K Kuchta

Olea europaea leaf (Ph.Eur.) extract as well as several of its isolated phenolics inhibit the gout-related enzyme xanthine oxidase

In Mediterranean folk medicine Olea europaea L. leaf (Ph.Eur.) preparations are used as a common remedy for gout. In this in vitro study kinetic measurements were performed on both an 80% ethanolic (v/v) Olea europaea leaf dry extract (OLE) as well as on nine of its typical phenolic constituents in order to investigate its possible inhibitory effects on xanthine oxidase (XO), an enzyme well known to contribute significantly to this pathological process. Dixon and Lineweaver-Burk plot analysis were used to determine K(i) values and the inhibition mode for the isolated phenolics, which were analysed by RP-HPLC for standardisation of OLE. The standardised OLE as well as some of the tested phenolics significantly inhibited the activity of XO. Among these, the flavone aglycone apigenin exhibited by far the strongest effect on XO with a K(i) value of 0.52 μM. In comparison, the known synthetic XO inhibitor allopurinol, used as a reference standard, showed a K(i) of 7.3 μM. Although the phenolic secoiridoid oleuropein, the main ingredient of the extract (24.8%), had a considerable higher K(i) value of 53.0 μM, it still displayed a significant inhibition of XO. Furthermore, caffeic acid (K(i) of 11.5 μM; 1.89% of the extract), luteolin-7-O-β-D-glucoside (K(i) of 15.0 μM; 0.86%) and luteolin (K(i) of 2.9 μM; 0.086%) also contributed significantly to the XO inhibiting effect of OLE. For oleuropein, a competitive mode of inhibition was found, while all other active substances displayed a mixed mode of inhibition. Tyrosol, hydroxytyrosol, verbascoside, and apigenin-7-O-β-D-glucoside, which makes up for 0.3% of the extract, were inactive in all tested concentrations. Regarding the pharmacological in vitro effect of apigenin-7-O-β-D-glucoside, it has to be considered that it is transformed into the active apigenin aglycone in the mammalian body, thus also contributing substantially to the anti-gout activity of olive leaves. For the first time, this study provides a rational basis for the traditional use of olive leaves against gout in Mediterranean folk medicine.

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.

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.

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.

1H-qNMR for direct quantification of stachydrine in Leonurus japonicus and L. cardiaca

(1)H-qNMR-spectroscopy was successfully applied to quantify the pharmacologically active alkaloid stachydrine ((2S)-1,1-dimethylpyrrolidinium-2-carboxylic-acid) in aerial parts of Leonurus japonicus (Leonuri herba, yimucao; Chin.Ph.2010, DAB2012) which are used in TCM and Kampo for the treatment of various gynaecological and cardiovascular disorders. Pharmacological publications on this betaine describe cardiovascular, hypotensive, and tissue-protective effects. However, its pharmacopeial analytics poses severe difficulties as it does not contain any chromophore suitable for HPLC-UV-detection. Nine samples from three countries were prepared as decoctions and freeze-dried. (1)H-NMR-spectra were recorded in D2O. The direct-quantitative (1)H-qNMR-procedure was carried out using the N-CH3-singlet at δ 3.03 ppm in comparison to the δ 6.18 ppm singlet of the two vinylic protons of maleic-acid, which was identified as a most favourable internal standard. The quantification limit of stachydrine was 0.44 mg/g drug material. Neither reference-compounds for calibration-curves nor sample-pre-purification was necessary. This protocol revealed stachydrine contents in the range from 0.09 up to 1.01% (w/w) for the tested yimucao samples. Furthermore, between 0.18 and 0.21% of stachydrine was found in the L. japonicus fruit-drug (Leonuri fructus, chongweizi; Chin.Ph.2010) which was examined for this constituent for the first time. In four co-investigated samples of the closely related and similarly used European herb Leonurus cardiaca Ph.Eur., even higher contents up to 1.55% were attested. The presented quantitative (1)H-qNMR-method was shown to be precise with respect to concentration, and yielded highly reproducible data in a series of inter-day repetitions. Methodically, (1)H-qNMR may be a powerful tool for quality assurance of stachydrine containing plants and herbal drugs, especially for industrial routine protocols.

GABAA Receptor Binding Assays of Standardized Leonurus cardiaca and Leonurus japonicus Extracts as Well as Their Isolated Constituents.

A main traditional use of European Leonurus cardiaca and East Asian Leonurus japonicus is in the treatment of neurological disorders such as anxiety, depression, nervousness, and as a sedative for insomnia. However, their mechanism of action is still under discussion. As anxiety and depressive disorders are increasingly being recognized as connected to dysfunctions of the gamma-aminobutyric acid system, the in vitro effects of standardized L. cardiaca and L japonicus extracts as well as five of their isolated constituents, namely, the labdane-type isoleosibirin, the novel iridoid 7R-chloro-6-desoxy-harpagide, the phenylethanoid lavandulifolioside, and the N-containing compounds stachydrine and leonurine, on this type of neuronal receptor were investigated for the first time. Extracts of L. cardiaca and L. japonicus, characterized by reversed-phase high-performance liquid chromatography determination, as well as their above named isolated, possible active constituents of different chemical nature were tested in several receptor binding assays at rat GABAA receptors using [(3)H]-SR95 531 and [(3)H]-Ro-15-1788 (flumazenil)/diazepam control. The L. cardiaca and L. japonicus extracts as well as leonurine inhibited the concentration-dependent binding of [(3)H]-SR95 531 to the gamma-aminobutyric acid site of the gamma-aminobutyric acid type A receptor with a high binding affinity: IC50s 21 µg/ml, 46 µg/ml, and 15 µg/ml, respectively. In contrast, binding to the benzodiazepine site of the rat gamma-aminobutyric acid type A receptor had a 15 to 30 times lower binding affinity than to the gamma-aminobutyric acid site. The presented experiments provide hints that the neurological mechanism of action of L. cardiaca and L. japonicus may essentially be based on their interaction to the gamma-aminobutyric acid site of the gamma-aminobutyric acid type A receptor, while the benzodiazepine site most probably does not contribute to this effect. In the case of L. japonicus, these effects can be at least partially explained by its leonurine constituent, whereas the active principle of L. cardiaca, which does not contain leonurine, is subject to further research as none of the other investigated individual constituents displayed significant activity in the applied test system.

HPLC quantification of all five ginkgolic acid derivatives in Ginkgo biloba extracts using 13 : 0 ginkgolic acid as a single marker compound.

An HPLC quantification method for ginkgolic acid derivatives in Ginkgo biloba leaf extracts was developed. Using 13 : 0 ginkgolic acid as a marker compound, the relative correlation factors of the four other ginkgolic acid derivatives - namely, 15 : 0 ginkgolic acid, 15 : 1 ginkgolic acid, 17 : 1 ginkgolic acid, and 17 : 2 ginkgolic acid - to 13 : 0 ginkgolic acid were determined by HPLC and subsequently used for calculating their contents in ten hydro-ethanolic refined extract samples. In other words, the content of 13 : 0 ginkgolic acid in the extracts was determined using the isolated compound as an external standard. Subsequently the now known concentration of this compound functioned as an internal standard for the quantification of the other four ginkgolic acid derivatives via the described correlation factors. This HPLC method was validated by two independent control measurements, one with an external standard for every individual compound and one based on the present method with the single marker compound alone. The results did not differ significantly in any of the 10 tested extract samples. The protocol presented here thus not only uses the same reference substance for G. biloba extracts as the current Chinese Pharmacopoeia method but also incorporates the advantages of the current European Pharmacopoeia approach. It is simple, reproducible, and can be used to determine the total contents of ginkgolic acid derivatives in G. biloba leaf extracts.

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.

Curcumin Promotes Apoptosis of Activated Hepatic Stellate Cells by Inhibiting Protein Expression of the MyD88 Pathway

Activation and proliferation of hepatic stellate cells (HSC) play an important role in the progress of liver fibrosis. HSC activation occurs in response to inflammatory cytokines, cellular interactions with immune cells, and morphogenetic signals. The literature hints to a role of the adaptor protein MyD88 in fibrosis. Although curcumin has been shown to exert inhibitory effects on the proliferation of HSC in vitro, its influence on the MyD88 pathway in HSC has remained unclear. Here, we investigated whether curcumin accelerates apoptosis of HSC through the MyD88 pathway. HSC (rat HSC T6) were divided into a control group, MyD88 small interfering RNA (siRNA) group, curcumin group, and curcumin + MyD88 siRNA group. The MyD88 siRNA groups were exposed to siRNA for 48 h. The curcumin groups were cultured in the presence of curcumin for 24 h. Apoptosis was detected by flow cytometry. For Toll-like receptor (TLR) 2 and 4 as well as MyD88 and the dependent factors NF-κB, TNF-α, and IL-1β, mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). For MyD88, protein expression was further observed by Western Blot. Both curcumin and MyD88 siRNA inhibited the mRNA expression of MyD88 pathway-related effectors (TLR2, TLR4, NF-κB, TNF-α, IL-1β) in HSC. Furthermore, both treatments reduced the expression of MyD88 protein in HSC and promoted their apoptosis. These effects were more obvious in the curcumin + MyD88 siRNA group. This study demonstrates that curcumin promotes apoptosis of activated HSC by inhibiting the expression of cytokines related to the MyD88 pathway. It elucidates the possible mechanisms of curcumin in inducing apoptosis of HSC through the MyD88 pathway.

Curcumin, the main active constituent of turmeric (Curcuma longa L.), induces apoptosis in hepatic stellate cells by modulating the abundance of apoptosis-related growth factors.

Abstract In order to elucidate the mechanism of action of curcumin against hepatic fibrosis, cultured rat hepatic stellate cells (HSC) (HSC-T6) were incubated with curcumin for 24 h, after which apoptosis was measured by flow-cytometry. The protein levels of the pro-apoptotic factors Fas and p53b as well as of the anti-apoptotic factor Bcl-2 were monitored by immunocytochemical ABC staining after incubation with curcumin for 24 h. In the case of 20 μM curcumin, not only was the respective apoptosis index increased, but also the abundance of the pro-apoptotic factors Fas and p53 were amplified, whereas that of the anti-apoptotic factor Bcl-2 decreased. All these effects were highly reproducible (P<0.05). Consequently, curcumin has an up-regulating effect on pro-apoptotic factors like Fas and p53 as well as a down-regulating effect of the anti-apoptotic factor Bcl-2, thus inducing apoptosis in HSC.