Bioactive compounds from Artemisia dracunculus L. activate AMPK signaling in skeletal muscle.

An extract from Artemisia dracunculus L. (termed PMI-5011) improves glucose homeostasis by enhancing insulin action and reducing ectopic lipid accumulation, while increasing fat oxidation in skeletal muscle tissue in obese insulin resistant male mice. A chalcone, DMC-2, in PMI-5011 is the major bioactive that enhances insulin signaling and activation of AKT. However, the mechanism by which PMI-5011 improves lipid metabolism is unknown. AMPK is the cellular energy and metabolic sensor and a key regulator of lipid metabolism in muscle. This study examined PMI-5011 activation of AMPK signaling using murine C2C12 muscle cell culture and skeletal muscle tissue. Findings show that PMI-5011 increases Thr172-phosphorylation of AMPK in muscle cells and skeletal muscle tissue, while hepatic AMPK activation by PMI-5011 was not observed. Increased AMPK activity by PMI-5011 affects downstream signaling of AMPK, resulting in inhibition of ACC and increased SIRT1 protein levels. Selective deletion of DMC-2 from PMI-5011 demonstrates that compounds other than DMC-2 in a "DMC-2 knock out extract" (KOE) are responsible for AMPK activation and its downstream effects. Compared to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, the phytochemical mixture characterizing the KOE appears to more efficiently activate AMPK in muscle cells. KOE-mediated AMPK activation was LKB-1 independent, suggesting KOE does not activate AMPK via LKB-1 stimulation. Through AMPK activation, compounds in PMI-5011 may regulate lipid metabolism in skeletal muscle. Thus, the AMPK-activating potential of the KOE adds therapeutic value to PMI-5011 and its constituents in treating insulin resistance or type 2 diabetes.

Dimeric Proanthocyanidins on the Stability of Dentin and Adhesive Biointerfaces.

A dentin biomodification strategy with selective proanthocyanidin (PAC)-enriched extracts reinforces dentin and dentin-resin interfaces. Enrichment of the extracts according to the degree of polymerization allows exploration of bioactive principles of PACs and structure-activity relationships. This study investigated the sustained dentin matrix biomodification and dentin-resin bioadhesion of 2 fractions consisting exclusively of B-type PAC dimers with or without a single galloyl motif (specifically, DIMERG and DIMERNG) and their precursor material, enriched grape seed extract (e-GSE; Vitis vinifera). The biomodification potential was determined by long-term evaluation of the apparent modulus of elasticity and collagen solubility (hydroxyproline release). Chemical characterization of the dentin matrix was performed by attenuated total reflectance-Fourier-transform infrared spectroscopy. The bioadhesive properties were assessed by a microtensile bond strength test at different time points, and macro-hybrid layers were produced to verify the degree of conversion of the adhesive resin. Fractions consisting of DIMERG, DIMERNG, and their precursor, e-GSE, increased the modulus of elasticity at all time points and reduced collagen degradation. Specimens treated with DIMERNG remained stable throughout 12 mo of storage, whereas a significant drop in the modulus of elasticity was observed for the DIMERG and e-GSE groups at 6 mo. The fractions and precursor did not affect the degree of resin conversion at the hybrid layer. Changes in infrared resonances corresponding to collagen cross-links in the dentin matrix occurred for all treatments. Higher bond strength was observed for dentin treated with e-GSE as compared with DIMERG and DIMERNG; all biointerfaces remained stable after 12 mo. Nongalloylated PACs mediate stable dentin biomodification, which includes protective activity against collagen degradation and reinforcement of the anchoring dentin matrix. Collectively, PACs with a higher degree of oligomerization offer a robust bioadhesion between the hydrophilic dentin matrix and the hydrophobic adhesive.

Biostability of the Proanthocyanidins-Dentin Complex and Adhesion Studies.

Oligomeric proanthocyanidins (OPACs) are potent and renewable natural bioactives possible to be refined into chemically standardized mixtures for biological applications. Herein, we found that multiscale interactions of OPACs with the dentin matrix create tight biointerfaces with hydrophobic methacrylate adhesives on wet surfaces. An enriched mixture of OPACs, with a known phytochemical profile, was produced from grape seed crude extract ( Vitis vinifera; enriched grape seed extract [e-GSE]) and applied to dentin matrices to determine changes to the mechanical properties and biodegradability of the dentin matrix and favorable resin adhesion mechanisms. Methods included a 3-point flexural test, quantification of hydroxyproline (collagen solubilization), static and dynamic nanomechanical analyses, resin-dentin microtensile bond strength, and micropermeability at the adhesive interface. The e-GSE-modified dentin matrix exhibited remarkably low collagen solubilization and sustained the bulk elastic properties over 12 mo. Tan δ findings reveal a more elastic-like behavior of the e-GSE-modified dentin matrix, which was not affected by H-bond destabilization by urea. Dentin-methacrylate biointerfaces with robust and stable adhesion were created on e-GSE-primed dentin surfaces, leading to a dramatic decrease of the interfacial permeability. Standardized OPAC mixtures provide a new mechanism of adhesion to type I collagen-rich tissues that does not rely on hydrophilic monomers. The bioadhesion mechanism involves physicochemical modifications to the dentin matrix, reduced tissue biodegradation, and bridging to methacrylate resins.

Effect of Bioactive Primers on Bacterial-Induced Secondary Caries at the Tooth-Resin Interface.

Secondary caries at the tooth-resin interface is the primary reason for replacement of resin composite restorations. The tooth-resin interface is formed by the interlocking of resin material with hydroxyapatite crystals in enamel and collagen mesh structure in dentin. Efforts to strengthen the tooth-resin interface have identified chemical agents with dentin collagen cross-linking potential and antimicrobial activities. The purpose of the present study was to assess protective effects of bioactive primer against secondary caries development around enamel and dentin margins of class V restorations, using an in vitro bacterial caries model. Class V composite restorations were prepared on 60 bovine teeth (n=15) with pretreatment of the cavity walls with control buffer solution, an enriched fraction of grape seed extract (e-GSE), 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide/N-hydroxysuccinimide, or chlorhexidine digluconate. After incubating specimens in a bacterial model with Streptococcus mutans for four days, dentin and enamel were assessed by fluorescence microscopy. Results revealed that only the naturally occurring product, e-GSE, significantly inhibited the development of secondary caries immediately adjacent to the dentin-resin interface, as indicated by the caries inhibition zone. No inhibitory effects were observed in enamel margins. The results suggest that the incorporation of e-GSE into components of the adhesive system may inhibit secondary caries and potentially contribute to the protection of highly vulnerable dentin-resin margins.

Dentin biomodification potential depends on polyphenol source.

Although proanthocyanidins (PACs) modify dentin, the effectiveness of different PAC sources and the correlation with their specific chemical composition are still unknown. This study describes the chemical profiling of natural PAC-rich extracts from 7 plants using ultra high pressure/performance liquid chromatography (UHPLC) to determine the overall composition of these extracts and, in parallel, comprehensively evaluate their effect on dentin properties. The total polyphenol content of the extracts was determined (as gallic acid equivalents) using Folin-Ciocalteau assays. Dentin biomodification was assessed by the modulus of elasticity, mass change, and resistance to enzymatic biodegradation. Extracts with a high polyphenol and PAC content from Vitis vinifera, Theobroma cacao, Camellia sinensis, and Pinus massoniana induced a significant increase in modulus of elasticity and mass. The UHPLC analysis showed the presence of multiple types of polyphenols, ranging from simple phenolic acids to oligomeric PACs and highly condensed tannins. Protective effect against enzymatic degradation was observed for all experimental groups; however, statistically significant differences were observed between plant extracts. The findings provide clear evidence that the dentin bioactivities of PACs are source dependent, resulting from a combination of concentration and specific chemical constitution of the complex PAC mixtures.

Pharmacokinetics of 23-epi-26-deoxyactein in women after oral administration of a standardized extract of black cohosh.

Dietary supplements containing black cohosh are alternatives to conventional hormone replacement therapy in menopause. This study investigates the maximum tolerated dose of a 75% ethanol extract of black cohosh and determines the pharmacokinetics of one of its most abundant triterpene glycosides, 23-epi-26-deoxyactein. Single doses of black cohosh extract containing 1.4, 2.8, or 5.6 mg of 23-epi-26-deoxyactein were administered to 15 healthy, menopausal women. Serial blood samples and 24-h urine samples were obtained; blood chemistry, hormonal levels, and 23-epi-26-deoxyactein levels were determined. No acute toxicity or estrogenic hormone effects were observed. Pharmacokinetic analyses of 23-epi-26-deoxyactein in sera indicated that the maximum concentration and area under the curve increased proportionately with dosage, and that the half-life was ~2 h for all dosages. Less than 0.01% of the 23-epi-26-deoxyactein was recovered in urine 24 h after administration. No phase I or phase II metabolites were observed either in clinical specimens or in vitro.

Binary concepts and standardization in counter-current separation technology.

Counter-current separation (CS) technology is currently faced with the challenge of being fit for the purpose of omics analysis, which involves highly complex samples and digitized research environments. Resembling a network of binary decisions, CS requires standardization of operation parameters in order to be efficient. While recent CS engineering solutions uniformly involve centrifugal force designs to overcome the limitation of the earth's 1xg force, factors of instrument design, operation, and graphical representation of the outcome are equally important targets for standardization. For example, chromatograms that emphasize the unique K-based nature of CS, such as reciprocal symmetry (ReS) plots, foster the fundamental understanding of CS operation. Because significant differences exist in underlying mechanism (e.g., stationary phase volume), outcome (e.g., construction of chromatograms), and scale (e.g., factors affecting overall method sensitivity) of solid-liquid vs. liquid-liquid chromatography technologies, standardization will enable the systematic exploration of the differential properties of the two LC technologies, and will be key to making CS fit for the digital omics age.

The vasodepressor function of the kidney: prostaglandin E2 is not the principal vasodepressor lipid of the renal medulla.

AIM: Whereas prostaglandin E2 has been characterized as the principal vasodepressor lipid, medullipin remains a hypothetical vasodepressor principle of the renal medulla. Representing the first step towards the isolation of medullipin as a pure compound, the aim of the present study was to determine whether or not the known vasodilator and antihypertensive action of prostaglandins play a role in the antihypertensive activity of renal medulla. METHODS: A chloroform extract of porcine kidney medulla was fractionated by gradient vacuum liquid chromatography (VLC) and analysed by capillary GC-MS for the presence of prostaglandins (detection limit: 2.2 ppm). The biological activity was determined in spontaneously hypertensive Wistar rats. The particle size of injectable colloids prepared from extract and fractions was controlled by photon correlation spectroscopy. RESULTS: The extract caused a pronounced blood pressure decline (29.6 +/- 6.3/24.9+/- 5.5 mmHg; P = 0.0078; 10 mg kg(-1) body weight; particle size of 143 +/- 18 nm; n = 7) lasting for more than 1 h. The heart rate remained stable, showing only a slightly decrease. All fractions were shown to be devoid of vasodilator prostanoid substances. The VLC procedure allowed the successful separation of endogenous emulsifiers from the active principle. An extract from the renal cortex did not exhibit a similar vasodepressor effect. CONCLUSION: Prostaglandins are excluded as the blood pressure-lowering active principle of a total lipid kidney medulla extract. The vasodepressor principle is contained in the kidney medulla, but not in the cortex. It can be separated from endogenous emulsifying substances, is chromatographically stable, and is amenable to purification and chemical characterization.

Development of an extraction method for mycobacterial metabolome analysis.

As a prerequisite for studying the intracellular metabolome of mycobacteria, several methods were evaluated for efficient breakage of the cell using Mycobacterium bovis (BCG) as a model microorganism. Several pulping methods, treating with an Ultra-Turax, deep-freezing in liquid nitrogen followed by mechanical grinding, sonicating with probe head or cup horn and bead beating prior to solvent extraction were applied and compared. Gravimetry, electron microscopy, and nuclear magnetic resonance spectrometry were used to analyze the extracts. All analytical methods prove that sonicating is superior to mechanical grinding of deep-frozen cells. Two methods indicated that sonicating with a probe head enhances the efficiency of cell disruption compared to sonicating with a cup horn. The highest extract yield and chemical diversity were achieved by a combination of mechanical grinding and sonicating. Within the scope of a metabolomic analysis, the method of choice to treat mycobacterial cells is a combination of deep-freezing in liquid nitrogen and mechanical grinding followed by sonicating with a probe head.

The pharmacognosy of Humulus lupulus L. (hops) with an emphasis on estrogenic properties.

As the population ages, there is an ever-increasing need for therapeutic agents that can be used safely and efficaciously to manage symptoms related to postmenopausal estrogen deficiency. Endogenous estrogens, e.g., 17beta-estradiol, of exogenous mammalian origin, e.g., horses, have long been used to manage such symptoms. There are more than 20 different classes of phytochemicals that have demonstrated affinity for human estrogen receptors in vitro. Some studies on exogenous estrogenic substances of botanical origin (phytoestrogens), such as standardized formulations of plant extracts with in vitro and in vivo estrogenic activity from soy (Glycine max Merill.) and red clover (Trifolium pratense L.), suggest clinical efficacy. Few clinical data for phytoestrogens other than isoflavonoids are available. In an exhaustive review of the literature through 2003, only two clinical trials were identified that were designed to evaluate the effect of hops (Humulus lupulus L.) on symptoms related to menopause. Folkloric, chemical, and biological literature relating primarily to the use of hops for their estrogenic activity, and two human clinical trials, are reviewed.

qNMR--a versatile concept for the validation of natural product reference compounds.

The validation of reference compounds for natural products is a domain of the same physico-chemical methods that are used for their isolation, especially those techniques involving coupled high-resolution chromatography. Acknowledging the great problem of co-eluting impurities contained in the 'biogenetic cocktail' of a plant extract, there is a strong demand for non-chromatographic alternatives in the quality assessment of reference compounds. Because of this, the concept of qNMR is introduced as a versatile tool based on qualitative and quantitative 1H-NMR allowing the precise and simultaneous determination of both the compound content as well as the amount and nature of the impurities. As opposed to measuring carbons, 1H-NMR benefits from much higher sensitivity and is far more versatile for routine analysis with respect to time and cost. Since quantification of impurities is reliant upon their identification and, therefore, limited by knowledge about their structure, the concept emphasizes the high demand for qualitative reference dossiers including quality NMR data for profiling potential impurities which may be analogues, isomers, or degradation or oxidation products of the reference compounds. The qNMR concept is developed with focus on its potential in the certification and quality control of reference compounds. Taking into account published work in the field of quantitative NMR, selected natural products are analysed in order to elaborate suitable experimental parameters and to obtain preliminary validation data. The method is discussed with respect to sensitivity, precision and selectivity. Typical relative errors are found to be below 2% for the quantification of both the major analyte and the minor impurities even when the latter are contained at the 1% level only. Documentation of the conformity of signal integration and precision is based on measurements of a certified reference standard. Determination of the natural 13C isotope is suggested as an elegant method of validation because the content values could be reproduced with errors below 1%. The qNMR concept offers a rapid and efficient way to assess the purity of natural products in a single analytical step without the need of performing multiple analyses, while still offering the option to retain the substance. Thus, qNMR pays tribute to the increasing demands in reference compound certification, but also holds out the prospect of easy access to the valid characterisation of natural products tested in vitro or in vivo for their biological and pharmacological effects.

alpha-Onocerin chloroform hemisolvate.

The triterpenoid natural product alpha-onocerin [8, 14-secogammacera-8(26),14(27)-diene-3,21-diol], determined here as the chloroform hemisolvate, C(30)H(50)O(2).0.5CHCl(3), consists of two independent symmetric trans-decalin C(15) building blocks. Hydrogen bonds between the hydroxyl groups form an infinite two-dimensional network perpendicular to the c axis.

Higher order and substituent chemical shift effects in the proton NMR of glycosides.

The full analysis of (1)H NMR spin systems and the charting of substituent chemical shifts offer great potential in the structure elucidation of glycosides. Due to the chiral nature of sugar residues, asymmetric induction causes diastereotopic shifts of aglycon resonance, while on the other hand aromatic substituent-induced shifts affect the sugar portion of the molecule. Therefore, it is possible to deduce critical structural information, such as the site of sugar linkage in bisdesmosides and oligoglycosides, through the analysis of exact chemical shifts, J patterns, and signal multiplicity. The potential of the method is demonstrated for three kaempferol bisdesmosides (1-3) by establishing the shift characteristics for 3-O-, 7-O-, and 6' '-O-glycosidation. Spectral simulation and soft-pulse 1D NMR turn out to be indispensable tools in the course of refined signal and structure assignment.

Comprehensive spectroscopic investigation of alpha-onocerin.

A detailed NMR and MS analysis of alpha-onocerin was performed in order to provide precise phytochemical reference for structure identification and quality control of this analytical lead compound from Ononis spinosa the roots of which are widely used as a diuretic drug. Unambiguous 1H- and 13C-NMR assignments revealed that the linkage of two subunits were confirmed to possess identical stereochemistries in this triterpenoid.

Major flavonoids from Arabidopsis thaliana leaves.

The three major flavonoids isolated from Arabidopsis thaliana plants grown in the greenhouse were identified by means of spectroscopic analysis (UV, NMR, MS) and chiral capillary zone electrophoresis as the novel kaempferol 3-O-beta-[beta-D-glucopyranosyl(1-->6)D-glucopyranoside]-7-O-alpha-L- rhamnopyranoside (1), kaempferol 3-O-beta-D-glucopyranoside-7-O-alpha-L-rhamnopyranoside (2), and kaempferol 3-O-alpha-L-rhamnopyranoside-7-O-alpha-L-rhamnopyranoside (3). Comprehensive NMR studies including selective 1D and gradient-enhanced 2D techniques were applied in order to achieve full signal assignment and definitive proof of linkage for compound 1.

Fukiic and piscidic acid esters from the rhizome of Cimicifuga racemosa and the in vitro estrogenic activity of fukinolic acid.

Hydroxycinnamic acid esters of fukiic acid and piscidic acid were isolated from a 50% ethanolic extract obtained from the rhizomes of Cimicifuga racemosa (Ranunculaceae). Besides 2-E-caffeoylfukiic acid (fukinolic acid), 2-E-feruloylfukiic acid (cimicifugic acid A), 2-E-isoferuloylfukiic acid (cimicifugic acid B), 2-E-feruloylpiscidic acid (cimicifugic acid E) and 2-E-isoferuloylpiscidic acid (cimicifugic acid F), free caffeic, ferulic and isoferulic acids were isolated. The estrogenic activity of fukinolic acid was shown by increased proliferation (126% at 5 x 10(-8) M) of an estrogen dependent MCF-7 cell system with reference to estradiol (120% at 10(-10) M).

The cardenolides of Speirantha convallarioides.

The cardenolide pattern of Speirantha convallarioides has been investigated and compared with Convallaria majalis, a close relative with active cardenolides and used extensively as a cardiac remedy. Five cardenolides were isolated from small amounts (25 g) of plant material by application of an efficient isolation procedure. Their identification was achieved by an easy concept mainly based on detailed analysis of 1H-NMR and DCI-NH3-mass spectral data. Rhodexin A(1) and lokundjoside (2) are the major cardenolides (42% and 31% of the total cardenolides in the plant), while digitoxigenin-glucoside (3), periplorhamnoside (4), and periplogenin-glucoside (5) are minor constituents of the cardenolide complex (3 to 4% each). Similarities in cardenolide patterns (structures of the aglycones, variability of the sugar portion) along with morphological and biogenetical characteristics support the classification of S. convallarioides as belonging in the same tribe Convallarieae as C. majalis within the family Convallariaceae.

Adoligoses, oligosaccharides of rare sugars from Adonis aleppica.

Five novel tri-, tetra-, and penta-saccharides named adoligoses A-E [1-5], consisting of rare didesoxy sugars and their 3-O-Me ethers, have been isolated from Adonis aleppica. Their structures were determined by dcims and nmr measurements. Full establishment of the proton spin-systems was based on 2D nmr data, spectral simulation procedures, and force-field calculations. Sarmentose and cymarose moieties were found in both anomeric configurations and represent a major difference from alepposides A-D, biogenetically related cardenolide oligoglycosides isolated previously from the same plant source. This is the first report on complex, long-chained oligosaccharides of 2,6-didesoxy sugars found typically in cardenolide and pregnane glycosides.

Alepposides, cardenolide oligoglycosides from Adonis aleppica.

The structures of novel oligoglycosidic cardenolides, alepposide A (C55H86O23) [1] and alepposide B (C48H74O20) [2], have been deduced mainly by nmr methods. Based on homonuclear (1H and 13C nmr, 1H COSY) and proton-detected heteronuclear shift correlation experiments [HMQC both for 1J(C,H) and for long-range couplings], alepposide A [1] was shown to have the structure strophanthidin-3-O-beta-glucopyranosyl-(1-->4)-O-beta-diginopyranosyl -(1-->4)-O - beta-oleandropyranosyl-(1-->4)-O-beta-digitoxopyranosyl-(1-- >4)-O-beta- digitoxopyranoside. The structure of alepposide B [2] was established as strophanthidin-3-O-beta-glucopyranosyl-(1-->4)-O-beta-oleandropyranos yl- (1-->4)-O-beta-digitoxopyranosyl-(1-->4)-O-beta-digitoxopyranos ide.