Hydroxylated cyclopamine analogues from Veratrum californicum and their hedgehog pathway inhibiting activity.

Cyclopamine (1), the teratogenic steroidal alkaloid isolated from corn lily (Veratrum californicum), has recently gained renewed interest due to its anticancer potential, that has been translated into the FDA approval of three Hedgehog (Hh) pathway inhibiting antitumor drugs. A chemical analysis of mother liquors obtained from crystallization of cyclopamine, extracted from roots and rhizomes of V. californicum, resulted in the isolation of two unprecedented cyclopamine analogues, 18-hydroxycyclopamine (2) and 24R-hydroxycyclopamine (3), the first compounds of this class to show modifications on rings D-F. The stereostructures of these new natural compounds have been established based on a detailed MS and 1D/2D NMR investigation. The isolated compounds were evaluated with the dual-luciferase bioassay for their inhibition of the hedgehog pathway in comparison to cyclopamine, providing new insights into the structure-activity relationships for this class of compounds.

Shedding light on curcumin stability.

The stability of molecular curcumin (purcumin, 1a) in solution is strongly light-dependent. Under laboratory artificial light, a relative stability is observed only at neutral pH, while more intense light and/or solar light can trigger degradation via a combination of hydrolytic and oxidative fragmentation of the heptadiendione moiety. Minor curcuminoids in commercial curcumin (purcuminoids) can improve the stability of molecular curcumin, but only under conditions of low irradiation. While confirming earlier observations alerting to the instability of purcumin, our results provide new rationales for unexplained differences between previous studies, question the biological relevance of a non-enzymatic degradation for the bioactivity profiles that have been reported for purcumin, and highlight the need of a better characterization of the degradation of purcuminoids under visible light irradiation.

Cannabitwinol, a Dimeric Phytocannabinoid from Hemp, Cannabis sativa L., Is a Selective Thermo-TRP Modulator.

Cannabitwinol (CBDD, 3), the second member of a new class of dimeric phytocannabinoids in which two units are connected by a methylene bridge, was isolated from a hemp (Cannabis sativa L.) industrial extract. The structural characterization of cannabitwinol, complicated by broadening of 1H NMR signals and lack of expected 2D NMR correlations at room temperature, was fully carried out in methanol-d4 at -30 °C. All the attempts to prepare CBDD by reaction of CBD with formaldehyde or its iminium analogue (Eschenmoser salt) failed, suggesting that this sterically congested dimer is the result of enzymatic reactions on the corresponding monomeric acids. Analysis of the cannabitwinol profile of transient receptor potential (TRP) modulation evidenced the impact of dimerization, revealing a selectivity for channels activated by a decrease of temperature (TRPM8 and TRPA1) and the lack of significant affinity for those activated by an increase of temperature (e.g., TRPV1). The putative binding modes of cannabitwinol with TRPA1 and TRPM8 were investigated in detail by a molecular docking study using the homology models of both channels.

A New Food-grade Coenzyme Q10 Formulation Improves Bioavailability: Single and Repeated Pharmacokinetic Studies in Healthy Volunteers.

BACKGROUND: Coenzyme Q10 is a fundamental endogenous factor involved in cell energy production that shows protective properties in oxidative stress, mainly in skeletal and heart muscle. Coenzyme Q10 supplementation appears to benefit athletes in strenuous training and in the elderly, demonstrating ant-inflammatory properties by reducing inflammatory cytokines. Improved absorption of coenzyme Q10 via a new delivery system would represent an important step forward in the use of coenzyme Q10 as a dietary supplement. OBJECTIVE: The aim of the study was to evaluate the solubility and oral absorption in human healthy volunteers of a new food grade coenzyme Q10 phytosome formulation. METHODS: Solubility studies were performed in vitro in simulated gastrointestinal fluids; human studies were conducted in healthy volunteers to evaluate oral absorption in a Single dose study, in comparison with the coenzyme Q10 capsules, and in a repeated study at two increasing doses. RESULTS: The highest solubility shown by coenzyme Q10 phytosome in simulated intestinal fluids results in an improvement in oral absorption of coenzyme Q10 in healthy volunteers, three times more than the coenzyme Q10 according to AUC (area under the time/concentration curve) values. When two increasing doses (one and two capsules) were administered to healthy volunteers within a two-week schedule, the plasmatic levels of coenzyme Q10 resulted in 0.864±0.200 µg/ml (Mean±S.D.+41%) and 1.321±0.400 µg/ml (+116%), respectively versus baseline (0.614±0.120 µg/ml one capsule, 0.614±0.160 µg/ml two capsules). This detected dose-related bioavailability of coenzyme Q10 phytosome was even observed with no alterations in vital signs, neither in the physical examination nor in ECG, and no changes of clinical and biochemical parameters were observed. CONCLUSION: These findings, taken together, support the safety profile and significantly improved coenzyme Q10 oral absorption in humans with this new phytosome delivery formulation.

Combined use of isotopic fingerprint and metabolomics analysis for the authentication of saw palmetto (Serenoa repens) extracts.

Saw palmetto (Serenoa repens, SP) is the most expensive oil source of the pharmaceutical and healthfood market, and its high cost and recurrent shortages have spurred the development of designer blends of fatty acids to mimic its phytochemical profile and fraudulently comply with the current authentication assays. To detect this adulteration, the combined use of isotopic fingerprint and omic analysis has been investigated, using Principal Component Analysis (PCA) to handle the complex databases generated by these techniques and to identify the possible source of the adulterants. Surprisingly, the presence of fatty acids of animal origin turned out to be widespread in commercial samples of saw palmetto oil.

Metabolomics study of Saw palmetto extracts based on 1H NMR spectroscopy.

Preparations containing Saw palmetto extracts are used in traditional medicine to treat benign prostatic hyperplasia. According to the European and the American Pharmacopoeias, the extract is obtained from comminuted Saw palmetto berries by a suitable extracting procedure using ethanol or supercritical carbon dioxide or a mixture of n-hexane and methylpentanes. In the present study an approach to metabolomics profiling using nuclear magnetic resonance (NMR) has been used as a finger-printing tool to assess the overall composition of the extracts. The phytochemical analysis coupled with principal component analysis (PCA) showed the same composition of the Saw palmetto extracts obtained with carbon dioxide and hexane with minor not significant differences for extracts obtained with ethanol. In fact these differences are anyhow lower than the batch-to-batch variability ascribable to the natural-occurring variability in the Saw palmetto fruits' phytochemical composition. The fingerprinting analysis combined with chemometric method, is a technique, which would provide a tool to comprehensively assess the quality control of Saw palmetto extracts.

In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.

The molecular characterization of bioactive food components is necessary for understanding the mechanisms of their beneficial or detrimental effects on human health. This study focused on γ-conglutin, a well-known lupin seed N-glycoprotein with health-promoting properties and controversial allergenic potential. Given the importance of N-glycosylation for the functional and structural characteristics of proteins, we studied the purified protein by a mass spectrometry-based glycoproteomic approach able to identify the structure, micro-heterogeneity and attachment site of the bound N-glycan(s), and to provide extensive coverage of the protein sequence. The peptide/N-glycopeptide mixtures generated by enzymatic digestion (with or without N-deglycosylation) were analyzed by high-resolution accurate mass liquid chromatography-multi-stage mass spectrometry. The four main micro-heterogeneous variants of the single N-glycan bound to γ-conglutin were identified as Man2(Xyl) (Fuc) GlcNAc2, Man3(Xyl) (Fuc) GlcNAc2, GlcNAcMan3(Xyl) (Fuc) GlcNAc2 and GlcNAc 2Man3(Xyl) (Fuc) GlcNAc2. These carry both core ß1,2-xylose and core α1-3-fucose (well known Cross-Reactive Carbohydrate Determinants), but corresponding fucose-free variants were also identified as minor components. The N-glycan was proven to reside on Asn131, one of the two potential N-glycosylation sites. The extensive coverage of the γ-conglutin amino acid sequence suggested three alternative N-termini of the small subunit, that were later confirmed by direct-infusion Orbitrap mass spectrometry analysis of the intact subunit.

New liquid chromatography method with ultraviolet detection for analysis of anthocyanins and anthocyanidins in Vaccinium myrtillus fruit dry extracts and commercial preparations.

The Vaccinium myrtillus fruits (bilberry) are a well-known anthocyanins source, and their extracts are widely used in dietary botanicals and pharmaceutical products for the treatment of vascular and vision disorders. Different analytical methods used for standardization of the bilberry extracts and their preparations are available from pharmacopeias and from the literature. However, the methods reported in the literature do not allow the detection of free anthocyanidins, which are markers of poor product quality. A new liquid chromatography method was developed and validated for the identification and quantification of both anthocyanins and anthocyanidins present in bilberry extracts and products. The method shows a good reproducibility and, due to its high specificity, is suitable to identify unequivocally the botanical raw materials used for manufacturing and to evaluate the extract composition, thus ensuring a high degree of product consistency and quality. Forty typical bilberry preparations belonging to 24 different brands were purchased in the marketplace and evaluated for their quality by using the developed method. Results revealed marked differences among the brands despite a common origin and labeling.