Use of an electrophysiological technique for stepwise detection of trace agonist constituents of Hochuekkito in Xenopus oocytes injected with serotonin 2C receptor mRNA.

An electrophysiological bioassay was used to isolate the active compound from Hochuekkito (HET), which the current authors previously described as having potent agonist action against serotonin 2C receptors (5-HT2CR). Synthetic 5-HT2CR mRNA was injected into Xenopus oocytes to specifically express these receptors. Crude extracts and purified products were subjected to an electrophysiological bioassay using the voltage clamp method. HET stimulated a 5-HT2CR-induced current response, whereas Juzentaohoto (JTT), which has anti-depressive action similar to that of HET, did not. Current responses were not observed with an extract mixed with five types of herbal medicines common to HET and JTT but were detected with an extract with the five types of herbal medicines found in HET alone (Hoc5). When the responses to each of the five types of Hoc5 were examined, current responses were noted with Cimicifugae rhizoma (CR) and Citrus unshiu Markovich extracts. Since efficacy and the EC50 value were higher for CR, its constituents were separated using three-dimensional high-performance liquid chromatography and the current response at each of the isolated peaks was examined. One constituent displayed a strong response and was identified as a single substance with a molecular weight of 283.1393 based on liquid chromatography/mass spectrometry. These results will contribute to the isolation of 5-HT2CR-stimulating constituents in HET and the identification of trace constituents with agonist action.

Characterization of metabolites in Saposhnikovia divaricata root from Mongolia.

Saposhnikoviae Radix (SR), derived from the dried root and rhizome of Saposhnikovia divaricata, is a popular crude drug used in traditional Chinese and Japanese medicine. To evaluate the metabolites of S. divaricata roots from Mongolia and to investigate their geographical variation, we developed the HPLC method, determined the contents of 9 chromones and 4 coumarins, and conducted multivariate statistical analysis. All Mongolian specimens contained prim-O-glucosylcimifugin (1) and 4'-O-ß-D-glucosyl-5-O-methylvisamminol (3), and their total amount (5.04-25.06 mg/g) exceeded the criterion assigned in the Chinese Pharmacopoeia. Moreover, the content of 1 (3.98-20.79 mg/g) was significantly higher in the Mongolian specimens than in Chinese SR samples. The specimens from Norovlin showed the highest contents of 1 and 3. The total levels of dihydropyranochromones were higher in the specimens from Bayan-Uul. The orthogonal partial least squares-discriminant analysis revealed that the Mongolian specimens tended to be separated into three groups based on growing regions, in which several chromones contributed to each distribution. Furthermore, 1H NMR analysis revealed that Mongolian specimens had less amount of sucrose and a substantial amount of polyacetylenes. Thus, in this study, the chemical characteristics of Mongolian S. divaricata specimens were clarified and it was found that the specimens from the northeast part of Mongolia, including Norovlin, had the superior properties due to higher amounts of major chromones.

Metabolomic profiling of Saposhnikoviae Radix from Mongolia by LC-IT-TOF-MS/MS and multivariate statistical analysis.

Saposhnikoviae Radix (SR) is a commonly used crude drug that is obtained from the root and rhizome of Saposhnikovia divaricata which is distributed throughout China, Korea, Mongolia, and Russia. To evaluate the quality of Mongolian S. divaricata, metabolomic profiling of 43 plant specimens from different regions of Mongolia, as well as 8 SR samples and 2 plant specimens from China, were conducted by liquid chromatography-ion-trap-time-of-flight-mass spectrometer (LC-IT-TOF-MS). LC-MS profiles of the specimens showed uniformity and 30 compounds were tentatively identified, including 13 chromones and 17 coumarins. Among them, 16 compounds were isolated and unambiguously verified by comparing them with the spectroscopic data of standard compounds. Orthogonal partial least squares-discriminant analysis (OPLS-DA) based on LC-MS data from 7 Mongolian specimens and 8 Chinese SR samples as well as 2 plant specimens revealed that these 2 groups were clearly distinguishable and that Mongolian specimens were characterized by an abundance of prim-O-glucosylcimifugin (1). Moreover, the OPLS-DA of the Mongolian specimens showed that they can be discriminated by their growing regions based on the content of 8 chromones. The total content of dihydrofurochromones 1-3 was relatively higher in the specimens from Khalkhgol in the far eastern part of Mongolia, while contents of 10, 11, 15, and 16 were higher in those from Holonbuir in the eastern part. Based on this research, the roots of S. divaricata from Mongolia have potential as a new resource of SR in Kampo medicine.

Cloning and Functional Analysis of Three Chalcone Synthases from the Flowers of Safflowers Carthamus tinctorius.

The flowers of safflowers (Carthamus tinctorius L.) are very important as they are the sole source of their distinct pigments, i.e. carthamus-red and -yellows, and have historically had strong connections to the cultural side of human activities such as natural dyes, rouge, and traditional medicines. The distinct pigments are quinochalcone C-glucosides, which are found specifically in the flowers of C. tinctorius. To investigate the biosynthetic pathways of quinochalcone C-glucosides, de novo assembly of the transcriptome was performed on the flowers using an Illumina sequencing platform to obtain 69,312 annotated coding DNA sequences. Three chalcone synthase like genes, CtCHS1, 2 and 3 were focused on and cloned, which might be involved in quinochalcone C-glucosides biosynthesis by establishing the C6-C3-C6 chalcone skeleton. It was demonstrated that all the recombinant CtCHSs could recognize p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA as starter substrates. This is the first report on the cloning and functional analysis of the three chalcone synthase genes from the flowers of C. tinctorius.

Monoterpene derivatives with anti-allergic activity from red peony root, the root of Paeonia lactiflora.

The methanolic extract and its subfractions from red peony root, the dried roots of Paeonia lactiflora Pallas showed potent antiallergic effects, as inhibition of immunoglobulin E (IgE)-mediated degranulation in rat basophil leukemia (RBL)-2H3 cells. Bioassay-guided fractionation led to the isolation of 16 monoterpene derivatives, including 3 new compounds, paeoniflorol (1), 4'-hydroxypaeoniflorigenone (2) and 4-epi-albiflorin (3), together with 13 known ones (4-16). The chemical structures of the new compounds were elucidated on the basis of spectroscopic and chemical evidences. Among the isolated monoterpene derivatives, nine compounds showed potent anti-allergic effects and compound 1 was the most effective. A primary structure-activity relationship of monoterpene derivatives was discussed.

Crataegusins A and B, New Flavanocoumarins from the Dried Fruits of Crataegus pinnatifida var. major (Rosaceae).

Crataegusins A (1) and B (2), new flavanocoumarins, were isolated from the crude drug Crataegus Frictus, i.e., the dried fruits of Crataegus pinnatifida var. major..Their structures were determined by spectroscopic methods. They were unique in terms of carrying a 3-(or 4-)substituted coumarin substructure while a flavanocoumarin generally does not carry any substituents in the 2-pyron ring. They showed a significant DPPH reducing activity compared with epicatechin Their production would be biosynthetically regulated considering the results of an LC-MS analysis of the dried and fresh fruits, fruit skin, hypanthia, and leaves. Their structures led the authors to consider a hypothetical general biosynthetic pathway of the flavanocoumarins, to which a flavan-3-ol is converted through a Michael addition and successive oxidative decarboxylation or dehydration pathway.

Active Constituents from Drynaria fortunei Rhizomes on the Attenuation of Aß(25-35)-Induced Axonal Atrophy.

Axonal regeneration might contribute to the restoration of damaged neuronal networks and improvement of memory deficits in a murine Alzheimer's disease (AD) model. A search for axonal regenerative drugs was performed to discover novel therapeutic options for AD. In this study, an aqueous extract of Drynaria fortunei rhizomes reversed Aß25-35-induced axonal atrophy in cultured cortical neurons of mice. Bioassay-guided fractionation of this extract led to the isolation and identification of compounds 1-5. Among them, (2S)-neoeriocitrin (2) and caffeic acid 4-O-glucoside (4) showed significant axonal elongation effects on Aß25-35-induced atrophy.

The anti-inflammatory secoiridoid glycosides from gentianae scabrae radix: the root and rhizome of Gentiana scabra.

Gentianae Scabrae Radix is a well-known traditional medicine that is used for the treatment of hepatitis, cholecystitis and inflammatory diseases. It consists mainly of secoiridoid glycosides, with representatives of gentiopicroside, sweroside and swertiamarin. In the present study, a chemical investigation of the CHCl3 extract of Gentianae Scabrae Radix derived from Gentiana scabra Bunge yielded seven new (1-7) and ten known (8-17) secoiridoid glycosides. Their structures were elucidated by extensive spectroscopic analyses and comparison with literature data. All 17 compounds were evaluated for their inhibitory effects against NO, IL-6 and TNF-α productions induced by lipopolysaccharide (LPS) in RAW264 cells. Among them, 8-epi-kingiside derivatives 1-3; kingiside derivatives 4, 5 and 10; and a sweroside derivative 6 showed inhibition activity against IL-6 production with IC50 values of 51.70-61.10 µM, whereas sweroside derivatives 12 and 15-17 and a swertiamarin derivative 13 showed inhibition effects on both NO and IL-6 productions with IC50 values of 64.74-94.95 and 48.91-75.45 µM, respectively. All the compounds exhibited weak inhibitory activity (IC50 > 100 µM) in a TNF-α bioassay. Finally, a primary structure-activity relationship of these secoiridoid glycosides is discussed.

Biosynthesis of malonylated flavonoid glycosides on the basis of malonyltransferase activity in the petals of Clitoria ternatea.

The crude malonyltransferase from the petals of Clitoria ternatea was characterized enzymatically to investigate its role on the biosynthetic pathways of anthocyanins and flavonol glycosides. In C. ternatea, a blue flower cultivars (DB) and mauve flower variety (WM) accumulate polyacylated anthocyanins (ternatins) and delphinidin 3-O-(6''-O-malonyl)-beta-glucoside which is one of the precursors of ternatins, respectively. Moreover, WM accumulates minor delphinidin glycosides - 3-O-beta-glucoside, 3-O-(2''-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(2''-O-alpha-rhamnosyl-6''-O-malonyl)-beta-glucoside of delphinidin. These glycosidic patterns for minor anthocyanins in WM are also found among the minor flavonol glycosides in all the varieties including a white flower variety (WW) although the major flavonol glycosides are 3-O-(2''-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(6''-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(2'',6''-di-O-alpha-rhamnosyl)-beta-glucoside of kaempferol, quercetin, and myricetin. How do the enzymatic characteristics affect the variety of glycosidic patterns in the flavonoid glycoside biosynthesis among these varieties? While the enzyme from DB highly preferred delphinidin 3-O-beta-glucoside in the presence of malonyl-CoA, it also has a preference for other anthocyanidin 3-O-beta-glucosides. It could use flavonol 3-O-beta-glucosides in much lower specific activities than anthocyanins; however, it could not utilize 3-O-(2''-O-alpha-rhamnosyl)-beta-glucosides of anthocyanins and flavonols, and 3,3'-di- and 3,3',5'-tri-O-beta-glucoside of delphinidin - other possible precursors in ternatins biosynthesis. It highly preferred malonyl-CoA as an acyl donor in the presence of delphinidin 3-O-beta-glucoside. The crude enzymes prepared from WM and WW had the same enzymatic characteristics. These results suggested that 3-O-(2''-O-alpha-rhamnosyl-6''-O-malonyl)-beta-glucosides of flavonoids were synthesized via 3-O-(6''-O-malonyl)-beta-glucosides rather than via 3-O-(2''-O-alpha-rhamnosyl)-beta-glucosides, and that malonylation proceeded prior to glucosylation at the B-ring of delphinidin in the early biosynthetic steps towards ternatins. It seemed that the substrate specificities largely affected the difference in the accumulated amount of malonylated glycosides between anthocyanins and flavonols although they are not simply proportional to the accumulation ratio. This enzyme might join in the production of both malonylanthocyanins and flavonol malonylglycosides as a result of broad substrate specificities towards flavonoid 3-O-beta-glucosides.