Song You

Development of a sensitive LC-MS/MS method for the determination of bilobalide in rat plasma with special consideration of ex vivo bilobalide stability: application to a preclinical pharmacokinetic study.

The ex vivo instability of bilobalide containing three γ-lactone rings has been paid less attention by researchers who developed bioanalytical methods for bilobalide. In the present study, a sensitive LC-MS/MS method for the determination of bilobalide in rat plasma was developed with special consideration of ex vivo bilobalide stability. Several important factors affecting the stability of bilobalide in sampling and handling procedures were investigated. To prevent the ex vivo degradation of bilobalide, EDTA instead of heparin was used as an anticoagulant as well as an esterase inhibitor for blood collection and the separation of plasma was performed at 4 °C. 20 μL of plasma sample was acidified with 0.1 M hydrochloric acid, and then extracted with ethyl ether-methylene chloride (2:1, v/v). The extract was chromatographed on a Thermo Hypersil GOLD (100 mm × 2.1 mm, 5 μm) column using acetonitrile-10mM ammonium acetate-formic acid (90:10:0.4, v/v/v) as the mobile phase. The analyte and the internal standard (ginkgolide B) were detected by selected reaction monitoring mode via negative electrospray ionization. The method was fully validated and proved to be linear over a concentration range of 5.0-5000 ng/mL. The intra- and inter-day precisions were less than 5.2% and the accuracy was within 92.5-101%. The extraction recoveries ranged from 80.7% to 86.7%. The proposed method was successfully applied to a preclinical pharmacokinetic study of bilobalide in rats after intragastric administration of a single dose of bilobalide at 7, 14 and 28 mg/kg.

Identification and characterization of three impurities in clocortolone pivalate

Clocortolone pivalate is a synthetic corticosteroid that can be used to cure corticosteroid-responsive dermatoses. Three previously unknown impurities detected by HPLC were isolated by semi-preparative LC. Based on the NMR and MS spectral data, these were identified as (6R,9R,16R)-9-chloro-6β-fluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione-21-pivalate (Impurity I), (9R,16R)-9-chloro-4-fluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione-21-pivalate (Impurity II) and (9R,16R)-9-chloro-6α-fluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione-11,21-dipivalate (Impurity III). The possible mechanism of the formation of the impurities is discussed.

Localization, induction and characterization of a novel carbon–carbon double bond reductase from Aspergillus versicolor

Abstract A novel reductase has been detected in cell-free extracts from growing/resting cultures of the fungus Aspergillus versicolor D-1, which specifically catalyzes NADPH-dependent reduction of the γ,δ-double bond of the lactone-conjugated unsaturated system in securinine to form 14,15-dihydrosecurinine. The localization of the reductase has been investigated using differential centrifugation techniques. It was found that the securinine reductase is a cytosolic enzyme. The reductase was highly inducible in growing/resting cultures when securinine was used as the substrate and inducer. Optimal incubation conditions for assay of the securinine reductase were determined by using the enzyme preparation from resting cultures of A. versicolor D-1. The optimum temperature and pH for the reductase activity were in the range of 20–24°C and 8.0–8.5 in 0.05 M Tris–HCl buffer, respectively. The thermal stability of the securinine reductase was poor.

Stereoselective hydrogenation on the exocyclic and conjugated double bond of sesquiterpene lactones by Aspergillus versicolor D-1.

Aspergillus versicolor D-1 was employed to convert dehydrocostuslactone (1) and 3-hydroxy-1(10),3,11(13)-guaiatriene-12,6-olide-2-one (5) stereoselectively. The reactions occurring were specific hydrogenation on the exocyclic α,β-double bond of sesquiterpene lactones with excellent conversion. Products were identified by the analysis of their spectra such as UV, IR, MS, 1H, 13C NMR, and NOESY, and the structure of one new compound was elucidated. The characteristic of the stereoselective hydrogenation was also discussed and suggested.

Selective oxidations of cyperenoic acid by slightly reshaping the binding pocket of cytochrome P450 BM3

The selective hydroxylation of unactivated C−H bonds in complex natural products represents a formidable challenge in synthetic organic chemistry. Cyperenoic acid, a sesquiterpenoid isolated from Croton crassifolius, possesses an antiangiogenic‐promoting effect. Its C7‐ and C9α‐hydroxylated products can significantly inhibit the release of vascular endothelial growth factor (VEGF). To prepare these hydroxylated products, cytochrome P450 BM3 monooxygenase was chosen as a catalyst for the hydroxylation of cyperenoic acid. A simple and fast strategy, slightly reshaping the binding pocket of P450 BM3, was described to expedite the development of highly regio‐ and stereoselective P450 catalysts. P450 BM3 was evolved through one or two generations of mutations, and a highly enriched mutant library that contained fewer than 30 variants was then constructed. The obtained P450 BM3 variants achieved selective hydroxylation at positions C7 (94 % selectivity in the case of the F87A/A330W/F331L mutant) and C9 (90 % regioselectivity and 100 % stereoselectivity in the case of the L75V/F87A/T88F/A330W mutant) of cyperenoic acid and were also used to prepare the desired hydroxylated products at a preparative scale with high isolated yields.

The isolation and identification of a light-induced protein in alfalfa sprouts and the cloning of its specific promoter

We used 2D-PAGE to isolate a light-induced protein (AL-A) that is expressed abundantly in light-growth alfalfa sprouts. The seven amino acids of the N-terminal region of the protein were identified, and we searched for the protein in GenBank using the BLAST program. The results of the homology analysis showed that the amino acid sequence of the isolated protein is most similar to one from a pea plastocyanin. To identify the protein, we amplified and sequenced the DNA fragment encoding AL-A from genomic alfalfa DNA. We found that the AL-A gene was highly homologous (90%) to the sequences from the pea plastocyanin via multiple alignments, and the deduced protein precursor was predicted to be chloroplast-specific via the ChloroP computer program. The protein was named alfalfa-plastocyanin (AL-P). It was characterized as being a light-inducible protein, and RT-PCR analysis showed that AL-P mRNA transcription only occurred in the leaves of the alfalfa plant and the alfalfa seedlings growth in lighted conditions. PCR was also used to amplify the DNA fragment encoding the AL-P promoter (AL-Pp) from genomic alfalfa DNA. PlantCARE analysis of the promoter sequence indicated that both a typical TATA box and a CAAT box were located in the promoter sequence, and some of the cis-elements that are responsible for light responsiveness were also identified within this promoter region. The AL-P gene promoter fused to the β-glucuronidase (GUS) reporter gene has been examined for expression in transgenic alfalfa seedlings. Our findings have a potential application in plant genetic engineering; the AL-Pp may be used to drive the expression of heterologous genes in transgenic alfalfa plants.