Protective effect of ginsenosides Rk3 and Rh4 on cisplatin-induced acute kidney injury in vitro and in vivo.

BACKGROUND: Nephrotoxicity is the major side effect in cisplatin chemotherapy. Previously, we reported that the ginsenosides Rk3 and Rh4 reduced cisplatin toxicity on porcine renal proximal epithelial tubular cells (LLC-PK1). Here, we aimed to evaluate the protective effect of ginsenosides Rk3 and Rh4 on kidney function and elucidate their antioxidant effect using in vitro and in vivo models of cisplatin-induced acute renal failure. METHODS: An enriched mixture of ginsenosides Rk3 and Rh4 (KG-KH; 49.3% and 43.1%, respectively) was purified from sun ginseng (heat processed Panax ginseng). Cytotoxicity was induced by treatment of 20µM cisplatin to LLC-PK1 cells and rat model of acute renal failure was generated by single intraperitoneal injection of 5 mg/kg cisplatin. Protective effects were assessed by determining cell viability, reactive oxygen species generation, blood urea nitrogen, serum creatinine, antioxidant enzyme activity, and histopathological examination. RESULTS: The in vitro assay demonstrated that KG-KH (50 µg/mL) significantly increased cell viability (4.6-fold), superoxide dismutase activity (2.8-fold), and glutathione reductase activity (1.5-fold), but reduced reactive oxygen species generation (56%) compared to cisplatin control cells. KG-KH (6 mg/kg, per os) also significantly inhibited renal edema (87% kidney index) and dysfunction (71.4% blood urea nitrogen, 67.4% creatinine) compared to cisplatin control rats. Of note, KG-KH significantly recovered the kidney levels of catalase (1.2-fold) and superoxide dismutase (1.5-fold). CONCLUSION: Considering the oxidative injury as an early trigger of cisplatin nephrotoxicity, our findings suggest that ginsenosides Rk3 and Rh4 protect the kidney from cisplatin-induced oxidative injury and help to recover renal function by restoring intrinsic antioxidant defenses.

Solid-phase extraction assisted dispersive liquid-liquid microextraction based on solidification of floating organic droplet to determine sildenafil and its analogues in dietary supplements.

A novel analytical method for the simultaneous determination of the concentration of sildenafil and its five analogues in dietary supplements using solid-phase extraction assisted reversed-phase dispersive liquid-liquid microextraction based on solidification of floating organic droplet combined with ion-pairing liquid chromatography with an ultraviolet detector was developed. Parameters that affect extraction efficiency were systematically investigated, including the type of solid-phase extraction cartridge, pH of the extraction environment, and the type and volume of extraction and dispersive solvent. The method linearity was in the range of 5.0-100 ng/mL for sildenafil, homosildenafil, udenafil, benzylsildenafil, and thiosildenafil and 10-100 ng/mL for acetildenafil. The coefficients of determination were ≥0.996 for all regression curves. The sensitivity values expressed as limit of detection were between 2.5 and 7.5 ng/mL. Furthermore, intraday and interday precisions expressed as relative standard deviations were less than 5.7 and 9.9%, respectively. The proposed method was successfully applied to the analysis of sildenafil and its five analogues in complex dietary supplements.

Simultaneous Determination of Volatile Organic Compounds in Commercial Alcoholic Beverages by Gas Chromatography with Flame Ionization Detection.

A simple and fast method was developed for the determination of volatile organic compounds in alcoholic beverages. Eleven volatile organic compounds (acetaldehyde, methanol, 2-propanol, tert-butanol, 1-propanol, ethyl acetate, 2-butanol, isobutanol, 1-butanol, 3-methyl-1butanol, and 2-methyl-1-butanol) in alcoholic beverages were analyzed with a simple direct-injection method using GC with flame ionization detection. These compounds should be monitored in the QC of production processes because they are detrimental to human health. The method was validated with four types of alcoholic beverages (beers, fruit wines, rice wines, and spirits) to confirm the versatility of the method. Linearity showed r2 values from 0.9986 to 0.9995, with LODs ranging from 0.010 to 1.000 mg/L. Precision and accuracy showed acceptable results, proving the effectiveness of the method. The developed method was applied to 40 commercial samples representing the four types of alcoholic beverages, and principal component analysis was performed to determine profiles of the volatile organic compounds, depending on the type of alcoholic beverage.

Systematic development of a group quantification method using evaporative light scattering detector for relative quantification of ginsenosides in ginseng products.

The determination for the contents of multi-components in ginseng products has come to the fore by demands of in-depth information, but the associated industries confront the high cost of securing pure standards for the continuous quality evaluation of the products. This study aimed to develop a prospective high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method for relative quantification of ginsenosides in ginseng products without a considerable change from the conventional gradient analysis. We investigated the effects of mobile phase composition and elution bandwidth, which are potential variables affecting the ELSD response in the gradient analysis. Similar ELSD response curves of nine major ginsenosides were obtained under the identical flow injection conditions, and the response increased as the percentage of organic solvent increased. The nine ginsenosides were divided into three groups to confirm the effect of elution bandwidth. The ELSD response significantly decreased in case of the late eluted ginsenoside in the individual groups under the isocratic conditions. With the consideration of the two important effects, stepwise changes of the gradient condition were carried out to reach a group quantification method. The inconsistent responses of the nine ginsenosides were reconstituted to three normalized responses by the stepwise changes of the gradient condition, and this result actualized relative quantification in the individual groups. The availability was confirmed by comparing the ginsenoside contents in a base material of ginseng products determined by the direct and group quantification method. The largest difference in the determination results from the two methods was 8.26%, and the difference of total contents was only 0.91%.

UPLC-QTOFMS based metabolomics followed by stepwise partial least square-discriminant analysis (PLS-DA) explore the possible relation between the variations in secondary metabolites and the phylogenetic divergences of the genus Panax.

Phylogenetic and metabolomic approaches have long been employed to study evolutionary relationships among plants. Nonetheless, few studies have examined the difference in metabolites within a clade and between clades of the phylogenetic tree. We attempted to relate phylogenetic studies to metabolomics using stepwise partial least squares-discriminant analysis (PLS-DA) for the genus Panax. Samples were analyzed by ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QTOFMS) to obtain metabolite profiles. Initially, conventional principal component analysis was subsequently applied to the metabolomic data to show the limitations in relating the expression of metabolites to divisions in the phylogenetic tree. Thereafter, we introduced stepwise PLS-DA with optimized scaling methods, which were properly applied according to the branches of the phylogenetic tree of the four species. Our approach highlighted metabolites of interest by elucidating the directions and degrees of metabolic alterations in each clade of the phylogenetic tree. The results revealed the relationship between metabolic changes in the genus Panax and its species' evolutionary adaptations to different climates. We believe our method will be useful to help understand the metabolite-evolution relationship.

Expeditious discrimination of four species of the Panax genus using direct infusion-MS/MS combined with multivariate statistical analysis.

A practical approach based on direct infusion-MS/MS (DI-MS/MS) was demonstrated for metabolomic classification of four species in the Panax genus. The species Panax ginseng, Panax notoginseng, Panax quinquefolius and Panax vietnamensis were analyzed to develop an efficient tool for authenticating ginseng. Four target ions (m/z 783.5, 945.5, 1107.5 and 1149.2) were selected from LC-MS screening results for DI-MS/MS analysis. The target ions served as classifiers of the four species. As a targeted analysis, DI-MS/MS provided the structural identities of the target ions, clear spectral data and high sensitivity in a shorter time than routine LC-MS analysis. Principal component analysis and partial least squares-discriminant analysis of the DI-MS/MS fingerprinting revealed distinct grouping of the data. The results were validated by cross-validation and a permutation test to examine the utility of the statistical models. The spectral intensities of each species were compared with one another using box plots, which allowed straightforward authentication of the Panax species. The proposed method showed improved efficiency over other current methods for discrimination of large quantities of plant material. Additionally, to the best of our knowledge, this is the first study in which DI-MS/MS has been used to classify plant samples.

Conformational studies of dammarane-type triterpenoids using computational and NMR spectroscopic methods.

Natural triterpenoids are of great interest to researchers of various fields as they possess diverse physicochemical and biological properties. In medicinal chemistry, detailed information about the chemical structures of bioactive triterpenoids often helps find new lead compounds. Herein, the low-energy structures of (20S)-protopanaxadiol and (20S)-protopanaxatriol, the aglycones of various triterpenoid saponins found in Panax ginseng, and their (20R)-epimers have been predicted by the geometry optimization of the conformers extracted from molecular dynamics simulations with the self-consistent-charge density functional tight-binding method. By performing quantum mechanical calculations on the low-energy conformers, we have estimated the NMR chemical shifts of the compounds, which display good agreement with the most recently reported experimental values within an expected range of errors. Our results indicate that theoretical estimation of the NMR parameters of a relatively large molecule with a molecular mass of 500 is feasible.

A simple double quantum coherence ESR sequence that minimizes nuclear modulations in Cu(2+)-ion based distance measurements.

Double quantum coherence (DQC) ESR is a sensitive method to measure magnetic dipolar interactions between spin labels. However, the DQC experiment on Cu(2+) centers presents a challenge at X-band. The Cu(2+) centers are usually coordinated to histidine residues in proteins. The electron-nuclear interaction between the Cu(2+) ion and the remote nitrogen in the imidazole ring can interfere with the electron-electron dipolar interaction. Herein, we report on a modified DQC experiment that has the advantage of reduced contributions from electron-nuclear interactions, which enhances the resolution of the DQC signal to the electron-electron dipolar modulations. The modified pulse-sequence is verified on Cu(2+)-NO system in a polyalanine-based peptide and on a coupled Cu(2+) system in a polyproline-based peptide. The modified DQC data were compared with the DEER data and good agreement was found.

A new naphthalene glycoside from Chimaphila umbellata inhibits the RANKL-stimulated osteoclast differentiation.

A new naphthalene glycoside was isolated from the leaves and stems of Chimaphila umbellata Barton. Its chemical structure was elucidated to be 2,7-dimethyl-1,4-dihydroxynaphthalene-1-O-ß-D-glucopyranoside (DMDHNG), based on spectroscopic evidence. DMDHNG significantly inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP) activity and the formation of multinucleated osteoclasts in a dose-dependent manner. In addition, the new glycoside inhibited the RANKL-induced mRNA expression of osteoclast-associated genes that encode TRAP, cathepsin K, and another transcription factor-nuclear factor of activated T-cells c1. We believe that the inhibitory effects of DMDHNG on the osteoclast differentiation may be exploited for a therapeutic benefit.

Chemical diversity of ginseng saponins from Panax ginseng.

Ginseng, a perennial plant belonging to the genus Panax of the Araliaceae family, is well known for its medicinal properties that help alleviate pathological symptoms, promote health, and prevent potential diseases. Among the active ingredients of ginseng are saponins, most of which are glycosides of triterpenoid aglycones. So far, numerous saponins have been reported as components of Panax ginseng, also known as Korean ginseng. Herein, we summarize available information about 112 saponins related to P. ginseng; >80 of them are isolated from raw or processed ginseng, and the others are acid/base hydrolysates, semisynthetic saponins, or metabolites.

A simple quasi-analytical method for the deconvolution of Voigtian profiles.

In electron spin resonance spectroscopy, spectral lineshapes are often assumed to be Voigtian. A number of researchers have suggested ways to approximate the Voigtian profile. Herein, we have devised a new quasi-analytical method to deconvolve it. In particular, we have derived an equation that relates the Lorentzian-to-Gaussian linewidth ratio directly to the product of the linewidth and the maximum value of a normalized Voigtian profile. Our calculations show that the Lorentzian and Gaussian linewidths obtained by the quasi-analytical deconvolution of computer-generated Voigtian absorption spectra are accurate within an error range of 1% in the absence of noise. Also, simulations with noise-added spectra reveal that, in the presence of white noise, our method is valid to a certain extent that depends on several factors such as the number of data points and the spectral sweep width. The new deconvolution method is valuable in that it estimates the Lorentzian and Gaussian linewidth in a rapid manner. The method may be also useful in other fields of science, such as optical spectroscopy, especially if some a priori knowledge about the lineshape is given.

Insight into potential Cu(II)-binding motifs in the four pseudorepeats of tau protein.

Tau protein and Cu(II) are believed to be associated with the pathogenesis of Alzheimer's disease. However, little is known about atomic-level interactions between tau protein and Cu(II). Herein, we suggest, on the basis of electron spin resonance (ESR) data, that the four pseudorepeats of tau protein in the microtubule-binding region play an important role in Cu(II) binding. We use a number of tau protein fragments in order to examine Cu(II)-binding site(s) and binding affinities. Continuous-wave (CW) ESR experiments on the four highly conserved octadecapeptides, each of which is a segment of one of the four pseudorepeats, reveal that the equimolar Cu(II) complexes of the four octadecapeptides are similar to one another in terms of the coordination environment and binding affinity. The spectra obtained with pulsed ESR techniques such as electron spin-echo envelope modulation and hyperfine sublevel correlation provide direct evidence that a histidine residue and a backbone amide group coordinate to Cu(II) in each Cu(II)-octadecapeptide complex. The results of CW and pulsed ESR experiments on some chemically modified peptides indicate that the cysteine residues in the second and third pseudorepeats are unlikely to be involved in Cu(II) binding. On the other hand, similar experiments on tau fragments of the second pseudorepeat with different lengths lead to the conclusion that the affinity for Cu(II) decreases as the octadecapeptide is either truncated or elongated. The high Cu(II)-binding affinity of the octadecapeptide is presumably due to the N-terminal amino group stabilizing the Cu(II)-octadecapeptide complex. Finally, the ESR data for a longer tau fragment that contains two octadecapeptides suggest that the Cu(II) binding site(s) of even longer fragments of tau protein is similar to that of a single octadecapeptide.

Substantial contribution of the two imidazole rings of the His13-His14 dyad to Cu(II) binding in amyloid-ß(1-16) at physiological pH and its significance.

The interaction of amyloid-ß (Aß) peptide with Cu(II) appears to play an important role in the etiology of Alzheimer's disease. At physiological pH, the Cu(II) coordination in Aß is heterogeneous, and there exist at least two binding modes in which Cu(II) is coordinated by histidine residues. Electron spin resonance studies have revealed a picture of the Cu(II) binding at a higher or lower pH, where only one of the two binding modes is almost exclusively present. We describe a procedure to directly examine the coordination of Cu(II) to each histidine residue in the dominant binding mode at physiological pH. We use nonlabeled and residue-specifically (15)N-labeled Aß(1-16). For quantitative analysis, the intensities of three-pulse electron spin-echo envelope modulation (ESEEM) spectra are analyzed. Spectral simulations show that ESEEM intensities provide information about the contribution of each histidine residue. Indeed, the ESEEM experiments at pH 6.0 confirm the dominant contribution of His6 to the Cu(II) coordination as expected from the work of other researchers. Interestingly, however, the ESEEM data obtained at pH 7.4 reveal that the contributions of the three residues to the Cu(II) coordination are in the order of His14 ≈ His6 > His13 in the dominant binding mode. The order indicates a significant contribution from the simultaneous coordination by His13 and His14 at physiological pH, which has been underappreciated. These findings are supported by hyperfine sublevel correlation spectroscopy experiments. The simultaneous coordination by the two adjacent residues is likely to be present in a non-ß-sheet structure. The coexistence of different secondary structures is possibly the molecular origin for the formation of amorphous aggregates rather than fibrils at relatively high concentrations of Cu(II). Through our approach, precise and useful information about Cu(II) binding in Aß(1-16) at physiological pH is obtained without any side-chain modification, amino acid residue replacement, or pH change, each of which might lead to an alteration in the peptide structure or the coordination environment.

The second Cu(II)-binding site in a proton-rich environment interferes with the aggregation of amyloid-beta(1-40) into amyloid fibrils.

The overall morphology and Cu(II) ion coordination for the aggregated amyloid-beta(1-40) [Abeta(1-40)] in N-ethylmorpholine (NEM) buffer are affected by Cu(II) ion concentration. This effect is investigated by transmission electron microscopy (TEM), atomic force microscopy (AFM), and electron spin echo envelope modulation (ESEEM) spectroscopy. At lower than equimolar concentrations of Cu(II) ions, fibrillar aggregates of Abeta(1-40) are observed. At these concentrations of Cu(II), the monomeric and fibrillar Abeta(1-40) ESEEM data indicate that the Cu(II) ion is coordinated by histidine residues. For aggregated Abeta(1-40) at a Cu(II):Abeta molar ratio of 2:1, TEM and AFM images show both linear fibrils and granular amorphous aggregates. The ESEEM spectra show that the multi-histidine coordination for Cu(II) ion partially breaks up and becomes exposed to water or exchangeable protons of the peptide at a higher Cu(II) concentration. Since the continuous-wave electron spin resonance results also suggest two copper-binding sites in Abeta(1-40), the proton ESEEM peak may arise from the second copper-binding site, which may be significantly involved in the formation of granular amorphous aggregates. Thioflavin T fluorescence and circular dichroism experiments also show that Cu(II) inhibits the formation of fibrils and induces a nonfibrillar beta-sheet conformation. Therefore, we propose that Abeta(1-40) has a second copper-binding site in a proton-rich environment and the second binding Cu(II) ion interferes with a conformational transition into amyloid fibrils, inducing the formation of granular amorphous aggregates.

An ESR analysis of the mechanism of pericyclic reactions of bicyclobutane.

Experimental and simulated ESR data are in good agreement with a biradical mechanism for the intramolecular pericyclic reactions of bicyclo[1.1.0]butanes.

Direct evidence that all three histidine residues coordinate to Cu(II) in amyloid-beta1-16.

We provide direct evidence that all three histidine residues in amyloid-beta 1-16 (Abeta 1-16) coordinate to Cu(II). In our approach, we generate Abeta 1-16 analogues, in each of which a selected histidine residue is isotopically enriched with (15)N. Pulsed electron spin resonance (ESR) experiments such as electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectroscopy clearly show that all three histidine imidazole rings at positions 6, 13 and 14 in Abeta 1-16 bind to Cu(II). The method employed here does not require either chemical side chain modification or amino acid residue replacement, each of which is traditionally used to determine whether an amino acid residue in a protein binds to a metal ion. We find that the histidine coordination in the Abeta 1-16 peptide is independent of the Cu(II)-to-peptide ratio, which is in contrast to the Abeta 1-40 peptide. The ESR results also suggest tight binding between the histidine residues and the Cu(II) ion, which is likely the reason for the high binding affinity of the Abeta peptide for Cu(II).