Ginkgo biloba Food Supplements on the European Market - Adulteration Patterns Revealed by Quality Control of Selected Samples.

The aim of this study was to prove whether Ginkgo biloba food supplements on the European market comply with pharmaceutical quality, and whether their composition satisfies the European Pharmacopoeia criteria. Medicinal products containing a standardised Ginkgo leaf extract are used for the improvement of cognitive impairment and quality of life in mild dementia. Further, Ginkgonis folium is used for the treatment of peripheral circulation disorders. Pharmacopoeial Ginkgo dry extract contains 22.0 - 27.0% flavonoids and 5.4 - 6.6% terpene lactones (ginkgolides, bilobalide). In addition to its widespread use as an herbal medicine (herbal medicinal product), the same extract can be an ingredient in food supplements. The content of active secondary metabolites was quantified in a number of European food supplements containing Ginkgo dry extract or Ginkgo leaf. Flavonoids were quantified using a modified pharmacopoeial HPLC-UV method, and terpene lactones (ginkgolides A, B, C, and bilobalide) using LC-MS/MS. Some Ginkgo leaf supplement samples were also analysed by microscopy. The quality of food supplements on the European market is dubious. In this paper, we present selected examples of several methods of adulteration and falsification, including higher/lower doses of Ginkgo dry extract or Ginkgo leaf than declared and the addition of undeclared extraneous materials. These examples reveal several patterns in the manufacturing of adulterated products.

Bioactivity-Guided Investigation of the Anti-Inflammatory Activity of Hippophae rhamnoides Fruits.

According to modern ethnobotanical records, the fruit of Hippophae rhamnoides is effective in the treatment of different allergic symptoms. In order to obtain pharmacological evidence for this observation, the fruit was investigated for anti-inflammatory activity using in vivo animal models. Aqueous and 70% MeOH extracts were tested in 48/80-induced rat paw edema assay after oral administration, and it was found that the 70% MeOH extract (500 mg/kg) reduced significantly edema volume (0.660 ± 0.082 mL vs. control 0.935 ± 0.041 mL). Extracts of different parts of the fruit (pulp, peel, seed) were investigated in the same assay, and the peel extract was shown to exhibit maximum edema-reducing effect (0.470 ± 0.124 mL vs. control 0.920 ± 0.111 mL). This extract was used to elucidate the mode of action. Different inflammation inducers (serotonin, histamine, dextran, bradykinin, and carrageenan) were applied in the rat paw model, but the extract inhibited only the compound 48/80 elicited inflammation. The active extract was then fractionated by solvent-solvent partitioning and chromatographic methods with the guidance of the 48/80-induced anti-inflammatory assay, and the main compounds responsible for the activity were identified as ursolic acid and oleanolic acid. Our data suggest that the activity is most probably based on a membrane stabilizing effect caused by the inhibition of degranulation of mast cells. Moreover, previously unknown 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignans, nectandrin B, fragransin A2, and saucernetindiol were isolated and identified from H. rhamnoides for the first time.

Penetration of lycopsamine from a comfrey ointment through human epidermis.

Mutagenic and teratogenic pyrrolizidine alkaloids (PAs) have been identified in several plant species. The industrially most important PA-containing plant is Symphytum officinale (common comfrey). The application of its root is restricted in several countries due to its PA content. In medicines, the daily alkaloid quantity and duration of treatment may be limited even in case of topical application. Due to the confirmed good absorption of PAs from the gastrointestinal tract, the prohibition of oral use is justified, however the limitation of external application is not supported by relevant data. Penetration experiments on human skin are not available to be a rational basis for limitation. The aim of our work was to carry out pharmacokinetic studies on the diffusion and penetration of lycopsamine (a main PA of comfrey) from a Symphytum product through a synthetic membrane and human skin. Investigations were carried out on vertical Franz diffusion cell and lycopsamine was quantified by a validated LC-MS method. The amount of lycopsamine diffused through a synthetic membrane varied between 0.11% and 0.72% (within 24 h). On human epidermis, the rate of penetration was lower (0.04-0.22%). Our results may contribute to the more realistic toxicological assessment of externally applied PA-containing products.

Evaluation of lignans from Heliopsis helianthoides var. scabra for their potential antimetastatic effects in the brain.

Two new arylbenzofuran-type neolignans, 1"-dehydroegonol 3"-methyl ether (1) and egonol 3"-methyl ether (2), and four known lignan derivatives, namely, helioxanthin (3), (7E)-7,8-dehydroheliobuphthalmin (4), heliobuphthalmin (5), and 7-acetoxyhinokinin (6), were isolated from a chloroform-soluble partition of the methanol extract of the fresh roots of Heliopsis helianthoides var. scabra. These six compounds were evaluated in vitro in terms of their ability to inhibit the various steps involved in brain tumor metastasis formation. Compounds 3 and 4 inhibited the migration of both melanoma and brain endothelial cells, and 3 also reduced the adhesion of melanoma cells to the brain endothelium. Furthermore, 3 and 4 additionally enhanced the barrier function of the blood-brain barrier and the expression of the tight junction protein ZO-1 at the junctions of the endothelial cells. These findings suggest that 3 and 4 may have the potential to interfere with different steps of brain metastasis formation and to enhance the barrier function of cerebral endothelial cells.

1H and 13C NMR investigation of 20-hydroxyecdysone dioxolane derivatives, a novel group of MDR modulator agents.

The synthesis, structure elucidation and the complete (1)H and (13)C signal assignment of a series of dioxolane derivatives of 20-hydroxyecdysone, synthesized as novel modulators of multidrug resistance, are presented. The structures and NMR signal assignment were established by comprehensive one-dimensional and two-dimensional NMR spectroscopy supported by mass spectrometry.

Isolation of Chemical Constituents of Centaurea virgata Lam. and Xanthine Oxidase Inhibitory Activity of the Plant Extract and Compounds.

BACKGROUND: Centaurea virgata Lam. is a species widely used in the traditional medicine in Turkey for the treatment of diabetes, allergy and gastric ulcers. The rationale of its use in the therapy has not been studied previously, therefore the present work aimed at the chemicalpharmacological evaluation of the plant. OBJECTIVE: The xanthine oxidase (XO) inhibitory activity of the MeOH extract and its subextracts (n-hexane, CHCl3 and remaining MeOH-H2O) prepared from C. virgata was investigated in vitro. Moderate activity was exerted in case of the CHCl3 extract (98.9 ± 15.8 µg/mL), therefore constituents of this extract were analysed. METHOD: Different purification steps, such as VLC, CPC, PLC and crystallization were used for the isolation, and ESIMS, NMR, LC-MS and authentic standards were applied for identification of the compounds. XO inhibitory and DPPH assays were used for evaluation of the bioactivities. RESULTS: Sesquiterpenes [8α-hydroxysonchucarpolide, 8α-(3,4-dihydroxy-2-methylenebutanoyloxy)- dehydromelitensine, and cnicin], flavones (apigenin, hispidulin, salvigenin, eupatorin, 3'-methyleupatorin), and the flavonol isokaempferide were isolated from the active extract. The XO-inhibitory activity of these compounds was analyzed using allopurinol as a positive control (IC50 7.49 ± 0.29 µM). It was found that sesquiterpenes and flavonoids, containing 7- OMe group, are inactive. CONCLUSION: 7-Hydroxyflavones (apigenin and hispidulin) exerted significant XO inhibitory effect with IC50 values of 0.99 ± 0.33 µM and 4.88 ± 1.21 µM, respectively. Therefore, these compounds are responsible for the XO-inhibitory effect of the extract. The free radical scavenging activity of the isolated flavonoids was determined by DPPH assay, and it was stated that none of the compounds have substantial antioxidant activity, therefore the reduced generation of reactive oxygen species may be the consequence only of XO inhibition.

Theanine and Caffeine Content of Infusions Prepared from Commercial Tea Samples.

BACKGROUND: Caffeine and L-theanine are pharmacologically important constituents of tea, especially due to their effects on the central nervous system. The effects of these two compounds are opposite: While caffeine is a well-known stimulant, theanine has a relaxing effect. Tea processing may influence the caffeine and theanine content of tea leaves. OBJECTIVE: The aim of our work was to quantify these constituents from a set of commercial products to reveal the possible correlations of caffeine and theanine content and processing methods. MATERIALS AND METHODS: Theanine and caffeine contents of 37 commercial white, green, oolong, black, and pu-erh tea samples were quantified by high-performance liquid chromatography-diode array detector. RESULTS: The mean L-theanine content of white, green, oolong, and black teas were 6.26, 6.56, 6.09, and 5.13 mg/g, respectively. The same values for caffeine content were 16.79, 16.28, 19.31, and 17.73 mg/g. CONCLUSION: Though the effect of processing on theanine content was evident, quantification for these analytes does not seem to be a good criterion to discriminate the different types of tea. Caffeine content provided no information on the effect of processing, and the theanine content of the samples was rather variable, independently from the type of the tea. The quantitative analysis of caffeine and theanine is essential to assess the stimulating effect of the tea, however, for chemical profiling further secondary metabolites have to be determined. SUMMARY: Thirty-seven commercial white, green, oolong, black, and pu-erh tea samples were analyzed for caffeine and theanine contentWhile the caffeine content was similar, the theanine contents of black teas were slightly lower and practically zero in pu-erhThe great variability of these two compound within the tea categories allows no discrimination of tea types based solely on theanine and caffeine quantificationContrary to the previous data, the way of processing has no determining effect on theanine content. FigureAbbreviations used: CZE: Capillary zone electrophoresis, DAD: Diode array detector, EEG: Electroencephalography, GC: Gas chromatography, HPLC: High-performance liquid chromatography, IR: Infrared spectroscopy, MEKC: Micellar electrokinetic capillary chromatography, MS: Mass spectrometry, RP: Reversed phase, RSD: Relative standard deviation, SD: Standard deviation, TLC: Tile liquid chromatography, UV: Ultraviolet.

A Validated, Rapid HPLC-ESI-MS/MS Method for the Determination of Lycopsamine.

The aim of the present work was to develop and validate an HPLC-MS/MS method for the determination of a major pyrrolizidine alkaloid of comfrey (lycopsamine) in aqueous samples as a basis for the development of a method for the determination of absorption of lycopsamine by human skin. A linear calibration curve was established in the range of 1.32-440 ng. The intraday precision during the 3-day validation period ranged between 0.57 and 2.48% while the interday precision was 1.70% and 1.95% for quality control samples. LOD was 0.014 ng and recovery was above 97%. The lycopsamine content of the samples stored for 9 and 25 days at 22 degrees C, 10 degrees C and -25 degrees C did not vary. These results underline the good repeatability and accuracy of our method and allow the analysis of samples with very low lycopsamine content.

Dual excitatory and smooth muscle-relaxing effect of Sideritis montana extract on guinea-pig ileum.

The neuronal and smooth muscle effects of a methanol extract prepared from the air-dried flowering aerial parts of Sideritis montana L. (SME) was tested in vitro on Guinea-pig ileum. The chemical composition of the investigated extract was analysed by HPLC-MS, and chrysoeriol, chlorogenic acid and caffeic acid were detected as main constituents. The isolated organ assay showed that S. montana extract caused an immediate contraction and a more slowly developing inhibitory response in the ileum. The SME-induced contractions were strongly inhibited by the acetylcholine muscarinic receptor antagonist atropine (0.5 µM), but not by either the Na+ channel blocker tetrodotoxin (TTX; 0.5 µM) or the histamine H1 receptor antagonist chloropyramine (0.5 µM). Selective desensitization of capsaicin-sensitive neurons by the sensory neuron stimulant and blocker capsaicin did not influence the contractile effect of SME. As to the spasmolytic effect, SME inhibited the effects of electrical field stimulation, exogenous acetylcholine, and histamine. These smooth muscle-relaxing effects were reversible in 40 min by repeated renewals of the bathing solution.

Effects of Chelidonium majus extracts and major alkaloids on hERG potassium channels and on dog cardiac action potential - a safety approach.

Chelidonium majus or greater celandine is spread throughout the world, and it is a very common and frequent component of modern phytotherapy. Although C. majus contains alkaloids with remarkable physiological effect, moreover, safety pharmacology properties of this plant are not widely clarified, medications prepared from this plant are often used internally. In our study the inhibitory effects of C. majus herb extracts and alkaloids on hERG potassium current as well as on cardiac action potential were investigated. Our data show that hydroalcoholic extracts of greater celandine and its alkaloids, especially berberine, chelidonine and sanguinarine have a significant hERG potassium channel blocking effect. These extracts and alkaloids also prolong the cardiac action potential in dog ventricular muscle. Therefore these compounds may consequently delay cardiac repolarization, which may result in the prolongation of the QT interval and increase the risk of potentially fatal ventricular arrhythmias.

Possible role of fat tissue in the pharmacokinetics of Dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides after oral administration of Echinacea angustifolia extract in rats.

Alkamides are one of the most important constituents of lipophilic extracts of Echinacea angustifolia roots. These compounds play an important role in the versatile pharmacological actions of this plant. The present study aimed to compare the concentrations of isomeric dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (DTAI) in brain and periepididymal fat tissues and blood plasma of rats. Thirty, 60, 240 and 720 min after the oral administration of E. angustifolia root extract, tissue and plasma concentrations were determined by reversed-phase HPLC with ESI-MS/MS detection. The calculated terminal t1/2 of the mixture of DTAI was 8.28 h, which indicates a relatively slow elimination. In the 0.5-4 h period the brain/plasma and fat/plasma concentration ratios were continuously above 3 and 18, respectively, followed by equilibrium at 12 h. Our results indicate substantial accumulation of alkamides in lipid-rich tissues, which presumably contributes to a maintained pharmacological action.

Identification of diterpene alkaloids from Aconitum napellus subsp. firmum and GIRK channel activities of some Aconitum alkaloids.

Diterpene alkaloids neoline (1), napelline (2), isotalatizidine (3), karakoline (4), senbusine A (5), senbusine C (6), aconitine (7) and taurenine (8) were identified from Aconitum napellus L. subsp. firmum, four (2-4, 6) of which are reported for the first time from this plant. The structures were determined by means of LC-MS, 1D and 2D NMR spectroscopy, including (1)H-(1)H COSY, NOESY, HSQC and HMBC experiments. Electrophysiological effects of the isolated compounds, together with nine diterpene alkaloids previously obtained from Aconitum toxicum and Consolida orientalis were investigated on stable transfected HEK-hERG (Kv11.1) and HEK-GIRK1/4 (Kir3.1 and Kir3.4) cell lines using automated patch clamp equipment. Significant blocking activity on GIRK channel was exerted by aconitine (7) (45% at 10 µM), but no blocking activities of the other investigated compounds were detected. The tested compounds were inactive on hERG channel in the tested concentration. The comparison of the previously reported metabolites of A. napellus subsp. firmum and compounds identified in our experiment reveals substantial variability of the alkaloid profile of this taxon.

Exposure of chlorpromazine to 266 nm laser beam generates new species with antibacterial properties: contributions to development of a new process for drug discovery.

INTRODUCTION: Phenothiazines when exposed to white light or to UV radiation undergo a variety of reactions that result in degradation of parental compound and formation of new species. This process is slow and may be sped up with exposure to high energy light such as that produced by a laser. METHODS: Varying concentrations of Chlorpromazine Hydrochloride (CPZ) (2-20 mg/mL in distilled water) were exposed to 266 nm laser beam (time intervals: 1-24 hrs). At distinct intervals the irradiation products were evaluated by spectrophotometry between 200-1500 nm, Thin Layer Chromatography, High Pressure Liquid Chromatography (HPLC)-Diode Array Detection, HPLC tandem mass spectrometry, and for activity against the CPZ sensitive test organism Staphylococcus aureus ATCC 25923. RESULTS: CPZ exposure to 266 nm laser beam of given energy levels yielded species, whose number increased with duration of exposure. Although the major species produced were Promazine (PZ), hydroxypromazine or PZ sulfoxide, and CPZ sulfoxide, over 200 compounds were generated with exposure of 20 mg/mL of CPZ for 24 hrs. Evaluation of the irradiation products indicated that the bioactivity against the test organism increased despite the total disappearance of CPZ, that is due, most probably, to one or more new species that remain yet unidentified. CONCLUSIONS: Exposure of CPZ to a high energy (6.5 mJ) 266 nm laser beam yields rapidly a large number of new and stable species. For biological grade phenothiazines (in other words knowing the impurities in the samples: solvent and solute) this process may be reproducible because one can control within reasonably low experimental errors: the concentration of the parent compound, the laser beam wavelength and average energy, as well as the duration of the exposure time. Because the process is "clean" and rapid, it may offer advantages over the pyrogenically based methods for the production of derivatives.