Validation of a microscale extraction and high-throughput UHPLC-QTOF-MS analysis method for huperzine A in Huperzia.

Herein we report on an improved method for the microscale extraction of huperzine A (HupA), an acetylcholinesterase-inhibiting alkaloid, from as little as 3 mg of tissue homogenate from the clubmoss Huperzia squarrosa (G. Forst.) Trevis with 99.95% recovery. We also validated a novel UHPLC-QTOF-MS method for the high-throughput analysis of H. squarrosa extracts in only 6 min, which, in combination with the very low limit of detection (20 pg on column) and the wide linear range for quantification (20-10,000 pg on column), allow for a highly efficient screening of extracts containing varying amounts of HupA. Utilization of this methodology has the potential to conserve valuable plant resources.

Antioxidant properties of extracts of wild medicinal mushroom species from Croatia.

Antioxidant activity and total phenol (TP) content of methanol and water extracts of three wild Croatian mushroom species Auricularia auricula-judae (Bull.) Quél., Sarcoscypha austriaca (Sacc.) Boud., and Strobilurus esculentus (Wulfen) Singer were determined and compared with the values obtained for extracts of four cultivated mushrooms Agaricus bisporus (J.E. Lange) Imbach (brown and white strains), Pleurotus ostreatus (Jacq.) P. Kumm., and Lentinus edodes (Berk.) Singer. Spectrophotometric determination of the TP content was performed using the Folin-Ciocalteu method, while antioxidant activity was measured in a reaction with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH assay) and ferric-tripyridyltriazine (Fe3+-TPTZ) complex (FRAP assay). On the average, 5.8-fold higher TP content was observed for water in comparison to methanol extracts of all analyzed mushrooms. Consequently, antioxidant activity was also higher for water extracts, which is evident from the obtained higher values in the FRAP assay and lower EC50 values in the DPPH assay. Among the three tested wild species, the water extract of S. esculentus exhibited the highest concentration of TP, 8.12 mg/g gallic acid equivalents (GAE), the highest reducing power, 19.42 mmol Fe2+/kg, and the best radical scavenging properties, EC50= 13.5 mg/mL.

Free radical-scavenging activity and DNA damaging potential of auxins IAA and 2-methyl-IAA evaluated in human neutrophils by the alkaline comet assay.

Auxins, of which indole-3-acetic acid (IAA) is the most widespread representative, are plant hormones. In addition to plants, IAA also naturally occurs in humans in micromolar concentrations. In the presence of peroxidase, indolic auxins are converted to cytotoxic oxidation products and have thus been proposed for use in gene-directed enzyme/prodrug tumor therapy. Since data on the genotoxicity of IAA and its derivatives are not consistent, here we investigate the early DNA damaging effects (2-h treatment) of the auxins, IAA, and 2-methyl-indole-3-acetic acid (2-Me-IAA) by the alkaline comet assay and compare them with their free radical-scavenging activity measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Human neutrophils are chosen as the test system since they possess inherent peroxidase activity. The results of the comet assay indicate an increase in DNA damage in a dose-dependent manner up to 1.00 mM of both auxins. Generally, IAA applied in the same concentration had greater potential to damage DNA in human neutrophils than did 2-Me-IAA. The genotoxicities of the two examined auxins are negatively correlated with their antioxidant activities, as measured by the DPPH assay; 2-Me-IAA showed a higher antioxidant capacity than did IAA. We assume that differences in the molecular structure of the tested auxins contributed to differences in their metabolism, in particular, with respect to interactions with peroxidases and other oxidative enzymes in neutrophils. However, the exact mechanisms have to be elucidated in future studies.

Polysaccharides and Antioxidants from Culinary-Medicinal White Button Mushroom, Agaricus bisporus (Agaricomycetes), Waste Biomass.

Agaricus bisporus, also known as the white button mushroom or champignon, is the most cultivated mushroom species worldwide. In addition to its favorable nutrient profile, it contains a number of compounds with antioxidant, antibacterial, anti-inflammatory, antitumor, and immunomodulatory activities. Waste biomass is a secondary product obtained from A. bisporus during the harvesting stage. It is underused, although it could be a cheap source of polysaccharides and antioxidants for use in food and feed production, or a source of nutraceuticals and cosmeceuticals. In this study, waste biomass was used as raw material for extraction of crude polysaccharides. The mean amount of crude polysaccharides extracted was 106 g/kg dry weight debris-free mushroom waste biomass. The crude polysaccharides recovered contained 11.57% α-glucan and 16.37% ß-glucan. Total carbohydrates composed 44.18%. No significant differences were found in the Fourier transform infrared spectra, which confirmed the presence of protein, α-glucan, and ß-glucan in all samples; phenols were detected only in waste biomass and market-ready A. bisporus fruiting bodies. The total phenol content in methanol extracts of waste biomass and A. bisporus fruiting bodies was 6.16 and 11.25 mg gallic acid equivalents/g extract, respectively. Antioxidant capacities of methanol extracts from waste biomass, as determined by spectrophotometric techniques, were 22.67 µmol Trolox/g extract (ABTS radical scavenging), 51.77 µmol Fe2+/g extract (ferric-reducing antioxidant power), and 51.52% (DPPH radical scavenging). Although these values were lower than those for A. bisporus fruiting bodies, the waste biomass has great potential for use in food, feed, and other bioproducts of economic importance.

Genetic and phytochemical variability of six Teucrium arduini L. populations and their antioxidant/prooxidant behaviour examined by biochemical, macromolecule- and cell-based approaches.

Teucrium arduini L., an Ilyric-Balcanic endemic species, has been reported for decades as a valuable plant used in traditional medicine for treating digestive disorders. The present study evaluated genetic and phytochemical variability of six T. arduini populations in order to determine factors that influence an accumulation of polyphenolic compounds. Results strongly suggest that a phytochemical variation was caused by environmental rather than genetic factors. T. arduini leaf extract from the locality Ucka, which accumulated significantly more polyphenolic phytochemicals in comparison to others, showed antioxidant activity in DNA and lipid bioassays. Furthermore, the same extract exhibited prooxidant behaviour at protein level and induce formation of reactive oxygen species in human laryngeal carcinoma cells causing cytotoxic activity, in a dose dependent manner. All the results of the present study suggested that T. arduini extract could be responsible for antioxidative/prooxidative mechanisms and would help in determination of optimal conditions for their ethnopharmacological use.

Molecular and cellular approach in the study of antioxidant/pro-oxidant properties of Micromeria croatica (Pers.) Schott.

Phytochemical composition and antioxidant activity of three wild populations of endemic Illyric-Balcanic species Micromeria croatica (Pers.) Schott have been evaluated with respect to plant organ and growing location. Multivariate analysis (principal component analysis) was performed to visualise (dis-)similarity among samples and identify the correlations between phytochemical variables that explain the most variability. The tested leaf extract from BaCic kuk locality exhibited protective effects against reactive oxygen species-induced damage of DNA and inhibition of lipid peroxidation, while it caused oxidative degradation of protein in the bovine serum albumin assay at higher concentrations. This extract also exhibited cytotoxic activity and facilitated the formation of reactive oxygen species in the HEp2 cell line, in a dose-dependent manner.

Accurate mass-time tag library for LC/MS-based metabolite profiling of medicinal plants.

We report the development and testing of an accurate mass-time (AMT) tag approach for the LC/MS-based identification of plant natural products (PNPs) in complex extracts. An AMT tag library was developed for approximately 500 PNPs with diverse chemical structures, detected in electrospray and atmospheric pressure chemical ionization modes (both positive and negative polarities). In addition, to enable peak annotations with high confidence, MS/MS spectra were acquired with three different fragmentation energies. The LC/MS and MS/MS data sets were integrated into online spectral search tools and repositories (Spektraris and MassBank), thus allowing users to interrogate their own data sets for the potential presence of PNPs. The utility of the AMT tag library approach is demonstrated by the detection and annotation of active principles in 27 different medicinal plant species with diverse chemical constituents.

Irinotecan side effects relieved by the use of HI-6 oxime: in vivo experimental approach.

Some compounds, although not primarily designed as supportive drugs in chemotherapy, are promising candidates for clinical use. The ability of HI-6 oxime to relieve the side effects of irinotecan was recently determined in vitro. In this animal study, we investigated the efficacy of HI-6 in vivo, when given as a pre-treatment and concomitantly with irinotecan. We evaluated the cholinesterase (ChE)/acetylcholinesterase (AChE) activity, the levels of oxidative stress markers, DNA damage and the radical scavenging capacity of HI-6. Both HI-6 and irinotecan inhibited ChE/AChE activity but showed different levels of ChE inhibition in plasma and AChE inhibition in the liver and brain tissue. We also observed a weak antioxidant capacity of HI-6, undiscovered until now, and found an acceptable genotoxicity profile in three types of somatic cells in rats. The in vivo erythrocyte micronucleus assay showed that HI-6 did not significantly change either the frequency of micronuclei or the ratio of polychromatic and normorchromatic erythrocytes. Taken together, our results provide a good argument in favour of HI-6 as a promising molecule for further studies and eventual use in humans.

Successful treatment of chronic decubitus ulcer with 0.1% dirhamnolipid ointment.

BACKGROUND: Previous investigations of the biologic activities of dirhamnolipid alpha-L-rhamnopyranosyl-(1-2)alpha-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid have demonstrated that it could be a novel therapeutic agent for wound healing and various immunologic and dermatologic conditions. OBJECTIVE: The aim of this article is to report the successful treatment of a decubitus ulcer with dirhamnolipid. METHODS: The patient was a 90-year-old woman who developed a decubitus ulcer on her right buttock. The patient was initially treated by a standard procedure using pressure reduction, wound management, surgical intervention, and nutrition. The open, full-thickness wound progressed to a size of 10 x 7 cm, with evidence of tissue deterioration. The draining ulcer reached a dimension of 1 x 1.5 x 3 cm. The 0.1% dirhamnolipid ointment was administered at regular intervals, three times daily, by applying a thin layer of ointment directly to the wound area. Photographs were taken at regular 5-day intervals. RESULTS: The standard therapy gave no improvement. Subsequently, therapy with topical dirhamnolipid ointment resulted in a completely healed wound on day 48 of the treatment. CONCLUSION: This case demonstrates that application of dirhamnolipid resulted in the healing of a chronic decubitus ulcer in an elderly, debilitated patient and might be a useful therapy to improve healing of decubitus ulcers.