayama 2004]. least 10 days) of single doses of a methanolic extract containing 250, 750 and 1500 µg of hypericin and 526, 1578 and 3135 µg Male Sprague-Dawley rats were given a methanolic extract of pseudohypericin, respectively gave peak plasma levels of (80%; 1000 mg/kg/day p.o.) for 14 days. On day 15, were 1.3, 7.2 and 16.6 µg/L for hypericin and 3.3, 12.2 and 29.7 µg/L isolated and perfused in a recirculating system with fexofenadine for pseudohypericin. Cmax and AUC values for the lowest dose (FEXO; 2 µg/mL), either alone or following addition of CyA (0.5 were disproportionately lower than those for the higher doses. µg/mL) 5 min before the application of FEXO. Administration Lag-times were determined as 1.9 hours for hypericin and 0.4 of the extract significantly (p<0.05) increased biliary clearance hours for pseudohypericin. Mean half-lives for absorption, with respect to perfusate, and biliary clearance with respect to distribution and elimination were 0.6, 6.0 and 43.1 hours after the concentration in the liver, by 74% and 71% respectively. 750 µg of hypericin, and 1.3, 1.4 and 24.8 hours after 1,578 µg This was reversed by the Pgp inhibitor CyA [Turkanovic 2009]. of pseudohypericin. After 14 days of oral treatment with 250 µg of hypericin and 526 µg of pseudohypericin, steady state levels In male Wistar rats i.p. administration of 100 mg/kg/day of a of 7.9 µg/L for hypericin and 4.8 µg/L for pseudohypericin were methanolic (80%) extract for 10 days resulted in significant achieved. Kinetic parameters in two subjects after intravenous induction of CYP2D2 (p<0.001) and CYP3A2 (p<0.001) as administration resembled those after oral administration. well as inhibition of CYP2C6 (p<0.001) and CYP1A2 (p<0.001) Hypericin and pseudohypericin were initially distributed into activity in an isolated perfused rat model [Dostalek 2011; volumes of 4.2 and 5.0 L respectively; at steady state the mean Dostalek 2005]. distribution volumes were 19.7 L for hypericin and 39.3 L for pseudohypericin; systemic bioavailability from the methanolic A methanolic (80%) extract was given to male Wistar rats for extract was about 14 and 21% respectively [Kerb 1996]. 10 days at an i.p. dose of 100 mg/kg prior to liver preparation. Paracetamol formation from (12.5 mg/L) was used as Hypericin levels in serum and skin blister fluid were determined marker for CYP1A2 activity in this isolated perfused liver model. in volunteers after oral administration of a hydromethanolic The formation rate of acetaminophen was significantly inhibited extract as a single dose of 6 tablets or 3 x 1 tablet daily for 7 by pre-treatment with the extract (p<0.001) [Dostalek 2011]. days. Each tablet contained 300 mg extract, standardised to 900 µg of total hypericin. Six hours after the single high dose, A methanolic (80%) extract at a dose of 300 mg was given mean levels of total hypericin were 43 ng/mL in serum and 5.3 orally to four dogs every 24 h for 14 days. A single dose of CyA ng/mL in skin blister fluid. After 3 tablets daily for one week, (5 mg/kg) was given orally 7 days before and 7 and 14 days mean levels were 12.5 ng/mL in serum and 2.8 ng/mL in skin after start of the extract administration. Control dogs received a blister fluid [Schempp 1999]. single oral dose of CyA (5 mg/kg). The maximum whole-blood concentration and AUC(0-infinity) of the extract group were Plasma levels of hyperforin were measured over a 24-hour significantly lower and the CLtot /F and Vd /F were significantly period in volunteers treated with 300 mg of an ethanolic higher (all p<0.05) than those in the control group 7 and 14 extract containing 14.8 mg of hyperforin. Maximum plasma days after the start of extract administration [Fukunaga 2011]. levels of about 150 ng/mL were reached 3.5 hours after oral administration. Half-life and mean residence time of hyperforin Pharmacokinetic parameters of (EXB) were signifi- were 9 and 12 hours respectively. Up to 600 mg of the extract, cantly altered by oral co-administration to rats of an unspecified hyperforin kinetics were linear [Biber 1998]. extract (25 mg/kg/day) alongside EXB (15 mg/kg/day) for 3 weeks. The steady state peak plasma concentration and terminal In a phase I dose escalation study, patients received oral doses half-life were reduced by 32% and 91% respectively, due to a of hypericin once daily for 8 weeks, at either 0.05 mg/kg more than 3-fold increase in its apparent clearance which was (n=12) or 0.1 mg/kg (n=7). At the end of the 8 week period, a concentration and time dependent effect [Radwan 2012]. pharmacokinetic data revealed a long elimination half-life (mean values of 36.1 and 33.8 h respectively) and mean AUC Rats were given single oral doses of methotrexate (MTX; 5 mg/ determinations of 1.5 and 3.1 mg×h/mL respectively [Jacobson kg), alone or co-administered with either an unspecified extract 2001]. (300 or 150 mg/kg) or (25 mg/kg). The extract at a dose of 300 mg/kg significantly increased (both p<0.05) the In a placebo-controlled, randomised, double-blind study,

AUC(0-t) and Cmax of MTX, by 163% and 60% respectively, and 33 healthy volunteers received a methanolic (80%) extract at 150 mg/kg significantly (p<0.05) increased the AUC(0-t) of alongside various co-medications (placebo, cimetidine and

MTX by 55%. Diclofenac enhanced the Cmax of MTX by 110% ) for 7 days, after a run-in period of 11 days [Yang 2013]. with extract alone. No significant differences in AUC(0-24),

Cmax, and tmax values for hypericin and pseudohypericin were Pharmacokinetics in humans observed between the treatment groups [Johne 2004]. A study of the bioavailability of hypericin in 2 healthy volunteers after oral administration of an extract (300, 600 and 1200 mg at The bioavailability of five constituents from an ethanolic (80%) intervals of 7 days) demonstrated that plasma levels of hypericin extract was determined in two open clinical trials in 18 healthy were dose-dependent. After ingestion of a single dose of 600 male volunteers. Subjects received the extract (612 mg/day p.o.) mg of the extract by 12 volunteers, the following parameters either as a single oral dose or as a daily dose over a period of 14 were determined for hypericin: tmax 2.5 hours, Cmax 4.3 ng/mL days. Concentration/time curves were determined for hypericin, and a plasma half-life of about 6 hours [Weiser 1991]. pseudohypericin, hyperforin, quercetin and isorhamnetin for 48 h after single dosing and for 24 h on day 14 at the end of 2 weeks A methanolic extract was administered orally as single doses of continuous daily dosing. After single dose administration, the of 900, 1800 and 3600 mg, containing 2.81, 5.62 and 11.25 key pharmacokinetic parameters were determined as follows: x mg of total hypericin. Maximum plasma concentrations of total hypericin: AUC(0-infinity) = 75.96 ng h/mL, Cmax = 3.14 ng/mL,

45 HYPERICI HERBA

tmax = 8.1 h, t1/2 = 23.76 h; pseudohypericin: AUC(0-infinity) = to-dose ratios, with a median decrease to 47% of the original x 93.03 ng h/mL, Cmax = 8.50 ng/mL, tmax = 3.0 h, t1/2 = 25.39 h; concentration (range: 19% - 60% of the original concentration). x hyperforin: AUC(0-max) = 1009.0 ng h/mL, Cmax = 83.5 ng/mL, Two patients reported symptoms that suggested a withdrawal tmax = 4.4 h, t1/2 = 19.64 h. Quercetin and isorhamnetin showed syndrome [Eich-Hochli 2003]. two peaks of maximum plasma concentration separated by x about 4 h. Quercetin: AUC(0-infinity) = 318.7 ng h/mL, Cmax, Twelve healthy men, 6 extensive metabolisers of CYP2C19 1 = 47.7 ng/mL, tmax, 1 = 1.17 h, Cmax, 2 = 43.8 ng/mL, tmax, 2 (2C19*1 / 2C19*1) and 6 poor metabolisers (4 with 2C19*2 / = 5.47 h, t1/2 = 4.16 h; isorhamnetin: AUC(0-infinity) = 98.0 2C19*2 and 2 with 2C19*2 / 2C19*3), were enrolled in a two- x ng h/mL, Cmax, 1 = 7.6 ng/mL, tmax, 1 = 1.53 h, Cmax, 2 = 9.0 ng/ phase, randomised, crossover study. All subjects took oral doses mL, tmax, 2 = 6.42 h, t1/2 = 4.45 h. Similar results were obtained of either an unspecified extract (0.3% hypericin and a minimum under the steady-state conditions reached during multiple dose 4% hyperforin; 300 mg) or placebo three times daily for 14 days, administration [Schulz 2005]. and the activities of CYP2C19 and CYP1A2 were measured using mephenytoin and caffeine. The extract significantly increased Interactions CYP2C19 activity in CYP2C19 wild-genotype subjects, with The acute effects of an unspecified extract on CYP2D6 and urinary 4'-hydroxymephenytoin excretion raised by 151.5% CYP3A4 activity were studied in 7 healthy volunteers given a dose ± 91.9% (p=0.0156), whereas no significant alteration was of 3×300 mg/day p.o. for 3 days. Enzyme activity was assessed observed for CYP2C19 poor metabolisers. Repeated extract by phenotyping, using as probes 30 mg (DM) administration did not affect the CYP1A2 phenotypic ratio for for CYP2D6 and (ALPZ) for CYP3A4. Each probe either CYP2C19 genotype subjects [Wang 2004b]. was given before and during co-administration with the extract. Pharmacokinetics of ALPZ were measured in plasma, while DM In a trial involving 340 outpatients with major depression, treated and its metabolite (DX) were measured in urine with either a methanolic extract (80%; 900-1500 mg/day), SERT after collection for 8 hours, and the metabolic ratio DM-DX was (50-100 mg/day) or placebo for 8 weeks, treatment adherence determined. Compared to baseline, no significant differences was assessed by measuring plasma hyperforin. Samples from were observed for ALPZ or DM-DX ratios when probes were the placebo and extract groups were assayed for hyperforin, given during treatment with the extract [Markowitz 2000]. and samples from the SERT group for SERT/N-desmethyl-SERT. Of the 104 patients randomised to placebo, 18 (17%) had The effects of a methanolic extract (80%, V/V; 3 × 300 mg/ detectable plasma hyperforin. Of the 97 patients randomised day for 14 days) on the activity of CYP3A4 and CYP2D6 were to the extract, 17 (17%) had no detectable plasma hyperforin. assessed in 13 healthy volunteers by determination of the urinary All the assayed SERT patients (n = 91) had detectable plasma 6-b-hydrocortisol/cortisol and dextromethorphan–dextrorphan SERT/N-desmethyl-SERT plasma concentrations. The clinical (DM/DX) ratios at baseline and at the end of treatment [Roby trial conclusions remained unchanged when only patients with 2001]. The CYP2D6 participants received an additional 30 plasma assay consistent with random assignment were included mg of dextromethorphan hydrobromide on day 15. On the in the analyses [Vitiello 2005]. last day of both studies urine was collected over 24 hours and concentrations of 6-b-hydroxycortisol and cortisol and urinary Healthy volunteers (n = 18) received a methanolic (80%, DM–DX ratios were determined. The mean urinary 6-b-hydroxy- V/V) extract at a dose of 3 × 300 mg daily for 14 days. The cortisol/cortisol ratio significantly increased (p=0.003) from a CYP2D6 substrate debrisoquine (5 mg) was administered baseline value of 7.1 ± 4.5 to 13 ± 4.9. No significant change before and at the end of treatment. CYP2D6 activity was was seen for DM-DX ratios [Roby 2000]. assessed using 8-hour debrisoquine urinary recovery ratios (DURR): [4-hydroxydebrisoquine/(debrisoquine + 4-hydroxy- The effects of a methanolic (80%) extract on the urinary excretion debrisoquine)]. No significant effect on CYP2D6 activity was of D-glucaric acid, 6b-hydroxycortisol and free cortisol were found [Gurley 2008a]. investigated in order to assess its impact on the activity of hepatic xenobiotic metabolising enzymes. Forty-eight healthy volunteers The effects of an unspecified extract (3 x 300 mg/day p.o.) (25 males and 23 females) received a daily dose of 1800 mg of on oral contraceptive (OC) therapy (1 mg norethindrone and extract for 14 days. Urinary excretion of D glucaric acid was 20 µg ethinyl oestradiol), ovarian activity and break-through unaffected after the 14-day treatment, whereas excretion of 6b bleeding were studied in sixteen healthy women treated for -hydroxycortisol significantly increased from a mean baseline two consecutive 28-day cycles in a single-blind sequential value of 254 μg/day to 369 μg/day (p<0.0001). The excretion of trial. Treatment with the extract resulted in a significant 13-15% free cortisol was unaltered, but the ratio of 6b-hydroxycortisol (p=0.021) reduction in the dose exposure to the contraceptive. to free cortisol changed significantly (p<0.001) from 9.9 at Breakthrough bleeding increased in the treatment cycles, as did baseline to 14.3. Thus, high-dose treatment with the extract evidence of follicle growth and probable ovulation [Murphy induced CYP3A activity in healthy volunteers as evidenced by 2005]. increased 6b-hydroxycortisol excretion [Bauer 2002]. To investigate whether administration of a methanolic (80%) In a patient under long-term treatment with CyA (125 – 150 extract at an oral dose of 3×300 mg together with an OC (20 mg/day) and (5 mg/day), stable blood levels of µg ethinyl oestradiol and 1 mg norethindrone acetate) modified CyA between 100 and 130 pg/L suddenly decreased to around elevated serum androgen levels in OC treatment of hirsutism 70 pg/L, despite the daily dose being raised to 250 mg. The and acne, 15 healthy women received OC alone for 2 menstrual patient had started to drink a containing St. John’s cycles, followed by two cycles with addition of the extract. wort to control seasonal depressive symptoms. Five days after No significant differences in testosterone, free testosterone, cessation of the tea, CyA blood levels increased from 70 to 170 androstendione, dehydroepiandrosterone, 3a-diol-glucuronide, µg/L (250 mg/day), and a subsequent dose reduction to 175 mg/ dehydrotestosterone and or sex hormone binding globulin were day resulted in blood levels of around 130 pg/L [Alscher 2003]. observed during the co-administration [Fogle 2006].

Treatment of four patients on methadone medication with an The time for recovery to baseline activity of CYP3A after induction unspecified extract (900 mg/d) for between 14 and 47 days by an unspecified extract was studied in 12 healthy males. The resulted in a major reduction of (R,S)-methadone concentration- volunteers received an oral dose of 5 mg midazolam and from

46 HYPERICI HERBA

the next day 3 × 300 mg/day of the extract p.o. for 14 days. On baseline value (p<0.05). After cessation of the co-administered the last day of treatment, and 3 and 7 days later, they received the drugs, the AUC(0–6h) of orally administered MDZ decreased same dose of midazolam. Compared to baseline, oral clearance to 6% of the level observed during combined administration, of midazolam was significantly (p=0.001) increased after the and the AUC(0–8h) of i.v. administered midazolam decreased extract, from 65.3 ± 8.4 L/h to 86.8 ± 17.3 L/h. It returned to to 33% of the values observed during combined administration the control level 7 days after the last dose of extract (day 7; (p<0.001 for each) [Hafner 2010]. 59.7 ± 3.8 L/h) [Imai 2008]. A male patient with ADHD who had been successfully treated Five cancer patients received irinotecan (IT) at a dose of 350 with (20 mg/day), as measured by a reduction mg/m2 i.v. once every 3 weeks. An unspecified extract was in Conner’s score (CS) from 75.3 to 68.4, began additional given at a daily dose of 3 x 300 mg for 14 days, from before self-medication with an unspecified extract (600 mg/day) six the first IT administration until 4 days after IT administration. months after starting on the methylphenidate. Treatment with the Myelosuppression was clearly more pronounced with IT alone extract for 4 months diminished the efficacy of methylphenidate, (decrease in leukocyte and neutrophil counts of 56% and 63% demonstrated by an increase in the CS from 68.4 to 74.9. Three respectively) than after co-administration with the extract (8.6% weeks after discontinuation of the extract, the CS dropped back and 4.3% respectively). The AUC of the active metabolite down again to 70.3 [Niederhofer 2007]. decreased by 42% (p=0.033) [Mathijssen 2002]. The interaction of (10 – 40 mg daily) and an In a study involving 16 healthy volunteers, the effects of unspecified extract (300 mg twice daily) was studied in a a methanolic extract (80%, V/V; 3 × 300 mg/day p.o. for controlled, randomised, open, crossover study involving 16 14 days) on the activities of CYP1A2, CYP2D6, CYP3A4, patients with hypercholesterolaemia. After a 4-week run-in N-acetyltransferase 2 (NAT2) and xanthine oxidase (XO) were period, patients on a stable atorvastatin dose were randomised assessed by phenotyping before and at the end of treatment. to additional treatment with either the extract or control for 4 Probes for enzyme activities were ratios of metabolites weeks. The extract significantly increased the serum level of 17MX/137MX from caffeine (CYP1A2), DM/DX (CYP2D6), LDL cholesterol compared with control (2.66 mmol/L vs. 2.34 endogenous 6-hydroxycortisol/cortisol (CYP3A4), AFMU/1MX mmol/L; p=0.004) and of total cholesterol (5.10 mmol/L vs. from caffeine (NAT2) and 1MU/1MX from caffeine (XO), found 4.78 mmol/L; p=0.02). No significant changes were observed in saliva or urine. From these metabolic ratios an induction of in HDL cholesterol or in triglycerides [Andren 2007]. CYP3A4 by the extract was confirmed. CYP1A2 was induced only in females. The activities of CYP2D6, NAT2 and XO were The interaction of simvastatin (10 – 40 mg daily) and an not affected [Wenk 2004]. unspecified extract (300 mg twice daily) was investigated in a similar study involving 24 patients with hypercholesterolaemia. In a controlled, open-label study, acute and long-term effects of a After a 4-week run-in period, patients on a stable simvastatin methanolic extract (80%, V/V; 3 × 300 mg/day p.o. for 15 days) on dose received either the extract or control for 4 weeks. Compared the pharmacokinetics of voriconazole (VCZ) were investigated to control, total cholesterol, LDL-cholesterol and triglycerides in 16 healthy male subjects. Since the extensive metabolism increased significantly in the extract group after 4 weeks: 4.56 of VCZ is primarily mediated by CYP2C19 and CYP3A4, and vs. 5.08 mmol/L (p<0.001), 2.30 vs. 2.72 mmol/L (p<0.0001) to a minor extent by CYP2C9, subjects were genotyped for the and 1.30 vs. 1.54 mmol/L (p=0.0155) respectively. No significant CYP2C19 alleles *2 and *3. A single dose of VCZ (400 mg) was differences were observed for HDL-cholesterol [Eggertsen 2007]. given on days -3 (baseline), 1 and 15. On days 1 and 15, the extract was administered 60 min before VCZ adminis¬tration. Table 2 below summarises the interactions of St. John’s wort On day 1 of extract administration the AUC(0-10h) of VCZ was extracts with other drugs from clinical studies: increased by 22% compared with control (15.5 ± 6.84 h×µg/mL versus 12.7 ± 4.16 h×µg/mL; p=0.02). After 15 days intake, the Preclinical safety data AUC(0-10h) was reduced by 59% compared with control (9.63 Systematic studies on single dose toxicity, reproductive toxicity ± 6.03 h×µg/mL versus 23.5 ± 15.6 h×µg/mL; p=0.0004), with and carcinogenicity of St. John’s wort extracts have been carried a corresponding increase in oral VCZ clearance (CL/F) from 390 out by major manufacturers, but not published. ± 192 to 952 ± 524 mL/min (p=0.0004). The baseline CL/F of VCZ and the absolute increase in CL/F were smaller in carriers Acute toxicity of 1 or 2 deficient CYP2C19*2 alleles compared with wild-type No data available individuals (p<0.03) [Rengelshausen 2005]. Repeated dose and chronic toxicity studies The net effect of simultaneous administration of an inducer No significant tissue lesions or adverse events were observed in of CYP3A4 (St. John’s wort) and an inhibitor (ritonavir) was male Long-Evans rats fed St. John’s wort (SJW; locally collected in investigated in 12 healthy volunteers in an open, fixed sequence the fully blooming stage) at 5% of their diet for 119 days. Feeding study design. The CYP3A4 probe midazolam (MDZ) was given SJW in conjunction with Senecio jacobaea did not influence orally (4 mg) as a single dose in 4 different study phases: (i) at the chronic toxicity of Senecio as assessed by rat survival time. baseline, (ii) after a single oral dose of either a methanolic extract SJW did not affect hepatic levels [Garrett 1982]. (80%, V/V; 300 mg) or ritonavir (3 × 100 mg) (n = 6 each), (iii) after 14 days of co-administration of ritonavir (2 × 300 mg daily) and No signs of toxicity were observed following administration of the extract (3 × 300 mg daily), and (iv) at 2 days after cessation dried herb to rats at up to 2 g/kg/day for one year, at 3 g/kg/day of both the extract and ritonavir. Each oral MDZ administration for 28 days, and to dogs at 2 g/kg/day for 1 year. Tolerability was followed by an intravenous MDZ injection of 2 mg 6 h later levels of < 10 g/kg wet weight, eaten at the flowering stage, (‘semi-simultaneous administration’). Combined administration and < 2.65 mg/kg for hypericin were demonstrated in sheep. of inducer and inhibitor resulted in a predominance of enzyme Non-specific toxic symptoms, including slight load damage inhibition: co-administration of the extract and ritonavir with to liver and kidneys, were observed after administration of 0.9 i.v. administration of MDZ showed an increase in the MDZ and 2.7 g/kg of a methanolic extract (80%, V/V) for 26 weeks AUC(0–8h) to 180% of baseline value (p<0.05), whereas with in pivotal toxicological models in rats and dogs respectively orally administered MDZ, the AUC0–6h increased to 412% of [Morgan 2005].

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TABLE 2: CHANGES IN THE PHARMACOKINETICS OF THE FOLLOWING DRUGS BY CO-ADMINISTRATION OF ST. JOHN’S WORT

Drug Subjects Type of preparation, Effect on blood levels Presumed References posology (mg/day) and (AUC) Mechanism duration of administration Alprazolam Healthy subjects 900 mg of a methanolic (80%) 53.6% decrease CYP3A4 [Markowitz 2003] extract for 12-14 days Amitriptyline Healthy subjects 900 mg of a methanolic (80%) 22% decrease CYP3A4 [Johne 2002] (n = 12) extract for 12-14 days Boceprevir Healthy subjects 600 mg of an ethanolic (60%) No significant effect [Jackson 2014] (n = 17) extract for 14 days Bupropion Healthy subjects 975 mg of an unspecified 14.3% decrease CYP2B6 [Lei 2010] (n = 18) extract for 14 days Caffeine Healthy subjects 900 mg of a methanolic (80%) No significant effect [Wang 2001] (n = 12) extract for 2 weeks Carbamazepine Healthy subjects 900 mg of a methanolic (80%) No significant effect [Burstein 2000] (n = 8) extract for 14 days Ciclosporin Transplant patients 900 mg of a methanolic (80%) Increased clearance CYP3A4, Pgp [Ruschitzka 2000] (n=7) extract for 14 days [Mai 2000] [Karliova 2000] [Mandelbaum 2000] [Barone 2001] Ciclosporin 1 patient Unspecified extract Significant decrease CYP3A4, Pgp [Barone 2000] Ciclosporin Kidney transplants 600 mg of a methanolic 42% decrease CYP3A4, Pgp [Bauer 2003] (n = 11) extract Ciclosporin Kidney transplant Unknown 47% decrease CYP3A4, Pgp [Breidenbach 2000] (n = 30) Ciclosporin Healthy subjects 900 mg of a methanolic (80%) 38.6% decrease CYP, Pgp [Dresser 2003] (n = 21) extract for 10 days Ciclosporin Transplant patients 900 mg of a methanolic (80%) 51.9% decrease CYP, Pgp [Mai 2004] (n = 10) extract Clozapine 1 Patient 900 mg unspecified extract 65.2% decrease in CYP1A2 [Van Strater 2011] for 3 weeks trough levels CYP3A4 Dextromethorphan Healthy subjects 900 mg of methanolic (80%) No significant effect CYP2D6 [Wang 2001] (n = 12) extract Healthy women 600 and 900 mg of a 43.9% decrease CYP3A4 [Pfrunder 2003] (n = 18) methanolic (80%) extract for 4 weeks Digoxin Healthy subjects 900 mg of a methanolic (80%) 25% decrease Pgp [Johne 1999] (n = 13) extract for 10 days Digoxin Healthy subjects Different extracts for 10 days mg/day hyperforin / % Pgp [Mueller 2004] (n = 96) decrease 28.9 / 24.8 21.1/ 26.6 10.6/ 17.7 5.3 /1.8 3.56/ 7.3 2.6/ -1.5 0.83/ -5.6 0.13/ 5.2 0.3/ 9 0.04/0.3 Placebo/6.4 Digoxin Healthy subjects 900 mg of a methanolic (80%) 23% decrease Pgp [Gurley 2008b] (n = 18) extract for 14 days Digoxin Healthy subjects 900 mg of a methanolic 18% decrease Pgp [Dürr 2000] (n = 8) extract (80%) for 14 days Ethinyloestradiol Healthy women 900 mg of an unspecified No significant effect [Hall 2003] (n = 12) extract (3% hyperforin) for 28 days Ethinyloestradiol Healthy women 600 and 900 mg of a No significant effect [Pfrunder 2003] (n = 18) methanolic (80%) extract for 4 weeks 48 HYPERICI HERBA

Drug Subjects Type of preparation, posology Effect on blood levels Presumed References (mg/day) and duration of (AUC) Mechanism administration Ethinyloestradiol Healthy women 900 mg of an unspecified 14.1% decrease CYP, Pgp [Murphy 2005] (n = 16) extract for two times 28 days Fexofenadine Healthy subjects 900 mg of a methanolic (80%) 14% decrease, Pgp [Wang 2002] (n = 12) extract for 14 days not significant Fexofenadine Healthy subjects 900 mg of a methanolic (80%) 48.5% decrease Pgp [Dresser 2003] (n = 21) extract for 10 days Finasteride Healthy volunteers 600 mg of a methanolic (80%) 58% decrease CYP3A4 [Lundahl 2009] (n = 12) extract for 2 weeks Gliclazide Healthy volunteers 900 mg/of a methanolic (80%) 33% decrease CYP2C9 [Xu 2008] (n = 21) extract for 2 weeks Ibuprofen Healthy volunteers 900 mg of an methanolic (80%) No significant decrease [Bell 2007b] (n = 21) extract Imatinib Healthy volunteers 900 mg of a methanolic (80%) 30% decrease CYP3A4, [Frye 2004] (n = 12) extract for 2 weeks Pgp, BCRP Imatinib Healthy volunteers 900 mg of an unspecified extract 32% decrease CYP3A4, Pgp, [Smith 2004a; Smith (n = 10) for 2 weeks 2004b] Indinavir Healthy volunteers 900 mg of a methanolic extract 57% decrease CYP3A4, Pgp [Piscitelli 2000] (n = 8) for 2 weeks Irinotecan Cancer patients 900 mg of an unspecified extract 42% decrease of SN-38 CYP3A4 [Mathijssen 2002] (n = 5) for 18 days Ivabradine Healthy subjects 900 mg of a methanolic (80%) 61.7% decrease CYP3A4, Pgp [Portoles 2006] (n = 12) extract for 14 days Methadone 2 Patients 900 mg of a methanolic extract 47% decrease CYP3A4 [Eich-Hochli 2003] (> 3% hyperforin) for 31 days Midazolam Healthy subjects 900 mg of a methanolic (80%) 52.3% decrease CYP3A4 [Wang 2001] (n = 12) extract for 2 weeks Midazolam Healthy subjects 900 mg of an unspecified extract Significant decrease CYP3A4, Pgp [Gurley 2002] (n = 12) for 28 days Midazolam Healthy subjects 900 mg of a methanolic (80%) 30.6% decrease CYP3A4 [Dresser 2003] (n = 21) extract for 10 days Midazolam Healthy women 900 mg of an unspecified extract 35% decrease CYP3A4 [Hall 2003] (n = 12) (3% hyperforin) for 28 days Midazolam Healthy subjects Different extracts for 14 days mg/day hyperforin / % CYP3A4 [Mueller 2006] (n = 42) decrease 41.25 / 80.0 12.06 / 48.0 8.04 / 36.0 5.36 / 32 2.68 / 22 0.13 / 21 Mycophenolate Transplant patients 600 mg of a methanolic (80%) No significant effect [Mai 2003] (n = 10) extract for 14 days Nevirapine HIV patients (n = 5) unknown decrease CYPs, Pgp [De Maat 2001] Nifedipine Healthy subjects 900 mg of a methanolic extract 45.2% decrease CYP3A4, Pgp [Wang 2007] (n = 12) for 14 days Norethindrone Healthy women 900 mg of unspecified extract About 14% decrease CYP3A4 [Hall 2003] (n = 12) (3% hyperforin) for 28 days Norethindrone Healthy women 900 mg of unspecified extract 12.6% decrease CYP, Pgp [Murphy 2005] (n = 16) for two times 28 days Healthy subjects 900 mg of a methanolic (4% 37.9% decrease CYP3A4, CYP2C19 [Wang 2004c] (n = 12) hyperforin) extract for 14 days Oxycodon Healthy subjects 900 mg of a methanolic (80%) 50% decrease CYP3A4 [Nieminen 2010] (n = 12) extract for 15 days Phenprocoumon Healthy subjects 900 mg of a methanolic (4% 17.4% decrease CYP3A4 [Maurer 1999] (n = 10) hyperforin) extract for 14 days

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Drug Subjects Type of preparation, posology Effect on blood levels Presumed References (mg/day) and duration of (AUC) Mechanism administration Healthy subjects 900 mg methanolic (4% Increased metabolism CYP2C19 [Wang 2004b] (n = 12) hyperforin) extract for 14 days Pravastatin Healthy subjects 900 mg of unspecified extract No significant effect [Sugimoto 2001] (n = 16) for 14 days Prednison Healthy subjects 900 mg of a methanolic extract No significant effect [Bell 2007a] Prednisolone (n = 8) (> 3% hyperforin) for 4 weeks

Quazepam Healthy subjects 900 mg of an unspecified extract 28.5% decrease CYP2C9 [Kawaguchi 2004] (n = 13) for 14 days Repaglinide Healthy subjects 975 mg of an unspecified extract No significant effect [Fan 2011] (n = 15) for 14 days Simvastatin Healthy subjects 900 mg of unspecified extract 48% decrease CYP3A4, Pgp [Sugimoto 2001] (n = 16) for 14 days Tacrolimus Transplant patients 600 mg of a methanolic (80%) 57.8% decrease CYP3A4, Pgp [Mai 2003] (n = 10) extract for 14 days Tacrolimus Healthy subjects 900 mg of a methanolic (80%) 35.3% decrease CYP3A4, Pgp [Hebert 2004] (n = 10) extract for 18 days Talinolol Healthy subjects 900 mg of a methanolic (80%) 31% decrease Pgp [Schwarz 2007] (n = 9) extract for 12 days Healthy subjects 900 mg of an unspecified extract No significant effect [Morimoto 2004] (n = 12) for 15 days Verapamil Healthy subjects 900 mg of an unspecified extract 78% (R-verapamil) CYP3A4, Pgp [Tannergren 2004] (n = 8) for 14 days and 80% (S-verapamil) decrease Voriconazole Healthy subjects 900 mg of a methanolic (80%) 59% decrease CYP3A4, CYP2C9, [Rengelshausen (n = 16) extract for 15 days CYP2C19 2005] Warfarin Healthy subjects 3000 mg of an unspecified 27% and 24.6% CYP3A4, CYP2C9, [Jiang 2004] (n = 12) extract for 14 days decrease for S- and R- CYP1A2 warfarin Healthy subjects 900 mg of a methanolic (80%) 30.3% decrease CYP3A4, CYP2C9 [Hojo 2011] (n = 14) extract for 14 days CYP1A2

Low-dose hyperforin

Alprazolam Healthy subjects 240 mg of a low hyperforin No significant effect [Arold 2005] (n = 28) extract for 10 days Caffeine Healthy subjects 240 mg of a low hyperforin No significant effect [Arold 2005] (n = 28) extract for 10 days Ciclosporin Transplant Patients 900 mg of an unspecified low No significant effect CYP, Pgp [Mai 2004] (n = 10) hyperforin extract Desogestrel Healthy women 500 mg of a low hyperforin No significant effect [Will-Shahab 2008] (n = 16) ethanolic extract (57.9%) for 14 days Digoxin Healthy subjects 240 mg of a low hyperforin No significant effect [Arold 2005] (n = 28) extract for 10 days Healthy women 500 mg of a low hyperforin No significant effect [Will-Shahab 2008] (n = 16) ethanolic extract (57.9%) for 14 days Midazolam Healthy subjects 900 mg of an unspecified low No significant effect [Mueller 2006] (n = 42) hyperforin extract for 14 days (Hyperforin 0.13 mg/day) Midazolam Healthy subjects 1000 mg of an unspecified No significant effect [Mueller 2009] (n = 20) low hyperforin extract or 14 days Tolbutamide Healthy subjects 240 mg of a low hyperforin No significant effect [Arold 2005] (n = 28) extract for 10 days

50 HYPERICI HERBA

Mutagenicity and carcinogenicity decrease of the levels of mdr1a, mdr1b, mrp1 and mrp2 in the livers of foetuses, and an increase (p<0.01) in the levels of Genotoxicity mdrla, mdrlb, mrp2 and cyp3A2 in the mothers [Garrovo 2006]. No increase in the extent of DNA fragmentation was observed on addition of a methanolic (80%, V/V) extract to rat cortical High concentrations of hyperforin (> 1 µM) inhibited mouse cells. The cells were incubated for 18 hours with concentrations embryonic stem (ES) cell population growth (p<0.05) and up to 40 µg/mL [Perovic 1995]. induced apoptosis (p<0.05) in fibroblasts (representing adult tissues). Hyperforin affected ES cell differentiation into Genotoxicity of a standardised aqueous ethanolic extract was cardiomyocytes in a dose-dependent manner (p<0.05). Analysis investigated in various in vivo and in vitro test systems. No of tissue-specific marker expression also revealed that hyperforin suggestion of mutagenicity was found. The testing involved: at high concentrations partially inhibited ES cell differentiation A) guanidine phosphoribosyl transferase-test into mesodermal and endodermal lineages [Nakamura 2013]. in Chinese hamster V79 cells with extract concentrations up to 0.50 µg/mL (without S9-mix) or 4 µg/mL (with S9-mix); Effects on offspring B) Unscheduled DNA synthesis assay in primary rat hepato- Treatment of male CD rats for 14 days with 500 mg/kg daily cytes with extract concentrations up to 1.37 µg/Ml; C) Cell of a methanolic (80%, V/V) extract, hypericin 0.1 mg/kg, or transformation test using S9-mix activated Syrian hamster embryo hypericin combined with procyanidin B2 1:25, did not lead to cells, the extract was added in concentrations up to 10 µg/Ml; any changes in the reproductive organs [Butterweck 2001a]. D) Fur spot test in NMRI mice with oral doses of up to 10 mL/kg of test substance; and E) aberration test with bone The cognitive impact of prenatal exposure to an extract (0.3% marrow cells of Chinese hamsters receiving oral doses of up to hypericin, solvent not specified, 182 mg/kg/day) was studied 10 mL/kg of the extract [Okpanyi 1990]. in CD1 mice. The extract or a placebo was consumed in food bars for 2 weeks before mating and throughout gestation. One Carcinogenicity offspring per gender from each litter (verum 13, placebo 12) No data are available was tested using the juvenile runway with adult memory, adult Morris spatial water maze, adult passive avoidance and adult Reproductive toxicity straight water runway, followed by a dry Cincinnati maze. Learning occurred in both genders in all tasks (p<0.003) with In vitro experiments no significant differences between treatments at the final Hamster oocytes were incubated with an extract before sperm/ trial. Female offspring exposed to the extract, rather than to oocyte interaction. Penetration was prevented by a very high dose placebo, required more time to learn the MWM task (p<0.05). of 0.6 mg/mL, but 0.06 mg/mL had no effect [Vandenbogaerde Post-learning sessions did not show any significant differences 1998]. [Rayburn 2001a].

In an explanted Sprague-Dawley rat embryo model, enabling Female mice were treated orally with 180 mg/kg/day of the the study of embryonic development during the critical period same extract or placebo for 2 weeks before conception and of organogenesis from 9.5 to 11.5 gestational days, exposures throughout gestation. Gestational age at delivery and litter size to high concentrations of hypericin (71.0 and 142.0 ng/mL) did not differ between the groups. Body weight, body length demonstrated a significantly lower total morphological score and head circumference from postnatal day 3 to adulthood did (p=0.002) and number of somites (p<0.001) compared to not differ from controls regardless of gender. The only difference control. A significant negative linear trend in total morphological between the groups was a temporary delay in the eruption of score (p<0.001), yolk sac diameter (p=0.01), and number of upper incisors in male offspring exposed to the extract. The somites (p<0.01) was observed with increasing concentration extract did not affect reproductive capacity, perinatal outcomes, of hypericin. No statistical difference was detected in crown- or growth and development of second-generation offspring rump length [Chan 2001]. [Rayburn 2001b].

In vivo experiments A methanolic extract (0.3% total hypericin) given to Wistar rats Exposure of Sprague-Dawley rats to diets containing 0, 180, at doses of 100 and 1000 mg/kg/day by gavage during pregnancy 900, 1800 or 4500 ppm of an unspecified extract beginning on and lactation revealed severe damage in the livers and kidneys gestational day 3 and ending at off-spring weaning on postnatal of new-born animals euthanised post-natally on days 0 and 21 day (PND) 21 did not reveal any effects on maternal weight gain, at both doses. The lesions were more severe at the higher dose duration of gestation or extract-related behavioural alterations. and in animals that were breastfed for 21 days [Gregoretti 2004]. Brain weights of offspring at adulthood indicated no significant effects of the extract [Cada 2001]. A methanolic extract (80%, V/V) at a dose of 36 mg/kg was given to 30 pregnant Wistar rats during organogenesis (days 9 A methanolic (80%) extract was administered to female rats - 15 of pregnancy). No clinical signs of maternal toxicity were during the period of organogenesis (gestational days 9-15). observed in the necropsy on day 21. The indices of implantation Thirty inseminated Wistar rats received oral doses of saline or and resorption, and toxicological parameters (such as foetus 36 mg/kg of the extract. No clinical signs of maternal toxicity and placenta weight), were not significantly different compared were observed and none of the variables analysed in foetuses to control [Borges 2005]. and placentas showed significant differences [Borges 2005]. In a systematic review on the safety of St. John’s wort during The effect of treatment with high doses of an unspecified pregnancy and lactation, no impact on maternal weight gain methanolic extract (100 and 1000 mg/kg/day), administered or duration of gestation were observed in Sprague-Dawley rats prenatally and during breastfeeding, on the level of transcripts who were exposed to oral doses of St. John’s wort 1 to 25 times of mdrla, mdr1b, mrp1, mrp2 and cyp3A2 genes was studied in the recommended human dose. Offspring body weights were Wistar rats. All transcripts were detected in the liver, and their similar to controls, although there was a tendency towards lower level of expression increased from foetuses to adults. Extract weight after treatment with St. John’s wort. No alterations in administration, at both dosages, caused a significant (p<0.01) behaviour were found [Dugoua 2006].

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Organ mRNA expression and protein content of Abcb1 were administration with the extract diminished the phototoxicity of determined following oral administration of an 80% metha- the latter [Wilhelm 2001]. nolic extract (hypericin 0.3%) to pregnant female Wistar rats at 1 g/kg for 9 days during late pregnancy. Abcb1 expression The phototoxic and apoptosis-inducing capacity of pseudo- was substantially lower in foetal than in maternal organs and hypericin was compared to hypericin in human leukaemic significantly induced in the maternal jejunum and placenta lymphoma cells (Jurkat). Treatment with both photoactivated [Saljé 2012]. hypericin and pseudohypericin resulted in a dose-dependent inhibition of cell proliferation, whereas compounds without An extract (0.3% hypericin; not further specified) was admin- photoactivation had no effect at the concentrations tested. The istered orally to pregnant Wistar rats at 36, 72 or 144 mg/kg IC50 of hypericin was lower (100 ng/mL) than pseudohypericin per day.The highest dose demonstrated greater head-dipping (200 ng/mL) (p<0.05). A dose-dependent increase of DNA activity in the hole-board test (p<0.001) and reduced immobility fragmentation was observed following treatment with both in the tail suspension test (p<0.001) and FST (p<0.001), at 10 photoactivated compounds in an apoptosis assay [Schempp and 60 days post-treatment [Vieira 2013b]. 2002b].

Maternal exposure to FLU (7.5 mg/kg) or an unspecified extract Phototoxicity of hypericin to the eye was evaluated in human (100 mg/kg), given daily by gavage during pregnancy and lens epithelial cells. Cells were exposed to 0.1 – 10 µM hypericin lactation, showed that FL, but not the extract interfered with and irradiated with 4 J/cm2 UVA or 0.9 J/cm2 visible light. reproductive parameters in adult male rats [Vieira 2013a]. Neither hypericin alone nor light exposure alone reduced cell viability. In contrast, cells exposed to hypericin in combination Hepatotoxicity with UVA or visible light underwent necrosis and apoptosis. Hypericin and pseudohypericin at a dose of 50 µg (oral, i.v., The ocular antioxidants lutein and N-acetyl cysteine did not i.p.) exhibited low toxicity in BALB/c mice and prevented viral- prevent damage [He 2004]. induced manifestations following retrovirus infection. Neither hypericin nor pseudohypericin had an impact on alkaline The cytotoxicity of various extracts (prepared with ethanol, water, phosphatase, lactate dehydrogenase, AST, ALT or cholesterol. chloroform, hexane) and purified compounds was examined in Haematology and blood biochemistry were unaffected [Meruelo three cell lines. All extracts exhibited significant cytotoxicity; 1988]. those prepared in ethanol (no hyperforin, 3.6 μM hypericin, 134.6 μM flavonoids) showed between 7.7 and 77.4% cell Intraperitoneal administration of 280 mg/kg/day of an extract survival (p<0.0001 and 0.01), whereas the chloroform and (0.29% total hypericins; 2.29% hyperforin; solvent not specified), hexane extracts (hyperforin, hypericin and flavonoids not 1 mg/kg/d of hypericin or 10 mg/kg/d of hyperforin to male Swiss detected) showed approximately 9.0% (p<0.0001) and 4.0% Webster mice for periods ranging from 4 days to 3 weeks did (p<0.0001) survival. Light-sensitive toxicity was observed not induce changes in ALT, and no histological changes in the primarily with the ethanol extracts sequentially extracted livers were observed [Bray 2002a; Bray 2002b]. following removal of material extracted in either chloroform or hexane. The absence of light-sensitive toxicity with the extracts Phototoxicity suggests that the hypericins were not playing a prominent role Photosensitization caused by St. John’s wort has mainly been in the toxicity of the extracts [Schmitt 2006]. reported in veterinary studies, especially in unpigmented skin of grazing animals [James 1992]. Dose-dependent phototoxic The phototoxic effects of 0.1 - 10 µM hypericin were investigated symptoms were observed in calves within 4 hours after single further in combination with visible light on the human retina. oral doses of dried St. John’s wort at 3 – 5 g/kg [Araya 1981]. Fluorescence emission confirmed hypericin uptake by human retinal pigment epithelial cells (hRPE). When irradiated with Merino ewes (an unpigmented breed, freshly shorn of wool), 0.7 J/cm2 of visible light (λ > 400 nm) loss of cell viability as dosed orally with 5.7, 4.0 or 2.85 g/kg of dried St. John’s wort measured by mitochondrial activity and cell membrane damage (corresponding to 5.3, 3.7 or 2.65 mg/kg of hypericin) and then was observed. Hypericin in irradiated hRPE cells significantly exposed to bright sunlight, had a tolerance level for hypericin changed the equilibrium of glutathione and decreased the of less than 2.65 mg/kg [James 1992]. activity of glutathione reductase. Increased lipid peroxidation was correlated to hypericin concentration in hRPE cells and Foetal calf serum or albumin strongly inhibited the photocyto- visible light radiation [Wielgus 2007]. toxic effects of pseudohypericin, but not those of hypericin, in A431 tumour cells [Bernd 1999]. Human keratinocytes were Mild phototoxicity was observed after topical administration cultured in the presence of various concentrations of an extract of hypericin at a dose of 0.01 to 1% [Boiy 2008]. and irradiated with UVA or UVB. A phototoxic effect was only seen with high hypericin concentrations (≥ 50 µg/mL) after Neither hypericin exposure nor UVA irradiation alone reduced irradiation with UVA [Rayburn 2001b]. the cell viability of human pigmented and unpigmented mela- noma cells, immortalised keratinocytes and melanocytes. An The phototoxic potential of 3 unspecified extracts from exposure to 1 µM UVA-activated hypericin did not change cell various sources, as well as some of their main constituents, viability, while 3 µM activated hypericin induced necrosis in was investigated in an immortalised human keratinocyte cell pigmented melanoma cells and melanocytes and apoptosis in line (HaCaT cells) under UVA irradiation (250 and 700 mJ/ cells without melanosomes (non-pigmented melanoma cells cm2). Known phototoxic compounds like chloropromazine and keratinocytes). Hypericin localisation in the endoplasmic and psoralenes like 5-MOP served as controls. The extracts reticulum in these cells, shown by fluorescent microscopy, demonstrated cytotoxicity and photocytotoxicity in a dose- further supported a disruption in cellular processing and and UVA-dose dependent manner (IC50 without irradiation induction of cell death. It was demonstrated that intracellular 35 to 74 µg/mL, with irradiation 24 to 84 µg/mL). Hypericin accumulation of hypericin induced mitochondrial-associated also evoked severe phototoxic effects and was the main caspase-dependent apoptosis [Davids 2008]. phototoxic constituent. Among the tested flavonoids quercitrin and rutin were found to be cytotoxic. However, quercitrin co- The photobiochemical properties of an extract and 19 known

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constituents were characterised with a focus on generation (AEs) among patients between 0% and 6%. Of these studies, of ROS, lipid peroxidation and DNA photocleavage as the four large-scale surveillance studies with a total of 14,245 indicators of photosensitive, photoirritant and photogenotoxic patients recorded a rate of AEs ranging from 0.1% to 2.4% and potentials. The ROS assay revealed the photoreactivity of the a drop-out rate due to AEs of 0.1-0.9%. AEs associated with extract and some constituents as evidenced by type I and/or II St. John’s wort treatment were mild and transient in nearly all photochemical reactions under light exposure. Only hypericin, cases [Schulz 2006b]. pseudohypericin and hyperforin exhibited in vitro photoirritant potential [Onoue 2011]. In an observational study 1778 patients with depressive symptoms received an extract (3.5-6:1; 425 – 1700 mg) for Fluorescence confocal microscopy was used to visualise binding 12 weeks. The incidence of ADRs was 3.54% and decreased between hypericin and a-crystallin, a major lens protein, in continuously from the first visit onwards; serious ADRs did not a human lens epithelial (HLE) cell line. UVA irradiation of occur [Melzer 2010]. hypericin-treated HLE cells resulted in a decrease in a-crystallin detection concurrent with an accumulation of the tryptophan Spontaneous ADR reports (n = 84) reported to the Australian oxidation product N-formylkynurenine (NFK). Examination of Therapeutic Goods Administration (TGA) between 2000 - actin in HLE cells indicated that it accumulated NFK resulting 2013 were obtained for St. John’s wort (SJW). The majority of from hypericin-mediated photosensitisation. Wavelengths < reported ADRs for SJW were concerning females aged 26-50 400nm provided an incomplete protection against a-crystallin years (28.6%). The central nervous system (45.2%) was most modification and NFK accumulation [Ehrenshaft 2013]. affected by ADRs to SJW [Hoban 2015].

Clinical safety data Pregnancy and lactation At therapeutic dose levels (up to the equivalent of 6 g of A prospective, observational, controlled cohort study followed drug), occasional mild gastrointestinal disturbances, nausea, three groups of pregnant women who contacted the Motherisk restlessness, fatigue, headache, insomnia or allergic reactions Program (a teratogen information service in Canada): 54 have been reported [Albrecht 1994; Bergmann 1993; Bernhardt women using any St. John's wort product at any time during 1996; De Maat 2001; Engesser 1996; Grube 1999; Halama 1991; their pregnancy, a disease-match group of 54 women with Hänsgen 1996; Häring 1996; Harrer 1994; Harrer 1991; Harrer depression under conventional antidepressant therapy and 54 1999; Hoffmann 1979; Holsboer-Trachsler 1999; Hübner 1994; healthy women not exposed to any known teratogen. Pregnancy Kaehler 1999; Kalb 2001; Kasper 2010; Knüppel 2004; Kugler outcomes were similar in the three groups. There were 3, 2, and 1990; Laakmann 1998; Lecrubier 2002; Lehrl 1993; Lenoir 0 malformations in the St. John’s wort, disease-matched and 1999; Martinez 1994; Meier 1997; Mueller 1998; Pieschl 1989; healthy groups respectively. The rate of spontaneous Quandt 1993; Rahimi 2009; Reh 1992; Röder 2004; Schakau was higher in the St. John's wort (n = 11) and disease-matched 1996; Schlich 1987; Schmidt 1989; Schrader 2000; Schrader control (n = 7) groups compared to the healthy control group (n 1998; Schulz 2001; Sepehrmanesh 1999; Sommer 1994; = 5). Neither finding reached statistical significance. The rate of Vorbach 1994; Warnecke 1986; Werth 1989; Wheatley 1997; malformation was as expected in the population [Moretti 2009]. Witte 1995; Woelk 2000a; Woelk 1994]. Onset of mania and hypomania were reported in several patients with latent bipolar In a cohort study, 33 breastfeeding women presenting to the disorders [Barbenel 2000; Fahmi 2002; Moses 2000; Nierenberg teratogen/toxicant counselling service taking St. John's wort 1999; O'Breasail 1998; Saraga 2005; Schneck 1998; Stevinson preparations (not further specified) at an average dose of 2004]. In one patient taking up to 3600 mg of a methanolic 704.9 mg/day (range 225 - 2150 mg/day) (group 1) were extract acute psychotic symptoms were observed that recovered compared to 101 disease-matched controls (group 2) and 33 completely after termination of treatment [Shimizu 2004]. age- and parity-matched healthy controls (Group 3). Whereas only 1 infant each in groups 2 and 3 was reported to be A detailed overview of the pharmacological, toxicological colicky, there were 2 cases of "colic", 2 of "drowsiness" and 1 of and clinical literature on St. John’s wort is provided in several "lethargy" in group 1. No significant difference was observed in reviews [Ernst 1995; Greeson 2001; Nathan 2001; Russo 2014]. the frequency of maternal reports of decreased milk production among the groups, nor a difference in infant weight over the In a meta-analysis of 35 double-blind randomised trials and first year of life [Lee 2003]. observational studies of St. John's wort mono-preparations, drop-out and adverse event rates were extracted from 17 In a case report, hypericin and hyperforin were measured in the observational studies including 35,562 patients. Drop-out rates breast milk of a woman receiving 300 mg of an 80% methanolic ranged between 0% and 5.9%. No serious adverse effects were extract 3 times a day. Only hyperforin was excreted at minimal reported. In 24 double-blind, randomised trials (n = 1,334 St. levels into breast milk, but not into infant´s plasma [Klier 2002]. John's wort, n = 1,292 placebo) drop-out and rates were similar to placebo (OR = 0.82 (CI = 0.64 to 1.06) An excretion of hyperforin into breast milk was confirmed in and 0.79 (CI = 0.51 to 1.03) respectively). In 7 studies versus 5 other breastfeeding women. Low plasma concentrations of older antidepressants, drop-out and adverse effect rates were hyperforin were detected in breastfed infant plasma, with a significantly lower in the St. John's wort group (OR = 0.25 (CI = milk-to-plasma ratio of 0.04 to 0.13 corresponding to relative 0.14 to 0.45) and 0.39 (CI = 0.31 to 0.50)). In 6 studies versus doses of 0.9% to 2.5%. No adverse effects were observed in SSRIs, drop-out and adverse effect rates were slightly but not mothers or infants [Klier 2006]. significantly lower in the St. John's wort group (OR = 0.60 (CI = 0.31 to 1.15) and 0.75 (CI = 0.52 to 1.08)) [Knüppel 2004]. Phototoxicity and photosensitization A double-blind, placebo-controlled study with 40 volunteers The clinical efficacy of some standardised methanolic and showed that photosensitivity was not induced by therapeutically ethanolic extracts in the treatment of mild, moderate and severe relevant dosages of total hypericin, i.e. up to 1 mg daily for 8 major depression has been demonstrated in 38 controlled clinical days [Wienert 1991]. trials and two recent meta-analyses. Sixteen post-marketing surveillance studies with such preparations, based on a total In a study involving i.v. administration of synthetic hypericin of 34,804 patients, recorded an incidence of adverse events to HIV-infected patients, reversible symptoms of phototoxicity

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were observed at the highest dosage regime, which was 35-times REFERENCES higher than the highest oral dosage of total hypericin used in the therapy of depressive disorders [James 1992]. Abdali K, Khajehei M, Tabatabaee HR. Effect of St John's wort on severity, frequency, and duration of hot flashes in premenopausal, perimenopausal In a placebo-controlled, randomised clinical trial (n = 13), high and postmenopausal women: a randomized, double-blind, placebo- daily oral doses of up to 3600 mg of a methanolic extract (80%, controlled study. Menopause 2010; 17(2): 326-31. V/V; corresponding to 11.25 mg hypericin) were administered http://dx.doi.org/10.1097/gme.0b013e3181b8e02d to volunteers. No dose-related trend in light sensitivity to solar stimulated irradiation (SSI) was observed. Sensitivity to UVA light Abdel-Salam OM. Anti-inflammatory, antinociceptive, and gastric effects was slightly but significantly (p=0.03) increased only after the of Hypericum perforatum in rats. Scientific World J 2005; 5: 586-95. highest dose (from 10.8 J/cm2 (placebo) to 8.7 J/cm2 (extract)). http://dx.doi.org/10.1100/tsw.2005.78 After multiple doses of 5.6 mg total hypericin for 15 days, Al-Akoum M, Maunsell E, Verreault R, Provencher L, Otis H, Dodin S. the minimal erythema dose (MED) was assessed. A small but Effects of Hypericum perforatum (St. John's wort) on hot flashes and significantly increased SSI sensitivity was observed (decrease quality of life in perimenopausal women: a randomized pilot trial. in MED from 0.17 to 0.16 J/cm2, p=0.005) and an increased 2 Menopause 2009; 16(2): 307-14. sensitivity to UVA light (decrease in MED from 9.9 to 7.8 J/cm , http://dx.doi.org/10.1097/gme.0b013e31818572a0 p<0.0001). In spite of the high doses used, frequency of side effects was equal to placebo medication and light sensitivity Albert D, Zündorf I, Dingermann T, Müller WE, Steinhilber D, Werz O. was only marginally increased [Brockmöller 1997]. Hyperforin is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase. Biochemical 2002; 64(12): 1767-75. After topical application of St. John’s wort oil (110 µg/mL http://dx.doi.org/10.1016/S0006-2952(02)01387-4 hypericin; n = 8) and St. John’s wort ointment (30 µg/mL hypericin; n = 8) to the volar forearms of sixteen volunteers Albrecht M, Hübner W-D, Podzuweit H, Schmidt U. Johanniskraut- (with skin types II and III), solar simulated radiation was applied. Extrakt zur Behandlung der Depression. Therapiebegleitende No change of the MED was detected on visual assessment after Anwendungsbeobachtung mit Jarsin® Dragees. Der Kassenarzt 1994; either treatment (p>0.05). Using photometric measurement of 41: 45-54. skin erythema, an increase of the erythema-index was observed after treatment with the oil (p≤ 0.01) [Schempp 2000]. Alscher DM, Klotz U. of herbal tea containing St. John's wort with cyclosporine. Transpl Int 2003; 16(7): 543-4. In a prospective randomised study the effect of a methanolic http://dx.doi.org/10.1007/s00147-003-0560-z (80%) extract on skin sensitivity to ultraviolet B (UVB), ultraviolet A (UVA), visible light (VIS) and solar simulated radiation was Amini Z, Boyd B, Doucet J, Ribnicky DM, Stephens JM. St. John's determined. Volunteers of skin types II and III received either a wort inhibits adipocyte differentiation and induces insulin resistance single dose equivalent to 5.4 or 10.8 mg hypericin (n = 48) or in adipocytes. Biochem Biophys Res Commun 2009; 388(1): 146-9. an initial dose equivalent to 5.4 mg hypericin, and subsequently http://dx.doi.org/10.1016/j.bbrc.2009.07.137 3 x 2.7 mg hypericin per day for 7 days (n = 24). No significant Andersen DO, Weber ND, Wood SG, Hughes BG, Murray BK, North JA. influence on the erythema-index or melanin-index was detected In vitro virucidal activity of selected and anthraquinone after either dose regimen, with the exception of a marginal derivatives. Antiviral Res 1991; 16(2): 185-96. influence on UVB induced pigmentation (p=0.0471) in the http://dx.doi.org/10.1016/0166-3542(91)90024-L single-dose study [Schempp 2003b]. Andren L, Andreasson A, Eggertsen R. Interaction between a commercially Volunteers received 3 x 340 mg of an ethanolic (60%, V/V) available St. John's wort product (Movina) and atorvastatin in patients extract daily corresponding to 3 mg hypericin over a period of with hypercholesterolemia. Eur J Clin Pharmacol 2007; 63(10): 913-6. 10 days. Even at this high hypericin concentration, and in spite http://dx.doi.org/10.1007/s00228-007-0345-x of the observed decrease in visual and objective MED reduction from 20 to 14 J/cm2 (p=0.047) and from 33.73 to 22.0 J/cm2 Anghelescu IG, Kohnen R, Szegedi A, Klement S, Kieser M. Comparison (p=0.014) respectively, and an increase in erythema intensity of Hypericum extract WS 5570 and paroxetine in ongoing treatment (in 73% of subjects), the maximum slope of the dose-response after recovery from an episode of moderate to severe depression: curve (which is an indicator of an individual’s susceptibility results from a randomized multicenter study. Pharmacopsychiatry to burning) was not increased by the extract [Beattie 2005]. 2006; 39(6): 213-9. http://dx.doi.org/10.1055/s-2006-951388 The effect of two different ethanolic extracts (50% and 80%) was assessed on photosensitivity with respect to MED after 14 Araya OS, Ford EJ. An investigation of the type of photosensitization days of treatment. Both open, multiple-dose, phase I studies caused by the ingestion of St John's Wort (Hypericum perforatum) by were conducted in 20 healthy men receiving 612 and 900 mg calves. J Comp Pathol 1981; 91(1): 135-41. of the extracts respectively. Mean MED at 24 h, in both studies, http://dx.doi.org/0021-9975(81)90053-0 [pii] was not significantly different between baseline and after 14 days of treatment [Schulz 2006a]. Arborelius L, Owens MJ, Plotsky PM, Nemeroff CB. The role of corticotropin-releasing factor in depression and anxiety disorders. The Journal of endocrinology 1999; 160(1): 1-12. The effect of extract use on cataractogenesis was investigated utilising data from a large, population-based sample. Self- Arndt S, Haag SF, Kleemann A, Lademann J, Meinke MC. Radical reported data on St. John’s wort use in the past 12 months protection in the visible and infrared by a hyperforin-rich cream--in and cataracts were obtained from the 2002 National Health vivo versus ex vivo methods. Experimental dermatology 2013; 22(5): Interview Survey, a nationally representative population-based 354-7. http://dx.doi.org/10.1111/exd.12124 sample. Crude and adjusted OR and 95% CI between SJW use and cataracts were estimated using logistic regression. After Arokiyaraj S, Balamurugan R, Augustian P. Antihyperglycemic effect of adjusting for potential confounding characteristics, participants Hypericum perforatum ethyl acetate extract on streptozotocin-induced that reported having cataracts were 59% more likely to report diabetic rats. Asian Pac J Trop Biomed 2011; 1: 386-90. St. John’s wort use (OR 1.59; 95% CI 1.02-2.46) [Booth 2009]. http://dx.doi.org/10.1016/S2221-1691(11)60085-3Arokiyaraj AsianPacJ

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Arold G, Donath F, Maurer A, Diefenbach K, Bauer S, Henneicke-von Beattie PE, Dawe RS, Traynor NJ, Woods JA, Ferguson J, Ibbotson SH. Zepelin HH, Friede M, Roots I. No relevant interaction with alprazolam, Can St John's wort (hypericin) ingestion enhance the erythemal response caffeine, tolbutamide, and digoxin by treatment with a low-hyperforin during high-dose ultraviolet A1 therapy? Br J Dermatology 2005; 153(6): St John's wort extract. Planta medica 2005; 71(4): 331-7. 1187-91. http://dx.doi.org/10.1111/j.1365-2133.2005.06946.x http://dx.doi.org/10.1055/s-2005-864099 Beck AT. Depression: Causes and Treatment: Philadelphia: University Avato P, Raffo F, Guglielmi G, Vitali C, Rosato A. Extracts from St John's of Pennsylvania Press; 1972. Wort and their antimicrobial activity. Phytother Res 2004; 18(3): 230-2. http://dx.doi.org/10.1002/ptr.1430 Behnke K, Jensen GS, Graubaum HJ, Gruenwald J. Hypericum perforatum versus fluoxetine in the treatment of mild to moderate Aydin A, Sakrak O, Yilmaz TU, Kerem M. The effects of Hypericum depression. Adv Ther 2002; 19(1): 43-52. perforatum on hepatic ischemia - reperfusion injury in rats. Bratislavske lekarske listy 2014; 115(4): 209-15. Beijamini V, Andreatini R. Effects of Hypericum perforatum and parox- etine in the mouse defense test battery. Pharmacol Biochem Behav 2003; Bach-Rojecky L, Kalodjera Z, Samarzija I. The antidepressant activity 74(4): 1015-24. http://dx.doi.org/10.1016/S0091-3057(03)00034-0 of Hypericum perforatum L. measured by two experimental methods on mice. Acta pharmaceutica 2004; 54(2): 157-62. Bell EC, Ravis WR, Chan HM, Lin YJ. Lack of Pharmacokinetic Interaction between St. John's Wort and Prednisone. Ann Pharmacother 2007a; Barbenel DM, Yusufi B, O'Shea D, Bench CJ. Mania in a patient receiving 41: 1819-24. testosterone replacement postorchidectomy taking St John's wort and sertraline. Journal of 2000; 14(1): 84-6. Bell EC, Ravis WR, Lloyd KB, Stokes TJ. Effects of St. John's wort supplementation on ibuprofen pharmacokinetics. Ann Pharmacother Barliya T, Mandel M, Livnat T, Weinberger D, Lavie G. Degradation 2007b; 41(2): 229-34. http://dx.doi.org/10.1345/aph.1H602 of HIF-1alpha under hypoxia combined with induction of Hsp90 polyubiquitination in cancer cells by hypericin: a unique cancer therapy. Benedi J, Arroyo R, Romero C, Martin-Aragon S, Villar AM. Antioxidant PloS one 2011; 6(9): e22849. properties and protective effects of a standardized extract of Hypericum http://dx.doi.org/10.1371/journal.pone.0022849 perforatum on -induced oxidative damage in PC12 cells. Life Sci 2004; 75(10): 1263-76. Barnard DL, Huffman JH, Morris JL, Wood SG, Hughes BG, Sidwell RW. http://dx.doi.org/10.1016/j.lfs.2004.05.001 Evaluation of the antiviral activity of anthraquinones, anthrones and anthraquinone derivatives against human cytomegalovirus. Antiviral Res Berghöfer R, Hölzl J. Biflavonoids in Hypericum perforatum; Part 1. 1992; 17(1): 63-77. http://dx.doi.org/10.1016/0166-3542(92)90091-I Isolation of I3, II8-Biapigenin. Planta medica 1987; 53(2): 216-7.

Barnes J, Barber N, Wheatley D, Williamson EM. A pilot randomised, Bergmann R, Nüßner J, Demling J. Behandlung leichter bis mittelschwerer open, uncontrolled, clinical study of two dosages of St John's wort Depressionen. Vergleich von Hypericum perforatum mit Amitriptylin. (Hypericum perforatum) herb extract (LI-160) as an aid to motivational/ TW Neurol Psychiatr 1993; 7: 235-40. behavioural support in smoking cessation. Planta medica 2006; 72(4): 378-82. http://dx.doi.org/10.1055/s-2005-916219 Bergonzi MC, Bilia AR, Gallori S, Guerrini D, Vincieri FF. Variability in the content of the constituents of Hypericum perforatum L. and Barone GW, Gurley BJ, Ketel BL, Abul-Ezz SR. Herbal supplements: a some commercial extracts. Drug Dev Ind Pharm 2001; 27(6): 491-7. potential for drug interactions in transplant recipients. Transplantation http://dx.doi.org/10.1081/ddc-100105173 2001; 71(2): 239-41. Berlanda J, Kiesslich T, Oberdanner CB, Obermair FJ, Krammer B, Barone GW, Gurley BJ, Ketel BL, Lightfoot ML, Abul-Ezz SR. Drug inter- Plaetzer K. Characterization of apoptosis induced by photodynamic action between St. John's wort and cyclosporine. Ann Pharmacother treatment with hypericin in A431 human epidermoid carcinoma cells. 2000; 34(9): 1013-6. http://dx.doi.org/10.1345/aph.10088 J Environ Pathol Toxicol Oncol 2006; 25(1-2): 173-88. http://dx.doi.org/5d84548f012e18bf,04443efd0aff3b6d [pii] Bauer S, Stoermer E, Kerb R, Johne A, Brockmoeller J, Roots I. Differential effects of Saint John's Wort (Hypericum perforatum) on the Bernd A, Simon S, Ramirez Bosca A, Kippenberger S, Diaz Alperi J, Miquel urinary excretion of D-glucaric acid and 6b-hydroxycortisol in healthy J, Villalba Garcia JF, Pamies Mira D, Kaufmann R. Phototoxic effects of volunteers. Eur J Clin Pharmacol 2002; 58: 581-5. Hypericum extract in cultures of human keratinocytes compared with http://dx.doi.org/10.1007/s00228-002-0527-5 those of psoralen. Photochem Photobiol 1999; 69(2): 218-21. http://dx.doi.org/10.1111/j.1751-1097.1999.tb03276.x Bauer S, Störmer E, Johne A, Krüger H, Budde K, Neumayer HH, Roots I, Mai I. Alterations in cyclosporin A pharmacokinetics and metabolism Bernhardt M, Liske E. Antidepressive Therapie mit Johanniskraut- during treatment with St John's wort in renal transplant patients. Br J Extrakt unter besonderer Berücksichtigung von zwei unterschiedlichen Clin Pharmacol 2003; 55(2): 203-11. Dosierungsschemata. Jatros Neurologie 1996; 1996(12): 43-8.

Baureithel KH, Buter KB, Engesser A, Burkard W, Schaffner W. Inhibition Bhattacharya SK, Chakrabarti A, Chatterjee SS. Activity profiles of of benzodiazepine binding in vitro by amentoflavone, a constituent two hyperforin-containing hypericum extracts in behavioral models. of various species of Hypericum. Pharmaceutica acta Helvetiae 1997; Pharmacopsychiatry 1998; 31 Suppl 1: 22-9. 72(3): 153-7. http://dx.doi.org/10.1055/s-2007-979342

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Booth JN, 3rd, McGwin G. The association between self-reported Budzinski JW, Foster BC, Vandenhoek S, Arnason JT. An in vitro evaluation cataracts and St. John's Wort. Curr Eye Res 2009; 34(10): 863-6. of human cytochrome P450 3A4 inhibition by selected commercial http://dx.doi.org/10.3109/02713680903144692 herbal extracts and tinctures. Phytomedicine 2000; 7(4): 273-82.

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Tian R, Koyabu N, Morimoto S, Shoyama Y, Ohtani H, Sawada Y. Uzbay IT, Coskun I, Kayir H, Ozturk N, Ozturk Y. Extract of Hypericum Functional induction and de-induction of P-glycoprotein by St. John's perforatum blocks caffeine-induced locomotor activity in mice: a wort and its ingredients in a human colon adenocarcinoma cell line. possible role of nitric oxide. Phytother Res 2007; 21(5): 415-9. Drug Metab Dispos 2005; 33(4): 547-54. http://dx.doi.org/10.1002/ptr.2085 http://dx.doi.org/10.1124/dmd.104.002485 van Gurp G, Meterissian GB, Haiek LN, McCusker J, Bellavance F. St Timoshanko A, Stough C, Vitetta L, Nathan PJ. A preliminary investigation John's wort or sertraline? Randomized controlled trial in primary care. on the acute pharmacodynamic effects of hypericum on cognitive Can Fam Physician 2002; 48: 905-12. and psychomotor performance. Behavioural pharmacology 2001; 12: 635-40. Van Strater AC, Bogers JP. Interaction of St John's wort (Hypericum perforatum) with clozapine. International clinical psychopharmacology Treiber K, Singer A, Henke B, Müller WE. Hyperforin activates 2011; 27(2): 121-4. nonselective cation channels (NSCCs). British journal of pharmacology 2005; 145(1): 75-83. Vandenbogaerde AL, Kamuhabwa A, Delaey E, Himpens BE, Merlevede WJ, de Witte PA. Photocytotoxic effect of pseudohypericin versus Trifunovic S, Vajs V, Macura S, Juranic N, Djarmati Z, Jankov R, hypericin. J Photochem Photobiol B 1998; 45(2-3): 87-94. Milosavljevic S. Oxidation products of hyperforin from Hypericum perforatum. Phytochemistry 1998; 49(5): 1305-10. Vantieghem A, Xu Y, Assefa Z, Piette J, Vandenheede JR, Merlevede http://dx.doi.org/10.1016/S0031-9422(97)00903-5 W, De Witte PA, Agostinis P. Phosphorylation of Bcl-2 in G2/M phase- arrested cells following photodynamic therapy with hypericin involves Tripathi YB, Pandey E. Role of alcoholic extract of shoot of Hypericum a CDK1-mediated signal and delays the onset of apoptosis. J Biol Chem perforatum Linn on lipid peroxidation and various species of free radicals 2002; 277(40): 37718-31. in rats. Indian J Exp Biol 1999; 37(6): 567-71. Vieira ML, Hamada RY, Gonzaga NI, Bacchi AD, Barbieri M, Moreira Trofimiuk E, Braszko JJ. Alleviation by Hypericum perforatum of the EG, Mesquita Sde F, Gerardin DC. Could maternal exposure to the stress-induced impairment of spatial working memory in rats. Naunyn- antidepressants fluoxetine and St. John's Wort induce long-term Schmiedeberg's Arch Pharmacol 2008; 376(6): 463-71. reproductive effects on male rats? Reproductive toxicology 2013a; 35: http://dx.doi.org/10.1007/s00210-007-0236-9 102-7. http://dx.doi.org/10.1016/j.reprotox.2012.07.006

Trofimiuk E, Holownia A, Braszko JJ. Activation of CREB by St. John's Vieira VA, Campos LV, Silva LR, Guerra MO, Peters VM, Sá RdCS. wort may diminish deletorious effects of aging on spatial memory. Arch Evaluation of postpartum behaviour in rats treated with Hypericum

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perforatum during gestation. Rev Bras Farmacogn 2013b; 23: 796-801. Wang Z, Gorski JC, Hamman MA, Huang SM, Lesko LJ, Hall SD. The http://dx.doi.org/10.1590/S0102-695X2013000500012 effects of St John's wort (Hypericum perforatum) on human cytochrome P450 activity. Clin Pharmacol Ther 2001; 70(4): 317-26. Vitiello B, Shader RI, Parker CB, Ritz L, Harlan W, Greenblatt DJ, Gadde http://dx.doi.org/10.1016/S0009-9236(01)00127-8 KM, Krishnan KR, Davidson JR. Hyperforin plasma level as a marker of treatment adherence in the National Institutes of Health Hypericum Wang Z, Hamman MA, Huang SM, Lesko LJ, Hall SD. Effect of St John's Depression Trial. J Clin Psychopharmacol 2005; 25(3): 243-9. wort on the pharmacokinetics of fexofenadine. Clin Pharmacol Ther http://dx.doi.org/00004714-200506000-00008 [pii] 2002; 71(6): 414-20. http://dx.doi.org/10.1067/mcp.2002.124080

Volak LP, Court MH. Role for protein kinase C delta in the functional Warnecke G. Beeinflussung klimakterischer Depressionen. Therapie- activity of human UGT1A6: implications for drug-drug interactions ergebnisse mit Hypericin (Johanniskraut-Extrakt). Z Allg Med 1986; between PKC inhibitors and UGT1A6. Xenobiotica 2010; 40(5): 306- 62: 1111-3. 18. http://dx.doi.org/10.3109/00498251003596817 Watkins RE, Maglich JM, Moore LB, Wisely GB, Schroeder M, Davis- Volz HP. St John's wort extract (Li 160) in somatoform disorder: results Searles PR, Lambert MH, Kliewer SA, Redinbo MR. 2.1 Å crystal of a placebo-contolled trial. Psychopharmacology 2002; 164: 1-7. structure of human PXR in complex with the St. John's wort compound hyperforin. Biochemistry 2003; 42: 1430-8. Volz HP, Zeller K. Hochdosiertes Johanniskrautextract als Einmalgabe. http://dx.doi.org/10.1021/bi0268753 Nervenheilkunde 2000: 1-4. Weber CC, Kressmann S, Fricker G, Müller WE. Modulation of von Zerssen D. Beschwerdenskalen bei Depressionen. Therapiewoche P-glycoprotein function by St John's wort extract and its major 1973; 46: 4426-40. constituents. Pharmacopsychiatry 2004; 37(6): 292-8. http://dx.doi.org/10.1055/s-2004-832686 Vorbach EU, Arnoldt KH, Hübner WD. Efficacy and tolerability of St. John's wort extract LI 160 versus imipramine in patients with severe Weber W, Vander Stoep A, McCarty RL, Weiss NS, Biederman J, depressive episodes according to ICD-10. Pharmacopsychiatry 1997; McClellan J. Hypericum perforatum (St John's wort) for attention-deficit/ 30 Suppl 2: 81-5. hyperactivity disorder in children and adolescents: a randomized controlled trial. JAMA 2008; 299(22): 2633-41. http://dx.doi. Vorbach EU, Hübner WD, Arnoldt KH. Effectiveness and tolerance of the org/10.1001/jama.299.22.2633 Hypericum extract LI 160 in comparison with imipramine: randomized double-blind study with 135 outpatients. J Geriatr Psychiatry Neurol Weiser D. Pharmakokinetik von Hypericin nach oraler Einnahme des 1994; 7 Suppl 1: S19-23. Johanniskraut -Extraktes LI 160. Nervenheilkunde 1991; 10: 318-9.

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Widy-Tyszkiewicz E, Piechal A, Joniec I, Blecharz-Klin K. Long term Wonnemann M, Singer A, Siebert B, Müller WE. Evaluation of administration of Hypericum perforatum improves spatial learning and synaptosomal uptake inhibition of most relevant constituents of John's memory in the water maze. Biological & pharmaceutical bulletin 2002; Wort. Pharmacopsychiatry 2001; 34(Suppl 1): S148-51. 25: 1289-94. http://dx.doi.org/10.1248/bpb.25.1289 Wright CW, Gott M, Grayson B, Hanna M, Smith AG, Sunter A, Neill JC. Wielgus AR, Chignell CF, Miller DS, Van Houten B, Meyer J, Hu DN, Correlation of hyperforin content of Hypericum perforatum (St John's Roberts JE. Phototoxicity in human retinal pigment epithelial cells Wort) extracts with their effects on alcohol drinking in C57BL/6J mice: a promoted by hypericin, a component of St. John's wort. Photochem preliminary study. Journal of psychopharmacology 2003; 17(4): 403-8. 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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.

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