Samnamul (Shoots of Aruncus dioicus) Inhibit Adipogenesis by Downregulating Adipocyte-Specific Transcription Factors in 3T3-L1 Adipocytes

Adipocyte-specific transcription factors and antioxidants are considered the best target of obesity. Aruncus dioicus var. kamtschaticus (A. dioicus, Samnamul) is easily available owing to edible and inexpensive. However, the anti-adipogenic effects of the underlying mechanism of A. dioicus extract (ADE) have not yet been reported. In the present study, we evaluate anti-adipogenic pathway in 3T3-L1 adipocytes, antioxidant activities and quantified phenolics using high-performance liquid chromatography of ADE. The results revealed ADE had reduced adipocyte differentiation (0.72-fold vs. MDI (media of differentiation) control), triglyceride (TG; 0.50-fold vs. MDI control, p < 0.001), and total cholesterol contents (0.77-fold vs. MDI control) by regulating adipocyte-specific transcription factors (C/EBPα, PPARγ, and SREBP1) and their downstream mRNA (AdipoQ, Ap2, SREBP1-c, and FAS) levels. Furthermore, ADE has higher total phenol and flavonoid contents and scavenging assay in the DPPH and ABTS+. In particularly, ADE contains chlorogenic acid (7.04 mg/kg), caffeic acid (20.14 mg/kg), ferulic acid (1.74 mg/kg), veratric acid (29.31 mg/kg), cinnamic acid (4.70 mg/kg), and quercetin (4.18 mg/kg). In conclusion, since these phenols, especially quercetin, in the ADE appear to reduce differentiation, TG and cholesterol content by regulating adipocyte-specific transcription factors in adipocytes, ADE has the potential to be developed into a new antioxidant and anti-obesity therapeutics.

Bioanalytical Method Development and Validation of Veratraldehyde and Its Metabolite Veratric Acid in Rat Plasma: An Application for a Pharmacokinetic Study

A simple, sensitive, and rapid UHPLC-MS/MS method was developed for the simultaneous determination of veratraldehyde and its metabolite veratric acid in rat plasma. Cinnamaldehyde was used as an internal standard (IS) and the one-step protein precipitation method with 0.2% formic acid in acetonitrile (mobile phase B) was used for the sample extraction. Reversed C18 column (YMC-Triart C18 column, 50 mm × 2.0 mm, 1.9 µm) was used for chromatographic separation and was maintained at 30 °C. The total run time was 4.5 min and the electrospray ionization in positive mode was used with the transition m/z 167.07 → 139.00 for veratraldehyde, m/z 183.07 → 139.00 for veratric acid, and m/z 133.00 → 55.00 for IS. The developed method exhibited good linearity (r2 ≥ 0.9977), and the lower limits of quantification ranged from 3 to 10 ng/mL for the two analytes. Intra-day precision and accuracy parameters met the criteria (within ±15%) during the validation. The bioanalytical method was applied for the determination of veratraldehyde and veratric acid in rat plasma after oral and percutaneous administration of 300 and 600 mg/kg veratraldehyde. Using the analytical methods established in this study, we can confirm the absorption and metabolism of veratraldehyde in rats for various routes.

MASS SPECTROMETRIC CHARACTERIZATION OF PLASMA MEBEVERINE METABOLITES AND ITS SYNTHESIS

Mebeverine is a musculotropic antispasmodic drug that is widely used in the treatment of irritable bowel syndrome. As an ester of mebeverine alcohol and veratric acid, mebeverin is quickly metabolized and is practically undetectable in blood plasma. The main goal of this work was establishing the structure of the main metabolites of mebeverin in human blood plasma. The study was conducted by time-of-flight mass spectrometry (LC-IT-TOF MS), metabolites of mebeverine were extracted from plasma with acetonitrile. When comparing chromatograms of blood plasma obtained before and after drug administration, four main peaks of metabolites were detected. To establish the structure of the compounds, mass spectra of the first and second order were taken. The first-order spectra were used to calculate the metabolite formula and the structure was determined from the fragmentation spectra, as well as by comparing the fragmentation spectra of mebeverine and its proposed metabolites. The proposed compounds were synthesized, and their structure was confirmed using NMR and chromatography-mass spectrometry. Four main metabolites were found in this study: desmethylmebeverine acid (DMAC), glucuronide product of DMAC (DMAC-Glu), mebeverine acid (MAC) and mebeverine alcohol (MAL). The results complement the available literature data about the veratric acid metabolism, urine, and microsomal studies. According to the data obtained, the main metabolite of mebeverine in the blood is DMAC. The concentration of MAC after mebeverine administration is almost ten times less than DMAC, the content of MAL and DMAC-Glu is insignificant, and probably does not affect the pharmacological effect of mebeverine. Therefore, the concentration of DMAC is the main parameter to be monitored in pharmacokinetics studies.

Carbon Source-Dependent Inducible Metabolism of Veratryl Alcohol and Ferulic Acid in Pseudomonas putida CSV86

ABSTRACT Pseudomonas putida CSV86 degrades lignin-derived metabolic intermediates, viz., veratryl alcohol, ferulic acid, vanillin, and vanillic acid, as the sole sources of carbon and energy. Strain CSV86 also degraded lignin sulfonate. Cell respiration, enzyme activity, biotransformation, and high-pressure liquid chromatography (HPLC) analyses suggest that veratryl alcohol and ferulic acid are metabolized to vanillic acid by two distinct carbon source-dependent inducible pathways. Vanillic acid was further metabolized to protocatechuic acid and entered the central carbon pathway via the β-ketoadipate route after ortho ring cleavage. Genes encoding putative enzymes involved in the degradation were found to be present at fer, ver, and van loci. The transcriptional analysis suggests a carbon source-dependent cotranscription of these loci, substantiating the metabolic studies. Biochemical and quantitative real-time (qRT)-PCR studies revealed the presence of two distinct O-demethylases, viz., VerAB and VanAB, involved in the oxidative demethylation of veratric acid and vanillic acid, respectively. This report describes the various steps involved in metabolizing lignin-derived aromatic compounds at the biochemical level and identifies the genes involved in degrading veratric acid and the arrangement of phenylpropanoid metabolic genes as three distinct inducible transcription units/operons. This study provides insight into the bacterial degradation of lignin-derived aromatics and the potential of P. putida CSV86 as a suitable candidate for producing valuable products. IMPORTANCE Pseudomonas putida CSV86 metabolizes lignin and its metabolic intermediates as a carbon source. Strain CSV86 displays a unique property of preferential utilization of aromatics, including for phenylpropanoids over glucose. This report unravels veratryl alcohol metabolism and genes encoding veratric acid O-demethylase, hitherto unknown in pseudomonads, thereby providing new insight into the metabolic pathway and gene pool for lignin degradation in bacteria. The biochemical and genetic characterization of phenylpropanoid metabolism makes it a prospective system for its application in producing valuable products, such as vanillin and vanillic acid, from lignocellulose. This study supports the immense potential of P. putida CSV86 as a suitable candidate for bioremediation and biorefinery.