Ultra-Micro-Scale-Fractionation (UMSF) as a Powerful Tool for Bioactive Molecules Discovery.

Herein is detailed the development and validation of an ultra-micro-scale-fractionation (UMSF) technique for the discovery of plant-based, bioactive molecules, coupling the advantages of ultra-performance liquid chromatography mass spectrometry (UPLC-MS) separations with microtiter plate-based bioassay screens. This novel one-step approach simultaneously uses UPLC to collect chemical profile information, while performing high-resolution fractionation, greatly improving workflow compared to methods relying on high-performance liquid chromatography (HPLC), solid phase extraction or flash systems for chromatographic separations. Using the UMSF technique, researchers are able to utilize smaller quantities of starting materials, reduce solvent consumption during fractionation, reduce laborious solvent dry down times, replace costly single-use solid-phase-extraction cartridges with reusable analytical-sale UPLC columns, reduce fractionation times to less than 10 min, while simultaneously generating chemical profile data of active fractions and enjoying superior chromatographic resolution. Using this technique, individual bioactive components can be readily purified, identified, and bioassayed in one step from crude extracts, thereby eliminating ambiguous synergistic effects often reported in plant-based natural products research. A successful case-study is presented illustrating the versatility of this technique in identifying lupulone as the principal cytotoxic component from hops (Humulus lupulus L.), using a brine shrimp (Artemia franciscana) model. These results confirm and expand upon previous cell-based bioassay studies using a more complex, multicellular organism, and add to our understanding of structure-function activity relationships for secondary metabolites in hops and the Cannabaceae plant family.

Discovery of an Isothiazolinone-Containing Antitubercular Natural Product Levesquamide.

Antitubercular agent levesquamide is a new polyketide-nonribosomal peptide (PK-NRP) hybrid marine natural product isolated from Streptomyces sp. RKND-216. The structure contains a rare isothiazolinone moiety which has only been reported in collismycin SN. Structure elucidation by NMR spectroscopy was a significant challenge due to a deficiency of protons in this aromatic moiety. Therefore, the genome of Streptomyces sp. RKND-216 was sequenced to identify the levesquamide biosynthetic gene cluster (BGC). Analysis of the BGC provided structural insights and guided stable-isotope labeling experiments, which led to the assignment of the fused pyridine-isothiazolinone moiety. The BGC and the labeling experiments provide further insights into the biosynthetic origin of isothiazolinones. Levesquamide exhibited antimicrobial activity in the microplate alamarBlue assay (MABA) and low oxygen recovery assay (LORA) against Mycobacterium tuberculosis H37Rv with minimum inhibitory concentration (MIC) values of 9.65 and 22.28 µM, respectively. Similar activity was exhibited against rifampicin- and isoniazid-resistant M. tuberculosis strains with MIC values of 9.46 and 9.90 µM, respectively. This result suggests levesquamide has a different mode of action against M. tuberculosis compared to the two first-line antitubercular drugs rifampicin and isoniazid. Furthermore, levesquamide shows no cytotoxicity against the Vero cell line, suggesting it may have a useful therapeutic window.

NMR characterization of novel pyranoanthocyanins derived from the pulp of Panax quinquefolius L. (North American ginseng).

Three major pigments (one natural and two derived) were determined to be present in the berry pulp of Panax quinquefolius L. (North American ginseng). The first was a simple anthocyanin (pelargonidin 3-O-lathyroside) along with two novel pyranoanthocyanins, structurally similar to those recently discovered in Staghorn sumac. The three anthocyanins were structurally characterized using NMR (1 H, gCOSY, gHSQC, gHMBC, TOCSY, ROESY, and 13 C DEPTq135) and High Resolution MS. All three anthocyanins had the disaccharide lathyrose (2-O-(ß-D-xylopyranosyl)-ß-D-galactopyranoside) attached at the 3-O position. In the tradition of naming novel anthocyanin aglycones based on botanical origin, the new pyranoanthocyanin aglycones have been given the common names Panaxidin A (pelaragonidin-4-vinylcatechol) and Panaxidin B (pelargonidin-4-vinylphenol). Copyright © 2015 Her Majesty the Queen in Right of Canada Magnetic Resonance in Chemistry © 2015 John Wiley & Sons, Ltd.

Prebiotic effects of diet supplemented with the cultivated red seaweed Chondrus crispus or with fructo-oligo-saccharide on host immunity, colonic microbiota and gut microbial metabolites.

BACKGROUND: Gastrointestinal microbial communities are diverse and are composed of both beneficial and pathogenic groups. Prebiotics, such as digestion-resistant fibers, influence the composition of gut microbiota, and can contribute to the improvement of host health. The red seaweed Chondrus crispus is rich in dietary fiber and oligosaccharides, however its prebiotic potential has not been studied to date. METHODS: Prebiotic effects were investigated with weaning rats fed a cultivated C. crispus-supplemented diet. Comparison standards included a fructo-oligo-saccharide (FOS) diet and a basal diet. The colonic microbiome was profiled with a 16S rRNA sequencing-based Phylochip array. Concentrations of short chain fatty acids (SCFAs) in the feacal samples were determined by gas chromatography with a flame ionization detector (GC-FID) analysis. Immunoglobulin levels in the blood plasma were analyzed with an enzyme-linked immunosorbent assay (ELISA). Histo-morphological parameters of the proximal colon tissue were characterized by hematoxylin and eosin (H&E) staining. RESULTS: Phylochip array analysis indicated differing microbiome composition among the diet-supplemented and the control groups, with the C. crispus group (2.5% supplementation) showing larger separation from the control than other treatment groups. In the 2.5% C. crispus group, the population of beneficial bacteria such as Bifidobacterium breve increased (4.9-fold, p=0.001), and the abundance of pathogenic species such as Clostridium septicum and Streptococcus pneumonia decreased. Higher concentrations of short chain fatty acids (i.e., gut microbial metabolites), including acetic, propionic and butyric acids, were found in faecal samples of the C. crispus-fed rats. Furthermore, both C. crispus and FOS supplemented rats showed significant improvements in proximal colon histo-morphology. Higher faecal moisture was noted in the 2.5% C. crispus group, and elevated plasma immunoglobulin (IgA and IgG) levels were observed in the 0.5% C. crispus group, as compared to the basal feed group. CONCLUSIONS: The results suggest multiple prebiotic effects, such as influencing the composition of gut microbial communities, improvement of gut health and immune modulation in rats supplemented with cultivated C. crispus.

Distinguishing Vaccinium species by chemical fingerprinting based on NMR spectra, validated with spectra collected in different laboratories.

A method was developed to distinguish Vaccinium species based on leaf extracts using nuclear magnetic resonance spectroscopy. Reference spectra were measured on leaf extracts from several species, including lowbush blueberry (Vaccinium angustifolium), oval leaf huckleberry (Vaccinium ovalifolium), and cranberry (Vaccinium macrocarpon). Using principal component analysis, these leaf extracts were resolved in the scores plot. Analysis of variance statistical tests demonstrated that the three groups differ significantly on PC2, establishing that the three species can be distinguished by nuclear magnetic resonance. Soft independent modeling of class analogies models for each species also showed discrimination between species. To demonstrate the robustness of nuclear magnetic resonance spectroscopy for botanical identification, spectra of a sample of lowbush blueberry leaf extract were measured at five different sites, with different field strengths (600 versus 700 MHz), different probe types (cryogenic versus room temperature probes), different sample diameters (1.7 mm versus 5 mm), and different consoles (Avance I versus Avance III). Each laboratory independently demonstrated the linearity of their NMR measurements by acquiring a standard curve for chlorogenic acid (R(2) = 0.9782 to 0.9998). Spectra acquired on different spectrometers at different sites classifed into the expected group for the Vaccinium spp., confirming the utility of the method to distinguish Vaccinium species and demonstrating nuclear magnetic resonance fingerprinting for material validation of a natural health product.

Isolation and structural characterization of unusual pyranoanthocyanins and related anthocyanins from Staghorn sumac (Rhus typhina L.) via UPLC-ESI-MS, (1)H, (13)C, and 2D NMR spectroscopy.

The six major anthocyanins found in the burgundy coloured fruits of Staghorn sumac (Rhus typhina L.) were isolated and the structures of four compounds were determined by NMR spectroscopic methods as being: 7-O-methyl-delphinidin-3-O-(2″galloyl)-ß-d-galactopyranoside; 7-O-methyl-cyanidin-3-O-(2″galloyl)-ß-d-galactopyranoside; 7-O-methyl-delphinidin-3-O-(2″'galloyl)-ß-d-galactopyranoside-4-vinyl-catechol-3″-O-ß-d-glucopyranoside; and 7-O-methyl-cyanidin-3-O-(2″'galloyl)-ß-d-galactopyranoside-4-vinyl-catechol-3″-O-ß-d-glucopyranoside, respectively. Additionally, two related anthocyanin compounds, cyanidin-3-O-(2″galloyl)-ß-d-galactopyranoside and 7-O-methyl-cyanidin-3-O-ß-d-galactopyranoside were also recovered, with NMR spectroscopic values closely matching previous reports from other plant species. The prevalence of 7-O-methyl anthocyanins and their galloylated derivatives in sumac is highly unusual, and warrants special attention. Additionally, the in planta occurrence of two 7-O-methyl-pyranoanothocyanin-vinyl-catechol aglycones, Sumadin A and Sumadin B, and their derivatives is noted. To our knowledge, E-ring glycosylated vinyl-catechol pyranoanthocyanins were previously unknown.

Metal exchange in metallothioneins: a novel structurally significant Cd(5) species in the alpha domain of human metallothionein 1a.

Metallothioneins (MTs) are cysteine-rich, metal-binding proteins known to provide protection against cadmium toxicity in mammals. Metal exchange of Zn(2+) ions for Cd(2+) ions in metallothioneins is a critical process for which no mechanistic or structural information is currently available. The recombinant human alpha domain of metallothionein isoform 1a, which encompasses the metal-binding cysteines between Cys33 and Cys60 of the alpha domain of native human metallothionein 1a, was studied. Characteristically this fragment coordinates four Cd(2+) ions to the 11 cysteinyl sulfurs, and is shown to bind an additional Cd(2+) ion to form a novel Cd(5)alpha-MT species. This species is proposed here to represent an intermediate in the metal-exchange mechanism. The ESI mass spectrum shows the appearance of charge state peaks corresponding to a Cd(5)alpha species following addition of 5.0 molar equivalents of Cd(2+) to a solution of Cd(4)alpha-MT. Significantly, the structurally sensitive CD spectrum shows a sharp monophasic peak at 254 nm for the Cd(5)alpha species in contrast to the derivative-shaped spectrum of the Cd(4)alpha-MT species, with peak maxima at 260 nm (+) and 240 nm (-), indicating Cd-induced disruption of the exciton coupling between the original four Cd(2+) ions in the Cd(4)alpha species. The (113)Cd chemical shift of the fifth Cd(2+) is significantly shielded (approximately 400 p.p.m.) when compared with the data for the Cd(2+) ions in Cd(4)alpha-MT by both direct and indirect (113)Cd NMR spectroscopy. Three of the four original NMR peaks move significantly upon binding the fifth cadmium. Evidence from indirect (1)H-(113)Cd HSQC NMR spectra suggests that the coordination environment of the additional Cd(2+) is not tetrahedral to four thiolates, as is the case with the four Cd(2+) ions in the Cd(4)alpha-MT, but has two thiolate ligands as part of its ligand environment, with additional coordination to either water or anions in solution.

Bymixer system can measure O2 uptake and CO2 elimination in the anesthesia circle circuit.

BACKGROUND: The ability to measure carbon dioxide elimination (Vco(2)), oxygen uptake (Vo(2)), and R (respiratory exchange ratio, Vco(2)/Vo(2)) during anesthesia may help the non-invasive detection of critical events (e.g., abrupt decrease in cardiac output) and metabolic upset (e.g., onset of anaerobic metabolism). METHODS: We have developed a new clinical bymixer (inline mixing chamber) that can measure mixed inspired and expired gas fractions in the anesthesia circle circuit. The addition of a standard anesthesia gas analyzer and flowmeter, and a new airway temperature and humidity sensor, allow determinations of Vco(2) and Vo(2) at the airway opening of the circle circuit. Over a range of tidal volume and frequency, Vco(2) and Vo(2) were compared to reference values generated by the combustion of metered liquid ethanol in a new metabolic lung simulator. RESULTS: By linear regression, bymixer-flow measurements of Vco(2) (slope = 1.02, Y-intercept = -5.31, coefficient of determination, R(2) = 0.998) and Vo(2) (slope = 1.05, Y-intercept = -4.34, R(2) = 0.993) correlated closely to the reference values generated by the metabolic lung simulator. Limits of agreement analysis generated percent errors (mean +/- 1.96 SD) of -1.2 +/- 7.2% for Vco(2) and 2.5 +/- 9.8% for Vo(2). CONCLUSIONS: The new clinical bymixer is compact, lightweight, disposable, inexpensive, and has a fast and adjustable response time (time constant about 14 sec). Anesthesia circle circuit integrity is maintained. Bymixer-flow measurements of Vco(2) and Vo(2) are accurate and may add to clinical monitoring under anesthesia and surgery.

Activation of C-H bonds of arenes: selectivity and reactivity in bis(pyridyl) platinum(II) complexes.

The reaction of [PtMe2(NN)] and B(C6F5)3/H2O in CF3CH2OH with arenes Ar-H gives [PtAr{HOB(C6F5)3}(LL)] if the bis(pyridyl) ligand NN forms a six-membered, but not five-membered, chelate ring; methyl-substituted arenes give selectivity for metalation of meta > para > ortho, but methoxy-substituted arenes give ortho > meta, para.

Tetraphosphinitoresorcinarene complexes: dynamic clusters with silver(I) and copper(I) halides.

Silver(I) and copper(I) halide derivatives of several tetrakis(diphenylphosphinito)resorcinarene ligands are reported. The complexes [resorcinarene(O(2)CR)(4)(OPPh(2))(4)(M(5)X(5))], with resorcinarene = (PhCH(2)CH(2)CHC(6)H(2))(4), R = C(6)H(11), 4-C(6)H(4)Me, C(4)H(3)S, OCH(2)CCH, or OCH(2)Ph, M = Ag, X = Cl, Br, or I, M = Cu, and X = Cl or I, contain a crownlike [P(4)M(5)X(5)] metal halide cluster. These crown clusters were found to be dynamic in solution, as studied by variable-temperature NMR, and easily fragment to give the corresponding complexes containing [P(4)M(4)X(5)](-) and [P(4)M(2)(micro-X)](+) units. Reaction of pentasilver crown clusters with triflic acid gave the corresponding disilver complexes [resorcinarene(O(2)CR)(4)(OPPh(2))(4)]Ag(2)(micro-Cl)]]CF(3)SO(3). Thus, these resorcinarene-based ligands act as a platform for the easy and reversible assembly of copper(I) and silver(I) clusters with novel structures.

Rebaudioside F, a diterpene glycoside from Stevia rebaudiana.

The sweet diterpenoid glycoside, rebaudioside F, was isolated from leaves of a high rebaudioside C producing line of Stevia rebaudiana, and its structure was established by chemical and spectral studies.