Chia Ying Li

A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2–4D motif

SUMMARY Geranyl diphosphate (GDP) is the precursor of monoterpenes, which are the major floral scent compounds in Phalaenopsis bellina. The cDNA of P. bellina GDP synthase (PbGDPS) was cloned, and its sequence corresponds to the second Asp-rich motif (SARM), but not to any aspartate-rich (Asp-rich) motif. The recombinant PbGDPS enzyme exhibits dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP), and a yeast two-hybrid assay and gel filtration revealed that PbGDPS was able to form a homodimer. Spatial and temporal expression analyses showed that the expression of PbGDPS was flower specific, and that maximal PbGDPS expression was concomitant with maximal emission of monoterpenes on day 5 post-anthesis. Homology modelling of PbGDPS indicated that the Glu-rich motif might provide a binding site for Mg(2+) and catalyze the formation of prenyl products in a similar way to SARM. Replacement of the key Glu residues with alanine totally abolished enzyme activity, whereas their mutation to Asp resulted in a mutant with two-thirds of the activity of the wild-type protein. Phylogenetic analysis indicated that plant GDPS proteins formed four clades: members of both GDPS-a and GDPS-b clades contain Asp-rich motifs, and function as homodimers. In contrast, proteins in the GDPS-c and GDPS-d clades do not contain Asp-rich motifs, but although members of the GDPS-c clade function as heterodimers, PbGDPS, which is more closely related to the GDPS-c clade proteins than to GDPS-a and GDPS-b proteins, and is currently the sole member of the GDPS-d clade, functions as a homodimer.

A rapid and simple determination of protoberberine alkaloids in Rhizoma Coptidis by 1H NMR and its application for quality control of commercial prescriptions

Simple, convenient, sensitive and accurate analytical methods are needed for the analysis of alkaloid components in Rhizoma Coptidis in traditional Chinese herbal medicine, which has important bioactivity. In the present study, a highly specific and sensitive method using (1)H NMR has been developed for the quantitative determination of protoberberine alkaloids berberine, palmatine, coptisine and jatrorrhizine in Coptis species and their commercial traditional Chinese medicine prescriptions. A (1)H NMR analysis of the H-13 signals of target protoberberine alkaloids was performed. By comparing the solvent effects on the resolution of these signals, methanol-d(4)-benzene-d(6) (75:25) is selected as an optimal (1)H NMR solvent. The quantity of the compounds is calculated by the relative ratio of the integral values of the target peak for each compound to the known amount of the internal standard anthracene. This method allows rapid and simple quantitation of protoberberine alkaloids from Coptis species and the more complex commercial prescriptions in 5 min without any pre-purification steps. The recoveries of these alkaloids from Coptis chinensis are in the range of 93-105%. Limit of detection of berberine in the plant material or prescription is 0.03 mg/mL. The advantages of this method are that no reference compounds are required for calibration curves, the quantification can be directly realized on a crude extract, and the better selectivity for protoberberine alkaloids and a very significant time-gain can be achieved, in comparison to conventional HPLC methods, for instance.

Limonoids and alkaloids of the root bark of Dictamnus angustifolius

Abstract Six limonoids, limonin, limonin diosphenol, limonexic acid, obacunone, fraxinellone and fraxinellonone; nine alkaloids, dictangustine-A, iso- γ -fagarine, dictamine, γ -fagarine, skimmianine, isodictamine, isomaculosidine, N -methylflindersine and preskimmianine; five coumarins, angustifolin, umbelliferone, herniarin, scopoletin and scoparone; three steroids, sitosterol, stigmast-4-ene-3,6-dione and stigmast-4-ene-3-one, together with dictafolin-A and dictafolin-B were isolated from the root bark of Dictamnus angustifolius . Their structures were elucidated by spectral analysis. Among them, dictangustrine-A, iso- γ -fagarine, dictafolin-A and dictafolin-B are reported for the first time from a natural source.

Quality assessment of Radix Codonopsis by quantitative nuclear magnetic resonance.

Radix Codonopsis (Dangshen) is a famous traditional Chinese medicine and has long been used for replenishing energy deficiency, strengthening the immune system, lowering blood pressure and improving appetite in China, Japan and Korea. A highly specific quantification method using (1)H NMR has been developed for the simultaneous determination of novel quaternary ammonium alkaloids codotubulosine A and B, adenosine and 5-(hydroxymethyl)furfural in Radix Codonopsis materials Codonopsis pilosula, C. pilosula var. modesta, C. tangshen, C. tubulosa, C. subglobosa, C. clematidea, C. lanceolota and Campanumoea javanica collected from different regions of China and Taiwan. A solid-phase extraction with C-18 cartridge followed by elution with water can easily remove sugars the major components that may affect the determination of target constituents. In the (1)H NMR spectrum, the signals of N-CH(3) of codotubulosine A (delta 2.75) and B (delta 2.83), H-8 of adenosine (delta 8.15), and CHO signal of 5-(hydroxymethyl)furfural (delta 9.49) are well separated from other signals in [(2)H(4)]methanol. The quantity of the compounds was calculated by the relative ratio of the integral values of the target peaks of each compound to the known amount of internal standard pyrazine. The described NMR method is found to be relatively simple, specific, precise and accurate for the quality control of Radix Codonopsis herbs and no reference compounds are required for calibration curves, in comparison to conventional HPLC methods, for instance.

New diterpenoids and the bioactivity of Erythrophleum fordii.

A phytochemical investigation of the leaves of Erythrophleum fordii Oliv. has led to the isolation of three new cassaine-type diterpenoids, erythrofordin A (1), erythrofordin B (2) and erythrofordin C (3), as well as a norcassaine diterpenoid with a novel skeleton, norerythrofordin A (4), and 27 known compounds (5-31). The structures of 1-4 were elucidated on the basis of spectroscopic analysis. Selected compounds from this plant were examined for anti-inflammatory activity. Taraxerol (16) displayed potent NO-reducing activity in microglial cells, and gallic acid (27) exhibited excellent DPPH radical-scavenging effects.

Delayed Treatment with Nicotinamide Inhibits Brain Energy Depletion,Improves Cerebral Microperfusion, Reduces Brain Infarct Volume, but does not Alter Neurobehavioral Outcome Following Permanent Focal Cerebral Ischemia in Sprague Dawley Rats

Delayed treatment with nicotinamide (NAm) reduces infarction induced by middle cerebral artery occlusion (MCAO) in rats. This study explored some potential mechanisms by which delayed NAm treatment may confer protection in the brain of Sprague-Dawley rats following permanent MCAO (pMCAO). NAm (500 mg/kg) or vehicle was given 2 h after the onset of pMCAO. Cortical microperfusion, brain and rectal temperature were serially measured. Neurobehavioral examinations were performed at 24 h post-ischemia followed by sacrifice for histologic assessment. Some rats were also sacrificed at 4 h post-ischemia for analyses of ATP, ADP, AMP, and adenosine. Permanent MCAO induced spontaneous hyperthermia and a sharp decrease in cortical microperfusion, ATP concentration, and the sum of adenine nucleotides (p < 0.05). At 4 h post-ischemia, NAm improved ATP recovery, the sum of adenine nucleotides (p < 0.05) and attenuated the ischemia-induced systemic hyperthermia (p < 0.05) without affecting brain temperature or cortical microperfusion. At 24 h, NAm improved cortical microperfusion in the ischemic hemisphere and reduced total infarct volume (p < 0.05), but did not affect behavioral scores. The data suggest that NAm attenuated brain damage following pMCAo initially by improving cerebral bioenergetic metabolism during the sub-acute phase of ischemia, followed by a delayed improvement in microvascular perfusion.

Cytotoxic phenanthroindolizidine alkaloids from the roots of Ficus septica.

Activity-monitored fractionation of the CHCl3 solubles of the MeOH extract of the roots of Ficus septica using cytotoxicity assay led to the isolation of twenty phenanthroindolizidine alkaloids, including ten new ficuseptines E-N and ten known compounds. The cytotoxic effects of some of the isolates were tested on gastric adenocarcinoma (NUGC) and nasopharyngeal carcinoma (HONE-1) cell lines and the results demonstrated that, with the exception of dehydrotylophorine, all the tested compounds displayed pronounced cytotoxic activity with significant cell growth inhibitory effects. In addition, 13a R-antofine was found to be highly effective with ED(50) values of < 0.1 microg/mL against L-1210, P-388, A-549 and HCT-8 cell lines in another assay.

Constituents of leaves of Phellodendron chinense var. glabriusculum

The leaves of Phellodendron chinense var. glabriusculum yielded three new flavanones, phellodensin D (1), phellodensin E (2), and phellodensin F (3) along with five known compounds, amurensin (4), phellamurin (5), methyl caffeate (6), beta-sitosterol (7), and pheophytin-a (8). The structural assignment of new compounds was based on spectroscopic studies.

A secoiridoid and other constituents of Gonocaryum calleryanum

Investigation of the leaves, branch, stem and root bark of Gonocaryum calleryanum resulted in the isolation of a new secoiridoid glycoside, gonocaryoside E, together with 14 known compounds. Their structures were elucidated by spectral analysis and chemical transformation. Gonocaryoside E was shown to be a derivative of kingiside in which the lactone ring was open, and with the 8-hydroxy group esterified with tiglic acid. Alkaline hydrolysis of these secoiridoids is discussed.

Severibuxine, a new quinolin-2,4-dione and other constituents from Severinia buxifolia

Abstract A new quinolin-2,4-dione alkaloid, severibuxine, together with 23 known compounds were isolated from the root bark of Severinia buxifolia . The structure of these compounds were determined by spectral and chemical methods. Most of them showed cytotoxic activity against P-388.