Two Triterpene Synthases from Imperata cylindrica Catalyzing the Formation of a Pair of Diastereoisomers through Boat or Chair Cyclization.

Imperata cylindrica is known to produce a pair of triterpenes, isoarborinol and fernenol, that exhibit identical planar structures but possess opposite stereochemistry at six of the nine chiral centers. These differences arise from a boat or a chair cyclization of the B-ring of the substrate. Herein, we report the characterization of three OSC genes from I. cylindrica. IcOSC1 and IcOSC5 were identified as isoarborinol and fernenol synthases, respectively, while IcOSC3 was characterized as a multifunctional enzyme that produces glutinol and friedelin as its major products. Mutational studies of isoarborinol and fernenol synthases revealed that the residues surrounding the DCTAE motif partially affected the conformation of the B-ring during cyclization. Additionally, the IcOSC1-W255H mutant produced the rare triterpene boehmerol. The introduced histidine residue presumably abstracted a proton from the intermediary carbocation at C18 during the 1,2-rearrangement. Expression analysis indicated that all OSC genes were highly expressed in stems.

Squalene Cyclases and Cycloartenol Synthases from Polystichum polyblepharum and Six Allied Ferns.

Ferns are the most primitive of all vascular plants. One of the characteristics distinguishing them from flowering plants is its triterpene metabolism. Most cyclic triterpenes in ferns are hydrocarbons derived from the direct cyclization of squalene by squalene cyclases (SCs). Both ferns and more complex plants share sterols and biosynthetic enzymes, such as cycloartenol synthases (CASs). Polystichum belongs to Dryopteridaceae, and is one of the most species-rich of all fern genera. Several Polystichum ferns in Japan are classified as one of three possible chemotypes, based on their triterpene profiles. In this study, we describe the molecular cloning and functional characterization of cDNAs encoding a SC (PPH) and a CAS (PPX) from the type species Polystichum polyblepharum. Heterologous expression in Pichia pastoris revealed that PPH and PPX are hydroxyhopane synthase and CAS, respectively. By using the PPH and PPX sequences, we successfully isolated SC- and CAS-encoding cDNAs from six Polystichum ferns. Phylogenetic analysis, based on SCs and oxidosqualene cyclase sequences, suggested that the Polystichum subclade in the fern SC and CAS clades reflects the chemotype-but not the molecular phylogeny constructed using plastid molecular markers. These results show a possible relation between triterpenes and their biosynthetic enzymes in Polystichum.

Development of an immunoassay using an anti-wogonin glucuronide monoclonal antibody.

Wogonin 7-O-ß-D-glucuronide (Wgn) is a bioactive flavone present in the dried root of Scutellaria baicalensis Georgi. To generate a monoclonal antibody (MAb) against Wgn, BALB/c mice injected with Wgn-bovine serum albumin yielded splenocytes that we fused with SP2/0 myeloma cells using the polyethylene glycol method. We obtained a hybridoma designated 315A that produced a MAb reactive to Wgn. The anti-Wgn MAb 315A was applied to an indirect competitive enzyme-linked immunosorbent assay (icELISA) to quantify Wgn. Subsequent validation revealed that icELISA using the 315A anti-Wgn MAb is an accurate and reliable method for the quantification of Wgn in S. baicalensis.

Impallidane skeleton as a novel triterpenoid methyl ether from rhizomes of Imperata cylindrica var. koenigii f. pallida.

ABSTRUCT: n-Hexane extract of rhizomes of Imperata cylindrica var. koenigii f. pallida yielded five novel skeletal triterpenoids, designed as impallidin (1), impallidol (2), impallidin ozonide (3a, 3b), trisnorimpallidin aldehyde (4), tetranorimpallidin aldehyde (5). Structures of novel compounds were elucidated by mainly 2D NMR and other spectroscopic analysis and chemical correlations. Alternatively, compound 3a, 3b was derivatized from 1 under ozone oxidation condition.

Ecological and pharmacognostical characteristics in Ephedra saxatilis in the Yarlung Zangbo River Valley of southeastern Tibetan Plateau, China.

In the Yarlung Zangbo River Valley of the southeastern Tibetan Plateau, China (29°07'49.5"N, 92°41'11.0"E, 3256 m above sea level), we found an Ephedra saxatilis community in the xeric steppe with shrubland vegetation habitat of the broad alluvial plain of the river with soil having relatively higher water-soluble cation (Ca2+, 8.62; K+, 1.94; Mg2+, 2.38 mmol/100 g dry soil weight) and nitrogen (NO3-, 21.78; NH4+, 1.82 mmol/100 g dry soil weight) content. The ranges of ephedrine and pseudoephedrine in 13 E. saxatilis samples were as follows: ephedrine, not detected-3.03 of dry weight (%DW) and pseudoephedrine, not detected-1.36%DW. The 13 E. saxatilis plants collected in the study area showed intraspecific variability of ephedrine and pseudoephedrine with 6 samples containing ephedrine and pseudoephedrine, 6 samples containing only ephedrine, and 1 sample containing only pseudoephedrine.

Essential oil composition of Curcuma species and drugs from Asia analyzed by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry.

Essential oils (EOs) comprised of various bioactive compounds have been widely detected in the Curcuma species. Due to the widespread distribution and misidentification of Curcuma species and differences in processing methods, inconsistent reports on major compounds in rhizomes of the same species from different geographical regions are not uncommon. This inconsistency leads to confusion and inaccuracy in compound detection of each species and also hinders comparative study based on EO compositions. The present study aimed to characterize EO compositions of 12 Curcuma species, as well as to detect the compositional variation among different species, and between the plant specimens and their related genetically validated crude drug samples using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. The plant specimens of the same species showed similar EO patterns, regardless of introducing from different geographical sources. Based on the similarity of EO compositions, all the specimens and samples were separated into eight main groups: C. longa; C. phaeocaulis, C. aeruginosa and C. zedoaria; C. zanthorrhiza; C. aromatica and C. wenyujin; C. kwangsiensis; C. amada and C. mangga; C. petiolata; C. comosa. From EOs of all the specimens and samples, 54 major compounds were identified, and the eight groups were chemically characterized. Most of the major compounds detected in plant specimens were also observed in crude drug samples, although a few compounds converted or degraded due to processing procedures or over time. Orthogonal partial least squares-discriminant analysis allowed the marker compounds to discriminate each group or each species to be identified.

Genetic analysis of Curcuma species from Asia based on intron regions of genes encoding diketide-CoA synthase and curcumin synthase.

Intron length polymorphism (ILP) markers in genes encoding diketide-CoA synthase (DCS) and curcumin synthase (CURS) showed high identification rates in 13 Curcuma species from Asia. However, the sequences of the intron regions have not yet been analyzed. To elucidate the sequence differences in intron regions of the DCS and CURS genes and to search for specific sequences suitable for the identification of Curcuma species, a large number of sequences were determined through subcloning coupled with sequencing analysis of six Curcuma plant specimens belonging to five species that showed distinct ILP patterns. More than 30 sequences of each region from each specimen were grouped into genes DCS1, DCS2, or CURS1-3 and subsequently the sequences of the same genes were compared. Sequences belonging to the same gene showed inter-species similarity, and thus, these intron sequences were less informative within each single-gene region. The determined sequences from each specimen showed 3-5 kinds of sequence lengths in DCS intron I region, and 5-7 kinds of sequence lengths in CURS intron region. The length of determined sequences and the fragment number in each intron region were different among species, or specimens in C. longa, which were in accordance with the fragment lengths and numbers in their corresponding ILP patterns.

Relationship between ephedrine alkaloid profile in Ephedra gerardiana and soil characteristics of glacial landforms in southeastern Tibetan Plateau, China.

In the Kaluxung River catchment of the southeastern Tibetan Plateau in China, we identified three Ephedra gerardiana communities on different soils and glacial landforms from 4842 to 4899 m above sea level: a moraine community located on constantly collapsing sandy gravel alpine steppe slopes with exposed bedrock on the outer slope of the terminal moraine of the Qiangyong Glacier on Mt. Kaluxung; an outwash plain community located on a gentle alpine steppe slope with exposed bedrock at the terminal end of the outwash plain in the glacial valley of the southeast side of Mt. Noijinkangsang; and a river terrace community located in an alpine meadow on a rock-scattered flat river terrace along a glacier-fed river in the outwash plain in the glacial valley of the southeast side of Mt. Noijinkangsang. Based on the finding of identical DNA sequences of the intergenic spacers of chloroplast trnT-trnF and trnS-trnfM regions for all Ephedra specimens examined in this study, the E. gerardiana in this study were considered to comprise a genetically homogeneous population. Analysis of the relationship between ephedrine alkaloid profiles of these three communities and soil characteristics showed that the river terrace community in wet alpine meadow had significantly lower ephedrine content than did the moraine and outwash plain communities in dry alpine steppe (moraine community, 1.52 ± 0.44; outwash plain community, 1.42 ± 0.68; river terrace community, 0.33 ± 0.65%DW), but pseudoephedrine content showed the reverse pattern (moraine community, 0.86 ± 0.30; outwash plain community, 0.73 ± 0.60; river terrace community, 1.50 ± 0.71%DW). In addition, total alkaloid (ephedrine and pseudoephedrine) content in the river terrace community (1.83 ± 0.24%DW) was significantly lower than that in the moraine community (2.38 ± 0.64%DW) and outwash plain community (2.15 ± 0.55%DW).

Discrimination of Curcuma species from Asia using intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase.

Recently, Curcuma rhizome-related foods with claimed health benefits have been used worldwide; however, correct identification and quality assessment have not been conducted. Due to the wide distribution and morphological similarities of Curcuma species, the classification of some species is debated and nomenclature is inconsistent among countries. In this study, to elucidate specific molecular markers of medicinally used Curcuma species in Asia, and to solve the confusion on the reported botanical origin of crude drugs, molecular analysis based on the intron length polymorphism (ILP) in genes encoding diketide-CoA synthase and curcumin synthase and the trnK intron sequences was performed using 59 plant specimens and 42 crude drug samples from 13 Curcuma species, obtained from Asian countries. The ILP patterns of the respective species from both plant specimens and crude drug samples revealed high consistency in C. aromatica, C. zedoaria, C. phaeocaulis, C. aeruginosa, C. wenyujin, and C. zanthorrhiza, but showed intraspecies polymorphism in C. longa, C. kwangsiensis, C. amada, C. mangga and C. comosa. The C. longa specimens and samples were separated into three subgroups which were highly consistent with their geographical origins. Based on the ILP markers and the trnK intron sequences, the botanical origins of "Khamin oi" from Thailand were correctly determined to be C. longa or a hybrid between C. longa and other species, and "Wan narn kum" from Thailand and "Kasturi manjal" from India were correctly determined to be C. zanthorrhiza.

Capnographic monitoring during endoscopic submucosal dissection with patients under deep sedation: a prospective, crossover trial of air and carbon dioxide insufflations.

BACKGROUND AND AIMS: The safety of carbon dioxide (CO(2)) insufflation has not been fully established for esophageal and gastric endoscopic submucosal dissection (ESD) under deep sedation, because CO(2) retention is not only caused by CO(2) insufflation but also by the sedation level and the patient's respiratory status. To clarify the clinical safety of CO(2) insufflation, we conducted a crossover trial of air and CO(2) insufflations. METHODS: A total of 60 patients with early esophageal or gastric cancers underwent ESD during which insufflation was switched from CO(2) to air or from air to CO(2); transcutaneous partial pressure CO(2) (PtcCO(2)) was monitored in all patients. We also assessed respiratory function, arterial blood gas analysis, and smoking history. RESULTS: Although significant increases in PtcCO(2) from baseline were observed, there were no significant differences in PtcCO(2) levels during CO(2) insufflation compared with levels during air insufflation in groups that received CO(2) preceding air or air preceding CO(2). All patients underwent ESD safely without adverse events, including 20 patients with subclinical respiratory dysfunction. The sedation protocol was the only significant predictor of CO(2) retention, independent of CO(2) insufflation. CONCLUSIONS: CO(2) insufflation can be used as safely as air insufflation during ESD under deep sedation.

Composite constituent: lactucenyl acetate, a novel migrated lupane triterpenoid from Lactuca indica revision of structure of tarolupenyl acetate.

Lactucenyl acetate (1), a new member of migrated lupane triterpenoids was isolated from Lactuca indica and its structure was elucidated on the basis of spectral analyses. The structure of tarolupenyl acetate was revised as lup-19(21)-en-3ß-yl acetate (2).

Environmental and soil characteristics in Ephedra habitats of Uzbekistan.

In Uzbekistan, Ephedra distachya L., E. equisetina Bunge, E. foliata Boiss. ex C. A. Mey., E. lomatolepis Schrenk, and E. strobilacea Bunge show species specificity for habitat environments and physical and chemical characteristics of habitat soils. Furthermore, the relationship between soil characteristics and ephedrine and pseudoephedrine contents was examined. E. distachya was found growing from 80 to 200 m above sea level (a.s.l) in the Plateau Ustyurt on the desert steppe of cliffs on soil having relatively higher loss on ignition (19.8-33.8%) and water-soluble cations (Ca2+, 5.14-133.13; Mg2+, 0.85-3.18; and Na+, 2.27-8.33 mmol/100 g dry soil weight) than for other Ephedra habitats. E. strobilacea was found growing on the flat sandy Kyzylkum desert at 94 m a.s.l. and had habitat soil that was the driest with the lowest loss on ignition (2.9%) and highest Na+ (9.05 mmol/100 g dry soil weight) of all the Ephedra habitat soils. On dry steppe from 1054 to 1819 m a.s.l., E. foliata, E. lomatolepis, and E. equisetina formed not only a single community but also a complex community on constantly collapsing sandy gravel slope with relatively higher Ca2+ (3.40-17.44 mmol/100 g dry soil weight) soil content. Notably, E. equisetina grew on the dry steppe of constantly collapsing sandy gravel slopes, in rocky areas, on sandy gravel floodplains of rivers, and on stable humus soil at the base of coniferous trees in a wide range of habitats from dry steppe to coniferous forest zones at altitudes ranging from 1392 to 1819 m a.s.l., as reflected in the greater variability than for other Ephedra habitats in the parameters of loss on ignition (1.4-34.8%), pH (7.1-9.6), NO3- (0.08-35.17 mmol/100 g dry soil weight), Ca2+ (0.24-17.44 mmol/100 g dry soil weight), Mg2+ (not detected-1.25 mmol/100 g dry soil weight), and Na+ (0.13-5.19 mmol/100 g dry soil weight). Ephedrine alkaloids were not detectable in E. strobilacea, E. foliata, and E. lomatolepis. Almost all E. distachya contained only pseudoephedrine (1.25-1.59% of dry weight, %DW), while E. equisetina contained from 1.31 to 2.05%DW ephedrine and from 1.29 to 2.80%DW pseudoephedrine. Ephedrine and pseudoephedrine in E. equisetina showed a statistically significant negative correlation with soil Cl- and Mg2+, respectively.

Physical and chemical characteristics of soils in Ephedra gerardiana and E. pachyclada habitats of Kali Gandaki Valley in Central Nepal.

In the Kali Gandaki Valley in Central Nepal, Ephedra gerardiana and E. pachyclada show species specificity for physical and chemical characteristics of soils. Here, the relationship between soil characteristics and ephedrine and pseudoephedrine contents was examined. E. gerardiana grew in moist alpine scrub and upper alpine meadow from 3735 to 4156 m a.s.l., while E. pachyclada grew in the lower Caragana steppe and dry alpine scrub from 2629 to 3671 m a.s.l. The soil texture of E. gerardiana and E. pachyclada collection sites were classified as loam or sandy loam mainly composed of sand and silt. Loss on ignition (%) of soil in E. gerardiana habitats (28.4-35.0%) was markedly higher than for that in E. pachyclada habitats (14.2-17.2%). E. pachyclada soil (pH 8.4-9.2) was more alkaline than that for E. gerardiana (pH 8.5). The five ions (Cl-, SO42-, Ca2+, Mg2+, and Na+) in soil of E. pachyclada (Cl-, 0.01-18.97 mmol/100 g dry soil weight; SO42-, 1.95-83.33; Ca2+, 3.79-77.91; Mg2+, 1.28-27.9; Na+, 0.94-34.49) were markedly higher than those of E. gerardiana (Cl-, 0.18-0.29; SO42-, 0.07-0.08; Ca2+, 4.19-4.59; Mg2+, 0.22-0.58; Na+, 0.93-1.40). The main factor contributing to strongly alkali soils for each species was different between E. gerardiana and E. pachyclada: CaCO3 for E. gerardiana and CaSO4, MgSO4, NaCl, or a combination of these for E. pachyclada. The total ephedrine and pseudoephedrine content in E. gerardiana and E. pachyclada ranged from 1.67-1.88%DW and 1.95-4.80%DW, respectively. Both E. gerardiana and E. pachyclada were amenable for use a raw material source for extraction of ephedrine and pseudoephedrine, and the ephedrine content of both species showed a statistically significantly positive correlation with Mg2+ and Na+ contents of the soil.

Metabolome analysis of Ephedra plants with different contents of ephedrine alkaloids by using UPLC-Q-TOF-MS.

Metabolome analysis of four varieties of Ephedra plants, which contain different amounts of ephedrine alkaloids, was demonstrated in this study. The metabolites were comprehensively analyzed by using ultra performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF-MS) and the ephedrine alkaloids were also profiled. Subsequently, multivariate analyses of principal component analysis (PCA) and batch-learning self-organizing mapping (BL-SOM) analysis were applied to the raw data of the total ion chromatogram (TIC). PCA was performed to visualize the fingerprints characteristic for each Ephedra variant and the independent metabolome clusters were formed. The metabolite fingerprints were also visualized by BL-SOM analysis and were displayed as a lattice of colored individual cells which was characteristic for each Ephedra variant. BL-SOM analysis was also used for identification of chemical marker peaks because the information assigned to a cell represented either increases or decreases in peak intensities. Using this analysis, ephedrine alkaloids were successfully selected from the TICs as chemical markers for each Ephedra variant and this result suggested that BL-SOM analysis was an effective method for the selection of marker metabolites. We report our study here as a practical case of metabolomic study on medicinal resources.

Screening for Plant Volatile Emissions with Allelopathic Activity and the Identification of L-Fenchone and 1,8-Cineole from Star Anise (Illicium verum) Leaves.

One hundred and thirty-nine medicinal plant species were screened for their allelopathic activity through volatile emissions using Lactuca sativa as a test plant. Volatile emissions from the leaves of star anise (Illicium verum) showed the highest inhibition (100%) on the radicle and hypocotyl growth. Using headspace gas collection and gas chromatography-mass spectrometry (GC-MS), seven major volatile compounds from the leaves of star anise, including α-pinene, ß-pinene, camphene, 1,8-cineole, D-limonene, camphor, and L-fenchone were detected. To determine volatile compounds that may contribute to the inhibitory activity of star anise, the allelopathic potential of individual volatiles from star anise was evaluated using the cotton swab bioassay. The EC50 was calculated for each of the seven identified compounds. L-fenchone showed the strongest growth inhibitory activity (EC50 is 1.0 ng/cm3 for radicle and hypocotyl growth of lettuce), followed by 1,8-cineole, and camphene. This is the first report that L-fenchone could be an important volatile allelochemical from the leaves of star anise. From the actual concentration of each volatile compound in headspace and EC50 value, we concluded that the four volatile compounds, including L-fenchone, 1,8-cineole, ß-pinene, and camphene are the most important contributors to the volatile allelopathy of star anise.

Effects of two terpene alcohols on the antibacterial activity and the mode of action of farnesol against Staphylococcus aureus.

We have studied changes in the antibacterial activity and the mode of action of farnesol against Staphylococcus aureus when two terpene alcohols with an aliphatic carbon chain were added, individually, to a bacterial suspension that contained farnesol. Geraniol increased the growth-inhibitory activity of farnesol, but suppressed its ability to damage cell membranes, which is one of the predominant features of the growth-inhibitory activity of farnesol. Geranylgeraniol decreased the growth-inhibitory activity of farnesol and also suppressed its cell-damaging activity. It is possible that the presence of a terpene alcohol can both enhance and suppress the antibacterial activity of farnesol, and even change its mode of action. Thus, it is important to study not only the antibacterial activity of each constituent of an essential oil but also the interactions between them in efforts to characterize the antibacterial activity of the essential oil.

A new bis-seco-abietane diterpenoid from Hyptis crenata Pohl ex Benth.

A new, highly oxidized, bis-seco-abietane diterpenoid named hyptisolide A (1) was isolated from Hyptis crenata Pohl ex Benth. Its structure and stereochemistry were elucidated on the basis of data obtained by HRESIMS, NMR, and X-ray diffraction analyses, and its absolute configuration was determined with vibrational circular dichroism spectroscopy. By reporter gene assay, 1 was demonstrated to induce cAMP-responsive element-dependent transcription in Neuro2A cells.

The identification of vitamin E homologues in medicinal plant samples using ESI(+)-LC-MS3.

The aim of this study was to elucidate the presence of vitamin E homologues in medicinal plants. To identify various homologues in the matrix of medicinal plant samples, a method for simultaneous determination was developed using ESI(+)-LC-MS3. A complete separation of each homologue was achieved within 20 min using a PFP column and an isocratic elution system of water/methanol (10:90, v/v) at a flow rate of 0.5 mL/min. The ESI-MS condition for each homologue was optimized, and the m/z value and the fragmentation pathway of each homologue were summarized. This LC-MS3 method made it possible to detect the homologues without the effect of matrix; therefore, high sensitive analysis was established, and then, the MS3 makes it possible to extract from plants with methanol only. The LC-MS3 method was applied to identify the eight vitamin E homologues in 11 medicinal plants.