Cephalotaxus Sinensis Research Articles

Functional Characterization and Cyclization Mechanism of a Diterpene Synthase Catalyzing the Skeleton Formation of Cephalotane‐Type Diterpenoids

AbstractCsCTS, a new diterpene synthase from Cephalotaxus sinensis responsible for forming cephalotene, the core skeleton of cephalotane‐type diterpenoids with a highly rigid 6/6/5/7 tetracyclic ring system, was functionally characterized. The stepwise cyclization mechanism is proposed mainly based on structural investigation of its derailment products, and further demonstrated through isotopic labeling experiments and density functional theory calculations. Homology modeling and molecular dynamics simulation combined with site‐directed mutagenesis revealed the critical amino acid residues for the unique carbocation‐driven cascade cyclization mechanism of CsCTS. Altogether, this study reports the discovery of the diterpene synthase that catalyzes the first committed step of cephalotane‐type diterpenoid biosynthesis and delineates its cyclization mechanism, laying the foundation to decipher and artificially construct the complete biosynthetic pathway of this type diterpenoids.

Functional Characterization and Cyclization Mechanism of a Diterpene Synthase Catalyzing the Skeleton Formation of Cephalotane-Type Diterpenoids.

CsCTS, a new diterpene synthase from Cephalotaxus sinensis responsible for forming cephalotene, the core skeleton of cephalotane-type diterpenoids with a highly rigid 6/6/5/7 tetracyclic ring system, was functionally characterized. The stepwise cyclization mechanism is proposed mainly based on structural investigation of its derailment products, and further demonstrated through isotopic labeling experiments and density functional theory calculations. Homology modeling and molecular dynamics simulation combined with site-directed mutagenesis revealed the critical amino acid residues for the unique carbocation-driven cascade cyclization mechanism of CsCTS. Altogether, this study reports the discovery of the diterpene synthase that catalyzes the first committed step of cephalotane-type diterpenoid biosynthesis and delineates its cyclization mechanism, laying the foundation to decipher and artificially construct the complete biosynthetic pathway of this type diterpenoids.

Natural product drupacine acting on a novel herbicidal target shikimate dehydrogenase

Natural products are one of the important sources for the creation of new pesticides. Drupacine ((1R,11S,12S,13R,15S)-13-methoxy-5,7,21-trioxa-19-azahexacyclo[11.7.1.02,10.04,8.011,15.015,19]henicosa-2,4(8),9-trien-12-ol), isolated from Cephalotaxus sinensis (Chinese plum-yew), is a potent herbicidal compound containing an oxo-bridged oxygen bond structure. However, its molecular target still remains unknown. In this study, the targets of drupacine in Amaranthus retroflexus were identified by combining drug affinity responsive target stability (DARTS), cellular thermal shift assay coupled with mass spectrometry (CETSA MS), RNA-seq transcriptomic, and TMT proteomic analyses. Fifty-one and sixty-eight main binding proteins were identified by DARTS and CETSA MS, respectively, including nine co-existing binding proteins. In drupacine-treated A. retroflexus seedlings we identified 1389 up-regulated genes and 442 down-regulated genes, 34 up-regulated proteins, and 194 down-regulated proteins, respectively. Combining the symptoms and the biochemical profiles, Profilin, Shikimate dehydrogenase (SkDH), and Zeta-carotene desaturase were predicted to be the drupacine potential target proteins. At the same time, drupacine was found to bind SkDH stronger by molecular docking, and its inhibition on ArSkDH increased with the treatment concentration increase. Our results suggest that the molecular target of drupacine is SkDH, a new herbicide target, which lay a foundation for the rational design of herbicides based on new targets from natural products and enrich the target resources for developing green herbicides.

Molecular Networking Accelerated Discovery of Biflavonoid Alkaloids from Cephalotaxus sinensis.

Four undescribed biflavonoid alkaloids, sinenbiflavones A-D, were isolated from Cephalotaxus sinensis using a MS/MS-based molecular networking guided strategy. Their structures were elucidated by series of spectroscopic methods (HR-ESI-MS, UV, IR, 1D, and 2D NMR). Sinenbiflavones A-D are the first examples of amentoflavone-type (C-3'-C-8'') biflavonoid alkaloids. Meanwhile, sinenbiflavones B and D are the unique C-6-methylated amentoflavone-type biflavonoid alkaloids. Sinenbiflavone D showed weak SARS-CoV-2 3CLpro inhibitory activity with 43 % inhibition rate at 40 μM.

Cytotoxic alkaloids from the twigs and leaves of Cephalotaxus sinensis.

Twelve new Cephalotaxus alkaloids (1-12) and nine known analogues (13-21) were isolated and identified from the twigs and leaves of Cephalotaxus sinensis. The structures of the new compounds (1-12) were elucidated by extensive spectroscopic analysis and single-crystal X-ray diffraction analysis. Cephalosine H (8) is the third example of an alkaloid containing the cephalolancine skeleton. Cephalosines J and K (10 and 11) are the rare natural Δ(2)1-alkene-6-hydroxyl homoerythrina-type alkaloids isolated from the Cephalotaxus genus. The racemization of cephalotaxine-type alkaloids is discussed. Alkaloids 6, 7, 11, 16, 18 and 19 exhibited broad and potent cytotoxicities against five human cancer cell lines, with IC50 values ranging from 0.053 to 10.720 μM, highlighting these compounds as promising leads for the development of new antitumor agents.

Exploring Contact Toxicity of Essential Oils against Sitophilus zeamais through a Meta-Analysis Approach.

Sitophilus zeamais is a primary pest of maize. Our aim was to perform a qualitative review and meta-analyses with 56 scientific articles published from 1 January 2000 to 1 October 2022 dealing with direct (topical application) and indirect (impregnation of essential oils, EOs, onto filter paper or maize grains) contact toxicity of EOs against S. zeamais. Three independent meta-analyses of single means of LD50 (direct contact) and LC50 (indirect contact) were conducted using a random effect model. Essential oils more frequently evaluated were those belonging to Asteraceae, Apiaceae, Lamiaceae, Myrtaceae, Piperaceae, and Rutaceae. The LC50 global mean values were 33.19 µg/insect (CI95 29.81-36.95) for topical application; 0.40 µL/cm2 (CI95 0.25-0.65) for filter paper indirect contact; and 0.50 µL/g maize (CI95 0.27-0.90) for maize grains indirect contact. The species Carum carvi, Salvia umbratica, Ilicium difengpi, Periploca sepium, Cephalotaxus sinensis, Murraya exotica, Rhododendron anthopogonoides, Ruta graveolens, Eucalyptus viminalis, Ocotea odorifera, Eucalyptus globulus, Eucalyptus dunnii, Anethum graveolens, Ilicium verum, Cryptocarya alba, Azadirachta indica, Chenopodium ambrosioides, Cupressus semperivens, Schinus molle, Piper hispidinervum, Mentha longifolia, and Croton pulegiodorus showed LC50 or LD50 values lower than the global means, indicating good insecticidal properties. Our results showed that EOs have great potential to be used as bioinsecticides against S. zeamais.

Two novel cephalotaxine-type alkaloids from Cephalotaxus sinensis

Silica gel, octadecyl-silica(ODS), Sephadex LH-20, and semi-preparative high performance liquid chromatography(HPLC) was performed to isolate nine cephalotaxine-type alkaloids from Cephalotaxus sinensis: 8-oxodeoxyharringtonine(1), 8-oxonordeoxyharringtonine(2), cephafortunine A(3), 8-oxocephalotaxine(4), deoxyharringtonine(5), acetylcephalotaxine(6), cephalotaxine(7), epicephalotaxine(8), and cephalotaxinone(9). Compounds 1 and 2 were identified for the first time and their structures were determined by high-resolution-electrospray ionization-mass spectrometry(HR-ESI-MS), nuclear magnetic resonance(NMR), and electronic circular dichroism(ECD). Compounds 1-3 and 5 significantly inhibited the transcription of nuclear factor kappa B(NF-κB), with the half-maximal inhibitory concentration(IC_(50)) of(3.91±0.70),(2.99±0.45),(7.84±0.51), and(1.46±0.17) μmol·L~(-1), respectively.

Recent Advances in the Total Synthesis of Cephalotane-Type Norditerpenoids from Cephalotaxus sinensis

Abstract Cephalotaxus diterpenoids are well known for their unique structures and biological activities. Cephanolides, as new cephalotane-type norditerpenoids isolated from Cephalotaxus sinensis, have attracted considerable attention from the synthetic community. The present Short Review summarizes strategic approaches toward the total synthesis of cephanolides from 2018 to 2021.1 Introduction2 Synthetic Approaches toward Cephalotane-Type Norditerpenoids2.1 First Total Synthesis of Cephanolides B and C by Zhao (2018)2.2 Total Synthesis of Cephanolides A–D by Sarpong (2021)2.3 Total Synthesis of Cephanolide B by Yang (2021)2.4 Asymmetric Total Synthesis of Cephanolide A by Gao (2020)2.5 Asymmetric Total Synthesis of Cephanolide B by Gao (2021)2.6 Asymmetric Total Synthesis of Cephanolides A and B by Cai (2021)3 Conclusion and Perspectives

The genera Coccus and Prococcus (Hemiptera: Coccomorpha: Coccidae) in China, with two new combinations and descriptions of two new species.

The Chinese soft scale species in the genus Coccus Linnaeus, 1758 (Hemiptera: Coccomorpha: Coccidae) were studied. Coccus cambodiensis Takahashi and the Malaysian species C. cameronensis Takahashi are considered to be non-congeneric with Coccus hesperidum Linnaeus, 1758, the type species of Coccus, and are transferred to Prococcus Avasthi, 1993, as Prococcus cambodiensis (Takahashi, 1942), comb. n. and Prococcus cameronensis (Takahashi, 1952), comb. n. The generic diagnosis of Prococcus is revised. In the genus Coccus, two new species are described and illustrated based on adult females: Coccus nanningensis Cao Feng, sp. n. from Guangxi, China, on Ficus carica (Moraceae), and Coccus cephalotaxus Cao Feng, sp. n. from Shannxi, China, on Cephalotaxus sinensis (Taxaceae). Identification keys to separate adult females of Prococcus from Coccus, the 14 species of Coccus found in China, and all three species of Prococcus are provided.

Asymmetric Total Synthesis of Cephanolide A

AbstractThe first asymmetric total synthesis of cephanolide A, a complex hexacyclic C18 dinorditerpenoid from cephalotaxus sinensis, was achieved. The synthesis features a convergent strategy, which provides a flexible approach to prepare the biogenetically cephalotaxus diterpenoids and structurally related derivatives for biological studies. A mild intramolecular Prins cyclization was developed to construct the central hexahydrofluorenol skeleton (A‐B‐C ring), which relies on the originally proposed hydroacylation strategy. A remote hydroxy group directed hydrogenation was applied to stereospecifically reduce the tetra‐substituted enone unit. A sequence of ring forming steps, including lactonization, cation mediated etherification and Friedel–Crafts cyclization, was efficiently utilized to forge the cage‐like skeleton.

Asymmetric Total Synthesis of Cephanolide A.

The first asymmetric total synthesis of cephanolide A, a complex hexacyclic C18 dinorditerpenoid from cephalotaxus sinensis, was achieved. The synthesis features a convergent strategy, which provides a flexible approach to prepare the biogenetically cephalotaxus diterpenoids and structurally related derivatives for biological studies. A mild intramolecular Prins cyclization was developed to construct the central hexahydrofluorenol skeleton (A-B-C ring), which relies on the originally proposed hydroacylation strategy. A remote hydroxy group directed hydrogenation was applied to stereospecifically reduce the tetra-substituted enone unit. A sequence of ring forming steps, including lactonization, cation mediated etherification and Friedel-Crafts cyclization, was efficiently utilized to forge the cage-like skeleton.

Insecticidal activity of essential oil from Cephalotaxus sinensis and its main components against various agricultural pests

Plant essential oils (EOs) could be used as new alternatives of synthetic insecticides due to their environmental friendliness, safety to non-target organisms, and low-level resistance. In this study, the essential oil of Cephalotaxus sinensis (CSEO), an important Chinese medicinal species, showed potent insecticidal activity against Megoura japonica, Plutella xylostella and Sitophilus zeamais in laboratory bioassays; CSEO 20 % emulsifiable concentrate exhibited the control efficacy of 85.67 % against M. japonica after 7 days treatment at concentration of 0.25 % (w/v) in the greenhouse experiment. The GC-MS results showed that the main components of CSEO were α-pinene (37.89 %), β-caryophyllene (16.78 %) and germacrene D (10.79 %). Five active compounds, β-elemene (1), β-caryophyllene (2), β-phellandrene (3) caryophyllene oxide (4) and α-pinene (5) were isolated from CSEO. β-Caryophyllene (2) exhibited highest contact toxicity against M. japonica and P. xylostella, with the median lethal dose (LD50) values of 0.072 μg/adult and 0.32 μg/larva, respectively. α-Pinene (5) showed the highest fumigant toxicity against M. japonica and P. xylostella, with the median lethal concentration (LC50) values of 6.74 and 7.35 mg/L, respectively. Furthermore, β-phellandrene (3) had the potent contact and fumigant toxicity against S. zeamais with the LD50 and LC50 value of 2.19 μg/adult and 3.57 mg/L, respectively. Thus, CSEO and their major components have the potential to be developed as novel botanical insecticides for the control of agricultural pests.

Cephasinenoside A, a new cephalotane diterpenoid glucoside from Cephalotaxus sinensis

A new cephalotane diterpenoid glucoside, cephasinenoside A (1), was isolated from the twigs and leaves of Cephalotaxus sinensis (Rehd. et Wils.) Li. The structure of 1 was established on the basis of spectroscopic analyses and ECD calculations. Compound 1 represents the first cephalotane diterpenoid glycoside which inhibits the proliferation of human acute myeloid leukemia HL-60 cells with a GI50 value of 7.17 ± 1.03 μM.

Characterization of the complete chloroplast genome of Cephalotaxus sinensis (Cupressales: Taxaceae), an endangered Tertiary relict tree endemic to China

Cephalotaxus sinensis is an endangered Tertiary relict tree endemic to China with high medicinal values. To contribute to its conservation genetics studies, its complete chloroplast (cp) genome was assembled using Illumina high-throughput sequencing technology in this study. The cp genome is 135,646 bp long with an asymmetric base composition (32.5% A, 17.6% C, 17.5% G and 32.4% T). It encodes a set of 113 unique genes (incl. 82 protein coding, 27 tRNA and 4 rRNA genes) with one of them (trnQ-UUG) being duplicated. Besides, 14 genes (atpF, ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, rps12, rps16, trnA-UGC, trnG-UCC, trnI-GAU and trnK-UUU) possess a single intron, and another gene (ycf3) has a couple of introns. Phylogenetic analysis generally supported the current family-level taxonomy of the order Cupressales, and suggested that C. sinensis is closely related to the congeneric C. oliveri.

Characterization of the complete chloroplast genome of Cephalotaxus sinensis (Cephalotaxaceae), an endangered species endemic to China

Cephalotaxus sinensis, is an endangered species endemic to China. Here, the complete chloroplast genome (plastome) of C. sinensis was obtained by using Illumina pair-end sequencing. The total genome size is 134,621 bp in length, and encodes 114 genes, including 82 protein-coding genes, 28 transfer RNA genes and four ribosomal RNA genes. The nucleotide composition is asymmetric (32.5% A, 17.7% C, 17.5% G and 32.9% T) with an overall G+C content of 35.2%. Unexpectedly, we observed the plastome lacks typical inverted repeat regions, which is consistent with C. oliveri plastome in the genus Cephalotaxus. Furthermore, a total of 38 simple sequences repeats were identified in the plastome of C. sinensis. The phylogenetic analysis based on 14 plastomes revealed that C. sinensis is closely related to C. oliveri.

Cephanolides A-J, Cephalotane-Type Diterpenoids from Cephalotaxus sinensis.

Ten new cephalotane-type diterpenoids, cephanolides A-J (1-10), and two known analogues were isolated and characterized from Cephalotaxus sinensis. Compounds 1-3 represent the first examples of A-ring-contracted cephalotane-type dinorditerpenoids, and compound 4 is an A-ring-contracted norditerpenoid. The biosynthetic pathways for compounds 1-4 are postulated with the coexisting cephalotane-type troponoids as the precursors. Compounds 11 and 12 showed significant cytotoxicities against a panel of tumor cell lines (A549, KB, HL-60, and HT-29) with IC50 values ranging from 0.464 to 6.093 μM.

A new bisabolane sesquiterpenoid and a new abietane diterpenoid from Cephalotaxus sinensis

Phytochemical investigation of Cephalotaxus sinensis has led to the isolation of a new bisabolane sesquiterpenoid (1), a new abietane diterpenoid (2), and 13 known compounds (3–15). Their structures were elucidated by extensive spectroscopic analysis (MS, UV, IR, and NMR).

Antiphytoviral activity of alkaloids from Cephalotaxus sinensis

The evaluation of pesticidal activity and the isolation of plant allelochemicals can lead to the discovery of new pesticides. This study confirmed that Cephalotaxus sinensis, an important Chinese medicinal species, has excellent activities against tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) both indoors and outdoors; its control of the TMV is comparable to that of a commercialized antiviral agent (VA; active ingredient=a mixture of moroxydine hydrochloride and copper acetate). Using bioassay-guided fractionation, we isolated eight active alkaloids, which were identified as drupacine, 11-hydroxycephalotaxine, cephalancetine A, isocephalotaxine, cephalotaxine β-N-oxide, 4-hydroxycephalotaxine, wilsonine and cephalotaxine. At the same dosage, the inactivation activities against TMV were not significantly different among drupacine, 11-hydroxycephalotaxine, cephalotaxine and VA (inhibition ratio=50.76% to 53.41%). The inhibiting TMV replications of all tested compounds were inferior to that of VA, but the inhibition ratios of drupacine and cephalotaxine were still greater than 50%. Thus, C. sinensis, in which drupacine and cephalotaxine are the main active constituents, has the potential for further development as a biological antiviral agent. This is the first report of the antiviral activities of eight alkaloids from C. sinensis.

Cephalotanins A–D, Four Norditerpenoids Represent Three Highly Rigid Carbon Skeletons from Cephalotaxus sinensis

Four polycyclic norditerpenoids, cephalotanins A-D (1-4) representing three unprecedented carbon skeletons with highly rigid ring systems, were isolated from Cephalotaxus sinensis and structurally characterized by a combination of various methods. Compounds 1 and 2 are new skeletal norditerpenoid trilactones, while 3 and 4 are two norditerpenoids featuring different new carbon skeletons. Biosynthetic pathways for 1-4 were proposed by involving diverse and very fascinating chemical events with the coexisting cephalotane troponoids as the precursors. Compound 1 exhibited good NF-κB inhibition with an IC50 value of 4.12±0.61 μΜ.

Insights from the pollination drop proteome and the ovule transcriptome ofCephalotaxusat the time of pollination drop production

Many gymnosperms produce an ovular secretion, the pollination drop, during reproduction. The drops serve as a landing site for pollen, but also contain a suite of ions and organic compounds, including proteins, that suggests diverse roles for the drop during pollination. Proteins in the drops of species of Chamaecyparis, Juniperus, Taxus, Pseudotsuga, Ephedra and Welwitschia are thought to function in the conversion of sugars, defence against pathogens, and pollen growth and development. To better understand gymnosperm pollination biology, the pollination drop proteomes of pollination drops from two species of Cephalotaxus have been characterized and an ovular transcriptome for C. sinensis has been assembled. Mass spectrometry was used to identify proteins in the pollination drops of Cephalotaxus sinensis and C. koreana RNA-sequencing (RNA-Seq) was employed to assemble a transcriptome and identify transcripts present in the ovules of C. sinensis at the time of pollination drop production. About 30 proteins were detected in the pollination drops of both species. Many of these have been detected in the drops of other gymnosperms and probably function in defence, polysaccharide metabolism and pollen tube growth. Other proteins appear to be unique to Cephalotaxus, and their putative functions include starch and callose degradation, among others. Together, the proteins appear either to have been secreted into the drop or to occur there due to breakdown of ovular cells during drop production. Ovular transcripts represent a wide range of gene ontology categories, and some may be involved in drop formation, ovule development and pollen-ovule interactions. The proteome of Cephalotaxus pollination drops shares a number of components with those of other conifers and gnetophytes, including proteins for defence such as chitinases and for carbohydrate modification such as β-galactosidase. Proteins likely to be of intracellular origin, however, form a larger component of drops from Cephalotaxus than expected from studies of other conifers. This is consistent with the observation of nucellar breakdown during drop formation in Cephalotaxus The transcriptome data provide a framework for understanding multiple metabolic processes that occur within the ovule and the pollination drop just before fertilization. They reveal the deep conservation of WUSCHEL expression in ovules and raise questions about whether any of the S-locus transcripts in Cephalotaxus ovules might be involved in pollen-ovule recognition.