3-4-dihydroxyphenyllactic-acid and catechol

D-danshensu (D-DSS), extracted from the plant Salvia miltiorrhiza (Danshen), is widely used to treat cardiovascular and cerebrovascular diseases. Here we engineered Escherichia coli strains to produce D-DSS from catechol, pyruvate and ammonia by one-pot biotransformation. Tyrosin-phenol lyase (TPL), L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD) and glucose dehydrogenase (gdh) genes were overexpressed in Escherichia coli strain. First, the expression of genes was regulated by different copy number plasmids combination, the result of E. coli TALG6, with strong overexpression of TPL, aadL, ldhD and moderate overexpression of gdh, exhibited 253.7% increase D-DSS production compared to E. coli TALG1. Second, the optimum concentration of catechol was found to be 50 mM. Finally, a fed-batch biotransformation strategy was proposed, namely the amount of catechol was added to 50 mM every 2 h. The total production of D-DSS reached 55.35 mM within 14 h, which was 1.7 times that without feeding.

Topics: Ammonia; Batch Cell Culture Techniques; Biotransformation; Catechols; Escherichia coli; Escherichia coli Proteins; Gene Expression; Glucose 1-Dehydrogenase; L-Amino Acid Oxidase; L-Lactate Dehydrogenase; Lactates; Metabolic Engineering; Plasmids; Pyruvic Acid; Tyrosine Phenol-Lyase

A fast solid phase extraction-high performance liquid chromatography-ion trap mass spectrometry (SPE-HPLC-MSn) method is presented for quantitative determination of 3-(3,4-dihydroxyphenyl)-2-hydroxylpropionic acid (Danshensu, DSS) from Compound Danshen Dripping Pills (CDDP) in rabbits' heart, liver, kidney, and lung and for qualitative analysis of the bioactive metabolites of Radix Salvia miltiorrhizae (DS) from CDDP in these tissues. The proposed method was validated with a detection limit of 0.06 ng/mL and a linear range of 5.0-200 ng/mL for DSS. The main metabolites of DS were pyrocatechol, vanillic acid, isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, and 2-hydroxyl-3-(3,4-dihydroxyphenyl)propinonic sulfuric anhydride. In vitro pharmacological experiments demonstrated that isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate had a distinct vasodilatating action.

Topics: Animals; Catechols; Chromatography, High Pressure Liquid; Drug Stability; Drugs, Chinese Herbal; Female; Lactates; Male; Mesenteric Arteries; Molecular Structure; Plant Extracts; Plant Roots; Rabbits; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Salvia miltiorrhiza; Solid Phase Extraction; Spectrometry, Mass, Electrospray Ionization; Tissue Distribution; Vanillic Acid; Vasodilator Agents