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.

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.

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.

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).

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.

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.

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.

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.

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.

High-performance liquid chromatography analysis of capsaicin content in 16 Capsicum fruits from Nepal.

Capsicum fruit, a popular spice as chili pepper, is an important ingredient of the formulations used in traditional medicines. Moreover, Capsicum fruit is listed as an official drug in several pharmacopoeias. Capsaicin, the most abundant component in Capsicum fruit, exhibits its therapeutic and adverse effects in a dose-dependent manner. Therefore, the known capsaicin content is the prerequisite for optimizing any formulation based on Capsicum fruit as a crude drug. We studied 16 samples of Capsicum fruits grown at different altitudes in Nepal and determined their capsaicin content by high-performance liquid chromatography. The capsaicin content was found to range from 2.19 to 19.73 mg/g of dry weight of Capsicum fruits. Capsaicin content in pericarp was found to be higher than in seeds. No correlation was found between the shape or size of the fruits and its capsaicin content. Our findings indicate that many of the formulations prepared from Capsicum fruit, even as described in pharmacopoeias, may vary in their strength, therapeutic activity, and possible side effects if the capsaicin content in Capsicum fruit is not standardized.

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.

Leishmanicidal active constituents from Nepalese medicinal plant Tulsi (Ocimum sanctum L.).

In the course of screening leishmanicidal active compounds from Asian and South American medicinal plants, a Nepalese medicinal plant, Tulsi (Ocimum sanctum L.), showed strong activity. We therefore studied the isolation and structural elucidation of the active constituents from O. sanctum L. From the ethyl acetate soluble fraction of the plant, seven new novel neolignan derivatives were isolated along with 16 known compounds. The structures of the new compounds (1-7) were elucidated as 6-allyl-3',8-dimethoxy-flavan-3,4'-diol (1), 6-allyl-3-(4-allyl-2-methoxyphenoxy)-3',8-dimethoxyflavan-4'-ol (2), 5-allyl-3-(4-allyl-2-methoxyphenoxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran (3), 1,2-bis(4-allyl-2-methoxyphenoxy)-3-(4-hydroxy-3-methoxyphenyl)-3-methoxypropane (4), 1-(4-hydroxy-3-methoxyphenyl)-1,2,3-tris(4-allyl-2-methoxyphenoxy)propane (5), 1-allyl-4-(5-allyl-2-hydroxy-3-methoxyphenoxy)-3-(4-allyl-2-methoxyphenoxy)-5-methoxybenzene (6), and 3-(5-allyl-2-hydroxy-3-methoxyphenyl)-1-(4-hydroxy-3-methoxyphenoxy)-prop-1-ene (7) by means of (1)H-NMR, (13)C-NMR, and 2D-NMR spectral data. Some of these compounds showed leishmanicidal activity.

Molecular identification of "Chuanxiong" by nucleotide sequence and multiplex single base extension analysis on chloroplast trnK gene.

Chloroplast trnK gene sequences of Cnidium officinale and Ligusticum chuanxiong were determined to establish an effective method for identifying Japanese Senkyu and Chinese Chuanxiong, the two which have the same drug name in Chinese characters, similar external feature, but different botanical origins. Three sites of nucleotide differences were found between these 2 species at positions 767,924 and 964 from upstream in trnK gene sequence, allowing molecular identification of the two plants and crude drugs. Further, three kinds of specific primers of 14 mer, 23 mer and 30 mer long were designed to detect these 3 sites of marker nucleotides. By using multiplex single base extension (MSBE) analysis with the 3 specific primers, C. officinale and L. chuanxiong could be distinguished clearly by the electrophoretograms, where 3 peaks with different color of ddTMP, ddCMP and ddTMP were observed in case of C. officinale and those of ddGMP, ddAMP and ddGMP in L. chuanxiong. Moreover, trnK gene sequence of "Dongxiong," a kind of Chuanxiong cultivated in Northeast China, suggested that its botanical origin was C. officinale.

Studies on nepalese crude drugs. XXVIII. Chemical constituents of Bhote Khair, the underground parts of Eskemukerjea megacarpum HARA.

From the underground parts of Eskemukerjea megacarpum HARA, two new stilbenes (14, 15) were isolated, together with a known coumarin, 5,7-dihydroxycoumarin (1), a tyramine derivative, trans-feruloyltyramine (2), two pyrogallol derivatives, gallic acid (3) and beta-glucogallin (4), four flavonoids, trifolin (5), hyperin (6), myricetin 3-O-beta-D-galactopyranoside (7), and myricitrin (8), five stilbenes, resveratorol (9), astringenin (10), piceid (11) astringin (12), and resveratorol 3-O-beta-D-(6-O-galloyl)glucopyranoside (13), a flavan-3-ol, (-)-epigallocatechin 3-O-gallate (16), two proanthocyanidins, catechin-(4alpha-->8)-epigallocatechin 3-O-gallate (17) and epicatechin 3-O-gallate-(4beta-->8)-epigallocatechin 3-O-gallate (18), and an anthocyanin, idaein (19). Compounds 14 and 15 were identified as (E)-3,5,3',4'-tetrahydroxystilbene 3-O-beta-D-(6-O-galloyl)glucopyranoside and (E)-3,5,4'-trihydroxystilbene 3-O-beta-D-(6-O-galloyl)glucopyranoside, respectively, based on spectral and chemical data.

Glycosides of Atractylodes lancea.

Five sesquiterpenoid glycosides (two guaiane-type glycosides and three eudesmane-type glucosides) and a glucoside of an acetylene derivative were newly isolated from the water-soluble portion of the methanolic extract of Atractylodes lancea rhizome together with 26 known compounds. Their structures were characterized as atractyloside A 14-O-beta-D-fructofuranoside, (1S,4S,5S,7R,10S)-10,11,14-trihydroxyguai-3-one 11-O-beta-D-glucopyranoside, (5R,7R,10S)-isopterocarpolone beta-D-glucopyranoside, cis-atractyloside I, (2R,3R,5R,7R,10S)-atractyloside G 2-O-beta-D-glucopyranoside, and (2E,8E)-2,8-decadiene-4,6-diyne-1,10-diol 1-O-beta-D-glucopyranoside on the basis of chemical and spectroscopic investigation. The presence of six characteristic guaiane-type glucosides in both rhizomes of A. lancea and Atractylodes japonica suggested a close chemotaxonomic relationship between them.

Glycosides of Atractylodes japonica.

From the water-soluble portion of the methanol extract of the fresh rhizome of Atractylodes japonica, five new sesquiterpenoid glycosides, including a compound having a secoguaiane skeleton, and a new aromatic compound glycoside were isolated together with ten known compounds. Their structures were clarified by spectral investigation.