Phytochemical analysis of Ficus carica L. active compounds possessing anticonvulsant activity.

The anticonvulsant potential of Ficus carica methanol-extract (Fc) has been studied. It was found that Fc most active fraction is rich in oligosaccharides (OFG). 1H, 13C NMR and Nano-ESI, MALDI MS, and LC-MS techniques proved that OFG contains alpha-glucopyranoside oligomer in high amounts. Both Fc and OFG reduced strychnine (STR) convulsion-action. Fc and OFG fully protected the experimental-animals from STR-lethality. The intracerebroventricular-administration (ICV) of Fc or OFG in combination with glycine in ethanol-treated mice caused a dose-dependent returning to a 2nd-loss of righting-reflex (LORR), and was antagonized by STR. FC and OFG ICV injection counteracted STR-inhibition, confirming that Fc/OFG anticonvulsant mechanism of action was mediated by potentiation of glycine receptor. These results support Fc and OFG potential anticonvulsant-activity with good safety-profile.

Prunella vulgaris L. active components and their hypoglycemic and antinociceptive effects in alloxan-induced diabetic mice.

Prunella vulgaris L. (Lamiaceae) (PV) is a herbaceous plant traditionally utilized in management of diabetes and it has immunomodulatory activity. In this study, acute and subchronic antidiabetic, in-vivo antioxidant and antinociceptive effects of PV were evaluated in alloxan-induced type 1 diabetes (T1D) in a mouse model. Bio-guided fractionation, isolation, RP-HPLC, and 1H and 13C NMR identification of the active components responsible for PV effects were determined. RP-HPLC analysis showed that PV contained rosmarinic acid (RA) 4.5%, caffeic acid (CA) 9.8% and p-coumaric acid (pCA) 11.6%. Bio-guided fractionation showed that PV most active fraction was rich in caffeic acid, hence named, caffeic acid-rich fraction (CARF). RP-HPLC, and 1H and 13C NMR experiments showed that CARF contained CA (93.4%) and RA (6.6%). CARF reduced blood glucose levels and improved in-vivo oxidative-stress. It also inhibited the carbohydrate-hydrolyzing enzymes (alpha-amylase and alpha-glucosidase) and reduced HbA1c levels more significantly (p≤0.05) than that of PV and equivalent amounts of CA or RA. For longer times, CARF had significantly (p≤0.05) increased serum-insulin, ameliorated thermal hyperalgesia and tactile allodynia more significantly (p≤0.05) than the effects of PV and equivalent amounts of CA or RA. Moreover, the tested compounds showed potential restoration of the lipid peroxide levels. Consequently, CARF and PV observed increase in serum-insulin, attenuation of alpha-amylase and alpha-glucosidase, and their antioxidant potentials might be responsible for their antidiabetogenic and antinociceptive properties. In conclusion, CARF isolated from PV could be a potential therapeutic agent to ameliorate T1D and related complications.

Pharmacokinetic properties of MH84, a γ-secretase modulator with PPARγ agonistic activity.

Alzheimer's disease (AD) is the most common cause of dementia. Since no causative treatment is available, new therapeutic options are utmost needed. Several pirinixic acid derivatives, including MH84 (2-((4,6-bis(4-(trifluoromethyl)phenethoxy)pyrimidin-2-yl)thio)hexanoic acid), have shown promising in vitro results as γ-secretase modulators as well as PPARγ activators as potential pharmacological compounds against AD. Using a newly developed and validated sensitive LC-MS (APCI-qTOF mass analyzer) method, the pharmacokinetic and long-term accumulating properties as well as the blood-brain-barrier permeability of MH84 were evaluated in a preclinical animal study. MH84 was administered to mice by oral gavage with a dose of 12 mg/kg. Nine time points from 0.5 to 48 h with 6 animals per point were investigated. Additionally 6 animals were fed daily, for 21 days with an identical dose to determine possible long-term accumulation in plasma and brain tissue. The sample preparation was performed by a liquid-liquid extraction on Extrelut(®) columns whereas the LC separation was operated on a MulthoHigh 100 RP 18-5 µ column (125 × 4 mm) using an isocratic mobile phase of formic acid (0.1% (v/v))-methanol mixture (11:89 (v/v)) at a flow rate of 1 ml/min. The validation confirmed the new LC-MS method to be precise, accurate and reliable. After oral application, Cmax and Tmax of unmetabolized MH84 was determined to be 10.90 µg/ml and 3h in plasma. In brain tissue a constant level of 300 to maximum 320.64 ng/g was found after 1.5-6h. Daily gavage for 21 days did not lead to a long-term drug accumulation in the brain. The efficacy of the obtained MH84 levels needs to be investigated in further preclinical pharmacodynamic animal studies.

Molecular pharmacological profile of a novel thiazolinone-based direct and selective 5-lipoxygenase inhibitor.

BACKGROUND AND PURPOSE: The potency of many 5-lipoxygenase (5-LOX) inhibitors depends on the cellular peroxide tone and the mechanism of 5-LOX enzyme activation. Therefore, new inhibitors that act regardless of the mode of enzyme activation need to be developed. Recently, we identified a novel class of thiazolinone-based compounds as potent 5-LOX inhibitors. Here, we present the molecular pharmacological profile of (Z)-5-(4-methoxybenzylidene)-2-(p-tolyl)-5H-thiazol-4-one, compound C06. EXPERIMENTAL APPROACH: Inhibition of 5-LOX product formation was determined in intact cells [polymorphonuclear leukocytes (PMNL), rat basophilic leukaemia-1, RAW264.7] and in cell-free assays [homogenates, 100, 000×g supernatant (S100), partially purified 5-LOX] applying different stimuli for 5-LOX activation. Inhibition of peroxisome proliferator-activated receptor (PPAR), cytosolic phospholipase A(2) (cPLA(2) ), 12-LOX, 15-LOX-1 and 15-LOX-2 as well as cyclooxygenase-2 (COX-2) were measured in vitro. KEY RESULTS: C06 induced non-cytotoxic, direct 5-LOX inhibition with IC(50) values about 0.66 µM (intact PMNL, PMNL homogenates) and approximately 0.3 µM (cell-free PMNL S100, partially purified 5-LOX). Action of C06 was independent of the stimulus used for 5-LOX activation and cellular redox tone and was selective for 5-LOX compared with other arachidonic acid binding proteins (PPAR, cPLA(2) , 12-LOX, 15-LOX-1, 15-LOX-2, COX-2). Experimental results suggest an allosteric binding distinct from the active site and the C2-like domain of 5-LOX. CONCLUSIONS AND IMPLICATIONS: C06 was identified as a potent selective direct 5-LOX inhibitor exhibiting a novel and unique mode of action, different from other established 5-LOX inhibitors. This thiazolinone may possess potential for intervention with inflammatory and allergic diseases and certain types of cancer.

Isoquercitrin provides better bioavailability than quercetin: comparison of quercetin metabolites in body tissue and brain sections after six days administration of isoquercitrin and quercetin.

In the present study, over a period of 8 days 12 mg/kg/d quercetin aglycone and 18 mg/kg/d isoquercitrin were orally given to rats, respectively. Four hours after administration, plasma samples were taken as well as tissue samples of liver, lung, heart, kidney and the brain sections hippocampus, cerebellum, striatum, cortex and the remaining brain. A HPLC-FD method with in-line post-column complexation was employed to quantify the quercetin metabolites (QM) in plasma and tissues. Compared to the quercetin gavage the isoquercitrin gavage consistently produced higher levels of QM in tissues (double to five-fold) as well as in plasma (double to three-fold). In body tissues, the highest amounts of QM were observed in the lung. In brain tissue, the highest levels of QM were found in the cerebellum, while the striatum contained the lowest levels of QM. In conclusion, this study clearly demonstrates that orally given isoquercitrin leads to higher levels in plasma and in all investigated tissue than quercetin aglycone.

A pirinixic acid derivative (LP105) inhibits murine 5-lipoxygenase activity and attenuates vascular remodelling in a murine model of aortic aneurysm.

BACKGROUND AND PURPOSE Arachidonic acid derivatives play a central role in inflammation processes. Arachidonic acid is metabolized by several enzymes, particularly cyclooxygenases (COX), 5-lipoxygenase (5-LOX) and microsomal prostaglandin E-synthase-1 (mPGES-1) to pro-inflammatory mediators. EXPERIMENTAL APPROACH We determined the effect of LP105, a pirinixic acid derivative which acts as inhibitor of 5-LOX, COX and mPGES-1, on aortic aneurysm development in mice and on 5-LOX activity in murine monocytes. KEY RESULTS In a monocyte cell line (RAW264.7), LP105 inhibited 5-LOX in whole cells (IC(50) : 1-3 µM) and in supernatants (IC(50) : ∼10 µM). Oral administration of LP105 to mice resulted in therapeutic tissue and plasma levels. Aortic aneurysms were induced in ApoE(-/-) mice by angiotensin II (AngII) and LP105 (5 mg·day(-1) per animal) was co-administered to a subgroup. Compared with animals receiving AngII alone, the LP105+AngII group showed a lower heart rate, a trend towards reduced heart to body weight ratio but similar hypertensive responses. AngII alone significantly increased aortic weight and diameter but co-treatment with LP105+AngII prevented these changes. LC/MS-MS studies revealed increased 15-hydroxytetraenoic acid (15-HETE) and 14,15-epoxyeicosatrienoic acid (14,15-EET) plasma levels in LP105-treated animals. In the murine kidney, mRNAs of EET-generating or metabolizing enzymes and of 5-LOX and 15-LOX were unaffected by LP105. LP105 also did not inhibit the EET-metabolizing soluble epoxide hydrolase. CONCLUSIONS AND IMPLICATIONS LP105 was a potent inhibitor of monocyte 5-LOX and reduced AngII-induced vascular remodelling in mice. A shift of arachidonic acid metabolism to the protective EET pathway may contribute to the beneficial effects of LP105.

St. John's wort flavonoids and their metabolites show antidepressant activity and accumulate in brain after multiple oral doses.

Over the last few years many data have been published suggesting a participation of quercetin flavonoids in the antidepressive effect of St. John's wort (SJW) extract. To elucidate these data more deeply we performed two animal behavioural studies examining the antidepressant effects of SJW extract, rutin and, in addition, the quercetin metabolite isorhamnetin. The substances were in all cases compared to imipramine using Porsolt's forced swimming test (FST) after oral gavage of the substances over a 9 day period. All three compounds were found to be effective, with isorhamnetin exhibiting the strongest effect. In addition to this pharmacological study, we carried out two pharmacokinetic studies to examine the CNS level time-curve of the quercetin flavonoids after a single oral dose of SJW extract (1600 mg/kg) and isoquercitrin (100 mg/kg), respectively, and to observe the cumulative effects after daily repeated oral doses of SJW extract over 8 days. After a single dose the maximal CNS levels for quercetin (340 ng/g) and isorhamnetin/tamarixetin (50 ng/g) were found at 4 h. With repeated doses the maximal cumulation for quercetin (367 ng/g) occurred after 5 days whilst isorhamnetin/tamarixetin (640 ng/g) did not reach its maximal cumulation level within the 8 day test period.

Screening of herbal extracts for activation of the human peroxisome proliferator-activated receptor.

The peroxisome proliferator-activated receptors play a pivotal role in metazoan lipid and glucose homeostasis. Synthetic activators of PPARalpha (fibrates) and PPARgamma (glitazones) are therefore widely used for treatment of dislipidemia and diabetes, respectively. There is growing evidence for herbal compounds to influence nuclear receptor signalling e.g. the PPARs. We recently reported carnosic acid and carnosol, both being diterpenes found in the labiate herbs sage and rosemary, to be activators of PPARgamma. The subsequent screening of a variety of ethanolic extracts, obtained from traditionally used herbs, for PPAR activation, led to an exceptionally high hit rate. Among 52 extracts nearly the half significantly activated PPARgamma and 14 activated PPARalpha in addition, whereas three of them were pan-PPAR activators, which also activated PPARdelta. The most active extracts, for which a concentration dependent effect could be shown, were the extracts of Alisma plantago aquatica (ze xie/european waterplantain), Catharanthus roseus (madagascar periwinkle), Acorus calamus (sweet calamus), Euphorbia balsamifera (balsam spurge), Jatropha curcas (barbados nut), Origanum majorana (marjoram), Zea mays (corn silk), Capsicum frutescens (chilli) and Urtica dioica (stinging nettle). The results of the present study provide a possible rationale for the traditional use of many herbs as antidiabetics.

Development of a high-performance liquid chromatographic method with electrochemical detection for the determination of hyperforin.

An HPLC method with electrochemical detection for the determination of hyperforin extracts without using additional sample precleaning has been developed and validated. The hyperforin solutions were separated isocratically using a mobile phase consisting of 10% ammonium acetate buffer (0.5 M, pH 3.7)-MeOH-acetonitrile (10:40:50, v/v) at a flow rate of 0.8 mL/min. Hyperforin was detected amperometrically with a glassy carbon electrode at a potential of +1.1 V versus Ag/AgCl/3 M potassium chloride reference electrode. Under these conditions, a plot of integrated peak area versus concentration of hyperforin was found to be linear over the range of 0.054-5.4 microg/mL, with a relative standard deviation of 2.2-8.6%. The limit of detection was 0.050 ng on column. The determination of the hyperforin content in a commercially available St. John's Wort preparation exhibited a mean content of 1.56 mg. Recovery experiments led to a mean recovery rate of 97 +/- 5.8%. The proposed method is not time-consuming, sensitive and reproducible and is therefore suitable for routine analysis of hyperforin in herbal medicinal products.

Comparison of the synaptosomal uptake inhibition of serotonin by St John's wort products.

Although the number of prescriptions for psychotropic drugs has decreased in recent years, prescriptions for antidepressants are still increasing (Fritze 2002). Hypericum perforatum (St John's wort) is the main psychotherapeutic herbal medicinal product used for treatment of mild-to-moderate depression. The lipophilic constituent hyperforin (2-5% of the extract) demonstrated, similarly to chemical antidepressants, a significant effect on the synaptosomal uptake inhibition of several neurotransmitters in in-vitro assays. In Germany, St John's wort products are distributed via two different markets: products that are pharmacy restricted are only allowed to be distributed in pharmacies; traditionally used products, which do not claim to have a curative character, are allowed to be sold in supermarkets. Depending on the market wherein a St John's wort product is offered, it needs to fulfill the legal requirements regarding pharmaceutical quality, safety and efficacy. Our goal was to compare the quality of St John's wort products distributed in pharmacies with that of those available from supermarkets. Therefore, the quantity of the pharmaceutical active ingredients (the phloroglucinol derivate hyperforin, the flavonoids rutin, hyperoside, isoquercitrin, quercitrin and the biflavonoid biapigenin) was determined by high-performance liquid chromatography (HPLC). The naphthodianthrones hypericines and pseudohypericines were quantified by differential pulse polarography (DPP). The efficacy of the products was investigated by measuring their activity to inhibit serotonin (5-HT) uptake in-vitro using a radio ligand uptake assay. It could be demonstrated that the products were different not only in the concentration of pharmaceutically relevant ingredients but also in showing individual IC50 values (concentration producing half-maximal inhibition) in the serotonin reuptake assay (IC50 values between 3.07 and 17.9 microg extract mL(-1)). The results of our study confirm the assumption that the potency of St John's wort products in inhibiting the uptake of serotonin depends on the amount of hyperforin in their dosage forms. St John's wort products having greater hyperforin content and potency on synaptosomal serotonin uptake inhibition are restricted to be sold only in pharmacies.

Development of a high-performance-liquid-chromatographic method for the determination of biapigenin in biorelevant media.

A new precise, rapid and selective high-performance-liquid-chromatographic (HPLC) method has been developed to quantify biapigenin in St. John's Wort (SJW) preparations and to investigate its release characteristics in the dissolution test using both compendial and biorelevant media. Experiments were carried out on a LiChroCart 125-4, RP-18 (5 microm) column, using gradient elution at a flow rate of 1 ml/min. The binary mobile phase consisted of solvent A (acetic acid, 5:100, w:w) and B (a mixture of acetonitrile and methanol (3:1, v:v)). Detection was performed at a wavelength of 270 nm using a photodiodearray detector. The limit of detection was 0.05 microg/ml, the injection volume 20 microl. Five SJW preparations were chosen to determine the amount of biapigenin in the dosage form and to investigate their release characteristics. Best results in terms of release as well as discriminating the tested products were obtained, using fed state simulated intestinal fluid (FeSSIF), where over 80% of biapigenin was dissolved after 20 min comparing to 70% using simulated gastric fluid sine pepsin (SGF(sp)) as compendial medium. Experiments in fasted state simulated intestinal fluid (FaSSIF) show 80% release of biapigenin within 80 min.

Development of a high-performance liquid chromatographic method for the determination of 11-keto-beta-boswellic acid in human plasma.

A validated HPLC method for the determination of 11-keto-beta-boswellic acid (KBA) in human plasma was developed. The method involves the solid-phase extraction of KBA from plasma followed by a separation with reversed-phase HPLC. Calibration was based on external standardisation and ranged between 0.1 and 2.0 microg KBA per ml plasma. Linearity was established over the entire calibration range and in each case the coefficient of correlation (r2) was above 0.99. The recovery of KBA from plasma was 85.7%. It was further demonstrated that the method can be applied successfully to monitor the level of KBA in plasma.

Comparison of German St. John's wort products according to hyperforin and total hypericin content.

OBJECTIVES: To compare the hyperforin and hypericin content of currently available St. John's wort products and to determine their batch-to-batch reproducibility. DESIGN: Representative products were obtained either directly from the manufacturer or purchased from pharmacies in and around Frankfurt, Germany. For five batches from each of the eight manufacturers, 10 individual dosage forms (tablets or capsules) were analyzed for both hyperforin and hypericin content. SETTING: Laboratories of the Institute of Pharmaceutical Chemistry at Johann Wolfgang Goethe University, Frankfurt, Germany. PRODUCTS: Eight German St. John's wort products containing from 250 mg to 612 mg dry extract were studied. Three of these products are capsules, four are film-coated tablets, and one is a sugar-coated tablet. Two of the products (Jarsin 300 and Neuroplant 300) are also available in the United States. METHODS: Hyperforin concentrations were analyzed by high-performance liquid chromatography. Total hypericin concentrations were determined by polarography, an electrochemical method. Concentrations were compared among different batches of the same product and among products from different manufacturers. RESULTS: The products contained widely differing amounts of hypericin and hyperforin, even after correcting for differences in the amount of extract per dose. Some products demonstrated consistent concentrations of hyperforin and hypericin from batch to batch, others exhibited pronounced interbatch variability. CONCLUSION: The St. John's wort preparations studied exhibited large differences in hypericin and hyperforin content and are not interchangeable for the treatment of mild-to-moderate depression. Pharmacists should take this variability into account when counseling patients on the use of St. John's wort products.

Batch-to-batch reproducibility of St. John's wort preparations.

Over the last few years, St. John's Wort products have enjoyed a tremendous surge in interest and sales for the therapy of mild to moderate depression. Although the complete spectrum of active substances in this herbal extract has not yet been elucidated, it is certain that hyperforin is an important component. Further, it appears that the hypericins may also contribute to the antidepressive activity. In this study, the content uniformity of eleven St. John's Wort products sold exclusively in pharmacies was determined. The main objective was to determine the batch-to-batch reproducibility of the various products. The hyperforin was analysed according to a previously published HPLC method, while the total hypericin content was determined by an electrochemical method. The results indicate that some, but not all, products show very reproducible batch-to-batch properties. Also, individual products have different hypericin and hyperforin levels, and are therefore not switchable--even when products are manufactured under similar extraction and processing conditions, have the same raw material:extract ratios (on a dry basis) and contain the same amount of extract per unit dosage form.

Lack of correlation between surface and interfacial activities of saponins and their hemolytic properties.

For a series of model compounds (digitonin, aescine, tomatine, stevioside and ginsenoside Rg1) it was demonstrated that neither the surface nor the interfacial tension (n-decane/water) lowering properties of saponins can be correlated with their ability to induce hemolysis. Furthermore, no correlation was observed between the surface and interfacial activities of saponins and their hemolytic properties.