ABSTRACT
Tryptophan hydroxylase I (TPH1) catalases the 5-hydroxylation reaction of L-Trp, which is the rate-limiting step in the synthesis of serotonin. Serotonin, a major component of Bufonis venenum, is involved in numerous physiological functions as an important neurotransmitter. In this study, BbgTPH1 cDNA was cloned from the parotid gland of Bufo bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pMAL-BbgTPH1, and the induced conditions to express the recombinant BbgTPH1 in E. coli TB1 cells were optimized. The full length of BbgTPH1 is 1984 bp (GenBank accession No. JQ768313) with a 1443 bp open reading frame (ORF) encoding a 480 amino acid residues. The deduced protein molecular weight is 55.2 kDa and its theoretical isoelectric point is 5.58. The sequence includes conserved domain and special signal sequence of the aromatic amino acid hydroxylase (AAAH) superfamily. Homologous alignment showed that BbgTPH1 shared a high homology with other species. Phylogenetic tree showed the closest relative to BbgTPH1 was Xenopus tropical-TPH1. The best induction conditions of recombinant BbgTPH1 were 0.5 mmol·L-1 IPTG at 20℃ for 8 h. The function of BbgTPH1 was identified by in vitro enzymatic reaction and the recombinant BbgTPH1 was able to produce 5-hydroxytryptophan by catalyzing tryptophan. This study represents the first time of cloning and identification of the function of TPH1 in Bufo genus. The results of this study will be an important foundation for future studies of biosynthesis of bufotenines in the parotid gland of B. bufo gargarizans.
ABSTRACT
Tongmai granule (TM) is composed of Puerariae Lobatae Radix (Gegen), Salviae Miltiorrhizae Radix et Rhizoma(Danshen) and Chuanxiong Rhizoma(Chuanxiong). It has been used to treat ischemic cardio-cerebrovascular diseases for decades. For the purpose of elucidating its pharmacodynamic material foundation, the absorption and pharmacokinetic property of TM were investigated in acute myocardial ischemic model beagles. All serum samples were extracted before analysis with ethyl acetate after being acidified by hydrochloric acid. Under negative ESI detection mode, the chromatographic separation was carried out with monolithic C₁₈ column for gradient elution. A simultaneous quantitative analysis was made on 15 polyphenols, including 8 from Gegen, 5 from Danshen and 2 from Chuanxiong, in 8.5 min. The validation result demonstrated the specificity, accuracy and precision of the method in line with the bioanalysis requirements. After TM solution was administrated to acute myocardial ischemic model beagles through duodenum injection, serum samples were collected after 6 h. The quantitative detection proved the prompt absorption of TM, all of the components were detectable in the blood samples 5 min later, and reached peak respectively at 0.18-3.83 h after administration. The components presented large variabilities. The most components were exposed in serum with puerarin and salvianic acid A, followed by 3'-methoxypuerarin, mirificin, and 3'-hydroxypuerarin. The study proves puerarin and salvianic acid A are dominating active components of TM in acute myocardial ischemic model beagles.