Nematicidal Activity of 20-Deoxyingenol-3-angelate from Euphorbia peplus Latex Through Protein Kinase C Isotype TPA-1.

The latex of Euphorbia peplus and its major component 20-deoxyingenol-3-angelate (DI3A) displayed significant nematicidal activity against Caenorhabditis elegans and Panagrellus redivivus. DI3A treatment inhibited the growth and development of nematodes and caused significantly negative effects on locomotion behavior, reproduction, and accumulation of reactive oxygen species. Transcriptome analysis indicated that differential expression genes in DI3A-treated C. elegans were mainly associated with the metabolism, growth, and development process, which were further confirmed by RT-qPCR experiments. The expression level of TPA-1 gene encoding a protein kinase C isotype was obviously upregulated by DI3A treatment, and knockdown of TPA-1 by RNAi technology in the nematode could relieve the growth-inhibitory effect of DI3A. Metabolic analysis indicated that DI3A was hardly metabolized by C. elegans, but a glycosylated indole derivative was specifically accumulated likely due to the activation of detoxification. Overall, our findings suggested that DI3A from E. peplus latex exerted a potent nematicidal effect through the gene TPA-1, which provides a potential target for the control of nematodes and also suggests the potential application value of E. peplus latex and DI3A as botanical nematicides.

New triterpenoids from the aerial parts of the Uygur medicine Salvia deserta.

Phytochemical investigation on the aerial parts of Salvia deserta led to the isolation of eight new pentacyclic triterpenoids including three oleanane- (1 - 3) and five ursane-type (4 - 8) triterpenoids, whose structures were elucidated based on extensive spectroscopic analysis and quantum chemical calculation. Weak immunosuppressive potency was observed for compounds 1, 2, and 4 - 8 via inhibiting the secretion of cytokines TNF-α and IL-6 in LPS-induced macrophages RAW264.7 at 20 µM. In addition, compounds 1, 2, and 4 - 6 exhibited moderate protective activity on t-BHP-induced oxidative injury in HepG2 cells.

Leucosceptrane Sesterterpenoids as a New Type of Natural Immunosuppressive Agents in Treating Sepsis.

Intragastric administration of the total sesterterpenoid extract (TSE) of medicinal plant Leucosceptrum canum at 2.5 g/kg dose protected mice from LPS-induced sepsis. Phytochemical investigation led to the isolation and identification of 47 leucosceptrane sesterterpenoids (1-47) including 30 new compounds (1-30) with complicated oxygenation patterns. Biological screening indicated their immunosuppressive activity via inhibiting IFN-γ secretion and/or proliferation of T cells with different potencies. Mechanism study of compounds 9, 25, and 32 revealed that they inhibited the activations of AKT-mTOR, JNK, p38 MAPK or ERK pathway in T cells and macrophages. In addition, compounds 9 and 25 induced G0/G1 cell arrest of T cells. The major component, leucosceptroid N (32), significantly lowered the levels of IL-6 and TNF-α in peripheral blood serum, and ameliorated the multiorgan damages of LPS-induced sepsis mice at 25 mg/kg dose. These findings suggest that leucosceptrane sesterterpenoids are a new type of potential immunosuppressive agents for sepsis treatment.

Discovery of Unusual Sesterterpenoids from Colquhounia coccinea var. mollis and Their Metabolic Implications.

Three unusual sesterterpenoids featuring unprecedented rearranged colquhounane (C25) and tetranorcolquhounane (C21) frameworks, colquhounoids E (1) and F (3) and norcolquhounoid F (2), were isolated from a Lamiaceae medicinal plant Colquhounia coccinea var. mollis. Their structures were elucidated by spectroscopic analysis and quantum chemical calculations. A biomimetic inspired regioselective cyclopropane cleavage was achieved under acidic conditions. The immunosuppressive activities of these new sesterterpenoids were also evaluated.

Defensive furofuran lignans localized to the oil cells of Neocinnamomum delavayi and their metabolism by a specialist insect.

Neocinnamomum delavayi (Lauraceae) leaves with abundant oil cells are seldom attacked by insects, but their chemical constituent and biological function remain obscure. Three furofuran lignans, including (+)-eudesmin (3), (+)-magnolin (4), and demethoxyaschantin (5), were identified to be the major specialized metabolites in the oil cells of N. delavayi leaves through laser microdissection coupled with NMR analysis. Compounds 3 and 4 exhibited obvious antifeedant activity against a generalist insect Spodoptera exigua, and their natural contents in the leaves could effectively defend against generalist insects. Intriguingly, three specific metabolites 9-11, the O-demethylation derivates of compounds 3-5, were identified from a native specialist insect Dindica polyphaenaria feeding with N. delavayi leaves, implying an adaptation mechanism of specialist insects to plant defensive compounds. The results revealed a chemical connection between plants and insects, which would contribute to our understanding of plant-insect interaction and insect management.

Clerodane diterpenoids from the Uygur medicine Salvia deserta with immunosuppressive activity.

Six undescribed clerodane diterpenoids along with five known ones were isolated from the aerial parts of Salvia deserta, a traditional Uygur medicine. Their chemical structures including absolute configurations were elucidated by extensive spectroscopic analysis (including 1D and 2D NMR, HRESIMS, and IR), combined with calculated ECD method and single-crystal X-ray diffraction analysis. All the compounds possessed a terminal α,ß-unsaturated-γ-lactone moiety, and were assayed for their immunosuppressive activity via inhibiting the secretion of cytokines TNF-α and IL-6 in macrophages RAW264.7. Among them, (5R,8R,9S,10R)-18-nor-cleroda-2,13-dien-16,15-olide-4-one obviously suppressed the secretion of TNF-α and IL-6 with IC50 values of 8.55 and 13.65 µM, respectively.

A new sesquiterpene synthase catalyzing the formation of (R)-ß-bisabolene from medicinal plant Colquhounia coccinea var. mollis and its anti-adipogenic and antibacterial activities.

The sesquiterpene ß-bisabolene possessing R and S configurations is commonly found in plant essential oils with antimicrobial and antioxidant activities. Here, we report the cloning and functional characterization of a (R)-ß-bisabolene synthase gene (CcTPS2) from a Lamiaceae medicinal plant Colquhounia coccinea var. mollis. The biochemical function of CcTPS2 catalyzing the cyclization of farnesyl diphosphate to form a single product (R)-ß-bisabolene was characterized through an engineered Escherichia coli producing diverse polyprenyl diphosphate precursors and in vitro enzyme assay, indicating that CcTPS2 was a high-fidelity (R)-ß-bisabolene synthase. The production of (R)-ß-bisabolene in an engineered E. coli strain harboring the exogenous mevalonate pathway, farnesyl diphosphate synthase and CcTPS1 genes was 17 mg/L under shaking flask conditions. Ultimately, 120 mg of purified (R)-ß-bisabolene was obtained from the engineered E. coli, and its structure was elucidated by detailed spectroscopic analyses (including 1D and 2D NMR, and specific rotation). Four chimeric enzymes were constructed through domain swapping, which altered the product outcome, indicating the region important for substrate and product specificity. In addition, (R)-ß-bisabolene exhibited anti-adipogenic activity in the model organism Caenorhabditis elegans and antibacterial activity selectively against Gram-positive bacteria.

Chemical investigation of Buddleja officinalis leaves and localization of defensive triterpenoids to its glandular trichomes.

Buddleja officinalis is a traditional Chinese medicinal plant covered with glandular and non-glandular trichomes on leaves. Phytochemical investigation of its leaves led to the identification of one undescribed tetranorcycloartane 3-oxo-25,26,27,29-tetranorcycloartan-24-oic acid (1) and one first identified natural product tetranorcycloartane 3-oxo-25,26,27,29-tetranorcycloartan-24-oic methyl ester (2), along with an undescribed megastigmane glucoside (3) and 14 known constituents (4-17). Structures of undescribed chemicals were elucidated by comprehensive 1D and 2D NMR, MS and CD analysis. Further chemical investigation resulted in six triterpenoids (4-9) being localized to the trichomes of B. officinalis. The major trichome components cycloeucalenone (4) and 24-oxo-29-norcycloartan-3-one (5) showed potent antifeedant activity against a generalist insect cotton bollworm (Helicoverpa armigera), but no obvious activity against the specialist herbivore Hyphasis inconstans. Compounds 4 and 7 also displayed inhibitory effects on seed germination of Arabidopsis thaliana. In addition, 1 and 4 exhibited moderate antibacterial activity toward three gram-positive bacteria.

Labdane diterpenoids from the heartwood of Leucosceptrum canum that impact on root growth and seed germination of Arabidopsis thaliana.

Eleven undescribed diterpenoids possessing labdane, 3,18-cyclo-labdane, 19 (4 â†’ 3)-labdane and 12-nor-labdane skeletons, named leucolactones A-K, were isolated from the heartwood of a large woody Lamiaceae plant, Leucosceptrum canum. Their structures were determined by NMR, MS, and in the case of leucolactones A by single crystal X-ray diffraction analysis. Plausible biosynthetic pathway of leucolactones were proposed. Leucolactones showed significant inhibitory effects against seed germination and root elongation of Arabidopsis thaliana in the Petri dish bioassay. Among them, the diastereomeric leucolactones G and H were the most potent, with EC50 values for root elongation of 6.53 ± 1.35 and 9.75 ± 1.25 µM, respectively. The preliminary structure-activity relationship of leucolactones was discussed. The increase of auxin reporter activity in A. thaliana DR5::GUS roots by leucolactone H was observed, indicating that leucolactones altered auxin accumulation and distribution. These findings suggested that leucolactones might be involved in regulation of plant growth and development through altering auxin accumulation and distribution, presumably contributing to the heartwood formation in L. canum.

An extremely promiscuous terpenoid synthase from the Lamiaceae plant Colquhounia coccinea var. mollis catalyzes the formation of sester-/di-/sesqui-/mono-terpenoids.

Terpenoids are the largest class of natural products with complex structures and extensive bioactivities; their scaffolds are generated by diverse terpenoid synthases (TPSs) from a limited number of isoprenoid diphosphate precursors. Promiscuous TPSs play important roles in the evolution of terpenoid chemodiversity, but they remain largely unappreciated. Here, an extremely promiscuous terpenoid synthase (CcTPS1) of the TPS-b subfamily was cloned and functionally characterized from a leaf-specific transcriptome of the Lamiaceae plant Colquhounia coccinea var. mollis. CcTPS1 is the first sester-/di-/sesqui-/mono-TPS identified from the plant kingdom, accepting C25/C20/C15/C10 diphosphate substrates to generate a panel of sester-/di-/sesqui-/mono-terpenoids. Engineered Escherichia coli expressing CcTPS1 produced three previously unreported terpenoids (two sesterterpenoids and a diterpenoid) with rare cyclohexane-containing skeletons, along with four sesquiterpenoids and one monoterpenoid. Their structures were elucidated by extensive nuclear magnetic resonance spectroscopy. Nicotiana benthamiana transiently expressing CcTPS1 also produced the diterpenoid and sesquiterpenoids, demonstrating the enzyme's promiscuity in planta. Its highly leaf-specific expression pattern combined with detectable terpenoid products in leaves of C. coccinea var. mollis and N. benthamiana expressing CcTPS1 suggested that CcTPS1 was mainly responsible for diterpenoid and sesquiterpenoid biosynthesis in plants. CcTPS1 expression and the terpenoid products could be induced by methyl jasmonate, suggesting their possible role in plant-environment interaction. CcTPS1 was localized to the cytosol and may differ from mono-TPSs in subcellular compartmentalization and substrate tolerance. These findings will greatly aid our understanding of plant TPS evolution and terpenoid chemodiversity; they also highlight the enormous potential of transcriptome mining and heterologous expression for the exploration of unique enzymes and natural products hidden in plants.

A Cryptic Plant Terpene Cyclase Producing Unconventional 18- and 14-Membered Macrocyclic C25 and C20 Terpenoids with Immunosuppressive Activity.

A versatile terpene synthase (LcTPS2) producing unconventional macrocyclic terpenoids was characterized from Leucosceptrum canum. Engineered Escherichia coli and Nicotiana benthamiana expressing LcTPS2 produced six 18-/14-membered sesterterpenoids including five new ones and two 14-membered diterpenoids. These products represent the first macrocyclic sesterterpenoids from plants and the largest sesterterpenoid ring system identified to date. Two variants F516A and F516G producing approximately 3.3- and 2.5-fold, respectively, more sesterterpenoids than the wild-type enzyme were engineered. Both 18- and 14-membered ring sesterterpenoids displayed significant inhibitory activity on the IL-2 and IFN-γ production of T cells probably via inhibition of the MAPK pathway. The findings will contribute to the development of efficient biocatalysts to create bioactive macrocyclic sesterterpenoids, and also herald a new potential in the well-trodden territory of plant terpenoid biosynthesis.

Immunostimulatory 6/6/6/6 Tetracyclic Triterpenoid Saponins with the Methyl-30 Incorporated Cyclization from the Root of Colquhounia elegans.

Two novel triterpenoid saponins, colqueleganoids A (1) and B (2), with the first methyl-30 incorporated 6/6/6/6-cyclized carbon skeleton (named colquelegane), were isolated from the root of Colquhounia elegans. Their structures including absolute configuration were determined by spectroscopic methods and X-ray crystallographic analyses. Interestingly, both compounds significantly enhanced TNF-α production and 1 also increased the IL-6 production in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS), suggesting their potential application as immunostimulants in immunotherapy and vaccination.

Immunosuppresive Sesterterpenoids and Norsesterterpenoids from Colquhounia coccinea var. mollis.

A pair of new C-14 epimeric sesterterpenoids, colquhounoid D (1) and 14-epi-colquhounoid D (2), and five degradation products featuring new C20 and C21 frameworks, norcolquhounoids A-E (3-7), were isolated from Colquhounia coccinea var. mollis. Their structures were elucidated by comprehensive spectroscopic analysis and single-crystal X-ray diffraction. Degradation of the C25 skeleton to the C21 skeleton was also achieved using aqueous NaIO4 and RuCl3. Compounds 1 and 2 showed significant immunosuppressive activity on the cytokine IFN-γ secretion of mouse splenocytes induced by anti-CD3/CD4 monoclonal antibodies, with IC50 of 8.38 and 5.79 µM, respectively, and compounds 5 and 6 were moderately active.

Characterization of defensive cadinenes and a novel sesquiterpene synthase responsible for their biosynthesis from the invasive Eupatorium adenophorum.

Eupatorium adenophorum is a malignant invasive plant possessing extraordinary defense potency, but its chemical weaponry and formation mechanism have not yet been extensively investigated. We identified six cadinene sesquiterpenes, including two volatiles (amorpha-4,7(11)-diene and (-)-amorph-4-en-7-ol) and four nonvolatiles (9-oxo-10,11-dehydroageraphorone, muurol-4-en-3,8-dione, 9-oxo-ageraphorone and 9ß-hydroxy-ageraphorone), as the major constitutive and inducible chemicals of E. adenophorum. All cadinenes showed potent antifeedant activity against a generalist insect Spodoptera exigua, indicating that they have significant defensive roles. We cloned and functionally characterized a sesquiterpene synthase from E. adenophorum (EaTPS1), catalyzing the conversion of farnesyl diphosphate to amorpha-4,7(11)-diene and (-)-amorph-4-en-7-ol, which were purified from engineered Escherichia coli and identified by extensive nuclear magnetic resonance (NMR) spectroscopy. EaTPS1 was highly expressed in the aboveground organs, which was congruent with the dominant distribution of cadinenes, suggesting that EaTPS1 is likely involved in cadinene biosynthesis. Mechanical wounding and methyl jasmonate negatively regulated EaTPS1 expression but caused the release of amorpha-4,7(11)-diene and (-)-amorph-4-en-7-ol. Nicotiana benthamiana transiently expressing EaTPS1 also produced amorpha-4,7(11)-diene and (-)-amorph-4-en-7-ol, and showed enhanced defense function. The findings presented here uncover the role and formation of the chemical defense mechanism of E. adenophorum - which probably contributes to the invasive success of this plant - and provide a tool for manipulating the biosynthesis of biologically active cadinene natural products.

Bioactive tigliane diterpenoids from the latex of Euphorbia fischeriana.

Latex is a type of sticky endogenous fluids derived from diverse plants including Euphorbia fischeriana, and is of great scientific and commercial values. In the current study, it was demonstrated that the latex extracted from E. fischeriana strongly respelled the growth of cotton bollworm, Helicoverpa armigera. Using spectroscopic methods, HPLC, and GC-MS analyses, six aliphatic tigliane diterpenoids were isolated from the latex of E. fischeriana, among which three compounds (2, 3, and 5) were new. Two major compounds (1 and 4) exhibited remarkable antifeedant activity against H. armigera, with EC50 values at 2.59 and 15.32 µg/cm2, respectively. In addition, the quantification analysis of diterpenoids in different organs indicated that 4 was the most abundant constituent and was highly accumulated in the latex. Collectively, the current study highlighted that the diterpenoids in latex of E. fischeriana had a considerable antifeedant function against H. armigera, which might be employed for the future development of natural insecticides for organic farming.

[Precise investigation of digital guide plates applied to implant surgery of anterior teeth].

OBJECTIVE: To study the precision of digital guide plates applied to the implant surgery of anterior teeth. METHODS: Fifty patients scheduled to receive implant restoration treatment in anterior teeth were enrolled in this study and divided into two groups (n=25, each group): those who were given routine implant restoration treatment (control group, 45 implants) and those who received implant restoration treatment using a digital guide plate (test group, 51 implants). After implantation, planned and placed implants were superimposed using digital software, and deviations (corona, apex, depth, degree) were analyzed. Esthetic parameters were assessed at 1 week (baseline), 6 month, and 1 year post final restoration. Pink esthetic (PES) and white esthetic (WES) scores were respectively used to evaluate the soft tissue and restoration esthetic outcome. RESULTS: The deviation parameters in the test group were significantly lower than those in the control group (P<0.05). PES and WES values recorded for the control group at 1 week, 6 month, and 1 year post final restoration were significantly lower than those in the test group (P<0.05). CONCLUSIONS: The digital guide plate can improve the accuracy of the three-dimensional position of implants in the maxillary esthetic zone. As such, this device may play an important role in obtaining the ideal aesthetic effects of maxillary anterior teeth.

Peltate glandular trichomes of Colquhounia vestita harbor diterpenoid acids that contribute to plant adaptation to UV radiation and cold stresses.

Plant glandular trichomes (GTs) are adaptive epidermal structures that synthesize and accumulate diverse specialized metabolites well-known as defense chemicals against biotic attacks, but their roles against abiotic challenges including UV radiation and cold climates remain largely unexplored. Colquhounia vestita Wall is a Chinese-Himalayan Lamiaceae plant with dense peltate and capitate GTs on its leaf and stem surfaces under a scanning electron microscope. Three diterpenoid acids, including a clerodane 5-epi-hardwickiic acid and two labdanes polyalthic acid and E-communic acid, were identified from the peltate GTs of C. vestita through laser microdissection coupled with UPLC-MS/MS. Under UV radiation and cold stresses, the major GT component polyalthic acid increased the biomass of Arabidopsis thaliana seedlings and decreased their malondialdehyde content. Furthermore, polyalthic acid promoted photosynthetic efficiency and the expression of genes encoding peroxidative enzymes under UV radiation, and stimulated Ca2+ elevation and the expression of calmodulin binding transcription activator gene CAMTA3 and two downstream cold-responsive genes CBF3 and RD29A under cold stress. Therefore, polyalthic acid in GTs is likely to endow the plant with enhanced tolerance to UV radiation and cold stresses, which extends the current understanding of the function of GT compounds in plant adaptation to abiotic environments.

Detoxification of Plant Aromatic Abietanoids via Cleavage of the Benzene Ring into 11,12-Seco-diterpene Polyenes by a Specialist Insect of Leucosceptrum canum.

Leaves of Leucosceptrum canum harbor abundant toxic aromatic abietanoids, and they are rarely attacked by insect herbivores, except for the larvae of Nacna malachitis. The excrements of the insect that fed on L. canum leaves were investigated, leading to the isolation and identification of two unprecedented 11,12-seco-abietane diterpene polyenes: nacnabietanins A (1) and B (2). This discovery heralds a unique detoxification mechanism of plant aromatic abietanoids by insects through enzymatic cleavage of stable benzene rings into more easily degraded polyenes.

Antibacterial harziane diterpenoids from a fungal symbiont Trichoderma atroviride isolated from Colquhounia coccinea var. mollis.

Fungal endophytes from plants are an important source for discovery of novel bioactive natural products. In this study, five undescribed harziane diterpenoids with a 4/7/5/6 tetracyclic scaffold, harzianols F‒J and three known derivatives, were obtained from the liquid fermentation of an endophytic fungus Trichoderma atroviride B7, which was isolated from the healthy flower of a Lamiaceae plant Colquhounia coccinea var. mollis. Their structures were elucidated by comprehensive spectroscopic analyses and X-ray crystallographic diffraction in the case of harzianol F. Harzianol I exhibited significant antibacterial effect against the growth of Staphylococcus aureus (EC50 = 7.7 ± 0.8 µg/mL), Bacillus subtilis (EC50 = 7.7 ± 1.0 µg/mL), and Micrococcus luteus (EC50 = 9.9 ± 1.5 µg/mL). Meanwhile, cytotoxic activity of harzianol I against three cancer cell lines was also observed. A plausible biosynthetic pathway for harziane diterpenoids was proposed.

Oral health in patients with stroke: a meta-analysis of comparative studies.

Objective: This meta-analysis systematically analyzed and compared oral health between stroke patients and controls.Data source: The electronic databases of PubMed, EMBASE, PsycINFO, Medline and Web of Science were independently searched by two authors from their inception to 14 June 2018.Study selection: Eleven studies comparing oral health between stroke patients (n = 1,742) and controls (n = 1,193) were analyzed.Data extraction: The full texts of the 11 studies were independently reviewed. Data on oral health were independently extracted by two authors.Data synthesis: Mean differences (MD) and 95% confidence intervals (CI) were calculated and synthesized using fixed or random-effects models, as appropriate. Compared to controls, stroke patients had poorer oral health: they had more Dental Caries (5 studies; MD = 2.89, 95% CI: 0.91-4.88, p= .04), but less Remaining Teeth scores (6 studies; MD = -2.93, 95% CI: -3.91, -1.95; p < .00001). Both the Plaque Index (3 studies; MD = 0.21, 95% CI: 0.14, 0.28; p < .00001) and Gingival Index scores (4 studies; MD = 0.22, 95% CI: 0.14, 0.30; p < .00001) were significantly higher in stroke patients, indicating worse periodontal status.Conclusions: Stroke patients had poorer overall oral health status compared to controls. Given the importance of oral health to overall health, further research on screening for oral health problems after stroke should be conducted and effective management strategies should be devised and implemented.