Hans W. Rauwald

Cardiac and electrophysiological effects of primary and refined extracts from Leonurus cardiaca L. (Ph.Eur.).

Although several antiarrhythmic drugs of chemical origin are in clinical use since decades, their application is often limited by their adverse effects and especially by their inherited proarrhythmic risk, which can lead to a significantly increased mortality in patients receiving these compounds. On the other hand, aqueous extracts from the aerial parts of the European Lamiaceae Leonurus cardiaca (Ph.Eur.) have been used for centuries as a remedy against tachyarrhythmia and other cardiac disorders. Nevertheless, a scientific basis for the claim of direct cardiac electrophysiological, antiarrhythmic, or functional effects of Leonurus cardiacae herba (LCH) preparations has not been established until now. In order to enrich the active constituents from the primary extract which was tested as the most cardioactive, namely the aqueous Soxhlet extract, and to eliminate undesired substances such as the dichloromethanic fraction or potassium, a bioassay guided fractionation procedure was applied, resulting in the development of a Leonurus cardiaca refined extract (LCRE) which was characterised together with Leonurus crude extracts by a newly developed gradient elution HPLC fingerprint analysis for separation and quantification of six major phenolics as well as by qNMR for determining the stachydrine content. This refined extract was applied intracoronarily in isolated rabbit hearts perfused according to the Langendorff technique. Mapping experiments with 256 electrodes on the heart surface showed a reduction of left ventricular pressure and an increase of relative coronary flow at concentrations of 1.0 and 2.0 mg/mL LCRE. Furthermore, the PQ-interval was prolonged and both the basic cycle length and the activation recovery interval increased. In addition, voltage-clamp measurements were performed on the following cell models in order to characterise the electrophysiological profile of LCRE: neonatal rat ventricular cardiomyocytes to investigate the effect on I(Na) and I(Ca.L), sinoatrial node cells and ventricular myocytes isolated from adult guinea pigs to test effects on I(f) and action potential (AP) duration, as well as HERG-transfected HEK 293 cells to analyse the influence on the I (K.r). In these voltage clamp experiments LCRE exerted a calcium-antagonistic activity by I(Ca.L) blockade, reduced the repolarising current I(K.r), and prolonged the AP-duration, while I(Na) was not affected. Although LCRE displayed only weak effects on the I(f) amplitude and voltage dependence, it significantly prolonged the activation time constant of I(f). Thus, LCRE acts on multiple electrophysiological targets, specifically I(Ca.L), I(K.r), and I(f), observed both at whole organ and single cell level.

High-performance liquid chromatographic separation and determination of diastereomeric anthrone-C-glucosyls in Cape aloes

Abstract Isocratic high-performance liquid chromatography using reversed-phase packing (C 18 ) and the solvent system methanol-water (1:1) was successful in separating and determinating the anthrone-C-glucosyls in Cape aloes, allowing a rapid and high resolution of each diastereomer for the first time. By applying this method to fifteen commercial Cape and East African aloes of different origin, all Cape aloes samples showed the same anthrone-C-glucosyl pattern comprising 5-hydroxyaloin A, aloins A/B and aloinosides A/B. For differentiating non-official East African aloes from Cape aloes, 5-hydroxyaloin A proved to be the most specific marker of the official drugs in high-performance liquid and also thin-layer chromatography, replacing the aloinosides. Previous assumptions of chemical races of Aloe ferox MILL. based on the aloinosides have to be revised.

Effects of Bergamot (Citrus bergamia (Risso) Wright & Arn.) Essential Oil Aromatherapy on Mood States, Parasympathetic Nervous System Activity, and Salivary Cortisol Levels in 41 Healthy Females

Background: Bergamot essential oil (BEO) is commonly used against psychological stress and anxiety in aromatherapy. The primary aim of the present study was to obtain first clinical evidence for these psychological and physiological effects. A secondary aim was to achieve some fundamental understanding of the relevant pharmacological processes. Methods: Endocrinological, physiological, and psychological effects of BEO vapor inhalation on 41 healthy females were tested using a random crossover study design. Volunteers were exposed to 3 experimental setups (rest (R), rest + water vapor (RW), rest + water vapor + bergamot essential oil (RWB)) for 15 min each. Immediately after each setup, saliva samples were collected and the volunteers rested for 10 min. Subsequently, they completed the Profile of Mood States, State-Trait Anxiety Inventory, and Fatigue Self-Check List. High-frequency (HF) heart rate values, an indicator for parasympathetic nervous system activity, were calculated from heart rate variability values measured both during the 15 min of the experiment and during the subsequent 10 min of rest. Salivary cortisol (CS) levels in the saliva samples were analyzed using ELISA. Results: CS of all 3 conditions R, RW, and RWB were found to be significantly distinct (p = 0.003). In the subsequent multiple comparison test, the CS value of RWB was significantly lower when compared to the R setup. When comparing the HF values of the RWB setup during the 10 min of rest after the experiment to those of RW, this parameter was significantly increased (p = 0.026) in the RWB setup for which scores for negative emotions and fatigue were also improved. Conclusion: These results demonstrate that BEO inhaled together with water vapor exerts psychological and physiological effects in a relatively short time.

Allium ursinum L.: Bioassay-Guided Isolation and Identification of a Galactolipid and a Phytosterol Exerting Antiaggregatory Effects

Aims: We wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adenosine diphosphate (ADP)-induced aggregation of human platelets by wild garlic, Allium ursinum L. Methods: For this purpose, a bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. For the bioassay, blood was taken from healthy human volunteers and platelet-rich plasma was prepared for turbidimetric platelet aggregation tests. Platelet-rich plasma, stimulated with 20 µmol/l of ADP, was treated with extracts of different polarities, fractions and isolated single compounds from A. ursinum. The extracts were investigated by thin-layer chromatography (TLC), HPLC, mass spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and 1/2-dimensional 1H/13C-nuclear magnetic resonance (NMR) spectroscopic techniques. Results: Fresh A. ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited ADP-induced aggregation of human platelets. This ethanolic extract was subjected to liquid-liquid partition. Whilst the aqueous phase, containing the moiety of cysteine sulphoxide and thiosulphinate derivatives, showed only weak activity on platelet aggregation, the ethyl-acetate and particularly the chloroform partitions showed the highest aggregation-inhibiting potency. Thus, in our bioassay, the effects of alliins/allicins could be neglected. The chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient-elution open column chromatography on silica gel. The most active fractions 11–17 were separated again, yielding 10 subfractions. This afforded 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol and β-sitosterol-3-O-β-D-glucopyranoside, the structures of which were determined by ESI-MS and 1/2-dimensional 1H/13C NMR spectroscopic techniques. Furthermore, the minute amounts of volatile oil of A. ursinum leaves obtained by steam distillation according to Ph. Eur. could be evaluated as a third aggregation-inhibiting principle. Conclusion: In our study, for the first time, 2 active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on ADP-induced aggregation in human blood platelets. As a major constituent, the galactolipid, 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol, not yet found in Allium sp., appears as a new, highly useful marker substance for A. ursinum drugs, or their pharmaceutical or food preparations, as shown by our orientating TLC analyses.

5-Hydroxyaloin A in the Genus Aloe Thin Layer Chromatographic Screening and High Performance Liquid Chromatographic Determination

By a comparative thin layer chromatographic screening of the methanol-soluble leaf exudates from more than 400 Aloe plants (183 species), 5-hydroxyaloin A was identified in 20 species. Whilst 13 of the 20 species revealed interindividual variations concerning to the occurrence of 5-hydroxyaloin A, this anthrone-C-glucosyl was unambiguously detected in each individual of 6 Aloe species. In the leaf exudates from A. marlothii Berger 5-hydroxyaloin A was only traceable in the aloin-containing chemivars. The complete anthrone-C-glucosyl pattern of these 7 clearly characterized species has been determined additionally by qualitative and quantitative high performance liquid chromatography: The results obtained demonstrate that 5-hydroxyaloin only occurs in the more stable A-configuration (10 R, 1′S), thus being till now the only anthrone-C-glycosyl which has not been found as diastereomeric pair genuinely in plants. As well, 5-hydroxyaloin A characterizes a quantitatively significant hydroxylating pathway in biosynthesis of anthranoids. It is discussed as a chemotaxonomic marker of the genus Aloe, especially of the sections Pachydendron and Eualoe.

First proof of anthrone aglycones and diastereomeric anthrone-C-glycosyls in flowers and bracts of Aloe species

Abstract Anthranoids were surveyed in flowers and leaves of 11 Aloe species. Flower extracts of five species contained diastereomeric anthroce- C -glycosyls (aloins, homoanatloins, 7-hydroxyaloins, 8-hydroxylaloin acetates). Anthrona aglycones (aloeemodinathrone, chrysophanolanthrone) were present in flowers of six species but were not detectable in leaves of the same plants. In Aloe flowers anthranoids are not localized in aloin cells. However, those cells could be identified in the bracts of A. succotrina storing anthrone- C -glycosyls of the 7-hydroxyaloin-type.

Efficacy of a Novel Herbal Multicomponent Traditional Chinese Medicine Therapy Approach in Patients with Atopic Dermatitis

Background: In Western medicine, the application of topical steroids and oral antihistaminic or antiallergic agents is the main treatment option for atopic dermatitis (AD). However, instead of these therapies the disease may remain intractable in some patients, resulting in long-term exposure to these chemical agents and consequently leading to concerns about possible adverse drug reactions. Methods: In the present open-label clinical study, the efficacy and safety of a novel multi component TCM therapy approach for AD was investigated. Therefore, 94 patients received the formula I (10 crude drugs) orally, combined with both the lotion II (7 crude drugs), and the ointment III (8 crude drugs). Each crude drug was extracted with boiling water in a defined manner, concentrated, and reworked into the preparations. Standardized scores were used for evaluating the severities of AD (clinical severity 0-4) and pruritus (pruritus score 0-4). Results: Both scores had significantly improved at the end of a 12 month treatment (P<0.001). Eosinophil ratio and serum IgE levels, which were elevated in AD patients, were significantly reduced at the end of therapy (P<0.01). In 32 of 94 treated patients the condition markedly improved, in 59 cases AD improved, and in 3 patients there was a slight improvement with no case of ineffective treatment. There was no hint of renal or hepatic toxicity or any other adverse effects. Conclusion: The present study confirms that the 3 newly developed herbal TCM combination preparations are clinically efficacious on AD, accomplishing a significant reduction in both clinical and pruritus scores as well as in eosinophil ratios and serum IgE levels.

Aloe Plants Accumulate Anthrone-Type Anthranoids in Inflorescence and Leaves, and Tetrahydroanthracenes in Roots

Abstract The accumulation and distribution of characteristic secondary products in the different organs of an Aloe plant (A. succotrina Lam.) were studied by high performance liquid chromatography for the first time. In the leaves of the Aloe plant, only anthrone-C-glycosyls of the 7-hydroxyaloin type and, for the first time in plant material, the free anthraquinone 7-hydroxyaloeemodin were found. In contrast to previous reports on the distribution of secondary products in Aloe plants, anthrone-C-glycosyls were also detected in flowers, bracts and the inflorescence axis of the species examined. Aloesaponol I, a tetrahydroanthracene aglycone, was only present in the underground organs and in the stem. The 2-alkylchromone-C-glucosyl aloeresin B showed no specific occurrence as it was found in every type of organ. Based on these results and the findings of recent studies on Aloe roots and flowers, a distribution scheme of polyketide types in the Aloe plant was established. It suggests a separate and independent anthranoid metabolism for underground Aloe organs and stem on the one hand, and for leaves and inflorescence organs on the other hand. In the latter structures anthranoid metabolism seems to be additionally compartmentalized as the anthranoid pro files of inflorescence organs and leaves differ in two points relevant to anthranoid biosynthe sis: firstly, the occurrence of anthrone aglycones and secondly, the individual content of corresponding anthrone-C-glucosyl diastereomers.

Tetrahydroanthracenes as Markers for Subterranean Anthranoid Metabolism in Aloe Species

Summary Anthranoids in the roots of 14 Aloe species were surveyed by a novel high-performance liquid chromatographic method and photodiode-array detection and, in leaves of the same species, by thin-layer chromatography. Whereas diastereomeric anthrone-C-glycosyls were detectable in the majority of leaf samples, these compounds as well as anthrone aglycones were shown to be absent in root material. The anthraquinone aglycone chrysophanol was found in every root extract. Roots of ten species contained the tetrahydroanthracene aglycone aloesaponol I, which was isolated as a major anthranoid from root and rhizome material of A. succotrina Lam. Three of the remaining four species exhibited compounds with UV-VIS spectra analogous to aloesaponols III/IV. The substitution pattern of the compounds detected indicates striking differences between subterranean and aerial anthranoid biosynthesis in Aloe . With regard to distribution and accumulation of tetrahydroanthracenes within the individual plant as well as in the species examined here, these anthranoids are suggested to be specific markers for subterranean Aloe metabolism.

Neue Untersuchung über Inhaltsstoffe aus Aloe- und Rhamnus-Arten, XIII [1] / A New Investigation on Constituents of Aloe and Rhamnus Species, XIII [1]

Rhamnus purshianus D. C., 10-Hydroxyaloins A/B, Oxanthrone Glucosyls, Quinonoids, Anthranoids The absolute configurations of the diastereomeric 10-hydroxyaloins, which may be regarded as parent structures for other naturally occurring oxanthrone-C-glucosyls, have been established as 10R, 16 R (A) and 10 S, 16 R (B) by an X-ray structure analysis of the A-octaacetyl derivative (C 16 is the anomeric glucosyl carbon atom). The determination was confirmed by CD spectroscopic comparison with the structural analogues aloins A and B, which should prove useful for making future configurational assignments within this class of compounds. A conformational analysis by the use of a molecular modeling method based on force-field calculations reveals the presence of an extra- and an intra-form, the extra-form of which is energetically preferred.