Liquid chromatographic determination of magnolol in urine collected from volunteers after a single dose of Saiboku-To,an oriental herbal medicine for bronchial asthma

Abstract— Saiboku-To is an anti-asthmatic herbal remedy which consists of ten herbal extracts. To investigate the clinical relationship between the effects and chemical components of Saiboku-To, a simple and sensitive high-performance liquid chromatographic method (HPLC) for determination of magnolol, one of the major urinary products, was developed. Organic solvent extraction of urinary magnolol was conducted by diatomaceous earth column rapid-flow fractionation using ethanol/dichloromethane (8/92, v/v). Recovery rates of magnolol were more than 99% with coefficient of variations less than 6% in the concentration range 9·7–970 ng mL^?1. Subsequent HPLC determination of magnolol was achieved using a conventional silica-gel column, a mobile phase mixture of acetic acid/diethyl ether/n-hexane (0·2/17·0/82·8, v/v), and a UV-absorption detector set at 290 nm. Calibration was on the basis of peak height ratio between magnolol and flavone as an internal standard. The method was used to demonstrate excretion profiles of magnolol in healthy and asthmatic subjects following single administration of Saiboku-To.     

Dipeptide synthesis catalyzed by aminoacyl-tRNA synthetases from Bacillus stearothermophilus

A new approach to enzymatic peptide synthesis by using aminoacyl-tRNA synthetase (ARS) as a catalyst has been investigated. Four ARSs (AspRS, HisRS, LeuRS and TyrRS) have been purified from a thermophilic bacterium, Bacillus stearothermophilus. By using TyrRS as a catalyst, tyrosine and leucinamide were shown to be condensed in the presence of ATP to give tyrosylleucinamide. In this manner, all of the ARSs investigated catalyzed the peptide synthesis reactions. TyrRS did not have strict specificity for the amino acid derivatives used as substrates and even D-amino acids were incorporated into peptides fairly easily in this enzymatic reaction. Preparative scale synthesis of L-Tyr-L-LeuNH₂ was carried out and from this the scope and limitation of this new enzymatic reaction as a tool to the peptide synthesis has been described.     

Hydrolysis of various types of ester substrates with linear,cyclic and polymeric peptides containing His,Ser and Asp residues

Catalytic activities of linear, cyclic and polymeric peptides having the sequences of -Asp-ßAla-Gly-His-ßAla-Gly-(nonapeptide series) and -Asp-eAhx-Ser-eAhx-His-eAhx- (hexapeptide series) in the hydrolysis of various types of ester substrates were compared with one another and with that of poly (-His-ßAla-Gly) (Poly-3) which has no Ser or Asp residues. Hydrolytic activity of the hexapeptide series, the cyclic form in particular, was larger than that of the nonapeptide series in many cases such as in the hydrolysis of N-protected amino acid p-nitrophenyl esters (Z-Gly-ONp, Boc-Ala-ONp, Z- (l and d ) -Leu-ONp, Boc- (l and ) -Phe-ONp) and anionic 3-nitro-4-acetoxybenzoic acid ( NABA ). The activities of the polymeric peptides were generally smaller than those of the linear or cyclic peptides. None of the peptides showed the remarkable activity to the hydrophobic substrate with long chain, p-nitrophenyl laurate ( PNPL ), and they were almost inactive to bulky p-nitrophenyl trimethylacetate ( PNPTMA ) and cationic 3-acetoxy-N-trimethylanilinium iodide ( ANTI ). Enantiomer-selectivity in the hydrolysis of N-protected amino acid p-nitrophenyl esters and solvent isotope effect in the hydrolysis of p-nitrophenyl acetate ( PNPA ) are also reported.