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.

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.

Secoiridoids from the traditional Chinese medicine Swertia pseudochinensis.

Detailed phytochemical investigation on the traditional Chinese medicine Swertia pseudochinensis Hara led to the isolation of ten undescribed secoiridoids and fifteen known analogs. Their structures were elucidated by extensive spectroscopic analysis (including 1D and 2D NMR, and HRESIMS). Selected isolates were assayed for their anti-inflammatory and antibacterial activities, and moderate anti-inflammatory activity via inhibiting the secretion of cytokines IL-6 and TNF-α in macrophages RAW264.7 induced by LPS were observed. Antibacterial activity against Staphylococcus aureus was not found at 100 µM.

Secondary metabolites from the Mongolian medicine Lomatogonium carinthiacum.

Systematic phytochemical investigation on the Mongolian medicinal herb Lomatogonium carinthiacum led to the isolation of 12 monoterpenoids including three new secoiridoids (1, 2 and 4) and one new iridoid glycoside (13), one new monoterpenoid alkaloid (3), and three new sesquiterpenoids (14-16). Comprehensive spectroscopic analysis (including 1D and 2D NMR, and HRESIMS) and quantum chemistry computations (including ECD and NMR calculations) were applied to elucidate their structures. Weak immunosuppressive activities were observed for the new isolates via inhibiting T cell proliferation and cytokine IFN-γ secretion in vitro.

Secoiridoids and triterpenoids from the traditional Tibetan medicine Gentiana veitchiorum and their immunosuppressive activity.

Two undescribed secoiridoids (dehydroxyl-swerimilegenin H and 9-oxo-swerimuslactone A) with eight known ones, and two undescribed triterpenoids [28-O-(3,4-dihydroxyl-benzyl)-lupeol and 17-hydroperoxide-28-norurs-12-en-3-one] with seven known ones, were isolated from the aerial parts of Gentiana veitchiorum, a traditional Tibetan medicine. The structures of these compounds were elucidated by detailed spectroscopic analyses (including 1D and 2D NMR, HRMS, IR, and specific rotation) and comparison with structurally related known compounds. The isolates were selected to evaluate for their immunosuppressive activity via inhibiting the proliferation of T cells and cytokine IFN-γ production in T cells. Among them, 28-O-(3,4-dihydroxyl-benzyl)-lupeol exhibited significant effect by inhibiting the proliferation of T cells (IC50, 20.08 µM) and T cell IFN-γ production (IC50, 7.29 µM).

Immunosuppressive gentianellane-type sesterterpenoids from the traditional Uighur medicine Gentianella turkestanorum.

Whole plants of Gentianella turkestanorum are commonly used as a traditional Uighur medicine. A phytochemical investigation led to the isolation of eight undescribed gentianellane-type sesterterpenoids (18-epi-nitidasin, gentianelloids D-F, and 18-epi-gentianelloids C-F), one undescribed 11,12-seco-gentianellane (18-epi-alborosin), and three known analogs (nitidasin, gentianelloid C and alborosin) among which gentianelloid C was found for the first time from a natural source. The structures of these compounds were elucidated by extensive spectroscopic analyses (including 1D and 2D NMR, HRMS, IR, and specific rotation) and in the case of 18-epi-gentianelloid C by the single-crystal X-ray diffraction analysis. A putative biosynthetic route for these sesterterpenoids was proposed. The immunosuppressive activity of the isolated compounds was also evaluated by their ability to inhibit the proliferation of T cells and T cell cytokine IFN-γ production. Nitidasin suppressed IFN-γ production with an IC50 value of 16.50 µM, while gentianelloid F and alborosin inhibited the proliferation of and IFN-γ production in T cells with IC50 values of 12.40-14.66 µM.

Antifeedant, cytotoxic, and anti-inflammatory neo-clerodane diterpenoids in the peltate glandular trichomes and fresh leaves of Ajuga forrestii.

The Lamiaceae plant Ajuga forrestii Diels is a traditional Chinese herbal medicine with abundant glandular trichomes (GTs), but their chemistry and biological functions remain uninvestigated. Here, a panel of six highly functionalized neo-clerodane diterpenoids was localized to the peltate GTs of A. forrestii using laser microdissection coupled with HPLC analysis, indicating that the GTs of A. forrestii are an excellent material for the elucidation of the yet unclear biosynthetic pathway of natural neo-clerodane diterpenoids. In addition, four undescribed neo-clerodane diterpenoids with an acyclic C-9 side chain including two pairs of 1:1 mixture of inseparable diastereomers, ajuforrestins D-G, were isolated from the fresh leaves of A. forrestii together with six known compounds. The structures of the undescribed compounds were elucidated by spectroscopic (including 1D and 2D NMR and HR-ESI-MS) analyses. Biological assays indicated that the major GT compound ajugacumbin B and undescribed ajuforrestins D/E showed antifeedant activity against Helicoverpa armigera, suggesting that neo-clerodanes in A. forrestii should be involved in plant defence against insects. Moreover, the abietane diterpenoid ajuforrestin B exhibited significant anti-inflammatory activity on the secretion of interleukin-2 (IL-2) and cytotoxicity against three cancer cell lines, NCI-H1975, HepG2 and MCF-7, suggesting that ajuforrestin B could positively contribute to the therapeutic effects of this traditional Chinese medicine.

An integrated strategy toward comprehensive characterization and quantification of multiple components from herbal medicine: An application study in Gelsemium elegans.

Objective: To develop a powerful integrated strategy based on liquid chromatography coupled with mass spectrometry (LC-MS) systems for the comprehensive characterization and quantification of multiple components of herbal medicines. Methods: Firstly, different mobile phase additives, analysis time, and MS acquisition modes were orthogonally tested with liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) in order to detect as many components of Gelsemium elegans as possible with high peak intensity. Secondly, several data mining strategies, including database searching, diagnostic ion filtering and neutral loss filtering, were utilized to perform chemical profiling. Subsequently, this study focused on the quantification and validation of the performance of a liquid chromatography-triple mass spectrometry (LC-QqQ/MS) assay based on derivative multiple reaction monitoring (DeMRM). Results: A total of 147 components from G. elegans were characterized, among them 116 nontarget components were reported for the first time. A sensitive and reproducible LC-QqQ/MS method was successfully developed and validated for the simultaneous relative quantification of 41 components of G. elegans. This LC-QqQ/MS method was then applied to compare the contents of components in the roots, stems and leaves. Conclusion: The present integrated strategy would significantly contribute to chemical studies on herbal medicine, and its utility could be extended to other research fields, such as metabolomics, quality control, and pharmacokinetics.

Diterpenoids and Flavonoids from the Twigs of Cephalotaxus fortunei var. alpina.

Three new diterpenoids (a cephalotane, an abietane and a 9(10→20)-abeo-abietane) and one new flavonoid, together with 11 known compounds, were isolated from the twigs of Cephalotaxus fortunei var. alpina. The new compounds were identified by comprehensive spectroscopic (including 1D and 2D-NMR and HR-ESI-MS) analysis. Anti-inflammatory, immunosuppressive and cytotoxic activities of three new compounds were evaluated. 3ß,20-epoxyabieta-8,11,13-triene-3α,12-diol showed weak cytotoxicity against tumor cell lines NCI-H1975, HepG2, MCF-7, while fortalpinoid R and 3-acetonyl-3,5,7,4'-tetrahydroxy-2-methoxyflavanone were not active at 80 µM. None of these compounds showed anti-inflammatory and immunosuppressive activities.

Characterization of absorbed and produced constituents in goat plasma urine and faeces from the herbal medicine Gelsemium elegans by using high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal medicine contains hundreds of natural products, and studying their absorption, metabolism, distribution, and elimination presents great challenges. Gelsemium elegans (G. elegans) is a flowering plants in the Loganiaceae family. The plant is known to be toxic and has been used for many years as a traditional Chinese herbal medicine for the treatment of rheumatoid arthritis, neuropathic pain, spasticity, skin ulcers and cancer. It was also used as veterinary drugs for deworming, promoting animal growth, and pesticides. At present, studies on the metabolism of G. elegans have primarily focused on only a few single available reference ingredients, such as koumine, gelsemine and gelsedine. MATERIAL AND METHODS: The goal of this work is to elucidate the overall metabolism of whole G. elegans powder in goats using high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC/QqTOF-MS). RESULTS: Analyses of plasma, urine and fecal samples identified or tentatively characterized a total of 44 absorbed natural products and 27 related produced metabolites. Gelsedine-type, sarpagine-type and gelsemine-type alkaloids were the compounds with the highest metabolite formation. In the present study, most natural products identified in G. elegans were metabolized through glucuronidation and oxidation. Hydrogenation, dehydrogenation and demethylation also occurred. CONCLUSION: To our knowledge, this is the first report of the metabolite profiling of the G. elegans crude extract in goats, which is of great significance for a safer and more rational application of this herbal medicine.

Leucosceptroid B from glandular trichomes of Leucosceptrum canum reduces fat accumulation in Caenorhabditis elegans through suppressing unsaturated fatty acid biosynthesis.

Obesity that is highly associated with numerous metabolic diseases has become a global health issue nowdays. Plant sesterterpenoids are an important group of natural products with great potential; thus, their bioactivities deserve extensive exploration. RNA-seq analysis indicated that leucosceptroid B, a sesterterpenoid previously discovered from the glandular trichomes of Leucosceptrum canum, significantly regulated the expression of 10 genes involved in lipid metabolism in Caenorhabditis elegans. Furthermore, leucosceptroid B was found to reduce fat storage, and downregulate the expression of two stearoyl-CoA desaturase (SCD) genes fat-6 and fat-7, and a fatty acid elongase gene elo-2 in wild-type C. elegans. In addition, leucosceptroid B significantly decreased fat accumulation in both fat-6 and fat-7 mutant worms but did not affect the fat storage of fat-6; fat-7 double mutant. These findings indicated that leucosceptroid B reduced fat storage depending on the downregulated expression of fat-6, fat-7 and elo-2 and thereby inhibiting the biosynthesis of the corresponding unsaturated fatty acid. These findings provide new insights into the development and utilization of plant sesterterpenoids as potential antilipemic agents.

The Difference in Cytotoxic Activity between Two Optical Isomers of Gelsemine from Gelsemium elegans Benth. on PC12 Cells.

Two optical isomers, +/- gelsemine (1, 2), together with one known compound were isolated from the whole plant of G. elegans. The structures of the separated constituents were elucidated on 1D and 2D (1H-1H COSY, HMBC, HSQC) NMR spectroscopy and high-resolution mass spectrometry (HRMS). The isolated alkaloids were tested in vitro for cytotoxic potential against PC12 cells by the MTT assay. As a result, (+) gelsemine (compound 1) exhibited cytotoxic activity against PC12 cells with an IC50 value of 31.59 µM, while (-) gelsemine (compound 2) was not cytotoxic.

Medicinal plants of the genus Macleaya (Macleaya cordata, Macleaya microcarpa): A review of their phytochemistry, pharmacology, and toxicology.

In the genus Macleaya, Macleaya cordata and Macleaya microcarpa have been recognized as traditional herbs that are primarily distributed in China, North America, and Europe and have a long history of medicinal usage. These herbs have been long valued and studied for detumescence, detoxification, and insecticidal effect. This review aims to provide comprehensive information on botanical, phytochemical, pharmacological, and toxicological studies on plants in the genus Macleaya. Plants from the genus of Macleaya provide a source of bioactive compounds, primarily alkaloids, with remarkable diversity and complex architectures, thereby having attracted attention from researchers. To date, 291 constituents have been identified and/or isolated from this group. These purified compounds and/or crude extract possess antitumor, anti-inflammatory, insecticidal, and antibacterial activities in addition to certain potential toxicities. Macleaya species hold potential for medicinal applications. However, despite the pharmacological studies on these plants, the mechanisms underlying the biological activities of active ingredients derived from Macleaya have not been thoroughly elucidated to date. Additionally, there is a need for research focusing on in vivo medical effects of Macleaya compounds and, eventually, for clinical trials.

Identification of gelsemine metabolites in rat liver S9 by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

RATIONALE: Gelsemine has been extensively studied because of its anti-tumor, immunomodulatory, insecticidal itching and other significant effects. However, limited information on the pharmacokinetics and metabolism of gelsemine has been reported. Therefore, the purpose of the present study was to investigate the in vitro metabolism of gelsemine in rat liver S9 by using rapid and accurate high-performance liquid chromatography/ quadrupole-time-of-flight mass spectrometry (HPLC/QqTOF-MS). METHODS: The incubation mixture was processed with 15% trichloroacetic acid. Multiple scans of gelsemine metabolites and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only 30 min. The structural elucidations of these metabolites were performed by comparing their changes in accurate molecular masses and product ions with those of the parent drug. RESULTS: Five metabolites of gelsemine were identified in rat liver S9. Of these, four metabolites of gelsemine were identified for the first time. The present results showed that the metabolic pathways of gelsemine are oxidation, demethylation, and dehydrogenation in rat liver S9. CONCLUSIONS: In this study, metabolites of gelsemine in liver S9 were identified and elucidated firstly using the HPLC/QqTOF-MS method. The proposed metabolic pathways of gelsemine in liver S9 will provide a basis for further studies of the in vivo metabolism of gelsemine in animals and humans.

Fingerprint analysis of Gelsemium elegans by HPLC followed by the targeted identification of chemical constituents using HPLC coupled with quadrupole-time-of-flight mass spectrometry.

Gelsemium elegans, which is a genus of the family Loganiaease, is commonly used as a traditional medicine for promoting animal growth and treating rheumatoid arthritis pain and neuropathic pain, among others. In this study, we first established a valid high-performance liquid chromatography (HPLC) method for the fingerprint analysis of Gelsemium elegans samples. Then, the comprehensive detection of chemical constituents from the samples was performed using HPLC coupled with quadrupole-time-of-flight mass spectrometry. The similarity evaluation results showed that location and area differences influenced the quality of the samples. An efficient strategy for the rapid targeted identification of chemical components was matching with a developed Gelsemium database. As a result, the accurate elemental compositions and known structures of compounds are found as hits. This process facilitated the structural identification of compounds combined with the accurate mass measurement of product ions and fragmentation behaviors. Consequently, 41 components including six alkaloids and non-alkaloids were systematically identified from Gelsemium elegans. The results showed that at least seven relatively major components existing in Gelsemium elegans may be useful for its quality control. The present analytical method combined with the developed Gelsemium database was shown to be a useful tool for investigating the chemical components of Gelsemium products.

Localisation of Two Bioactive Labdane Diterpenoids in the Peltate Glandular Trichomes of Leonurus japonicus by Laser Microdissection Coupled with UPLC-MS/MS.

INTRODUCTION: Glandular trichomes of plants are biochemical factories that could produce, store and secrete copious pharmaceutically important natural products. The Labiatae plant Leonurus japonicus is an important traditional Chinese medicine used to treat gynecological diseases, and has abundant peltate glandular trichomes (PGTs), in which the secondary metabolites accumulated are still unknown. OBJECTIVE: To study the secondary metabolites specifically accumulated in the PGTs of L. japonicus and their biological activities, and provide a new way to pinpoint bioactive natural products from plants. METHODOLOGY: Morphology of the trichomes on L. japonicus were observed under a scanning electron microscope. The PGTs of L. japonicus were precisely collected using laser microdissection (LMD) and analysed for their secondary metabolites with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Targeted compounds were isolated with classical phytochemical methods, and their structures were elucidated by spectroscopic analysis. Biological activities were evaluated by in vitro assays. RESULTS: Two labdane diterpenoids, leoheterin (1) and galeopsin (2), were localised in the PGTs of L. japonicus. Antithrombotic activity of 1 in anti-platelet aggregation assay induced by arachidonic acid was observed. Both compounds showed potential anti-inflammatory activity by inhibiting proinflammatory cytokine TNF-α. In addition, anti-proliferative effect of both compounds on several cancer cell lines was also detected. CONCLUSION: Two bioactive labdane diterpenoids were localised in the PGTs of L. japonicus. The findings suggested that it might be an efficient approach to explore bioactive natural products from the glandular trichomes of medicinal plants with LMD-UPLC/MS/MS. Copyright © 2017 John Wiley & Sons, Ltd.

Comprehensive identification and structural characterization of target components from Gelsemium elegans by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry based on accurate mass databases combined with MS/MS spectra.

This study reports an applicable analytical strategy of comprehensive identification and structure characterization of target components from Gelsemium elegans by using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QqTOF MS) based on the use of accurate mass databases combined with MS/MS spectra. The databases created included accurate masses and elemental compositions of 204 components from Gelsemium and their structural data. The accurate MS and MS/MS spectra were acquired through data-dependent auto MS/MS mode followed by an extraction of the potential compounds from the LC-QqTOF MS raw data of the sample. The same was matched using the databases to search for targeted components in the sample. The structures for detected components were tentatively characterized by manually interpreting the accurate MS/MS spectra for the first time. A total of 57 components have been successfully detected and structurally characterized from the crude extracts of G. elegans, but has failed to differentiate some isomers. This analytical strategy is generic and efficient, avoids isolation and purification procedures, enables a comprehensive structure characterization of target components of Gelsemium and would be widely applicable for complicated mixtures that are derived from Gelsemium preparations. Copyright © 2017 John Wiley & Sons, Ltd.

The Effect of Salt Intake and Potassium Supplementation on Serum Gastrin Levels in Chinese Adults: A Randomized Trial.

Excess dietary salt is strongly correlated with cardiovascular disease, morbidity, and mortality. Conversely, potassium likely elicits favorable effects against cardiovascular disorders. Gastrin, which is produced by the G-cells of the stomach and duodenum, can increase renal sodium excretion and regulate blood pressure by acting on the cholecystokinin B receptor. The aim of our study was to assess the effects of altered salt and potassium supplementation on serum gastrin levels in humans. A total of 44 subjects (38-65 years old) were selected from a rural community in northern China. All subjects were sequentially maintained on a relatively low-salt diet for 7 days (3.0 g/day of NaCl), a high-salt diet for 7 days (18.0 g/day of NaCl), and then a high-salt diet supplemented with potassium for another 7 days (18.0 g/day of NaCl + 4.5 g/day of KCl). The high-salt intake significantly increased serum gastrin levels (15.3 ± 0.3 vs. 17.6 ± 0.3 pmol/L). This phenomenon was alleviated through potassium supplementation (17.6 ± 0.3 vs. 16.5 ± 0.4 pmol/L). Further analyses revealed that serum gastrin was positively correlated with 24 h urinary sodium excretion (r = 0.476, p < 0.001). By contrast, gastrin level was negatively correlated with blood pressure in all dietary interventions (r = -0.188, p = 0.031). The present study indicated that variations in dietary salt and potassium supplementation affected the serum gastrin concentrations in the Chinese subjects.

Obscurumines H-P, new Lycopodium alkaloids from the club moss Lycopodium obscurum.

Seven new fawcettimine-type (1-7) and two new lycopodine-type (8 and 9) Lycopodium alkaloids, as well as 10 known compounds, were isolated from the club moss, Lycopodium obscurum L. The structures of obscurumines H-P (1-9) were determined based on high-resolution MS and 1D and 2D NMR data. Compounds 1 and 2 include a new skeleton that is formed via the linkage of C-9-N-2', which is rarely present in Lycopodium alkaloids. The in vitro acetylcholinesterase (AChE) inhibitory activity assay showed that 5 exhibited weak anti-AChE activity with an IC50 value of 81.0 µM. Compound 8 exhibited inhibition of the secretion of IL-2 in phytohemagglutinin (PHA) and phorbol myristate acetate (PMA) stimulated Jurkat cells, and the IC50 value was 17.2 µM.