Tetracyclic Triterpene Research Articles

Pharmacokinetics and cytotoxic study of euphol from Euphorbia umbellata (Bruyns) Pax latex

BackgroundMedicinal plants are an important source to identify new active pharmaceutical compounds. Traditionally, the sap of Euphorbia umbellata is widely used to treat cancer and inflammatory conditions. These effects have been attributed to the presence of terpenes and phenolic compounds in the extracts of this plant. Euphol, a tetracyclic triterpene alcohol, is one of the major compounds present in Euphorbia species, and some biological activities have been attributed to this compound. PurposeThis study aimed to evaluate the in vitro cytotoxicity of euphol against Jurkat, HL-60, K-562, B16F10, and HRT-18 cells lines, as well as the biological stability, distribution, metabolism properties in vitro, and the determination of the concentration of euphol in the plasma and liver of rats. MethodsThe MTT reduction assay was used to evaluate the cytotoxicity of euphol against cancer cell lines, and the selectivity index, the morphology and cell cycle assays to evaluate the death mechanisms in K-562 and B16F10 lineages. UHPLC-MS was applied for the in vivo evaluation of the concentration of euphol in plasma and liver, and in vitro metabolic stability in human liver microsomes and S9 fraction, plasma protein binding, and stability in simulated gastric and intestinal fluids assays. ConclusionsThis study demonstrated that euphol exhibited cytotoxic effects against a variety of cancer cells lines, selectivity against leukemia and possibly, the mechanism involved is apoptosis. The evaluation of stability, distribution, and metabolism properties showed that euphol was unstable in gastric and intestinal fluids, presenting moderate plasma protein binding with two hours elimination half-life and possible phase II liver metabolism. All the results suggested that further studies could be developed to prove the viability of euphol as an anticancer agent.

Identification of key amino acid residues determining product specificity of 2,3-oxidosqualene cyclase in Oryza species.

Triterpene synthases, also known as 2,3-oxidosqualene cyclases (OSCs), synthesize diverse triterpene skeletons that form the basis of an array of functionally divergent steroids and triterpenoids. Tetracyclic and pentacyclic triterpene skeletons are synthesized via protosteryl and dammarenyl cations, respectively. The mechanism of conversion between two scaffolds is not well understood. Here, we report a promiscuous OSC from rice (Oryza sativa) (OsOS) that synthesizes a novel pentacyclic triterpene orysatinol as its main product. The OsOS gene is widely distributed in indica subspecies of cultivated rice and in wild rice accessions. Previously, we have characterized a different OSC, OsPS, a tetracyclic parkeol synthase found in japonica subspecies. Phylogenetic and protein structural analyses identified three key amino acid residues (#732, #365, #124) amongst 46 polymorphic sites that determine functional conversion between OsPS and OsOS, specifically, the chair-semi(chair)-chair and chair-boat-chair interconversions. The different orientation of a fourth amino acid residue Y257 was shown to be important for functional conversion The discovery of orysatinol unlocks a new path to triterpene diversity in nature. Our findings also reveal mechanistic insights into the cyclization of oxidosqualene into tetra- and pentacyclic skeletons, and provide a new strategy to identify key residues determining OSC specificity.

New cycloartane-type ester triterpenes from Euphorbia pterococca and biological evaluation

From acetonic extract of the whole plant Euphorbia pterococca Brot. (Euphorbiaceae), four new cycloartane-type ester triterpenes named cycloartenyl-2′E,4′E-decadienoate (1), cycloartenyl-2′E,4′Z-decadienoate (2), 24-methylenecycloartanyl-2′E,4′Z-tetradecadienoate (3), and 24-oxo-29-norcycloartanyl-2′E,4′Z-hexadecadienoate (4) were obtained along with nine known tetracyclic triterpenes (5–13). Their structures were established mainly by extensive use of spectroscopic techniques, including 1D (1H and 13C) and 2D homo- and heteronuclear NMR experiments (COSY, HSQC, HMBC and NOESY), and mass spectrometry (HRESIMS), and by comparison with data reported in the literature. In addition, the new compounds 1–3 have been tested for cytotoxicity, trypanocidal effects and on enzymes involved in endocannabinoid degradation. While inactive in all assays up to 100 μM, 1 showed selective inhibition of α/β-hydrolase 12 with an IC50 of 11.6 ± 1.9 μM.

Hydrophobic Constituents Extracted from Chaga by Ethylacetate

Chaga can be extracted with EtOAc to afford ~0.88% extracted substances, of which ~58% are lipophilic compounds with the remainder being phenolic compounds. Tetracyclic triterpenes, sterols and their esters, sesquiterpenes (azulenes), bicyclic monoterpenes (iridoids), phenolcarboxylic acids, simple phenols, and flavonoids were identified in the extract. Stilbene-type compounds, a phenanthrene derivative, and 9(11)-dihydroergosteryl benzoate were observed in chaga for the first time.

Natural product gelators and a general method for obtaining them from organisms.

Since the late 1980s, low molecular weight gelators (LMWGs) based on different classes of natural products have been reported. Until 2011, pure natural LMWGs (i.e., natural product gelators, NPGs) were not found. However, today only five NPGs are reported. We think that this may be due to the lack of awareness about the importance of NPGs and suitable methods to discover new NPGs. Here we illustrate the potential significance of NPGs, and present a general and efficient method for obtaining NPGs from organisms, which provides specific and important guidance to researchers for easy discovery of new NPGs from organisms in the future. Using this method, we screened a total of 64 kinds of organisms (including plants, animals and fungi), and 6 extracts with a gelation ability were tracked and isolated to yield six new NPGs. These new NPGs include new types of NPGs such as tricyclic triterpenes (1) and tetracyclic triterpenes (2), and new classes of NPGs such as steroids (4 and 5) and glycosides (6), which greatly expand the class of NPGs in the LMWG field.

Brucein D, a Naturally Occurring Tetracyclic Triterpene Quassinoid, Induces Apoptosis in Pancreatic Cancer through ROS-Associated PI3K/Akt Signaling Pathway.

Brucein D (BD), a major active quassinoid in Brucea javanica, has exhibited pronounced anticancer activities. However, the biologic mechanisms have not been fully explored. In this study, BD exhibited more potent cytotoxic effect on pancreatic cancer (PanCa) cell lines, while exerted weaker cytotoxic effects on GES-1 cells (non-tumorigenic). BD was shown to elicit apoptosis through inducing both the intrinsic and extrinsic mitochondria-mediated caspase activations. Furthermore, the BD-induced apoptotic effects were dependent on the accumulated reactive oxygen species (ROS) and inactivation of PI3K/Akt signaling pathway. Pretreatment with tempol completely prevented the cellular apoptosis induced by BD, and recovered the inactivation of AKT, which suggested ROS essentially involved in BD-elicited apoptosis and down-regulation of PI3K/Akt pathway. In addition, the results obtained from orthotopic xenograft in nude mice were congruent with those of the in vitro investigations. These results support the notion that BD held good potential to be further developed into an effective pharmaceutical agent for the treatment of PanCa.

Состав гидрофобных веществ чаги, извлекаемых этилацетатом

Показано, что этилацетатом из чаги можно извлечь около 0,88 % экстрактивных веществ, из которых около 58 % приходится на липофильные вещества, а остальная часть представлена фенольными соединениями. В экстракте определены тетрациклические тритерпены, стерины и их эфиры, сесквитерпены (азулены), бицикличекие монотерпены (иридоиды), фенолкарбоновые кислоты, простые фенолы и флавоноиды. Впервые в чаге обнаружены соединения, относящиеся к классу стильбенов, производное фенантрена и 9(11)-дигидроэргостерил бензоат.

Cucurbitacin B and SCH772984 exhibit synergistic anti-pancreatic cancer activities by suppressing EGFR, PI3K/Akt/mTOR, STAT3 and ERK signaling.

Cucurbitacin B (CuB) is a natural tetracyclic triterpene product and displays antitumor activity across a wide array of cancers. In this study, we explored the anti-pancreatic cancer activity of CuB alone and in combination with SCH772984, an ERK inhibitor, in vitro and in vivo. CuB inhibited proliferation of pancreatic cancer cells by arresting them in the G2/M cell cycle phase. This was associated with inhibition of EGFR expression and activity and downstream signaling, including PI3K/Akt/mTOR and STAT3. Interestingly, ERK activity was markedly enhanced by activating AMPK signaling after 12 h of CuB treatment. SCH772984 potentiates the cytotoxic effect of CuB on pancreatic cancer cells through complementary inhibition of EGFR, PI3K/Akt/mTOR, STAT3 and ERK signaling, followed by an increase in the pro-apoptotic protein Bim and a decrease in the anti-apoptotic proteins Mcl-1, Bcl-2, Bcl-xl and survivin. Furthermore, combined therapy with CuB and SCH772984 resulted in highly significant growth inhibition of pancreatic cancer xenografts. These results may provide a basis for further development of combining CuB and ERK inhibitors to treat pancreatic cancer.

Simultaneous determination of cucurbitacin B and cucurbitacin E in rat plasma by UHPLC-MS/MS: A pharmacokinetics study after oral administration of cucurbitacin tablets.

Cucurbitacin B (CuB) and cucurbitacin E (CuE) are tetracyclic triterpene compounds from Cucurbitaceae, and the main bioactive compounds of cucurbitacins tablets that used to treatment of chronic hepatitis. Pharmacological research has been very comprehensive, and there are few studies on pharmacokinetics, especially about CuE. An Ultra High Performance Liquid Chromatography-tandem Mass Spectrometry (UHPLC-MS/MS) method with high selectivity, simplicity and sensitivity has been used for quantitative analysis of Cucurbitacin B (CuB) and cucurbitacin E (CuE). Plasma samples were pretreatment by Liquid-liquid extraction (LLE) method with dichloromethane. The chromatographic separation was achieved on a C18 column (Agilent Eclipse Plus, 1.8μm, 50×2.1mm) using gradient elution with water - methanol at a flow rate of 0.3mL/min and the column temperature was set at 30°C. The method was validated according to FDA guidelines. Lower limit of quantification (LLOQ) was 1.60ng/mL for CuB and 1.58ng/mL for CuE. Correlation coefficients of CuB and CuE were more than 0.99 in rat plasma. All values of intra-day and inter-day precision (RSD%) were not exceeded 15%, the accuracy (RE%) were within -5.57 to 5.20% for CuB and -3.33 to 7.37% for CuE. The mean extraction recoveries were more than 80%. Pharmacokinetic parameters were also evaluated by UHPLC-MS/MS method. The results suggestion that this method was successfully applied to pharmacokinetic study of CuB and CuE in rat plasma after oral administration cucurbitacin tablets.

Integrating transcriptomics and metabolomics for the analysis of the aroma profiles of Saccharomyces cerevisiae strains from diverse origins

BackgroundDuring must fermentation thousands of volatile aroma compounds are formed, with higher alcohols, acetate esters and ethyl esters being the main aromatic compounds contributing to floral and fruity aromas. The action of yeast, in particular Saccharomyces cerevisiae, on the must components will build the architecture of the wine flavour and its fermentation bouquet. The objective of the present work was to better understand the molecular and metabolic bases of aroma production during a fermentation process. For such, comparative transcriptomic and metabolic analysis was performed at two time points (5 and 50 g/L of CO2 released) in fermentations conducted by four yeast strains from different origins and/or technological applications (cachaça, sake, wine, and laboratory), and multivariate factorial analyses were used to rationally identify new targets for improving aroma production.ResultsResults showed that strains from cachaça, sake and wine produced higher amounts of acetate esters, ethyl esters, acids and higher alcohols, in comparison with the laboratory strain. At fermentation time T1 (5 g/L CO2 released), comparative transcriptomics of the three S. cerevisiae strains from different fermentative environments in comparison with the laboratory yeast S288c, showed an increased expression of genes related with tetracyclic and pentacyclic triterpenes metabolism, involved in sterol synthesis. Sake strain also showed upregulation of genes ADH7 and AAD6, involved in the formation of higher alcohols in the Ehrlich pathway. For fermentation time point T2 (50 g/L CO2 released), again sake strain, but also VL1 strain, showed an increased expression of genes involved in formation of higher alcohols in the Ehrlich pathway, namely ADH7, ADH6 and AAD6, which is in accordance with the higher levels of methionol, isobutanol, isoamyl alcohol and phenylethanol observed.ConclusionsOur approach revealed successful to integrate data from several technologies (HPLC, GC-MS, microarrays) and using different data analysis methods (PCA, MFA). The results obtained increased our knowledge on the production of wine aroma and flavour, identifying new gene in association to the formation of flavour active compounds, mainly in the production of fatty acids, and ethyl and acetate esters.

Inducement of apoptosis by cucurbitacin E, a tetracyclic triterpenes, through death receptor 5 in human cervical cancer cell lines

Cervical cancer is the most common malignancy in women, for which conization or hysterectomy are the main therapy. Curcubitacin E (Cu E) is a natural compound-based drug which from the Guadi (climbing stem of Cucumic melo L). Previously shown to be an anti-tumor as well as a potent chemopreventive agent against several types of tumors. The present study, investigated anti-proliferation and apoptosis induced by Cu E in cervical cancer cell lines (HeLa and Ca Ski). The results indicate that the cytotoxicity is associated with accumulation in apoptosis but not necrosis. Cu E produced apoptosis as well as the up-regulation the expression of death receptor 5 (DR5). In addition, the DR5 gene activation in apoptosis, both effects increased proportionally with the dose of Cu E; however, mitosis delay was also dependant on the amount of Cu E treatment in the cancer cells. These results indicate that Cu E may delay cancer cell growth by apoptosis via upregulation of DR5 gene expression.

Convergence and divergence of bitterness biosynthesis and regulation in Cucurbitaceae.

Differentiation of secondary metabolite profiles in closely related plant species provides clues for unravelling biosynthetic pathways and regulatory circuits, an area that is still underinvestigated. Cucurbitacins, a group of bitter and highly oxygenated tetracyclic triterpenes, are mainly produced by the plant family Cucurbitaceae. These compounds have similar structures, but differ in their antitumour activities and ecophysiological roles. By comparative analyses of the genomes of cucumber, melon and watermelon, we uncovered conserved syntenic loci encoding metabolic genes for distinct cucurbitacins. Characterization of the cytochrome P450s (CYPs) identified from these loci enabled us to unveil a novel multi-oxidation CYP for the tailoring of the cucurbitacin core skeleton as well as two other CYPs responsible for the key structural variations among cucurbitacins C, B and E. We also discovered a syntenic gene cluster of transcription factors that regulates the tissue-specific biosynthesis of cucurbitacins and may confer the loss of bitterness phenotypes associated with convergent domestication of wild cucurbits. This study illustrates the potential to exploit comparative genomics to identify enzymes and transcription factors that control the biosynthesis of structurally related yet unique natural products.

Production of a new tetracyclic triterpene sulfate metabolite sambacide by solid-state cultivated Fusarium sambucinum B10.2 using potato as substrate

The aim of this work is to explore integracide analogues from secondary metabolites of microorganisms. A new tetracyclic triterpene sulfate was produced by solid-state fermentation (SSF) with Fusarium sambucinum B10.2. The tetracyclic triterpene sulfate was identified as (3S,5R,10S,11S,12S,13R,17R,20R)-4,4-dimethylergosta-8,14,24-triene-3,11,12-triol-12-acetate, 3-sulfate on the basis of HRESIMS, NMR and electronic circular dichroism (ECD) spectra and named sambacide (1). The antibacterial and antifungal assays of sambacide (1) showed significant antibacterial activities against Staphylococcus aureus and Escherichia coli. The fermentation conditions including culture media, fermentation temperature and time, were optimized. And potato was selected as the fermentation substrate, 28°C was used as the fermentation temperature, and 20-days fermentation time was determined for F. sambucinum-SSF to produce sambacide (1) with a high yield of 19.04±0.82g/kg. This paper provides an efficient approach to produce the antibacterial and antifungal agent sambacide (1) in a very high yield.

Cucurbitacin E Potently Modulates the Activity of Encephalitogenic Cells.

Cucurbitacin E (CucE) is a highly oxidized steroid consisting of a tetracyclic triterpene. It is a member of a Cucurbitacin family of biomolecules that are predominantly found in Cucurbitaceae plants. CucE has already been identified as a potent anti-inflammatory compound. Here, its effects on CD4(+) T helper (Th) cells and macrophages, as the major encephalitogenic cells in the autoimmunity of the central nervous system, were investigated. Production of major pathogenic Th cell cytokines: interferon-gamma and interleukin-17 were inhibited under the influence of CucE. The effects of CucE on CD4(+) T cells were mediated through the modulation of aryl hydrocarbon receptor, STAT3, NFκB, p38 MAPK, and miR-146 signaling. Further, production of nitric oxide and reactive oxygen species, as well as phagocytic ability, were inhibited in macrophages treated with CucE. These results imply that CucE possesses powerful antiencephalitogenic activity.

Isolation and Structure of Cancer Cell Growth Inhibitory Tetracyclic Triterpenoids from the Zimbabwean Monadenium lugardae.

The Zimbabwean medicinal plant Monadenium lugardae was evaluated as a potential source of new anticancer constituents. Four new tetracyclic triterpene (1-4) were isolated, accompanied by four previously known triterpenes (5-8). Against a panel of human tumor cell lines, lugardstatins 1 (1) and 2 (2) had good cancer cell growth inhibitory activity. All of the triterpene structures (1-8) were established by 1D and 2D NMR spectrometric and HR mass spectrometric analysis.

Protostane alisol derivatives from the rhizome of Alisma orientale

Alisma orientalis is a well-known traditional Chinese medicine with diuretic, hypopietic, hypolipemic, and antiatherosclerotic activities. In the present study, we investigated triterpenoids from the rhizome of A. orientalis, identifying four new alisol derivatives, namely, Alismanol M (1), Alismanol Q (2), Alismanol O (3) and Alismanol P (4). The structures of these compounds were characterized using detailed spectroscopic analysis (UV, HRESIMS, 1D and 2D NMR data including HSQC, HMBC, 1H–1H COSY, and NOESY). These four new alisol derivatives were all protostane triterpenoids, belonging to a group of tetracyclic triterpenes. A 17, 23- or 20, 24-five-membered oxide ring is the primary structural characteristic of these alismanols (1–4). Furthermore, in vitro bioassays demonstrated that the isolated compounds could not inhibit nitric oxide production in LPS-induced macrophages.

Tetra- and Penta-Cyclic Triterpenes Interaction with Lipid Bilayer Membrane: A Structural Comparative Study.

The effect of tetracyclic (cortisol, prednisolone, and 9-fluorocortisol acetate) and pentacyclic (uvaol and erythrodiol) triterpenes (TTPs) on the fluidity of dipalmitoyl phosphatidyl choline (DPPC) liposome membrane was investigated by differential scanning calorimetry, Raman spectroscopy, and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Liposomes were prepared in the absence and presence of TTPs at molar ratios DPPC:TTP 100:1, 100:2.5, and 100:10. All the studied TTPs abolished the pre-transition and modified the intensity of the Raman peak at 715cm(-1) proving the interaction of TTP molecules with the choline head group of phospholipids. An increase in the Raman height intensity ratios of the peaks I 2935/2880, I 2844/2880, and I 1090/1130, giving information about the ratio disorder/order of the alkyl chains, and a decrease of the main transition temperature demonstrated the interaction of TTPs with the alkyl chains. The tetracyclic TTPs produced broadening of the phase transition profile. Besides, a scarcely splitting of the main transition peak was obtained with prednisolone and 9-fluorocortisol acetate. The results of fluorescence depolarization of DPH showed that the studied molecules fluidized the liposomal membrane at 25, 41, and 50°C. Pentacyclic TTPs, being more hydrophobic than tetracyclic ones, demonstrated higher fluidizing effect than tetracyclic TTPs in the liquid crystalline phase suggesting a deeper incorporation in the lipid bilayer. The presence of a free polar head group at the ring D seems to control the TTP incorporation in the bilayer and consequently its effect on the membrane fluidity.

Cucurbitacins from the Leaves of Citrullus colocynthis (L.) Schrad.

Two new tetracyclic cucurbitane-type triterpene glycosides were isolated from an ethyl acetate extract of Citrullus colocynthis leaves together with four known cucurbitacins. Their structures were established on the basis of their spectroscopic data (mainly NMR and mass spectrometry). Evaluation of the in vitro cytotoxic activity of the isolated compounds against two human colon cancer cell lines (HT29 and Caco-2) and one normal rat intestine epithelial cell line (IEC6), revealed that one of the isolated compounds presented interesting specific cytotoxic activity towards colorectal cell lines.

The antinociceptive effects of the tetracyclic triterpene euphol in inflammatory and neuropathic pain models: The potential role of PKCε

Evidences suggest protein kinase C epsilon (PKCε) activation is involved in both inflammatory and neuropathic pains. We have previously shown that tetracyclic triterpene euphol produces antinociception in different models of persistent pain, an action associated with its anti-inflammatory properties. Among these properties are the cannabinoid system activation and different PKC isozymes modulation. Herein, we sought to explore the potential role of PKCε modulation on euphol antinociceptive effect, in inflammatory and neuropathic pain models, in rodents. Also, we investigated further mechanisms associated with euphol effects. Oral treatment with euphol (30mg/kg) prevented the putative effect of PGE2-induced acute and persistent mechanical hypersensitivity in mice and rats, respectively. In the PGE2-induced acute mechanical hypersensitivity euphol promoted an inhibitory effect similar to a PKCε inhibitor peptide. Likewise, in rats it prevented the mechanical hypersensitivity induced by a PKCε activator. Conversely, euphol effectiveness was not observed in a cAMP/PKA-induced mechanical hypersensitivity in mice. Single (1h prior) or repeated (twice daily during 3 or 13days) treatments with euphol ameliorated painful peripheral neuropathy induced by paclitaxel and also the mechanical hypersensitivity induced by B16F10 melanoma cells injection, in mice. Additionally, in both inflammatory and neuropathic pain models, euphol consistently prevented PKCε up-regulation, as well as, inhibited the up-regulation of PKCε-activated intracellular pathways; namely nuclear factor-κB (NF-κB), cyclic AMP response element binding protein (CREB) and cyclo-oxygenase-2 (COX-2). The present results suggest the antinociceptive effect on persistent pain caused by euphol is likely dependent on the inhibition of pro-inflammatory mediators modulated by PKCε.

Transcriptome profiling and analysis of Panax japonicus var. major

The rhizome of Panax japonicus var. major have been used as the natural medicinal agent by Chinese traditional doctors for more than thousand years. Most of the therapeutic effects of P. japonicus var. major had been reported due to the presence of tetracyclic or pentacyclic triterpene saponins. In this study, Illumina pair-end RNA-sequencing and de novo splicing were done in order to understand the pathway of triterpenoid saponins in this species. The valid reads data of 15. 6 Gb were obtained. The 62 240 unigenes were finally obtained by de novo splicing. After annotation, we discovered 19 unigenes involved in ginsenoside backbone biosynthesis. Additionally, 69 unigenes and 18 unigenes were predicted to have potential function of cytochrome P450 and UDP-glycosyltransferase based on the annotation results, which may encode enzymes responsible for ginsenoside backbone modification. This study provides global expressed datas for P. japonicus var. major, which will contribute significantly to further genome-wide research and analysis for this species.