Diterpenoids from Torreya grandis and their cytotoxic activities.

Eight previously undescribed diterpenoids, along with eleven previously reported analogues, were obtained from the supercritical CO2 extracts of Torreya grandis aril. The structures of these compounds were elucidated based on HRESIMS, NMR, ECD, and single-crystal X-ray diffraction data. In the MTT assay, compound 18 exhibited significant inhibitory effects on two human colon cancer cell lines, HT-29 and HCT 116 cells, with IC50 values of 7.37 µM and 6.55 µM, respectively. It was found that compound 18 induced apoptosis and significantly inhibited the migration of HCT 116 colon cancer cells in a concentration-dependent manner.

Amentotaxus×hybridia (Taxaceae), a new natural Amentotaxus hybrid from southeast Yunnan province, China.

During floristic surveys of Taxaceae in Hekou County, Yunnan Province, China, a putative natural hybrid between A.yunnanensis H.L. Li and A.hekouensis L.M. Gao was collected. Morphological and molecular evidence confirms its status as a natural hybrid. Amentotaxus×hybridia L.M. Gao has linear or linear-lanceolate leaves 6-13 cm × 1.0-1.5 cm, white stomatal bands with 34-40 rows on abaxial side, 2.5-3.5 mm, slightly wider than leaf margins; 3-6 seeds borne at the base of the branchlet, peduncle 1.3-1.6 cm long with 3-4 rows of persistent basal bracts.

Amenyunnaosides A-C, Three New Neolignans Isolated from Amentotaxus yunnanensis and Their Anti-inflammatory Activities.

Phytochemical study on the leaves of Amentotaxus yunnanensis led to the isolation of seventeen phenolic compounds including sixteen neolignans and lignans, and one flavone glycoside. Three among the isolates were previously unreported neolignans and named as amenyunnaosides A-C, respectively. Their structures were elucidated by extensive analyses of HR-ESI-MS, 1D and 2D NMR, and ECD spectra. The isolated neolignans potentially inhibited NO production in LPS-activated RAW264.7 cells with their IC50 values ranging from 11.05 to 44.07 µM, compared to that of the positive control compound, dexamethasone, IC50 value of 16.93 µM. Additionally, amenyunnaoside A dose-dependently reduced production of IL-6 and COX-2 but did not effect to that of TNF-α at concentrations of 0.8, 4, and 20 µM.

Antimicrobial abietane-type diterpenoids from Torreya grandis.

Twelve undescribed abietane-type diterpenoids, along with ten known analogues were isolated from the twigs and leaves of Torreya grandis var. merrillii Hu. Their structures were characterized by spectroscopic data analyses, single-crystal X-ray diffraction, and ECD spectra. Torgranols A-C possess three different architectures shaped via a common 6,7-seco-procedure and subsequent ring formations. In particular, torgranol A represents the first example of a 6,7-seco-abietane diterpenoid featuring a unique oxygen bridge between C-3 and C-6. The biosynthetic pathways for torgranols A-C were proposed. Some compounds displayed antimicrobial activities against Mycobacterium tuberculosis and/or Staphylococcus aureus.

Phytochemical and biological studies on rare and endangered plants endemic to China. Part XXII. Structurally diverse diterpenoids from the leaves and twigs of the endangered conifer Torreya jackii and their bioactivities.

A phytochemical investigation on the MeOH extract of the leaves and twigs of the endangered conifer Torreya jackii Chun led to the isolation and characterization of 21 structurally diverse diterpenoids. Among them, six are previously undescribed, including four abietane-type (torreyins A-D, resp.) and two labdane-type diterpenoids (torreyins E and F). Their structures and absolute configurations were determined by a combination of spectroscopic methods, calculated/experimental electronic circular dichroism (ECD) data, and single-crystal X-ray diffraction analyses. In particular, torreyins A-C are rare 11,12-seco-abietane type diterpenoids possessing a dilactone moiety, and their biosynthetic pathway starting from a co-occurring abietane derivative (i.e., cyrtophyllone B) was briefly proposed. Among the isolates, 7-oxo-dehydroabietic acid and 15-methoxy-7,13-abietadien-18-oic acid showed considerable inhibitory effects against acetyl-coenzyme A carboxylase 1 (ACC1) and protein tyrosine phosphatase 1 B (PTP1B), with IC50 values of 3.1 and 6.8 µM, respectively.

Carotenoid profiles of red- and yellow-colored arils of cultivars of Taxus baccata L. and Taxus × media Rehder.

The botanical delimitation of Taxus species and cultivars may be facilitated by characterizing the pigment profiles of their red- and yellow-fleshed arils. Therefore, we determined genuine carotenoid profiles of differently colored arils of seven defined cultivars of Taxus baccata L. and Taxus × media Rehder. In-depth HPLC-DAD-ESI/APCI-MSn analyses revealed the presence of 43 carotenoids. Exceptional retro-carotenoids dominated the profiles of all samples assessed. Rhodoxanthin (E/Z)-isomers were predominant in the red-colored arils, resulting in a rather unusual abundance of carotenoid isomers as expressed by ratios of up to 1.3:2.0:0.9 between (all-E)-, (6Z)-, and (6Z,6'Z)-rhodoxanthin, respectively. By contrast, the uncommon yellow arils of Taxus baccata L. 'Lutea' mainly contained eschscholtzxanthin (E/Z)-isomers and esters. Total carotenoid concentrations ranged from 17.00 to 58.78 µg/g fresh weight across all samples assessed. Highest total rhodoxanthin concentrations of 51.33 ± 0.46 µg/g fresh weight were obtained from the red arils of Taxus × media Rehder 'Hicksii'. Overall, Taxus arils represent a promising source of carotenoids and, in particular, of retro-carotenoids with exceptional molecular structures and extraordinary absorption properties.

Plastome phylogenomics of Cephalotaxus (Cephalotaxaceae) and allied genera.

BACKGROUND AND AIMS: Cephalotaxus is a paleo-endemic genus in East Asia that consists of about 7-9 conifer species. Despite its great economic and ecological importance, the relationships between Cephalotaxus and related genera, as well as the interspecific relationships within Cephalotaxus, have long been controversial, resulting in contrasting taxonomic proposals in delimitation of Cephalotaxaceae and Taxaceae. Based on plastome data, this study aims to reconstruct a robust phylogeny to infer the systematic placement and the evolutionary history of Cephalotaxus. METHODS: A total of 11 plastomes, representing all species currently recognized in Cephalotaxus and two Torreya species, were sequenced and assembled. Combining these with previously published plastomes, we reconstructed a phylogeny of Cephalotaxaceae and Taxaceae with nearly full taxonomic sampling. Under a phylogenetic framework and molecular dating, the diversification history of Cephalotaxus and allied genera was explored. KEY RESULTS: Phylogenetic analyses of 81 plastid protein-coding genes recovered robust relationships between Cephalotaxus and related genera, as well as providing a well-supported resolution of interspecific relationships within Cephalotaxus, Taxus, Torreya and Amentotaxus. Divergence time estimation indicated that most extant species of these genera are relatively young, although fossil and other molecular evidence consistently show that these genera are ancient plant lineages. CONCLUSIONS: Our results justify the taxonomic proposal that recognizes Cephalotaxaceae as a monotypic family, and contribute to a clear-cut delineation between Cephalotaxaceae and Taxaceae. Given that extant species of Cephalotaxus are derived from recent divergence events associated with the establishment of monsoonal climates in East Asia and Pleistocene climatic fluctuations, they are not evolutionary relics.

Characterization of the complete chloroplast genome of Taxus wallichiana as the medicinal plant from China.

Taxus wallichiana is a member of the family Taxaceae, which is a unique and endangered species in China and is widely used for ornamental, material and medicinal purposes. The complete chloroplast genome of T. wallichiana was found to possess a total size of 128,168 bp. The GC content of T. wallichiana chloroplast genome sequence is 37.3%, the overall nucleotide composition of chloroplast genome sequence is: A of 30.7%, T of 32.0%, C of 19.0% and G of 18.3%. The total of 116 genes were successfully annotated, which contained 83 protein-coding genes, 29 transfer RNA genes, and 4 ribosomal RNA genes. The ML phylogenetic analysis result showed that T. wallichiana was closely related to Taxus baccata in the phylogenetic relationship using the neighbour-joining (NJ) method in this study.

Exploiting the Biosynthetic Potency of Taxol from Fungal Endophytes of Conifers Plants; Genome Mining and Metabolic Manipulation.

Endophytic fungi have been considered as a repertoire for bioactive secondary metabolites with potential application in medicine, agriculture and food industry. The biosynthetic pathways by fungal endophytes raise the argument of acquisition of these machineries of such complex metabolites from the plant host. Diterpenoids "Taxol" is the most effective anticancer drug with highest annual sale, since its discovery in 1970 from the Pacific yew tree, Taxus brevifolia. However, the lower yield of Taxol from this natural source (bark of T. brevifolia), availability and vulnerability of this plant to unpredicted fluctuation with the ecological and environmental conditions are the challenges. Endophytic fungi from Taxus spp. opened a new avenue for industrial Taxol production due to their fast growth, cost effectiveness, independence on climatic changes, feasibility of genetic manipulation. However, the anticipation of endophytic fungi for industrial Taxol production has been challenged by the loss of its productivity, due to the metabolic reprograming of cells, downregulating the expression of its encoding genes with subculturing and storage. Thus, the objectives of this review were to (1) Nominate the endophytic fungal isolates with the Taxol producing potency from Taxaceae and Podocarpaceae; (2) Emphasize the different approaches such as molecular manipulation, cultural optimization, co-cultivation for enhancing the Taxol productivities; (3) Accentuate the genome mining of the rate-limiting enzymes for rapid screening the Taxol biosynthetic machinery; (4) Triggering the silenced rate-limiting genes and transcriptional factors to activates the biosynthetic gene cluster of Taxol.

Sites of biosynthesis and storage of Taxol in Taxus media (Rehder) plants: Mechanism of accumulation.

Taxol is a cytotoxic agent against various types of cancers. The cytotoxic activities of Taxol can be extended to its synthesizing plant. Here, Taxol is shown to have special synthesis, storage and transport mechanisms that avoid the toxic effects on its source plant. The sites of Taxol biosynthesis, transport and storage were revealed by quantification of plant Taxol, its intermediate baccatin III, the polyphenol side chain precursor , gene expression analysis of the major Taxol biosynthetic genes and in situ immuno-labeling. Although the biosynthesis of Taxol was limited by the expression of its biosynthetic genes and the presence of baccatin III, its presence did not correlate to baccatin III accumulation, nor to the expression of biosynthetic genes. However, Taxol presence positively correlated to polyphenol accumulation (late stage in Taxol assembly) and the resin-like hydrophobic bodies (HB, storage organelles). These results indicate that the presence of Taxol requires two complementary steps, biosynthesis followed by storage. Each step is limited by the availability of different precursors, which differ in their localization within the plant. Thus, the sites of biosynthesis, transport and storage of Taxol are different. Taxus media (Rehder) plant wood showed high concentrations of baccatin III and the expression of biosynthetic genes. However, the concentrations of Taxol, polyphenol and HB were very high in the plant outer layers including phloem and dead bark (rhytidome). Furthermore, in situ immuno-labeling showed that taxadiene synthase (the rate-limiting enzyme in Taxol biosynthesis) was mainly found in the wood, while Taxol primarily localized to the outer tissues. Conclusively, wood can be considered as the site of Taxol biosynthesis. Our data also propose that Taxol then accumulates into HB in order to permit its transport within the living plant tissues without causing toxic effects. This is followed by Taxol storage in the outer tissues including phloem and dead bark.

A Review of Conifers in Iran: Chemistry, Biology and their Importance in Traditional and Modern Medicine.

Cupressus sempervirens, Platycladus orientalis, Juniperus communis, J. excelsa, J. foetidissima, J. polycarpos var. turcomanica, J. sabina, and Taxus baccata are conifers in Iran. These plants have a long reputation in different systems of traditional medicines for a variety of diseases. This review aims to provide comprehensive and up-to-date information about the ethnopharmacological uses, chemical constituents, and pharmacology of these conifers. Furthermore this study comprises a bibliographical survey of major Islamic Traditional Medicine (ITM) books regarding different medical aspects of these species. A literature search was conducted on the applications of these conifers both in traditional and modern medicines by referencing traditional textbooks and scientific databases. Ethnobotanical literature review indicates that various parts of the plants including cones, berries, leaves, bark, wood, and resin have been used for a broad spectrum of applications. In Iran, C. sempervirens, J. sabina, J. communis, and T. baccata have been used traditionally for the treatment of urinary, digestive, nervous, respiratory, and integumentary systems-related problems. The phytochemical constituents of these plants can be divided into two main categories: volatile and non-volatile components, all dominated by terpenes. Considering the pharmacological and clinical evidence, while some of the traditional applications of these plants are supported by modern medicine, implying the value of the traditional and folklore knowledge for finding new lead compounds in drug discovery, some have remained unexamined showing the need for much more studies in this regard.

The complete chloroplast genome of Torreya nucifera (Taxaceae) and phylogenetic analysis.

Torreya nucifera (L.) Siebold & Zucc. (Taxaceae) is a tertiary relict tree species with a distribution that is limited to South Korea and Japan. In the present study, the complete chloroplast (cp) genome of T. nucifera was sequenced and analyzed. The genome was 136,985 bp in length and contained 118 genes, including 82 protein-coding genes, 33 tRNA genes, and 4 rRNA genes. Fifteen of the genes contained a single intron, whereas ycf3 contained two introns and there were no inverted repeat sequences in the genome. Phylogenetic analysis supported the monophyly of Torreya species within the Taxaceae and T. nucifera was closely related to its congener T. grandis.

Development of microsatellite markers for a Chinese endemic plant, Torreya yunnanensis (Taxaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed in Torreya yunnanensis (Taxaceae) to investigate the genetic diversity, phylogeography, and population structure of the species. METHODS AND RESULTS: Sixteen primer sets were identified using Illumina 2 × 100-bp paired-end sequencing and bioinformatic screening. Most primers also amplified in T. fargesii, T. grandis, T. jackii, and T. nucifera. CONCLUSIONS: These results indicate the utility of these microsatellite markers in T. yunnanensis for future studies of molecular ecology as well as their applicability across the genus.

Development and characterization of 23 polymorphic microsatellite loci for Amentotaxus argotaenia (Taxaceae), a relict vulnerable species.

PREMISE OF THE STUDY: New microsatellite markers were developed for the vulnerable conifer species Amentotaxus argotaenia (Taxaceae) to investigate population genetic variation and the effects of environmental heterogeneity on genetic structure. METHODS AND RESULTS: A total of 27 microsatellite loci were developed from A. argotaenia through a Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, of which 23 were polymorphic. These markers yielded 1-13 alleles and 1.0-7.9 effective alleles per locus; levels of observed and expected heterozygosity varied from 0.000-1.000 and 0.000-0.873, respectively. In total, 18 of the markers were transferable to the related species A. yunnanensis. CONCLUSIONS: These polymorphic markers are a valuable genetic resource for investigating population genetic variation and the potential for local adaptation in A. argotaenia.

Diversity and evolution of leaf anatomical characters in Taxaceae s.l.-fluorescence microscopy reveals new delimitating characters.

Taxaceae s.l. comprise six genera (including Cephalotaxus) and about 35 species; The present study aims to give new insights into the evolution of this family, especially into the phylogenetic position of Cephalotaxus. Moreover, only little is known about comparative leaf anatomy of this family and this study aims to expose and interpret the diversity and evolution of leaf anatomical characters and to assess their applicability to identify taxa at the generic and species level. A detailed phylogeny was reconstructed, using both maximum likelihood and Bayesian inference, with a combined dataset of four molecular markers from the plastid and nuclear genomes. Leaf sections from 132 specimens, representing 32 species and four varieties (fresh and herbarium material) were inspected, using fluorescence microscopy. Ancestral characters were reconstructed using Mesquite. The phylogenetic analyses provided full support for Cephalotaxus as sister group to Taxaceae s.str. Within the latter, two monophyletic tribes Taxeae (comprising Austrotaxus, Pseudotaxus, and Taxus) and Torreyeae (comprising Amentotaxus and Torreya) were fully supported. Fluorescence microscopy was shown to be very useful for identifying leaf tissues and their constitution. We were able to show that particularly sclerified tissues have highest potential for the discrimination of both freshly collected samples and rehydrated herbarium specimens at the generic and species level. A correlation between the presence of different sclereid types could be shown and sclereids were hypothesized to pose a primitive trait in the evolution of Taxaceae s.l. New identification keys were generated on the basis of leaf anatomical characters. The microscopic method presented here is applicable for further studies within gymnosperms and probably in angiosperms, as well.

The complete chloroplast genome sequence of an conifer plant Torreya grandis (Pinales, Taxaceae).

Chinese Nutmeg Tree (Torreya grandis) is a species of conifer in Taxaceae, which has a wide range in eastern and south-eastern China. Torreya grandis, endemic to eastern and south-eastern China, is a large tree, which own important economic value. Here, we report the complete chloroplast (cp) genome sequence and the cp genomic features of T. grandis. The genome was 136,949 bp long with 117 genes comprising of 82 protein-coding genes, 31 tRNA genes, and 4 rRNA genes. Phylogenetic analysis suggested that Torreya species formed a monophyletic clade within the Taxaceae family, and T. jackii is at the base of the tree and T. grandis and T. fargesii are sister groups of the inner clade.

Chloroplast microsatellite markers for Pseudotaxus chienii developed from the whole chloroplast genome of Taxus chinensis var. mairei (Taxaceae).

PREMISE OF THE STUDY: Pseudotaxus chienii (Taxaceae) is an old rare species endemic to China that has adapted well to ecological heterogeneity with high genetic diversity in its nuclear genome. However, the genetic variation in its chloroplast genome is unknown. METHODS AND RESULTS: Eighteen chloroplast microsatellite markers (cpSSRs) were developed from the whole chloroplast genome of Taxus chinensis var. mairei and successfully amplified in four P. chienii populations and one T. chinensis var. mairei population. Of these loci, 10 were polymorphic in P. chienii, whereas six were polymorphic in T. chinensis var. mairei. The unbiased haploid diversity per locus ranged from 0.000 to 0.641 and 0.000 to 0.545 for P. chienii and T. chinensis var. mairei, respectively. CONCLUSIONS: The 18 cpSSRs will be used to further investigate the chloroplast genetic structure and adaptive evolution in P. chienii populations.

Development of polymorphic microsatellite markers for Japanese yew, Taxus cuspidata, and T. cuspidata var. nana (Taxaceae).

PREMISE OF THE STUDY: Taxus cuspidata (Taxaceae), which is well known for the effective anticancer metabolite paclitaxel (e.g., taxol), is an evergreen needle-leaved tree widely distributed in eastern Eurasia including Japan. We developed 15 microsatellite markers from this species and confirmed their utility for the dwarf variety nana, which is common in alpine regions along the Sea of Japan. METHODS AND RESULTS: Thirteen polymorphic loci were characterized for genetic variation in three populations of T. cuspidata. The number of alleles per locus ranged from 11 to 31, with an average of 18.5; the expected heterozygosity ranged from 0.78 to 0.95, with an average of 0.89. All loci were successfully amplified in T. cuspidata var. nana and showed high polymorphism. CONCLUSIONS: These markers will be useful for investigating speciation and range formation of T. cuspidata in Japan, and the results will provide crucial information for the conservation of Taxus species.

Src/Syk/IRAK1-targeted anti-inflammatory action of Torreya nucifera butanol fraction in lipopolysaccharide-activated RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Seed of Torreya nucifera (L.) Siebold & Zucc is used to treat several diseases in Asia. Reports document that T. nucifera has anti-cancer, anti-inflammatory, anti-oxidative activities. In spite of numerous findings on its pharmacological effects, the understanding of the molecular inhibitory mechanisms of the plant remains to be studied. Therefore, we aimed to explore in vitro anti-inflammatory mechanisms of ethyl acetate fraction (Tn-EE-BF) prepared from the seed of T. nucifera in LPS-stimulated macrophage inflammatory responses. MATERIALS AND METHODS: For this purpose, we measured nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated macrophages. Additionally, using RT-PCR, luciferase reporter gene assay, immunoblotting analysis, and kinase assay, the levels of inflammatory genes, transcription factors, and inflammatory signal-regulatory proteins were investigated. Finally, the constituent of Tn-EE-BF was identified using HPLC. RESULTS: Tn-EE-BF inhibits NO and PGE2 production and also blocks mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in a dose dependent manner. Tn-EE-BF reduces nuclear levels of the transcriptional factors NF-κB (p65) and AP-1 (c-Jun and FRA-1). Surprisingly, we found that Tn-EE-BF inhibits phosphorylation levels of Src and Syk in the NF-κB pathway, as well as, IRAK1 at the protein level, part of the AP-1 pathway. By kinase assay, we confirmed that Src, Syk, and IRAK1 are suppressed directly. HPLC analysis indicates that arctigenin, amentoflavone, and quercetin may be active components with anti-inflammatory activities. CONCLUSION: Tn-EE-BF exhibits anti-inflammatory activities by direct inhibition of Src/Syk/NF-κB and IRAK1/AP-1.

The complete chloroplast genome of Torreya fargesii (Taxaceae).

The complete chloroplast genome sequence of Torreya fargesii (Taxaceae), a relic plant endemic to China, is presented in this study. The genome is 137 075 bp in length, with 35.47% average GC content. One copy of the large inverted repeats is lost from this genome. The T. fargesii chloroplast genome encodes 118 unique genes, in which trnI-CAU, trnQ-UUG, trnN-GUU are duplicated. Protein-coding, tRNA and rRNA genes represent 54.7%, 1.9% and 3.4% of the genome, respectively. There are 17 intron-containing genes, of which 6 are tRNA genes. A maximum likelihood phylogenetic analysis revealed a strong sister relationship between Torreya and Amentotaxus.