Andreas Hensel

SAFFRON IN PHYTOTHERAPY: PHARMACOLOGY AND CLINICAL USES

ZusammenfassungSafran (Stigmata von Crocus sativus L.) wird seit Jahrtausenden für medizinische Zwecke verwendet. Im Verlauf der Geschichte lässt sich regelmäßig die Verwendung gegen Krebsleiden und bei depressiver Verstimmung identifizieren. Diese Anwendungen waren auch Schwerpunkt der modernen Forschung. Vielversprechende und selektive Effekte gegen Krebs wurden in vitro und in vivo beobachtet, während antidepressive Effekte sowohl in vivo als auch in klinischen Pilot-studien bestätigt wurden. Safranextrakte haben daher das Potenzial, einen wesentlichen Beitrag zur rationalen Phytotherapie zu leisten.SummarySaffron (stigmates of Crocus sativus L.) has been used for medicinal purposes for millenaries. Throughout history, uses against cancer and depressive mood can regularly be identified. These applications have also been in the focus of modern research. Promising and selective anti-cancer effects have been observed in vitro and in vivo, but not yet in clinical trials. Antidepressant effects were found in vivo and in clinical pilot studies. Saffron extracts thus have the potential to make a major contribution to rational phytotherapy.

High molecular compounds (polysaccharides and proanthocyanidins) from Hamamelis virginiana bark: influence on human skin keratinocyte proliferation and differentiation and influence on irritated skin.

Although extracts from Hamamelis bark have long been used in therapy of skin diseases and in cosmetic formulas there are only few pharmacological investigations verifying the activity of distinct Hamamelis bark constituents. Therefore two major classes of constituents, namely polymeric proanthocyanidins and polysaccharides were isolated from Hamamelis bark and tested concerning their influence on proliferation and differentiation of cultured human keratinocytes. While the polysaccharide fraction, consisting mainly of arabans and arabinogalactans, did not effect human keratinozytes, the proanthocyanidins strongly increased the proliferation of the cells, while the differentiation was not influenced significantly. Within a preliminary cumulative in vivo study on SLS-irritated skin, proanthocyanidins (ProcyanoPlus) were proven to reduce transepidermal water loss and erythema formation. Furthermore, a clinical scoring indicated that procyanidins can influence irritative processes significantly.

Quality and Functionality of Saffron: Quality Control, Species Assortment and Affinity of Extract and Isolated Saffron Compounds to NMDA and σ1 (Sigma-1) Receptors

Extracts from saffron, the dried stigmata from Crocus sativus L., are being used more and more in preclinical and clinical trials for the treatment of cancer and depression. Because of the known quality problems of saffron, HPLC methods on RP(18) 2.5 microm and monolithic RP(18) material have been developed and validated for quality control including the quantification of crocins 1 to 5, crocetin, picrocrocin and the degradation products, the CIS-crocins. Additionally, a GC-MS method has allowed detection and quantification of the volatile compounds from the pentane extract of saffron. Both systems together allowed the comprehensive characterisation of saffron herbal material and extracts for clinical/preclinical trials. For effective preparation of the respective reference standards, a fast centrifugal partition chromatography (FCPC) method was developed allowing the quick isolation of crocins 1, 2, 5 and picrocrocin in good yields. Using these chromatographic methods and the reference standards, a representative survey of saffron from the global market indicated a high variability of quality, especially concerning the amounts of volatile compounds in saffron samples. A specification for high-quality saffron of >20% crocins, >6% picrocrocin and not less than 0.3% of volatiles, calculated as sum of safranal, isophorone and ketoisophorone, was developed. Because no detailed pharmacological studies are available to explain the clinical effects of saffron for the treatment of cancer and depression, receptor binding studies were performed. Saffron extracts and crocetin had a clear binding capacity at the PCP binding side of the NMDA receptor and at the sigma(1) receptor, while the crocins and picrocrocin were not effective. These data could give biochemical support for the above-mentioned pharmacological effects of saffron.

Genotoxic and antigenotoxic effects of catechin and tannins from the bark of Hamamelis virginiana L. in metabolically competent, human hepatoma cells (Hep G2) using single cell gel electrophoresis

The genotoxic and antigenotoxic activities of catechin, hamamelitannin and two proanthocyanidin fractions prepared from the bark of Hamamelis virginiana L. were investigated in a human derived, metabolically competent hepatoma cell line (Hep G2) using single cell gel electrophoresis (SCGE) for the detection of DNA-damage. DNA-migration was calculated as Olive tail moment (OTM). Catechin and a low-molecular weight proanthocyandin fraction (W(M)) caused only slight increases of OTM up to concentrations of 166 microg/ml whereas hamamelitannin and the proanthocyandin fraction with higher molecular weight (W(A)) led to a two-fold enhancement of OTM at the same concentrations. These effects were dose-independent. Treatment of the cells with the test compounds in a dose-range of 2-166 microg/ml prior to the exposure to benzo(a)pyrene (B(a)P, 10 microM, 2.5 microg/ml) led to a significant reduction of induced DNA damage which was dose-dependent for all test compounds, except for hamamelitannin. The inhibitory effects of proanthocyanidins were stronger than those of catechin and hamamelitannin; the lowest effective concentrations were about 2 microg/ml. In order to clarify the mechanisms of protection, possible effects of the test compounds on enzymes involved in toxification and detoxification of B(a)P were investigated. While B(a)P toxification by cytochrome P450 was not inhibited by the test compounds, detoxification by glutathion-S-transferase (GST) was induced by catechin and W(M). Combination experiments with the ultimate metabolite of B(a)P, (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE; 5 microM, 1.5 microg/ml), revealed strong inhibitory effects, indicating that the observed protective effects were caused by scavenging of the ultimate mutagen by the test compounds. Exposure of Hep G2 cells to the test compounds after B(a)P treatment did not influence B(a)P induced DNA damage, demonstrating that repair mechanisms were not affected.

Caffeic acid derivatives from Eupatorium perfoliatum L.

From the ethyl acetate soluble fraction of a methanol/water extract of the herb Eupatorium perfoliatum L. (Asteraceae) six caffeic acid derivatives have been isolated and identified by 1D- and 2D-NMR spectroscopic data. Besides the common quinic acid derivatives 5-caffeoylquinic acid (chlorogenic acid), 3-caffeoylquinic acid (neochlorogenic acid) and 3,5-dicaffeoylquinic acid, three up to now unknown depsides of caffeic acid with glucaric acid have been isolated: 2,5-dicaffeoylglucaric acid, 3,4-dicaffeoylglucaric acid, and 2,4- or 3,5-dicaffeoylglucaric acid.

Phytochemical characterization of Rhododendron ferrugineum and in vitro assessment of an aqueous extract on cell toxicity.

Within a systematic phytochemical investigation of the leaves of RHODODENDRON FERRUGINEUM L. (Ericaceae), the volatile oil was isolated (0.7u200a%) and its constituents were characterized. Eleven flavonoids were isolated and characterized, with quercetin 3- O-[6- O-(2-methyl-1-oxobutyl)]-β- D-glucopyranoside and 2 R,3 R-dihydromyricetin 3- O- β- L-arabinopyranoside as new natural products. Beside monomeric flavan-3-ols (catechin, epicatechin, gallocatechin, epigallocatechin) from the tannin fraction (about 3.5u200a% calculated as pyrogallol), the dimeric procyanidins B1 to B7 were identified, as well as the trimeric compounds procyanidin C1, epicatechin-(4 βu200a→u200a8)-epicatechin-(4 βu200a→u200a8)-catechin and the trimeric A type-linked cinnamtannin B1. Additionally, phloroacetophenon 4- O- β- D-glucopyranoside and chlorogenic acid were isolated. Water-soluble carbohydrates comprised about 13.5u200a% of the dried leaves, including fructans (3u200a%), polysaccharides (1u200a%) (mainly type II arabinogalactans), glucose, fructose, sucrose, stachyose and raffinose. The IN VITRO effects on cellular vitality (MTT test), proliferation (BrdU incorporation) and necrosis (LDH release) of an aqueous extract were investigated. The extract did not exert any toxic effects, while the vitality and the proliferation rates of epithelial HaCaT keratinocytes were significantly increased at 100u2009µg/mL, indicating that the aqueous extract does not have negative effects against cellular activity.

Procyanidins from Myrothamnus flabellifolia

From the ethyl acetate soluble fraction of an acetone–water extract of the twig tips of Myrothamnus flabellifolia Welw. (Myrothamnaceae), a variety of flavan-3-ols (epicatechin, epigallocatechin and their 3-O-galloylated analogues) and procyanidins (DP ≤ 3) were isolated and identified for the first time: Procyanidin B3 [catechin-(4α → 8)-catechin], B4 [catechin-(4α → 8)-epicatechin], B6 [catechin-(4α → 6)-catechin] and catechin-(4α → 8)-epigallocatechin along with the galloylated analogues B4-3′-O-gallate, procyanidin B2-3′-O-gallate [epicatechin-(4β → 8)-epicatechin-3-O-gallate], B2-3,3′-di-O-gallate, procyanidin B5-3,3′-O-gallate [epicatechin-3-O-gallate-(4β → 6)-epicatechin-3-O-gallate], catechin-(4α → 8)-epigallocatechin-3-O-gallate, the trimer procyanidin C1-3″-O-gallate[epicatechin-(4β → 8)-epicatechin-(4β → 8)-epicatechin-3-O-gallate] and the new epicatechin-3-O-gallate-(4β → 6)-epicatechin-3-O-p-hydroxybenzoate. The structures were elucidated by 1D- and 2D-NMR experiments of their peracetylated derivatives, partial acid-catalysed degradation with phloroglucinol, ESI-MS and CD spectra.

In Vivo Consumption of Cranberry Exerts ex Vivo Antiadhesive Activity against FimH-Dominated Uropathogenic Escherichia coli: A Combined in Vivo, ex Vivo, and in Vitro Study of an Extract from Vaccinium macrocarpon.

For investigation of the molecular interaction of cranberry extract with adhesins of uropathogenic Escherichia coli (UPEC), urine from four volunteers consuming standardized cranberry extract (proanthocyanidin content = 1.24%) was analyzed within ex vivo experiments, indicating time-dependent significant inhibition of 40-50% of bacterial adhesion of UPEC strain NU14 to human T24 bladder cells. Under in vitro conditions a dose-dependent increase in bacterial adhesion was observed with proanthocyanidin-enriched cranberry Vaccinium macrocarpon extract (proanthocyanidin content = 21%). Confocal laser scanning microscopy and scanning electron microscopy proved that V.m. extract led to the formation of bacterial clusters on the outer plasma membrane of the host cells without subsequent internalization. This agglomerating activity was not observed when a PAC-depleted extract (V.m. extract(≠PAC)) was used, which showed significant inhibition of bacterial adhesion in cases where type 1 fimbriae dominated and mannose-sensitive UPEC strain NU14 was used. V.m. extract(≠PAC) had no inhibitory activity against P- and F1C-fimbriae dominated strain 2980. Quantitative gene expression analysis indicated that PAC-containing as well as PAC-depleted cranberry extracts increased the fimH expression in NU14 as part of a feedback mechanism after blocking FimH. For strain 2980 the PAC-containing extract led to up-regulation of P- and F1C-fimbriae, whereas the PAC-depleted extract had no influence on gene expression. V.m. and V.m. extract(≠PAC) did not influence biofilm and curli formation in UPEC strains NU14 and 2980. These data lead to the conclusion that also proanthocyanidin-free cranberry extracts exert antiadhesive activity by interaction with mannose-sensitive type 1 fimbriae of UPEC.

Bioassay-Guided Fractionation of a Leaf Extract from Combretum mucronatum with Anthelmintic Activity: Oligomeric Procyanidins as the Active Principle.

Combretum mucronatum Schumach. & Thonn. is a medicinal plant widely used in West African traditional medicine for wound healing and the treatment of helminth infections. The present study aimed at a phytochemical characterization of a hydroalcoholic leaf extract of this plant and the identification of the anthelmintic compounds by bioassay-guided fractionation. An EtOH-H2O (1:1) extract from defatted leaves was partitioned between EtOAc and H2O. Further fractionation was performed by fast centrifugal partition chromatography, RP18-MPLC and HPLC. Epicatechin (1), oligomeric proanthocyanidins (OPC) 2 to 10 (mainly procyanidins) and flavonoids 11 to 13 were identified as main components of the extract. The hydroalcoholic extract, fractions and purified compounds were tested in vitro for their anthelmintic activity using the model nematode Caenorhabditis elegans. The bioassay-guided fractionation led to the identification of OPCs as the active compounds with a dose-dependent anthelmintic activity ranging from 1 to 1000 μM. Using OPC-clusters with a defined degree of polymerization (DP) revealed that a DP ≥ 3 is necessary for an anthelmintic activity, whereas a DP > 4 does not lead to a further increased inhibitory effect against the helminths. In summary, the findings rationalize the traditional use of C. mucronatum and provide further insight into the anthelmintic activity of condensed tannins.

The plant cell wall–A potential source for pharmacologically active polysaccharides

Four main crude polysaccharide fractions, which represented the complete polymeric carbohydrate pool, were isolated by sequential extraction from Digitalis purpurea leaves. These crude fractions were fractionated further by ion exchange and gel permeation chromatography in order to obtain homogenous polysaccharide fractions. The following polymers were identified: the water soluble reserve polysaccharides were mainly composed of neutral and acidic arabinogalactans, neutral and acidic glucomannans and starch. Pectic material from a sodium carbonate extract was identified as rhamnogalacturonan. Hemicellulosic cell wall polysaccharides consisted of a neutral, low substituted arabinoxyloglucan and several acidic xylans. Hemicellulosic polymers associated with cellulose were shown to be highly branched xyloglucans. Extracellular polysaccharides from Digitalis lanata suspension-cultured cells were fractionated and arabinoxyloglucans, as well as neutral and acidic arabinogalactans were identified. 2,6-Dideoxy sugars, the typical constituents of cardiac glycosides in Digitalis, are not part of the polysaccharide pool of both systems investigated.