Structure Elucidation of the Main Tetrahydroxyxanthones of Hypericum Seeds and Investigations into the Testa Structure.

Seeds from Hypericum species have recently been identified as an interesting source of xanthone derivatives. Extraction of seeds from H. perforatum with MeOH and subsequent concentration via polyamide adsorption yielded a fraction enriched in tetrahydroxyxanthones (THX), which were further semipurified by silica gel chromatography. Based on tentative structure assignment of the two main THX X1 and X2 by NMR a total synthesis was performed for both compounds (THX 1 and 2, respectively), starting with an Ullmann ether synthesis. The synthesized 1 and 2 were characterized via 1D- and 2D-NMR methods as well as by LC/HR-MS analysis and proven to be 1,4,6,7-THX (1) and 1,2,6,7-THX (2). Final structure assignment of the natural Hypericum THX constituents was accomplished by comparing chromatographic and spectroscopic data (LC/MSn and GC/MS) with those of 1 and 2 which were obtained by synthesis. Beyond, investigations into the seeds of H. perforatum and H. tetrapterum by scanning electron microscopy (SEM) provided insights of the structure of the testa (seed coat), which is established by two cell layers, with the lignified sclerenchyma presumably being the depository of the xanthones.

Comprehensive Characterisation of n-Alkylresorcinols and Other Lipid Constituents of Mercurialis tomentosa L. from Alicante, Spain.

Mercurialis tomentosa L. has been used in Spanish ethnomedicine. In the present study the first phytochemical characterisation of a lipid fraction from M. tomentosa was performed. The CHCl3 extraction of aerial parts from M. tomentosa and GC/MS investigations revealed the occurrence of cuticular lipid and wax constituents, like long chain n-alcohols and n-aldehydes (C22  - C30 ), besides several aromatic constituents, i.e., phenylpropanoids and n-alkylresorcinols. The latter were further purified by CC and analysed by LC/MSn . In contrast to other Mercurialis species, i.e., M. annua, M. perennis, which exclusively contain 5-n-alkylresorcinols (1a - j, Cn ), mainly 5-n-alkyl-2-methylresorcinols (2a - j, Cn *) with side chain lengths of C15  - C25 were found in M. tomentosa, in addition to 1a - j. Thus, the latter compounds may be utilised for analytical characterisation and authentication of M. tomentosa based on fingerprinting methods. For structure elucidation a novel facile total synthesis of one representative 5-n-alkyl-2-methylresorcinol homologue (2d, C19 *) was developed, starting with a Grignard reaction from a substituted benzoic acid chloride (19). The compound obtained by synthesis was identical to the natural product 2d in terms of its chromatographic and spectroscopic features. Futhermore, 2d exhibited satisfactory DPPH free radical scavenging activity (IC50  = 37.8 µm) when compared to trolox (IC50  = 21.0 µm), corroborating the antioxidant features of these amphipathic molecules.

Lipid and Phenolic Constituents from Seeds of Hypericum perforatum L. and Hypericum tetrapterum Fr. and their Antioxidant Activity.

Seeds of Hypericum perforatum and H. tetrapterum were extracted with dichloromethane and methanol and investigated by chromatographic and mass spectrometric methods. Both species yielded a fatty oil fraction amounting to 30.5% and 18.0% of the seed weight, respectively. Linoleic acid (C18:2n-6) was shown to be the predominant fatty acid constituent. Moreover, xanthone derivatives, i.e. tetrahydroxyxanthones (THX), xanthone-glycosides and xanthone-sulfonates, were assigned in methanolic extracts. For structure elucidation, one representative xanthone, namely 1,3,6,7-THX, was synthesized and analyzed via HPLC-DAD/MSn and GC/MS. Total THX contents were quantitated applying a validated HPLC-DAD method, resulting in 1.25 g/kg (H. perforatum) and 0.27 g/kg (H. tetrapterum), respectively. Moreover, the free radical scavenging capacity of the methanol extracts was tested using the DPPH antioxidant assay. Both, H. perforatum (IC50 = 8.7 mg/l) and 1,3,6,7-THX (IC50 = 3.0 mg/l), exhibited good DPPH free radical scavenging activity compared to Trolox (IC50 = 6.6 mg/l).

Aquatic testing guidelines insufficiently control the influence of dilution water toc and hardness on cationic polymer toxicity - A proposal to improve standardized test procedures.

Cationic polymers (CPs) are widely used chemicals for wastewater treatment applications and in various "down-the-drain" household products. The aquatic toxicity of CPs results from an electrostatic interaction with negatively charged cell surfaces. These effects are greatly mitigated by the binding affinity of CPs to total organic carbon (TOC) in surface water. Consequently, baseline aquatic toxicity tests of CPs using clean lab water (TOC < 2 mg/L) typically overestimate toxicity and risk which is greatly mitigated at higher environmentally relevant OC levels. However, the point at which mitigation begins is not well defined and low-level TOC in lab water may influence the baseline toxicity outcome. Similarly, divalent cations, quantified as water hardness, may modulate the electrostatic binding between OC and CP. Although standard guidelines define limits for lab water hardness and TOC, the consequences of variability within those limits on test outcome is unknown. We investigated the impact of part-per-billion (ppb) additions of TOC to lab water at different hardness levels on CP acute toxicity to Daphnia magna and Raphidocelis subcapitata. In both species, the acute toxicities of CPs with different molecular weight and charge density varied by > 10-fold in response to slight changes in TOC and water hardness, although parameters were maintained within guideline limits. When determining the baseline aquatic toxicity of CPs, the lab water should be standardized at the lowest biologically tolerable hardness and TOC at a reliably measurable level (>1 - < 2 mg/L) to reduce variability and increase the reliability of the toxicity estimate.

Constituents from oak bark (Quercus robur L.) inhibit degranulation and allergic mediator release from basophils and mast cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Oak bark has been used since ancient times in Europaen ethnomedicine because of its adstringent, antimicrobial and hemostatic features, e.g. as a remedy for the treatment of wounds and skin diseases. PURPOSE: Oak bark tannins are considered as bioactive natural products, interacting with surface proteins of mucous membranes and might be beneficial for the treatment of allergic diseases. This study investigated the effect of an oak bark decoction (OBD) and isolated tannin fractions on the degranulation capacity and cytokine/chemokine release from rat basophilic cells and human mast cells in vitro, which are essential for the initiation of early- and late-phase allergic reactions. METHODS AND METHODS: By chromatographic separation on Sephadex® LH-20 high- and low-molecular weight tannins were separated from OBD and the tannin composition analyzed by HPLC(DAD)-MSn. Then, the OBD and its fractions were tested in degranulation (ß-hexosaminidase activity) of allergen-specific-activated basophilic cells in a photometric assay. RESULTS: The OBD and the high-molecular tannin fraction showed a dose-dependent inhibition of cell degranulation. Furthermore, the OBD and particularly its high molecular weight tannin fraction exhibited an inhibitory activity on the IL-8-, IL-6- and TNF-α-secretion from stimulated human mast cells, detected and quantified by ELISA. CONCLUSION: The OBD and its high-molecular weight tannins revealed an impact on allergic mediator release of basophilic cells and human mast cells and thereby provide a rationale for the topical treatment with OBD preparations.