Molecular cloning and characterization of copper amine oxidase from Huperzia serrata.

A cDNA encoding a novel copper amine oxidase (CAO) was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae), which produces the Lycopodium alkaloid huperzine A. A 2043-bp open reading frame encoded an Mr 76,854 protein with 681 amino acids. The deduced amino acid sequence shared 44-56% identity with the known CAOs of plant origin, and contained the active site consensus sequence of Asn-Tyr-Asp/Glu. The phylogenetic tree analysis revealed that HsCAO from the primitive vascular plant H. serrata is closely related to Physcomitrella patens subsp CAO. The recombinant enzyme, heterologously expressed in Escherichia coli, catalyzed the oxidative deamination of aliphatic and aromatic amines. Among them, the enzyme accepted cadaverine as the best substrate to catalyze the oxidative deamination to Δ(1)-piperideine, which is the precursor of the Lycopodium alkaloids. Furthermore, a homology modeling and site-directed mutagenesis studies predicted the active site architecture, which suggested the crucial active site residues for the observed substrate preference. This is the first report of the cloning and characterization of a CAO enzyme from the primitive Lycopodium plant.

[Cloning, expression and functional identification of a type III polyketide synthase gene from Huperzia serrata].

A cDNA encoding novel type III polyketide synthase (PKS) was cloned and sequenced from young leaves of Chinese club moss Huperzia serrata (Thunb.) Trev. by RT-PCR using degenerated primers based on the conserved sequences of known CHSs, and named as H. serrata PKS2. The terminal sequences of cDNA were obtained by the 3'- and 5'-RACE method. The full-length cDNA of H. serrata PKS2 contained a 1212 bp open reading frame encoding a 46.4 kDa protein with 404 amino acids. The deduced amino acid sequence of H. serrata PKS2 showed 50%-66% identities to those of other chalcone synthase super family enzymes of plant origin. The recombinant H. serrata PKS2 was functionally expressed in Escherichia coli with an additional hexahistidine tag at the N-terminus and showed unusually versatile catalytic potency to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, the enzyme accepted bulky starter substrates N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 1, 3-dihydroxy-N-methylacridone. Interestingly, H. serrata PKS2 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).

Pharmacokinetic behavior of huperzine A in plasma and cerebrospinal fluid after intranasal administration in rats.

The aim of this study was to investigate the pharmacokinetic behavior of huperzine A (Hup A) in plasma and cerebrospinal fluid (CSF) after intranasal administration (0.5 mg/kg) in male Sprague-Dawley rats. A pharmacokinetic study of intravenous Hup A (0.5 mg/kg) was also performed. The concentrations of Hup A in the biological samples were measured by high performance liquid chromatography-mass spectrometry. Blood samples were taken from the tail vein and CSF was sampled by cisternal puncture using a stereotaxic frame. The contribution of the olfactory pathway to the uptake of Hup A into CSF was determined by comparing the AUC(CSF)/AUC(plasma) ratios after intranasal and intravenous administration. The AUC ratios of intranasal to intravenous administration in CSF and plasma were 104% and 118%, respectively. No significant difference was observed between the AUC(CSF)/AUC(plasma) ratios of Hup A after intranasal administration (20%) and after intravenous infusion (23%). This indicated that approximately 20% of the Hup A level in plasma reached the CSF after both nasal and intravenous administration, and that no direct transport of Hup A from nose to CSF was found in rats.

Distribution and metabolism of gastrodin in rat brain.

Gastrodin is the major and bioactive component in Tianma (Gastrodia elata Bl.) and has sedative, anticonvulsive and neuroprotective effects. Since little is known about its neuropharmacokinetics and brain metabolism, this study was undertaken to investigate the kinetic inter-relationship of gastrodin in rat plasma, cerebrospinal fluid (CSF) and brain microdialysate (frontal cortex, hippocampus, thalamus and cerebellum). Gastrodin was administered via the femoral vein at a dose of 200mg/kg, and blood, CSF and brain microdialysate were collected at timed intervals for the measurement of gastrodin concentrations by high-performance liquid chromatography. The samples were analyzed on a Diamonsil C18 column (5 microm, 250 mm x 4.6mm i.d.) with a mobile phase consisting of acetonitrile-water (5% acetonitrile for brain microdialysate, 2.5% acetonitrile for plasma and CSF), and detected with a UV detector at 221 nm. The distribution of gastrodin in rat showed that levels of gastrodin declined rapidly after drug administration, and the entry of gastrodin into the brain was rapid. However, the ratios of AUC(brain)/AUC(plasma) were not high. The individual ratios of the AUC in the CSF, frontal cortex, hippocampus, thalamus and cerebellum to the AUC in the plasma were 4.8+/-2.4%, 3.3+/-1.2%, 3.0+/-0.7%, 3.3+/-1.3% and 6.1+/-1.9%, respectively. The AUC in the cerebellum was significantly higher than that in other brain regions (P<0.05). The concentrations of p-hydroxybenzyl alcohol, the main metabolite of gastrodin, were very low both in the CSF and plasma.

Pharmacokinetics of Gastrodin in rat plasma and CSF after i.n. and i.v.

The pharmacokinetic behavior of Gastrodin in rat plasma and cerebrospinal fluid (CSF) after intranasal and intravenous administration (50mg kg(-1)) was investigated. Intranasal administration of Gastrodin provided a comparable AUC in CSF compared with the intravenous administration. But Gastrodin level in plasma was very low. The ratios of AUC values of intranasal to intravenous administration were 8.85% and 105.5% in plasma and CSF, respectively. The drug targeting index (DTI) was 12.34. In conclusion, intranasal administration of Gastrodin is a promising alternative to traditional administration. Olfactory mucosa did present another pathway for transport Gastrodin to the brain.

An acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata.

A cDNA encoding a novel plant type III polyketide synthase was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae). The deduced amino acid sequence of Hu. serrata polyketide synthase 1 showed 44-66% identity to those of other chalcone synthase superfamily enzymes of plant origin. Further, phylogenetic tree analysis revealed that Hu. serrata polyketide synthase 1 groups with other nonchalcone-producing type III polyketide synthases. Indeed, a recombinant enzyme expressed in Escherichia coli showed unusually versatile catalytic potential to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, it is remarkable that the enzyme accepted bulky starter substrates such as 4-methoxycinnamoyl-CoA and N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 4-methoxy-2',4',6'-trihydroxychalcone and 1,3-dihydroxy-N-methylacridone, respectively. In contrast, regular chalcone synthase does not accept these bulky substrates, suggesting that the enzyme has a larger starter substrate-binding pocket at the active site. Although acridone alkaloids have not been isolated from Hu. serrata, this is the first demonstration of the enzymatic production of acridone by a type III polyketide synthase from a non-Rutaceae plant. Interestingly, Hu. serrata polyketide synthase 1 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).

Ion-pair reverse-phase high performance liquid chromatography method for determination of Huperzine-A in beagle dog serum.

Huperzine-A (Hup-A), a biologically potent, reversible acetylcholinesterase inhibitor for the treatment of Alzheimer disease (AD) in China, has very low blood concentration. In order to study the pharmacokinetics of newly developed Hup-A transdermal patches in animal, a rapid and sensitive ion-pair reverse-phase high performance liquid chromatography (RP-HPLC) method for the determination of Hup-A in beagle dog serum using mebendazole as internal standard has been developed and validated. The analyte and internal standard were extracted from serum using chloroform-isopropanol (95:5, v/v), analyzed on a C (18) column (5 microm, 150 mm x 4.6 mm i.d.) with a mobile phase consisting of methanol-water-glacial acetic acid (50:48.5:1.5, v/v/v), using sodium dodecylsulfonate as an ion-pair reagent, and detected with UV detector at 313 nm. The chromatographic run time was within 15 min. The assay was linear over the concentration range of 1-12 ng/ml and intra- and inter-day precision over this range was not more than 12.8%. The limit of quantification in serum was 1 ng/ml. The method was successfully applied to characterize the Hup-A concentration-time profiles and study the single and multiple doses phamacokinetics of Hup-A transdermal patches in beagle dogs. The pharmacokinetic study results showed that Hup-A patches has the characteristic of sustained or controlled drug release in vivo.