German Kava Ban Lifted by Court: The Alleged Hepatotoxicity of Kava (Piper methysticum) as a Case of Ill-Defined Herbal Drug Identity, Lacking Quality Control, and Misguided Regulatory Politics.

Kava, the rhizome and roots of Piper methysticum, are one of the most important social pillars of Melanesian societies. They have been used for more than 1000 years in social gatherings for the preparation of beverages with relaxing effects. During the colonial period, extract preparations found their way into Western medicinal systems, with experience especially concerning the treatment of situational anxiety dating back more than 100 years. It therefore came as a surprise when the safety of kava was suddenly questioned based on the observation of a series of case reports of liver toxicity in 1999 and 2000. These case reports ultimately led to a ban of kava products in Europe - a ban that has been contested because of the poor evidence of risks related to kava. Only recently, two German administrative courts decided that the decision of the regulatory authority to ban kava as a measure to ensure consumer safety was inappropriate and even associated with an increased risk due to the higher risk inherent to the therapeutic alternatives. This ruling can be considered as final for at least the German market, as no further appeal has been pursued by the regulatory authorities. However, in order to prevent further misunderstandings, especially in other markets, the current situation calls for a comprehensive presentation of the cardinal facts and misconceptions concerning kava and related drug quality issues.

What is the best strategy for preclinical testing of botanicals? A critical perspective.

The development of a new drug is generally marked by a number of preclinical investigations in a sequential order with regard to contents and logic. However, ethnopharmacology often uses the "reverse pharmacology" approach, which is based on anecdotal therapeutic effects of plants in ancient texts or based on the empirical knowledge of traditional healers. While this approach could successfully lead to new therapeutic applications by using sophisticated techniques and appropriate bioassays in a logical order, unfortunately there is an exponentially increasing number of reports of pharmacological effects of botanical extracts with insignificant bioactivities obtained in often irrelevant in vitro bioassays. The interpretation based on in vitro data can only be misleading since the pharmacokinetic properties of a compound are ignored, unacceptable high dosages of extracts are tested, or metabolism to inactive metabolites is not considered. Further, many natural products are prodrugs that need to be metabolized in vivo by the intestinal microflora or by mammalian phase I/II metabolism. Frequently, attempts are made to master poor pharmacokinetics by administering the extract intraperitoneally or intravenously, clearly moving away from the traditional oral application. In this review article, it is proposed that preclinical testing strategies of botanicals should start with the in vivo examination of extracts in relevant animal models to substantiate the ethnopharmacological/ethnopharmaceutical use, followed by bioguided fractionation processes using an adequate in vitro model, further followed by pharmacokinetic studies and final in vivo testing of isolated compounds. With our article we would like to encourage authors, reviewers and editors to implement this strategy for the design of experiments and for the reviewing and editing process of manuscripts.

Proanthocyanidins from Ginkgo biloba leaf extract and their radical scavenging activity.

CONTEXT: Ginkgo biloba L (Ginkgoaceae) is a traditional herbal medicinal plant for the treatment of mild to moderate cognitive disorders, tinnitus, and dementia. These uses may be correlated with the presence of radical scavenging compounds. OBJECTIVE: The chemical composition and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of the flavan-3-ols and proanthocyanidins from G. biloba were studied. MATERIAL AND METHODS: The compounds have been isolated using column chromatography on Sephadex LH-20 and MCI gel and the structures were determined on the basis of 1D- and 2D-NMR (HSQC, HMBC) experiments of their peracetylated derivatives, MALDI-TOF-MS and by acid-catalyzed degradation with phloroglucinol. The DPPH radical scavenging activities of the compounds were investigated. RESULTS: The new trimeric prodelphinidin, epigallocatechin-(4ß→8)-epigallocatechin-(4ß→8)-catechin (compound 7), has been isolated from the air-dried leaves of the title plant, in addition to catechin, epigallocatechin, gallocatechin, and three dimeric proanthocyanidins. The dimeric prodelphinidin epigallocatechin-(4ß→8)-epigallocatechin (compound 6) showed the strongest DPPH radical scavenging activity, with IC(50) 1.7 µg/mL, 10 times more active than the positive control, BHT (IC(50) 17.3 µg/mL), followed by the new trimeric proanthocyanidin epigallocatechin-(4ß→8)-epigallocatechin-(4ß→8)-catechin with IC(50) 2.1 µg/mL. The crude extract exhibited high DPPH radical scavenging activity with IC(50) 15.5 µg/mL comparable with that of BHT. DISCUSSION AND CONCLUSION: The results showed that all the isolated compounds from the tannin fraction exhibited potent free radical scavenging activities, which were higher than that of BHT, suggesting that the condensed tannins from G. biloba leaves strongly contribute to the overall antioxidant effects.

Kava (Piper methysticum) Extract for the Treatment of Nervous Anxiety, Tension and Restlessness.

AIM: Prior to the kava ban of 2002, the indication for kava (Piper methysticum) extracts defined by the German Commission E was "nervous anxiety, tension and restlessness". In 2000, an observational trial was started in Germany with the aim of defining symptoms of these indications best treated with kava extract. The trial was interrupted and archived "unevaluated" in 2001 due to the upcoming safety debate on kava. The data from this study has now been analyzed in order to identify symptoms best treated with kava. METHODS: Documentation was available from 156 patients. Twelve typical symptoms of nervous anxiety, tension and restlessness were assessed on a five-item rating scale, together with the therapeutic context, the perceived time of onset of effects and the safety of application. RESULTS: The median duration of treatment was 28 days. All individual symptoms showed significant and clinically relevant improvements. The most effective results were seen for nervous tension and restlessness, with better effects in patients with acute versus chronic complaints. The safety of the treatment was found to be excellent, which included the assessment of laboratory data. CONCLUSIONS: Overall, the study confirms the effective and safe short-term use of kava in the Commission E-defined indication of "nervous anxiety, tension and restlessness", especially in other than chronic cases. The clinical use of kava might be translated into context-related phobias according to ICD-10 F40, or to nervous tension (ICD10 R45.0) or restlessness and excitation (ICD-10 R45.1).

Lessons learned from herbal medicinal products: the example of St. John's Wort (perpendicular).

The example of St. John's wort offers convincing evidence for the concept that modern methods of pharmacological and phytochemical research are effective in advancing the development of traditional herbal remedies. As a consequence of these efforts, it is known today that several compounds from different structural groups and with different mechanisms of action seem to be responsible for the observed antidepressant efficacy of St. John's Wort. Co-effectors in the extract improve the bioavailability of active constituents such as hypericin (1) (pharmacokinetic synergy). Unwanted side effects are preventable without remarkable loss of activity when the responsible constituent(s) are carefully removed during the extraction process, as demonstrated for hyperforin (3), which is responsible for the induction of cytochrome P450 (CYP)-metabolizing enzymes (CYP3A4, in particular). On the basis of our findings, it is likely that positive interactions between single compounds occur more frequently in traditionally used herbal preparations than is known presently.

Synthesis and characterisation of α-Glycosyloxyamides derived from cyanogenic glycosides.

INTRODUCTION: After exposure to oxidative stress, the leaves of some cyanogenic plants contain primary α-glycosyloxyamides with structures corresponding to their original cyanogenic glycosides. OBJECTIVES: The aim of this study was to prepare such amides from their nitrile precursors and to characterise the new substances in order to facilitate their early identification in forthcoming studies. Methods - A simple but highly specific method is described for the in-vitro synthesis of the amides from their nitrile glycoside precursors using the Radziszewski reaction with hydrogen peroxide and a single-step purification of the reaction product. A TLC method is presented for the preliminary and fast identification of the α-glycosyloxyamides. RESULTS: Following this procedure, seven representative α-glycosyloxyamides, five of them new, were obtained and analytically characterised by means of (1) H, (13) C NMR and ATR-IR spectroscopy, highlighting the differences from their respective nitrile glycoside precursors. CONCLUSION: Thus, α-glycosyloxyamides can be obtained in sufficient amounts and purity to serve as references for further studies on the catabolism of cyanogenic glycosides and the detoxification of cyanogenic foodplants using the new aspect of nitrile hydrolysis with (endogenous) hydrogen peroxide.

A bioactive prodelphinidin from Mangifera indica leaf extract.

A new trimeric proanthocyanidin, epigallocatechin-3-O-gallat-(4beta-->8)-epigallocatechin-(4beta-->8)-catechin (1), was isolated together with three known flavan-3-ols, catechin (2), epicatechin (3), and epigallocatechin (4), and three dimeric proanthocyanidins, 5-7, from the air-dried leaves of Mangifera indica. Their chemical structures were determined on the basis of 1D- and 2D-NMR spectra (HSQC, HMBC) of their peracetylated derivatives, MALDI-TOF-mass spectra, and by acid-catalyzed degradation with phloroglucinol. The isolated compounds 1-7 were in vitro tested for their inhibitory activities against COX-1 and COX-2. Compound 1 was found to have a potent inhibitory effect on COX-2, while compounds 1 and 5-7 exhibited moderate inhibition against COX-1.

Generation of primary amide glucosides from cyanogenic glucosides.

The cyanogenic glucoside-related compound prunasinamide, (2R)-beta-d-glucopyranosyloxyacetamide, has been detected in dried, but not in fresh leaves of the prunasin-containing species Olinia ventosa, Prunus laurocerasus, Pteridium aquilinium and Holocalyx balansae. Experiments with leaves of O. ventosa indicated a connection between amide generation and an excessive production of reactive oxygen species. In vitro, the Radziszewski reaction with H(2)O(2) has been performed to yield high amounts of prunasinamide from prunasin. This reaction is suggested to produce primary amides from cyanogenic glycosides in drying and decaying leaves. Two different benzoic acid esters which may be connected to prunasin metabolism were isolated and identified as the main constituents of chlorotic leaves from O. ventosa and P. laurocerasus.

Cyanogenic and non-cyanogenic pyridone glucosides from Acalypha indica (Euphorbiaceae).

Seven cyanopyridone derivatives and one corresponding seco compound have been isolated from a methanolic extract of the inflorescences and leaves of Acalypha indica L. (Euphorbiaceae). The absolute configuration of the main cyanogenic glucoside acalyphin, (-)-(5R,6S)-5-cyano-5-beta-d-glucopyranosyloxy-6-hydroxy-4-methoxy-1-methyl-2(5,6-dihydro)-pyridone, was deduced from an X-ray crystallographic study. In addition, the 6R-epimer of acalyphin, epiacalyphin, and the corresponding pair of N-demethyl derivatives were isolated. The corresponding amide of acalyphin and a 1',2'-glucosyl-fused epiacalyphin amide were isolated from air-dried material. Structural elucidation was performed by means of (1)H and (13)C NMR-spectra, chiroptical methods such as CD-spectroscopy and optical rotation. Two further corresponding derivatives, an aromatized compound and an open-chain structure, were isolated from the aqueous phase.

Anti-inflammatory, analgesic and wound healing activities of the leaves of Memecylon edule Roxb.

AIM OF THE STUDY: To determine the anti-inflammatory, analgesic and antioxidant activities of the leaves of Memecylon edule Roxb. used traditionally in Thailand. MATERIALS AND METHODS: Hexane, (Hex), ethyl acetate (EtOAc), methanol (MeOH) and 50% methanol (MeOH50) fractions of the dry leaves were tested in vitro for their interleukin-10 production; the most active fraction was further studied in vivo for its anti-inflammatory and analgesic activities using the ethylphenylpropiolate (EPP)-induced mouse ear edema and the writhing test with mice. All fractions except Hex were tested for their radical scavenging activity towards 1'-diphenyl-2-picrylhydrazyl radical (DPPH). RESULTS: The EtOAc showed the highest stimulation for interleukin-10 production. In the EPP test, this fraction was significantly active 30 min after topical application at all doses used (0.5, 1.0, 2.0mg/ear); after 4h and at 1.0mg/ear EtOAc was slightly less active (inhibition 47.8%) than the reference, indomethacin, at the same dose (62.4%). At 200mg/kg orally, the EtOAc caused a significant inhibition of the writhing response by 56.6% which was like indomethacin at 10mg/kg. EtOAc, MeOH and MeOH50 exhibited radical scavenging activity. The order of IC(50) values was: ascorbic acid (9.1 microg/mL)>trolox (11.6 microg/mL)>MeOH (46.9 microg/mL)>MeOH50 (152.1 microg/mL)>EtOAc (1742.2 microg/mL). CONCLUSION: The results provide support for the traditional use of Memecylon edule leaves in relieving inflammation and pain.

Antioxidant oligomeric proanthocyanidins from Cistus salvifolius.

The purified proanthocyanidin oligomers of Cistus salvifolius herb extract accounted for 78% of the total proanthocyanidins and 73% of the total antioxidant activity of this extract. To elucidate the structure of the oligomer, it was depolymerized by acid catalysis in the presence of phloroglucinol. The structures of the resulting flavan-3-ols and phloroglucinol adducts were determined on the basis of 1D- and reverse 2D-NMR (HSQC, HMBC) experiments of their peracetylated derivatives, MALDI-TOF-MS and CD spectroscopy. These observations resulting from the degradation with phloroglucinol were confirmed by 13C NMR spectroscopy of the oligomer. The mean molecular weight of the higher oligomeric fraction was estimated to be 5-6 flavan-3-ol-units.

In vitro receptor screening of pure constituents of St. John's wort reveals novel interactions with a number of GPCRs.

RATIONALE: Hypericum perforatum L. (St. John's wort; SJW) is one of the leading psychotherapeutic phytomedicines and great effort has been devoted to clarifying its mechanism of action. OBJECTIVE: We have undertaken a comprehensive analysis of several pure compounds isolated from the crude extract to gain further insight into the molecular actions of various substituents of SJW. METHODS: We characterized the in vitro pharmacology of the naphthodianthrones hypericin and pseudohypericin, the phloroglucinol derivative hyperforin, and several flavonoids at 42 biogenic amine receptors and transporters using the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. RESULTS: The biflavonoid amentoflavone significantly inhibited binding at serotonin (5-HT(1D), 5-HT(2C)), D(3)-dopamine, delta-opiate, and benzodiazepine receptors. The naphthodianthrone hypericin had significant activity at D(3)- and D(4)-dopamine receptors and beta-adrenergic receptors. With the exception of the D(1)-dopamine receptor, the phloroglucinol derivative hyperforin was less active than other SJW constituents tested on all screened receptors. CONCLUSION: Our present in vitro data clearly show that several pure substances in SJW are potential CNS psychoactive agents and may contribute to the antidepressant efficacy of the plant in a complex manner. Our data also reveal novel and heretofore unexpected interactions of pure compounds in SJW at a number of GPCRs, transporters, and ion channels. We hypothesize that additive or synergistic actions of different single compounds may be responsible for the antidepressant efficacy of SJW. These results and this general approach may impact our understanding of phytomedicines in general and H. perforatum specifically.

Cyanogenic allosides and glucosides from Passiflora edulis and Carica papaya.

Leaf and stem material of Passiflora edulis (Passifloraceae) contains the new cyanogenic glycosides (2R)-beta-D-allopyranosyloxy-2-phenylacetonitrile (1a) and (2S)-beta-D-allopyranosyloxy-2-phenylacetonitrile (1b), along with smaller amounts of (2R)-prunasin (2a), sambunigrin (2b), and the alloside of benzyl alcohol (4); the major cyanogens of the fruits are (2R)-prunasin (2a) and (2S)-sambunigrin (2b). The major cyanogenic glycoside of Carica papaya (Caricaceae) is 2a; only small amounts of 2b also are present. We were not able to confirm the presence of a cyclopentenoid cyanogenic glycoside, tetraphyllin B, in Carica papaya leaf and stem materials. In detailed 1H NMR studies of 1a/b and 2a/b, differences in higher order effects in glucosides and allosides proved to be valuable for assignment of structures in this series. The diagnostic chemical shifts of cyanogenic methine and anomeric protons in 1a/b are sensitive to anisotropic environmental effects. The assignment of C-2 stereochemistry of 1a/b was made in analogy to previous assignments in the glucoside series and was supported by GLC analysis of the TMS ethers.

Step by step removal of hyperforin and hypericin: activity profile of different Hypericum preparations in behavioral models.

Herbal extracts of Hypericum perforatum L. (St. John's wort, SJW) are now successfully competing for status as a standard antidepressant therapy. Because of this, great effort has been devoted to identifying the antidepressive active compounds. In the present study we used the following strategy to evaluate the relative pharmacological importance of various extract components: 1. preparation of an hydroalcoholic SJW extract containing both hyperforin (3.2%) and hypericin (0.15%) (extract A); 2. step by step removal of hyperforin and hypericin led to the following extracts: Extract B, devoid of hyperforin but still containing hypericin (0.14%) and Extract C, free of hypericin and hyperforin but enriched in flavonoids ( approximately 12%). We characterized the in vivo activity profile of all three preparations using the tail suspension test (TST) in mice and the forced swimming test (FST) in rats as screening models. We further investigated the activity of pure hyperforin. Extract B and C (500 mg/kg each) as well as pure hyperforin (8 mg/kg) significantly shortened immobility time in the TST after acute pre-treatment whereas extract A was inactive. In the FST all three extracts decreased immobility time in a dosage of 500 mg/kg after acute as well as after repeated treatment. The present results clearly show that an SJW extract free of hyperforin and hypericin exerts antidepressant activity in behavioral models, supporting our working hypothesis that flavonoids are part of the constituents responsible for the therapeutic efficacy of SJW extracts. We also could show that hyperforin contributes to the beneficial properties of SJW extract, confirming the hypothesis that the crude SJW extract contains several constituents with antidepressant activity.

Hydroxycinnamic Acid Derivatives Obtained from a Commercial Crataegus Extract and from Authentic Crataegus spp.

Eleven hydroxycinnamic acid derivatives were isolated from a 70% methanolic Crataegus extract (Crataegi folium cum flore) and partly verified and quantified for individual Crataegus species (C. laevigata, C. monogyna, C. nigra, C. pentagyna) by HPLC: 3-O-(E)-p-coumaroylquinic acid (1), 5-O-(E)-p-coumaroyl-quinic acid (2), 4-O-(E)-p-coumaroylquinic acid (3), 3-O-(E)-caffeoylquinic acid (4), 4-O-(E)-caffeoylquinic acid (5), 5-O-(E)-caffeoylquinic acid (6), 3,5-di-O-(E)-caffeoylquinic acid (7), 4,5-di-O-(E)-caffeoylquinic acid (8), (-)-2-O-(E)-caffeoyl-L-threonic acid (9), (-)-4-O-(E)-caffeoyl-L-threonic acid (10), and (-)-4-O-(E)-p-coumaroyl-L-threonic acid (11). Further, (-)-2-O-(E)-caffeoyl-D-malic acid (12) was isolated from C. submollis and also identified for C. pentagyna and C. nigra by co-chromatography. The isolates 10 and 11 were not found in the authentic fresh specimen, indicating that they may be formed during extraction by acyl migration from the 2-O-acylderivatives. Also, 9 and 11 are described here for the first time. All structures were assigned on the basis of their spectroscopic data ((1)H-, (13)C-NMR, MS, optical rotation).

Polyphenols from Ginkgo biloba.

By Sephadex LH-20 gel chromatography of an extract from Gingko biloba leaves, polymeric proanthocyanidins were eluted after the fractions of flavonol glycosides and biflavone glycosides. A purified proanthocyanidin polymer accounted for 86.6% of the total proanthocyanidins, and for 37.7% of the total antioxidant activity of this leaf extract. For structure elucidation, the polymer was submitted to acidic depolymerization in the presence of phloroglucinol. The structures of the resulting flavan-3-ols and phloroglucinol adducts were determined on the basis of 1D-and reverse 2D-NMR (HSQC, HMBC) spectra of their peracetylated derivatives, MALDI-TOF-MS and CD-spectroscopy. The observations resulting from the degradation with phloroglucinol were confirmed by (13)C-NMR spectroscopy of the polymer. The mean molecular weight of the polymeric fraction was estimated to be 9â10 flavan-3-ol-units.

Cinchonain Ib isolated from Eriobotrya japonica induces insulin secretion in vitro and in vivo.

AIMS OF THE STUDY: Eriobotrya japonica leaves had been used traditionally for the treatment of diabetes mellitus by immersing the dried leaves in a hot water drink. Few studies have shown the hypoglycemic effect of Eriobotrya japonica using crude alcoholic extract and isolated methanolic compounds. These studies proposed that the mechanism of action could be by stimulating the beta-islets of Langerhans to secrete insulin, however with no scientific evidence. METHODS: Eriobotrya japonica water extract (EJWE) and the compounds derived from it: cinchonain Ib, procyanidin B-2, chlorogenic acid and epicatechin, were tested for their effects on insulin secretion from INS-1 cells and following oral administration in rats. RESULTS: The present study showed that EJWE increased significantly (p<0.05) insulin secretion from INS-1 cells in dose-dependent manner. Oral administration of EJWE at 230 mg/kg to rats, however, decreased plasma insulin level for as long as 240 min post-administration and caused a transient drop of blood glucose at 15 and 30 min post-administration. On the other hand, cinchonain Ib enhanced significantly (p<0.05) insulin secretion from INS-1 cells, whereas epicatechin inhibited significantly (p<0.05) insulin secretion from INS-1 cells. In addition, cinchonain Ib enhanced significantly (150%: p<0.05) plasma insulin level in rats for as long as 240 min after 108 mg/kg oral administration but did not induce any change in blood glucose level. CONCLUSION: These data indicate that cinchonain Ib has an insulinotropic effect and suggest the possible use of cinchonain Ib for managing type 2 diabetes.

Petasiphenone, a phenol isolated from Cimicifuga racemosa, in vitro inhibits proliferation of the human prostate cancer cell line LNCaP.

Extracts of Cimicifuga racemosa (L.) Nutt. (syn.: Actaea racemosa L.) (CR) inhibit the proliferation of the human prostate cancer cell line LNCaP. Recently, the phenylpropanoid ester 3,4-dihydroxyphenacyl caffeate (petasiphenone, 1) was isolated from CR. This substance is a structural homologue to petasiphenol ([3-(3,4-dihydroxyphenyl)-2-oxopropyl caffeate]), a compound produced by Petasites japonicus Sieb. & Zucc. which inhibits the growth of various human leukemia cell lines. Because of the structural similarity, we examined whether 1 affects the proliferation of LNCaP cells and the secretion of prostate-specific antigen (PSA). Under basal conditions as well as under co-incubation with 10 nM estradiol [E2 or 1 nM dihydrotestosterone (DHT)], 1 dose-dependently inhibited proliferation of LNCaP cells while PSA release per cell was not altered. We report for the first time that a defined compound isolated from CR inhibits the growth of the human prostate cancer cells LNCaP.

Willow bark extract (BNO1455) and its fractions suppress growth and induce apoptosis in human colon and lung cancer cells.

BACKGROUND: Recently, there have been extensive efforts to evaluate the chemopreventive role of substances present in natural products. The aim of this study was to examine the effects of the main groups of compounds (salicylalcohol derivates, flavonoids, proanthocyanidins), and salicin isolated from willow bark extract BNO 1455 on proliferation and apoptosis in human colon and cancer cells. METHODS: We used human colon cyclooxygenase-2 (COX-2)-positive HT 29 and (COX-2)-negative HCT 116 or lung COX-2 proficient A 549 and low COX-2 expressing SW2 cells. After treatment for 72 h with various concentrations of single substances and acetylsalicylic acid (ASA) as control, inhibition of cell growth and cytotoxicity were measured by colorimetric WST-1 assay and propidium iodide uptake by flow cytometry, respectively. Apoptotic cells were identified by annexin V adhesion using flow cytometry. RESULTS: Studies on dose-dependent effects of BNO 1455 and its fractions showed anti-proliferative activity of all compounds with 50% maximal growth inhibitory concentrations (GI(50)) between 33.3 and 103.3 microg/ml for flavonoids and proanthocyanidins fractions and 50.0-243.0 microg/ml for salicylalcohol derivates and extract. Apoptosis induction was confirmed by annexin V adherence and analysis of cell morphology based on light scattering characteristics using flow cytometry in all cell lines at GI(50). CONCLUSIONS: We showed that willow bark extract BNO 1455 an its fractions inhibit the cell growth and promote apoptosis in human colon and lung cancer cell lines irrespective of their COX-selectivity.

Willow bark extract: the contribution of polyphenols to the overall effect.

The efficacy of willow bark extract in the treatment of painful mobility disorders, such as back pain and arthritis, has been attributed to the content of salicin and its derivatives as pro-drugs of salicylates. However, based on clinical experience and the evidence of experimental pharmacological studies, the fraction of total salicin cannot satisfactorily explain the clinical efficacy of willow bark. In addition, salicins and their metabolites lack the acetylating potential of ASA and must therefore possess a different mechanism of action. A detailed pharmacological screening of the aqueous willow bark extract STW 33-I addressed the question of the identification of fractions contributing to the overall effect. All in vivo and in vitro models studied pointed to relevant contributions of the fraction of polyphenols and flavonoids. The single compounds or their combinations responsible for the effect remain to be elucidated.